1. science
2. chemistry

ISBN: 978-9974-0-0911-0
Abstracts and Proceedi n gs of the
12th Inter national Sym posi u m on
Fe
Cu
Zn
Ru
Pt
Au
Pb
Si
Hg
Cr
Metal Ion s i n Biology and Medici ne
María H. Torre and Dinorah Gambino (Eds.)
11, 12 and 13 March 2013
Pu nta del Este, Uruguay
March 2013, Montevideo, Uruguay
ISBN: 978-9974-0-0911-0
Editors:
María H. Torre and Dinorah Gambino
General Chairpersons
Dr. Dinorah Gambino and Dr. María H. Torre
Inorganic Chemistry, Faculty of Chemistry
Organizing committee
Dr. Alicia Cuevas
Inorganic Chemistry, Faculty of Chemistry
Dr. Gianella Facchin
Inorganic Chemistry, Faculty of Chemistry
Ing. Eduardo Kremer
Inorganic Chemistry, Faculty of Chemistry
Dr. William Manzanares
Intensive Care Unit, Hospital de Clínicas, Faculty of Medicine
Dr. Nelly Mañay
Toxicology, Faculty of Chemistry
Dr. Lucía Otero
Inorganic Chemistry, Faculty of Chemistry
Dr. Mariela Pistón
Analytical Chemistry, Faculty of Chemistry
Dr. Ana Rey
Radiochemistry, Faculty of Chemistry
Dr. Inés Viera
Inorganic Chemistry, Faculty of Chemistry
Scientific Committee
Philippe Collery (France)
Susana Etcheverry (Argentina)
Gérard Bastian (France)
Antonio José Costa-Filho (Brazil)
Laetitia Pele (UK)
Alzir Azevedo Batista (Brazil)
Alejandro Vila (Argentina)
Ademir Neves (Brazil)
Etelka Farkas (Hungary)
Enrique J. Baran (Argentina)
Lena Ruiz-Azuara (Mexico)
Virtudes Moreno (Spain)
Victor Deflon (Brazil)
Ana María da Costa Ferreira (Brazil)
Paul Tchounwou (USA)
Declared of interest by:
Ministerio de Turismo, Uruguay
Edited by:
Cristina Álvarez
Gold Sponsors:
Bronze Sponsor:
Other sponsors:
Plenary Lectures ...........................................................................................................................................................6
Keynotes .......................................................................................................................................................................9
Oral Presentations ......................................................................................................................................................28
Poster Presentations ..................................................................................................................................................70
Proceedings ..............................................................................................................................................................145
Areas
MBD
Metal based drugs
AAMMIBBS
Advanced analytical methods for metal ions in biochemical and biological systems
MIEH
Metal ions in environmental health
TEMIBS
Toxicological effects of metal ions in biological systems
NATME
Nutritional aspects of trace and major elements
SBM
Structural biology of metalloproteins and metal-based redox processes
Plenary Lectures
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 149
OPENING LECTURE
Designing metalloproteins with hydrolytic, redox and electron transfer centers
Pecoraro, V.L.; Zastrow, M.; Cangelosi, V.; Tegoni, M.; Yu, F.; Tebo, A.; Plegaria, J.
Department of Chemistry, University of Michigan, Ann Arbor, MI, USA 48109-1055
We will use de Novo Protein Design to provide synthetic constructs to develop metalloproteins capable of hydrolytic
and redox catalysis. Examples will include hydrolases containing mononuclear Zn centers and reductases
containing Cu. We will discuss the approach and advantages of this strategy to understand the functioning of
metals in biology. We will show a mimic of carbonic anhydrase that precisely mimics the first coordination sphere of
this enzyme and is capable of efficient, multiturnover hydrolysis of nitrophenylacetate or carbón dioxide in aqueous
solution over the pH range 7.5 to 9.5. We will examine how changing the location of the site impacts the catalytic
reaction and effects substrate recognition. We will then discuss using the same peptide, now substituted with
copper, which can reduce nitrie to NO mimicking the Cu Nitrite Reductase. We will show how surface modifications
can influence the copper reduction potential and modify the reaction rate. If time permits, we will also discuss
progress on preparing rubredoxin and cupredoxin centers within designed systems. These studies illustrate how
advances in solid phase peptide synthesis and high level gene expression allow chemists to probe fundamental
questions in metallobiochemistry.
6
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 150
CLOSING LECTURE
Reactions of Mn-superoxide dismutase and manganese porphyrins with peroxynitrite:
relevance to mitochondrial dysfunction and redox-based pharmacology
Radi, R.
Departamento de Bioquímica and Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la
República, Montevideo, Uruguay
e-mail: [email protected]
Excess biological formation rates of free radicals and oxidants are associated to biomolecular oxidative damage
and disease development. Among the key endogenous antioxidant mechanisms is the mitochondrial, manganesecontaining, superoxide dismutase (MnSOD). MnSOD is an essential protein in mammals with its primary
functionbeing the detoxification of mitochondrial-derived superoxide radical (O2−). The diffusion-controlled reaction
of O2− with nitric oxide (NO) leads to the formation of peroxynitrite (ONOO−), an oxidizing and nitrating cytotoxin,
known to cause mitochondrial dysfunction. Thus, in mitochondria, MnSOD is a key element controlling the
formation of peroxynitrite and therefore, provides protection from its mitochondrial-damaging effects. However,
5
-1 -1
peroxynitrite readily reacts with MnSOD (k ~ 10 M s ) and therefore, sustained mitochondrial levels of
peroxynitrite promote its inactivation via a Mn-catalyzed nitration reaction at the active site Tyr34 both in vitro and in
vivo. Then, peroxynitrite-dependent MnSOD nitration and inactivation triggers a pro-oxidant vicious cycle, that can
ultimately cause severe alteration in mitochondrial, and subsequently cellular, homeostasis. As a way to provide
further antioxidant protection to mitochondria under pathophysiologically-relevant conditions involvingperoxynitrite,
we have studied a series of Mn-based compounds, manganese-porphyrins (MnP), originally conceived to act as
2+
6
7
-1 -1
“superoxide dismutase-mimics”. We have found that Mn P react fast with peroxynitrite (k ~ 10 -10 M s ) and
reduce it to nitrite; in turn, the oxidized Mn species (Mn4+, Mn3+) can be reduced back to the Mn2+ redox state by
mitochondrial reductants and the electron transport chain to complete a catalytic cycle of peroxynitrite
detoxification. These redox interactions of MnP with peroxynitrite and mitochondrial reductants provide
pharmacological protection in vitro and in vivo, and have opened promising opportunities for mitochondrial- and
redox-based pharmacology aimed to cope with the toxic effects of peroxynitrite.
7
Keynotes
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 016
AREA: AAMMIBBS
KEYNOTE
Trace elemental and speciation analysis based on volatile species generation and on
atomic absorption/fluorescence detectors - current state and perspectives
1
1
1
1
1
2
Dědina, J. ; Kratzer, J. ; Matoušek, T. ; Musil, S. ; Svoboda, M. ; Currier, J.M. ; Stýblo, M.
2
1
Department of Trace Element Analysis, Institute of Analytical Chemistry of the ASCR, v. v. i., Veveří 97, Brno, Czech Republic,
Prague, Czech Republic
2
Department of Nutrition, University of North Carolina at Chapel Hill, Chapel Hill, USA [email protected]
Detection power of atomic spectrometry detectors can be substantially improved employing generation of volatile
species (VSG) of elements since separation from the sample matrix and high efficiency analyte transfer from
sample to spectrometer are involved. In addition, it makes analyte preconcentration easy. All these factors result in
a superior sensitivity. VSG is therefore ideally suited for trace and ultratrace elemental/speciation analysis.
This presentation will briefly overview the recent developments and perspectives of individual approaches to trace
and ultratrace elemental/speciation analysis based on atomic absorption (AAS) and atomic fluorescence (AFS)
detection coupled to VSG. It will be shown that the combination of VSG with AAS or AFS can bring substantial
benefits for elemental as well as speciation analysis of those elements that can be converted to analytically useful
volatile species. VSG with AAS or AFS can thus substitute or even surpass conventional approaches to elemental
and speciation analysis based on the liquid phase sampling with inductively coupled plasma mass spectrometry
which generally serves as a trademark of unparalleled sensitivity.
The applicability of the combination of VSG with AAS or AFS will be illustrated on our recent results:
- hydride trapping in quartz tube atomizers for AAS for ultratrace elemental determination of hydride forming
elements, namely in the presence of very high concentrations of other hydride forming elements;
- generation of substituted arsines followed by cryogenic trapping and atomization in the multiatomizer (AAS
detector) for arsenic speciation analysis in extremely difficult matrices and in liver homogenate slurry;
- generation of substituted arsines followed by cryogenic trapping and atomization in the flame-in-gas-shield
atomizer for AFS for ultratrace analysis of toxicologically important arsenic species.
Acknowledgment: The institutional support RVO:6808171 as well as support from ASCR (m200311202), MŠMT
program Kontakt II (LH12040) and UNC Gillings School of Global Public Health is gratefully acknowledged.
9
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 132
AREA: AAMMIBBS
KEYNOTE
Metalloproteomics and chemical speciation: towards to a perfect couple
Zezzi Arruda, M.A.
Spectrometry, Sample preparation and Mechanization Group – GEPAM
Institute of Science and Technology for Bioanalytics, Institute of Chemistry, Department of Analytical Chemistry, UNICAMP
Campinas, Brazil
[email protected]
Metalloproteomics encompasses the inorganic elements content and assemble of their complexes with proteins [1].
Although metalloenzymes and metalloproteins are responsible for nitrogen fixation, photosynthesis and respiration,
and for catalyzing molecular oxygen reduction [2], the information related to proteins and metals is frequently found
fragmented in the literature. This lack of information contributes to remain allusive some mechanisms involving
metal incorporated as a cofactor in a cell [3], metal-sensing related to metalloproteins [4], among others. In this
way, this presentation outcome, besides brief definitions and contextualization of the area, some studies which are
being carried out in our research group encompassing comparative metalloproteomics involving plant stress as well
as some preliminary experiments related to chemical speciation involving metalloproteins.
References
[1] Garcia, J. S., Magalhães, C. S., Arruda, M. A. Z., Talanta 69 (2006) 1-15
[2] Lu, Y., Yeung, N., Sierack, N., Marshall N. M., Nature 460 (2009) 855-862
[3] Szpunar, J., Anal. Bioanal. Chem. 378 (2004) 54-56
[4] Waldron, K. J., Rutherford, J. C., Ford, D., Robinson, N. J., Nature 460 (2009) 823-830
Acknowledgements: Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP, São Paulo, Brazil),
Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brasília, Brazil) and Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brasília, Brazil) are greatly acknowledged for financial
support and fellowships.
10
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 014
AREA: MBD
KEYNOTE
Metal complexes based on oxindolimine ligands: antitumor and antiparasite activities
Da Costa Ferreira, A.M.1; de Paula, Q.A.1; Sabino, G.L.1; Dario, B.S.1; Ribeiro, G.A.2; Vieira, L.Q.2
1
2
Department of Chemistry, University of Sao Paulo, Sao Paulo, Brasil
Institute Biological Sciences, Federal University Minas Gerais, Belo Horizonte, Brasil [email protected]
Some oxindolimine compounds have been designed and prepared, based on oxindole derivatives already tested
clinically. These compounds were then metallated with essential ions (copper, zinc or vanadium) given rise to very
stable complexes, isolated and characterized by different spectroscopic techniques. Copper complexes showed
stability constants with log K = 15-17. Their reactivities towards fundamental biomolecules were verified in order to
investigate its possibilities as new therapeutical agents. In a process modulated by the ligand, monitored by CD
and EPR measurements, the corresponding copper(II) complexes interact with human serum albumin, inserting
selectively the metal ion at the N-terminal site, and changing remarkably the α-helix structure of the protein. They
can also effectively bind to DNA, with binding constants determined in the range 102 for plasmidial DNA, probably
at minor or major grooves, depending on the ligand. As cationic lipophilic species, these complexes can cross
membranes and enter into cells, where in the presence of common oxidant agents are able to generate reactive
oxygen species (ROS). The cytotoxicity of these complexes were tested against different tumor cells
(neuroblastoma, melanoma, and sarcoma), indicating that some of them are very effective on causing oxidative
damage to intracellular organelles, and on inducing apoptosis. The corresponding zinc(II) complexes also showed
interaction with HSA, in different metal binding sites, being less cytotoxic against tumor cells. Analogous vanadyl
complexes were also isolated and characterized. Furthermore, these metal complexes showed good activity
against T. cruzi, modulated both by the metal ion and the ligand.
Acknowledgements: FAPESP / INCT Redoxoma / Nanobiomed
11
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 069
AREA: MBD
KEYNOTE
Cell and tissue distribution of rhenium and selenium after a rhenium diselenate treatment
Collery, P.1; Santoni, F.2; Mohsen, A.3; Desmaele, D.4; D'Angelo, J.5; Wei, M.6; Collery, T.7; Bastian, G.8
1
Corsica, Polyclinique Maymard, Bastia, France
Office d'Equipement Hydraulique, Bastia, France
3
Cairo, Egypt
4
Université Paris Sud, Paris, France
5
Université Paris Sud, Paris, France
6
Cellvax Laboratory, Paris, France
7
Polyclinique Maymard, Bastia, France
8
Laboratoire de Pharmacologie, Paris, France
[email protected]
2
Rhenium diselenate is a new anticancer agent, combining an atom of rhenium (Re) and two atoms of selenium
(Se).
We studied the Re and Se cell uptake in different cancer cells, with several doses and after different times of
exposure and also after a period of wash-out.
The tissue distribution was studied after an oral daily administration (5 days a week for 4 weeks) of either 10 or 40
mg/kg of rhenium diselenate in mice bearing a MDA- MB 231 tumour, with a comparison with controls receiving a
vehicle. We also studied the interactions with essential metals, magnesium (Mg), zinc (Zn), iron (Fe).
We observed a correlation between the Re cell uptake and the inhibitory effects of rhenium diselenate in cancer
cells, depending on the type of cancer cells. We also noted an efflux from the cells after a period of 72 h after the
end of the exposure time.
In mice, the Re and Se concentrations increased from 10 to 40 mg/kg but much more in the healthy tissues than in
the tumour. The main concentrations were noted in the liver.
The results on the effects of the rhenium diselenate compound on the concentrations of essential metals will be
presented, but the data are not yet available and cannot be included in this abstract.
We conclude that pharmacological data are essential to determine the best schedule of treatment of metal
compounds.
12
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 126
AREA: MBD
KEYNOTE
Consequences of metal coordination to nucleobases: from alkali ions to platinum
Lippert, B.
Prof. of Inorganic Chemistry, Faculty of Chemistry, Technische Universitaet Dortmund, Germany
[email protected]
Potassium, sodium, and magnesium ions are the dominant natural counter ions of the negatively charged nucleic
acids, binding usually in a purely electrostatic or outer-sphere fashion, with the capacity, however, to influence the
overall structure (e.g. DNA curvature) and/or to stabilize particular structures (e.g. nucleobase quartets). Innershere binding, hence coordination of exogenous (transition) metal ions to nucleic acid constituents in general has
more profound consequences on the electronic properties of the nucleobases. These include effects on acid-base
properties of the nucleobases, on tautomerism and hydrogen bonding properties, on base pairing, and on overall
structure. Occasionally the metal can also cause irreversible modifications of the nucleobase or the oligonucleotide.
The lecture will provide an overview on metal binding patterns to nucleic acids in general and to nucleobases in
particular, and will focus especially on X-ray structural work as well as solution studies of Pt complexes of model
nucleobases carried out in the author's laboratory during the past three decades. The relationship and relevance to
biological findings (e.g. antitumor activity of Cisplatin, mutagenicity of heavy metals) will be pointed out and
discussed.
13
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 134
AREA: MBD
KEYNOTE
Mixed chelates copper(II) compounds Casiopeínas, first and second generation:
Antitumor activity and mechanism of action
Ruiz-Azuara, L.1*; Mejía, C.2; Bravo Gómez, M.E.1; García Ramos, J.C.1; Cortés, F.3; Galindo, R.4; Moreno1
5
6
1
1
7
8
Esparza, R. ; Hernández-Lemus, E. ; Alemón, R. ; Ballinas, G. ; Reina, M. ; Pessoa, J. ; Gambino, D. ; Moreno,
9
V.
1
2
3
4
Facultad de Química; Instituto de Investigaciones Biomédicas; Instituto de Química, UNAM, Mexico, University of Utah,
6
7
8
INMEGEN Mexico, INP Mexico, Instituto Superior Tecnico, Lisboa, Portugal, Universidad de la República, Montevideo,
9
Uruguay, Universidad de Barcelona, España.
*
[email protected]
5
Metal complexes have gained a growing interest as pharmaceuticals for their use as diagnostic agents or as
chemotherapeutic drugs [1]. In our group some efforts have been done in the development of anticancer agents
employing essential metals such as the family of copper (II) compounds known as Casiopeínas®, with a general
formula [Cu (N-N) (X-Y) H2O] NO3 where N-N is a diimine (phen or bipy) and X-Y is a bidentate ligand (acac, salal,
aminoacidate, peptide, benzimidazol). These compounds have shown cytostatic, cytotoxic and antineoplastic
activity in vitro and in vivo [2]. Comparison between first and second generation of Casiopeina´s activity is
presented. Also albumin/Casiopeinas interaction is discussed.
The mechanism of action is still not completely elucidated. However, experimental evidence suggests the
interaction of coordination compounds with DNA (nuclear or mitochondrial) and their components and the
generation of reactive oxygen species (ROS) as the main action pathways. Induction of apoptosis has been proved
to be the main death tumoral cell pathway.
DNA interaction has been done by several methods such as comet assay, AFM, electrophoresis, CD [3] and the
®
nuclease activity studies of 4 Casiopeínas employing plasmidic DNA in several reaction conditions is presented as
well as the microarray assay of isolated RNA to visualize the complete pathways implicated in the cell death.
Comparison of the results shown that compounds with 4,7-dimethyl phenanthroline in the copper (II) coordination
sphere have an extraordinary DNA cleavage capacity, compared with the 4, 4´-diimine analogs, then is suggested
an intercalation process as the first step for the DNA damage. Also calculations have been done in order to
modeling the interaction between Casiopeínas and DNA [4, 5].
References
[1] S. H. van Rijt, P. J. Sadler, Drug Discov. Today, 14 (2009) 1089-1097.
[2] L. Ruiz-Azuara, M.E. Bravo-Gómez, Curr. Med. Chem., 17 (2010) 3606-3615.
[3] L. Becco, A. Rodríguez, M. E. Bravo, M. J. Prieto, L. Ruiz-Azuara, B. Garat, V. Moreno, D. Gambino J. Inorg.
Biochem., 109 (2012) 49-56.
[4] R. Galindo-Murillo, J. Hernandez-Lima, M. González-Rendón, F. Cortés-Guzmán, L. Ruíz-Azuara, R. MorenoEsparza Phys. Chem. Chem. Phys., 13 (2011) 14511-14516.
[5] R. Galindo-Murillo, L. Ruíz-Azuara, R. Moreno-Esparza, F. Cortés-Guzmán Phys. Chem. Chem. Phys., 14
(2012) 15539-15546.
14
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 135
AREA: MBD
KEYNOTE
Second coordination-sphere effects increase the catalytic efficiency of hydrolases
biomimetics
Neves, A.
LABINC – Laboratory of Bioinorganic and Crystallography, Department of Chemistry – UFSC, 88040-900, Florianópolis – SC,
Brazil
[email protected]
Phosphatases, oxidases and a variety of other enzymes have been successfully modeled through metal complexes
that can reproduce their physico-chemical properties and even their catalytic activity, however with a much lower
1
efficiency .
As has been suggested, these discrepancies in catalytic efficiency between model compounds and true enzymes
are, by a large extent, due to the lack of many important intramolecular interactions in the second-coordination
sphere of the model systems in comparison to the enzyme2.
In this work we propose a new extension of dinuclear hydrolases models by binding the FeIIIMII catalytic unit to a
small polyethyleneimine chain (PEI, 1200 Da) that can emulate the enzyme microenvironment around the active
2
site (Figure 1) . In addition we also present the synthesis, characterization and hydrolase activity of conjugates in
which the well known DNA intercalator pyrene is anchored to the FeIIIMII catalytic center2.
Figure 1
Acknowledgment: CNPQ and INCT-catálise
References
1) Mitic´, N.; Smith, S. J.; Neves, A.; Guddat, L. W.; Gahan, L. R.; Schenk, G. Chem. Rev. 2006, 106, 3338. 2) Neves et al,
unpublished results.
15
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 140
AREA: MBD
KEYNOTE
New metal ions in cancer research
Bastian, G.
Oncopharmacology, CHU Pitié-Salpêtrière, 91 Bld de l’hôpital, 75013, Paris, France.
Metal compounds have been used in medicine for several hundreds of years. In oncology, the first, and still the
most used, metal ion is Platinum. More than three thousands platinum derivatives have been synthesized and
tested against cancer cells. In this talk, last development about this metal will be presented (attempt of oral
chemotherapy with Cis platinum, Micro and Nano particles containing various platinum derivativesa).
Rhodium belongs to the same group as Platinum and show interesting antitumor activity but with the same
nephrotoxicity. Clinical phase I will be discussed.
Gallium anticancer properties are known since 1971. The antiproliferative properties of Gallium could be related to
its competition with the iron metalGallium nitrate, gallium chloride have been tested successfully orally or
intravenously in different clinical trials. Gallium salicylate has been tested in preclinical trial. It was observed that
the inhibitory effects of gallium on malignant cells were dependent of the doses but also of the duration of
exposure.
Arsenic compounds are natural substances used in China for medical treatment. Arsenic trioxide is the most widely
used in cancer research for treating patients with acute promyelocytic leukemia
Copper complexes have been extensively investigated those past years as potential anticancer agents.
Cassiopeins have shown very promising activity and are ready to start clinical trials. Copper complexes associated
with ligands like peptides (Alanin and Phenylalanin) seem to be good candidates after in vitro testing against
various human tumor cell lines with very low level of toxicity against normal cell lines. Cell cycle modifications after
Copper treatment will be presented.
Rhenium complexes associated with Selenium ligands show also, at fairly high concentration, antitumor properties
which are potentialized with Cisplatin or Gallium salts. No toxicity is observed on normal cells or in animals. In vivo
biodistribution in nude mice of Rhenium, Gallium and Selenium will be presented.
Palladium is also a promising metal ion in cancer research. Compounds like Padeliporphin or Padoporphinare
palladium bacteriopheophorbide photosensitizers and are currently in phase II and phase III clinical trial.
Padeliporphin, which is water soluble, given in patients with low-risk prostate cancer yielded negative biopsies in
more than 80% of patients treated. Further clinical trial will be presented.
Gold complexes were used in Chinese and Arabic medicine. They bind to DNA and have more cytotoxicity than
Cisplatin. They tend also to be active against Cisplatin resistant cell lines. Organogold compound like Auranophin
is currently in clinical trial and results will be discussed.
Despite the tremendous amount of research done on metal chelating agents, very few data concerning the
molecular basis of the mechanisms underlying their antitumor activity are available and need to be investigated at
the time where cancer treatment is more and more targeted.
16
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 019
AREA: MIEH
KEYNOTE
Biomonitoring of lead exposure: Experience in Uruguay
Mañay, N.; Cousillas, A.; Heller, T.; Pereira Testa, L.
Toxicology and Environmental Hygiene, Faculty of Chemistry, Montevideo, Uruguay
[email protected]
Lead (Pb) is a well known bioavailable toxic environmental chemical pollutant, thus it can be absorbed and cause
adverse health effects on susceptible living organisms. The biomonitoring for lead human exposure reflects an
individual’s current body burden, which is a function of recent and/or past exposure activities. Lead in whole blood
(BLL, B-Pb) showed to be the best biomarker for screening, biomonitoring and diagnostic purposes to assess lead
exposure.
In Uruguay, lead pollution problem became of public concern on 2001, and it has been multidisplinary approached
since then. This work highlights the importance of lead biomonitoring of human and animal populations illustrated
by the Uruguayan experience.
Uruguayan studies in human populations (infants, children, adults, workers) and animals (dogs) lead studies are
reviewed to show the incidence of different variables on BLL, and the fact that animals can be "sentinels" of
environmental lead human health hazards.
Results of thousands of analyses done in a 10 ys period, indicated that almost 50% children had BLL above
reference intervention limits (>10ug/dL) initially, but a significant decrease in BLL was found over time in the
studies results, demonstrating the importance of medical intervention, nutrition, and environmental education. The
phase-out of leaded gasoline have also contributed to this improvement.
The severity of lead pollution discovered, required official governmental actions, both to reduce sources of lead
contamination and to address the health implications for lead exposed children. Besides, dogs provided key
diagnostic elements to consider that children, living with them, may be contaminated by lead because these pet
species are susceptible to develop early severe intoxication symptoms to lead exposure.
In conclusion, there has been a significant change in preventing lead exposure in Uruguay due not only to
biomonitoring research work, but also to the public sensitization together with the integration of multidisciplinary
actions promoted.
17
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 045
AREA: MIEH
KEYNOTE
Synthesis of resorcin[4]arenes as potential receptors for metal ions
Botta, B.; D’Acquarica, I.; Ghirga, F.; Santi, L.
Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza University of Rome, Rome, Italy
Calixarenes and/or resorcinarenes are a suitable alternative to cyclodextrins for the ability of mimicking the
molecular recognition in the living systems.
Three main features play a fundamental role in the recognition process:
i) variability in the cavity size and shape
ii) chirality
iii) solubility in water, where the biological processes occur.
Two well separated main coordination regions are generally identified in the structure of these macrocycles when
they act as receptors of transition metal cations: the so-called upper rim, the π-basic cavity corresponding to the
wide side of the aromatic rings system, and the lower rim, localized in the region limited by the substituents.
As a part of our studies on modifications of lower rim substituents of resorcin[4]arenes octamethyl ethers, we
prepared tetramine- and the corresponding 4-(aminomethyl)pyridine-derivatives and we will discuss during this
lecture the interaction with CuII, Co+2 and Hg+2 of these compounds as host.
Moreover, we have shown also that resorcin[4]arenes, bearing carboalkyloxy groups in the side chains, are able to
1
interact with FeIII in organic media. H-NMR studies, carried out using GaIII instead of FeIII, suggested that these
systems have two active sites for interaction, the first one located at the aromatic rim and the other in the vicinity of
the carbonyl groups. As a confirmation, resorcin[4]arenes without carbonyl groups in the side chains have been
found to exhibit only one active site. Notably, in the latter case the interaction resulted in configurational changes.
Finally we synthesized three resorcin[4]arene-capped porphyrins that differ in the porphyrin skeleton, in the linking
arms and in the cavity dimensions, as well as for their capability to inhibit the oxidation of Co(II) to Co(III).
18
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 141
AREA: MIEH
KEYNOTE
Cognition and behavior in children: are they worse off when undernutrition and toxic
metal exposures occur together?
Kordas, K.
3
Nutritional Sciences, Pennsylvania State University, United States, University Park, PA
Dr. Kordas will discuss the potential interactions between undernutrition and toxic metal exposures in terms of their
effects on cognition and behavior in children. She will use iron deficiency/anemia and lead exposure as case
studies to outline the reasons why additive or multiplicative effects of both conditions are possible, to examine the
available research evidence and to make recommendations for future studies. She will also discuss her ongoing
research collaborations in Uruguay and the evidence they are generating to address the question posed in the
talk's title.
19
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 131
AREA: NATME
KEYNOTE
Correlations between trace metal ions (TEs) and the development of infectious diseases
in home parenteral nutrition patients
Carver, P.
University of Michigan College of Pharmacy, Department of Clinical, Social, and Administrative Sciences, and University of
Michigan Hospitals, Ann Arbor, MI, USA
[email protected]
Background and Objectives: Although patients with ↑ plasma concentrations of Fe have an ↑ risk of certain
bacterial and fungal infections, the effect of ↑ or ↓ plasma concentrations of other metal ions is poorly understood.
Metal ions are important components of nutrition, and are required for cellular growth and metabolism, and may
play a role patients' susceptibility to infection. We evaluated patients receiving long-term (≥1 year) home TPN, to
determine the relationship between bacterial and fungal infections and plasma concentrations of trace metal ions
(Zn, Cu, Se, Mn, and Cr).
Methodology: Data collection included all fungal and bacterial infections, doses and plasma concentrations of
TEs, and risk factors for infections. Descriptive statistics were used for quantitative data, and a recurrent events
survival analysis and a Glimmix procedure to analyze plasma concentrations of the trace metals, and their
correlation with infections.
Results: In long term home TPN patients, TE doses correlate significantly with plasma concentrations of Zn, Cr,
and Mn, but NOT Se, Cu, or Fe. Although % Fe saturation and IV dosages of Fe were predictive of an ↑ risk of
“first” infection, ↑ serum [Fe] and ferritin were not. Higher cumulative doses of Fe are highly predictive of an overall
↑ risk of gram negative or fungal infections; modest correlations were seen for Zn and Mn. A novel and significant
finding was that ↑ Mn doses correlated significantly with plasma [Mn] and with a ↓ rate of infections.
Conclusions: Correlations found between TE doses and plasma [TE]s, and with [TE]s and infections may help to
improve our dosing and monitoring of trace elements, and minimize the risk of inadequate nutrition or lifethreatening infections. The clinical significance of higher Mn doses should be explored further.
20
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 137
AREA: RMNM
KEYNOTE
Bioorganometallic technetium and rhenium chemistry for imaging and therapy:
progresses and perspectives
Alberto, R.; Can, D.; Suleiman, S.; Shen, Y.
University of Zürich, Institute of Inorganic Chemistry, Zürich, Switzerland
Visualization of molecular events on the cellular and subcellular level requires a careful design of highly specific
compounds. Their design does not only concern the biological carrier of the imaging agent as often believed, but
the label in particular. Consequently, the label cannot be considered as “just a tag”, but its properties and
opportunities have to be integrated into the entire design. Following this approach, we have introduced the phenylcyclopentadienyl analogy concept in which phenyl groups of a lead structure are replaced by a [(Cp-R)
moiety. Along a novel approach, we synthesized a wide variety of [(Cp-R)
99m
99m
Tc(CO)3 ]
Tc(CO)3] complexes in which “R”
represents a broad spectra of targeting functions.
The cyclopentadienyl complex may be an essential part of the overall structure. We have prepared
99m
Tc
analogues of carbonic anhydrase and histone deacetylase inhibitors but also “simple structures such as artificial
amino acids1. Thereby, the function “R” is not exclusively a carboxylate group but can combine different linkers and
coupling groups. For metal-labeled mimetics of pharmacophores, the
99m
Tc complex is used for imaging and the
homologous Re complex for therapy, a new form of theragnostics. Classical Werner type ligands are as important
as organometallic cyclopentadienyl complexes. We have synthesized bifunctional 2,3-diamino-propionic acid based
2
ligands which belong to the smallest tripods . They have been coupled to peptides and to glucose. Synthetic
pathways and biological behaviour will be discussed in the context of Cp-chemistry. The presentation will
emphasize the importance of fundamental chemistry. A focus will be on Cp-complexes to corroborate the 3D-space
occupation concept. Aspects of the theragnostics will be presented as well as the integration of Tc and Re in very
small biological molecules.
(1) Can, D., Spingler, B., Schmutz, P., Mendes, F., Raposinho, P., Fernandes, C., Carta, F.,Innocenti, A., Santos,
I., Supuran, C. T., and Alberto, R. [(Cp-R)M(CO)3] (M=Re or
99m
Tc) Arylsulfonamide, Arylsulfamide, and
Arylsulfamate Conjugates for Selective Targeting of Human Carbonic Anhydrase IX. Angew. Chem. Int. Edit. 2012,
51, 3354-3357.
(2) Liu, Y., Oliveira, B. L., Correia, J. D. G., Santos, I. C., Santos, I., Spingler, B., and Alberto, R. Syntheses of
bifunctional 2,3-diamino propionic acid-based chelators as small and strong tripod ligands for the labelling of
biomolecules with
99m
Tc. Org. Biomol. Chem. 2010, 8, 2829-2839.
21
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 010
AREA: SBMMBRP
KEYNOTE
Catalytic mechanism and evolutionary traits of Zn-dependent beta-lactamases
Meini, M.R.; González, M.; González, L.; Barh, G.; Palacios, A.; Llarrull, L.; Vila, A.
Biophysics Section, Instituto de Biología Molecular y Celular de Rosario (IBR), Rosario, Argentina
[email protected]
β-lactamases represent the prevalent resistance mechanism to β-lactam antibiotics. In the last decade, the
dissemination of genes coding for metallo-β-lactamases (MBL´s) has become an emergent clinical problem. MBL´s
are Zn(II)-dependent enzymes. The exponential growth of MBL sequences being characterized has revealed an
initially unforeseen structural diversity, that gives rise to the presence of mono- and dinuclear metal sites. MBL´s
have been recently subdivided into classes B1, B2 and B3, each of them displaying different zinc ligands and
coordination geometries (1).
We have studied the structural features of MBL´s from different subclasses with the aim of finding common
structural and catalytic features. By means of mutagenesis, functional and structural studies, we conclude that a Zn
site, previously regarded as non essential for catalysis, plays a major role in substrate binding and catalysis (2-4,
10-12). Non-steady state kinetic studies, aided by time-resolved electronic, EPR and Resonance Raman
spectroscopy have allowed us to trap a key intermediate in β-lactam hydrolysis, and to assess the role of each
metal binding site in the mechanism and stabilization of this intermediate (5,6).
Directed evolution was used as an evolutionary engineering tool to explore the effect of challenging MBLs towards
different antibiotics. BcII (the MBL from B.cereus) has been considered as a precursor of more efficient MBL’s
present in pathogenic bacteria (7,8). These results suggest that evolution of the chemical landscape can be
predicted by means of this approach. We have also observed that evolution acts by enhancing the Zn(II) binding
capability upon under metal-defficient conditions (9).
References
1. M. W. Crowder, J. Spencer and A.J.Vila Acc. Chem.Res, 39, 721-728 (2006).
2. Llarrull et al. J. Biol.Chem., 282, 18276-18285 (2007).
3. Morán-Barrio et al. J.Biol.Chem., 282, 18286-93 (2007).
4. Llarrull et al. J.Biol.Chem., 282, 30586-95 (2007).
5. Tioni et al. J.Am.Chem.Soc., 130, 15852-15863 (2008).
6. Llarrull et al. J.Am.Chem.Soc., 130, 15842-15851 (2008).
7. Tomatis et al. Proc.Natl.Acad.Sci.USA, 102, 13761-13766 (2005).
8. Tomatis et al. Proc.Natl.Acad.Sci.USA, 105, 20605-20610 (2008).
9. Gonzalez et al. Nature Chem. Biol., 8, 698-700 (2012).
10. Lisa et al. Antimicrobial Agents and Chem., 56, 1769-1773 (2012).
11. J.M.González, et al. Biochemistry, 49, 7930–7938 (2010).
12. Lisa et al. J. Biol.Chem., 285, 4570-7 (2010).
22
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 050
AREA: SBMMBRP
KEYNOTE
Electrostatically-driven switches in electron transfer reactions of metalloproteins
Murgida, D.
INQUIMAE-DQIAyQF, CONICET y Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires,
Argentina
[email protected]
Natural selection has evolved specialized redox proteins able to perform fast intra- and inter-molecular long range
electron transfer (ET) reactions, in the milliseconds time scale and below, in spite of involving nearly null
thermodynamic driving forces. Most of these nonadiabatic reactions are well described by the high temperature
limit expression of Marcus semiclassical theory. Accordingly, the ET rate is proportional to the square of the
electronic coupling matrix element, and to a Frank-Condon exponential term that accounts for the thermal
accessibility of such degeneracy. The latter is dominated by the reorganization energy (λ) that accounts for both
the energy required to distort the reactants towards the equilibrium configuration of the products and the
reorganization of the solvent around the redox center. Here we present a combined spectroscopic, electrochemical
and computational study showing that for heme and Cu proteins these parameters are strongly modulated by
electrostatic interactions which, in turn, activate either second-sphere ligand conformational switches or enable
alternative electronic ground states that provide optimized pathways for electron entry and exit, respectively. Based
on these results, we propose that electron transfer chains involved in electron-proton energy transduction are
strongly regulated by changes of the membrane potential, as well as by protein-protein interactions, which
eventually may lead to a change or gain of function.
23
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 138
AREA: SBMMBRP
KEYNOTE
Structural determinants of the reaction mechanism and stereoselectivity in heme
containing dioxygenases
Marti, M.A.
Departamento de Química Biológica e INQUIMAE, Facultad de Ciencias Exactas y Naturales - CONICET, Universidad de
Buenos Aires, Argentina [email protected]
Indoleamine 2,3 dioxygenase (IDO) and tryptophan dioxygenase (TDO) are two heme- proteins that catalyze the
oxidation reaction of tryptophan (Trp) to N-formylkynurenine (NFK). Human IDO (hIDO) has recently been
recognized as a potent anti-cancer drug target, a fact that triggered intense research on the reaction and inhibition
mechanisms of hIDO. In order to shed light into the IDO and TDO reaction mechanism we have used a
combination of theoretical QM/MM based methods with kinetic and spectroscopic studies. Our results show that the
dioxygenase reaction is initiated by addition of a ferric iron-bound superoxide to the C2=C3 bond of Trp to form a
ferryl and Trp-epoxide intermediate, whose presence was confirmed using Resonance Raman spectroscopy.
These data demonstrated that the two atoms of dioxygen are inserted into the substrate in a stepwise fashion,
challenging the paradigm of heme-based dioxygenase chemistry. For the second oxygen insertion step our results
+
show that the most energetically favored pathway involves proton transfer from Trp-NH3 to the epoxide oxygen,
triggering epoxide ring-opening and a concerted nucleophilic attack of the ferryl oxygen to the C2 of Trp that leads
to a meta-stable reaction intermediate that subsequently converts to NFK, following C2-C3 bond cleavage and the
associated back proton transfer from the oxygen to the amino group of Trp. Last but not least, we show how the
different enzymes select L-Trp over D-Trp highlighting how the active site structure and dynamics achieve this
challenging task.
References
1. Capece L. et. al., J Phys Chem B. 2012;116(4):1401-13.
2. Capece, L. et. al., Proteins, struct. Func. And Bioinformatics. 2010, 78(14) 2961-72.
3. Lewis-Ballester et. al. Proc. Natl. Acad. Sci USA. 2009, 106(41) 17371- 17376.
24
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 025
AREA: TEMIBSS
KEYNOTE
Iron homeostatic mechanisms in normal and pathological retina: its implication in aging
and retinal degeneration
Courtois, Y.1; Jeanny, J.C.1; Yefimova, M.2; Behar Cohen, F.1; Picard, E.1
1
2
Centre de recherches des Cordeliers team17, INSERM University Paris V, Paris, France
INSERM UMRS872 team17, Sechenov Institute of Evolutionary Physiology and Biochemistry Russian Academy of Sciences,
St-Petersburg, Russia - France
[email protected]
Background: Iron is an essential metabolic component in the retina required for the vision mechanisms. All the
enzymes necessary for the control of its level and distribution have been recently described to be in place in the
retina. Iron accumulation has been reported in the retina as a function of aging and/or occurring in inflammatory
conditions, under oxidative stress or due to genetic defects in several proteins involved in iron metabolism. Since
excess iron is toxic for retina, it is postulated that in all the previous conditions described above, iron regulation
level by Transferrin (Tf) the main iron binding transporter will help to protect this organ.
Methodology: Localization of iron on retinal sections by Perl’s staining, analysis of the role of Tf on rod outer
segment phagocytosis by retinal epithelial pigmented cells (RPE). Addition of Tf on Muller cells loaded with iron. In
vivo injection of Tf in several genetic animal models of retinal degeneration: rd10 mice, RCS rats or in light-induced
retinal degeneration in mice. Comparison of retinal degeneration rate in transgenic mice carrying both the rd10 and
the hTf gene with rd10 mice.
Results: Iron accumulates during retinal degeneration in these models. Tf enhances phagocytosis process. It
protects Muller cells against iron excess. In animal models we observed that the delivery into the eyes of human Tf
by different routes (iv, ivt or gene transfection) at high dose protects or delays retinal degeneration, and in some
cases such as in light-induced degeneration maintains its function.
Conclusions: Iron has multiple functions in retinal physiology and its level is controlled by autonomous
homeostasis mechanisms. Many conditions disturb this equilibrium. Iron in excess induces further oxidative stress.
We show that increasing the level of Tf in the retina has a potent neuroprotective effect and could have potential
pharmacological applications for Age-related macular degeneration and other degenerative diseases
Acknowledgments: This work was funded by ANR and INSERM. We thank Mohamed El Sanharawi, Marianne
Berdugo for their expertise in ophthalmologic techniques.
Reference: Picard E, et al. Mol Vis. 2010 ; 16:2612-25.
25
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 147
AREA: TEMIBSS
KEYNOTE
Assessing the public health import of children’s exposures to metals
Bellinger, D.C.
The impact of environmental chemicals on children’s neurodevelopment is sometimes dismissed as unimportant
because the impairments observed among individual childrenare viewed as “clinically insignificant.” This reflects a
failure to distinguish between individual and population risk. The population impact of a risk factor depends both on
the effect size associated with it and its distribution (or incidence/prevalence). A set of analyses will be discussed in
which the total number of IQ points lost, among U.S. children under 5 years of age, for a variety of medical
conditions and events (e.g., congenital heart disease, preterm birth, traumatic brain injury, iron deficiency, ADHD)
and certain environmental chemical exposures (lead, methylmercury). The calculations show that the numbers of
IQ points lost as a result of chemical exposures exceed those associated with other neurodevelopmental risk
factors, largely due to the high prevalence of these exposures. The relative importance of different risk factors likely
differs by region and culture. The strategy described provides a rational basis for establishing priorities for reducing
neurodevelopmental morbidities in children, suggesting that a “population-oriented” approach to risk assessment
provides a better sense of public health impact of an exposure than does a “disease-oriented” approach.
26
Oral Presentations
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 053
AREA: AAMMIBBS
ORAL
Developing techniques for determining the spatial distribution, speciation and chemical
environment of metallic elements in brain tissues affected by neurological disorders
1
1
1
1
2
Lankosz, M. ; Szczerbowska-Boruchowska, M. ; Wandzilak, A. ; Czyzycki, M. ; Adamek, D. ; Radwanska, E.
1
2
Faculty of Physics and Applied Computer Science, AGH-University of Science and Technology, Krakow, Poland
2
Chair of Pathomorphology, Faculty of Medicine, Jagiellonian University, Krakow, Poland
[email protected]
The minor and trace elements are playing an essential role in pathophysiology of neoplastic and
neurodegenerative disorders. Selected elements may contribute, directly or indirectly, on the cancerous and
neurodegenerative processes. Molecular medicine is in need of the application of structural methods which are
capable of monitoring biochemical processes and interactions within the tissues. The methods based on
synchrotron radiation i.e. synchrotron radiation X-ray fluorescence (SRXRF), X-ray absorption near edge structure
spectroscopy (XANES), and extended X-ray absorption fine structure spectroscopy (EXAFS) were utilized for
analysis of metals and other elements in human brain samples. The measurements for this study were carried out
at DLS on the beam line I18, on the bending magnet beam lines L and C at HASYLAB, and on beam line BM23 at
ESRF.
The SRXRF technique was applied to the quantitative evaluation of elemental changes in substantia nigra pars
compacta (SNc) in Parkinson’s disease (PD). Two-dimensional maps of elemental distribution show that the
locations of nerve cells in sections are precisely visualized by the high levels of most elements. It was found that Cl,
Fe, Ca and Zn are the most significant elements in the general discrimination between PD and control nerve cells.
The samples from brain gliomas (BG) of various grades of malignancy were collected intraoperatively. The SRXRF
and XANES micro-spectroscopies were performed in thin freeze-dried sections. The results of imaging of areas of
calcification in BG showed that a high level of Ca was accompanied by an increased level of Zn. The Fe, Zn and
Cu XANES spectra were collected at selected points of different regions of samples. Results of measurements
+
+
revealed that the Fe oxidation state in the studied samples was between 2 and 3 , while that of Cu was between
1+ and 2+ and that of Zn was 2+. The cryo-EXAFS method was used for studies of frozen BG. The key findings from
these measurements relate to the oxidation state and the chemical environment of Fe, Cu and Zn in the samples. A
2+
trend can be observed that the higher the tumor grade, the more Fe
3+
as compared to Fe
the sample contains.
Analysis of Fe EXAFS spectra showed that the distance at which the atoms of the first coordination zone are
located slightly increases with the increase in the tumor malignancy grade. The Cu and Zn EXAFS functions are
indicating slight differences in the paths of scattering obtained for measured samples. The evident contribution of
chemical elements to the pathophysiology of PD and BG was shown.
Acknowledgements: The research leading to these results has received funding from: the European Community's
Seventh Framework Programme (FP7/2007-2013) under grant agreement n° 226716, Diamond Light Source Ltd,
Didcot, Great Britain, European Synchrotron Radiation Facility, Grenoble, France, HASYLAB, Hamburg, Germany
and the Ministry of Science and Higher Education (Warsaw, Poland) grant no N N518 377 537
28
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 065
AREA: AAMMIBBS
ORAL
Protein and peptide labeling with metals in nano-HPLC-UV-ICP-MS detection
Lyrio Tenorio Correia, C.1; Holste, A.2; Tholey, A.3; Saint'Pierre, S.1; Schaumloffel, D.2
1
Department of Chemistry, PUC Rio, Rio de Janeiro, Brazil
Department of Analytical Chemistry, UPPA, Pau, France
3
Division Systematic Proteom Sechearch, Christian-Albrechts-University, Kiel, Germany
[email protected]
2
The new challenge of analytical chemistry, to identify and understand the mechanisms of interaction between metal
ions and macromolecules, in addition to separation techniques, analytical techniques are required that can identify
and quantify elemental species, present in biological tissues at concentrations in the ng L-1. The principal approach
of this study is based on the use of metals detection in ICP-MS and derivatization of protein and peptide by metal
labels as a new approach to protein quantification to generate a method that allows higher sensitivity and accuracy.
Reduction for all peptides was carried out using a two-fold molar excess of TCEP per cysteine residue. After that,
the model peptides were derivatized with 100-fold molar excess of NHS-DOTA ester to each free amino group.
Subsequently, the complexation of peptides labelled with NHS-DOTA ester was carried out with lanthanide metal
ions. The end concentration of peptide was 5 pmol/mL. Moreover, a mixture of five peptides was prepared and the
same procedure for reduction, derivatization and complexation was applied. Every samples was analyzed by nanoHPLC with UV detection and the metals detected by ICP-MS. Peptides were eluted onto a column separation C18
and performed with a flow rate of 0.3 mL/min and the following eluents: A (0.05% aqueous TFA) and B (80% ACN,
0.04% TFA, 20% deionized water. In order to study the MS and MS/MS behavior of NHS-DOTA-labelled peptides a
number of synthetic peptides containing lysine residues or primary amino groups were analyzed by MALDI-TOFMS. The data obtained demonstrates the suitability of MALDI-MS and -MS/MS for the characterization of metallabelled peptides. The benefits of the complementary application of molecular and elemental ICP-MS clearly show
that upon use of this approach a straightforward accurate and precise absolute quantification of the labelled
proteins is possible.
29
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 106
AREA: AAMMIBBS
ORAL
Post-mortem biochemistry: Determination of trace elements in vitreous humor by ICP-MS
Santos Júnior, J.C.1; Höehr Nelci, F.1; Mollo Filho, P.C.2; Felice Guidugli, R.B.2; Eberlin, M.N.3; Pessôa, G. de S.4;
5
5
da Silva, E.G. ; Zezzi Arruda, M.A.
1
Department of Clinical Pathology, School of Medical Sciences - University of Campinas — UNICAMP, Campinas/SP, Brazil
2
Team of Forensic Medicine West, Medico-Legal Institute - Police Technical Scientific Superintendence — SPTC, São
Paulo/SP, Brasil
3
ThoMSon Mass Spectrometry Laboratory, Institute of Chemistry - University of Campinas — UNICAMP, Campinas/SP, Brazil
4
Campinas/SP, Institute of Chemistry - University of Campinas — UNICAMP, Group of Spectrometry, Sample Preparation and
Mechanization (GEPAM), Brasil
5
Group of Spectrometry, Sample Preparation and Mechanization (GEPAM), Institute of Chemistry - University of Campinas —
UNICAMP, Campinas/SP, Brazil [email protected]
Background: Biochemistry is of great importance in forensic pathology [1]. The vitreous humor for its features and
sterility, was a good sample for our analyzes [2]. Mass spectrometry with inductively coupled plasma source allows
quantification multi-elemental metals and metalloids with a dynamic range of 6-7 orders of magnitude [3].
Objective: To investigate the pattern of metals (Fe, Sn, Mo and Mg) in vitreous humor bodies and the correlation
of decomposing and non-decomposing bodies for different periods.
Methodology: The vitreous humor was extracted using puncture site, packed in Vacutainer ® brand tubes BD
(REF 368521), separated and identified. For the decomposition system in mini- vials, 200 µL of samples were
added to 200 µL of HNO3 sub-distilled and 100 µL of H2O2. The concentrations were determined with an ICP-MS
(Elan DRC-e, PerkinElmer) under appropriate conditions. Methane was used to minimize interferences.
Results: There is an increase of iron concentration in the samples of decomposing bodies and there is no
correlation between time and concentration of Sn, Mo, and Mg ions.
Conclusions: The use of ICP / MS in biochemistry postmortem check is extremely valuable in correlating the
phenomena of decomposition, where the relationships can serve as indicative postmortem on body decomposing.
Acknowledgments: The FCM / UNICAMP (No1270/2010), the West IML (No 09/2011) and to CNPq for the
postgraduate scholarship.
References
[1] MAEDA H.; ISHIKAWA T.; MICHIUE T. Forensic biochemistry for functional investigation of death: Concept and practical
application. Legal Medicine. 2011; 13:55-67.
[2] Sanches et al. Determination of opiates in whole blood and vitreous humor: a study of the matrix effect and an experimental
design to optimize conditions for the enzymatic hydrolysis of glucuronides. Journal of analytical toxicology. 2012; 36:162-170.
[3] Sussulini A, Garcia JS, Arruda MA. Microwave- assisted decomposition of polyacrylamide gels containing metalloproteins
using mini-vials: an auxiliary strategy for metallomics studies. Anal Biochem. 2007; 361(1):146-8.
30
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 125
AREA: AAMMIBBS
ORAL
Microwave induced combustion for metals determination in biological matrices
Flores, E.M.M.1; Smanioto Barin, J.2; Bizzi, C.A.1; Foster Mesko, M.3; Severo Fagundes Pereira, J.1; de Azevedo
1
Mello, P.
1
Departamento de Química, Universidade Federal de Santa Maria, Santa Maria-RS, Brazil
Departamento de Tecnologia e Ciência dos Alimentos, Universidade Federal de Santa Maria, Santa Maria, RS, Brazil
3
Centro de Ciências Químicas, Farmacêuticas e de Alimentos, Universidade Federal de Pelotas, Pelotas, RS, Brazil
[email protected]
2
Determination of trace elements in biological and food samples has received increased concern due to nutritional
or toxic aspects. In this sense, the determination of metals in biological matrices as milk powder and bovine liver is
of great importance and an appropriate sample preparation method should be used in order to obtain an efficient
sample digestion. The use of concentrated reagents is hazardous, could require a dilution step of the digests
before analytes determination and generates high volumes of concentrated acids as effluents. Considering the
limitations of methods based on microwave-assisted wet digestion (MAWD), concentrated acids are generally
required, increasing the blank values or could cause interferences in the determination step by some analytical
techniques, such as ICP OES. Recently the microwave-induced combustion (MIC) method has attended several
premises related low reagent consumption, use of diluted acids and high digestion efficiency. In the present work
MIC method is proposed for biological digestion for further metals determination (Cd, Cr, Cu, Mn, Zn, etc) by ICP
OES and and ICP-MS. The concentration of absorbing solution for analytes retention was studied using a reflux
step after sample combustion.
For results comparison, milk powder digestion was also performed by MAWD and MIC in closed vessels. Accuracy
was evaluated using certified reference materials of milk powder and bovine liver and it was better than 95% for all
the analytes. Efficiency of digestion was evaluated by the residual carbon content in digests and values lower of
1% were obtained for all the samples. It was possible to use diluted nitric acid solution for analytes absorption and
digests were suitable for determination of metals by ICP OES or ICP-MS.
Acknowledgements: CNPq, INCT-Bioanalítica, FAPERGS, UFPel, UFSM
31
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 002
AREA: MBD
ORAL
Tungsten reacting with 5-hydroxymethylcytosine
Okamoto, A.
Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo, Japan [email protected]
5-Hydroxymethylcytosine (hmC) is a newly discovered natural nucleobase that may play an important intermediary
role in the active DNA demethylation pathway. An effective method to detect the presence and abundance of hmC
in DNA is required to elucidate the relationship between the generation of hmC and the mechanism of
demethylation. We have found that the oxidation using dinuclear peroxotungstate K2[{W(=O)(O2)2(H2O)}2(mO)]•2H2O is hmC selective, and is useful for the discrimination of hmC from its epigenetic precursors, unmethylated
cytosine (C) and 5-methylcytosine (mC) in DNA.1 A fluorescein-labeled model DNA containing CG, mCG, or hmCG
dinucleotides was prepared, and the dinuclear peroxotungstate was added to a DNA solution in a pH 7 sodium
phosphate buffer. The mixture was incubated at 50°C for 5 h. The reaction proceeded hmC-selectively. The mass
spectrum of the oxidation product obtained from the hmC-containing DNA indicated the formation of trihydroxylated
thymine in DNA. The tungsten oxidation products induced the incorporation of adenine into the complementary site
in a primer extension, and made it possible to detect hmC in a DNA sequence of interest in a conventional DNA
sequencing analysis. Tungsten oxidation worked as a simple chemical reaction for the effective detection of hmC,
and will provide beneficial information on the design of a powerful method for hmC scanning and typing to solve the
mystery of the initialization of gene function through demethylation.
References
Okamoto, A., et al. 5-Hydroxymethylcytosine-selective oxidation with peroxotungstate. Chem Commun, 2011, 47, 11231–
11233.
32
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 015
AREA: MBD
ORAL
Trisenox-based chemotherapy of acute promyelocytic leukemia: Molecular mechanisms
of action
Tchounwou, P.B.; Yedjou, C.G.; Brown, E.; Rogers, C.
1
Molecular Toxicology Research Laboratory, NIH-Center for Environmental Health, College of Science, Engineering and
Technology, Jackson State University, 1400 Lynch Street, Box 18540, Jackson, Mississippi, USA
[email protected]
Background: Acute promyelocytic leukemia (APL) is a blood cancer characterized by a rapid accumulation of
abnormal white blood cells in the bone marrow and blood resulting in anemia, susceptibility to infections, bleeding,
and hemorrhage. Trisenox (TX)-based chemotherapy has recently been approved by the FDA based on its
effectiveness in providing for a complete remission in de novo and relapsed APL patients. Although TX has been
reported to induce degradation of PML-RAR alpha protein in these patients, its molecular mechanisms of action
against cancer cells remain to be elucidated. In this research, we hypothesize that TX pharmacology is mediated
through oxidative stress leading to p53 activation, genotoxicity, and apoptosis in cancer cells.
Methods: To test this hypothesis, we performed the MTT-assay for cell viability, thiobarbituric acid test for lipid
peroxidation, Western Blot analysis for p53 expression, microgel electrophoresis (Comet) assay for genotoxicity,
flow cytometry analysis of annexin-5 and caspase-3, and DNA laddering assay for apoptosis, using the human
leukemia (HL60) cell line as a test model.
Results: MTT assay indicated a strong dose-response relationship with regard to the cytotoxic property of TX. The
24 hr-LD50 was 6.35 ± 0.57µg/mL. Western Blot analysis also demonstrated a strong dose-response relationship
with regard to the expression of tumor suppression protein, p53. Similarly, strong dose-response relationships
(p<0.05) were obtained in connection with TX- induced MDA formation, and DNA damage as characterized by the
percentages of DNA fragmentation, and the lengths of the comet tails in leukemic cells. AT-induced apoptosis was
characterized by a significant increase in the percentages of annexin-5 and caspase-3 positive cells, as well as in
the degree of DNA fragmentation.
Conclusions: TX pharmacotherapy is associated with a cytotoxicity that is mediated by oxidative stress, upregulation of p53, DNA damage and morphological changes leading to apoptotic death in cancer cells.
Aknowledgments: Research supported by NIH Grant # 1G12RR13459 and DoD Grant # W911NF-11-1-0123 at
Jackson State University.
33
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 017
AREA: MBD
ORAL
Synthesis, characterization and antitrypanosomal evaluation of ferrocenyl and
cyrhetrenyl thiosemicarbazone complexes of platinium (II)
Arancibia, R.1; Lapier, M.2; Maya, J.D.2; Klahn, H.1
1
2
Instituto de Quimica, PUCV, Valparaiso, Chile
Facultad de Medicina, U. de Chile, Santiago, Chile
[email protected]
In the last few years, our research group has been involved on bioorganometallic compounds with potential
antiparasitic activity, particularly as antimalarial1 and antitrypanosomal2. By considering that Chagas’ disease or
American trypanosomiasis is still an important health problem that affects around 20 million people in Central and
South America3 and with the aim to find out new and more effective antichagasic agents, we decided to investigate
new complexes derivatives from organometallic thiosemicarbazones (TSCs). The very well known organic TSCs
4
complexes are currently being investigated as potential chemotherapeutics, including as antichagasic , but
organometallic TSCs analogues have received considerably less attention, according to our acknowledge some
4
ferrocenic-TSCs complexes have been evaluated as antimalarial but their antitrypanosomal activity is still
unreported. In the present work, we would like to report the synthesis, characterization and anti-T. cruzi evaluation
of ferrocenyl and cyrhetrenyl thiosemicarbazone complexes of platinium (II) (Esqueme 1).
All compounds were characterized for IR and NMR. The
31
P–{1H} NMR spectra showed a singlet, shifted to higher
frequency from the spectrum of the free phosphine, in agreement with phosphorus coordination to metal center.
The results of an in vitro antitrypanosomal assay of the compounds against strains of T. cruzi using MTT, indicate
that they have moderated antichagasic activity.
References
[1] R. Arancibia, F. Dubar, B. Pradines, I. Forfar, D. Dive, A. H. Klahn, C. Biot ; Bioorg. Med. Chem.; 2010, 18, 8085.
[2] R. Arancibia, A. H. Klahn, G. Buono-Core, E. Gutierrez-Puebla, A. Monge, M. Medina, C. Olea-Azar, J. Maya, F. Godoy; J.
Organomet. Chem.; 2011, 696, 3238.
[3] http://www.who.int/healthinfo/statistics/healthinfoachmsreport2006.pdf
[4] (a) H. Cerecetto, M. Gonzalez; Mini Rev. Med. Chem.; 2008, 8, 1355; (b) C. Biot, B. Pradines, M. Sergeant, J. Gut, P.
Rosenthal, K. Chibale; Bioorg. Med. Chem. Lett.; 2007, 17, 6434.
Acknowledgements: We thank the FONDECYT-Chile (Project 1110669; Project 3120091) and D.I.-PUCV.
34
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 033
AREA: MBD
ORAL
Paramagnetic resonance studies on Ru(III) azole complexes of medicinal interest
Telser, J.1; Alamiri, A.1; Reisner, E.2; Arion, V.B.3; Büchel, G.3; Freitag, L.4; Tierney, D.L.5; Marts, A.R.5
1
Biological, Chemical and Physical Sciences, Roosevelt University, Chicago, IL, USA
2
Chemistry, Cambridge University, Cambridge, UK
3
Institute of Inorganic Chemistry, University of Vienna, Vienna, Austria
4
Institute of Physical Chemistry, University of Vienna, Vienna, Austria
5
Chemistry and Biochemistry, Miami University, Oxford, OH, USA [email protected]
Ruthenium complexes have recently emerged as candidates for anti-cancer drugs. These include organoruthenium
6
5
complexes (of diamagnetic Ru(II), 4d ) as well as coordination complexes of paramagnetic Ru(III) (4d ). The latter
class of Ru complexes, which are analogous to well-known Pt-based drugs, comprise chlorido ligands, in some
cases dmso (dimethylsulfoxide), and a variety of azole ligands such as imidazole (Him) and indazole (Hind). Two
such complexes are in clinical trials: H2im[trans-RuCl4(dmso)(Him)] (NAMI-A), and H2ind[trans-RuCl4(Hind)2],
(KP1019, also known as FFC14a). A spectroscopic advantage of Ru(III) is that its S = 1/2 ground state is suitable
for investigation by EPR, which provides information on electronic structure not available by other means. We
describe low temperature EPR studies at both X-band (~9 GHz) and Q-band (35 GHz) frequencies on a series of
Ru(III) tetrachlorido complexes in DMF, which solvent had already been used for electrochemical studies [1]. In
1
addition, paramagnetic H NMR spectra in DMF-d7 at 300 K were recorded, which provided fluid solution
information on these complexes for comparison with EPR. Electronic absorption spectra in DMF were also
recorded to provide data for computational determination of electronic energy levels and redox potentials. The
complexes studied were of three types: the anionic "A" series, [trans-RuCl4(dmso)L]–; the anionic "B" series, [transRuCl4L2]–; and the neutral, "C" series: [mer-RuCl3L3], where L = a wide range of azole ligands. For the A and B
series, the azolium ion served as cation, although other cations were also investigated. The A and C series of
complexes each showed a distinct type of electronic structure, with little variation within each series. The B series
showed the presence of two forms in solution, which we associate with cis/trans isomerism, which has been
investigated computationally. The implications of these structures on reactivity of these complexes will be
discussed.
References
[1] Reisner, E; Arion, VB; Guedes da Silva, MFC; Lichtenecker, R; Eichinger, A; Keppler, BK; Kukushkin, VY; Pombeiro, AJL;
Tuning of Redox Potentials for the Design of Ruthenium Anticancer Drugs - an Electrochemical Study of [trans- RuCl4L(DMSO)]and [trans-RuCl4L2] Complexes, where L = Imidazole, 1,2,4-Triazole, Indazole. Inorg Chem; 2004; 43; 7083-7093.
35
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 039
AREA: MBD
ORAL
Bioinduced investigation on Cobalt(II)/(III) – hydroxamate systems
Farkas, E.; Szabó, O.
Department of Inorganic and Analytical Chemistry, University of Debrecen, Egyetem tér, Debrecen, Hungary
[email protected]
Hydroxamic acids are among the most well studied compounds due to their significance in so many fields. For
example (i) due to their good metal binding capability, they are effective inhibitors of many metalloenzymes. Among
others, hydroxamic acids are well-known inhibitors of cobalt containing metallohydrolases. To get deeper insight
into the mechanism of inhibition, numerous dinuclear complexes (including Co(II) containing ones) as structural
and functional models for catalytic centers have been synthesized and characterized and their interaction with
different hydroxamic acids in the solid state has been investigated [1-2]. (ii) Remarkable results have recently been
published in the literature [3-4]. Namely, different kinetically inert Co(III) complexes have been synthesized as
carriers to deactivate hydroxamate-based MMP (Matrix Metalloproteinases) inhibitors by chelation through the
hydroxamate binding group. The goal in this study was the development of Co(III)-containing prodrugs for selective
delivering of MMP inhibitors to target tumor sites. Since the number of equilibrium results for the cobalt(II)- and
cobalt(III)-hydroxamate systems in the literature is very limited, the investigation of such kind of systems is in the
focus of interest nowadays in our lab in Debrecen.
Although many different hydroxamic acids have been involved in this study, the most interesting results have been
obtained on the complexes formed with the most well-known hydroxamate based siderophore desferrioaxamine B
(DFB), and also with desferricoprogen (DFC).
O
O
HO
HO
HO
O
N
O
O
H
N
N
H
+H 3 N
O
O
N
OH
O
O
NH
N
OH
O
N
OH
O
HN
NH
HO
N
O
HO
N
O
Desferricoprogen (DFC)
Desferrioxamine B (DFB) B (DFB (DFB) (DFB)
Surprisingly, oxidation of Co(II)-siderophore complexes at high pH resulted the formation of extremely high stability
complexes, higher stability than the those of the corresponding Fe(III) species. Whether does it mean the possibility
of disruption of the microbial iron-uptake in the presence of this metal ion? This question and also the possibility of
the application of the extremely high stability and inert Co(III)-siderophore complexes as pro-drugs are planned to
discuss in a presentation on the conference.
Acknowledgments:
The work was supported by OTKA-NKTH CK77586 and TAMOP 4.2.1/B-09/1/KONV-2010-0007 National
Hungarian Fund
References
[1] D.A. Brown, W. Errington, W.K. Glass, W. Haase, T.J. Kemp, H. Nimir, S.M.Ostrovsky, R. Werner, Inorg. Chem. 40 (2001)
5962.
36
12th International Symposium on Metal Ions in Biology and Medicine
[2] Z. Tomkowicz, S. Ostrovsky, H. Müller-Bunz, A.J. Hussein Eltmimi, M. Rams, D.A. Brown, W. Haase, Inorg. Chem. 47 (2008)
6956.
[3] M.D. Hall, T.W. Failes, N. Yamamoto, T.W. Hambley, Dalton Trans. (2007) 3983.
[4] T.W. Failes, C. Cullinane, C.I. Diakos, N. Yamamoto, J.G. Lyons, T.W. Hambley, Chem. Eur. J. 13 (2007) 2974.
37
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 041
AREA: MBD
ORAL
In vitro antitumor activities of complexes with the general formula: [Ru(N-S)(bipy)(PP)]PF6, (N-S = thiolates ligands; bipy = 2, 2’-bipyridine; P-P = diphosphines)
Lima, B.A.V.1; Graminha, A.E.1; Ribeiro, A. de S.B.B.2; Pereira, F. de C.2; de Lima, A.P.2; Lacerda, E de P.S.*2;
*1
Batista, A.A.
1
Department of Chemisty, Federal University of São Carlos, São Carlos(SP), Brazil
Institute of Biologycal Ciences, Federal University of Goiás, Goiânia, Goiás, Brazil
*
Corresponding author: [email protected] (A.A. Batista); [email protected], (E. de P. Silveira-Lacerda)
2
The
reaction
of
the
complexes
cis-[RuCl2
(bipy)(P-P)],
bipy
=
2,2’-bipyridine
and
P-P
=
1,3-
bis(diphenylphosphino)propane (dppp) and 1,2- bis(diphenylphosphino)etane (dppe), with the ligands pySH(2mercaptopyridine),
Hprm
(2-mercaptopyrimidine)
and
dmpm
(4,6-dimethyl-2-mercaptopyrimidine)
yielded
complexes of the type [Ru(N-S)(bipy)(P-P)]PF6, N-S = pyS, prm or dmpm. Preliminary in vitro tests for antitumour
activity against the MDA-MB-231 human breast tumor cell line, using the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide], were carried out for the new complexes, for the precursors complexes and for the free
ligands. The complexes tested against MDA-MB-231tumour cells showed high activity, with IC50 values lower than
-1
1 µmol L .
It was shown that the complexes are able to increase the population of cells in the G0/G1 phase. This G0/G1
population increase suggests that these complexes interfere in cell metabolism in a way that the cell cannot enter
the DNA duplication phase (S phase), thereby preventing cell proliferation. While S180 tumour cells already have a
larger cell population in the G0/G1 phase, which is characteristic of melanomas, the complexes enhance the
increase of this population, further reducing the efficiency of cell cycling and thus cell proliferation.
Acknowledgements
We thank FAPES, CNPq, CAPES, and CYTED for financial support.
38
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 051
AREA: MBD
ORAL
Interaction of divalent cations with protein PARK9
Zoroddu, M.A.1; Peana, M.1; Medici, S.1; Solinas, C.1; Juliano, C.1; Remelli, M.2
1
2
Chemistry and Pharmacy, University of Sassari, Sassari, Italy
Chemical and Pharmaceutical Sciences, University of Ferrara, Ferrara, Italy
[email protected]
Metals have been shown to play a role in the genesis and development of many neurodegenerative diseases.
Park9 encoded protein can protect cells from manganese poisoning, an environmental risk factor for a Parkinson’s
1,2
disease- like syndrome . Park9 belongs to a family of ATP-ases involved in metal coordination and transportation;
familial mutations of this gene may result in early development of PD. We tested two peptide sequences from
Park9, -P1D2E3K4H5E6L7- (1) and -F1C2G3D4G5A6N7D8C9G10- (2), for Mn(II), Zn(II) and Cu(II) binding. These
fragments are located from 1165 to 1171 and from 1184 to 1193 residues in Park9 sequence, and are highly
conserved in a number of organisms, from yeasts to humans. Experiments have been carried out at different pH
values and ligand/metal molar ratios with both potentiometric and spectroscopic (NMR, UV-vis) techniques,
showing that the three metals are able to effectively bind the examined peptides. Mn(II) and Zn(II) coordination with
peptide (1) involves imidazol of His5 and carboxyl γ-O of Asp2, Glu3 and Glu6 residues, in a distorted octahedral
geometry, possibly involving bidentate interaction of carboxyl groups; four donor atoms participate in Zn(II) binding,
resulting in a tetracoordinated geometry. Mn(II) and Zn(II) coordination involves the two cysteines in peptide (2);
Mn(II) accepts additional ligand bonds from D4 and D8 to complete the coordination sphere, together with some
water molecules. Details of Cu(II) coordination are under study.
References
[1] Gitler A.D., Chesi A., Geddie M.L., Strathearn K.E., Hamamichi S., Hill K.J., Caldwell K.A., Caldwell G.A., Cooper A.A.,
Rochet J.-C., Lindquist S., Alpha-synuclein is part of a diverse and highly conserved interaction network that includes PARK9
and manganese toxicity, Nat Genet, 2009, 41, 308-315.
2+
2+
2+
2+
[2] Schmidt K., Wolfe D.M., Stiller B., Pearce D.A., Cd , Mn , Ni and Se toxicity to Saccharomyces cerevisiae lacking
YPK9p the orthologue of human ATP13A2, Biochem Biophys Res Com, 2009, 383, 198-202.
39
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 070
AREA: MBD
ORAL
A novel Cu(II) ternary complex with iminodiacetate and dimethyl-bipyridine. Synthesis,
characterization and DNA interaction
Alvarez, N.1; Costa-Filho, A.2; Torre, M.H.1; Ellena, J.3; Facchin, G.1
1
2
Cátedra de Química Inorgánica, Facultad de Química, UdelaR, Montevideo, Uruguay
Departamento de Biofísica Molecular, Faculdade de Filosofia Ciências e Letras de Ribeirão Preto, USP, Ribeirão Preto, Brazil
3
Grupo de Cristalografía, Instituto de Física de São Carlos, USP, São Carlos, Brazil [email protected]
The importance of metal complexes in medicine dates from the XVIth century with reports on the use of
metallodrugs in anticancer therapy. Inorganic Medicinal Chemistry has made great advances in search of new
metal compounds presenting antitumor activity, for instance, by use of essential metals. In this work, in the search
of new antitumor agents, the ternary complex [Cu(iminodiacetate)(4,4’-dimethyl-2,2’-bipyridine)]•4H2O was
prepared employing the reaction of copper acetate with iminodiacetic acid in presence of dimethyl-bipyridine. The
obtained complex was characterized by elemental analysis, single crystal X-ray diffraction, Fourier Transformed
Infrared spectroscopy (FT- IR), Electron Paramagnetic Resonance (EPR) and UV-visible spectroscopy (UV-vis).
The complex crystallizes in a triclinic unit cell, in the centrosymmetric space group P-1. The Cu(II) center is bonded
to a bypiridine ligand -through its N atoms- and to the iminodiacetate ion -through the carboxylate O atoms and the
imine N atom-. The molecular structure was confirmed by FT-IR where bands involving the ligand and donor atomCopper bonds are observed. Knowing the atom array in solid state is considered fundamental for further aqueous
solution analysis and comprehension of the complexes behavior in biological media. UV-vis absorption bands in
aqueous solution are consistent with a square pyramidal Cu(II) environment, which suggests the maintenance of
the coordination sphere observed in solid state. The ability of the complex to interact with deoxyribonucleic acid
(DNA) was studied by Circular Dichroism (CD) in constant concentration of DNA with increasing concentration of
complex. The profile observed in the CD spectra evidences complex-DNA interaction which is consistent with an
intercalative interaction, as observed in other complexes containing dimethyl-bipyridine.
40
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 077
AREA: MBD
ORAL
Targeting human topoisomerase I with different vanadyl compounds
Vieira, S.1; Katkar, P.1; Castelli, S.1; Benítez, J.2; Costa Ferreira, A.M.3; Gambino, D.2; Desideri, A.1
1
Department of Biology, University of Roma Tor Vergata, Rome, Italy
Cátedra de Química Inorgánica, Facultad de Química, Montevideo, Uruguay.
3
Department of Chemistry, University of Sao Paulo, Sao Paulo, Brasil [email protected]
2
DNA topoisomerases are essential and ubiquitous enzymes, belonging to two classes (type Iand type II), both
characterized by a catalytic mechanism whichinvolves a nucleophilic attack of a DNA phosphodiester bondby a
tyrosyl residue from the enzyme, but type I cleaves only oneDNA strand, whereas type II cleaves both strands.
These enzymeshave been shown to be essential in nearly all processes of DNAmetabolism such as replication,
transcription, recombination andchromosomal segregation. The human topoisomerase I enzyme is composed of
765 amino acids and has four distinct domains: the N-terminal domain (1–214), the core domain (215–635), the
linker domain (636–712) and the C-terminal domain (713–765). The three-dimensional structure of the
reconstituted N-terminal truncated version of human topoisomerase in complex with a 22 bp DNA molecule shows
the enzyme organized in multiple domains which ‘clamp on’ the DNA molecule. Human topoisomerase I is of
significant medical interest since it is the unique cellular target of camptothecin a plant alkaloid with strong
anticancer activity, that rapidly blocks both DNA and RNA synthesis. Here we present some recent results
concerning the interaction of the enzyme with different ligands coordinated to the vanadyl cation. The efficiency of
the metal compounds in inhibiting the enzyme is different depending on t the ligand dimension and polarity and
such differences are discussed in the attempt to derive a structure-function correlation and select the best ligand to
target the enzyme.
41
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 079
AREA: MBD
ORAL
The studies of the interactions between metal complexes and DNA
Mao, Z.
MOE Key Laboratory of Bioinorganic and Synthetic Chemistry, School of Chemistry and Chemical Engineering, Sun Yat-Sen
University, Guangzhou, China [email protected]
DNA, the carrier of the genetic information and the substantial basis of the expression of genes, plays a significant
role in the metabolic processes of lives like growth and reproduction, and is involved in the excitation of the silent
genes of cancers. Due to the diversity of the structures of the DNA and the fact that the DNA can be easily targeted
in the therapy of cancers, the studies of the interactions between the small molecular metal complexes and the
DNA, has a great impact on both the discovery of the structures or the functions of nucleic acids and the
development of potential anti-cancer drugs. Thus, there are two aspects. The effective exploration of the mimic of
metallonucleases that cleave dsDNA under physiological conditions provides us not only a solid theoretical
foundation for the chemical process of lives but the estimated applications in the fields of diagnosis and therapy of
cancers in the future. And the synthesis of the metal complexes targeting and stabilizing the G-quadruplex DNA
brings about the hope of conquering cancers. Considering the structural features of the duplex DNA and the Gquadruplex DNA, we have designed and synthesized a series of Cu and Pt complexes with functional groups for
the research of the interactions in recent years, which is as follows:
1) By exploring the thermodynamics and the kinetics mechanism of the interactions between model compounds of
nuclease enzyme and the DNA systematically, we made a conclusion of the different abilities to shear the DNA
from many angles, including the structures of ligands and the properties of the electrons of substituents of main
ligands.
2) We found a fact that some Pt complexes composed of pyridines are able to induce the G-quadruplex DNA and
promote the stability of the specific structures, which is successfully confirmed by the data of PCR-stop, FRET and
CD. Based on the research above, in addition, we focus on the synthesis of some Pt complexes with unique
configurations via supermolecular self-assembly. And the very complexes can stabilize the G-quadruplex DNA
efficiently and may be used as an inhibitor of telomere. We tried to explain the mechanism of the interactions at the
level of cells or molecules to provide theoretical and experimental proofs for the further development of therapeutic
reagents with higher selectivities and lower toxic side effects.
Acknowledgements: Financial support from the National Natural Science Foundation of China, the Guangdong
Provincial Natural Science Foundation and National Basic Research Program of China.
References
1. An, Y.; Tong, M. L.; Ji, L. N; Mao, Z. W. Dalton Trans., 2006, 35, 2066.
2. An Y.; Lin, Y. Y.; Wang, H.; Sun, H. Z.; Tong, M. L.; Ji, L. N.; Mao, Z. W. Dalton Trans., 2007, 36, 1250.
3. He, J.; Hu, P.; Wang, Y. J.; Tong, M. L.; Sun, H. Z.; Mao, Z. W.; Ji, L. N. Dalton Trans., 2008, 38, 3207.
4. Wang, J. T.; Xia, Q.; Zheng, X. H.; Chen, H. Y.; Chao, H.; Mao, Z. W. Dalton Trans., 2010, 39, 2128.
5. Wang, J. T.; Zheng, X. H.; Xia, Q.; Mao, Z. W. Ji, L. N.; Wang, K. Dalton Trans., 2010, 39, 7214.
6. Wang, J. T.; Li, Y.; Tan, J. H.; Ji, L. N.; Mao, Z. W. Dalton Trans., 2011, 40, 564.
7. Zheng, X. H.; Zhong, Y. F.; Tan, C. P.; Ji, L. N.; Mao, Z. W. Dalton Trans., 2012, 41, 11807.
8. Zheng, X. H.; Chen, H. Y.; Tong, M. L.; Ji, L. N.; Mao, Z. W. Chem. Commun., 2012, 48, 7607.
42
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 084
AREA: MBD
ORAL
Dual mode binding of ruthenium arene anticancer complexes to DNA: Coordination and
intercalation
Liu, H.
Jiangsu Province Key Laboratory of Biofunctional Material, School of Chemistry and Environmental Science, Nanjing Normal
University, Nanjing, China [email protected]
Organometallic Ru(II) arene complexes of the type [(η6-arene)Ru(en)Cl]+ [1], are toxic to cancer cells, including
cisplatin-resistant cell lines. We have investigated the kinetics and thermodynamics of binding to DNA. We find that
Ru-arene complexes with extended rings can bind to DNA duplex by bi-functional binding modes (coordinative and
intercalative) and further work has provided evidence that such bifunctional interactions of Ru-bip can affect the
basepairing of duplex DNA [2-3]. Two-dimensional NMR experiments [1,4] show the presence of a novel
penetrating intercalation of Ru-tha into a DNA hexamer. The results presented here provide and may help to
explain why ruthenium arene complexes have a different mechanism of antitumour activity compared to cisplatin
[4]. We also relate these interactions to themechanism of anticancer activity and to structure-activity relationships.
In addition, we find that the interactions between these complexes and DNA show close similarities to those of
covalent polycyclic aromatic carcinogens [5]. It is the first time that the anticancer mechanism and carcinogenesis
mechanism are tied together based on the above researches..
References
1. Liu, H. -K., Sadler, P. J., (2011). "Interactions of Metallodrugs with DNA in NMR of Biomolecules: Towards Mechanistic
Systems Biology". I. Bertini, K. McGreevy (Ed), Wiley VCH., 16, 283.
2. Liu, H. -K., Burners-Price, S, Sadler, P. J.,* Angew. Chem., Int. Ed., 2006, 45, 8153.
3. Liu, H. -K., Sadler, P. J.,* Chem. Eur. J., 2006, 12, 6151.
4. Liu, H. -K.*, Sadler, P. J.,* Chem. Sci. 1 (2010), 258.
5. Liu, H. -K.,* Sadler, P. J.*, Acc. Chem. Res., 2011, 44, 349.
43
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 093
AREA: MBD
ORAL
Towards the development of new copper compounds for the treatment of cancer: Study
of the cytotoxic activity of [Cu(L-dipeptide)(1, 10-o-phenantroline)] complexes
Iglesias, S.1; Noble, C.1; Kramer, G.2; González, R.2; Torre, M.H.1; Kremer, E.1; Facchin, G.1
1
2
Cátedra de Química Inorgánica, Facultad de Química, UdelaR, Montevideo, Uruguay
Departamento de Desarrollo Biotecnológico, Instituto de Higiene, Facultad de Medicina, Montevideo, Uruguay
[email protected]
Coordination compounds of essential metals are being explored as new antitumor species, in the search of drugs
with expanded activity and/or reduced side effects. To date, several Cu(II) compounds have shown promising
antitumor activities in cellular lines, some of them are being studied in vivo. Their mechanism of action is not
completely understood; however, copper and its complexes, present the ability to participate in redox reactions.
This event generates reactive oxygen species (ROS) that are toxic to cancerous cells. In addition, many of the
complexes present the ability to intercalate DNA, interfering with DNA replication of dividing cells, which may also
be part of their mechanism of antitumor action.
We have synthesized several new [Cu(L-dipeptide)(1,10-phenanthroline)] compounds. In solid state they present a
pyramidal geometry, where the Cu(II) ion is coordinated both to the phenanthroline, through its nitrogen atoms, and
to the dipeptide, through the amine and the amide nitrogen atoms and the carboxylic oxygen (Figure). They are
stable in aqueous solution, where its coordination is maintained. This work focuses on the study of the lipophilicity
and antitumor activity of [Cu(L- dipeptide)(1,10-phenanthroline)] (were L-dipeptide= phe-phe, phe-val, phe-ala, alaphe, gly-val, ala- gly). The cancer cell lines utilized were: HeLa (human cervical cancer), NMU (rat breast cancer)
and 4T1 (mouse breast cancer). The lipophilicity, depends on the dipeptide, being the [Cu(L-Phe- Phe)(1,10phenanthroline)] complex the most lipophilic. The estimated IC50 varied from 1 to 30 µM in these tumor cells, being
the complexes containing the dipeptides phe-val, phe-ala and ala-phe the ones that showed best antitumor activity
in vitro. Therefore, complexes [Cu(L-dipeptide)(1,10- phenanthroline)] with dipeptides phe-val, phe-ala and ala-phe,
may be the candidates to test its antitumor activity in animal models of breast cancer.
44
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 104
AREA: MBD
ORAL
Synthesis, characterization and anti-malarial activity of a binuclear copper(II) acetatequinine complex
Pastrana Alta, R.Y.1; Aguilar, V.H.1; Katzin, A.M.2; Espósito, B.P.1; Valderrama Negron, A.C.3
1
Instituto de Química, Universidade de São Paulo, São Paulo, Brasil
Instituto de Biociencias, Universidade de São Paulo, São Paulo, Brasil
3
Facultad de Ciencias, Universidad Nacional de Ingeniería, Lima, Perú
[email protected]
2
Malaria is one of the tropical neglected disease whose therapy may benefit from a bioinorganic chemistry
approach.
In this work, we prepared a binuclear copper(II) derivative of the type [Cu(O2CCH3)(µ-Qn)]2 (Qn = quinine). Its
structure was resolved by X-ray diffraction and showed two copper metal centers each surrounded by three oxygen
atoms and one nitrogen atom (CuNO3). The complex has been further characterized by thermogravimetric and
spectroscopic (UV- Vis, IR and EPR) analysis, as well as magnetic measurements. The complex present geometric
and electronic similarities with Type 3 copper centers of the multicopper oxidases and hemocyanin/tyrosinase.
Quinine and acetate coordinate to Cu as a zwitterionic bidentade ligand (O,N) and monodentade ligand (O),
respectively.
The antimalarial activity of the complex and the starting ligand was evaluated in vitro against bloodstream schizonts
of Plasmodium falciparum. The complex (IC50 = 14.6 nM) was more active than quinine (IC50 = 60.15 nM). Both
present superior activity in comparison with other reported antimalarial complexes [1].
References
[1] Nikhil H. Gokhale a, Subhash B. Padhye, David C. Billington,Daniel L. Rathbone, Simon L. Croft, Howard D. Kendrick,
Christopher E. Anson, Annie K. Powell. Synthesis and characterization of copper(II) complexes of pyridine-2carboxamidrazones as potent antimalarial agents. Inorganica Chimica Acta 2003;349:23-/29.
45
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 115
AREA: MBD
ORAL
Rhenium and technetium complexes with a dithiocarbazate ligand as potential
radiopharmaceuticals
de Araujo Fernandes, A.G.1; Lafratta, A.E.2; Portela Luz, C.2; Navarro Marques, F.L.2; Ulrich, A.3; Deflon, V.M.4
1
Departamento de Ciências Exatas e Tecnológicas - DCET, Universidade Estadual de Santa Cruz - UESC, Ilhéus, Brazil
2
Faculdade de Medicina, Universidade de São Paulo, São Paulo, Brasil
3
Institute of Chemistry and Biochemistry, Free University of Berlin, Berlin, Germany
4
Instituto de Química de São Carlos, Universidade de São Paulo, São Paulo, Brazil
[email protected]
Introduction:
have
a
99m
Tc is a leading radionuclide in nuclear medicine, being used in diagnostic, while
potential
usage
in
radiotherapy.
The
compound
186
Re and
188
Re
5-hydroxy-3-methyl-5-phenyl-pyrazoline-1-(S-
benzyldithiocarbazate) (H2-BDTC) consist on a versatile free ligand, being able to coordinate mono-, bi- or
tridentate as well as mono- or dianionically. This work presents rhenium and technetium complexes with H2-BDTC
mono- and dianions as ligands, in different metal:ligand stoichiometric proportions (1:1 and 1:2) and coordination
modes (bi- and tridentate).
Material and methods: The distorted octahedral complexes [ReO(BDTC)(H-BDTC)] and [99TcOCl(BDTC)] were
prepared
by
reacting
[ReOCl3(PPh3)2]
and
(NBu4)[99TcOCl4]
with
H2-BDTC,
respectively,
in
dichloromethane/methanol. The complexes were fully characterized by standard methods and crystal structure
determinations.
The
99m
99m
Na
Tc-BDTC complex was obtained by addition of 0.3 mL (1mg/mL – H2-BDTC/DMSO) to 0.3 mL of
TcO4 solution (370 MBq) followed by 20 µL of acid solution of Sn2+ (0.25 mg/mL), and 0.640 mL of 0.1 N
phosphate buffer (pH=7.4). Labeling efficiency was assessed by W3MM paper chromatography using acetone or
saline, and by HPLC [C18 (4,6x250 mm) acetone 1 mL/min] using [ReO(BDTC)(H-BDTC)] as standard.
Electrophoresis was evaluated using W3MM paper and PBS (50 mM, pH 7.4):methanol (1:1), at 275V, partition
coefficient (LogP) was assessed by standard procedure.
Results: Labeling efficiency was over 95 %. The compound showed charge zero and logP = 1.36. HPLC analysis
gave a compound retained at 2.1 min (100%), but was being converted to another compound with retention time of
12.8 min (88% at 12h), closer to 11.7 min to[ReO(BDTC)(H-BDTC)2].
Conclusion: Labeling process of H2-BDTC with technetium is simple and in high yield. The compounds, analyzed
just after preparation, showed neutral and lipophilic characteristics suggesting its use for radiopharmaceutical brain
studies.
Acknowledgements: The authors thank to FAPESP, CNPq and CAPES for the financial support.
46
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 118
AREA: MBD
ORAL
PTA (1, 3, 5-triaza-7-phosphaadamantane) as co-ligand for metal complexes bearing anti
T. cruzi activity
Cipriani, M.1; Toloza, J.2; Curbelo, E.1; Olea Azar, C.2; Maya, J.D.3; Gambino, D.1; Otero, L.1
1
Cátedra de Química Inorgánica, Facultad de Química, Montevideo, Uruguay
Departamento de Química Inorgánica y Analítica, Facultad de Ciencias Químicas y Farmacéuticas, Santiago, Chile
3
Programa de Farmacología Molecular y Clínica, ICBM, Facultad de Medicina, Universidad de Chile, Santiago, Chile
2
Chagas disease is caused by the protozoan parasite Trypanosoma cruzi (T. cruzi). Being originally an
autochthonous Latin American endemic disease, it is becoming a global health problem because of immigration. In
the search of an effective and safe chemotherapy for this incurable illness, we have been working in the rational
design of metal complexes with active ligands against T. cruzi in order to improve their activity and pharmacological
properties. Different co-ligands have also been included in the new species with the aim of modulating their
physicochemical properties. In particular, PTA (1,3,5-triaza-7-phosphaadamantane) had been previously included
in metal complexes bearing antitumor activity leading to an improvement in their aqueous solubility and to a pH
dependent DNA interaction.
In this work, we report the development of palladium and platinum complexes with bioactive 5-nitrofuryl containing
thiosemicarbazones (L) and PTA as ligands. Complexes of the formula [MCl(PTA)L] with M = Pd or Pt were
synthesized and characterized by elemental analysis, conductivity measurements and 1H NMR and infrared
spectroscopies. Their activity in vitro against the trypomastigote form of T. cruzi. was evaluated. All complexes
resulted more active than the reference drug nifurtimox and than the previously obtained Ru-PTA-L complexes.
Lipophilicity of the obtained complexes was studied by reverse phase TLC and its relation to obtained biological
activity was stated. In addition, being DNA a potential target for PTA containing metal complexes, the ability of the
obtained compound to interact with this macromolecule was studied. The inclusion of co-ligands that confer desired
properties to metal complexes seems to be a valid strategy to rationally design potential antiparasitic metal
compounds.
47
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 133
AREA: MBD
ORAL
Redox non-innocent thioether ligands and their copper compexes
Castillo, I.1; Martínez-Alanis, P.1; Sánchez Eguía, B.1; Ugalde-Saldívar, V.2; Aullón, G.3
1
2
Instituto de Química, Universidad Nacional Autónoma de México, México DFMéxico
Facultad de Química, División de Estudios de Posgrado, Universidad Nacional Autónoma de México, México DF, México
3
Departament de Quimica Inorgànica, Universitat de Barcelona, Barcelona, Spain
[email protected]
The presence of thioether ligands in the active sites of copper-containing metalloenzymes has resulted in a
growing number of reports involving these sulfur-based donors. Intriguingly, methionine residues have also been
identified in several types of copper monooxygenases such as Dopamine-β-monooxygenase (DβM), although their
+
role has not been firmly established. This raises the need for Cu complexes with thioether-rich coordination
environments; the known examples usually get oxidized to Cu2+ analogues, but the coordination environment
provided by tripodal benzimidazolylamino-thioether ligands results in a HOMO level with predominant arylthioether
character. A combined experimental and DFT study demonstrates that oxidation of such complexes occurs at the
thioether groups, giving rise to cascade ligand decomposition reactions initiated by sulfur-based radical cations.
The implications of these results for bioinorganic systems will be discussed.
48
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 064
AREA: MIEH
ORAL
Determination of Cd in urine: validation of a routine methodology
Alvarez, C.; Pizzorno, P.; Pereira, N.; Mañay, N.
Cátedra de Toxicología - DEC, Facultad de Química, Montevideo, Uruguay
[email protected]
Cadmium toxicity is well known and one of its main sources of exposure for general population is tobacco smoke.
According to the International Agency for Research on Cancer (IARC) cadmium and cadmium compounds are
carcinogenic to humans (Group 1). Therefore standardized protocols, validated and reliable for monitoring this
metal in humans are needed.
Urinary cadmium (U-Cd) has been shown to accurately reflect the amount of cadmium in the body, whereas blood
cadmium (B-Cd) shows recent exposure to the metal [1]. Reference values for U-Cd in general population are 0-1
1.4 µg L [2].
A routine methodology for Cd determination in urine is proposed and validated.
The method is based in the extraction of Cd from urine with diethylammonium diethyldithiocarbamate (DDDC) /
Triton X-100 / 4-methylpentan-2-one (methyl isobutyl ketone, MIBK), and its subsequent determination by
electrothermal atomic absorption spectrometry (ET-AAS) at 228,8 nm.
Urine samples were stored at -20ºC until analysis. For the extraction, 1 mL of DDDC 0.2% (w/v), Triton X-100 1%
(w/v) in MIBK were added to 2.5 mL of urine. The mixture was shaked for 5 minutes and then centrifuged for 5
minutes at 3500 rpm. The supernatant was introduced into the graphite furnace to perform the Cd determination.
-1
-1
-1
2
The figures of merit were: LOD: 0.06 µg L (3s), LOQ: 0.19 µg L (10s), linearity: up to 5 µg L (r >0.99), precision:
RSD%>5 (n=5), recovery: 108% (spiked samples, n=5).
The proposed methodology was adequate to the purpose and can be implemented as a routine analysis of Cd in
urine.
References
1)
Agency
for
Toxic
Substances
and
Disease
Registry
(ATSDR)
ToxFAQs™
for
Cadmium.
http://www.atsdr.cdc.gov/toxfaqs/tf.asp?id=47&tid=15. Accessed November, 20, 2012.
2) Repetto, M.R. y Repetto, M. Tabla de concentraciones de xenobióticos en fluidos biológicos humanos como referencia para
el
diagnóstico
toxicológico
(actualización
2007).
http://www.esmed.com.ar/download/postgrado/medicina_legal/criminalistica/toxicologia_de_laboratorio_17-102009/modulo_1_tabla_valores_ref.pdf. Accessed November 20, 2012
49
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 071
AREA: MIEH
ORAL
Lung oxidative metabolism after an acute exposure to transition metals present in
ambient particulate matter
Magnani, N.1; Marchini, T.1; Mebert, A.1; Tasat, D.2; Desimone, M.1; Diaz, L.1; Alvarez, S.1; Evelson, P.1
1
Universidad de Buenos Aires, Buenos Aires, Argentina
Universidad Nacional de San Martín, Buenos Aires, Argentina
[email protected]
2
Transition metals present in particulate matter (PM) could alter lung oxidative metabolism due to their ability to
participate in Fenton reactions thus increasing reactive oxygen species production. The aim of this work was to
study lung oxidative metabolism after an acute exposure to Residual Oil Fly Ashes (ROFA) and transition metals
present in PM. Mice were intranasally instilled with a ROFA suspension (1.0 mg/kg body weight) or silica
nanoparticles (NP) containing Ni or Cr adsorbed to its surface (0.01-1.00 mg Ni/kg body weight). Lung samples
were analysed 1 h after instillation. Tissue O2 consumption, NADPH oxidase (Nox) activity and TBARS content
were evaluated after PM and NP exposure. Mitochondrial respiration, H2O2 and ATP production rates,
mitochondrial membrane potential and oxidative damage markers were assessed in isolated mitochondria after
ROFA treatment.ROFA exposure was found to be associated with 61% increased tissue O2 consumption, a 30%
increase in Nox activity, and a 33% increased mitochondrial active respiration (state 3). During state 3, decreased
mitochondrial membrane potential and a 53% decreased ATP production rate were observed. No changes were
found in either H2O2 production rate or oxidative damage markers in isolated mitochondria. Regarding NP
exposure, tissue O2 consumption, Nox activity and TBARS were found significantly increased in every Ni
concentration assessed, while NP coated with Cr only showed significantly increased O2 consumption and TBARS
content at 0.05 mg/kg. After ROFA exposure, increased tissue O2 consumption may account for an augmented Nox
activity and mitochondrial respiration. The mitochondrial function modifications observed may prevent oxidative
damage within the organelle. Transition metals like Ni or Cr present in PM seem to be important components
involved in the altered lung oxidative metabolism after ROFA exposure. These findings provide new insights to the
understanding of lung oxidative damage mechanisms triggered by metals present in environmental PM.
50
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 072
AREA: MIEH
ORAL
The exposure to an environmental particulate matter surrogate rich in transition metals
impairs cardiac mitochondrial function and contractile reserve
Marchini, T.1; Magnani, N.1; D’Annunzio, V.1; Tasat, D.2; Gelpi, R.J.1; Alvarez, S.1; Evelson, P.1
1
Universidad de Buenos Aires, Buenos Aires, Argentina
Universidad Nacional de San Martín, Buenos Aires, Argentina
[email protected]
2
The exposure to environmental particulate matter (PM) is associated with increased cardiovascular morbidity and
mortality. Transition metal content of PM is suggested to play a predominant role, owing to their ability to drive
Fenton- like reactions leading to oxidative metabolism alterations. A byproduct of fossil fuel combustion known as
residual oil fly ashes (ROFA) is particularly rich in iron, nickel and vanadium, and is frequently used as a PM
surrogate to evaluate the contribution of transition metals on PM biological effects. To address this hypothesis, with
focus on heart mitochondrial and cardiac function, an in vivo animal model of acute exposure to ROFA was used.
Female Swiss mice weighting 25 g were intranasally instilled with a ROFA suspension (1 mg/kg) and hearts were
removed after 3 hours. Tissue and isolated mitochondrial oxygen consumption was followed polarographically with
a Clark-type oxygen electrode. Mitochondrial membrane potential was evaluated by flow cytometry. Mitochondrial
ATP production rate was determined by a chemiluminescent assay. Cardiac contractile reserve was evaluated in
isolated perfused hearts at constant flow, as left ventricular developed pressure before and after a β-adrenergic
stimulus with isoproterenol.
Tissue oxygen consumption was significantly decreased by 38% in ROFA-exposed mice (control: 1180±70 ng-at
O/min g tissue, p 0.001). Likewise, mitochondrial respiration was decreased by 30% in state 4 (control: 87±5 ng-at
O/min mg prot, p 0.05) and by 24% in state 3 (control: 240±20 ng-at O/min mg prot, p 0.01). These findings were
associated with significant mitochondrial depolarization and deficient ATP production. When contractile reserve
was evaluated, basal contractility was not modified (control: 75±5 mmHg). However, isolated perfused hearts failed
to properly respond to isoproterenol in ROFA-exposed mice. The present results show an impaired mitochondrial
function associated with deficient cardiac contractile reserve, and may provide new insights to the mechanisms of
the adverse cardiovascular effects triggered by PM exposure.
51
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 096
AREA: MIEH
ORAL
Exposure to copper in a community chronically exposed to mining waste, Chile
Cortes, S.; Molina, L.
Departamento de Salud Pública, Pontificia Universidad Catolica de Chile, Santiago, Chile
[email protected]
Background: North of Chile has some waste mining’s deposits. Chañaral is one the most important due their high
environmental impact in the bay. Environmental studies show the principal metal associated is copper. We had
measured urinary copper in 2009 by ICP-MS at INTA (Nutrition and Food Technology Institute, University of Chile).
The urinary levels varied between 4,4 – 107,4 µg/L, assuming this group was exposed chronically to copper. We
have made measurement of serum copper and ceruloplasmin to establish the real exposure to copper in this
population.
Methodology: we interviewed 167 adults volunteers from our first study on exposure to metals (2009). In 2011
they gave blood sample in fast to measure in serum copper and ceruloplasmin. Ceruloplasmin was made by
enzymatic methods while copper by atomic spectrophotometry at INTA.
Results: our preliminary results shows:
Urinary copper µg/L1
Serum copper µg/L
Serum ceruloplasmin (%)
N
204
129
129
Medium ± DE
20,2 ± 11,5
85,2 ± 24,9
37,8 ± 13,1
Mínimum
4,4
38,4
14,7
Máximum
107,4
206,9
97,0
Percentile 95
41,9
115,7
References value
2
13,0
70 – 150
52,5
3
4
26 - 33 %
It is showed that exist urinary excretion of copper, according high levels in the waste. Medium levels of biomarkers
of internal function were inside normal values. Serum level of copper and ceruloplasmin showed maximum level
above the normality.
Conclusions: It is not possible to establish this population has a normal excretory pattern. Could be necessary to
collect more data to define exposure (in air, water and food). This information together with this will estimate about
deleterious effects on health.
Acknowledgements: Thank to Profesor Miguel Arredondo.
References
1) Universidad de Chile. Cortes S. Thesis Doctoral 2009.
2) J Trace Elem Med Biol. 2006;20(4):253-62.
3) Biol Trace Elem Res. 2008; 123(1-3): 261-269.
4) Anal Biochem. 1960; 3: 452-456.
52
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 055
AREA: NATME
ORAL
Chemical speciation of phytate at gastrointestinal level: nutritional quality of vegetablebased diets
Veiga, N.; Torres, J.; Kremer, C.
Departamento Estrella Campos, Facultad de Química, Universidad de la República, Montevideo, Uruguay
[email protected]
Myo-inositol phosphates are essential biomolecules in all eukaryotic cells. Among them, phytate (Figure 1, L12-) is
largely abundant in vegetable-based products and it possesses various beneficial effects in health. Conversely, it
has been categorized as an antinutrient, a precipitating agent which reduces the absorption of Fe, Zn and Ca,
causing a global nutritional problem. This dichotomy has raised a debate1, and chemical quantitative data are still
not available to give an answer to this problem.
The interaction of phytate with metal ions was studied by potentiometry. Besides, the solid metal phytates were
synthesized and fully characterized. Solubility measurements were also carried out. These data and all the
nutritional and physiological information2,3 were included in a mathematical model, allowing the calculation of the
gastrointestinal speciation of phytate for each tested intake.
n+
Soluble mononuclear ([M (HxL)]
(12-n-x)-
) and polynuclear ([Mx(HyL)]) metal complexes were detected. Under metal
ion excess conditions, scarcely soluble polynuclear species (Zn5(H2L)•14H2O and Fe4L•22H2O) were obtained. The
resulting model showed nutritional trends that are in agreement with those already reported. In the duodenum,
phytate is mostly precipitated with magnesium and calcium. Phytate reduces zinc and iron bioavailability by
disturbing their adsorption and solution equilibria. Among all, the citrate and glucosamine are the most important
solubilizant agents at gastrointestinal level.
In sum, a large gastrointestinal model was generated. It was successfully applied to predict nutritional tendencies in
the bioavailability of essential metal ions. The chemical information can be used to improve the nutritional quality of
vegetable-based diets.
Aknowledgments: The authors wish to thank ANII for financial support.
References
1. Harland, B. F., Morris, E. R. Phytate: A good or a bad food component? Nutrition Research; 1995; 15; 733-754.
2. Diem, K., Lentner, C. Tablas Científicas Documenta Geigy. Barcelona, España: Ciba-Geigy, 1975.
3. Tablas de composición de alimentos. El pequeño "Souci-Fachmann-Kraut". Zaragoza, España: Acribia S.A., 1999.
53
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 143
AREA: NATME
ORAL
Pharmaconutrition with selenium in the critically ill
Manzanares,W.; Galusso, F.; Tacchini, R.; Facchin, G.; Torre, M.H.; Biestro, A.; Hardy, G.
[email protected]
In critically ill patients, low plasma selenium (Se) levels are associated with systemic inflammatory response
syndrome (SIRS) and multiple organ failure. In Uruguay, we have found that Se and glutathione peroxidase-3
(GPX-3) are significantly decreased in SIRS patients. Furthermore, we demonstrated that plasma Se has a
relatively good predictive value for intensive care unit (ICU) mortality (P= 0.034). The Se cut-off value of 60 µg/L
and the GPx-3 cut-off value of 0.5 U/mL have high specificity for severity of critical illness.
The optimum safe dose and method of administration remains controversial but the best clinical outcomes in SIRS
have been reported from very high Se doses, administered first by bolus followed by continuous infusion. Selenite
is postulated to have a biphasic action; initially as a pro-oxidant and then as an antioxidant. A loading dose given
as a bolus could have a direct reversible inhibition of NF-κB binding to DNA controlling gene expression and thus
down-regulating the synthesis of pro-inflammatory cytokines. In 20 critically ill SIRS patients we performed a dosing
study comparing the pharmacokinetic profile of two high doses of selenite (as selenious acid). Data analysis
showed that the concentration/time curves for Se overlapped. However the pharmacodynamic profile of GPx-3
showed that optimal GPx-3 levels were only achieved with the higher dose of selenite (1600µg/d Se). In both
groups, GPx –3 decreased after day 7 of supplementation. This suggests that GPx-3 inhibition or saturation occurs
as a result of insufficient synthesis of precursors such as hydrogen selenide or selenocysteine, and/or limitation of
glutathione synthesis.
Finally, in a phase II randomized we have investigated high-dose Se, administered first as a bolus (2,000 µg over 2
h), and thereafter 1,600 µg/day Se. After 10 days, ICU patients exhibited less organ dysfunction (P = 0.0001) and
the incidence of hospital-acquired pneumonia was lower (P= 0.03). Additionally, Se monotherapy was not
associated with a higher incidence of adverse events during 28 days follow-up.
54
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 008
AREA: RMNM
ORAL
Influence of pretreatment with caspofungin in the early diagnosis of fungal infections
with 99mTc- tricarbonyl-caspofungin complex
Fernández, L.1; Vilche, M.2; Reyes, L.3; Terán, M.4; Rey, A.5
Departamento Estrella Campos, Facultad de Química, Montevideo, Uruguay
[email protected]
Opportunistic fungal infections occur primarily in immunesupressed hosts, the development of fast and accurate
diagnostic techniques is necessary for appropriate treatment. The aim of this work was the synthesis,
physicochemical and biological evaluation of 99mTc-tricarbonyl-Caspofungin as potential diagnostic agent. The
complex was synthesized using Tc-tricarbonyl precursor and substitution with ecaspofungin (Merck).
Physicochemical studies included stability over time, lipophilicity of the complex (coefficient octanol/water), % of
binding to yeast, challenge against cysteine and histidine, and plasmatic protein binding. Biodistributions and
scintigraphic images were performed in CD1 female mice 20 ± 5 g (n = 3/group). Before the administration of the
complex, animals were immunosuppressed with cyclophosphamide (6mg inicial/2mg maintainment) and divided
into 3 groups: Group 1 was inoculated into the right hind leg with Candida albicans (9x108UFC). Group 2 was
inoculated with sterile water. Group 3 control was immunosuppressed without infection. Groups 1 and 3 received
pre-treatment with caspofungin at therapeutic concentration (1mg/kg) and Group 2 only saline to match stress
conditions. To verify the efficacy of immunosuppression, total count of viable white cells in the spleens was
determined in 3 groups of animals (normal as control and immunosuppressed with and without infection). The
complex was obtained with radiochemical purity higher than 95%, was stable until 6 hours post labelling. The Log P
was 0.56, suitable for passage through biological membranes. Binding to yeast was 11%. Stability in plasma was 4
hours post incubation. The complex retained over 90% radiochemical purity when challenged against cysteine and
histidine and plasma protein binding ranged from 65% to 75%. The biological evaluation showed that there is clear
differentiation of tissue target/nontarget = 3.4 and that pre- treatment with caspofungin does not compete with the
radiolabeled compound at the infection site, so it could be used for detection mycosis in these conditions.
Acknowledgements: Merck, PEDECIBA, ANII.
55
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 011
AREA: RMNM
ORAL
Development and evaluation of transition metal complexes with nitroimidazole ligands
with potential application in Nuclear Medicine
Fernández, S.1; Giglio, J.2; Dematteis, S.3; Cerecetto, H.4; Rey, A.1
1
Cátedra de Radioquímica, Facultad de Química, UdelaR, Montevideo, Uruguay
2
Centro Uruguayo de Imagenología Molecular, Montevideo, Uruguay
3
Cátedra de Inmunología, Facultad de Química, UdelaR, Montevideo, Uruguay
4
Grupo de Química Medicinal, Laboratorio de Química Orgánica, Facultad de Ciencias-Facultad de Química, UdelaR,
Montevideo, Uruguay
[email protected]
Tumour perfusion and oxygenation status are important factors for poor treatment outcome after radio and
chemotherapy. Herein, we present the development of
99m
Tc and
68
Ga complexes bearing the 5-nitroimidazol-1-yl
moiety as potential radiopharmaceutical for imaging tumour hypoxia in Nuclear Medicine. 5-Nitroimidazol-1-yl
moiety has bioreductive capacity since it is irreversibly reduced in hypoxic tissue to metabolites that are entrapped
within the cells.
All ligands were synthesized using Metronidazole®, a pharmaceutical containing the pharmacophore, as starting
reagent, and introducing an adequate chelator. Identity of final products was confirmed by IR, NMR spectroscopy
and mass spectrometry.
Labelling with
99m
99m
Tc was successfully performed through the formation of
Tc-“4+1” complexes.
68
99m
Tc(I)-tricarbonyl,
Ga-labelling was achieved by the formation of the
68
99m
Tc(V)-nitride and
Ga(III)-complex with the chelator
DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) previously coupled to the 5-nitroimidazol-1-yl
moiety. Radiochemical purity was above 90% and compounds were stable in labelling milieu and human plasma
for up to 2-4 hours. Binding to plasma protein (studied by molecular exclusion) was between 2 and 85% and
lipophilicity, determined as Log of the partition coefficient octanol/phosphate buffer pH 7,4 ranged between -3,3 to
1,5.
Uptake in HCT-15 cells both in normoxia and hypoxia was assessed. Biodistribution in animals bearing induced
3LL Lewis murine lung carcinoma was studied.
68
Ga-complexes and some of
99m
Tc-complexes showed high uptake
in hypoxic cells in vitro and a very favourable biodistribution profile in mice bearing induced tumours. The highest
hypoxia/normoxia ratios were achieved for
68
Ga-complexes: 3,27±0,03 and 2,03±0,02. In vivo, tumour uptake was
similar for all complexes (1.0% per gram) but biodistribution profile showed significant differences. Selective uptake
and retention in tumour together with favourable tumour/muscle ratio (6,8±1,8 for the best complex at 2 h) make
some of these compounds promising candidates for further evaluation as potential hypoxia imaging agents.
Acknowledgements: ANII, Pedeciba-Química, Gramón-Bagó S.A., Covidien, M. Moreno, L. Reyes.
56
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 023
AREA: RMNM
ORAL
Development of a potential 99mTc-radiopharmaceutical for estrogen receptors imaging
Tejería, E.1; Cerecetto, H.2; Fernández, S.1; Giglio, J.1; Rey, A.1
1
Cátedra de Radioquímica, Facultad de Química - Universidad de la República, Montevideo, Uruguay
Grupo de Química Medicinal, Facultad de Química- Facultad de Ciencias - Universidad de la República, Montevideo, Uruguay
[email protected]
2
Breast cancer is the main type of neoplasia in women and adequate treatment requires assessment of the
presence of estrogen receptors. With the objective to develop a potential radiopharmaceutical for estrogen
receptors imaging we have prepared and evaluated an ethinylestradiol derivative labelled with
99m
Tc.
Ethinylestradiol was derivatized by the so-called “click chemistry”, by reaction (50 µL of a 1x10-2 M solution) with N-2
Boc-protected azidoalanine (65µL of a 1x10 M solution) for 60 minutes at 75ºC. Deprotection was achieved by
adding 1 µL of trifluoroacetic acid for 60 minutes at 75ºC.
99m
Tc- labelling was performed in 2 steps: preparation of fac[99mTc(CO)3(H2O)3] by reduction of 99mTc- pertechnetate
(40-50 mCi, 1.0 mL) with sodium borohydride (7.0 mg) in CO(g) atmosphere at 70 °C for 20 minutes; substitution
by mixing 0.5 mL of neutralized precursor with the derivatized ligand and incubation for 30 minutes at 75ºC.
Fig. 1 - Proposed structure of 99mTc-complex
Radiochemical purity (RP) was controlled by reverse phase HPLC. The precursor showed a retention time (rt) of 4
min and a RP > 90 %. The final complex had a rt of 20.3 min and a RP > 72 % which remained unchanged for at
least 6 hours. The HPLC purified complex was stable in human plasma at 37°C and when incubated with histidine
(100M excess) for at least 2 hours. Lipofilicity expressed as logP (partition coefficient between octanol and
phosphate buffer pH=7.4) was 1.43 ± 0.04. Plasma protein binding was 76.4 ± 0.6 %.
Biodistribution in normal rats at 30, 60 and 120 minutes post-injection showed high liver uptake (40.8 ± 2.4 %), as
expected from a lipophilic compound with high protein binding. Excretion occurred mainly by the hepatobiliary tract.
Uptake in other organs was negligible.
These results are promising for a potential oncological radiopharmaceutical.
Acknowledgements: ANII, Pedeciba-Química, Bayer Schering Pharma AG, Inés Sanz, Sylvia Dematteis.
57
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 102
68
AREA: RMNM
ORAL
Gallium and 177Lutetium for diagnosis and therapy of tumors expressing CCK-2
receptors
Trindade, V.1; Reyes, L.2; Vasilskis, E.2; Kreimerman, I.1; Oliver, P.2; Engler, H.3; Balter, H.1
1
Radiofarmacia, CUDIM, Montevideo, Uruguay
2
Preclínico, CUDIM, Montevideo, Uruguay
3
CUDIM, Dirección, Uruguay
[email protected]
Cholecystokinin Subtype 2 (CCK-2) receptors are present at high expression levels in medullary thyroid carcinoma
(MTC), as well as neuroendocrine gut tumors, stromal tumors, small cell lung cancer and others. Thus, there is a
high interest in finding CCK-2 analogues suitable for imaging (68Ga, β+, Eγ:511 keV, T1/2:68min) and treatment
(
177
Lu, Eβ-:497 keV, T1/2:6.7days) of these tumors. Both radiometals form very stable complexes with DOTA
(1,4,7,10- Tetraazacyclododecane-1,4,7,10-tetraacetic acid) which can be conjugated to biomolecules.
Minigastrin (His2-MG11)His-His-Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2 is a CCK-2 analog with high uptake in this
kind of tumor.
We optimized radiolabelling with
68
Ga and
177
Lu of DOTA-His2-MG11, quality control methods and studied in vitro
and in vivo stability as well as biological behavior.
Parameters evaluated included peptide to activity ratio and incubation time. For
177
Lu labelling also was evaluated
the addition of gentisic acid to avoid radiolysis.
Yield for 68Ga-DOTA-His2-MG11 was ˃90% for pH 4.0, 4MBq/µg of peptide, in sodium acetate 1.14M, incubation at
90ºC-9min, while for
177
Lu-DOTA-His2-MG11 was >94% at pH 4.5, 6.5MBq/µg of peptide, in sodium acetate 0.4M
with gentisic acid 0.24M, incubation at 80ºC-20min.
Higher temperatures or longer incubation times increased the amount of the Met-oxidized species in the
peptidewhich results in loss of receptor affinity.
Biodistribution of
177
Lu-DOTA-His2-MG11 was done in normal and in nude mice with tumors induced with AR42J
cells. Normal mice showed a rapid blood clearance, high urinary excretion (92% at 24h), very low renal uptake
(0.4% at 24h) and neglectable uptake by other organs and tissues, as expected for peptides.
Images obtained in a SPECT/PET/CT camera up to 9 days, showed high tumor uptake (tumor/blood:25), confirmed
by autoradiography.
These results are very promising to apply the theragnostic approach in tumors expressing CCK-2 receptors, due to
the possibility to reach a molecularly specific target, both with positrons and beta emitters.
58
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 136
AREA: RMNM
ORAL
Clinical applications of radiometals in diagnostic and treatment
Alonso, O.
Centro de Medicina Nuclear, Hospital de Clínicas, Montevideo, Uruguay
Nuclear medicine is a discipline that, applying the tracer principle, acts in the diagnosis and therapy of patients.
Radiopharmaceuticals are then administered and external devices, which generate a planar or tomographic
functional image, detect the emitted radiation. Thus, according to the nature of the radioactive sources and the
equipment involved we can acquire "SPECT" (single photon emission tomography) or "PET" (positron emission
tomography) images.
Currently, the Molecular Imaging (MI) concept has emerged, allowing the evaluation of the body’s biological activity
at the molecular level of organization that underlies the development of different diseases. For this purpose it is
necessary to develop labeled target-specific ligands that can be detected externally either by radioisotopes or by
other means. Although 90% of clinical MI images correspond to PET and SPECT, optical imaging and other
modalities have promising potential. Ligands should be developed taking into account the identification of
molecular targets of clinical relevance.
It is well known that molecular changes occur early in disease development. Genomic alterations are responsible
for changes in protein synthesis determining, for example, an over-expression of protein molecular targets such as
cell membrane receptors. Most neuroendocrine tumors express somatostatin receptors (integrated membrane
proteins). Somatostatin analogues (regulator peptide) may be labeled with either
or
68
99m
Tc (99mTc -HYNIC-Octreotide)
68
Ga ( Ga- DOTATATE), resulting in PET or SPECT imaging, respectively. Moreover, these molecules can also
be labeled with
90
Y or
177
Lu, which due to their physical characteristics (high-energy electron emission) lead to a
selective molecular-based therapy. Accordingly, this approach allows diagnosis and therapy based on a single
molecular target.
59
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 144
AREA: RMNM
ORAL
68
Ga an emergent radionuclide for molecular imaging
Balter, H.
Centro Uruguayo de Imagenologia Molecular (CUDIM), Montevideo - Uruguay
68
Ga is a positron emitter with 68 min half life and available from
long half life (T1/2 271 days) of
68
68
Ge/68Ga radionuclide generator system. The
Ge is attractive to operate the generator over a long period of time, ensuring daily
availability with high yield and radionuclidic purity.
3+
The coordination chemistry of Ga
is dominated by its hard acid character. A variety of mono and bifunctional
chelators have been developed which allow the formation of stable
biomolecules.
68
68
Ga3+ complexes and convenient coupling to
Ga coupling to small biomolecules has the potential to facilitate development of clinically practical
PET based on molecularly specific recognition. In particular, peptides targeting G-protein coupled receptors
overexpressed on human tumour cells have shown preclinically and clinically high and specific tumour uptake.
68
Radiopharmaceuticals based on
Ga based somatostatin analogues are attracting interest because of their
significant proven potential for the management of patients suffering from neuroendocrine tumours. Other
labelled peptides like
68
Ga-DOTA-Minigastin,
68
Ga-DOTA-RGD,
68
68
Ga
Ga-NOTA-UBI 29-41are promising for the
diagnosis of some cancer types, such as lung, breast and prostate, as well as for infection detection. Small nonpeptide molecules labelled with
68
Ga-BPPEN,
68
68
Ga-BPPED and
Ga have potential in PET imaging of other indications such as bone disease ie
68
Ga-BPAMD. Carbon nanoparticles labeled with 68Ga (Gallgas) are also an
interesting approach for lung ventilation studies. In order to ensure safe and efficacious use of
68
Ga based PET
agents, standardization of radiopharmaceutical preparation, quality assurance and preclinical trails are of utmost
priority.
68
Ga-based peptide tracers offer a superior vehicle for tumor imaging/diagnosis, chemo- and radiotherapy planning
and monitoring as well as pretherapeutical dosimetry for radiotherapy. Furthermore, after the diagnosis and
68
dosimetry Ga might be substituted in the same vector with a therapeutical radionuclide like
60
177
Lu or
90
Y.
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 145
AREA: RMNM
ORAL
Development of therapeutic radiopharmaceuticals based on 177Lu
Cabral, P.
Centro de Investigaciones Nucleares, Facultad de Ciencias, UdelaR, Uruguay
Lutetium-177 (177Lu) belongs to the same chemical group (3A) of the periodic table as yttrium (90Y). Lutetium
chemistry is comparable to that of other trivalent metals such as
111
In or
90
Y and has been widely investigated, used
in nuclear medicine therapy of different disorders.
177
Lu has favorable characteristics that makes it a very good candidate to label peptides and monoclonal antibodies
to use in radionuclide therapy.
Its physical characteristics are: semi-decay period of 6.71 days, negative beta emission with a maximum energy of
497 keV (78% abundance), negative beta energy of 0.134 MeV average with an average range of 2 mm in soft
tissue, gamma photon energy of 208 keV (11% abundance) suitable for in vivo scintigraphic imaging and for
custom dosimetric studies.
Chemically,
177
Lu is obtained with a specific activity of 20 Ci / mg or 45 Ci / mg depending on its method of
production. It is produced by direction (n, γ) thermal neutron using two methods: a. natural white or enriched
oxide or b. by irradiating a target of
175
The main radionuclide contaminant is
176
Lu
Yb.
177
mLu and its concentration is about 95 ppm. As trace metal contamination,
commercial products have less than 20µg of Fe / Ci of 177Lu. This impurity (Fe) is the most problematic, it interferes
significantly decreasing yields, as many researchers have experimentally verified.
61
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 032
AREA: SBMMBRP
ORAL
Biochemical and structural basis of iron-sulfur cluster coordination by trypanosomes
glutaredoxins: Implications in protein function
Manta, B.1; Fleitas, L.1; Sturlese, M.2; Pavan, C.2; Bellanda, M.2; Comini, M.A.1
1
Lab. Redox Biology of Trypanosomes, Institut Pasteur Montevideo, Montevideo, Uruguay
2
Dep. of Chemical Sciences, University of Padova, Padova, Italy
[email protected]
Background: Iron-sulfur clusters (ISCs) are versatile inorganic structures that plays regulatory and/or catalytic
roles as cofactors of several proteins. Glutaredoxins (Grxs) are small proteins belonging to the thioredoxin-fold
family with multiple functions provided by their capacity to act as thiol/disulfide oxidoreductases and as mediators
of ISC biogenesis. African trypanosomes possess five Grxs, two with a dithiol CXXC (2-C-Grxs) and three with a
monothiol CXXS (1-C-Grxs) active site. In this work, we have investigated the ability of trypanosomal Grxs to bind
an ISC as well as the biochemical and structural components implicated.
Methodology: ISC reconstitution assays were performed on recombinant wild-type and mutant versions of three
Trypanosoma brucei and one T. cruzi Grx using a chemo-enzymatic method and different low molecular mass
thiols (LMM-RSH). The apo- and holo-proteins were studied by chromatographic, spectroscopic (absorption and
circular dichroism) and structural approaches (NMR), while the interaction of ISC and LMM-RSH with the apoproteins was analyzed by spectroscopic and calorimetric techniques.
Results: All the Grxs studied are able to bind ISC but with different outputs in terms of oligomeric conformation and
stoichiometry. Structural analysis of ISC/LMM-RSH interaction with apo-proteins revealed different binding
mechanisms. Point and deletion mutants contributed to dissect the role of specific aminoacids and regions in
ISC/LMM-RSH coordination/binding. Interestingly, trypanothione (bis-glutathyonylspermidine, the main LMM-RSH
in trypanosomes) forms stable protein-free ISC complexes that could be incorporated to 2-C-Grx1 but not to 1-CGrx1. Gene function studies supported different roles for the ISC in these proteins and highlighted the in vivo
indispensability of the holo-1-C-Grx1.
Conclusion: Several structural features of 2-C-Grx1 and 1-C-Grx1 explain the functional specificity of these
proteins in the thiol- and iron-metabolism of trypanosomatids, and led us to propose a new mode of ISC
coordination.
62
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 081
AREA: SBMMBRP
ORAL
Hemin promotes tyrosine nitration in membranes
Bartesaghi, S.1,2,3; Radi, R.1,3
1
Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay
Departamento de Educación Médica, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay
3
Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay
[email protected]
2
Hemin is a redox-active bioinorganic complex, normally present as a prosthetic group in heme-proteins. However,
under oxidative stress and inflammatory conditions, it can be released and participate in uncontrolled oxidation
reactions to biomolecules. Indeed, in the presence of oxidants hemin promotes, for example, lipid peroxidation and
protein tyrosine oxidation reactions. In this work, we have assessed the role of hemin in nitration of tyrosine
residues present in lipid bilayers. For this, we have used a model system of membranes consisting in
phosphatidylcholine (PC) liposomes of different fatty-acid composition, in which we have incorporated a
hydrophobic tyrosine analog, N-t-BOC L-tyrosine tert-butyl ester (BTBE).We have observed that in saturated fattyacid containing liposomes hemin promoted peroxynitrite-mediated tyrosine nitration and the same was observed
when the peroxyl radical-donor 2, 2´-azobis (2-amidinopropane) hydrochloride (ABAP), was used in the presence
of nitrite (NO2-). These results are consistent with transition metal-catalyzed processes favoring the oxidation of
tyrosine (TyrH) to tyrosyl radical (Tyr) and the formation of nitrogen dioxide (NO2) by reactions of hemin as
follows:
hemin Fe
3+
hemin Fe
4+
4+
+ ONOOH →hemin Fe
+ TyrH →hemin Fe
3+
-
+ NO2 + OH
+
+ Tyr + H
Tyr + NO2 →NO2 Tyr
[1]
[2]
[3]
In unsaturated fatty-acid containing liposomes (e.g.1-palmitoyl, 2-linoleyl-PC) exposed to the same oxidizing
systems, hemin primarily induced lipid peroxidation and through the formation of lipid peroxyl (LOO) radicals,
secondarily promoted tyrosine oxidation and nitration:
LOO + TyrH →LOOH + Tyr
[4]
In summary, hemin hydrophobicity favors its penetration into membranes and facilitates tyrosine nitration via two
possible mechanisms, one through a direct metal- catalyzed nitration reaction (Eqs. 1-3) and another that is
connected to the lipid peroxidation process (Eq. 4). Further studies are being carried out in 23 amino acid
transmembrane tyrosine-containing peptides.
63
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 107
AREA: SBMMBRP
ORAL
Reactivity and cytoprotective capacity of the synthetic catalytic antioxidants
MnPorphyrins towards peroxynitrite
Carballal, S.1; Valez, V.1; Batinic-Haberle, I.2; Ferrer-Sueta, G.1; Radi, R.1
1
Universidad de la República, Facultad de Medicina and Center for Free Radical and Biomedical Research, Montevideo,
Uruguay
2
Duke University Medical Center, Department of Radiation Oncology, USA
[email protected]
–
Peroxynitrite (ONOO ) is a cytotoxic oxidant formed in vivo from the diffusion-controlled reaction between
superoxide (O2–) and nitric oxide (NO) radicals. Peroxynitrite- dependent oxidative damage promotes cell
dysfunction and/or death. In several of these conditions, excess mitochondrial formation of oxidants, including
peroxynitrite, has been related to the pathogenesis of multiple diseases. Thus, a major interest has been generated
in the search for potential synthetic catalytic antioxidants. Metalloporphyrins catalyze numerous redox reactions, in
particular manganese porphyrins (MnPorphyrins) can reach mitochondria and as reported previously can act as
efficient catalytic antioxidants accelerating the reduction of oxygen- and nitrogen reactive species. In adddition,
MnPorphyrins have been shown remarkably protective in different animal models of disease. The reactivity of
MnPorphyrins can be modulated by changes in charge and lipophilicity providing opportunities to adapt redox
activity and subcellular distribution. In this work we have studied a new generation of MnPorphyrins compounds
differing in their substituents where the enhanced property resides in its bioavailability.
We have characterized the reactivity of Mn(III)Porphyrins with ONOO– using stopped- flow, with rate constants
5
7
ranging from 1.35 x 10 to 3.5 x 10 M
-1
s
-1
(pH 7.4, 37 °C). We also extended the kinetic characterization of
Mn(III)Porphyrins reaction with another relevant biological oxidant, the myeloperoxidase-produced hypochlorite,
with similar rate constants. In an effort to provide the links between chemical reactivity and the obtained kinetic
information with action mechanisms in vivo, we performed cytoprotection experiments by exposing vascular
endothelial cells, in the presence of MnPorphyrins (5-50 M), to a nitro-oxidative stress conditions generated by the
peroxynitrite donor SIN-1. We observed an inhibition of cell damage as evaluated by a decrease in protein tyrosine
nitration and a protection in the apoptotic cell death. Thus, these MnPorphyrins serve to attenuate the peroxynitritemediated injury and represent a detoxification mechanism which can also be expanded to another oxidants in
biological systems.
64
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 020
AREA: TEMIBSS
ORAL
Histopathological effects of silver nanoparticle exposure to chick embryos
Gagnon, Z.E.1; Pavel-Sizemore, I.2; Trivedi, P.3; Isaac, L.4
1
School of Science, Marist College, Poughkeepsie, NY, United States
2
Chemistry, Wright State University, Dayton, OH, United States
3
University Hospital, Cincinnati, OH, United States
4
Pathology, St.Francis Hospital, Poughkeepsie, NY, United States
[email protected]
The presence of silver nanoparticles (AgNPs) in industrial and household products has become increasingly
prevalent in recent years. Six-day old fertile-specific pathogen free white leghorn strain chick embryos were
exposed to AgNP concentrations of 15, 30, 60, and 100 ppm via injections into the egg air sac on the 7th and 14th
days of incubation. A modified Creighton method was employed, using AgNO3 and NaBH4 to manufacture AgNPs.
In addition to no injection and deionized water injection (DI) controls, AgNO3 and NaBH4 control treatments were
established having the same concentrations as those used for AgNP synthesis. On the 20th day of incubation,
embryos were sacrificed and the brains, hearts, livers, and tibiotarsi were harvested. Histological analysis and
graphite furnace atomic absorption spectroscopy analysis (GFAAS) were conducted on the collected tissue
samples. Statistical analyses were performed to determine variation in silver absorption levels. Mortality was
experienced in all treatments except for the DI and no injection controls. Analysis revealed correlation between Ag
concentrations in the tissues and pathological changes observed in the histological preparations. No pathological
changes were observed in the liver tissues of embryos exposed to 15 or 30 ppm AgNP concentrations. At the 60
ppm exposure level, fatty vacuoles in the liver cells were evident. Proliferation of hepatocytes, hyperplasia and
increased cellular mitotic activity was also observed in liver samples. Additionally, Von Kossa staining revealed cell
calcification, especially in the brain tissues. At the 100 ppm exposure, apoptotic bodies with nuclear fragmentation
were evident, and silver precipitation was observed in the intercellular spaces of liver tissue. Pathological changes
in AgNP-exposed chick embryonic tissue, especially accelerated cell death, support further experimentation on
possible human health effects. In light of these findings, increasing AgNP exposure to the human population
suggests that environmental risk assessment of this emerging factor should be considered.
65
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 063
AREA: TEMIBS
ORAL
Blood lead, iron deficiency, IQ and executive functions in Uruguayan first-graders
Barg, G.1; Queirolo, E.1; Mañay, N.2; Roy, A.3; Kordas, K.3
1
Facultad de Psicología, Universidad Católica del Uruguay, Montevideo, Uruguay
2
Facultad de Química, UdelaR, Montevideo, Uruguay
3
Nutritional Sciences, Pennsylvania State University, United States, University Park, PA
[email protected]
Cognition in children with both iron deficiency (ID) and elevated blood lead levels (BLL) is not well studied. In 1stgrade children (n=150, age 6.4±0.6 y, 56.7% boys) from Montevideo, Uruguay we investigated associations among
BLL≥5µg/dL, serum ferritin (SF) 12µg/L), IQ and two executive function (EF) tasks (Internal/External Dimensional
Shift—theIED shift—and Stockings of Cambridge—the SOC—boh part of the CANTAB testing battery). Mean ± SD
IQ was 92.9±17.0 points. Two of the EF task outcomes were 1) the IED shift errors (mean ± SD 19.2 ±10.5, range
0-36) and 2) initial thinking time on the SOC (mean ± SD 3.1 ± 5.8 sec, 0 – 46.4 sec). Mean SF and BLL were
15.0±14.1 µg/L, 4.7±2.2µg/dL, respectively, with 48.8 and 30.2% of study children having BLL ≥5µg/dL and ID,
respectively, and 13.4% having both conditions. BLL and SF were not correlated. In unadjusted linear regressions,
BLL and SF were not independently associated with IQ or the two EF tasks; neither were BLL ≥5µg/dL nor ID.
However, children with both elevated BLL and ID had lower IQ 11.7 points lower than their ID-only peers (p=0.07).
Preliminary analyses suggest that elevated BLL and ID may result in poor general cognitive function in school
children.
66
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 066
AREA: TEMIBS
ORAL
Selenium-induced oxidative stress in the model organism zebrafish
Hauser-Davis, R.A.1,2; Ziolli, R.L.3; Zezzi Arruda, M.A.1,2
1
Instituto de Química/UNICAMP, Departamento de Química Analítica, Grupo de espectrometria, Preparo de amostras e
Mecanização-GEPAM, Campinas, SP, Brazil
2
Instituto Nacional de Ciência e Tecnologia de Bioanalítica/ UNICAMP, Grupo de espectrometria, Preparo de amostras e
Mecanização-GEPAM, Campinas, SP, Brazil
3
Universidade Federal do Estado do Rio de Janeiro – UNIRIO, Rio de Janeiro – RJ, Brazil.
[email protected]
Selenium toxicity has been suggested as a result of oxidative damage [1]. The zebrafish is a potentially useful
model organism for the study of selenium metabolism and its role in biology and medicine. Zebrafish were exposed
to Se to analyze effects on fish metabolism. A control and a Se-exposed group (selenium selenite, 1 mg L-1) were
maintained in a static system for 96hs. Brains, livers and gallbladders were removed. Pooled samples were used
for metallothionein and GSH determination: 10 brains, 10 livers and 4 gallbladders from the controls and 10 brains,
7 livers and 8 gallbladders from Se-exposed fish. MT extraction was conducted by heat treatment. Samples were
homogeneized in Tris containing PMSF and β-mercaptoethanol, centrifuged at 20.000 x g for 1 h at 4º C, heated at
70ºC for 10 min and centrifuged again at 20.000 x g for 30 min. Supernatants were treated with HCl/EDTA and
NaCl/ buffered DTNB, and incubated for 30 min. The samples were then centrifuged at 3000 x g for 5 min and the
absorbance was evaluated at 412 nm. MT content was estimated by assuming mol MT = 20 mols GSH. Samples
for GSH determination were homogenized in Na-phosphate buffer, pH 7.0 followed by centrifugation at 11.000 x g.
Absorbance was evaluated at 412 nm. MT in bile is lower than liver, as expected, and MT in Se-exposed bile was
higher than in controls, indicating MT induction and efficient excretion through the bile leading to less MT
accumulation in liver. The brain, with no detoxification route as quick as the liver-gallbladder route, accumulated
significantly higher MT in the Se-exposed group. Se-exposed fish showed significant GSH induction compared to
the controls. Thus, selenium exposure induced significant oxidative stress in this model organism, being of interest
in the understanding and further study of Se metabolism.
References
[1] Misra, S., Niyogi, S. Selenite causes cytotoxicity in rainbow trout (Oncorhynchus mykiss) hepatocytes by inducing oxidative
stress. Toxicol In Vitro; 2009; 23; 1249–1258.
67
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 108
AREA: TEMIBS
ORAL
Evaluation of the intellectual capacity of children in areas which are exposed and
unexposed to environmental pollution in the extreme south of Brazil
Dupont-Soares, M.; Muccillo-Baisch, A.L.; Garcia, E.; Baisch, P.R.; Soares, M.C.F.
Programa de Pós-Graduação em Ciências da Saúde, Universidade Federal do Rio Grande - FURG, Rio Grande, Brazil
[email protected]
The study area, located in the extreme south of Brazil, has been the focus of studies due to problems with
environmental pollution. This study evaluated the intellectual capacity of children living in areas considered
exposed (E) or not exposed (NE) to environmental pollution. 246 students between the ages of 8 years and 11
years 8 months were evaluated. Data was collected between May and June 2010 utilizing a semi-structured
questionnaire to investigate factors associated with compromised intellectual capacity. Intellectual capacity was
evaluated by Raven’s Progressive Matrices Scale. Study areas that were exposed or not exposed were compared
by the chi-squared test and raw and adjusted analyses were carried out using Poisson regression with a robust
error estimate. General occurrence of intellectual impairment was 28.9%, with 22.0% in the non-exposed area and
36.1% in the exposed area (p=0.01). The outcome showed association with: lack of companionship (RP= 2.77;
p<0.00); maternal education less than five years (RP=2.13; p<0.00); mother not Caucasian (RP=1.57; p<0.00);
residence in an exposed area (RP=1.88; p<0.00); nutritional risk/malnutrition (RP=2.83; p<0.00). Results showed a
high occurrence of intellectual impairment and in this study there was a significant difference between the areas.
Further study is needed in order to improve understanding of some results. The town’s priorities should include
measures related to environmental health.
68
Poster Presentations
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 036
AREA: AAMMIBBS
POSTER
Determination of Cd in soybean and wheat seeds using TS-FF-AAS and preconcentration
with cloud point extraction (CPE)
Morales, G.; Knochen, M.; Pistón, M.
*
Cátedra de Química Analítica, Facultad de Química, Montevideo, Uruguay
[email protected], [email protected]
Antecedents: Cadmium is a toxic element associated with the environment. Seeds can accumulate it, so it is
important to determine the concentration of this element.
Thermospray Flame Furnace (TS-FF) coupled to a flow-injection preconcentration system can be used to enhance
detectability in flame atomic absorption spectrometry (FAAS) providing an alternative to the use of ET-AAS or ICPMS.
Methodology: The method is based on the adsorption of a complex of Cd and methyl green, potassium iodide and
Triton X-114 in a minicolumn filled with cotton. The elution was carried out with HNO3. The system consists of a
peristaltic pump, an injection valve and, a nickel tube 10 cm long with 7 holes. The analytical determinations were
carried out by FAAS at 228.8 nm.
The seeds were milled, and 1.8 g of the obtained flour was digested by means of an acidic treatment with a mixture
of HNO3/H2O2. The resultant solution was neutralized with solid NaOH.
-1
Results: The figures of merit were: LD (3s) and LQ (10s): 0.6 and 2.1 µg L respectively, linearity: up to 10 µg L
-1
(r2= 0.999), precision: sr(%) = 2.8 (n = 5), sampling frequency: 30 h-1 with a preconcentration factor of 30 compared
with FAAS.
Accuracy was evaluated with the CRM Wheat Flour 1567a (NIST), the recoveries were between 98% and 105 %
(n = 10). There was no evidence of the influence of potential interferences.
Conclusions: This method can be implemented for the control of Cd levels in soybean and wheat seeds.
Acknowledgements: Agencia Nacional de Investigación e Innovación (ANII-2841). Dr. Harald Berndt for providing
the TS-FF-AAS system.
70
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 062
AREA: AAMMIBBS
POSTER
Determination of copper in honey by on-line flow-injection cloud-point extraction with
atomic absorption spectrometry (AAS) detection
Sixto, A.1; Knochen, M.1; Heinzen, H.2
1
Departamento Estrella Campos (DEC), Facultad de Química, Montevideo, Uruguay
2
Departamento de Química Orgánica, Facultad de Química, Montevideo, Uruguay
[email protected]
Background: the content of metals in honey is highly variable depending on the botanical and geographical origins
of the floral resources available for bees. Bioaccessibility of Cu from honey depends on different factors, including
physicochemical forms of Cu species and other dietary components present. It has been reported that Cu in honey
is highly bioaccesible. Considering the daily consumption of honey for adults, it can provide approximately 10% of
the recommended daily intake of Cu. Cu content is also an indicator of contamination during handling, processing
and storage. Determination of metals in honey usually involves tedious procedures such as ashing or wet
digestion. Methods based on flow analysis can be advantageous in terms of speed, ease, and possibility of
automation. A system based on the concept of multicommutated flow analysis (MCFA)1 with on-line cloud-point
extraction and AAS detection was developed for the automated determination of Cu in honey.
Methodology: the system scheme was as follows:
The operation of the system was carried out by means of a PC and software compiled in Quick Basic. The sample
was dissolved in water and analysed without further processing.
Results: the calibration curve was linear up to 0.9 mg L-1 (A = 0.0834 C + 0.0137), repeatability (sr (%)) was ≤ 5,
-1
-1
-1
LOD (3σ) was 0.07 mg L , LOQ (10σ) was 0.14 mg L , the sampling frequency was 20 samples h , recovery was
-1
over 80% (3 samples). The content of Cu of the samples analysed was <0.78 mg kg .
Conclusions: regarding sampling preparation the method is much simpler than the traditional methods.
References
1- Rocha, F.R.P.; Reis, B.F.; Zagatto, E.A.G.; Lima, J.L.F.C.; Lapa, R.A.S.; Santos, J.L.M. Multiconmutation in flow analysis:
concepts, applications and trends. Anal Chim Acta (2002) 468 119-131.
71
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 075
AREA: AAMMIBBS
POSTER
Determination of selenium in antioxidant softgels by Continuous Flow-Hydride
Generation-Atomic Absorption Spectrometry (CF-HG-AAS)
Barbato, S.1; Mollo, A.1; Knochen, M.1; Viera, S.2; Huertas, R.2
1
Analytical Chemistry, School of Chemistry, Montevideo, Uruguay
Atomic Spectrometry Department of Food and Environment, Technological Laboratory of Uruguay, Montevideo, Uruguay
[email protected]
2
Background: Selenium is an essential trace element for human health recognized as antioxidant and related with
the reduction of certain types of cancer. As frequently the dietary intake is not sufficient there is a great interest into
develop Se- enriched nutritional supplements prepared based on selenised enriched yeast Saccharomyces
cerevisiae, in which selenium is present mostly as selenomethionine.
Hydride Generation Atomic Absorption Spectrometry (HG-AAS) is a widespread technique for the determination of
selenium. New trends in quality control laboratories aim to automated processes offering a number of advantages
such as low cost, lower detection limits, better precision and high sample throughput. Continuous Flow (CF)
techniques enable to generate the hydride on line by continuous pumping of both reactant and sample through the
manifold.
Methodology: The aim of this work was to develop an automated system for the determination of selenium in
antioxidant softgel. Sample preparation was achieved by acidic-oxidizing wet digestion, both by means of
microwave oven and hot plate. The prepared sample was introduced in the automated manifold and selenium
determined by Atomic Absorption Spectrometry at 196.03 nm.
Results: Both digestion strategies exhibited results differing less than 1%.
Accuracy was assessed by comparison with a reference method (microwave-assisted acid digestion followed by
Inductively-Coupled Plasma Optical-Emission Spectrometry). Results from both methods differed 6 % and the
precision sr(%) ≤ 10, n=10 at 6 levels, fitting the purpose of the analysis.
Conclusion: The CF-HG-AAS system proposed provides an accurate determination of selenium by means of a
simple and low cost manifold suitable for implementing in quality control laboratories.
Acknowledgements: The authors are grateful to Mariela Pistón for useful discussion and Germán Morales for his
collaboration in the early stages of this work.
72
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 101
AREA: AAMMIBBS
POSTER
Differential metal binding behavior determined by LC(SEC)-ICP-MS of brain
metallothioneins in high- and low- freezing rats used as a genetic model of anxiety
Hauser-Davis, R.A.1; Gomes, V. de C.2; Rocha, R.3; Ziolli, R.4; Saint'Pierre, T.3; Zezzi Arruda, M.A.1; Landeira5
Fernandez, J.
1
UNICAMP, Brazil
Uniformed Services University of the Health Sciences, Department of Psychiatry, USA
3
Chemistry Department, PUC-Rio, Brazil
4
UNIRIO, Brazil
5
PUC-Rio, Brazil
[email protected]
2
Carioca high- and low- conditioned freezing (CHF and CLF) Wistar rat selected lines represent the most recent
genetic model in the study of anxiety disorders. Metal-binding behavior of brain metallothioneins (MT) by size
exclusion chromatography (SEC) coupled to liquid chromatography (LC) separation and ICP-MS detection was
investigated to verify differences between these groups. Seven CHF, CLF and randomly selected animals, males
and females (n=42), were sacrificed and whole brains removed. Samples were homogeneized in a Tris buffer
containing PMSF and β - mercaptoethanol, centrifuged at 20.000 g for 1 h, heated at 70ºC for 10 min and
centrifuged again at 20.000 g for 30 min. Elemental detection was performed on-line by a NexION 300X Perkin
Elmer ICP-MS coupled to a Shimadzu UV HPLC with a Superdex 75 SEC column. Results showed no differential
metal-binding behavior in males. Differential binding in females was observed, where a Zn and Cd peak presented
retention time between 10 and 15 min, and between 15 and 20 min, respectively, present in the control group but
absent from CHF and CLF females. Comparing males and females, females presented several differential metalbound peaks: CHF presented Cd and Pb peaks at 10 min and another Pb peak at ca. 12.5 min, absent in male
CHF; female CLF also presented these same peaks, and an extra Zn peak at 15 min, which was absent in male
CLF. Comparing male CHF and female CLF, and male CLF compared to female CHF both females presented an
extra Cd peak at 10 min, absent from the males. This indicates that female CHF and CLF animals may show
alterations in basic homeostasis, since Zn and Cd are essential trace elements, and that inter-gender differences
are present, with females presenting more metal-binding proteins than males in the central nervous system.
73
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 124
AREA: AAMMIBBS
POSTER
Microwave-assisted digestion of milk powder with diluted nitric acid using oxygen as
auxiliary reagent
Bizzi, C.A.1; Smanioto Barin, J.2; Nóbrega, J. de A.3; Foster Mesko, M.4; de Azevedo Mello, P.1; Flores, E.M.M.1
1
Departamento de Química, Universidade Federal de Santa Maria, Santa Maria-RS, Brazil
Departamento de Tecnologia e Ciência dos Alimentos, Universidade Federal de Santa Maria, Santa Maria, RS, Brazil
3
Departamento de Química, Universidade Federal de São Carlos, São Carlos, SP, Brazil
4
Centro de Ciências Químicas, Farmacêuticas e de Alimentos, Universidade Federal de Pelotas, Pelotas, RS, Brazil
[email protected]
2
The feasibility of using diluted HNO3 solutions under oxygen pressure for decomposition of whole and non-fat milk
powders and whey powder samples has been evaluated. Digestion efficiency was evaluated by determining the
carbon content in solution (digests) and the determination of Ca, Cd, Cu, Fe, K, Mg, Mn, Mo, Na, Pb and Zn was
performed by inductively coupled plasma optical emission spectrometry and Hg by chemical vapor generation
coupled to inductively coupled plasma mass spectrometry. Samples (up to 500 mg) were digested using HNO3
solutions (1 to 14 mol L-1) and the effect of oxygen pressure was evaluated between 2.5 and 20 bar. It was possible
-1
to perform the digestion of 500 mg of milk powder using 2 mol L HNO3 with oxygen pressure ranging from 7.5 to
20 bar with resultant carbon content in digests lower than 1700 mg L-1. Using optimized conditions, less than 0.86
-1
mL of concentrated nitric acid (14 mol L ) was enough to digest 500 mg of sample. The accuracy was evaluated by
determination of metal concentrations in certified reference materials, which presented an agreement better than
95% (Student t test, P < 0.05) for all the analytes.
Acknowledgements: CNPq, INCT-Analítica, INCT-Bioanalítica, FAPESP, FAPERGS, UFSM
74
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 012
AREA: MBD
POSTER
Bioactivity of a new polyoxometalate with copper. Anti-tumoral activity in a human
osteosarcoma model
Leon, I.E.1; Di Virgilio, A.L.1; Porro, V.2; Egusquiza, G.3; Cabello, C.3; Bollati- Fogolin, M.2; Etcheverry, S.B.1
1
Cátedra de Bioquimica Patológica, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata, Argentina
2
Unidad de Biologia Celular, Instituto Pasteur, Montevideo, Uruguay
3
Centro de Investigación y Desarrollo en Ciencias Aplicadas CINDECA, Universidad Nacional de La Plata, La Plata, Argentina
[email protected]
The polyoxometalates are transition metals clusters of oxide anions, usually d0 species (V(V), Mo(VI) y W (VI))
formed by a self assembly process. In addition, the heteropolyoxometalates (HPOMs) contain one or more
heteroatoms with electrons of valency p-, d - and f. The structural and electronic properties of this type of
compounds that are easy to modify and their characteristics makes them attractive for several applications in
different fields, especially as pharmaceutical drugs. The biomedical importance of the POMs is based principally on
its interaction with proteins, including enzymes and constituents of the cellular systems. The selected
heteropolytungstate belongs to an isomorfic serie [(PW 9O34)2M4]10- with M=Cu(II), was synthesized and completely
characterized in our laboratories.
In the frame of a research project devoted to study the potential pharmacological effects of heteropolytungstates
10-
with copper ([(PW 9O34)2Cu4] ), we present herein the results of its biological effects in an osteosarcoma human
model.
The effects on cell proliferation, mitochondria and lysosomal activity and morphology were investigated. Besides,
the mechanisms of action involved in the cytotoxicity were also studied (oxidative stress, level of GSH, GSH/GSSG
ratio and Cell cycle arrest).
10-
[(PW 9O34)2Cu4]
caused inhibition of cell proliferation in the range of 25-100 µM (p<0.01). Besides, similar results
were obtained by MTT and Neutral Red uptake cytotoxicity assays. Complementary, morphological features were
studied by DAPI/ Cell Mask staining and epifluorescence microscopy. These results were in parallel with those of
the viability studies. The compound increased the level of Reactive Oxigen Species (ROS) 350% over basal and
decreased the GSH/GSSG ratio in the same range (p<0.01). The complex arrested the cell cycle in G2 phase at 24
h and 48 h (p<0.05)
Taken together, these results suggest that this compound is potentially a good candidate for future use in
alternative cancer treatments.
75
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 013
AREA: MBD
POSTER
Vibrational spectra of three maltolato complexes with interesting pharmacological
properties
Baran, E.J.; Parajón-Costa, B.S.
Centro de Química Inorgánica (CEQUINOR, CONICET/UNLP), Facultad de Ciencias Exactas, Universidad Nacional de La
Plata, La Plata, Argentina
[email protected]
Maltol, 3-hydroxy-3-methyl-4-pyrone, is one of several hydroxypyrones long known for high bioavailability and
favourable toxicity profiles. It has a high affinity for different metal cations and is, therefore, an excellent choice for
1
the formulation of therapeutic and/or diagnostic metallopharmaceuticals .
2+
We have prepared three metallic complexes of this ligand, namely with VO , Zn(II), and Ga(III) respectively. The
1-3
first two show insulino mimetic activity
4
and the other one is a very promising antitumoral drug .
The FTIR and Raman spectra of polycrystalline samples of the three complexes were recorded and briefly
discussed on the basis of their crystal structures and by comparison with the spectroscopic behavior of free maltol
and of other, previously investigated, maltolato complexes.
References:
1 Thompson, K.H.; Barta, Ch.A.; Orvig, Ch. Metal complexes of maltol and close analogues in medicinal inorganic chemistry.
Chem Soc Rev; 2006; 35; 545-556.
2 Thompson, K.H.; Orvig, Ch. Vanadium: in diabetes: 100 years from phase to phase I. J Inorg Biochem; 2006; 100; 1925-1935.
3 Yoshikawa, Y., Ueda, E., Kawabe, K., Miyake, H., Sakurai, H., Kojima, Y. New insulin-mimetic zinc(II) complexes; bismaltolato zinc(II) and bis-2- hydroxypyridine-N-oxido zinc(II) with Zn(O4) coordination mode. Chem Lett; 2000; 874-875.
4 Jakupec, M.A., Keppler, B.K. Gallium in cancer treatment. Curr Top Med Chem; 2004; 4; 1575-1583
76
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 035
AREA: MBD
POSTER
Apoptosis and autophagy in neuroblastoma by copper compounds
Gutiérrez, A.G.1; Vázquez-Aguirre, A.1; Palma-Tirado, L.2; Ruiz-Azuara, L.3; Hernández-Lemus, E.4; Mejía, C.1
1
Medicina Genómica y Toxicología Ambiental, Instituto de Investigaciones Biomédicas, UNAM, Ciudad de México, México
2
Unidad de Microscopía, Instituto de Neurobiología, Campus Juriquilla, UNAM, Qurétaro,
3
Química Inorgánica y Nuclear, Facultad de Química, UNAM, Ciudad de México, México
4
Departamento de Genómica Computacional, Instituto Nacional de Medicina Genómica, Ciudad de México, México
[email protected]
1
Background: Neuroblastoma is a childhood solid cancer refractory to several anti-carcinogenic agents .
2
Casiopeínas® are a group of coordination complexes with a central Copper atom , designed to be an alternative to
cancer therapy by means of apoptosis activation3. Controversy exists about autophagy may be a caspase- and
4
apoptosis-independent cell death, or rather, is a self-limited process that protect cells from death .
Methodology: We determined apoptotic and autophagic molecules by Western blot and electronic microscopy,
and ROS expression by flow cytometry. Finally, all results were analyzed with an integrative computational model.
Results: We found that Cas produce early mitochondrial apoptosis with an increase of cytoplasmic apoptotic
molecules Bcl-2, cytochrome C, and caspase-3, followed by a decrease of Bax and a disruption of the
mitochondrial transmembrane potential; and later autophagic molecules Beclin-1 and LC3-II were expresed. Both
processes appear in ROS presence, concomitantly with a decrease in glutathione levels. The protein regulatory
network for mitochondrial apoptosis and autophagy in NB by effect of Casiopeínas, showed that most molecules
are highly connected, especially as a result of a pro-oxidant environment.
77
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 044
AREA: MBD
POSTER
Spectroscopical studies of the peroxidase activity of cytoglobin
Nascimento, O.R.1; Ferreira, J.C.2; Icimoto, M.Y.2; Marcondes, M.Y.2; Oliveira, V.2; Nantes, I.L.3
1
Instituto de Física de São Carlos, Universidade de São Paulo, São Carlos, SP, Brazil
Departamento de Bioquímica, Universidade Federal de São Paulo, São Paulo, SP, Brazil
3
Santo André, SP, Universidade Federal do ABC, Centro de Ciências Naturais e Humanas, Brasil
[email protected]
2
This paper reports spectroscopical analysis by UV-visible absorption, magnetic circular dichroism (MCD) and
electron paramagnetic resonance (EPR) of the structure of a recombinant Cygb and its reactivity with hydrogen
peroxide (HOOH), tert-butylhydroperoxide (t-BuOOH) and cumene hydroperoxide (CuOOH). UV-visible direct EPR
of heme iron. Cygb was converted to the high valence species by peroxides, promoted homolytic scission of the OO bond and generated alkoxyl radicals. The secondary methyl and peroxyl radicals resulted respectively from the
β-scission of the alkoxyl radical and from the reaction of methyl radical with molecular oxygen. From kinetic studies
of Cygb with different peroxide concentrations they were determined the catalytic constants kcat/KM for CuOOH
(0.0028 ± 0.0006), t- BuOOH (0.0032 ± 0,0006) and HOOH (0.0061± 0,0010 s-1.µM-1). EPR spectra obtained in the
course of the reactions corroborated the conversion of the hexacoordinated heme iron to compound II. These
results are consistent with the biological role of Cygb grounded on pro-oxidant signaling activity.
78
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 047
AREA: MBD
POSTER
On the thermal oxidation and denaturation of oxy-HbGp, in presence of SDS, at pH 7.0,
monitoring by optical absorption and CD
Carvalho, J.W.P.; Tabak, M.
Departamento de Físico-Química, Instituto de Química de São Carlos-IQSC/Universidade de São Paulo-USP, São Carlos-SP,
Brazil
[email protected]
HbGp is an oligomeric protein with a molecular mass of 3.6 MDa, presenting a high oligomeric stability, at pH 7, in
the presence of both surfactant and high temperature. At 20ºC, the oxy-HbGp, in the absence and presence of
SDS, is characterized by absorption spectra with an intense Soret band, at 415 nm, and two Q-bands, at 535 and
575 nm. Increase of temperature promotes the decrease of intensity and shifting of λmax from 415 nm (20ºC) to
411-409 nm (70ºC). The Q-bands also present a decrease in intensity. The appearance of two bands, at 500 and
540 nm, at high temperatures, is observed. SDS concentration, in the range from 0.2 to 0.6 mmol/L, induces
changes on the Soret and Q-bands, already at lower temperatures. Our data indicate the presence in solution of a
mixture of oxy-, met-HbGp and hemichrome, at higher temperatures. Met-HbGp shows a Soret band at 405 nm,
and Q-bands at 500 and 540 nm, while the hemichrome displays a Soret band at 413 nm, and Q-bands at 534 and
564 nm. CD data show similar results, suggesting the oxy-HbGp oligomeric dissociation, in the presence of SDS.
These changes on the iron oxidation and coordination promote a decrease in the unfolding temperature of HbGp
from 56±2 (absence of surfactant) to 33±3ºC (with 0.6 mmol/L of SDS). Thus, our data show clearly that the iron
oxidation state is very important for the oligomeric stability of HbGp.
Aknowledgments: The authors thank FAPESP and CNPq for financial support.
79
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 052
AREA: MBD
POSTER
Ni(II) binding to 429-460 peptide fragment from human toll-like receptor (hTLR4)
Zoroddu, M.A.1; Peana, M.1; Medici, S.1; Solinas, C.1; Potocki, S.2; Kozlowski, H.2
1
Department of Chemistry and Pharmacy, University of Sassari, Sassari, Italy
2
Department of Chemistry, University of Wroclaw, Wroclaw, Poland
[email protected]
Contact allergy, commonly induced by nickel, is the most frequent cause of contact hypersensitivity in industrialized
countries, with 30% of population being affected. Ni(II) seems to trigger an inflammatory response by activating
human Tool-like-Receptor 4 (hTLR4). Species-specific activation, as in this case, required distinct sequence motifs
that are present in human but not in mouse, a species not sensitive to nickel-induced allergies. The specific region
of human TLR4 responsible for nickel responses could be a sequence containing three histidine residues, H431, and
the non- conserved H456 and H458, localized in the C-terminus. It has been proposed that the imidazole side chains
of the histidine residues H456 and H458 provide a potential binding site for nickel because they were located at an
optimal distance to interact with Ni(II) ions, whereas H431 was further apart. We decided to verify the possibility of
metal binding to FQH431SNLKQMSEFSVFLSLRNLIYLDISH456TH458TR sequence, containing the three histidines
supposedly involved in nickel response, in order to study the binding properties of the peptide fragment and on the
thermodynamic stability of its metal complexes. Formation equilibria of Ni(II) complexes have been investigated in
aqueous solution and in a wide pH range. Protonation and complex-formation constants have been
potentiometrically determined; complex-formation models and species stoichiometry have been checked by means
of UV-Vis absorption and CD spectroscopy and investigation through NMR is currently being carried out. The
predominant species for a 1:1 peptide/Ni(II) molar ratio was obtained at physiological pH and showed an effective
binding of the metal to the target sequence.
80
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 057
AREA: MBD
POSTER
Synthesis, characterization and antibacterial activities of a novel Ag(I) complex with
sulfadoxin
Abbehausen, C.; Zanvettor, N.T.; Bergamini, F.R.G.; Lancellotti, M.; Corbi, P.P.
Inorganic Chemistry Department, University of Campinas, Campinas, Brazil
[email protected]
The antibacterial action of silver is known since ancient times. Later, the use of silver compounds in therapy was
significantly reduced by the discovery of more efficient and less toxic antibiotics. However, the development of
resistant bacterial strains brought the resurgence of silver based drugs. The most used is silver sulfadiazine, which
was approved for clinical treatment of burns and skin infections[1].
Sulfadoxin (C12H13N4SO4, SFX), as sulfadiazine, is a sulfonamide used in the treatment of malaria in combinatory
therapies. Sulfadoxin increases the activity of pyrimethamine against the malaria protozoa Plasmodium
falciparum[2]. The coordination of sulfadoxin to silver, similarly to the coordination of sulfadiazine to silver, could
generate a new and potent antibiotic. Studies of metal coordination to sulfadoxin were not described previously.
Here, we describe the synthesis, characterization and antibacterial studies of a silver complex with sulfadoxin.
The silver-sulfadoxin (Ag-SFX) complex was prepared by the reaction of an alkaline solution of sulfadoxin with an
aqueous solution of AgNO3. A white solid was formed within one hour of reaction. Elemental analysis led to the
composition AgC12H13N4SO4. Anal. Calc. for AgC12H13N4SO4 (%): C 34.5; H 2.64; N 13.4. Found (%): C 34.3; H
2.52; N 13.0.
-1
The absence of the ν(NH) band at 3240 cm in the infrared spectrum of Ag-SFX in comparison to the free ligand
suggests the coordination of the nitrogen atom of the sulfonamide group to silver. Nitrogen coordination was
confirmed by
13
C NMR in the solid state and by DFT studies. An antibiogram assay showed the significant
antibacterial activity of the silver complex over six pathogenic Gram-positive and Gram-negative strains.
References
[1] Bakhtiar, R; Ochiai, Ei-I Pharmacological applications of inorganic complexes Gen Pharmacol 1999, 32, 525-540.
[2] Chulay, JD; Watkins, WM; Sixsmith, DG, Synergistic antimalarial activity of pyrimethamine and sulfadoxine against
Plasmodium Falciparumin vitro. Am J Trop Med Hyg 1984, 33, 325-330.
81
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 059
AREA: MBD
POSTER
A new nytrosil ruthenium compound with some special features
Mondelli, M.1; Carnizello, A.1; Tavares, D.2; Corrêa, R.1; Ellena, J.3; Batista, A.1
1
Departamento de Química, Universidade Federal de São Carlos (UFSCar), São Carlos, Brazil
2
Laboratório de Mutagênese, Universidade de Franca (UNIFRAN), Franca, Brazil
3
Departamento de Física e Informática, Instituto de Física, Universidade de São Paulo (USP), São Carlos, Brazil
[email protected]
NO is a little molecule that in the last twenty five years has extensively been studied for their beneficial effects in
the body. In 1992 was chosen by Science as "molecule of the year" and in 1998 R. Furchgott, F. Murad and L.
Ignarro won the Medicine Nobel Prize "for their discoveries concerning nitric oxide as a signalling molecule in the
cardiovascular system". 4,6-dimethyl-2-mercaptopyrimidine (spymm) has antibacterial, antithyroideal, antitumor,
antiviral and fungicide activity1. A new compound, [RuCl(spymm)2(NO)] (1), was synthesized and characterized by
1
cyclic voltammetry, molar conductivity, H NMR and IR/UV-vis spectroscopies, and its structure was determined by
X-ray analysis. The complex showed an octahedral distorted structure where the nitrogen atoms were trans
between each other, and also the sulfur atoms, in an equatorial plane; whereas the nitrosonium (NO+) was trans
1
with respect to chloride in the axial plane (Figure 1). H NMR spectrum showed two singlets in 7,3 ppm and in 6,7
ppm attributed to the hydrogen of the aromatic ring, and also in 2,5 ppm and 2,3 ppm attributed to hydrogens of
methyl groups; this differences could be explained by evaluating how the intermolecular hydrogen bonds are
affecting the other atoms present in the molecule and in the nearest neighbourhood. In the infrared spectrum was
observed an intense peak at 1856 cm-1, corresponding to νNO. An electrolytic process demonstrated the liberation
+
of NO from the molecule, property that can be useful for medical applications, as for example in photodynamic
2
therapy . Besides, IC50 was evaluated in three cell lines V79 (chinese hamster fibroblast), HeLa (human epithelial
carcinoma) and U251 (glioblastoma), showing a promisor result in HeLa (18,70 µM).
References
1) H. Parveen; F. Hayat; A. Salahuddin and A. Azam, “Syntheses, characterization and biological evaluation of novel 6ferrocenyl-4-aryl-2-substituted pyrimidine derivatives”. Eur J Med Chem, 2010; 45;3497-3503.
2) G. Stochel; A. Wanat; E. Kulís and Z. Stasicka, “Light and metal complexes in medicine”. Coord Chem Rev, 1998; 171; 203220.
Acknowledgments: CAPES, CNPq, FAPESP.
82
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 060
AREA: MBD
POSTER
Synthesis, characterization and in vitro cytotoxic activity of the palladium(II) complexes
with the naphthaldehyde thiosemicarbazone ligands against various human tumor cell
lines
Hernández Gorritti, W.R.1; Paz Castillo, J.1; Vaisberg, A.2; Manzur, J.3; Spitridonova, E.4; Beyer, L.5
1
Facultad de Ingeniería Industrial, Universidad de Lima, Lima, Perú
Laboratorio de Investigación y Desarrollo, Facultad de Ciencias y Filosofía, Universidad Peruana Cayetano Heredia, Lima,
Perú
3
Facultad de Ciencias Físicas y Matemáticas, Universidad de Chile, CEDENNA, Santiago, Chile
4
Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile, CEDENNA, Santiago, Chile
5
Fakultät für Chemie und Mineralogie, Universität Leipzig, Leipzig, Germany
[email protected]
2
It has been reported that the ligands derived from α–(N)-heterocyclic carbaldehyde, benzaldehyde and furaldehyde
thiosemicarbazone, and their metal complexes inhibit tumor cell growth [1-2].
The present work describes the preparation, characterization and antitumor activity of palladium(II) bis-chelates
1-3
Pd(L )2 of naphthaldehyde thiosemicarbazone derivatives. The ligands and their palladium complexes have been
synthetized and characterized by elemental analysis and IR and NMR (1H, 13C) spectroscopy.
Analytical and spectroscopy data are consistent with the proposed structural formulas for the ligands and their
complexes (Fig. 1). The spectroscopy data revealed that the desprotonated naphthaldehyde thiosemicarbazone
derivatives are coordinated to the Pd(II) ion through the nitrogen and sulphur atoms.
X
N
N
N
H
S
Pd
S
H
H
N
N
X
H N
(X = H, NO2, OCH3)
Figure 1. Structural formulas of the synthetized palladium(II) complexes.
The in vitro antitumor activity of the ligands and their complexes was determined against six human tumor cell
lines: H460, DU145, M14, HT29, K562 and MCF-7. The antitumor studies revealed that the palladium(II)
complexes are more cytotoxic than their ligands with IC50 values at the range of 0.65–2.39 µM. The complex
Pd(L2)2, with the 4-phenyl-1-(1´-nitro-2´-naphthaldehyde) thiosemicarbazone ligand, showed higher antiproliferative
activity (CI50 = 0.65- 0.68 µM) than the other tested palladium(II) complexes against H460 and K562 tumor cell
lines.
Acknowledgements: W. H. thanks the Universidad de Lima Scientific Research Institute for financial support to
carry out the research work. We also thank the Research Laboratory of the Faculty of Sciences and Philosophy,
Universidad Peruana Cayetano Heredia, for the cytotoxic studies of the compounds. E. S. and J. M. thank
Financiamiento Basal FB0807 Project.
83
12th International Symposium on Metal Ions in Biology and Medicine
References
1.-J. Patole, S. Padhye, M.S. Moodbidri and N. Shirsat. “Antiproliferative activities of iron and platinum conjugates of
salicylaldehyde semi-/thiosemicarbazones against C6 glioma cells”.Eur J Med Chem; 2005; 40; 1052–1055.
2.-W. Hernández, J. Paz, J. Vaisberg, E. Spodine, R. Richter y L. Beyer.“Synthesis and characterization of new palladium(II)
complexes with ligands derived from furan-2-carbaldehyde and benzaldehyde thiosemicarbazone and their in vitro cytotoxic
activities against various human tumor cell lines”. Z Naturforsch; 2010; 65b; 1271-1278.
84
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 067
AREA: MBD
POSTER
Effects of copper-dipeptide complexes on lipid model membranes
Pianca Barroso, R.1; Noble, C.2; Facchin, G.2; Torre, M.H.2; Costa-Filho, A.1
1
Instituto de Física, Universidade de São Paulo, Ribeirão Preto, Brasil
Cátedra de Química Inorgánica, Universidad de la República, Montevideo, Uruguay
[email protected]
2
Several copper-dipeptide complexes have demonstrated pharmacological activity, such as antiproliferative agents
on cell cultures, interaction with DNA and superoxide dismutase activity. As part of the mechanism underlying their
pharmacological activity is certainly the interaction with membranes that regulate the flux of molecules in and out of
the cell. Surprisingly this is an issue that has received little attention over the years. Studies about the possible
interactions of copper-dipetides with lipid membranes can then significantly contribute to the understanding of basic
aspects of complex activity. In this context, we use differential scanning calorimetry (DSC) and electron spin
resonance (ESR) to investigate the effect of copper-dipeptide complexes on the lipid phase transition and acyl
chain dynamics of lipid model membranes. Different membrane regions were monitored by spin probes located at
different positions along the lipid chain. The spin labels used were 1-palmitoyl-2-stearoyl-(n-doxyl)-sn-glycero-3phosphocholine, where n = 5, 10 and 16. The lipid membranes were made from 1,2-dipalmitoyl-sn-glycero-3phosphocholine (DPPC) and from 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DPPG). The results
showed interactions between the complexes and the lipid model membranes. The presence of copper-dipeptide
complexes caused changes on the mobility (fluidity) of the lipid membranes, thus indicating that non-specific
complex-membrane interactions could help the entrance of the molecules.
Aknowledgments: Financial support: FAPESP and CNPQ.
85
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 068
AREA: MBD
POSTER
Development of new coordination complexes of Co(III) with antitumoral activity
Noble, C.1; Iglesias, S.1; Pianca Barroso, R.2; Torre, M.H.1; Costa-Filho, A.2; Facchin, G.1
1
Departamento Estrella Campos, Facultad de Química, UdelaR, Montevideo, Uruguay
Departamento de Biofísica Molecular, Faculdade de Filosofía, Ciências y Letras de Ribeirão Preto, Universidade de São Paulo,
Ribeirão Preto, Brasil
[email protected]
2
Nowadays society is in need of new antitumor drugs with less collateral damage to the human body and with
different spectra of action. The development of new compounds with essential elements, aims to find new
compounds with less severe side effects. There is a background in coordination compounds of essential metals
such as Mn, Co, Cu and Zn, among others, which present pharmacologic activity. For instance, different mixed
ligand Co(III) complexes including phenanthroline inhibit cancer cell proliferation. There are numerous records of
antineoplasic drugs whose action mechanisms involves interaction with DNA, causing damage that interfere with
the cellular proliferation process, and eventually resolve in cellular death. Because of this, the development of new
metallic complexes, capable of interacting with DNA is a potential path in finding new antineoplastic compounds.
Our research group has synthesized and characterized several Cu(II) coordination compounds with L-dipeptides
and with mixed ligands, including L-dipeptides and 1,10-phenathroline (phen) which showed very good antitumor
activity in vitro studies in HeLa cells. Other studies have yielded that in vitro the aforementioned complexes are
capable of interacting with DNA. To continue advancing on the development of better antitumor drugs with
essential elements, this work focused on the synthesis of new complexes of Co(III), analogues to [Cu(II)(Ldipeptide)(phen)]. The syntheses of these new complexes, [Co(III)(L-dipeptide)(phen)(H2O)], were made by
substitution in aqueous solution of Co(II) and further oxidation of the complexes. The structural elucidation in solidstate was made by infrared spectroscopy, elemental analysis, and in aqueous solution by EPR, UV-Vis and DC.
Their lipophilicity, DNA interaction and antiproliferative activity was assessed. These studies have demonstrated
that these new complexes present one L-dipeptide and one phen coordinated to the Co(III) ion, and that they
interact with DNA. Furthermore, they exhibit antitumor activity in HeLa cells, possible due to its ability to interact
with DNA.
86
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 076
AREA: MBD
POSTER
New heteroleptic copper complexes with saccharinate: synthesis, characterization and
cytotoxicity against osteoblast and osteosarcome cell lines
Santi, E.1; Viera, I.1; León, I.2; Baran, E.J.2; Etcheverry, S.2; Torre, M.H.1*
1
Cátedra de Química Inorgánica, DEC, Facultad de Química (UdelaR), Montevideo, Uruguay
Centro de Química Inorgánica (CEQUINOR/CONICET, UNLP), Facultad de Ciencias Exactas (UNLP), La Plata, Argentina
[email protected]
2
With the aim of providing efficacious and safe products, the pharmaceutical industry needs to incorporate different
substances to maintain their attributes and stability, to aid in the administration and to impede the contamination.
Usually, these compounds can form complexes with metals coming from the pharmaceutical formulations or food.
Saccharin (sac) is an important sweetening agent used in pharmaceutical vehicles, foods, beverages and in diets
for diabetics. Besides, it has three potential coordinating centers: the heterocyclic nitrogen and the carbonyl and
sulphonyl oxygen atoms. On the other hand, aminoacids (aa) are ligands that usually transport metals in blood. For
this reason it is interesting to study the interaction of essential cations with saccharin and aminoacids.
As a part of our work in the development of metal complexes with pharmacological activities or pharmacotechnical
1
interest , in this work we report the synthesis and characterization (elemental analysis, UV-Vis, IR and Raman
spectroscopies) of new mixed Cu(II) complexes with saccharin and aminoacids (gly, ser, gln, asp) with general
formula Na2[Cu(sac)2(aa)2]•nH2O. The aminoacids coordinated as bidentate ligands through the amino and
carboxylate groups and the sacharinate as monodentate through the heterocyclic nitrogen.
Besides, due to the antecedents that several coadjuvants showed biological adverse effect, the antiproliferative
activity against osteoblastic cells (MC3T3-E1) and human osteosarcoma cells (MG-63) for [Cu(sac)2(H2O)4]•2H2O
(Cu-sac) and Na2[Cu(sac)2(gln)2]•H2O (Cu-sac-gln) were tested. The results presented in the Figure showed that
the first complex is less cytotoxic than the second one in both cell lines, showing the importance of these mixed
complexes. On the other hand the heteroleptic complexes presented different superoxide dismutase-like activity
than the homoleptic one.
References
1 Santi E., Viera I., Mombrú A., Castiglioni J., Baran E.J., Torre M.H., Synthesis and characterization of heteroleptic copper and
zinc complexes with saccharinate and aminoacids. Evaluation of SOD-like activity of the copper complexes, Biol. Trace. Elem.,
2011, 143, 3, 1843-1855.
87
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 080
AREA: MBD
POSTER
Physicochemical study, crystal structure and antitumoral activity of a novel vanadium(V)
complex with an isoniazid-derived hydrazone ligand
González Baró, A.C.1; Ferraesi Curotto, V.2; Parajón-Costa, B.S.1; Pis Diez, R.1; Resende, J.A.L.C.3; Paula,
4
4
5
F.C.S. ; Pereira, M.E.C. ; Rey, N.A.
1
CEQUINOR- Departamento de Química, CONICET-UNLP (Facultad de Ciencias Exactas), La Plata, Argentina
2
Departamento de Química, Facultad de Ciencias Exactas y Naturales UNCa, Catamarca, Argentina
3
Departamento de Quimica, Universdidade Federal Fluminense, Nitrói, RJ, Brazil
4
Departamento de Quimica, Universidade Federal de Minas Gerais, Brazil, Belo Horizonte
5
LABSO-BIO, Departamento de Química, Rio de Janeiro, Brazil
[email protected]
Condensation reaction of the antituberculous agent isoniazid with o-vanillin leads to the formation of a stable and
active hydrazone, INHOVA1. The interaction of this compound with vanadium was studied in an attempt to obtain
metal complexes of therapeutic interest. The complex was prepared from ethanolic solutions of INHOVA and VOCl2
in a 1:1 molar ratio and obtained in the hydrochloride form, with protonation of the pyridine N atom. It crystallizes in
the monoclinic system, space group P21/c. INHOVA acts as a tridentate ligand and the distorted coordination
environment is completed by an oxo ligand, a disordered ethoxide and a water molecule.
The optimized geometry of the complex, the vibrational analysis and the vertical electronic transitions were
obtained within the context of the DFT. Basis sets of triple-z quality were used. Solvent effects were included
through the IEF- PCM.
The IR spectrum of the solid and the UV-Vis spectra in DMSO and ethanol were compared with those of the free
ligand. The assignments were accomplished with the help of theoretical calculations. Electronic spectra were
similar in both solvents, in accordance with theoretical predictions. The bands could be assigned to intraligand and
charge transfer transitions. The redox behavior of the complex and the ligand was studied by cyclic voltammetry in
the same solvents. Several irreversible oxidation and reduction processes involving the metal center and the
coordinated ligand were observed.
Both INHOVA and the complex showed activity against the chronic myelogenous leukemia K562 cell line in a
concentration-dependent manner. The IC50 (concentration required to inhibit 50% of cellular growth) values
-1
obtained were, respectively, 36.46 ± 4.13 and 28.80 ± 3.48 µmol.L .
88
12th International Symposium on Metal Ions in Biology and Medicine
References
1 Ana C. González-Baró, Reinaldo Pis-Diez, Beatriz S. Parajón-Costa, Nicolás A. Rey; Spectroscopic and theoretical study of
the o-vanillin hydrazone of the mycobactericidal drug isoniazid. J. Mol. Struct. 2012, 1007, 95–101.
89
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 085
AREA: MBD
POSTER
Synthesis, spectroscopic characterization and DFT studies of a novel Pd(II) complex with
rimantadine
Sucena, S.; de Paiva, R.E.F.; Formiga, A.L.B.; Corbi, P.P.
Department of Inorganic Chemistry, University of Campinas, Campinas, Brazil
[email protected]
Since the discovery of cisplatin, reports concerning the use of metal complexes in cancer treatment have
experimented an extraordinary increasing. Platinum(II) and palladium(II) complexes were shown to present a wide
range of pharmacological activities, such as antitumoral and antibacterial activities [1]. Despite the widespread use
of cisplatin, this compound is also a toxic substance. In order to reduce the toxic side effects of cisplatin, new
compounds based on the cisplatin structure have been synthesized. Among the non-platinum compounds with
potential for the clinical treatment of human malignancies, palladium derivatives have received considerable
interest, because of the structural and electronic analogy between the Pt(II) and Pd(II) complexes [2].
Rimantadine (C12H21N, rtd) is a diamantoid obtained by the functionalization of adamantine with an ethanamine
group. It is a bioactive compound used as an antiviral agent in the treatment of influenza A, depression and some
cases of parkinsonism [3]. No studies of coordination compounds with rimantadine was previously reported in the
literature, excepted in the case of the gold(I) rimantadine complex published by us [4]. Here, we describe the
synthesis, characterization and antibacterial studies of a new palladium complex with rimantadine.
The palladium(II) complex with rimantadine (Pd-rtd) was prepared by the reaction of an alkaline methanolic solution
of rimantadine with a methanolic solution of Li2[PdCl4], in a 2:1 ligand/metal ratio. A yellow solid was formed within
one hour of reaction. Elemental analysis led to the composition PdCl2C24H42N2. Anal. Calc. for PdCl2C24H42N2 (%):
C 53.79; H 7.37; N 5.23. Found (%): C 53.79; H 7.89; N 5.23.
-1
The shifted of the (NH2) deformation band in the IR spectra of the complex, which appears at 1728 cm for the free
rimantadine indicates nitrogen coordination of the ligand to Pd(II). Nitrogen coordination was confirmed by 1H and
13
C and 1H- 15N HMBC NMR. The second one shows a ∆δ 13C (complex – free ligand) = 3.5 ppm. Studies based on
DFT are in progress in order to optimize the structure of the compound.
References
[1] Iyidogan, A. K.; Tasdemir, D.; Emre, E. E. O.; Balzarini, Novel platinum(II) and palladium(II) complexes of
thiosemicarbazones derived from 5-substitutedthiophene-2-carboxaldehydes and their antiviral and cytotoxic activities. J. Eur. J.
Med. Chem.2011, 46, 5616-5624.
[2] Ghani, N. T. A.; Mansour, A. M. Novel palladium(II) and platinum(II) complexes with 1H-benzimidazol-2-ylmethyl-N-(4bromo-phenyl)-amine: Structural studies and anticancer activity. Eur. J. Med. Chem. 2012, 47, 399-411.
[3] Garcia, J. C.; Justo, J. F.; Machado, W. V. M.; Assali, L. V. C. J. Structural, Electronic, and Vibrational Properties of Aminoadamantane and Rimantadine Isomers Phys. Chem. A 2010, 114, 11977-11983.
[4] Sucena, S. F.; Paiva, R. E. F.; Abbehausen, C.; Mattos, I. B.; Lancellotti, M.; Formiga, A. L. B.; Corbi, P. P. Chemical,
spectroscopic characterization, DFT studies and antibacterial activities in vitro of a new gold(I) complex with rimantadine.
Spectrochim. Acta A. 2012, 89, 114-118.
90
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 086
AREA: MBD
POSTER
Interaction with proteins and antitumoral activity of a ruthenium semicarbazone complex
Otero, L.1; Demoro, B.1; De Almeida, R.2; Marques, F.3; Costa Pessoa, J.4; Tomaz, I.5; Gambino, D.1
1
DEC, Facultad de Química, UdelaR, Montevideo, Uruguay
CQB/DQB, Faculdade de Ciências da Universidade de Lisboa, Lisboa, Portugal
3
UCQR, Instituto Superior Técnico/Instituto Tecnológico e Nuclear, Lisboa, Portugal
4
CQE, Instituto Superior Técnico, UT Lisboa, Lisboa, Portugal
5
CCMM/DQB, Faculdade de Ciências da Universidade de Lisboa, Lisboa, Portugal
[email protected]
2
Ruthenium complexes hold great potential in cancer treatment as non-platinum alternatives. Some 5nitrofurylsemicarbazone derivatives have shown activity against Trypanosoma cruzi, the parasite responsible for
Chagas’ disease. A panel of [RuIICl2(DMSO)2L] complexes with these ligands was previously developed by some of
us and screened for the activity against T. cruzi [1]. Some metal-based anti-trypanosomal compounds have
exhibited anti- tumor properties as well, probably due to metabolic similarities between tumor cells and pathogenic
trypanosomes.
In this work, we evaluate the antiproliferative activity of [RuIICl2(DMSO)2(5-nitrofurylsemicarbazone)], RuNTF, in
three human cancer cell lines with different sensitivity to cisplatin (A2780 ovarian adenocarcinoma, MCF7 breast
adenocarcinoma and PC3 grade IV prostate carcinoma) and the effect of serum protein binding on the cytotoxic
activity in the A2780 cell line. Human serum albumin (HSA), the most abundant protein in human plasma, is the
major nonspecific transporter in the circulatory system and is known to play an important role in drug
pharmacokinetics. As a first approach to the pharmacokinetics of RuNTF, we assessed its interaction with HSA at
37ºC by Circular Dichroism (CD), UV-visible absorption and both steady-state and time-resolved Fluorescence in
simulated blood plasma conditions. The great quenching extent of the Fluorescence emission from the HSA
Trp214 aminoacid residue observed upon binding of RuNTF to HSA, indicates a strong complex-protein affinity,
which is supported by CD data, with an Induced CD broad band at ~450 nm observed after short incubation times.
Acknowledgements: Authors thank CYTED network RIIDFCM and Portuguese Foundation for Science and
Technology
(PTDC/QuiQui/101187/2008,
PEst-OE/QUI/UI0100/2011,
PEst-OE/QUI/UI0612/2011,
PEst-
OE/QUI/UI0536/2011, Ciência2007 and 2008 Initiatives). B.D. thanks ANII Uruguay for a postgraduate fellowship.
References
[1] L. Otero, P. Noblia, D. Gambino, H. Cerecetto, M. González, J. A. Ellena, O. E. Piro, Inorg. Chim. Acta 344 (2003) 85.
91
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 087
AREA: MBD
POSTER
[Ru(tpy)(adtpy)]2+ and [Ru(dtp)(adtpy)]2+ as dual topoisomerase I/II Inhibitors
Chao, H.
School of Chemistry and Chemical Engineering, Sun Yat-Sen University, Guangzhou, China
[email protected]
DNA topoisomerases are essential enzymes that control and modify the topological states of DNA. In accordance,
topoisomerase activities are activated in cancer cell growths, and thus are important cellular targets for
antineoplastic drugs.As modern therapeutics, ruthenium complexes have specifically received considerable interest
as potential anti- cancer drugs in recent years. Here two novel ruthenium(II) anthraquinone complexes
[Ru(tpy)(adtpy)]2+ (1) and [Ru(dtp)(adtpy)]2+ (2) have been synthesized and characterized. Spectroscopic and
viscosity measurementssuggested that two Ru(II) complexes bind to DNA in an intercalative mode. Topoisomerase
inhibition and DNA strand passage assay confirmed that two Ru(II) complexes acted as efficient dual inhibitors of
topoisomerases I and II by interference with the DNA religation. In MTT cytotoxicity studies, two Ru(II) complexes
exhibited antitumor activity against HeLa, BEL-7402, MCF-7 and HepG2 tumor cells. The AO/EB staining assay,
Alexa Fluor® 488 annexin V/PI double staining assay and alkaline single-cell gel electrophoresis (comet assay)
indicated that Ru(II) complexes could induce the apoptosis of HeLa cells.
Acknowledgements: This work was supported by NSFC(No. 21071155, 21172273, 21171177), GDNSFC
(9351027501000003), the National High Technology Research and Development Program of China (863 Program,
2012AA020305), StateKey Laboratory of Optoelectronic Materials and Technologies (2010-ZY-4-5) and Sun YatSen University.
References
[1] K. J. Du, J. Q. Wang, J. F. Kou, G. Y. Li, L. L. Wang, H. Chao, L. N. Ji, Synthesis, DNA binding and topoisomerase inhibitory
activity of ruthenium(II) polypyridyl complexes, Eur. J. Med. Chem., 2011, 46, 1056-1065.
[2] J. F. Kou, C. Qian, J. Q. Wang, X. Chen, L. L. Wang, H. Chao, L. N. Ji,Chiral Ruthenium(II) Anthraquinone Complexes as
Dual Inhibitors of Topoisomerases I and II, J. Biol. Inorg. Chem., 2012, 17, 81-96.
92
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 088
AREA: MBD
POSTER
A novel silver(I) complex with Ibuprofen: synthesis, spectroscopic characterization in the
solid-state and preliminary antibacterial assays
Silva, I.M.P.1; de Paiva, R.E.F.1; Profirio, D.M.1; Lancellotti, M.2; Corbi, P.P.1; Formiga, A.L.B.1
1
Department of Inorganic Chemistry, University of Campinas, Campinas, Brazil
2
Department of Biochemistry, University of Campinas, Campinas, Brazil
[email protected]
The wide-ranging biological properties associated with metal ions have recently stimulated the development of new
metal-based drugs. Silver complexes have been extensively studied due to the antibacterial properties exhibited by
this metal, and the combination of silver with biologically active molecules can enhance this effect even further [1].
Ibuprofen (C13H18O2, Ibu) is a non-steroidal anti-inflammatory drug used to treat headaches, rheumatoid arthritis,
fever and acute or chronic pain related with the inflammatory processes. The present work describes the synthesis,
spectroscopic characterization and preliminary antibacterial assays of a novel silver(I) complex with ibuprofen (AgIbu).
The Ag-Ibu complex was synthesized by the reaction of equimolar quantities of silver nitrate and sodium
ibuprofenate in aqueous solution. Elemental analysis indicates the composition AgC13H17O2. Anal. Calc for
AgC13H17O2(%): C, 49.9; H 5.47. Found(%): C, 49.6; H, 5.01. Thermal decomposition starts at 180°C and ends at
400°C, with a mass loss of 64,6%, leading to the formation of Ag0 as the final residue (calcd 34.5%; found 36.4%).
The CP/MAS
13
C NMR spectrum of the complex shows that, upon coordination, the carbon atom of the carboxylic
group is shifted upfield by 3.97 ppm. The IR spectrum shows that the value of separation Dn = νasym(COO) -1
-1
νsym(COO) for the complex (175 cm ) is higher than the value observed for sodium ibuprofenate (138 cm ),
suggesting a monodentate carboxylate coordination. However, according to the literature, a dimeric bidentate
bridging coordination cannot be discarded [2]. The DFT studies are being performed in order to further evaluate the
coordination mode.
The antibacterial activities of the complex were evaluated in aqueous suspension, against Gram-negative (E. coli
ATCC 25922, P. aeruginosa ATCC 27853 and P. aeruginosa PA IMI) and Gram-positive (S. aureus ATCC25923,
-1
Rib 1 and BEC9393) bacterial strains. The minimum inhibitory concentration values (MIC) were of ~25 mg mL for
all tested bacterial strains, confirming the antibacterial activity of the compound in the considered experimental
conditions.
References
[1] J. L. Clement, P. Ss. Jarret. Antibacterial Silver. Metal Bases Drugs 1994, 1, 467-482.
[2] V. Zelenak, Z. Vargová, K. Gyoryová. Correlation of infrared spectra of zinc(II) carboxylates with their structures.Spectrochim
Acta A 2007, 66, 262-272
93
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 089
AREA: MBD
POSTER
Synthesis, characterization and DNA interactions of two new ruthenium(II) compounds
Silva, P.P.; Pereira-Maia, E.C.
Department of Chemistry, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil
[email protected]
Recently, ruthenium compounds have received considerable attention because the complexes imidazolium-transdimethylsulfoxide-imidazole-tetrachlororuthenate(III) and indazolium-trans-bis(1H-indazole)-tetrachlororuthenate(III)
entered human clinical trials. This fact has stimulated us to search for new ruthenium compounds as antitumoral
agents. This work reports on the synthesis, characterization and DNA interactions of two new ligands and mixedligand complexes of ruthenium(II). Their general formula is [Ru(L)(phen)(dmso)(Cl)](PF6), in which L= pqdS [N'-(6oxo-1,10- phenanthrolin-5(6H)-ylidene)thiophene-2-carbohydrazide] for complex 1 and pqdO [N'-(6-oxo-1,10phenanthrolin-5(6H)- ylidene)furan-2-carbohydrazide] for complex 2 (Figure 1). The ligands pqdS and pqdO were
synthesized by reacting 1,10-phenanthroline-5,6-dione with thiophene-2-carbohydrazide (ligand pqdS) or furan-2carbohydrazide (ligand pqdO) in ethanol with 10% acetic acid. The ligands were characterized by elemental
analyses, vibrational, electronic, and 1H NMR spectrometries. The complexes were prepared by refluxing cis[RuCl2(dmso)4] with pqdS (1) or pqdO (2) in methanol for 1 hour. Afterwards, 1,10- phenanthroline and NH4PF6
were added and the mixture kept in reflux for more 3 hours. The complexes were characterized by elemental and
conductivity analyses, vibrational, electronic, and ESI-MS spectrometries. The presence of the complexes in
solution was confirmed by ESI-MS spectrometry in positive mode. Complex 1 give a main peak at m/z 728.6
assigned to [Ru(pqdS)(phen)(dmso)(Cl)]+ (calculated mass 729.2) and complex 2 at 712.6 due to
+
[Ru(pqdO)(phen)(dmso)(Cl)] (calculated mass 713.2). The calculated isotopic distribution for the proposed species
agrees with the experimental spectra. The molar conductivity values of 10-3 mol L-1 solutions of 1 and 2 in
nitromethane, at 25ºC, are ΛM= 89.6 and 77.9 µS·cm-1, respectively, indicating that they are 1:1 electrolytes. The
addition of calf thymus DNA to a solution of the complexes induces a hypochromic effect in their UV-Vis spectra
indicating the formation of a ternary complex with DNA. From the spectrophotometric data, the following binding
4
4
constants were calculated 6.1 × 10 for 1 and 1.4 × 10 for 2.
+
O
H3 C
CH3
O
S
N NH
N
N
Ru
N
N
S
C
O
Cl
Complex 1
Acknowledgments: CNPq, FAPEMIG, CAPES and INCT-Catálise.
94
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 090
AREA: MBD
POSTER
Antitumoral potential of platinum(II) compounds with 4-aminoquinoline derivatives
de Paula, F.C.S.1; Carmo, A.M.L.2; Fontes, A.P.S.2; Pereira-Maia, E.C.1
1
Department of Chemistry, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil
2
Department of Chemistry, Universidade Federal de Juiz de Fora, Juiz de Fora, Brazil
[email protected]
Quinoline is extensively used as a starting compound to synthesize molecules with pharmacological properties. In
this work, we examined the antitumoral potential of five platinum(II) compounds with a series of 4-amino-7chloroquinoline analogues (Figure 1). The synthesis and characterization of the complexes were performed as
1
previously described . The sensitivity to drug was evaluated in a chronic myelogenous leukemia cell line from the
drug concentration needed to inhibit cell growth by 50%, the IC50. Measurements of cellular accumulation were
made by incubating cells in the culture medium with various drug concentrations for 72 h. Partition coefficients of
the complexes were determined in an n-octanol/water system. Platinum content was determined by graphite
furnace atomic absorption spectrometry. All complexes inhibited cellular growth with the following IC50 values:
47.16 ± 0.8 for complex 1; 21.02 ± 0.4 for 2; 8.94 ± 0.4 for 3; 10.45 ± 1.1 for 4; and 2.02 ± 0.3 for 5. There is a
general trend of enhancing cytotoxic activity with the increase in the carbon chain length. Comparing compounds
carrying the same R and R1 groups, one observes that increasing “n” from 2 to 3, it doubles the cytotoxic activity in
the case of 1 and 2 (R=R1=H) and it leads to a 5-fold increase in the cytotoxic activity in the case of 4 and 5
(R=R1= CH2CCH). Incubation of cells with equitoxic complex doses leads to the same intracellular platinum
concentrations. Increasing lipophilicity enhances cellular uptake and, consequently, the cytotoxic activity.
Figure 1. Structures of complexes 1-5
Acknowledgments: CNPq, FAPEMIG, CAPES and INCT-Catálise.
References
[1] Carmo AML, Silva FMC, Machado PA, Fontes APS, Pavan FR, Leite CQF, Leite SRA, Coimbra ES, Silva AD.
Synthesis of 4-aminoquinoline analogues and their platinum(II) complexes as new antileishmanial and
antitubercular agents. Biomed Pharmacother; 2011; 65; 204–209.
95
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 092
AREA: MBD
POSTER
Novel ortho-substituted binucleating ligands and the mononuclear copper(II) complexes
of their partially hydrolyzed derivatives display potent antimicrobial activity
Cruz, W.S.1; Resende, J.A.L.C.2; Siqueira, S.A.3; Beraldo, H. de O.3; Rey, N.A.1
1
Department of Chemistry, PUC-Rio, Rio de Janeiro, Brazil
2
Department of Chemistry, UFF, Niterói, Brazil
3
Department of Chemistry, UFMG, Belo Horizonte, Brazil
[email protected]
The utility of copper ions, either alone or in combination with bioactive ligands, as a biocidal tool is well
documented in literature1. On the other hand, metal complexes of Schiff bases have shown antibacterial and
antifungal activities2. In the present work, we report the synthesis and characterization of a series of new osubstituted binucleating ligands, as well as the preparation and study of copper(II) complexes concerning their
partially hydrolyzed Schiff base products. The antimicrobial potential of all compounds was assayed against
bacteria (P. aerouginosa, S. aureus) and fungus (C. albicans).
Crystal structures of complexes containing methyl (1) and iodo (2) substituents were resolved by X-ray diffraction.
The compounds are mononuclear, with two partially hydrolyzed ligands coordinated through their phenolate oxygen
and Schiff base nitrogen atoms. Geometry is essentially square-planar in 2, while a heavily distorted tetrahedral
coordination sphere is observed for 1 since both methyl groups are on the same side of the ligands plane, causing
relevant steric repulsion.
When tested against bacteria, all ligands showed some activity, being the iodo-substituted compound the best of
them (MIC = 165 and 83 mmol L-1 for P. aerouginosa - ATCC: 9027 and S. aureus - ATCC: 6538, respectively).
Complexation only improved the activity profile against Staphylococcus, with complexes 1 and 3 (nitro-substituted)
-1
presenting MIC values equal to 87 and 76 mmol L , correspondingly.
In the case of C. albicans, methyl and nitro-substituted ligands were as active as the reference drug fluconazole,
showing MIC around 25 mmol L-1.
96
12th International Symposium on Metal Ions in Biology and Medicine
References
1) G. Borkow, J. Gabbay; Copper as a biocidal tool. Curr. Med. Chem. 2005, 12, 2163–2175.
2) S. Kumar, D. N. Dhar, P. N. Saxena; Applications of metal complexes of Schiff bases – A review. J. Sci. & Ind. Res. 2009, 68,
181–187.
Aknowledgments: CNPq, PUC-Rio
97
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 098
AREA: MBD
POSTER
Synthesis, characterization and antimycobaterial activity of Ag(I) complexes with N, N´bis[thiophenil-2-methylidene]-1, 2-diamine
da Silva, S.A.1; Cuin, A.1; Bergamini, F.2; Corbi, P.P. 2; Leite, C.Q.3; Pavan, F.3
1
Department of Chemistry, UFJF, Juiz de Fora, Brazil
Department of Chemistry, UNICAMP, Campinas, Brazil
3
Department of Biological Sciences, UNESP, Araraquara, Brazil
[email protected]
2
Tuberculosis, an infectious disease, occurs mainly in Africa, Asia and America, and is caused by the bacillus
Mycobacterium tuberculosis (MTB) [1]. It causes nearly 3 million deaths annually worldwide, and estimated 8.8
million of new cases every year [2]. The long period of treatment and co-infection with HIV, are one of the factors
responsible for the emergence of multidrug-resistant strains [2]. The WHO estimates some 500,000 cases of
multiresistant tuberculosis (MDR-TB) in 2007 [1]. Therefore, the discovery of new drugs to combat the MDR-TB is
necessary. In 2007, A. Cuin et al. synthesized Ag(I) complexes with alpha-hydroxy acids, with MIC values lower
than 8.0mg mL-1 against MTB [2]. Recently, Ag(I) complex with 2-(2-thienyl) benzothiazole, that showed effective
antituberculosis activity, was synthesized in our laboratory [3]. The present work aims the synthesis and
characterization of the ligand (BNH) and its Ag(I) complexes, AgBNH, Ag(BNH)2 and AgBNH(PPh3)2.
Methodology: The ligand was synthesized mixing 2-thiophenecarboxaldehyde and ethylenediamine in methanol
and recrystallized from n-hexane (yield: 97%). The complexes, AgBNH and Ag(BNH)2 were synthesized in
isopropyl alcohol solution (yield: 47% and 41% respectively). The complex AgBNH(PPh3)2 was synthesized adding
AgNO3 in BNH and PPh3 methanolic solutions, in proportion 1:1:2. The complex was precipitated in water (yield:
70%).
Results: IR bands of C=N group were assigned at 1634, 1632, 1629 and 1630 cm-1 for AgBNH, Ag(BNH)2,
AgBNH(PPh3)2 and BNH, respectively. The 1H- and
13
C-NMR of the compounds showed chemical shifts (ppm) for
2
N=C-H group at: BNH (8.40; 155.6), AgBNH (8.85, 159.0), Ag(BNH) (8.85, 158.7) and AgBNH(PPh3)2 (8.48,
155.9). The minimal formulas of Ag(I) complexes were obtained by elemental and thermal analyses. For AgBNH
(34.3%C, 2.68%H, 10.7%N, 25.8%Ag - C24H24Ag2N6O6S4), Ag(BNH)2 (42.2%C, 3.51%H, 10.4%N, 16.2%Ag C24H24AgN5O3S4) and AgBNH(PPh3)2 (65.2%C, 4.25%H, 2.38%N, 11.3%Ag - C48H42AgN3O3P2S2). Tests against
Mycobacterium tuberculosis are underway for the four compounds.
Conclusions: Three promising compounds antituberculosis and a ligand with excellent yield were synthesized.
Acknowledgements: The authors are thankful to FAPEMIG and FAPESP/Brazil process–CEXAPQ-00256/11 and
2012/08230-2 and UFJF/Proquali for financial supports.
References
[1] SouzaW. Doenças Negligenciadas. Rio de Janeiro: Academia Brasileira de Ciências, 2010, 43.
[2] Cuin A, Massabni AC, Leite CQF, Sato DN, Neves A, Szpoganicz B, Silva MS, Bortoluzzi AJ. Synthesis, X-ray structure and
antimycobacterial activity of silver complexes with a-hydroxycarboxylic acids. J Inorg Biochem; 2007; 101; 291-296.
[3] Pereira GA, Massabni AC, Castellano EE, Costa LAS, Leite CQF, Pavan FR, Cuin A. A broad study of two new promising
antimycobacterial drugs: Ag(I) and Au(I) complexes with 2-(2-thienyl)benzothiazole. Polyhedron; 2012; 38; 291-296.
98
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 099
AREA: MBD
POSTER
DNA structural changes on binding and photoreaction with water-soluble tricarbonyl
Re(I) complexes
Ragone, F.1; Yañuk, J.2; Cabrerizo, F.2; Wolcan, E.1; Ruiz, G.1
1
2
INIFTA-CONICET, UNLP, Universidad Nacional de La Plata, UNLP, La Plata, Argentina
IIB–INTECH–CONICET, Universidad Nacional de San Martín, CHASCOMUS, ARGENTINA
[email protected]
Metal complexes containing the fac-{ReI(CO)3} core derivatized with different diimine ligands have been known to
be efficient emitters for many years and, therefore, their photophysical properties have been widely studied1. In
particular, fac-Re(CO)3L1L2 complexes, with L1 = dipyridil[3,2-a:2’3’-c]phenazine (dppz) and L2 = pyridine
derivatives, have been recognized as good intercalators in double helix of DNA2. Intercalative binding can disrupt
the helical nature of DNA, causing profound effects on DNA integrity. Because of their photophysical and redox
properties, many of those complexes have the capacity to cause DNA damage by photoinduced oxidative strand
breakage3. Furthermore, the binding modes to DNA for Re(I)-Pterin complexes (L1 = 2-amine-4-pteridinone) have
been scarcely studied.
In this work we used UV-vis absorption and fluorescence spectroscopy to investigate the binding properties of a
4
new Re(I)-Pterin complex with DNA (plasmid DNA, double-stranded calf thymus DNA (CT-DNA) and synthetic
polynucleotides Poly[dAdT]2 and Poly[dGdC]2), in aqueous solutions. In addition, the consequences on the DNA
chemical structure and the excited state reactions of these complex and super-coiled plasmid DNA were
investigated by means of electrophoresis. Briefly, when solutions of the complex were incubated (30 min.) with
plasmid DNA, agarose gels showed morphological changes on circular DNA changing its conformation from
supercoiled to relaxed. Thermal processes show a progression of the conversion with increasing [Re(I)
complex]/[DNA] ratio. Irradiation with 350 nm light reverted the changes observed in darkness and no
photocleavage was observed.
On the other hand, recently we have demonstrated that a Re(I)-dppz complex, (4,4'-bpy)ReI(CO)3(dppz)+,
5
intercalates to Poly[dGdC]2 and with some selectivity to Poly[dAdT]2, Kb = 1.8 x 10 M
-1 5
. These studies are now
complemented with binding to CT-DNA and photocleavage to plasmid DNA after intercalation.
All the experimental data will be discussed in relation to the potential applications of those complexes as both
luminescent and conformational probes to DNA and molecular scissors to the double helix.
Acknowledgements: This work was supported in part by ANPCyT (PICT 1435), CONICET (PIP 0389),
Universidad Nacional de La Plata (UNLP 11/X533 y 11/X611) of Argentina. G.T.R., F.M.C. and E.W. are Research
Members of CONICET (Argentina).
References
1- A. Kumar, S. Sun and A. J. Lees.Photophysics and Photochemistry of Organometallic Rhenium Diimine Complexes. Top
OrganometChem (2010) 29: 1–35
2- K. K. Lo. Exploitation of Luminescent Organometallic Rhenium(I) and Iridium(III) Complexes in Biological Studies. Top
OrganometChem (2010) 29: 115–158
3- J. A. Smith, M. W. George and J. M. Kelly.Transient spectroscopy of dipyridophenazine metal complexes which undergo
photo-induced electron transfer with DNA.CoordChem Rev 255 (2011) 2666– 2675
99
12th International Symposium on Metal Ions in Biology and Medicine
4- F. Ragone, G. T. Ruiz, O. E. Piro, G. A. Echeverría, F. M. Cabrerizo, G. Petroselli, R. Erra-Balsells, K. Hiraoka, F. S. García
Einschlag and E. Wolcan. Water-Soluble (Pterin)rhenium(I) Complex: Synthesis, Structural Characterization, and Two
Reversible Protonation–Deprotonation Behavior in Aqueous Solutions. Eur J InorgChem, 2012, 4801–4810.
5- G.T. Ruiz, M.P. Juliarena, R.O. Lezna, E.Wolcan, M.R. Féliz and G. Ferraudi. Intercalation of fac-(4,4’- bpy)ReI(CO)3(dppz)+,
dppz = dipyridil[3,2-a:2’3’-c]phenazine, in polynucleotides. On the UV-visphotophysics of the Re(I) intercalate and the redox
reactions with pulse radiolysis-generated radicals. J ChemSoc, Dalton Trans, 2007, 20, 2020–2029.
100
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 103
AREA: MBD
POSTER
On the reaction between trans-[Ru(NH3)4(4- pic)(H2O)]3+ and cysteine
Franco, D.W.1; Souza, M.L.1; Castellano, E.E.2
1
Instituto de Química de São Carlos, Universidade de São Paulo, São Carlos, Brazil
2
Instituto de Física de São Carlos, Universidade de São Paulo, São Carlos, Brazil
[email protected]
Cysteine and glutathione (RSH) are the non protein thiols of higher concentration in the human body (0.1 – 1.0
mM) being part of an important redox buffer involving the oxidized form (RSSR)1. Furthermore they are very good
2
candidates to act as in vivo reductant of ruthenium nitrosyls trough a series of reactions in which is conceivable
the formation of the trans-[Ru(NH3)4(4-pic)(H2O)]
3+/2+
3
species . Aiming to better understand this system the complex
trans-[Ru(NH3)4(4-pic)(H2O)](CF3SO3)3.H2O has been isolated and studied through elemental analysis, UV-vis, Xray and EPR spectroscopies and cyclic voltammetry. The water molecule in trans-[Ru(NH3)4(4-pic)(H2O)]
-
-4
-1 -1
-4
-1 -1
is
substituted by Br and Cl ions at the rate constants of 1.5×10 M s and 2.5×10 M s (55 ± 0.5ºC, [H ] = 1.0×102
-
3+
+
M), respectively. The trans-[RuIII(NH3)4(4-pic)(OH2)]3+ did not reacts with cysteine while trans-[RuIII(NH3)4(42+
pic)(OH)]
-1 -1
does at the specific rate constant of 7.4 M s (pH = 4.8 µ = 0.2 M, 25ºC) yielding trans-[RuII(NH3)4(4-
pic)(OH2)]2+ and cystine, through an outer sphere mechanism. The calculated equilibrium constant for this reaction,
2
-1
Keq= 2,6×10 M , allows to calculate, in the above experimental conditions, Eº’RSSR/RSH equal to +0.088 vs.
NHE.
Acknowledgements: The authors are thankful to the Brazilian agencies FAPESP, CNPq and CAPES for financial
support.
References
[1] Schafer, F.Q.; Buettner. G.R.; Redox environment of the cell as viewed through the redox state of the
glutathione disulfide/glutathione couple. Free Radical Biology & Medicine; 2001; 30; 1191–1212.
[2] Tfouni, E; Truzzi, D.R.; Tavares, A.; Gomes, A.J.; Figueiredo, L.E.; Franco, D. W. Biological activity of ruthenium
nitrosyl complexes. Nitric Oxide; 2012; 26; 38-53.
[3] Roncaroli, F.; Olabe. J.A.; The Reactions of Nitrosyl Complexes with Cysteine. Inorganic Chemistry; 2005; 44;
4719 – 4727.
101
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 119
AREA: MBD
POSTER
New ruthenium(II) cyclopentadienyl thiosemicarbazone complexes with antitrypanosomal
activity
Sarniguet, C.1; Morais, T.S.2; Tomaz, A.I.2; Medeiros, A.3; Comini, M.4; Otero, L.1; Garcia, M.H.2; Gambino, D.1
1
Cátedra de Química Inorgánica, Facultad de Química, Montevideo, Uruguay
Faculdade de Ciências da Universidade de Lisboa, Centro de Ciências Moleculares e Materiais, Lisboa, Portugal
3
Departamento de Bioquímica, Facultad de Medicina, Universidad de la República / Group Redox Biology of Trypanosomes,
Institut Pasteur de Montevideo, Montevideo, Uruguay
4
Group Redox Biology of Trypanosomes, Institut Pasteur de Montevideo, Montevideo, Uruguay
2
Both classical octahedral ruthenium complexes and organoruthenium compounds have been widely explored for
their potential use in medicine as chemotherapeutics against different diseases. In particular, they have
demonstrated high potentiality as drug candidates for cancer therapy being promising alternatives to platinum
complexes. Indeed, two ruthenium compounds have already reached clinical trials for cancer therapy. The interest
on ruthenium stems on the fact that it shows chemical and biological properties that make its compounds
particularly suitable for the development of drug candidates. These properties make interesting the design of
1
antiparasitic ruthenium drugs .
Among the infectious illnesses designated by the World Health Organization as neglected tropical diseases, those
caused by genetically related parasites from the Trypanosomatidae family (genus Trypanosoma and Leishmania)
constitute major health concerns in the developing world. Our group has been working on the development of
prospective antitrypanosomal ruthenium complexes with bioactive 5-nitrofuryl containing semicarbazones and
thiosemicarbazones as ligands. Metal-drug combined effects could lead to enhance ligands´ activity and to
generate new mechanisms of action.
In this work, we describe the synthesis, characterization and evaluation on Trypanosoma brucei brucei (strain 427)
of a novel organoruthenium (II) cyclopentadienyl compound with 5-nitrofuryl thiosemicarbazone (TN) as co-ligand.
The compound [RuCp(PPh3)(TN)] was synthesized by ligand substitution from a suitable Ru-Cp precursor and
1
characterized by elemental analysis, infrared and H,
13
C and
31
P NMR spectroscopies. It shows to be a highly
selective anti-trypanosomal compound with parasiticidal effect (IC50 T. brucei brucei 3.2 < M; IC50 J774
macrophages < 100 < M; selectivity index < 31.2).
Acknowledgements: Authors thank CYTED network RIIDFCM and Portuguese national funds through FCT, the
Portuguese Foundation for Science and Technology (PTDC/QUI-QUI/101187/2008, PTDC/QUI-QUI/118077/2010
PEst- OE/QUI/UI536/2011, Ciência2008 Initiative and grant SFRH/BD/45871/2008).
References
1. D. Gambino, L. Otero, Perspectives on what ruthenium-based compounds could offer in the development of potential
antiparasitic drugs. Inorg Chim Acta 2012, 393, 103-114.
102
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 120
AREA: MBD
POSTER
Structure activity relationships of new prospective antiparasitic compounds based on
oxidovanadium(IV) complexes
Benítez, J.1; Fernández, M.1; Becco, L.2; Varela, J.3; Birriel, E.3; Correia, I.4; Lavaggi, M.L.3; González, M.3;
3
5
2
4
1
Cerecetto, H. ; Moreno, V. ; Garat, B. ; Costa Pessoa, J. ; Gambino, D.
1
Cátedra de Química Inorgánica, Facultad de Química, UDELAR, Montevideo, Uruguay.
Laboratorio de Interacciones Moleculares, Facultad de Ciencias, UDELAR, Montevideo, Uruguay.
3
Grupo de Química Medicinal, Laboratorio de Química Orgánica, Facultad de Ciencias-Facultad de Química, Universidad de la
República, Montevideo, Uruguay.
4
Centro de Química Estrutural, Instituto Superior Técnico, TU-Lisbon, Lisbon, Portugal.
5
Departamento de Química Inorgánica, Universitat Barcelona, Spain.
2
American trypanosomiasis, caused by Trypanosoma cruzi, is endemic throughout Latin America and causes more
deaths in this region than any other parasitic disease. The available chemotherapy is based on old non specific
drugs that give rise to undesirable toxic effects, show limited efficacy in the chronic phase of the disease and suffer
from development of resistance.
In the search for new therapeutic tools against this disease twenty five structurally related oxidovanadium(IV)
mixed- ligand complexes, [VIVO(L-2H)(NN)], including five tridentate salicylaldehyde semicarbazone derivatives (L)
and five polypyridyl NN derivatives (NN) as ligands were synthesized and characterized in the solid state and in
solution. The compounds were evaluated in vitro on Trypanosoma cruzi. Most of the complexes showed
significantly higher in vitro activities than the reference drug Nifurtimox and than the free semicarbazone and NN
ligands. In addition, most of the complexes showed high selectivity indexes towards the parasite in respect to
mammalian cells (J774 murine macrophages). The interaction of the complexes with DNA was demonstrated by
gel electrophoresis, atomic force microscopy and viscosity measurements, suggesting that this biomolecule may be
a parasite target.
Having a large number of structurally related VIVO-compounds structure-activity relationships may be established.
The lipophilicity was determined for the whole series of compounds. A nearly parabolic relationship between
biological response and lipophilicity was observed and an optimal lipophilicity value could be established as a guide
for further drug design. The nature of the NN coligand was determinant of the biological activity. The substitution on
the phenol moiety of the semicarbazone ligand seemed to have only a low incidence on the antitrypanosomal
activity.
Acknowledgements: Authors thank CYTED networks RIIDFCM and RIDIMEDCHAG and Portuguese Foundation
for
Science
and
Technology
(FCT):
(PTDC/QuiQui/101187/2008,
PEst-OE/QUI/UI0100/2011,
PEst-
OE/QUI/UI0612/2011, PEst-OE/QUI/UI0536/2011, Ciência2007 and 2008). M.F. thanks ANII Uruguay for a
fellowship.
103
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 121
AREA: MBD
POSTER
Synthesis, single crystal structure analysis and evaluation of apoptosis in vitro of the
trans-bis(3-aminoflavone)dichloroplatinum(II)
Ochocki, J.1; Fabijanska, M.1; Raber, G.2; Raab, A.3; Cebula-Obrzut, B.4; Smolewski, P.4; Krupp, E.M.5
1
Department of Bioinorganic Chemistry, Medical University of Lodz, Lodz, Poland
2
Institute of Analytical Chemistry, Karl-Franzens-University, Graz, Austria
3
Department of Chemistry, University of Aberdeen, United Kingdom
4
Department of Experimental Hematology, Medical University of Lodz, Lodz, Poland
5
Department of Chemistry, University of Aberdeen / ACES Aberdeen Centre for Environmental Sustainability, Aberdeen,
Scotland
[email protected]
Background: Currently, cisplatin (cis-DDP) is widely used in chemotherapy in many types of cancer.
Unfortunately, its application is limited because of serious side effects such as nephrotoxicity, myelotoxicity,
ototoxicity, allergy and the development of resistance in tumor cells. Therefore, studies are concentrated to search
for other platinum compounds, with at least equally effectiveness in chemotherapy and lower toxicity. Because of
the biological relevance of flavanone system, the derivatives of flavanone were used as the ligands in the synthesis
of novel cis Pt(II) and trans Pt(II) complexes, for which a possible application as antitumor agents is conceived.
Methodology:
The
compound
was
characterized
using
reversed
HPLC
(High
Performance
Liquid
Chromathography), Mass Spectrometry (MS) and Inductively Coupled Plasma Mass Spectrometry (ICP-MS),
Results: Our previous studies revealed that cis-bis(3-aminoflavone) dichloroplatinum(II) exhibited higher toxicity,
induced a higher degree of apoptosis and necrosis and a higher percentage of P53 and BAX expression in A549
human non small cell lung cancer in comparison with the classical antitumor agent cis-DDP. This complex
structurally related to cis-DDP, possesses flavones derivatives as non leaving ligands instead of amine with two
cis- bound labile chloride ligands. In this study, the crystal and molecular structure of trans-Pt[(af)2Cl2], (af = 3aminoflavone) shows a square planar geometry of PtL2Cl2, with two organic molecules and two chloride leaving
ligands in a trans configuration. The proapoptotic effect of platinum(II) complex in comparison with cisplatin in
leukemia cell lines (L1210, L1210R) and the mechanisms of apoptosis induction was examined using flow
cytometry analysis (detection of caspase-3 activity and Annexin V/PI assay).
Conclusions: Results suggested that trans-Pt[(af)2 Cl2] effectively induced programmed cell death in a dose- and
time- depended manner. Furthermore, the new compound revealed stronger apoptotic effect in contrast to cisDDP.
Acknowledgements: This work supported by Polish Ministry of Science and Higher Education grant No. N N405
674040
104
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 127
AREA: MBD
POSTER
Bis-thiazole copper (II) complex in the research of new cytotoxic drugs
1
Velluti, F. ; Guidali, F.2; Alvarez, N.1; Serra, G.2; Medeiros, A.3; Comini, M.3; Ellena, J.4; Scarone, L.2*; Torre, M.H.1*
1
Química Inorgánica (DEC), Facultad de Química, Universidad de la República, Montevido, Uruguay
Química Farmacéutica (DQO), Facultad de Química, Universidad de la República, Montevideo, Uruguay
3
Group of Redox Biology of Trypanosomes, Institut Pasteur, Montevideo, Uruguay
4
Laboratorio de Cristalografía, Instituto de Física de São Carlos, Universidade de São Paulo, Sao Paulo, Brasil
2
Small natural macrocyclic peptides have attracted significant attention due to their interesting biological activities
[1]. Their geometry and the set of possible donor atoms suggest that their biological function might involve the
binding of metal ions inside the organisms. Taking into account previous studies based on 2-amino-thiazole copper
(II) and manganese (II) complexes [2], as part of our research for new candidates with anticancer activity employing
molecular simplification, the bis-thiazole type BT (Figure a) and their copper(II) complex (Figure b), were
synthesized.
The characterization of the ligand was performed by NMR, HRMS and IR and for the complex by elemental
analysis, UV- Vis, IR and X-ray diffraction spectroscopies.
The stoichiometry of this new compound was [Cu2(BT)2(Cl)2]•H2O. It presented a dimeric structure where two BT
molecules acted as bridges between both Cu(II) ions (Figure b). Each BT molecule linked with one Cu(II) through
the thiazole and amide nitrogen atoms and with the other Cu(II) through the other thiazole N atom and the
carboxylic oxygen. The distorted square pyramidal geometry of each Cu(II) is completed by Cl- ions. The
spectroscopic measurements agreed with the structure found.
The biological activity of BT and the copper complex against tumor cell lines (MCF-7 and HT29) and the cytotoxicity
in Murine macrophage J774 were determined.
References
[1] Comba, P.; Gahan, L.R.; Haberhauer, G.; Hanson, G.R.; Noble, C. J.; Seibold, B.; van den Brenk, A.L. ”Copper(II)
Coordination Chemistry of Westiellamide and Its Imidazole, Oxazole, and Thiazole Analogues”. Chem. Eur. J. 2008, 14, 4393 –
4403.
[2] Alexandru, M.G.; Velickovic, T.C.; Jitaru, I.; Grguric-Sipka, S.; Draghici, C. “Synthesis, characterization and antitumor activity
of Cu(II), Co(II), Zn(II) and Mn(II) complex compounds with aminothiazole acetate derivative”. Cent. Eur. J. Chem. 2010, 8(3),
639–645.
105
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 128
AREA: MBD
POSTER
Antitumor activity of novel trans-platinum(II) complexes based on intercalating and
sulfonamide ligands
Navarro-Ranninger, C.1; Del Solar, V.1; Martín-Santos, C.1; Alemán, J.2
1
Inorganic Chemistry Department, Universidad Autónoma Madrid, Madrid, Spain
Organic Chemistry Department, Universidad Autónoma Madrid, Madrid, Spain
[email protected]
2
Cisplatin (CDDP) is one of the most successful and traditional antitumor metal compoundswhich has been used in
nearly 50% of all tumor therapies1. However, different adverse effects, such as dose-limiting, nephrotoxicity,
peripheral neuropathy, tinnitus and hearing loss are known.Thousands of platinum complexes have been tested
during the last half-century;therefore, the continuous search for new platinum complexes (and especially with trans
geometry in our group) is an interesting area which have directed to hundred of chemists to do huge efforts in this
topic.
At this moment, only few platinum complexes with sulfonamide ligands and cis-geometry have been synthesized
and none of them have been evaluated in biological assays. Therefore, we will present the synthesis and in vitro
evaluation of new trans-monosulfonamide platinum complexes will be presented (right, Scheme). In addition, we
2
will focus our attention in simple intercalating compounds , which can be incorporated into the platinum complex.
We will show how to prepare these compounds in one step from commercial available compounds in high yields
(50-95%), and their citoxicity studies (left, Scheme).
Acknowledgements: We acknowledge grants from the Spanish MICINN (SAF2009-09431, and CTQ2008-06806C02-01/BQU).J. A. thanks the Spanish MICINN for Ramón y Cajal contract. C. M. And V. S. thank Autónoma
University andSpanish government for pre-doctoral fellowships, respectively.
References
[1] For selected reviews in platinum anticancer complexes, see: (a) Bruyninx, M., Sadler, P. J., Curr Opin Chem Biol, 2008, 12,
197-206; (b) L. Kelland Expert Opin Investig Drugs, 2007, 16, 1009-1021.
[2] S. Neidle, Principles of Nucleic Acid Structure. Elsevier. Chapter 5, page 132, 2008.
106
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 129
AREA: MBD
POSTER
Iodido complexes “the second generation”: trans-[PtI2(amine)(amine´)] with different
aliphatic amines
Álvarez-Valdés, A.1; Parro, T.1; Medrano, M.A.1; Cubo, L.1; Carnero, A.2; Quiroga, A.G.1
1
Inorganic Chemistry, Universidad Autónoma Madrid, Madrid, Spain
2
IBIS. Hospital Universitario Virgen del Rocío, Sevilla, Spain
[email protected]
The research developed in our laboratories has demonstrated that the use of chemical modifications in metallic
based drugs structures (in particular trans platinum drugs) can result in improved anticancer activity [1]. Our work
regarding the importance of the leaving group has afforded very interesting results in the evaluation of iodido
complexes [2]. The potential of cis iodide complexes as antitumoral agent was firstly proven and the studies of the
trans parent complexes have not only afforded a higher cytotoxicity values, but also proven to have a peculiar
different reactivity (compared to cisplatin) versus sulfur donors and Cyt C [3].
Our experience on the design of non-conventional metallodrugs manifested in most of the cases, a higher
cytotoxicity using different aliphatic amines in the same structure [1].
Following these results, we are now exploring the activity of the iodido complexes bearing different aliphatic amines
in trans configuration. In this contribution, we will present the synthesis, characterization and cytotoxicity values in a
selected panel of cell lines of complexes with general formulae trans-[PtI2(amine)(amine´)]. The interaction with
biomolecules such as 5-GMP, supercoiled pBR322, N-AcCys and N-AcMet (sulphur donor models) will be also
presented, discussed and compared with the interaction. The interaction with sulphur donors is also investigated.
Acknowledgements: Financial support of Spanish MICINN: SAF 2009-09431 and SAF-2012-34424 and COST
CM1105 are acknowledged.
References
[1] Quiroga, A. G. Understanding trans platinum complexes as potential antitumor drugs beyond targeting DNA. J Inorg
Biochem2012 114. 106-112.
[2] Messori, L., Casini, A., Gabbiani, C., Micheluci, E., Cubo, L., Rios-Luci, C., Padrón, J. M., Navarro-Ranninger, C., Quiroga,
A. G. Cytotoxic Profile and Peculiar Reactivity with biomolecules of a Novel “Rule-Breaker” Iodidoplatinum(II) Complex.ACS
Med Chem Lett 2010;1, 381-385.
[3] Messori, L., Cubo, L., Gabbiani, C., Álvarez-Valdés, A., Michelucci, E., Pieraccini, G., Ríos-Luci,C., León, L.G., Padrón, J.M.,
Navarro-Ranninger, C., Casini, A., Quiroga, A.G. Reactivity and Biological Properties of a Series of Cytotoxic PtI2(amine)2
Complexes, Either cis or trans Configured.Inorg Chem 2012; 51, 1717-1726.
107
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 130
AREA: MBD
POSTER
Searching for gallium bioactive compounds: a new gallium(III) complex as prospective
antitumoral and anti-Trypanosoma cruzi compound
Machado, I.1; Fernández, M.1; Becco, L.2; Brissos, R.F.3; Zabarska, N.3; Gamez, P.3,4; Marques, F.5; Garat, B.2;
1
Gambino, D.
1
Cátedra de Química Inorgánica, Facultad de Química, UDELAR, Montevideo, Uruguay.
Laboratorio de Interacciones Moleculares, Facultad de Ciencias, UDELAR, Montevideo, Uruguay.
3
Departament de Química Inorgànica, QBI, Universitat de Barcelona, Barcelona, España.
4
Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, España.
5
Unidade de Ciências Químicas e Radiofarmacêuticas, Instituto Tecnológico e Nuclear, Sacavém, Portugal
2
Cancer is the second largest cause of death in developed countries. The need to overcome the drawbacks exerted
by cisplatin, oxaliplatin and carboplatin, the worldwide approved metal-based anti-tumoral compounds, has lead to
the search for other metallodrugs. Chagas disease (American Trypanosomiasis), caused by the protozoa
Trypanosoma cruzi, constitutes a major health problem concentrated in the poorest regions of Latin America.
The anti-neoplastic activity of simple Ga(III) salts was discovered in the 1970s and much work has been performed
since then to elucidate their mechanism of action. To prevent hydrolysis processes and to improve cell membrane
permeation and oral bioavailability, coordination compounds of this metal ion are currently being developed. So far,
tris(8-quinolato) gallium(III) and tris(maltolato) gallium(III) have entered the clinical trials phase as prospective oral
anti- tumor agents.
In the search for gallium bioactive compounds we have been developing Ga(III) complexes with tridentate ligands
having an N, N, O donor set. In this work we report the synthesis and characterization of the complex [GaIII(LH)2](NO3) with 2-methoxy-6-(pyridin-2-ylcarbohydrazonoyl) phenol as ligand. Its biological potential has been
explored. Highly- proliferative cells such as Trypanosoma parasites and tumor cells show metabolic similarities that
have lead in many cases to a correlation between antitrypanosomal and antitumor activities. Therefore, both
activities are evaluated in this work. Three human tumor cell lines were selected that show different degrees of
resistance to metallodrugs: ovarian A2780 (low resistance), breast MCF7 (medium resistance) and prostate PC3
(high resistance) cells. The complex shows IC50 values in the low micromolar range on T. cruzi epimastigotes
(Dm28c strain) and on A280 cells. Complexation with gallium leads to the enhancement of the cytotoxic potency of
the organic ligand.
Acknowledgements: I.M. and M.F. thank ANII-Uruguay for a research grant. Authors thank ICREA (Institució
Catalana de Recerca i Estudis Avançats) and Ministerio de Economía y Competitividad de España (Proyecto
CTQ2011-27929- C02-01).
108
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 139
AREA: MBD
POSTER
Antibacterial activity of silver(I) complexes with ligands having anti- inflammatory
properties
Azócar, M.I.*; Muñoz, H.; Levin, P.; Dinamarca, N.; Gómez, G.; Paez, M.A.
Laboratory of Bioinorganic Chemistry, Faculty of Chemistry and Biology, University of Santiago of Chile, Santiago, Chile.
During recent decades silver complexes have been extensively studied for their excellent antibacterial properties,
which have proven to be even more effective than silver salts. This metal is active at low concentrations and has a
low toxicity [1]. Antibacterial experiments have shown broader antimicrobial activity spectra for silver complexes
with Ag-O and Ag-N bonds than with Ag-P and Ag-S bonds [1]. Investigations on silver complexes to date have
attributed their enhanced antimicrobial properties to distinctive weak Ag-O and Ag-N bonds in their structure [3,4].
Figure 1: Ligands used in the synthesis of silver compounds.
Herein, we describe the preparation, characterization (by FTIR, NMR, UV-C radiation and X-ray diffraction) and
antibacterial activity (MIC and Viability test) of five silver (I) complexes: AgIbu, AgNap, AgMef, AgAsp and AgSal,
where
Ibu= iso-butyl-propanoic- phenolate, Nap=(+)-(S)-2-(6-methoxynaphthalen-2-yl) propanoate, Mef=2-(2,3-
dimethylphenyl)aminobenzoate, Asp=2-acetoxybenzoate and Sal= 2- hydroxybenzoate(See figure 1) against E.
coli and S. aureus.
The results show a tendency between the nature of Ag-X (X = Oxygen and Nitrogen) bonds and the rate or
efficiency of antibacterial behavior.
Acknowledgements
This work was supported by FONDECYT (Grants No 11080133 and 1100537), MINCYT- CONICYT(2010-188) and
CONICYT (Grant: 79090024). M.A. Paez and M. I. Azócar are also grateful to VRID USACH.
References
[1] Ahmad, S.; Isab, A.A.; Ali, S. and Al-Arfaj, A.R. Perspectives in bioinorganic chemistry of some metal based therapeutic
agents. Polyhedron, 2006, 25, 1633 and references therein.
[2] Kasuga, N. C.; Sugie, A. and Nomiya, K. Syntheses, structures and antimicrobial activities of watersoluble silver(I)–oxygen
bonding complexes with chiral and racemic camphanic acid (Hca) ligands. Dalton Trans., 2004, 3732.
[3] Abarca, R.; Gomez, G.; Velasquez, C.; Paez, M.A.; Gulppi, M.; Arrieta, A.; Azocar, M.I. Antibacterial behavior of
pyridinecarboxylate silver(I) complexes. Chin J Chem. 2012, 30, 1631.
109
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 152
AREA: MBD
POSTER
Antitumor activity of [Ru(O-O)(bipy)(dppb)]PF6 complexes and their interactions with
bovine serum albumin
[O-O=cinnamic acid derivatives; dppb = 1,4-bis(diphenylphosphino)butane; bipy = 2,2´bipyridine]
Graminha, A.E.*1; Honorato, J.2, Menezes, A.C.S.2; Cominetti, M.R.3, Batista, A.A.1
1
Department of Chemistry, Federal University of São Carlos, São Carlos(SP), Brazil
2
Department of Chemistry, Federal University of Goiás, Goiânia, Goiás, Brazil
Department of Gerontology, Federal University of São Carlos, São Carlos(SP), Brazil
3
1
Phenolic compounds are secondary metabolites that are capable of inhibiting DNA damage and protecting the skin
cells from damage caused by UV radiation2. Among the phenolic acids, cinammic acid found to be an important
bioactive substance. Moreover, ruthenium complexes have been showing antitumor activity, in different cancer
3
cells, including metastatic cancer . Therefore, in this work we aim to combine the antitumor properties of the
cinnamic acid derivatives, using it as ligand, with the ruthenium complex core, containing the ruthenium/dppb/bipy.
Hence, five new complexes of ruthenium (II) were synthesized from the precursor, cis- [RuCl2(bipy)(dppb)], with
ligands derivative of the cinnamic acid. The reaction with α- methylcinnamic acid (α-metcn); 4-methoxycinnamic
acid (4-metoxcn); 3-Hydroxy-4-methoxycinnamic acid (3,4-metoxocn) yielded complexes of the type [Ru(OO)(bipy)(dppb)]PF6, O-O = coordination by carboxylic group (Fig. 1), as products, by displacement of the chloride
ions from the precursor complex. The complexes were characterized by elemental analysis, spectroscopic and
electrochemical techniques. Furthermore, albumin binding studies with complexes showed good binding affinity to
BSA proteins giving relatively high binding constants. In vitro tests for antitumor activity against the MCF-7 human
breast tumor cell line were carried out for the new complexes, for the precursor complex (cis-[RuCl2(bipy)(dppb)]),
and for the free ligands as well. The complexes tested showed higher activities than the free ligands, and than the
reference drugs, cisplatin and doxorubicin.
Figure 1. Representative scheme of reaction between cis-[RuCl2(dppb)(bipy)] and cinnamic acid.
Acknowledgements
We thank CNPq, CAPES and FAPESP, for financial support.
References
1. Rice-Evans C, Spencer JP, Schroeter H, Rechner AR., Drug Metabol Drug Interact. 2000,17: 291-310.
2. Saija A, Tomaino A, Trombetta D, De Pasquale A, Uccella N, Barbuzzi T, Paolino D, Bonina F. Int J Pharm.,2000, 199: 39-47.
3. C.G. Hartinger, S. Zorbas-Seifried, M.A. Jakupec, B. Kynast, H. Zorbas, B.K. Keppler, J. Inorg. Biochem., 2006, 100: 891904.
110
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 153
AREA: MBD
POSTER
Synthesis of semi-sandwich complexes η6-C10H14-Ru(II) with nitrogen chelating ligands
and interaction with DNA
Colina-Vegas, L.*1; Villarreal-Peña, W.1; Navarro, M.2; Batista, A.A.1
1
Departamento de Química, Universidade Federal de São Carlos – UFSCar, São Carlos – SP, Brazil
2
School of Chemical and Mathematical Sciences, Murdoch University, Australia
The development of half-sandwich η6arene-Ru (II) has attracted particular attention and extensively studied over
the past 20 years because these complexes show applications in different areas of scientific and industrial interest
[1]. The synthesis, spectroscopic characterization, crystal structures and electrochemistry of π-arene piano-stool
ruthenium (II) complexes with bipyridine and derivatives as nitrogen donor ligands are described. A series of six
6
organometallic compounds of formula [RuCl(η -C10H14)(N∩N)]PF6 (N∩N = 1,10’-phenantroline, 4,7’-diphenyl-1,10’phenantroline, 2,2’-bipyridine, 5,5’-dimethyl-2,2’- bipyridine, 4,4’-dimethoxi-2,2’-bipyridine and 4,4’-di-t-buthyl-2,2’bipyridine) were synthesized and characterized. The solid state structures of the six complexes were determined by
X-ray crystallography (Figure 1) and their characterization were completed by IR spectroscopy, molar conductivity
and NMR studies (1H and
13
C). Additionally, were done spectroscopic titration with DNA, to estimate interaction
constants between the metal complex and this macromolecule and in vitro tests for antitumor activity against the
MDA- MB-231 human breast tumor cell line were carried out for the new complexes. The electrochemical
measurements in dichloromethane compounds do not exhibit a reversible process, is only observed after addition
-
of acetonitrile, which showed the exchange between the ligand Cl for CH3CN [2]. The crystallographic structures of
the compounds show a distorted octahedral geometry, the semi-sandwich, in all lengths and bond angles do not
vary significantly. In the study of metal complex and DNA interaction by spectroscopic titrations allowed us to
calculate constant interaction using two calculation methods well reported, the values of the interaction constants
are between 105 and 103 M-1 indicate a reversible interaction with DNA [3]. The complexes tested showed higher
activities.
Figure 1. ORTEP view of the complexes 1-6 with the thermal ellipsoids at the 50 % probability level.
Acknowledgements: We thank CAPES, CNPq and FAPESP for financial support.
References:
[1] Dutta, B., Scolaro, C., Scopelliti, R., Dyson, P. J., Severin, K., Importance of the π-Ligand: Remarkable Effect of the
Cyclopentadienyl Ring on the Cytotoxicity of Ruthenium PTA Compounds, Organometallics, 2008, 27, 1355-1357.
[2] Gollas, B., Speiser, B., Zagos, I., Maichle-Mössmer, C., Electrochemistry of ruthenium metallocenes: Part 3. Synthesis and
properties of ruthenium [22]paracyclophane complexes with methacrylic acid and methacrylate ester substitutents, J.
Organomet. Chem. 2000, 602, 75-90.
[3] Navarro, M., Castro, W., Martínez, A., Sánchez-Delgado, R. A., The mechanism of antimalarial action of
[Au(CQ)(PPh3)]PF6: Structural effects and increased drug lipophilicity enhance heme aggregation inhibition at lipid/water
interfaces, J. Inorg. Biochem., 2011, 105, 276–282.
111
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 154
AREA: MBD
POSTER
Study of the mechanism of action of Pt(CQ)2(Cl)2 through its interaction with nitrogenous
bases
Colina-Vegas, L.*1,2, Castro, W.2, Navarro, M.2
1
2
Universidad del Zulia, Maracaibo, Venezuela
Instituto Venezolano de Investigaciones Cientificas, Caracas, Venezuela
Transition metals in medicine is a field of growing chemical development which has enormous potential [1]. One
drug design strategy used in our laboratory was based on the binding of an organic compound with known
therapeutic value to a transition metal looking for a synergistic metal-drug effect [2]. Recently, we have found that
Pt(CQ)2(Cl)2 displayed good activity against various cancer cell lines and its mechanism of action seems to be
related with its interaction with DNA, through covalent binding [3]. In the present work, we show the synthesis of
Pt(CQ)2(Cl)2 and the study of the hydrolysis of this complex as a funtion of time using conductivity and UV-vis
spectroscopy was carried out. Additionally, we have studied the interaction of title complex with guanosine,
1
adenosine, guanosine 5'- monophosphate (5'-GMP) and adenosine 5'-monophosphate (5'-AMP) by NMR- H and
NMR-
31
P spectroscopic. The results indicated that both chloride ligands of the complex exchanges with water
molecules, these exchanges showed dependency with the time, temperature and solvent. Also, we have observed
that the interaction between this complex and guanosine and adenosine produced different changes in the
absorption spectrum of these nucleosides such as bathochromic shift and hyperchromic. The absorption spectra of
5'-GMP and 5'-AMP showed hypochromic (at 251 and 260 nm respectively), hyperchromic at 268, 330 and 345 nm,
and several isobestic points at 264 and 310 nm when they was tritrated with Pt(CQ)2(Cl)2. The study by NMR-1H
31
and NMR- P indicated that the compound interacts with the guanosine and adenosine via primary amine; the
reaction was warmed in the case of adenosine. 5'-GMP in order to promote the interaction with the oxygen of the
phosphate group.
Figure 1. From left to right: Molecular structure of complex Pt(CQ)2(Cl)2 and spectroscopic titration with 5'-GMP and
5' –AMP
Ackowledge: This work was funded by IVIC.
References:
[1] Farrel, N. Uses of inorganic chemistry in medicine, The Royal Society of Chemistry, Bodmic, 1999.
[2] Gill, M.R., Thomas, J.A,. Ruthenium(II) polypyridyl complexes and DNA—from structural probes to cellular imaging and
therapeutics. Chem. Soc. Rev. 2012, 41, 3179-3192.
[3] Navarro, M., Castro, W., Higuera-Padilla, A., Sierraalta, A., Abad, M., Taylor P., Sánchez-Delgado, R., Synthesis,
characterization and biological activity of trans-platinum(II) complexes with chloroquine. J. Inorg. Biochem. 2011, 105, 16841691.
112
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 155
AREA: MBD
POSTER
Adaptation of a novel class of proteasome inhibitors for treatment of multiple myeloma
Szenkier-Garcia, N.1; Navon, A.2; Sterchook, R.3, Gumienna-Kontecka, E.4; Szebesczyk, A.4; Kozlowski, H.4;
1
Shanzer, A.
1
Department of Organic Chemistry, Weizmann Institute of Science, Rehovot, Israel
Department of Biological Regulation, Weizmann Institute of Science, Rehovot, Israel
3
Biological Services, Weizmann Institute of Science, Rehovot, Israel
4
Faculty of Chemistry, University of Wroclaw.
2
The proteasome is a large complex protein present in eukaryotes and Archaea, responsible for the ubiquitinedependent degradation of intracelular proteins, envolved in the regulation of multiple physiological functions, and
an established target of anticancer drugs.
In this work we describe the proteasome inhibition of a thiosemicarbazate-Cu complex, which, in contrast to all
other known proteasme inhibitors, lacks an electrophillic moiety capable of covalently binding the active sites.
We explored the possible mechanism of inhibition, and present experimental evidence and in silico calculations
showing that catalysis of the formation of a disulfide bond in the proximity of the β-2 active site may be envolved.
A series of derivatives possesing different substituents was prepared, as well as complexes of other transition
metals, and their activities were compared to the original compound. Among them, for one derivative, we suceded
to diminish proteasome activity in one order of magnitude.
Physicochemical characterization, including species distribution curves is also presented.
In conclusion, we have explored the chemical, biochemical and biological aspects of proteasome inhibitory activity
of thiosemicatbazate-Copper complexes.
113
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 021
AREA: MIEH
POSTER
Arsenic environmental and health issues in Uruguay: a multidisciplinary approach
Mañay, N.1*; Pistón, M.2; Goso, C.3
1
Cátedra de Toxicología, Facultad de Química, Montevideo, Uruguay
Cátedra de Química Analítica, Facultad de Química, Montevideo, Uruguay
3
Departamento de Evolución de Cuencas, Facultad de Ciencias, Montevideo, Uruguay
[email protected]
2
Scientific research has been recently conducted in Uruguay to study the presence of geogenic arsenic (As) in
groundwater and preliminary results, showed As levels above those recommended by WHO for drinking water
(10 µgL-1) in different aquifers of the country. For example, in Raigon aquifer, water samples showed arsenic
-1
-1
concentrations ranged from 1,4 to 39,7 µgL with a median of 24,6 µgL .
Several laws and decrees regulate water sources quality and the drinking water is supplied only by a state
company that is controlled by a state regulation institute, being As new recommended levels < 20 µgL-1. In a 6
years period study of those data assessment, As concentration in drinking water samples taken from different
areas of the country, showed an increase from not detectable to quantifiable levels but within local regulation limits.
This environmental health issue is now becoming a matter of concern with a multidisciplinary approach, as similar
aims research teams and experts from geosciences and biosciences, joined to face those arsenic exposure risks
problems. The aim of this work is to review those environmental and health arsenic issues in Uruguay to show the
recent advances in our country.
As preliminary results, several multidisciplinary studies have been developed focusing on arsenic geological and
toxicological aspects. For this purpose, analytical and speciation methodologies were optimized and validated, for
arsenic analysis in water and urine as available tools in Uruguay. Arsenic biomonitoring and determination of urine
metabolites on general population and exposed workers, are currently ongoing research studies.
To assess population’s health impacts from chronic exposure to inorganic arsenic through drinking water and in the
workplace, it is important to continue developing those systematic studies and analytical tools because the risk of
the Uruguayan population has not yet been estimated.
114
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 048
AREA: MIEH
POSTER
Occupational exposure evaluation of workers of portland cement industry in Uruguay
Cousillas, A.; Pereira Testa, L.; Clavijo, G.; Mañay, N.
Cátedra de Toxicología, Departamento Estrella Campos, Facultad de Química, Montevideo, Uruguay
[email protected]
Portland cement is a light gray or white powder used as a building material in the production of concrete with strong
adhesive properties when mixed with water. Employees who work with Portland cement are at risk of developing
skin problems, ranging from mild and brief to severe and chronic. Portland cement becomes highly caustic (pH>12)
when it isin contact with moisture in eyes or on skin, or when mixed with water, and will damage or burn the eyes or
skin. Inhalation may cause irritation to the moist mucous membranes of the nose, throat and upper respiratory
system or may cause or may aggravate certain lung diseases or conditions.
The aim of this work is to evaluate occupational exposure of workers of three uruguayan Portland cement plants.
46 personal and zone airborne samples were collected in plants 1, 2 and 3 and analyzed to determine occupational
exposure. Samples of total and respirable dust were collected according to NIOSH methods 0500 and 0600.
3
2010 ACGIH TLVs were considered as reference values being 1 mg/m for respirable Portland cement dust and
10 mg/m3 for total dust (PNOS).
We found that in Plant 1: 50 % of the samples were above the TLV value and 25 % exceeded del action level. In
Plant 2: 43 % > TLV and 21% > action level. In Plant 3: 38 % > TLV and 8 % > action level.
We discuss the reasons we observed that could lead to this results and conclude that there is a need of improving
working conditions. Important measures should be taken to prevent occupational exposure.
115
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 049
AREA: MIEH
POSTER
Portland Cement and health impacts
Pereira Testa, L.; Cousillas, A.; Mañay, N.
Cátedra de Toxicología, Departamento Estrella Campos, Facultad de Química, Montevideo, Uruguay
[email protected]
Portland Cement is basically a finely ground cement clinker mixed with a small amount of calcium sulfate
dehydrate. It is obtained by heating to a high temperature a mixture of substances such as limestone, sand, clay
and shale. It is a light gray or white powder. Cement dusts are produced during blasting of raw materials, grinding
of cement clinker and packaging or loading of finished cement.
Although this material is widely used in building and construction works as an important ingredient in concrete
products, very scarce information on health effects of the particulate air pollution from cement factories, is
available. However, most cement dusts chemical elements are potentially harmful to the environmental biota in
general and children are the most susceptible affected human populations.
Some of the dangerous elements of cement dust include: Particulate Matter (PM2.5 & PM10), lead, arsenic,
chromium VI and chromium III, mercury, manganese, cadmium, crystalline silica among others.
It is well known that when Portland Cement, contacts with moisture in eyes or on skin, or when mixed with water, it
becomes highly caustic and will damage or burn the eyes or skin.
Uruguay has several Portland Cement kilns in different parts of the country and there are only few studies on
occupational exposure assessment.
This work describes the main health risks of populations living near a cement kiln and of occupationally exposed
workers. Risk assessment and safety conditions are explained with special emphasis on minimizing toxic effects of
cement pollutants.
We conclude that there must be a concern on risk management not only for health prevention but also for the
environment protection in cement quarrying sites and around cement factories.
116
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 054
AREA: MIEH
POSTER
Iron analysis from Octopus hubbsorum (Cephalopoda: Octopodidae) for human health
Palacios-Abrantes, J.1; Melo, V.1; Urbano, B.2
1
2
Departamento de Sistemas Biológicos, Universidad Autónoma Metropolitana, Xochimilco, Ciudad de México, México
Malacología, Instituto de Ciencias del Mar y Limnología, Universidad Nacional Autónoma de México, Ciudad de México,
México
[email protected]
The Food and Agriculture Organization of the United Nations declared that 12% of the world’s population is
undernourishand and for Mexico around 5% of the populations is included. Minerals are important components of
the daily diet. Hence, the important task of the scientific community is to augment information about the chemical
composition of edible organisms that human population consume on regular basis, this information will help to
improve world’s people health, nutrition, food security and ecological sustainability. Minerals are essential in
humans, the lack of them have been related to many degenerative diseases. Iron (Fe) is an important mineral on
human diet, and on the role of their function of the immune and central nervous systems, among others. Adult man
contains of iron on his body with a relation of 60 mg/kg, because the highly conservation rate (except menstruating
and pregnant woman), the daily need is considerably low for man. Despite this, iron anemia is the most common
nutritional deficiency worldwide, that lead governments to fight undernutrition, hunger and promote health. Typically
Mexican population gets the iron needs from vegetables (wich have poor absorption rates) and costal population
include fish every once in a while in their diet. Simpson et al. (1981) mention the impact of fiver on iron absorption It
is mention that that addition of 12g/day of bran to a meal decreases iron absorption by 51% to 74%, a considerable
fact for Mexican population who's basic food is rich in cereals. The aim of this work was to analyze the iron
contents of Octopus hubbsorum (Cephalopoda: Octopodidae) an animal reported at all mexican Pacific coast. Tree
specimens where analyzed, two from the north and one from central Pacific Ocean. Data obtained for organism of
central Pacific Octopus 0.008% and data for North Pacific was 0.00% and 0.011%. In conclusion, the aport of Iron
to the mexican diet provided by Octopus hubbsorum is significant, therefore is recommended to consume more of
this resource, to diminish iron deficiency, a world-wide problem and to improve human health.
117
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 056
AREA: MIEH
POSTER
Interactions of Mo(VI) oxyanions with environmentally relevant metal cations in natural
ground waters
Gonzatto, L.1; Tissot, F.2; Torres, J.1; Kremer, C.1; Kremer, E.1
1
Cátedra de Química Inorgánica, Facultad de Química, Montevideo, Uruguay
2
Cátedra de Química Analítica, Facultad de Química, Montevideo, Uruguay
[email protected]
Molybdenum is an essential trace element, whose mobility in the upper crust is highly dependant on the
hydrological cycle. In natural waters, speciation and concentration of the main anionic components are important in
determining its bioavailability and environmental impact. Its chemical speciation in natural aquatic scenarios has
been yet poorly studied. This work reports the potentiometric study of oxyanions of Mo(VI) and their interaction with
some divalent metal ions of special environmental relevance.
Experimental conditions were chosen to simulate the natural ground waters media of low depth (20.0 C, low ionic
2-
strength). The influence of the supporting electrolyte was also evaluated. In the absence of metal ions, MoO4 is
the predominant species for pH values above 4.4, HMoO4- predominates from pH 3.6 to pH 4.4and H2MoO4 is the
most abundant species below pH 3.6. Polymeric species such as [Mo8O26]4- and [Mo7O-]6- appear when the total
molybdenum concentration exceeds 1 mM.
The M
2+
cations are bound to molybdenum anions to a considerable extent. For total molybdenum concentration
below 1 mM, the predominant form of molybdenum (VI) is still MoO42- above pH 4.4, with a variable percentage of
[M(MoO4)]+, depending on the metal ion concentration. As the pH value becomes lower than 4.4, the most
-7
abundant species is [M(HMoO4)]. For example, for 10 M Mo(VI) in the presence of mean natural ground waters
concentrations of Ca2+ (16.3 mM) shows the predominance of [Ca(MoO4)]for pH values above 4.4, and
+
[Ca(HMoO4)] for more acidic media. For some transition metal ions, the low solubility of the MMoO4 salt prevents
the accurate detection of the soluble species [M(MoO4)] above pH 4.4.
Results show that in conditions simulating natural ground waters, the availability of Mo(VI) is strongly influenced by
pH media and the presence of +2 metal ions.
Aknowledgments: This work was partially supported by CSIC.
118
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 061
AREA: MIEH
POSTER
Metal ions binding and extraction using functionalized magnetic nanoparticles
Mendoza, C.1; Pericàs, M.2; Vilar, R.3; Jansat, S.2
1
DEC, Facultad de Química, Universidad de la República, Montevideo, Uruguay
2
Institute of Chemical Research of Catalonia (ICIQ), Tarragona, España
3
Department of Chemistry, Imperial College London, United Kingdom, London
[email protected]
Magnetic nanoparticles are being widely applied in catalysis, cellular delivery systems, MRI and ion recognition and
sensing1. They offer the advantage that their movement can be controlled as required by the application of an
external magnetic field.
These nanoparticles, functionalized with ligands that have the ability to selectively bind metal cations could be
applied in metal recovery process and decontamination of water samples or biological fluids. The ligand
coordinates the cations and the nanoparticles are then removed from the solution by the application of an external
magnetic field2-3.
Copper (I) catalyzed 1,3-dipolar cycloaddition of an azide and an alkyne (click chemistry) has received broad
attention for its convenient characteristics as high yields and no by products, and it has been employed in the
functionalization of a variety of materials: polymers, silica, or nanoparticles.
In this work click chemistry was used to anchor crown ether- or monoaza-crown ether- type macrocycles onto
azide-magnetite nanoparticles. The obtained nanoparticles (1) were characterized by infrared spectroscopy,
thermogravimetric analysis, TEM microscopy and microanalysis. They were used in the extraction of heavy metals
from aqueous solutions. Metal concentrations were determined by flame atomic absorption spectroscopy,
achieving high extractions percentages. The same binding moieties were attached to a variety of polystyrene (PS)
resins, and the resulting functional polymers have also been used for the selective removal of cations from
aqueous and organic solvents solutions (2). The merits of these two extraction systems for particular situations are
discussed.
References
1. Lu, A.-H.; Salabas, E. L.; Schüth, F., Magnetic nanoparticles: synthesis, protection, functionalization, and application. Angew
Chem Int Ed; 46; 2007; 1222-1244.
119
12th International Symposium on Metal Ions in Biology and Medicine
2. Wang, L.; Yang, Z.; Gao, J.; Xu, K.; Gu, H.; Zhang, B.; Zhang, X.; Xu, B., A biocompatible method of decorporation:
biophosphonate-modified magnetite nanoparticles to remove uranyl ions from blood. J Am Chem Soc; 128; 2006; 13358-13359.
3. Koehler, F. M.; Rossier, M.; Waelle, M.; Athanassiou, E. K.; Limbach, L.K.; Grass, R N.; Günther, D.; Stark, W.J., Magnetic
EDTA: coupling heavy metal chelators to metal nanomagnets for rapid removal of cadmium, lead and copper from contaminated
water. Chem Commun; 2009; 4862-4864.
120
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 082
AREA: MIEH
POSTER
Manipulation of metalloproteins for heavy metal bioremediation and detection
Zhao, J.1; Wei, W.2
1
School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, China
2
School of Life Sciences, Nanjing University, Nanjing, China
[email protected]
A gold-specific sensory protein GolS from Samonella gol regulon was incorporated into E. coli, which in conjunction
with an engineered downstream red fluorescence protein allowed the highly sensitive and selective whole-cell
detection of gold(III) ions by naked eyes. The putative gold-chaperone, GolB, in the gol regulon was next verified to
be specific to gold(I) ions over other metal ions including copper(I). The subsequent display of GolB on E. coli cell
surface permitted selective enrichment of gold ions from media containing various thiophilic metal ions. The cell
surface- enriched gold(I) was further shown to be easily recovered and the gold-deprived bacteria were capable for
re-usage. E. coli bacteria harboring these gold-specific elements from the gol regulon could be a valuable tool for
visual detection and facile recycling of gold ions from environmental resources. A similar approach to lead ions will
also be discussed.
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12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 110
AREA: MIEH
POSTER
Mechanisms of cell death induced by cytochrome c metal-substituted species
Prieto, T.1; Smaili, S.2; Nascimento, O.3; Nantes, I.1
1
Centro de Ciências Naturais e Humanas, Universidade Federal do ABC, Santo André, Brazil
2
Farmacologia, Universidade Federal de São Paulo, São Paulo, Brazil
3
Instituto de Física, Universidade de São Paulo, São Carlos, Brazil
[email protected]
Cytochrome c is a mitochondrial respiratory hemeprotein that participates of respiratory chain, elimination of
superoxide ion and apoptosis. The latter role is played by the protein in the cytosol as resulting of endogenous
mobilization or exogenous deliver to cells via microinjection or cell carriers. In the present study, apoptosis was
induced by the delivery of exogenous metal-substituted cytochrome c species and microperoxidases (hemepeptide
product of cytochrome c proteolytic digestion) to rabbit aortic smooth muscle cells. The delivery vehicles were
microinjection and dioctadecyl dimethylammonium bromide (DODAB) liposomes. It was observed that only zincsubstituted cytochrome c that retains the native cytochrome c structural features was efficient to activate caspase3. Although unable to active caspase 3, free base-, Fe3+- and Mn3+ -microperoxidases-11 as well as free basecytochrome c led to the occurrence of typical events of the apoptosis program, such as: nuclear fragmentation,
membrane blebbing and extracellular exposure of phosphatidylserine. In addition, it was also observed that the
delivery of free base and native iron cytochrome c led to lose of cellular adhesion simultaneously to the induction of
cell death. DODAB liposomes loaded with heme-peptides and proteins were less efficient than microinjection to
induce cell death. These results suggest that protein structure and surface rather than the redox center determine
the efficiency of cytochrome c to activate caspases, however the peroxidase activity of the redox centers promote
oxidative stress that could induce apoptosis via endogenous cytochrome c mobilization.
Aknowledgments: Supported by FAPESP and CNPq
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12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 113
AREA: MIEH
POSTER
Correlations among water, blood and hair lead concentrations in first-grade children
from Uruguay
Martínez Savio, G.1; Mañay, N.1; Guido, M.2; Kordas, K.3; Queirolo, E.4
1
Catedra de Toxicología e Higiene Ambiental, Facultad de Química (UdelaR), Montevideo, Uruguay
Catedra de Toxicología e Higiene Ambiental, Facultad de Química(UDELAR), UCUDAL, Montevideo, Uruguay
3
UCUDAL, Pensilvania, Estados Unidos
4
UCUDAL, Montevideo, Uruguay
[email protected]
2
Over the last decade, environmental lead exposure and its impact on public health have been one of the biggest
concerns in Uruguay, mainly within the low income populations. Lead does not play any physiological role in
humans. However, often significant body lead burdens are present in humans, especially children, in relation to the
recommended limit values. Children’s exposure to lead, depending on the concentration, can be related to
physiological disorders, intellectual impairment or behavior problems. Therefore, it is important to monitor lead
concentrations in children’s environment to understand its relationship to the accummulation of the metal in
chidlren’s bodies.
The aim of this study was to measure and correlate lead levels in biological and environmental samples in firstgrade Montevideo children (N=134, ages 6-7 years). Lead children’s whole bood, in household water used for daily
consumption, and children’s hair was measured using atomic absorption spectrometry and ICP-MS, respectively.
Mean blood lead concentrations (BLL) in the study sample were: (4,9 ±2.2) µg/dL (range 1,80-13,2), Pb-hair (3,5 ±
3.9) µg/g (range 0,17-23,8) and Pb-Water (4,6 ± 10.6) µg/L (range 0,10-57,2). 31.3 % of the children have BLL > 5
µg/L, 10.4 % of the household water was > 10 µg/L, and 25.4 % children had > 3.9 µg/g of Pb in hair. SPSS was
used for this Exploratory Data Analysis (EDA). None of the three variables studied were normally distributed,
therefore bivariate correlations for pairs of variables performed using Spearman Rank Correlation Test.
Hair and water Pb levels were significantly correlated (p =0.04), p<005. However there was no significative
correlation between Pb in water and Pb in blood (p > 0,05), and neither between Pb in hair and BLL.
Lead in blood represents environmental exposure over the short term while lead in hair may represent longer-term
exposure. Thus, the contribution of lead in water as an environmental health risk should be taken into account for
further studies.
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12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 122
AREA: MIEH
POSTER
Assessment of environmental exposure to methyl mercury in polish women of
childbearing age
Trzcinka-Ochocka, M.; Brodzka, R.
Biological Monitoring Laboratory, Nofer Institute of Occupational Medicine, Lodz, Poland
[email protected]
Background: Methyl mercury (MeHg) is well–documented neurotoxicant and primarily affects the central nervous
system. The main exposure pathway of Hg to humans is through the consumption of marine fishery products. The
MeHg exposure of 306 occupationally non-exposed women of childbearing age, inhabitants of three Polish regions,
with different levels of fish consumption: Lodz (n=100), Wladyslawowo (n=107) and Gizycko (n=99) was assessed.
Methodology: Total mercury concentrations in hair (THg-H) was determined by flow-injection cold vapor atomic
absorption spectrometry technique using Mercury Analizer after mineralization of hair samples in microwave
digestion system. Detection limit (DL 3s) of method is 0.052 µg/g and quantification limit (QL) 0.104 µg/g. Accuracy
of the method was verified by the using certified reference materials: BCR CRM 397 (accuracy = 0.8%) and NCS
DC 73347 (accuracy = 13.89%).
Results: The results showed that the geometric mean concentrations of THg-H among women of Lodz were
statistically significantly lower 0.114 ± 2.02 µg/g (2.6 fish/month) than in the other two regions (p<0.05). The
concentrations of THg- H of women in Wladyslawowo and Gizycko increased in proportion to the consumption of
fish per month and amounted 0.177 µg/g ± 2.89 (6.5 fish/month) and 0.235 µg/g ± 2.17 (10.4 fish/month)
respectively. We found a statistically significant positive correlation (p<0.05) between THg-H concentration and the
number of fish meals/month in all examined regions: for Lodz, y = 0.0087 x + 0.1223, r = 0.226; for Wladyslawowo,
y = 0.0215 x + 0.1456, r = 0.464, and for Gizycko, y = 0.0151 x + 0.1727, r = 0.433.
Conclusions: The obtained results indicated that THg-H were low but in accordance with data quoted in the
current literature. The hair mercury concentration increased with increasing of fish consumption.
124
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 040
AREA: NATME
POSTER
Trace elements in soybean seeds and their correlation with the in vitro quality
parameters
Machado, I.1; Cardoso, J.2; Irigoyen, J.O.3; Viera, I.2; Pistón, M.1*; Torre, M.H.2
1
Cátedra de Química Analítica, DEC, Facultad de Química, UdelaR, Montevideo, Uruguay
Cátedra de Química Inorgánica, DEC, Facultad de Química, UdelaR, Montevideo, Uruguay
3
Agropecuaria Valdense S.R.L., Colonia, Uruguay
*[email protected]
2
Soybean is an important food source in the world. To ensure a good harvest, country governments recommend the
evaluation of seed quality using several in vitro tests for viability and vigor, properties related to the potential for
rapid, uniform emergence, and development of normal seedlings under a wide range of field conditions. However,
the results of these in vitro tests do not always fully correlate with the actual development of plants in the crops. On
the other hand, it is well known that mineral content in plants and seeds are fundamental for plant growth and
health.
As a part of our studies, Fe, Cu, Zn, Mn and Ni levels in soybean seeds were assessed and correlated with vigor
and viability tests done on the same seeds, with the aim of understanding the poor performance observed for some
of them.
Sample treatment was performed according to AOAC 975.03. The trace elements were determined by Flame
Atomic Absorption Spectrometry in 10 representative lots of seeds with good and bad viability.
To ensure the accuracy a reference material was also analyzed (NIST 1567a). Analytical precision was better than
-1
-1
6%. Soybean seeds with viability higher than 95% presented ranges of: 47.0 - 90.6 mgkg , 10.7 - 15.0 mgkg , 33.9
- 51.5 mgkg-1, 19.5 - 24.1 mgkg-1, 3.9 - 5.8mgkg-1 for Fe, Cu, Zn, Mn and Ni respectively.
These results are in accordance with the reported ranges in the literature for soybean seeds. No significant
difference in metal level was observed between seeds with viability less than 95% and those with higher viability,
except for Zn levels that were higher in the former. On the other hand, a negative correlation between vigor results
and Fe levels was observed.
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12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 046
AREA: NATME
POSTER
Escamoles ant eggs Liometopum apiculatum M source of metal ions for human health
Melo Ruiz, V.1; Quirino, T.1; Macín, S.1; Calvo, C.2; Muñiz, I.1
1
Division de Ciencias Biologicas y de la Salud, Universidad Autónoma Metropolitana Unidad Xochimilco, Ciudad de México,
México
2
Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Ciudad de México, México
[email protected]
The earth crust is believed to be made of a mass of minerals and the participation of these elements in the physical
world is matched by their importance in human life. The human body like other leaving organisms depend on
several minerals as essential constituents of its existence. Metal ions in foodstuff are in different chemical forms, as
inorganic salts or organic molecules or complexes with other compounds such as proteins, amino acids, enzymes
and some vitamins, among others, that play an important role in human health. Entomophagy, insect consumption
by several ethnic groups in Mexico as cultural tradition since prehispanic era represent an option for population to
obtain the minerals needed by the body to keep a good health. Escamoles ant eggs of the Liometopum apiculatum
M genus, much appreciated either at rural communities as well as in urban cities, contain minerals with a favorable
effect in human health. The aim of this study was to investigate the mineral composition of Escamoles and the
benefits they can provide to the human body. Sampling was perform at an arid region of the Hidalgo state on April
2012. Minerals in dry basis were determined by Atomic Absorption Spectrophotometry, with the exception of
phosphorus, content was obtained from a triple acid digested extract and determined colorimetrically. Data of
mineral analysis in dry basis of Escamoles was: total minerals 5.92%; Na 0.079%; K 0.075%; Ca 0.097%; P
0.701%; Fe 0.021%; Zn 0.035%; Cu 0.009%; Mg 0.998%; Mn 0.002%. Minerals quantify are not equal to total ash
contained because not all of them were determined. Elements concentration depends not only of the total mineral
composition of foodstuff but also on their availability and lability on it. Minerals in Escamoles ant eggs have a
considerable influence in the condition of human health.
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12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 058
AREA: NATME
POSTER
Effect of zinc supplementation on the production of IL-17 in elderly individuals
Aguilar Cardenas, A.E.1; Sanchez, M.T.C.1; Muñoz, B.1; Espejel, G.2; Saldivar, L.2; Pastelin, R.1
1
Department of Biology, University of Mexico, Mexico DF, Mexico
Department of Analytical Chemistry, University of Mexico, Mexico DF, México
[email protected]
2
The suboptimal contribution of micronutrients may damage the immune response. In the particular case of zinc, this
microelement has shown their participation in various immunological processes around perinatal stages. In the
elderly, eating lesser amounts of food can cause a negative balance in the concentration of zinc and its possible
effects on immunity. In this regard, it is interesting to know the impact of zinc may have on proinflammatory
cytokines such as IL-17 for its role in delayed hypersensitivity and its possible effect in the induction of
autoimmunity. This study aimed to evaluate the effects of zinc supplementation on the production of IL-17 over
elderly stages. For this, we used an experimental model of zinc supplemented mice (500 microgram/mL zinc
acetate, BALB/c inbred mice). We measured the microelement concentration in lymphoid organs by Atomic
Absorption Spectrometry and also the cytokine production in serum by double antibody immunoenzime assay.
Results showed that female group of supplemented mice diminished the cytokine production 34.35 % compared
with control group whereas male group of supplemented animals showed a noticeable reduction of 54.34 %.
Further, zinc concentration increased in the spleen of female supplemented mice (80%). We concluded that
supplementation of zinc in mice has the effect of causing decreased production of IL-17 by Th17 cells, which far to
produce a problem of autoimmunity this means a regulatory response to supplemented individuals.
127
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 074
AREA: NATME
POSTER
Star fish source of calcium to prevent this metal ion deficiendy
Palacios, J.; Melo, V.; Quirino, T.; Dominguez, O.; Schettino, B.
Universidad Autónoma Metropolitana, Xochimilco, México
[email protected]
Calcium is the most abundant mineral in the body. It forms a functional reserve during mineralization of bone and is
required by essentially all body metabolic processes1. Therefore finely harmony homeostatic control mechanisms
to maintain constant blood levels of this mineral should be studied, since, it has complex cellular mechanisms to
control movement of intracellular calcium. Inadequate calcium consumption might cause on increased risk of
several diseases. Scientific community think Star fish (Echinodermata: Asteroidea) is high in minerals mainly in
calcium due to the calcareous osicles that forms the animal's body. Available all year, sometimes is consumed by
residents along the Atlantic coast. The objective of this research is to assess calcium content to inform people the
benefits of an adequate intake can provide in human health. Starfish obtained in Acapulco Bay last August was
analyzed by tirtation with EDTA method. Data obtained was 1.3%. In conclusion starfish is a good calcium source
to reduce the risk to several diseases such as osteoporosis, hypertension, cardiovascular dysfunction, cancer and
kidney stones in human health, it can also be an alternative food source of calcium for lactose intolerant
people2,3,4,5,6,7. Consumption is highly recommended suggesting no excessive calcium intake to avoid adverse
effect for human health and it's consumption is highly recommended. It can also be an alternative food source of
calcium for lactose intolerant people.
References
1. Crabb E. & Moore E (Ed.) Metals and life. RscPublishing 2010
2. Gebhardt L. & Silva E. Dairy augumentation of total and central fat loss in obese subjects. Int Jobes Relat Metab Disord.
2005 Vol 29 pp 391-397
3. Dawson-Hughes B. Interaction of dietary calcium and protein in bone health in humans. J Nutr. 2003 Vol 133 pp 8525-8545
4. Wu K., Willett W.C., Fuchs C., GOlditz G., Giovanuchi E. Calcium intake and risk of colon cancer in women and men. J Natl.
Cancer Inst. 2002 Vol 94 pp 437-446
5. Heaney R. Calcium diary products and osteoporosis. J am Coll Nutri, 2000 Vol. 19 pp 835-995
6. Heller J. The role of calcium in the preention of kidney stones. J Am Coll Nutr. 1999 Vol 19 pp 3375-3785
7. Bucher H., Cook R., Guyatt G., Lang J., Hatala R., Hunt D. Effects of dietary calcium supplementation on blood pressure: a
metaanalysis of randomized controlled trials. JAMA 1996 Vol 275 pp 1016-1022
128
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 116
AREA: NATME
POSTER
Preliminary study of superoxide dismutase activity in soybean seeds with good and poor
in vitro viability used in Uruguay
Cardoso, J.1; Machado, I.2; Irigoyen, J.O.3; Pistón, M.2; Viera, I.1; Torre, M.H.1*
1
Química Inorgánica, DEC, Facultad de Química, UdelaR, Montevideo, Uruguay
2
Química Analítica, DEC, Facultad de Química, UdelaR, Montevideo, Uruguay
3
Agropecuaria Valdense (J.J.Ferreira), Colonia, Uruguay
[email protected]
Reactive oxygen species (ROS) such as superoxide and hydroxyl radicals are present in seeds, plants and animals
as a result of normal aerobic metabolism. If the organisms are not able to control the intracellular ROS level, they
may suffer damage in membrane lipids, proteins and nucleic acids leading to the death of the cells. For
detoxification of ROS, efficient antioxidant systems both enzymatic and non-enzymatic ones are present in all cells.
Superoxide dismutases (SOD) can react with O2- radical to prevent the damage. They are metalloproteins where
Cu, Mn, Zn or Ni are linked to the peptide residues. Therefore when the metal levels are low the activity of these
enzymes decrease and different disorders can appear.
The SOD extraction from seeds was optimized by means of multivariate experiments (best conditions: stirring time
60 min using 10 mL of phosphate buffer pH=7).
The SOD activity was determined by the method based on the inhibitory effect of SOD over the reduction of NBT
by the O2- generated by xanthine/xanthine oxidase system.
SOD activity of soybean seeds used in our country was in the range 20-63 %, similar in all the analyzed seeds. The
results showed that no significant difference was observed in seeds with viability less than 95%. In these
conditions, the poor viability of soybean seeds used in our country is not explained by oxidative stress.
129
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 024
AREA: RMNM
POSTER
α-MSH analog cyclized through rhenium coordination as new potential agent for
melanoma imaging
Teixeira, V.1; Fernadez, M.1; Moreno, M.2; Chabalgoity, J.A.2; Gambini, J.P.3; Porcal, W.4; Quinn, T.5; Cabral, P.1
1
Radiofarmacia, Facultad de Ciencias, UdelaR, Montevideo, Uruguay
Biotecnología, Facultad de Medicina, UdelaR, Montevideo, Uruguay
3
Medicina Nuclear, Facultad de Medicina, UdelaR, Montevideo, Uruguay
4
Química Orgánica, Facultad de Química, UdelaR, Montevideo, Uruguay
5
Biochemistry, University of Missouri, Columbia, USA
[email protected]
2
Background: α-Melanocyte stimulating hormone (α-MSH) analogs, cyclized through site-specific rhenium metal
coordination (ReCCMSH) present high chemical stability and affinity for the MC1R receptors of melanoma cells. In
this way the radiolabelled ReCCMSH is a potential radiopharmaceutical for melanoma diagnosis and treatment.
99m
Tc is the radioisotope most used in nuclear medicine diagnostic imaging owing to its optimal nuclear properties,
ready availability, low cost and favourable dosimetry. The bifunctional chelators tetraamine (N4) forms octahedral
complexes with
99m
Tc as [Tc(V)O2](tetraamine)+.
99m
Tc-tetraamine (N4)]ReCCMSH, presented herein, and can be
used as a potential MC1R-targeting radiotracer based on
99m
Tc for non invasive in vivo imaging of melanoma.
Methodology: the synthesis of the peptide N4-ReCCMSH was performed in an automated peptide synthesizer. N4ReCCMSH was radiolabeled with pertechnetate in alkaline conditions (pH 11.5) in presence of citrate and stannous
chloride as reduceding agent. Radiochemical evaluation of conjugates, as well as studies of stability and partition
coefficient was performed. Biological studies included binding, internalization, blocking studies at 30, 60, 90 and
120 minutes in B16-F1 mouse melanoma cells, biodistribution at 1, 2 and 24 hours in normal mice and in tumorbearing mice (C57 BK with B16-F1 mouse melanoma cells).
Results: The radiolabeling was efficiently performed at 37ºC in 30 minutes.
99m
Tc-N4ReCCMSH was obtained with
a purity >99% and was stable up to 24 hours post incubation. The cell studies exhibit high binding, fast
internalization and inhibition of 70% against unlabeled peptide. Biodistribution studies in mice demonstrated a
favourable pharmacokinetics with rapid urinary excretion, very low hepatobiliary elimination, combined with a high
1
and specific uptake in the tumor ( H, 8% ID/g)
Conclusions: these promising preclinical results of
translation as a novel
99m
99m
Tc-N4ReCCMSH warrant its potential candidature for clinical
Tc based in vivo non invasive diagnostic melanoma agent.
Acknowledgements: ANII, PEDECIBA QUIMICA and CHLCC.
130
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 028
AREA: RMNM
POSTER
Development and optimization of 177Lu-DOTA-Tocilizumab: a potential Multiple Myeloma
theragnostic agent
Camacho, X.1; Calzada, V.1; Gutiérrez, E.1; Fernández, M.1; Moreno, M.2; Chabalgoity, J.A.2; Riva, E.3; Cabral, P.1
1
Nuclear Research Centre, University of the Republic, Faculty of Sciences, Montevideo, Uruguay
2
Institute of Hygiene, University of the Republic, Faculty of Medicine, Montevideo, Uruguay
3
Clinical Hematology, University of the Republic Faculty of Medicine, Montevideo, Uruguay
[email protected]
Antecedents: Interleukin 6 (IL-6) is one of the key molecules related to growth, survival and proliferation of Multiple
Myeloma (MM) cells. Tocilizumab is a humanized monoclonal antibody directed against the receptor both soluble
and membrane of IL-6. Lutetium is a lanthanide element, in the f-block of the periodic table. Its only common
oxidation state is +3.
177
Lu is a ß-particle emitter (497 keV, 90%), γ-rays (113 and 208 keV, 6% and 11%) and half-
life of 6.7 days. Tocilizumab labeled with 177Lu could be a potential MM theragnostic agent.
Methodology: Tocilizumab was purified by PD-10 column equilibrated and eluted with NaHCO3 0.1 M, pH=8.5 and
was derivatized with DOTA-NHS at 4ºC for 18 h. DOTA-Tocilizumab was radiolabeled with 177LuCl3 (15 MBq/mg) at
37ºC for 1 h. MALDI TOF/TOF was used to determine the level of DOTA conjugation to Tocilizumab.
Radiochemical purity was determined by ITLC-SG and by HPLC. In-vitro stability of
177
Lu-DOTA-Tocilizumab was
studied in solution, serum, DTPA and EDTA. In-vitro studies binding and competition were performed with U
266
MM
cells up to 120 minutes. Biodistribution studies were performed in normal Balb/C mice at 24 and 48 h (n = 5).
Results: DOTA-Tocilizumab was efficiently labeled with
177
LuCl3. The in-vitro stability of labeled product was
optimal over 72 h in solution and serum, being stable and don’t showing significant transchelation. In-vitro
experiments binding and displacement confirm the specificity of recognition of
177
Lu-DOTA-Tocilizumab by IL6R.
Biodistribution studies showed hepatic uptake and renal elimination.
Conclusions:
177
Lu-DOTA-Tocilizumab was easily and rapidly labeled. This is a potential new agent for MM
theragnostic agent.
Acknowledgements: ANII, Roche Laboratories, Pro.In.Bio, PEDECIBA Química.
131
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 031
AREA: RMNM
POSTER
Validation of an atomic absorption spectroscopic method for the quantification of copper
in tetrakis (2-methoxy isobutyl isonitrile) copper (I) tetrafluorborate
Leonardi, N.1; Fuda, J.2; Torti, H.2; Molinari, M.3; Zubata, P.4; Salgueiro, J.1; Zubillaga, M.1
1
Radioisotopes Laboratory, Physics Department, School of Pharmacy and Biochemistry, Buenos Aires, Argentina
2
Physics Department, School of Pharmacy and Biochemistry, Buenos Aires, Argentina
3
Quality Assurance, Bacon Laboratories, Buenos Aires, Argentina
4
Technical Direction, Bacon Laboratories, Buenos Aires, Argentina
[email protected]
Antecedents: The quantification of tetrakis (2-methoxy isobutyl isonitrile) copper (I) tetrafluorborate (CuTFB) is an
important issue when measuring samples of Sestamibi, a common radiopharmaceutical for cardiac imaging. The
aim of this work was to validate the copper determination in CuTFB by means of an atomic absorption
spectroscopic (AAS) methodology to be use as an indirect quantitative method for CuTFB.
Material and method: Validation characteristics assayed were linearity, precision, accuracy, specificity and range
according to the ICH Q2. All CuTFB and copper standard solutions were prepared using distilled water and
processed in order to be measure by AAS.
Results: The method proved to be linear showing a correlation coefficient (R2) for linearity of 0,9962. In precision
studies the results obtained for repeatability were 0% and for intermediate precision were 0,2%. There was no
significant difference for both analysts working under different conditions (different days). For accuracy, the
recovery percentages were between 99,5 % and 101,1 %. In specificity studies there were no noticeable
interferences of tin in copper determination. In robustness studies there was a lack of influence of operative
variables studied compared to results obtained without variables. For the purpose of this methodology, the
specified range is between 50 µg and 1,5 mg of CuTFB.
Conclusion: The results obtained showed that the proposed method for the quantification of CuTFB, by means of
copper detection in AAS, proved to be linear, precise, accurate and specific.
References: International Conference on Harmonization (ICH Q2), version 4, 2005.
Acknowledgments: This work was financial supported by project UBACYT 20020100100489 from the University
of Buenos Aires.
132
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 038
99m
AREA: RMNM
POSTER
Tc labelling of phenazine dioxides with recognized DNA interaction capacity. In vitro
and in vivo studies
Tejería, E.1; Berchesi, A.2; Fernández, S.1; Sanz, I.1; Cerecetto, H.2; González, M.2; Lavaggi, M.L.2; Rey, A.1
1
Cátedra de Radioquímica, Facultad de Química, UdelaR, Montevideo, Uruguay
Grupo de Química Medicinal, Facultad de Ciencias, UdelaR, Montevideo, Uruguay
[email protected]
2
Phenazine dioxides are drugs used for antitumor therapy that exhibit DNA-interaction and produce DNA-damage.
We have selected two of them that have demonstrated good in vitro DNA interaction, namely ML44 (2aminophenazine N,N’-dioxide) and ML84 (2- amino-7-fluorophenazine N,N’-dioxide) and we herein present their
labeling with
99m
Tc- and their evaluation as potential radiopharmaceuticals. Labeling through formation of
tricarbonyl complexes, was performed in two stages: 1) preparation of the precursor fac[
99m
99m
Tc(I)-
Tc(CO)3 (H2O)3]+ by
sodium pertechnetate reduction (40-50 mCi, 1.0 mL) with sodium borohydride (7.0mg) under CO (g) atmosphere, in
alkaline medium at 70°C, 20 minutes, 2) substitution with phenazine: ML44 or ML84 (2-3 mg in 0.5 mL of DMSO,
30 minutes at 70°C). The radiochemical purity (RP) of precursor and the final compounds were controlled by
reverse phase HPLC. The precursor showed a retention time (rt) of 4 min and a RP > 90 %. The labelled
phenazines had a rt of 21 and 22 min., respectively and a RP > 95 % and were stable for at least 4 h. Both
complexes were 100 % stable in plasma at 37°C. The
histidine solution (100-fold excess), while the
99m
99m
Tc(CO)3 -ML44 complex showed 100% stability against
Tc(CO)3 -ML84 complex suffered a 15 % transchelation. The
lipophilicity (partition coefficient octanol:phosphate buffer pH = 7.4) was log P = 0.12 ± 0.07 and 0.50 ± 0.06,
respectively. The plasma protein binding (PPB) was 35.2 ± 4.5% and 54.0 ± 0.9%, respectively. Attempts to reduce
the PPB through formation of more stable 2+1 complexes using acetylacetone as bidentate coligand were
unsuccessful. Biodistribution at 30 and 120 minutes in normal mice (females, CD1, 3 months, 3 animals per group)
showed high liver uptake: 28.1 ± 2.9% and 35.9 ± 3.6% at 2 h, respectively, consistent with the high PPB. Excretion
occurs mainly via the hepatobiliary tract and uptake in other organs is low. Studies in tumor bearing mice will
determine its potentiality as radiopharmaceuticals for Nuclear Oncology.
Acknowledgements: CSIC (Proy. 622), ANII, Pedeciba-Química
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12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 094
AREA: RMNM
POSTER
Value of 99mTc in the characterization of an anti-Tn chimeric antibody
Vasilskis, E.1; Trindade, V.1; Giglio, J.1; Reyes, L.1; Oliver, P.1; Balter, H.1; Osinaga, E.2; Engler, H.1
1
CUDIM, Montevideo, Uruguay
Medicine Faculty, Montevideo, Uruguay
[email protected]
2
Tc (I)-Tricarbonyl complexes synthesis leads to stable precursors in aqueous solution and wide pH range, useful in
the labeling of biomolecules by ligand exchange. Carboboranes as reducers and CO groups source in situ, allow
synthesize [
99m
+
Tc(CO)3(H2O)3] , as intermediary complex. H2O molecules in carbonyl complex have adequate Kd
for the exchange and transference to protein and peptides through histidine residues.
We report here the labeling optimization of the monoclonal chimeric anti-Tn with
99m
Tc through [
99m
+
Tc(CO)3(H2O)3]
complex, and its characterization by HPLC and ITLC-SG as well as biodistribution studies.
[99mTc(CO)3(H2O)3]+ was obtained by addition of 1mL of
99m
TcO4-(0.24-3.98 GBq) to the Isolink kit (given by
Mallinckrodt), 20m incubation at 100°C and controlled by RP-HPLC C18 column. Retention times (RT) were 3.96 ±
99m
0.70 min for [
99m
[
+
Tc(CO)3(H2O)3] and 12.70 ± 0.60 min for
99m
TcO- 4. Then 0.3- 2.0 nmol antiTn was added to
+
Tc (CO)3(H2O)3] , incubated 1 to 3 h at 37ºC and 45ºC to synthetize
99m
controlled by HPLC molecular exclusion, RT [
+
Tc (CO)3(H2O)3] : 5.6 min;
99m
99m
Tc(CO)3-anti-Tn (99mTcAb) and
Tc(CO)3-anti-Tn: 3.3 min;
99m
TcO4-
:11.8 min.
99m
Tc red-hidr was determined by ITLC-SG, pretreated with 5% BSA, in EtOH: NH4OH: H2O (2:1:5).
The radiolabeled antibody was purified by PD-10 gel filtration. Stability was assessed up to 48h post-labeling.
Biodistribution studies were done in Balb-C and NudehealthySPF at 4, 16 and 24h postintravenous injection of1 to
12MBq.
[99mTc(CO)3(H2O)3]+ yield was above 99%. Anti-Tn antibody was successfully labeled reaching 100% radiochemical
purity post purification. The antibody was stable up to 48h post labeling for up to 600 MBq/nmol.
Biexponential pharmacokinetics, with urinary (35.0±6.73; 50.8±3.28) and hepato-biliary (24.4±4.07; 28.9±4.93)
elimination was found for Balb-C and Nude mice, respectively.
Labeling through tricarbonyl complex formation is useful for anti-Tn labeling showing the typical profile of the
antibodies biological behavior.
134
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 105
AREA: RMNM
POSTER
Biological evaluation of two glucose derivates radiolabeled with 99m-Tc as potential
imaging agents for melanoma
Dapueto, R.1; Fernández, M.1; Gambini, J.P.2; Aguiar, R.3; Marques, F.4; Chammas, R.3; Cabral, P.1; Porcal, W.5
1
Radiofarmacia, Centro de Investigaciones Nucleares, Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay
2
Centro de Medicina Nuclear, Facultad de Medicina, Hospital de Clínicas, Montevideo, Uruguay
3
Radiologia e Oncologia, Facultade de Medicina, Universidade do Sao Paulo, Sao Paulo, Brazil
4
Centro da Medicina Nuclear, Facultade de Medicina, Universidade do Sao Paulo, Sao Paulo, Brazil
5
Departamento de Química Orgánica, Facultad de Química-Facultad de Ciencias, Universidad de la República, Montevideo,
Uruguay
[email protected]
Background: Malignant tumors can be detected with high sensibility by imaging the increased metabolism of
glucose, aminoacids and lipids. GLUT´s over- expression in tumor cells allows the detection of several cancer
types, using 18FDG with PET images, worldwide. Glucose derivates radiolabeled with
99m
Tc had been widely
studied to obtain a 18FDG analogue that substitute or complement 18FDG-PET images with SPECT.
Methodology: Two glucose derivatives, 11 and 2 (Figure 1) were synthesized. Radiolabeling was achieved by
99m
adding 5-20 mCi of Na
-
TcO4 to the corresponding compound and SnCl2 as reducing agent. After agitation, the
radiochemical purity was controlled using paper chromatography and HPLC. In vitro internalization was
accomplished incubating the radiolabeled compounds with B16F10 melanoma murine cells for 30, 60 and 120
minutes at 37ºC and 4ºC. Competition studies in vitro with 18FDG and the unlabeled compounds were done.
Images with C57BL6 mice bearing B16F1 melanoma were acquired at 60 and 120 minutes post- injection of the
radiolabeled compound, using a gamma camera.
Figure 1. Glucose derivates 1 and 2 used for radiolabeling with 99m-Tc and biological study
Results and conclusions:
99m
Tc radiolabeling of 1 and 2 was accomplished with high radiochemical purity
(>98%). In vitro studies of the radiolabeled compounds showed low cell accumulation (0,2- 0,5 %) at both
incubation temperatures and both unlabeled compounds did not alter 18FDG internalization. In vivo studies with
B16F1 melanoma bearing mice showed high accumulation in the tumor and renal bioelimination. These results
suggests that different mechanisms could be occurring in the live animal: in vivo accumulation could be nonspecific
due to an interaction with biomolecules in the tumor micro-environment.
Acknowledgements: PEDECIBA - Química, ANII, Espacio Interdisciplinario-UdelaR, CMN-FMUSP.
135
12th International Symposium on Metal Ions in Biology and Medicine
References
1. Dapueto, R; Castelli, R; Fernández, M; Chabalgoity, J;Moreno, M; Gambini, J.P; Cabral, P; Porcal,W. Biological evaluation of
99m
+
Glucose and Deoxyglucose derivatives radiolabeled with [ Tc(CO)3(H2O)3] core as potential melanoma imaging agents.
Bioorg Med Chem Lett. 23, 7102-7106.
136
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 112
AREA: RMNM
POSTER
Preparation and biologic evaluation of glucose derivates bearing a thiol group
radiolabeled with 99mTc as potential diagnostic agents for cancer
Castelli, R.1; Fernández, M.1; Gambini, J.P.2; Aguiar, R.3; Marques, F.4; Chammas, R.3; Cabral, P.1; Porcal, W.5
1
Laboratorio de Radiofarmacia, Centro de Investigaciones Nucleares, Facultad de Ciencias, Montevideo, Uruguay
2
Centro de Medicina Nuclear, Hospital de Clínicas, Facultad de Medicina, Montevideo, Uruguay
3
Departamento de Radiologia e Oncologia, Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil
4
Centro de Medicina Nuclear, Hospital das Clínicas da Facultade de Medicina da Universidade de São Paulo, São Paulo, Brasil
5
Laboratorio de Química Orgánica, Instituto de Química Biológica, Facultad de Ciencias-Facultad de Química, Montevideo,
Uruguay
[email protected]
Antecedents: PET-CT with FDG is a very important technique among the several existing of imaging melanoma
1-3
and non-Hodking lymphoma (LNH) . Many research groups worldwide are working on the development of imaging
agents based on the expression of glucose transporters (GLUT `s) that are over-expressed in most tumor cells.
Therefore, the development of novel analogs of FDG for use in SPECT imaging, adapted to non-invasively and in
vivo evaluation of patients with melanoma and non-Hodgkin lymphoma is very desirable.
Methodology: We performed organic synthesis, purification and characterization of two new glucose derivates
which incorporated a thiol group for coordination with
99m
carbon of the glucose. Labeling was performed using
99m
Tc (Figure) through an ethylene linker in the anomeric
TcO4- in presence of SnCl2 as reductive agent. In vitro
internalization was carried out using murine cellular lines of LNH (A20) and melanoma (B16F10 and B16F1). In
vivo images were performed in mice C57BL6 carrying of melanoma and LNH, at different times, using a gamma
camera.
OAc
AcO
AcO
O
OAc
OH
O
SH
O
HO
HO
1
O
OH
SNa
2
hc
c
T
i
h 9
m
w 9
st
h
ei
t
aw
v
i
n
r
o
i
et
da
en
i
s
d
r
oo
c
o
uc
l
gr
wo
f
ep
nu
or
o
wg
t
fl
oi
o
h
et
r
ua
t
c
d
ue
r
t
t
a
Sr
.o
er
p
r
uo
gc
i
n
Fi
Results and conclusions: Labeling of the compounds 1 and 2 with
99m
Tc was obtained with high purity. In vitro
studies showed a moderate percentage of cellular cellular internalization for both compounds. In vivo studies
showed high tumoral uptake and renal elimination in both tumoral models.
References
1. Friedman, KP.; Wahl, RL. Clinical use of positron emission tomography in the management of cutaneous melanoma. Semin
Nucl Med. 2004, 34, 242-253.
2. Karantanisa, D.; Durskia, J.; Lowea, V.; Nathana, M.; Mullana, B.; Georgioub, E.; Johnstonc, P.; Wisemana, G. 18F- FDG
PET and PET/CT in Burkitt’s lymphoma. Europ J of Radiol. 2010, 75, e68–e73.
3. Ho Shon, IA.; Chung, DK.; Saw, RP.; Thompson, JF. Imaging in cutaneous melanoma. Nucl Med Commun. 2008, 29, 847876.
Acknowledgements: CSIC-UdelaR, AUGM, Espacio Interdisciplinario-UdelaR, CMN-FMUSP.
137
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 117
AREA: RMNM
POSTER
Clinical application of two neutral 99mTc radiopharmaceuticals in diagnosis of brain
pathologies
Crócamo, N.
Servicio de Medicina Nuclear Dr. Touya y Dr. Gaudiano, Montevideo, Uruguay
[email protected]
The aim of this paper is to present the experience in the preparation and clinical application of two neutral
radiopharmaceuticals,
99m
Tc-etilendicisteína (ECD) and
99m
99m
Tc
Tc-TRODAT. These radiopharmaceuticals cross the
intact blood-brain barrier and are used to image cerebral perfusion and dopamine transporters in
neurodegenerative lesions (such as Parkinson's), respectively.
The preparation of
N'-
(1,2
99m
Tc-ECD requires the use of a kit formulation: one vial containing dihidroclorhidric ester of N,
ethylendiethyl)
bis-L-cysteine
(ligand),
stannous
chloride
dihydrate
(reductant),
disodium
ethylendiaminotetraacetic acid dihidrated (stabilizer) and mannitol (inert filler); vial 2 containing phosphate buffer
and water to reach the proper pH. Labelling is performed by addition of
99m
TcO4-(A 50mCi) at room temperature for
45 minutes. The radiochemical purity (PR) determined by paper chromatography on Whatman 1 paper with ethyl
acetate is above 90%.
The
99m
Tc-TRODAT is obtained by adding sodium pertechnetate (A 50mCi) to a single vial containing TRODAT
(the ligand), stannous chloride dihydrate and calcium glucoheptonate, as intermediate ligand and heated at 100ºC
for 30 minutes. The PR is also greater than 90%.
Both radiopharmaceuticals are administered intravenously at a dose between 27 and 30mCi. The images are
obtained using a SPECT camera The acquisition time should be less than 30 minutes and 360 ° rotation following
the contour of the body.
For
99m
Tc-ECD the normal biodistribution pattern is increased uptake in cerebellum and occipital lobe and a nearly
uniform and symmetrical distribution in the rest of the brain. Hypoperfusion or hyperperfusion in some of these
regions are considered pathological.
For
99m
Tc-TRODAT, images are acquired 4 hours post-injection, Areas of interest are drawn around caudate
nucleus, putamens, striatum and occipital cortex. Decreased uptake of the radiotracer in any of the previous zones
may denote a neurodegenerative disorder.
Acknowledgements: Dr. A. Rey
138
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 004
AREA: SBMMBRP
POSTER
pH effects on the iron oxidation states of the extracellular hemoglobin of Glossoscolex
paulistus (HbGp)
Carvalho, F.A.O.; Tabak, M.
Química e Física Molecular, Instituto de Química de São Carlos, Universidade de São Paulo, São Paulo, SP, Brazil, São
Carlos, Brazil
[email protected]
HbGp is characterized by a molecular mass (MM) of 3.6MDa, a high resistance to oxidation and high oligomeric
stability. The present work has as the main focus to study the pH effect on the HbGp iron oxidation states and the
characterization of the species in alkaline medium, by optical absorption and analytical ultracentrifugation (AUC). At
pH 7.0, oxy-HbGp displays an intense Soret band at 415nm, and other two bands (Q bands) at 576 and 540nm,
while met-HbGp presents a Soret band at 403nm, and Q bands at 500 and 540nm. At pH 8.0, for oxy-HbGp, Soret
and Q bands λabs values have no significant changes, suggesting that in this case the heme group remains intact.
For met-HbGp, pH 8.0 induces significant changes in the absorption spectrum due to the formation of the
hemichrome species. The hemichrome is characterized by a Soret band centered at 413nm, and Q bands at 534
and 565nm. The changes in the metal coordination sphere induce other phenomena in the HbGp structure. Thus,
oxy-HbGp, at pH 8.0 and 9.0, is partially dissociated, with 88 and 56% of native protein contribution, respectively.
The sedimentation coefficient c(S) curves for the oxy- form at these pH values show three species in the
equilibrium: tetramer with sedimentation coefficient ( s 020,w ) and MM of 4.9S and 68.4kDa, dodecamer with s 020,w
and MM of 9.3S and 204kDa, and whole protein with s 020,w and MM of 58.4S and 3600kDa. Met-HbGp, at pH
values 5.0 and 7.0, has a single species in the c(S) distribution, attributed to the native protein, while, at pH 8.0,
only smaller species, of MM between 16 and 70kDa, are observed in equilibrium. Our results show that the HbGp
stability depends on the iron oxidation state and on the ligand coordinated in heme group.
Support: Brazilian agency FAPESP.
139
12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 078
AREA: SBMMBRP
POSTER
Effect of cationic surfactant (DTAB) on the HbGp iron oxidation at neutral and acidic pH
values
Alves, F.R.; Carvalho, J.W.; Oliveira Carvalho, F.A.; Tabak, M.
Química e Física Molecular, USP, São Carlos, Brasil
[email protected]
HbGp has a hexagonal bilayer structure, with a high molecular mass of 3.6MDa, and a high oligomeric stability. In
the absence of DTAB, oxy-HbGp presents characteristic absorbances at 415, 540 and 575nm. At pH 5.0, the
absorption spectrum remains unchanged up to 0.5mmol/L of surfactant, indicating the stability of the heme group
iron. The increase of DTAB concentrations, above 0.5mmol/L, induces a blue-shift of the Soret band from 413 to
405nm, as well as of Q bands from 540 and 575nm to 533 and 568nm, suggesting the formation of met-HbGp and
hemichrome species. The appearance of the LMCT band at 618nm is also observed above 1.0mmol/L DTAB,
indicative of aquomet-HbGp species. However, at pH 7.0, the presence of low DTAB concentration (in the range
0.1-0.5mmol/L) alters the absorption spectrum of oxy-HbGp drastically. DTAB concentration required for the
formation of aquomet-HbGp and hemichrome species is smaller at pH 7.0, as compared to pH 5.0, despite the
similarity of absorption spectra. The HbGp fluorescence emission, at pH 5.0, changes slightly less, in the presence
of DTAB, when compared with pH 7.0, increasing, roughly, 30-fold. The protein structural changes and oxidation of
the heme groups occur at smaller DTAB concentrations, at pH 7.0 as compared to pH 5.0, due to the acidic pI of
protein (5.5) that favors the oxy-HbGp-cationic surfactant interaction at pH 7.0. In conclusion, our present data
show that differences and similarities are noticed for DTAB and CTAC (Santiago et al. BBA 2007) effects upon
HbGp spectroscopic features.
Aknowledgments: Financial support: FAPESP and CNPq.
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12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 009
AREA: TEMIBS
POSTER
A particular approach of the correlation between metals and health: arsenic removal from
groundwater by Fe-rich geo-materials
Botto, I.L.1; González, M.J.2; Gazzoli, D.3; Soto, E.L.4
1
Quimica Universidad Nacional de La Plata, CEQUINOR Facultad de Ciencias Exactas, La Plata, Argentina
2
Facultad de Ciencias Exactas, Univ. Nacional de La Plata, CEQUINOR-INREMI, La Plata, Argentina
3
Dipartimento di Chimica, Universitá La Sapienza di Roma, Roma, Italia
4
Facultad de Ciencias Exactas, UNLP, PLAPIMU, La Plata, Argentina
[email protected]
Arsenic is a very toxic element, observed in some groundwaters at levels above those suggested by the WHO (10
-1
µgL ). The geo-genetic origin is responsible of serious health problems, particularly the pathology known as
Chronic Endemic Regional Hydroarsenicism (CERHA). In Argentina the arsenical grounwaters are consumed in the
big Chaco- pampean area, affecting more than 4 million people. In general, several technological removing
strategies were used, but the adsorption arsenate/metal-oxide seem to be the most appropriate treatment for areas
of low population density and/or low income. In our case, the method was supported by the abundance of mineral
species naturally rich in iron or possible to modify with this metal. Chemical modification treatment was carried out
by using solutions of Fe (III) salts, through a hydrolytic process and adjustment of pH, promoting the formation of
Fe-O-H2O active clusters (precursors of nanoscopic ferrihydrite). Alumino-silicates such as clinoptilolite,
pyrophylite, pyroclastic material and kaolinite (Fe2O3 content lower or close to 5%) and Fe-rich clay minerals (~40%
Fe2O3), have been analyzed. The performance of the process was studied by means of Jar tests and
physicochemical techniques (micro-Raman, XR diffraction, chemical analysis (ICP-AAS), scanning electron
microscopy (SEM-EDS) and surface (BET) assays). It was noted that a small increase in the iron content (2-10%)
correlates with an increase in mesoporosity and a marked increase in the arsenate(V) adsorption, measured from
the number of treatment cycles. During the process, scorodite precursor is formed, whose stability in the clay matrix
ensures the As isolation. The results revealed that the trigger alternative, dependent on structural and surface
mineral properties, constitute a low-cost, effective, accessible and eco-friendly technology. Hence, several
prototypes were successfully installed in rural and/or vulnerable localities of the affected region, producing water
with As values lower than 10µgL-1.
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12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 097
AREA: TEMIBS
POSTER
Line of research: Effects of multiple metals on cognitive ability and behavior in children
from Montevideo Uruguay
Queirolo, E.1; Barg, G.1; Mañay, N.2; Kordas, K.3
1
Facultad de Psicología, Universidad Católica del Uruguay, Montevideo, Uruguay
2
Facultad de Química, UdelaR, Montevideo, Uruguay
3
Nutritional Sciences, Pennsylvania State University, United States, University Park, PA
[email protected]
Since 2006 an interdisciplinary and collaborative group formed by the Catholic University of Uruguay, Pennsylvania
State University and the Republic University of Uruguay is studying the effects of nutrition and heavy metals on
development, behavior and learning of children. We present a review of studies: In 2007 the authors screened lead
and hemoglobin levels in capillary blood of 222 preschool children from Montevideo, Uruguay. In 2008 the authors
examined metal exposures in 109 young children from Montevideo, Uruguay and their mothers participating in a
community-based study. In 2009, 150 children from first grade were enrolled. Underwent psychological and clinical
assessments (blood, urine and hair was collected for measuring metals), information was requested from teachers
and parents. The mothers were interviewed nutritional and psychological. There was also a home visit and
sampled water, soil and household dust. This year we are investigating the relationship between exposure to low
levels of arsenic and neurobehavioral disorders in children.
RESULTS
Study 1
Older child age, hemoglobin 10.5g/dL, and putting finges/toys in the mouth were associated with higher blood lead
level (BLLs). Young maternal age, less education, father's job with potential risk of lead exposure, and fewer family
possessions were also associated with higher BLLs.
Study 2
BLLs ≥ 5 µg/dL in mother or child were associated with lower maternal perceptions of being skilled at discipline
(panemia was associated with lower likelihood that mothers would let their children explore and play (panemia was
associated with maternal perception of lower emotional support (p<0.01).
Study 3
Mean IQ, SF and BLL were 92.9±17.0 points, 15.0±14.1 µg/L, 4.7±2.2 µg/dL, respectively. 48.8% and 30.2% of
children had BLL ≥5µg/dL and iron deficiency respectively, 13.4% had both. 31% of children had IQ ≤ 85 points.
Conclusions
The consolidation of an interdisciplinary group specializing in environmental pollution and the experience of a
collaborative work between different universities and institutes is a significant step in understanding the problem
and finding solutions to the most vulnerable populations: children and reproductive-age women.
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12th International Symposium on Metal Ions in Biology and Medicine
Abstract code: 109
AREA: TEMIBS
POSTER
The use of biomarkers in evaluation of mutagenicity risk in offspring of rats exposed to
contaminated soils - Contribution to the study of environmental contaminants
Garcia, E.M.1; Rodrigues da Silva Júnior, F.M.2; Dupont-Soares, M.1; Baisch, P.R.1; Muccillo-Baisch, A.L.1
1
Programa de Pós-Graduação em Ciências da Saúde, Universidade Federal do Rio Grande - FURG, Rio Grande, Brazil
Programa de Pós-Graduação em Ciências Fisiológicas, Universidade Federal do Rio Grande - FURG, Rio Grande, Brazil
[email protected]
2
Industrial advances have significantly increased the release of contaminants to various environmental
compartments, such as soil, which is static and accumulates substances. Frequent human exposure to
contaminated soil has led to more risks of mutagenic effects since environmental contaminants can be transferred
from mother to child. In order to measure irreversible DNA damage, micronucleus test was performed in the bone
marrow of rat offspring - in pre-pregnancy, pregnancy and lactation - exposed to soil affected by atmospheric
dispersion from the industrial complex in Rio Grande, a city located in the south of Brazil. Results showed an
increased micronucleus frequency in all periods, by comparison with the control animals. In addition, chemicals
released to the soil can induce mutagenicity in rat offspring whose mothers were exposed to it at different stages of
the reproductive period, leaving an alert so that fewer pollutants are emitted in order to avoid serious harm to
human health.
143
Proceedings
12th International Symposium on Metal Ions in Biology and Medicine
Tungsten reacting with 5-hydroxymethylcytosine
Okamoto, A.
Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904,
Japan
[email protected]
Abstract
5-Hydroxymethylcytosine (hmC) is a newly discovered natural nucleobase that may play an important intermediary
role in the active DNA demethylation pathway. An effective method to detect the presence and abundance of hmC
in DNA is required to elucidate the relationship between the generation of hmC and the mechanism of
demethylation. We have found that the oxidation using dinuclear peroxotungstate K2[{W(=O)(O2)2(H2O)}2(µO)]•2H2O is hmC selective, and is useful for the discrimination of hmC from its epigenetic precursors, unmethylated
cytosine (C) and 5-methylcytosine (mC) in DNA. A fluorescein-labeled model DNA containing CG, mCG, or hmCG
dinucleotides was prepared, and the dinuclear peroxotungstate was added to a DNA solution in a pH 7 sodium
phosphate buffer. The mixture was incubated at 50°C for 5 h. The reaction proceeded hmC-selectively. The mass
spectrum of the oxidation product obtained from the hmC-containing DNA indicated the formation of trihydroxylated
thymine in DNA. The tungsten oxidation products induced the incorporation of adenine into the complementary site
in a primer extension, and made it possible to detect hmC in a DNA sequence of interest in a conventional DNA
sequencing analysis. Tungsten oxidation worked as a simple chemical reaction for the effective detection of hmC,
and will provide beneficial information on the design of a powerful method for hmC scanning and typing to solve the
mystery of the initialization of gene function through demethylation.
Keywords: tungsten, osmium, epigenetics, 5-hydroxymethylcytosine
Introduction
5-Hydroxymethylcytosine (hmC) is a newly discovered natural nucleobase that is induced by modification of 5methylcytosine (mC) with TET proteins, which have potential roles in epigenetic regulation (Scheme 1).
1,2
Given the
critical role of mC in epigenetic regulation, hmC may play an important biological role in vivo, such as an
intermediary role in the pathway of active DNA demethylation.
Scheme 1. hmC in epigenetic modification.
An effective method to detect the presence and abundance of hmC in DNA is required to elucidate the relationship
between the generation of hmC and the mechanism of demethylation. Several assays have been attempted for
hmC detection.
1–5
Although these methods can confirm whether or not hmC is present, including within a DNA
sample, it is difficult to carry out a scan that can detect which mC in a sequence is hydroxylated and whether or not
a specific mC is hydroxylated. An effective chemical reaction is required for the easy detection of hmC, such as the
chemical methods used to detect mC. If such a reaction is available, then the reaction product could be detected as
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12th International Symposium on Metal Ions in Biology and Medicine
an hmC-positive signal using a conventional detection method, such as site-selective strand cleavage assay,
sequencing analysis, and mass spectroscopy. In this paper, we report on an hmC-positive chemical reaction that is
effective for the discrimination of hmC from C and mC in a DNA of interest.
6
Materials and Methods
Metal oxidation and hot piperidine treatment. The fluorescein-labelled DNA (5 µM) to be examined was
incubated in a solution of 5 mM of the metal oxidant (plus 50 mM hydrogen peroxide as an option) and 100 mM of
sodium chloride in 50 mM of sodium phosphate (pH 7.0) at 50°C for 5 h. The reaction solution was filtered to
deionize it using Micro Bio-Spin Columns with Bio-Gel P-6 (BioRad). After drying in vacuo, the precipitated DNA
was redissolved in 50 µL of 10% piperidine (v/v), heated at 90°C for 2 h, and then evaporated to dryness using a
vacuum rotary evaporator.
Sequencing. The DNA (3 ng) to be examined was incubated in a reaction mixture, and then it was deionized by
filtration. The reaction sample was analysed using capillary electrophoresis-based DNA sequencing technology
using BigDye® Terminator v1.1.
Results and Discussion
A fluorescein-labeled model sequence containing CG, mCG, or hmCG dinucleotides, DNA1(X) 5′-fluorescein7
AAAAAAG- XGAAAAAA-3′ (X = C, mC, or hmC), was prepared, and several metal oxidants have been tested as
oxidation agents of DNA1(X), considering their reactivity against nucleobases, reagent availability, and solubility in
water. The metal oxidant was added to a solution of DNA1(X) in a pH 7 sodium phosphate buffer, and the mixture
was incubated at 50 °C for 5 h, as described in Experimental section. After the solution was desalted through a
filter, the sample was treated with hot piperidine and analyzed using polyacrylamide gel electrophoresis (PAGE) to
find the oxidized nucleotides in DNA1(X) through the detection of DNA cleavage bands. The dinuclear
peroxotungstate K2[{W(=O)(O2)2(H2O)}2(µ-O)]•2H2O (1)8 was the most effective hmC-selective oxidant among
metal oxidants tested in this experiment (Fig. 1).
Fig. 1. Structure of dinuclear peroxotungstate.
PAGE analysis of the strand cleavage product after the reaction of DNA1(X) with 1 and hot piperidine treatment
showed a band at the hmC site of DNA1(hmC). The cleavage bands observed at the mC and C sites in DNA1(mC)
and DNA1(C), respectively, were negligible. The mass spectrum of the oxidation product of DNA1(hmC) indicated
the formation of trihydroxylated T in DNA, and the product increased in molecular mass by four when the oxidation
of DNA1(hmC) with 1 was carried out in a buffer solution prepared with H218O (Scheme 2).
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12th International Symposium on Metal Ions in Biology and Medicine
Scheme 2. Oxidation of hmC with 1.
The reactions of DNA1(X) with other metal oxidants, e.g., MeReO3,9 OsO4,10 and K2[{Mo(=O)(O2)2(H2O)}2(µ8
O)]•2H2O dinuclear peroxomolybdate, which are known to oxidize a carbon–carbon double bond, were much less
hmC-selective (Fig. 2). Methylrhenium trioxide was weakly reactive, and osmium tetroxide and molybdenum
peroxocomplex exhibited an hmC selectivity that was much lower than that of tungstic acid.
hmC in a single-stranded DNA was efficiently oxidized with 1, whereas the oxidation efficiency for hmC in a DNA
duplex
was
lower.
Oxidative
cleavage
of
the
fluorescence-labeled
DNA2,
5′-fluorescein-
GCAGGGCCCACTAChmCGCTTCCTCCAGATGA-3′, was observed at hmC (79% conversion). In contrast, the
oxidation of bases in a hybrid with the fully matched complementary DNA was suppressed (7% conversion).
The protection of the C5−C6 double bond by the stacking of the flanking base pairs inhibits the attack of 1.
Fig. 2. hmC selectivity of metal oxidations.
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12th International Symposium on Metal Ions in Biology and Medicine
Tungsten oxidation was applicable to detection of hmC in fluorescence-labeled human genome sequences. The
DNA fragments containing hmCG dinucleotides in the promoter regions of human STAT3, BCL2, OCT4, NANOG,
HOX5A and TNF-β genes were prepared and oxidized with 1. They exhibited cleavage bands at hmC after hot
piperidine treatment, suggesting that tungsten oxidation is effective for hmC-selective reaction and detection of
hmC in DNA.
The deamination of cytosines makes it possible to incorporate adenines at the complementary position in a strand
extension process. Therefore, the deaminated product from the oxidation of hmC may help the efficient detection of
hmC through PCR amplification and sequencing. A DNA fragment from the human TNF-β putative promoter
region11 including CG, mCG, and hmCG dinucleotides was prepared and incubated with 1 at 50 °C for 5 h, and
then the sequences were analysed using capillary electrophoresis-based DNA sequencing technology. The
sequencing profiles of the tungsten oxidation products indicated that adenine was incorporated into the position
opposite the original hmC group (Fig. 3), whereas only guanine was incorporated into the position opposite mC and
C. The degree of the oxidative damage of guanine, which was observed on reaction with 1 in the PAGE analysis,
seemed to have little effect on the sequencing efficiency and fidelity in the present sequence analysis. The
selective base conversion of hmC in the sequencing process facilitated the ability to distinguish hmC from C and
mC in the DNA sequencing analysis.
Fig. 3. Sequence analysis of
hm
C- containing DNA using 1 oxidation. The sequencing profiles of human TNF-β
promoter fragments containing CCChmC-GGGC.
In the present study, the tungsten oxidation system was effective for discrimination of hmC from C and mC. The
key point of the discrimination was the use of the oxidation of a carbon-carbon double bond. The structural
difference between hmC and mC is the presence of a hydroxyl group (O–H). However, there is a large number of
chemically active O–H, N–H, and S–H bonds in nucleic acids and proteins. Therefore, it may be difficult to make a
chemical reagent react only the hydroxyl group of hmC. On the other hand, considering the structure of hmC in a
6
5
wider view, hmC sites possess a characteristic allyl alcohol group (C = C –CH2OH), which is rare in biomolecules.
Tungsten-based oxidation systems are known to be effective for the oxidation of the carbon-carbon double bond of
allyl alcohol groups.
8
Therefore, the allyl alcohol group of hmC worked as a key feature in the hmC-selective reaction of tungsten
oxidants.
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12th International Symposium on Metal Ions in Biology and Medicine
Conclusions
Oxidation using tungsten oxidants, such as tungstic acid activated by hydrogen peroxide and dinuclear
peroxotungstate 1, is hmC selective, and is useful for the discrimination of hmC from C and mC in DNA. Tungsten
is a key element that can selectively access hmC, and is different from the other metallic elements that are close to
tungsten in the Periodic Table, such as osmium, molybdenum, and rhenium. Although the data shown here are
preliminary, and further optimization of the reaction conditions is required for improved reaction yield and
sequence-independent reactivity, the above reaction will provide beneficial information on the design of a powerful
method for hmC scanning and typing, and is undoubtedly a promising way to solve the mystery of the initialization
of gene function through demethylation.
References
1.
Kriaucionis, S. and Heintz, N. The nuclear DNA base 5-hydroxymethylacytosine is present in Purkinje
neurons and the brain. Science, 2009, 324, 929–930.
2.
Tahiliani, M. et al. Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL
partner TET1. Science, 2009, 324, 930–935.
3.
Münzel, M. et al. Quantification of the sixth DNA base hydroxymethylcytosine in the brain. Angew Chem Int
Ed, 2010, 49, 5375–5377.
4. Ito, S. et al. Role of Tet proteins in 5mC to 5hmC conversion, ES-cell self-renewal and inner cell mass
specification. Nature, 2010, 466, 1129–1133.
5.
Song,
C.-X.
et
al.
Selective
chemical
labeling
reveals
the
genome-wide distribution
of
5-
hydroxymathylcytosine. Nature Biotech, 2011, 29, 68–72.
6.
Okamoto, A., et al. 5-Hydroxymethylcytosine-selective oxidation with peroxotungstate. Chem Commun,
2011, 47, 11231–11233.
7. Sugizaki, K., Ikeda, S., Yanagisawa, H. and Okamoto, A. Facile synthesis of hydroxymethylcytosine-containing
oligonucleotides and their reactivity upon osmium oxidation. Org Biomol Chem, 2011, 9, 4176–4181.
8. Kamata, K., Yamaguchi, K. and Mizuno, N. Highly selective, recyclable epoxidation of allylic alcohols with
hydrogen peroxide in water catalyzed by dinuclear peroxotungstate. Chem Eur J, 2004, 10, 4728–4734.
9. Tetzlaff, H. R. and Espenson, J. H. Kinetics and mechanism of the epoxidation of allylic alcohols by hydrogen
peroxide with methyltrioxorhenium as catalyst. Inorg Chem, 1999, 38, 881–885.
10. Dizdaroglu, M., Holwitt, E., Hagan, M. P. and Blakely, W. F. Formation of cytosine glycol and 5, 6dihydroxycytosine in deoxyribonucleic acid on treatment with osmium tetroxide. Biochem J, 1986, 235, 531–536.
11. Nedwin, G. E. et al. Human lymphotoxin and tumor necrosis factor geges: structure, homology and
chromosomal localization. Nucleic Acids Res, 1985, 13, 6361–6373.
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Acknowledgments
We wish to thank Ms Kaori Sugizaki and Ms Akiko Nakamura (RIKEN) for the PAGE analysis. We are also grateful
to the Support Unit for Bio-material Analysis, RIKEN BSI Research Resources Center, for help with DNA
sequencing analysis.
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12th International Symposium on Metal Ions in Biology and Medicine
Silver nanoparticle exposure: Pathological changes in developing chick embryos
Gagnon, Z.E.1*; Pavel Sizemore, I.E.2*; Trivedi, P.3; Dagher, J.M.2; Monahan, J.L.2; Lam, T.2; Isaac; L.A.C.4
1
Marist College, School of Science, Department of Environmental Sciences, 3399 North Road, Poughkeepsie, NY 12601, USA;
e-mail: [email protected];
2
Department of Chemistry, Wright State University, Dayton, OH 45435: [email protected], [email protected], and
[email protected]
3
University Hospital, Cincinnati, OH 45291: [email protected]
4
St. Francis Hospital, Pathology Department, Poughkeepsie, NY 12601: [email protected]
Abstract
The presence of silver nanoparticles (AgNPs) in industrial and household products has become increasingly
prevalent in recent years. Six-day old chick embryos were exposed to AgNPs in concentrations of 15, 30, 60, and
-1
th
th
100 µg mL via injections into the egg air sac on the 7 and 14 days of incubation. The colloidal AgNPs were
synthesized according to the widely-used Creighton method through the aqueous reduction of silver nitrate
(AgNO3) with sodium borohydride (NaBH4). The control groups included no injection, highly purified water, AgNO3,
th
and NaBH4 injections (10 embryos per each control and treatment group). On the 20 day of incubation, embryos
were sacrificed, and the brains and livers were harvested. Histological analysis and flame atomic absorption
spectroscopy (FAAS) measurements were conducted on the collected tissue samples. The SPSS statistical
package was used to determine variation in serotonin and Ag bioaccumulation levels. Mortality occurred in all
treatments except for the water and no injection controls. A direct correlation was found between Ag
bioaccumulation levels in tissues and the pathological changes observed in chick embryos exposed to 60 and 100
µg mL-1. Fatty vacuoles were present in the liver cells for the 60 µg mL-1 of AgNP treatment. Proliferation of
hepatocytes, hyperplasia, and increased cellular mitotic activity was also observed in these samples. Additionally,
-1
Von Kossa staining revealed cell calcification, especially in brain tissues. At the 100 µg mL AgNP exposure level,
apoptotic bodies with nuclear fragmentation were evident, and Ag precipitation was detected in the intercellular
spaces of liver tissue. These pathological findings raise major health concerns about the increasing exposure of the
human population to AgNPs, and suggest that environmental risk assessment of this emerging factor should be
considered.
Key words: Creighton silver nanoparticles, chick embryos, liver and brain pathology, serotonin, flame atomic
absorption spectroscopy
Introduction
Although nanoparticles (NPs) are part of our natural environment, engineered NPs have recently become a
dominant factor in modern technology. Because of their unique physical, chemical and antimicrobial properties,
AgNPs are the most commonly used NPs for industrial and medical purposes, in particular in consumer products.
The Nanotechnology Consumer Product Inventory prepared in 2010 by the Woodrow Wilson Center’s Project on
Emerging Nanotechnologies [1] indicated that over 50% of the nanomaterial-based consumer products contain
nanosilver. Approximately 5% or over 1,200 tons of the total silver (Ag) that is produced worldwide is represented
by AgNPs. In spite of their numerous applications, AgNPs also have drawbacks. Questions regarding the
environmental fate of AgNPs in terrestrial and aquatic environments remain unanswered [2]. The potential effect of
AgNPs on human health is of even greater concern. Because there are currently no efficient methods for the
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removal of AgNPs from wastewater effluents [2], it is imperative to learn more about AgNP uptake into the food
chain and their long term effects on human health and animals.
Recent nanotoxicology studies on AgNPs, ranging in size between 6 and 25 nm diameter and exposure levels
between 5 to 400 µg mL-1, were found to increase production of reactive oxygen species (ROS) in human lung
fibroblast cells [3], increase cell membrane leakage in mouse embryonic stem cells and mouse embryonic
fibroblasts [4], diminish mitochondrial function in Buffalo rat liver cells [5], and cause chronic alveolar inflammation
in rats [6]. The main goal of this study was to quantify the amount of Ag accumulated in the liver and brain tissues
of developing chick embryos, and to determine the histopathological effects associated with the exposure to AgNPs
in these organs.
Materials and Methods
Experimental Organisms. One hundred forty fertilized specific pathogen free white leghorn strain chick embryos
(Gallus domesticus) were obtained from Charles River Laboratories, Inc., and were divided into 14 experimental
groups of 10 chicks each. The experimental setup, embryo acclimation, and embryo preparation followed the
procedure described by Gagnon et al. [7]. Embryos were incubated for 20 days in a forced air chamber with
automatic turner at 38.5ºC ± 1ºC and 50-55% relative humidity. Hamburger and Hamilton (HH) [8] nomenclature
was used to estimate embryo developmental stages. Embryological age was established based on a full 24 hours
from the time of day the eggs were first placed in the incubator.
Synthesis and Characterization of AgNPs. Colloidal AgNPs were synthesized according to the widely-used
Creighton approach through the reduction of 1 mM of AgNO3 solution (50 mL) with 2.0 mM of NaBH4 solution (300
mL) in water. The reaction mechanism and the characterization of the Creighton colloid were presented in our
previous work [9, 10, 11]. This method was shown to lead to the formation of round AgNPs with an average
diameter of approximately 11 nm and a moderate size distribution in the 1-100 nm size range. The UV-VIS
absorption spectrum of the yellow colloid exhibited a typical sharp surface plasmon peak at 390 nm. These AgNPs
have a negative surface charge and a shelf life time of up to 3-6 months at 10ºC. Literature shows that aqueous
suspensions of AgNPs release Ag+ ions over time in the presence of dissolved O2 and H+ through an oxidation
process. However, this oxidation process is slow [12] (e.g., 6-125 days for citrate-capped AgNPs) and temperature
dependent. In our study, the Creighton colloidal AgNPs were used immediately after fabrication.
In Ova Injections. The following experimental treatments were established: a) control with no injection; b) 0.0 µg
mL-1 control treatment with deionized water only; c) four AgNP treatment aqueous suspensions of 15.0, 30.0, 60.0
-1
and 100 µg mL
-1
each; d) four AgNO3 treatment solutions of 23.6, 47.2, 94.4 and 157.4 µg mL
-1
NaBH4 treatment solutions of 10.5, 21.0, 42.0 and 70.0 µg mL
each; e) four
each. There is some evidence that AgNPs in
colloidal solution can deaggregate to form their original compounds [13]. For this reason, AgNO3 and NaBH4
control treatments were established with the same concentrations as those for AgNP synthesis. These
concentrations were based on the equivalent amounts of AgNO3 and NaBH4 that used in the AgNP synthesis.
Stock solutions of AgNPs, AgNO3, and NaBH4 were prepared in sterile deionized water for subsequent 1 mL
injections. The pH of all solutions was adjusted to 7.2 with 0.1 N NaOH or HCl to compensate for minor treatment
variations (pH close to that of the stable, Creighton colloid). All solutions were filtered using a 0.22 µm filter (Fisher
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th
th
Scientific). All treatments were injected directly into the air sac on the 7 and 14 days of incubation. The internal
membrane surrounding the embryo was not punctured, allowing for slow diffusion of liquid through the membrane.
Tissue Sampling. The experiment was terminated on the 20th day of incubation. Embryos were sacrificed through
cervical decapitation within the first 5-10 seconds after removal from the egg shell to avoid any major stresses. A
gross visual examination was performed to identify potential external and internal abnormalities. Chick brains,
livers, and tibiotarsi were extracted by dissection. Collected samples were separated for histology, chemical
analysis by FAAS, and serotonin ELISA immunoassay.
Histological Analysis. Tissue samples for histological analysis were rinsed in saline solution and fixed in 10%
phosphate-buffered formalin solution. Tissue cassettes were processed in a Vacuum Infiltration Processor (VIP),
and dehydrated with formalin, ethanol, and americlear. Paraffin molds were sectioned at 3 µm using a Spencer
microtome. Prepared slides were stained using a hematoxylin-eosin (H&E) dye (LEICA Autostainer XL), and
examined for pathological changes via light microscopy. Following these analyses, a Von Kossa Stain Kit
(American Master Tech Scientific, Inc.) was used to identify tissue calcification.
Serotonin Analysis. Analyses were performed on homogenized tissue samples using modified ELISA
Immunoassay (LDN Labor Diagnostica Nord GmbH & Co. KG). Chick brain tissue samples (0.1 g) were
-1
homogenized in 2 mL of freshly prepared buffer (1mg mL of ascorbic acid added to 50 mM of Tris, pH of 7.5). In
the assay, acetylated serotonin was bound to the solid phase of the microtiter plate. Substrate TMB (3,3′,5,5′tetramethylbenzidine)/peroxidase reaction was determined by recording the difference in absorbance using a
BIOLOG Reader at 450 nm. A calibration curve was developed using six LDN Standard Controls of different,
known serotonin concentrations. Concentration of serotonin was obtained using the Datalogger program and curve
fitting techniques.
FAAS Analysis. Tissues were dehydrated at 80°C for 72 h. Samples (0.5 g) were then homogenized, and digested
in 5 mL of high purity nitric acid (HNO3) (Fisher Scientific, Optima Grade). Ag content analyses were conducted via
FAAS. An Ag stock standard solution of 1.0 x 103 µg mL-1 (Fisher Scientific, Lot Number: CL4-132AG, certified by
SPEX CertiPrep) was used for the preparation of seven Ag standards (0.01, 0.1, 0.2, 0.5, 1.0, 2.0, and 4.0 µg mL
1
-
). The pH of each sample solution was adjusted to 2.0 using high purity nitric acid. Samples corresponding to
higher Ag-exposure levels were diluted 2x or 4x using a blank solution before each measurement to fit within the
concentration range of the Ag calibration curve. A fast sequential spectrometer (model AA240 FS, Varian Inc.)
equipped with an air-acetylene burner and a Ag hollow cathode lamp (5 mA) was used to determine Ag content
within samples. The acquisition parameters were as follows: wavelength of 328.1 nm, slit width of 0.5 nm, air flow
rate of 13.50 L min-1, and acetylene flow rate of 2.00 L min-1. Ag amounts were determined through standard
external calibration using least-squares fit of regression curves.
Statistical Analysis. The effect of applied treatments on Ag accumulation and serotonin concentration in brain
tissues was analyzed using SPSS. One-way analysis of variance (ANOVA) by multi-comparison Student-NewmanKeuls procedure was used to examine sample variation at the probability level α ≤ 0.05.
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Results and Discussion
-1
Mortality. One hundred percent mortality was observed in NaBH4 exposures at the 30, 60 and 100 µg mL levels
and in AgNO3 exposures at the 94.4 and 157.4 µg mL-1 levels. The lower AgNO3 and NaBH4 treatment levels
exhibited mortality proportional to increasing compound concentrations, as recorded in Gagnon et al. [7]. The
chicks surviving the lower AgNO3 and NaBH4 exposure levels were smaller, unhealthy in appearance, and severely
underdeveloped in comparison to the no injection and water controls. Additionally, tissue damage was so extensive
that the structural integrity of the organs of interest rendered them unusable for analysis. Chick mortality for AgNP
treatments occurred at all exposure levels, but was lower in comparison to the paternal compound exposures. At
60 µg mL-1 of AgNP, 7 of 10 specimens died before harvest. However, at the highest AgNP concentration, only 2
specimens were dead at harvest. No mortality occurred in the no injection or water control groups.
FAAS Analysis. The results of FAAS analysis are presented in Table 1. Each sample was measured three times
2
and the absorbance values were averaged before interpolating the Ag concentration from the calibration curve (r >
0.998). Calibrations were performed every 20 samples, using acid-matched standard solutions [14]. Statistically
significant differences in Ag concentration were found in liver tissue samples. Ag bioaccumulation was found to
increase with the increase in the AgNP concentration of the injected solutions. The results demonstrate a
correlation between Ag concentrations in the tissues and the pathological changes observed in the histological
preparations. Additionally, there was higher Ag accumulation from AgNO3 sources than from AgNPs.
-1
Table 1. Total amount of Ag (in µg mL ) accumulated in chick embryo liver tissue as estimated by FAAS. Each
value is the mean of three measurements ± standard deviation.
Treatment
Total amount of Ag accumulated
-1
in 0.5 g of liver tissue (µg mL )
Control (no injection)
0.0
Control (water injection)
0.0
-1
1.6 ± 0.3
-1
3.0 ± 0.1
23.6 µg mL of AgNO3
47.2 µg mL of AgNO3
-1
15 µg mL of AgNPs
1.2 ± 0.4
-1
30 µg mL of AgNPs
2.4 ± 0.6
-1
5.5 ± 1.1
60 µg mL of AgNPs
-1
100 µg mL of AgNPs
6.2 ± 1.2
All NaBH4 samples
0.0
Liver Toxicity. AgNP exposures of 15 and 30 µg mL-1 resulted in no pathological changes in liver tissue. The 60
µg mL-1 treatment of AgNPs, however, had a dramatic effect on liver tissue integrity and health. Fatty vacuoles in
the liver cells were evident. Fatty vacuoles of individual cells coalesced and caused cell membrane to disintegrate.
There is a significant body of research demonstrating formation and accumulation of fat in the liver of animals,
including avian species, caused by exposure to heavy metals, mostly mercury and lead [15]. Identification of fatty
liver in the embryonic liver tissue of mammalian and avian models is not uncommon in many pathological
conditions, including human population.
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12th International Symposium on Metal Ions in Biology and Medicine
H&E stained liver tissue showed black pebbly structures (Figure 1a). Von Kossa staining was conducted confirming
cell calcification. Proliferation of hepatocytes, hyperplasia, and increased cellular mitotic activity was observed in
liver tissue. Proliferation of hepatocytes with undifferentiated hepatocytes was not uniformly dispersed but rather in
clusters spread out among normally differentiated hepatocytes.
Deposition of ultra-fine AgNPs in hepatocytes could cause inhibition of cell differentiation. It is known that liver
proliferation in experimental animals can be induced by a variety of external conditions such as hepatectomy or
heavy metals [16]. The liver is known to be a main target for heavy metal exposure. In this study, regenerative
hyperplasia was accompanied by liver enlargement. Therefore, increased mitotic activity and DNA synthesis could
be an adaptive response to AgNP toxicity.
Another possibly adaptive response to liver toxicity from AgNPs was demonstrated by vascular proliferation. It
seems that developing hypoxia was caused by two AgNP injections during embryonic development, and stimulated
th
th
pathological angiogenesis. The 7 day correlates with HH stages 30 and 31, when limb bud is developing. The 14
day corresponds to HH stage 40, when structural development is completed and only growth of existing structures
is taking place [17]. Widespread hypoxia, which induced vascular proliferation, was observed in the Ag-exposed
chick liver. In healthy tissue, the occurrence of vascular proliferation is extremely low [18]. Our study also revealed
angiogenesis, thick vessels in the proliferation area, which can be induced in response to several physiological and
pathological stimuli.
Ag deposits were observed within the liver cells and in the extracellular space. As indicated in the previous section,
the FAAS analysis confirmed the Ag bioaccumulation and quantified the total Ag amounts in tissue. Ham and
Tange [19] reported Ag deposition in liver of rats receiving a 0.25% solution of AgNO3 in distilled water as their
drinking water for several weeks. There is a large body of knowledge on Ag affinity to proteins, especially to the SH
groups. However, the composition of the Ag aggregates can be more diverse and requires further investigations.
Ag aggregates formed in cells and tissues can be composed of metallic Ag, silver oxide, or silver sulphide [19].
In this study, clusters of undifferentiated liver cells were observed. At the same time, however, cells undergoing
pyknosis and apoptotic bodies with nuclear fragmentation were also observed. Dramatic pathological changes
were noted at AgNP exposure level of 100 µg mL-1 with a ghost like appearance of cytoplasm. This type of
piecemeal necrosis in the liver also occurs in viral hepatitis and other autoimmune conditions.
Brain Toxicity. At an AgNP exposure level of 15 µg mL-1, no changes were observed in brain tissue. However, at a
-1
concentration level of 30 µg mL , black deposits of Ag were clearly identified in the subcortical space of the pia
matter under light microscopy at 400x (Figure 1b). The pia matter is a delicate membrane that covers the brain
surface and is impermeable to fluids. In addition, patchy loss of structural integrity was demonstrated by localized
dense nuclei and highly diffused cellular arrangement. Von Kossa staining revealed cell calcification most
prominent in brain tissue. The authors’ previous research on chick embryo exposed to platinum group metals
identified widespread brain tissue calcification [20]. In contrast, this experiment’s patchy spatial tissue calcification
could be correlated with target-like calcium distribution and is believed to indicate the onset of neuron cell death.
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12th International Symposium on Metal Ions in Biology and Medicine
b)
a)
Figure 1. a). Cross section of chick embryo liver exposed to 100 µg mL-1 of AgNPs. Visible pyknosis (arrow) shows
the irreversible condensation of chromatin in the nucleus of an apoptotic cell (100x). b) Brain tissue of a chick
embryo exposed to 60 µg mL-1 of AgNPs. The Ag appears in the tissues as small dark granules. Ag deposits form a
patchy pattern. Note the presence of very few neurons outside the area of Ag deposition (indicated by arrow).
Serotonin Content. There was a statistically significant difference in the level of serotonin in the brains of AgNP-1
exposed chick embryos with respect to the control groups. The significant serotonin increase at the 60 µg mL
-1
AgNP exposure level was followed by a decrease at the highest AgNP exposure level of 100 µg mL , possibly
indicative of an unconventional dose response. In comparison with no injection group, serotonin levels in the 10.5
-1
µg mL NaBH4 group experienced a significant decrease. Due to low survival rate, no AgNO3 exposed chicks were
available for serotonin examination.
In principal, the blood brain barrier (BBB) protects the brain from blood-borne exposure to heavy metals. During
embryonic development, however, the BBB is not fully developed. In humans, the placenta provides additional
protection to the embryo. Ag deposition in brain tissue indicates that the BBB is not an effective barrier for NPs
(including AgNPs). It has been demonstrated that serotonin plays a decisive role in cell motility initiation coupled
with the process of gastrulation. According to Wallace [21], serotonin is a common neurotransmitter in most animal
embryos during this early period of development. In his experiments, chick embryos deprived of normal access to
serotonin by either serotonin synthesis inhibitors or by serotonin receptor blockers showed typical morphological
disturbance of gastrulation, neurulation, and semitogenesis. Our results suggest that decreased levels of serotonin
in the chick brain tissue could have a significant effect on embryo development.
Conclusions
The exposure to Creighton AgNPs had multiple effects on developing chick embryos. Our results demonstrate that
there was significant bioaccumulation of Ag in the chick liver tissue and reduction of serotonin level in the brain
tissue for the highest AgNP exposure. In addition, our previous AgNP study [7] documented an effect on the
immune system. In our controlled experimental environment, bioaccumulation of AgNPs in tissues and repeatable
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results in 10 replicates suggest that the pathological changes in the immune system and serotonin level are the
effect of exposure to AgNPs. As part of a comprehensive investigation on AgNP exposure, our study presents
multiple pathological outcomes using the chick embryo model that could suggest similar responses in humans.
DNA damage, cell cycle arrest, morphological changes of cells, Ag tissue deposits, and brain calcification raise
concerns about the safety associated with widespread use of AgNPs.
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Trefry JC, Monahan JL, Weaver KM, Meyerhoeher AJ, Markopoulos MM, Arnold ZS, Wooley DP, Pavel IE.
Size selection and concentration of silver nanoparticles by tangential flow ultrafiltration for SERS-based
biosensors. J Am Chem Soc 2010 132:10970-10972.
11.
Anders CB, Baker JD, Stahler AC, Williams A, Sisco JN, Trefry JC, Wooley DP, Pavel Sizemore IE.
Tangential Flow Ultrafiltration: A “Green” Method for the Size Selection and Concentration of Colloidal Silver
Nanoparticles. Journal of Visualized Experiments 2012 e4167:1-9.
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12.
Liu J, Hurt RH. Ion release kinetics and particle persistence in aqueous nano-silver. Environ Sci Technol
2010 44:2169-2175.
13.
Jayabalan J, Singh A, Chari C, Srivastava H, Mukhopadhyay PK, Srivastava AK, Oak SM. Aggregated
nanoplatelets: Optical properties and optically induced disaggregation. J Phys-Condens Mat 2008
20(44):8pp.
14.
Creighton JA, Eadon DG. Ultraviolet-visible absorption spectra of the colloidal spectra elements. J Chem Soc
Faraday T 1991 87(24):3881-3992.
15.
Gomez-Munoz A, Rodriguez-Fernandez C. Fatty acid composition of liver lipids during ontogeny of the chick
embryo: effect of a single dose of triiodothyronine. Exp Clin Endocrinol Diabetes 1988 92(3):323-327.
16.
Columbano A, Ledda GM, Sirigu P, Perra T, Pani P. Liver cell proliferation induced by a single dose of lead
nitrate. Am J Pathol 1983 110(1):83-88.
17.
Bellaris R, Osmond M. The Atlas of Chick Development. New York: Academic Press, 1998.
18.
Humar R, Kiefer FN, Berns H, Resnik TJ, Battegay EJ. Hypoxia enhances vascular cell proliferation and
angiogenesis in vitro via rapamycin (mTOR) - dependent signaling. FASEB J. 2002 16(8):771-80
19.
Ham KN, Tange JD. Silver deposition in rat glomerular basement membrane. Immunol Cell Biol 1972
50:423–434.
20.
Gagnon ZE, Isaac LAC, Newkirk C. Neural damage in chick embryo brains exposed to platinum group
metals. In: Collery P, Maymard I, Theophanides T, Khassanova L, Collery T, Eds. Metal Ions in Biology and
Medicine, Volume 10. Paris: John Libbey Eurotext, 2008:82-87.
21.
Wallace JA. Monoamines in the early chick embryo: Demonstration of serotonin synthesis and the regional
distribution of serotonin-concentrating cells during morphogenesis. Am J Anat 1982 165(3):261-276.
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Determination of Cd in soybean and wheat seeds using TS-FF-AAS and preconcentration
with cloud point extraction (CPE)
Morales, G.; Knochen, M.; Pistón, M.*
Cátedra de Química Analítica, Facultad de Química, Universidad de la República
Montevideo, Uruguay
[email protected]
Abstract
Cadmium is a toxic element associated with the environment. Seeds can accumulate it, so it is important to
determine the concentration of this element.
Thermospray Flame Furnace (TS-FF) coupled to a flow-injection preconcentration system can be used to enhance
detectability in flame atomic absorption spectrometry (FAAS) providing an alternative to the use of ET-AAS or ICPMS.
The method is based on the adsorption of a complex of Cd and methyl green, potassium iodide and Triton X-114 in
a minicolumn filled with cotton. The elution was carried out with HNO3. The system consists of a peristaltic pump,
an injection valve and a nickel tube 10 cm long with 7 holes. The analytical determinations were carried out by
FAAS at 228.8 nm.
The seeds were milled, and 1.8 g of the obtained flour was digested by means of an acidic treatment with a mixture
of HNO3/H2O2. The resultant solution was neutralized with solid NaOH.
The figures of merit were: LD (3s) and LQ (10s): 0.6 and 2.1 µg L
2
-1
-1
(r = 0.999), precision: sr(%) = 2.8 (n = 5), sampling frequency: 30 h
respectively, linearity: up to 10 µgL
-1
with a preconcentration factor of 30
compared with FAAS.
Accuracy was evaluated with the CRM Wheat Flour 1567a (NIST), the recoveries were between 98% and 105 %
(n = 10). There was no evidence of the influence of potential interferences.
This method can be implemented for the control of Cd levels in soybean and wheat seeds.
Keywords: cadmium, thermospray, soybean, wheat
Introduction
Cadmium is one of the most toxic elements associated with environmental and industrial pollution, either as a
secondary product of metallurgic industry, where it reaches the soil by aerial deposition, or through the use of
phosphate fertilizers and the use of compost. Soybean and wheat seeds can accumulate this element from the
environment, and then it is very important to determine the concentration of Cd in the seeds because wheat is the
raw material of many foods [1].
Analytical determinations of Cd can be performed using different detection systems. Once the pretreatment of the
sample is performed (digestion, extraction, preconcentration), this element can be quantified by flame atomic
absorption spectrometry (FAAS), by atomic absorption spectrometry with electrothermal atomization (ET-AAS) or
by atomic emission inductively coupled plasma (ICP-OES, ICP-MS), among others [2].
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12th International Symposium on Metal Ions in Biology and Medicine
Thermospray Flame Furnace (TS-FF) coupled to a flow-injection preconcentration system can be used to enhance
detectability and sensitivity in flame atomic absorption spectrometry (AAS) of some volatile elements providing an
alternative to the use of ET-AAS or ICP-MS [3].
To further improve detectability of trace elements, strategies of on-line preconcentration can be used. Cloud point
extraction (CPE) is a separation/preconcentration procedure which has proved to be very efficient for determination
of trace elements in varied matrices and also in good agreement with the principles of Green Chemistry [4-7].
CPE is based on the principle that a surfactant containing aqueous solution becomes cloudy and separates into
two phases, if certain conditions are adjusted appropriately as temperature, pressure or if an adequate substance
is added. When the surfactant solution becomes cloudy it has reached the "cloud point". At this point, the original
layer of surfactant phase is separated into a small volume of solution which will be rich in the analyte of interest
(linked to organic or inorganic species by having high affinity), trapped by micellar type structures [4].
Flow injection systems with micelle mediated preconcentracion are made up with minicolumns filled with cotton,
this material is commonly used for the retention of micellar aggregates [5].
In Uruguay the local regulation establishes that the maximum concentration of cadmium admitted in solid foods is
0.2 mg kg-1 [8]. Thus analytical methods used for the determination of this element in foods should provide high
sensitivity.
The aim of this work is to propose an automated method for routinely Cd determination in soybean and wheat
seeds using a flow injection system (FI) with on-line CPE preconcentraton coupled to Thermospray Furnace (TSFF) and detection by flame atomic absorption spectrometry (FAAS).
Materials and Methods
All reagents were of analytical reagent grade. Purified water (ASTM Type I) was obtained from a Millipore (São
Paulo, Brazil) Simplicity 185 purifier fed with glass-distilled water.
All glassware was soaked overnight in 10% (v/v) nitric acid and then rinsed exhaustively with distilled water. For
-1
calibration, adequate dilutions in purified water were prepared from a standard stock solution of Cd 1000 mg L in
HNO3 4% (v/v) (SCP SCIENCE traceable to NIST).
The proposed method is based on micelle formation by the reaction between the reagent methyl green, and
potassium iodide (KI) using Triton X-114 surfactant. These micelles are retained in a Teflon PFA minicolumn (60 x
-1
2.5) mm packed with 40 mg of cotton. Elution is carried out with HNO3 1 mol L .
The system consists of a peristaltic pump (Rainin Dynamax) fitted with Tygon tubing, a 6-ports injection valve
(Valco Cheminert), connections coils were made from 0.8 mm internal diameter Teflon PFA tubing. The detection
system used consisted of an atomic absorption spectrometer Perkin Elmer AAnalyst 200, equipped with a
thermospray flame furnace system (TS-FF-AAS). A nickel tube of 10 cm long by 10 mm of diameter with 7 holes
was used above an adapted burner with ceramic supports. The measurements were carried out at a wavelength of
228.8 nm using a deuterium lamp for background correction.
-1
During the preconcentration step, the reaction occurs between the Cd present in the sample, KI 0.5 mol L , methyl
-1
green 0.005 mol L and Triton X-114 0.5% (v/v), to form micelles that are retained on the cotton minicolumn. After
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12th International Symposium on Metal Ions in Biology and Medicine
one minute of preconcentration (charge position), micelles are dissolved in HNO3 1 mol L
-1
in the elution step
(elution position).
Figure 1 ilustrates the system.
Figure 1. Flow injection system used for the preconcentration of Cd. P: peristaltic pump; V: 6-port injection valve;
C: Column, W: waste, S: sample, T: Triton X-114, MG: methyl green, KI: potassium iodide, HNO3: nitric acid. Solid
lines represent the valve position during the preconcentration step, dotted lines represents the valve position during
the elution step.
The soybean and wheat seeds were obtained from a local distributor (Agropecuaria Valdense S.R.L). The seeds
were milled, and 1.8 g of the obtained flour was digested by means of an acidic treatment with a mixture of 30 mL
of HNO3 and 4 mL of H2O2 (30% (v/v) heated in a hot plate for 30 minutes in a vessel with reflux and afterwards the
evaporated volume was completed to 25 mL with purified water. The resultant solution was neutralized with solid
NaOH.
Certificate reference material of wheat flour purchased from NIST (1567a) was also prepared as described above.
Reagent blanks were also run.
Results and discussion
The optimum concentrations of the reagents were obtained by means of a three-level central composite design [9].
It was decided to work with 1 minute as preconcentration time, because it is a reasonable time in agreement with
good results in terms of the best net signal. The operative parameters of the system were optimized using
multivariate experiments, where the net signal of a standard solution was monitored.
Once the operative conditions were optimized, the figures of merit were evaluated to complete the validation of the
method. The results obtained are show in Table 1.
Accuracy was evaluated by means of trueness and precision. Trueness was studied using a reference material of
wheat flour (NIST-1567a) by means of the recoveries of Cd. The precision of the method was evaluated taking into
account the intermediate precision (relative standard deviation between 3 days).
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12th International Symposium on Metal Ions in Biology and Medicine
Table 1. Figures of merit
Parameter
Result
Limit of detection (3s)
0.6 µg L-1
Limit of Quantification (10s)
2.1 µg L-1
-1
Linearity
up to 10 µg L
Precision (sr, n=5)
2.8 %
Accuracy (n=10)
98% - 105%
Sampling frequency
30 h
Enrichment factor
-1
30 respect to FAAS
Preconcentration factor
6 respect to TS-FF-AAS
Sample consumption (mL)
4
The validated method was applied to analyze 24 samples of wheat and 14 of soybean seeds, the obtained values
were in the range of 0.03 and 0.10 mg kg-1 and 0.02 and 0.11 mg kg-1 of Cd respectively.
Conclusions
All the analyzed samples met the requirements of the regulations and can be used as raw material for food.
The proposed method was successful for the application and can be implemented for the control of Cd levels in
wheat and soybean seeds.
References
[1] Da Matta Chasin, A.A., Cardoso, L. M. N. Cádmio. In De Azeredo, F.A. and Da Matta Chasin, A.A. (Eds.).
Metais, Geranciamento da Toxicidade. San Pablo, Editora Atheneu, 2003, San Pablo, 187-298.
[2] Ferreira, S. L.C., de Andrade J. B., María das Graças A. Korn, M., Pereira, M. G., Lemosc, V. A., dos Santos,
Walter N.L., de Medeiros Rodrigues, F. Frederico, Souza, A. S., Ferreira, H. S., da Silva E. G.P. Review of
procedures involving separation and preconcentration for the determination of cadmium using spectrometric
techniques. J Hazard Mater; 2007; 145; 358-367.
[3] Tarley, C.R.T., Arruda, M.A.Z. A sensitive method for cadmium determination using an on-line polyurethane
foam preconcentration system and thermospray flame furnace atomic absorption spectrometry. Anal Sci; 2004; 20;
961-966.
[4] de Almeida Bezerra, M., Zezzi Arruda, M.A., Ferreira, S. L. C. Cloud Point Extraction as a Procedure of
Separation and Pre-Concentration for Metal Determination Using Spectroanalytical Techniques: A Review. Appl
Spectrosc Rev; 2005; 40; 269-299.
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12th International Symposium on Metal Ions in Biology and Medicine
[5] Silva, E. L., dos Santos Roldan P. Simultaneous flow injection preconcentration of lead and cadmium using
cloud point extraction and determination by atomic absorption spectrometry. J Hazard Mater, 2009; 161; 142-147.
[6] Xiang G., Wen, S., Wu, X., Jiang, X. Lijun He, L., Liu, Y. Selective cloud point extraction for the determination of
cadmium in food samples by flame atomic absorption spectrometry. Food Chem, 2012; 132; 532-536.
[7] Silva, E. L., dos Santos Roldan P., Giné, M. F. Simultaneous preconcentration of copper, zinc, cadmium, and
nickel in water samples by cloud point extraction using 4-(2-pyridylazo)- resorcinol and their determination by
inductively coupled plasma optic emission spectrometry. J Hazard Mater, 2009; 171; 1133-1138.
[8] Reglamento Bromatológico Nacional. Características de los alimentos. Disposiciones Generales. Decreto
315/94; 1994.
[9] Massart, D. L., Vandeginste, B. G. M., Buydens, L. M. C., De Jong, S., Lewi, P.J., Smeyers-Verbeke, J.,
Handbook of Chemometrics and Qualimetrics: Part A, Amsterdam, Elsevier Science, 1997, 711-716.
Acknowledgements
Agencia Nacional de Investigación e Innovación (ANII) for Scientific Initiation Grant – ANII-2841). Dr. Harald Berndt
for providing the TS-FF-AAS system.
Dra. María H. Torre and Agropecuaria Valdense S.R.L for the seeds samples.
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12th International Symposium on Metal Ions in Biology and Medicine
Escamoles ant eggs of Liometopum apiculatum M source of metal ions for human health
Melo, V.1; Calvo, C.2; Quirino, T.1; Macín, S.1; Muñiz, I.1
1
Universidad Autónoma Metropolitana-X. Calzada del Hueso 1100, Edif. Central, 1er. Piso, Coyoacán, 04960, D.F. México.
[email protected]
2
Instituto Nacional de Ciencias Médicas y Nutrición SZ. Vasco de Quiroga 14. Tlalpan, 14000, D.F., México.
Abstract
The earth crust is believed to be made of a mass of minerals and the participation of these elements in the physical
world is matched by their importance in human life. The human body like other leaving organisms depend on
several minerals as essential constituents of its existence. Metal ions in foodstuff are in different chemical forms, as
inorganic salts or organic molecules or complexes with other compounds such as proteins, amino acids, enzymes
and some vitamins, among others, that play an important role in human health. Entomophagy, insect consumption
by several ethnic groups in México as cultural tradition since prehispanic era, represents an option for population to
obtain the minerals needed by the body to keep a good health. Escamoles ant eggs of the Liometopum apiculatum
M genus, much appreciated either at rural communities as well as in urban cities, contain minerals with a favorable
effect in human health. The aim of this study was to investigate the mineral composition of Escamoles and the
benefits they can provide to the human body. Sampling was perform at an arid region of the Hidalgo state on April
2012. Minerals in dry basis were determined by Atomic Absorption Spectrophotometry, with the exception of
phosphorus, content was obtained from a triple acid digested extract and determined colorimetrically. Data of
mineral analysis in dry basis of Escamoles was: total minerals 5.92%; Na 0.079%; K 0.075%; Ca 0.097%; P
0.701%; Fe 0.021%; Zn 0.035%; Cu 0.009%; Mg 0.998%; Mn 0.002%. Minerals quantify are not equal to total ash
contained because not all of them were determined. Elements concentration depends not only of the total mineral
composition of foodstuff but also on their availability and avail of it. Minerals in Escamoles ant eggs have a
considerable influence in the condition of human health.
Introduction
Entomophagy is traditional in México since ancient times, escamoles ant eggs of the Formicidae family are part of
the cultural diet in rural communities and urban restaurants, consider as delicacies in México. Some other coutries
of Latin America, Asia and Australia ant eggs are much appreciated as well by their delicate flavor. [1]. Ant eggs
provide a good amount of macronutrients and micronutrients such as minerals, these insects reproduced at
desertic regions at an altitud range from 1800 to 3000 mts. above sea level in underground nests depth 1 to 1.5
mts, to keep low humidity and warm environment for natural develop of eggs. Nests are at xerophit ticket, near by
maguey agave sp or nopal opuntia sp cactus, as source of food sugars, amino acids and minerals. Ants mantain a
relationship with their habitat diversity and preserve an equilibrium of the ecology where they leave. [2]
Poor nutrition is a problem worldwide and is getting worse every day [3]. The natural available sources of food are
often less considered, whereas research of new technologies for synthesis and conservation, raises prices and
make the products inaccesible to the majority of population, particularly social groups from rural communities and
urban cities. Poor nutrition affects an individual´s performance and makes him/her vulnerable to different chronic
degenerative diseases [4,5,6]. Mineral deficiency represents a serious problem in the development and behavior of
the human being [7,8,9]. Absence of knowledge of both the edible species and their nutritional value, determine
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12th International Symposium on Metal Ions in Biology and Medicine
lack of consumption and decreases potential food benefits from a large variety of organisms existing in the
environment.
The escamoles ant eggs of the Liometopum apiculatum M found at an arid regions of México and several other
countries worldwide, are consider as a food source with a good nutritional value of metal ions that could contribute
to improve human health of vulnerable groups. The aim of this is to assess mineral composition of the Liometopum
apiculatum M ant eggs and the benefits they can provide to the human body [10]
Material and Methods
Seasonal escamoles ant eggs reproduced from the second week of February to the end of May at Cardonal,
Hidalgo State, arid zone situated at 2100 masl, in a xerophit ticket environment of about 2,5 km, with a BS kw
climate, they were harvested early in the morning, with special techniques, by escamoleros, local country men. The
samples (450 g of eggs and few adults each time) were collected at February 11, March 30 and May 4, of 2012.
Samples captured from nests near by maguey cactus, were washed and transported by land in plastic containers
to the laboratory of Universidad Autónoma Metropolitana–X, to determin the taxonomy in adult insects and the
mineral analysis in eggs. Samples were analysed separately and by triplicate each one.
Material obtained was divided, into: adult ants and ant eggs. Adult ants were used to determine taxonomy and ant
eggs to perform analysis for chemical quantification of metal ions [11,12] The moisture content was measured by
simple drying in an oven at 80º C for 24 h. The dry product was powdered in a Willey Mill to 60 mesh size. The fine
powder so obtained was used for further analysis. Total minerals content were determined by incineration in a cold
muffle furnance set a 550º C by 2 h or until whitish/greyish ash was obtained. Individual ions, Na, K, Ca, Fe, Zn,
Cu, Mg and Mn, were analysed by atomic absorption spectrophotometry, and P content in acid digested extracts
was determined colorimetrically.
Results and Discussion
The Escamoles ant eggs are classified as an invertebrate, class insecta, order Hymenoptera, family Formicidae,
genus Liometopum, specie apiculatum M (Table 1) the moisture content is high (Table 2). The total minerals in
escamoles was 5.92 g/100g dry basis. There is a difference between the value of the total inorganic matter
obtained and the individual ions reported because not all elements content were assessed (Table 3, 4). The
availability of Escamoles ant eggs are seasonal (Table 5). The data obtained might change accorging biotic and
abiotic conditions of environment.
Table 1. Taxonomy determination of Escamoles
Class
Insecta
Order
Hymenoptera
Family
Formicidae
Genus
Liometopum
Specie
apiculatum M
Common name
Escamoles
Morón, M.A., Terrón, R. 1980. [22]
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12th International Symposium on Metal Ions in Biology and Medicine
Table 2. Moisture content of Escamoles %
Moisture
65.93
Dry matter
34.07
Table 3. Metal ions content in Escamoles ant eggs g/100g dry basis
Sodium
0.079
Potasium
0.075
Calcium
0.097
Phosphorus
0.701
Iron
0.021
Zinc
0.035
Copper
0.009
Magnesium
0.998
Manganesum
0.002
Tabla 4. Escamoles availability on year at Cardonal, Hidalgo State.
Season
Months
Winter
Jan
Spring
Feb
Mar
Apr
May
X
X
X
X
Summer
Jun
Jul
Aug
Autumm
Sep
Oct
Nov
Dec
The magnitude of the under-nutrition problem, due to lack of metal ions, has reached significant proportions in the
world population, mainly in developing countries like México. The inadequate consumption of foods containing
adequate amounts of metal ions is closely related to phisical and mental development and the good health of
population [7]. Sodium and potasium are important ions in the functioning of the sodium-potasium pump [13].
Copper fulfills key metabolic functions in different organs and systems [14,15]. Iron is important in avoiding different
degrees of anemia which reduce the capacity of the individual work, another characteristic is the incapacity to
mantain body temperatura in a cold environment. Low consumption of iron produces a deficiency in psychomotor
development and intelectual activity, changes in the behavior of breast-fed infants up to two years of age and a
lower resistence to infections. Iron deficiency induces a substantial increase in risk of greater absorption of lead,
however, excessive administration of iron competes with absorption of zinc and copper that fulfills key metabolic
functions in different organs and systems [16]. Zinc a cell component is important for regulatory capacity of cells
since its intracelular concentration can be homeostatically controlled in a specific way for each tissue. It also has
functional and structural actions for many metalloenzymes and macromolecules [17,18,19]. Calcium and
phosphorus are indispensable for the formation of bone. Although phosphorus also acts as a structural part of high
energy compounds, the amounts of calcium and phosphorus necessary to mantain metabolic equilibrium depend
on the physiological needs of these metal ions and the capacity of the intestine to absorb them and the possibility
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of the kidneys conserving them [20]. Magnesium is an enzyme cofactor that intervenes in the metabolism of
carbohydrates chalesterol and proteins [21].
Conclusion
The Escamoles ant eggs are seasonal but for a further consumption can be storaged refrigerated douring one
month. After that period of time they should be frozen. Most people intake Escamoles by their sensory
characteristics not for their nutritional value. It is very well accepted by population and can be intake either raw or in
different preparations. Consumption of this foodstuff is recomended for all population groups in low and high
density population regions, to void degenerative diseases and improve health.
References
1. Hopkins, J. 2004. Extreme Cusine. Periplus Editions Ltd. Singapore.
2. Speight, M.R., Hunter, M.D. and Wat, A. 1999. Ecology of Insects. Blacwell Science Ltd. London, UK.
3. Pelletier, D.L., Olson, C.M., and Fronguillo, EA. 2006. Food Insecurity. Hunger and undernutrition in: B.A.
Bowman and R.M. Russell (eds) Present Knowledge in Nutrition. ILSI Press, Washington DC. USA.
4. Chavez, A. and Chavez M. M. 2009. Nutrición su impacto en la salud humana y en la capacidad funcional.
Instituto Nacional de Ciencias Médicas y Nutricion Salvador Zubiran. México.
5. Chavez M. M. et al, 2010. Tablas de valor nutritivo de los alimentos en México. International Mc Graw Hill.
México.
6. FAO 1995. Conferencia Mundial Cumbre 50 Aniversario. FAO World Conference Report, Quebec, Canada.
7. Reilly, C. 2004. The Nutritional Trace Metals. Blackwell Publishing Ltd. Oxford, UK.
8. Conor, R 2006. The nutritional trace metals. Blackwell Publishing Ltd. Oxford, UK.
9. Crabb, E and More, E. 2010. Metals and Life. RSC Publishing. Cambridge, UK
10. Melo, V. Reyes, J, Castejon, E., Nogueda, N. 2006. Metal in three species of edible insects in México. Metal
Ions in Biology and Medicine Proceedings of the 9th International Symposium on Metal Ions in Bology and
Medicine Vol 9. Editions John Libbey Eurotext, France.
11. AOAC 2003. Official Methods of Analysis of AOAC. International. 17th Ed. Association of oficial Analitical
Chemists Publication. Washington D.C. USA.
12. Cantle, J.E. 1982. Techniques and Instrumentation in: Analytical Chemistry Vol 5 Atomic absorption
spectrophotometry, Elsevier Amsterdam.
13. Preus, H.G. 2006. Electrolites: Sodium Chloride and Potasium In: B.A. Bowman and R.M. Russell (Eds)
Present Knowledge in Nutrition ILSI Press. Washington D.C. USA.
14. Wapnier, R.A. 1998. Copper absorption and bioavailability. American J. Clin. Nutr 67: 1054s – 1060s.
15. Frausto da Silva, J.J.R., and Williams, R.J.P. 2001. The Biological Chemistry of the Elements, Oxford
University Press. Oxford, UK.
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16. Pennintongton, J.A. and Yong, T.B. 1990. Iron, Zinc, Copper, Magnesium, Selenium and iodine. In: Foods from
The United State Total Diet Study. J. Food Comp. Anal. 13:495-503.
17. Mc Call, K.A.H., Chin, C. and Fierke, C.A. 2000. Function and Mechanism of Zinc metalloenzymes. J. Nutr.
130:1437s-146s.
18. Greger, J.L. 1987. Mineral bioavailability/New Concepts. Nutrition today 22,4-9.
19. Food and Nutrition Board, Institute of Medicine 1997. Dietary Reference Intakes for Calcium, Phosphorus,
Magnesium, Vitamin D and Fluoride. Accademic Press Washington, D.C. USA.
20. Weaver, C. 2006. Calcium distribution and function in Body. In: B.A. Bowman, and R. M. Russell (Eds). Present
Knowledge in Nutrition. ILSI Press. Washington D.C. USA.
21. Franz K. B. 1989. Influence of Phosphorus on intestinal absorption of Calsium and Magnesium. In: Y. Itokawa
and J. Durlach. (Eds.) Magnesium in health and disease. John Libbey and Co. London.
22. Morón, M.A., Terrón, R. 1980. Entomología Práctica. Instituto de Ecología, A.C. México.
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Park9 interaction with Manganese and other divalent cations
Zoroddu, M.A.a*; Remelli, M.b; Peana, M.a; Medici, S.a; Solinas, C.a
a
Department of Chemistry and Pharmacy, University of Sassari, Sassari (Italy),
Department of Chemical and Pharmaceutical Sciences, University of Ferrara, Ferrara (Italy),
*
Email: [email protected]
b
Abstract
Two peptide sequences from Park9 Parkinson’s disease gene, P1D2E3K4H5E6L7 (1) and F1C2G3D4G5A6N7D8C9G10
(2) have been studied in their interaction with Mn(II) and Zn(II) ions. These fragments lie from residue 1165 to 1171
and from 1184 to 1193 in the Park9 encoded protein, that can protect cells from manganese poisoning, an
environmental risk factor for a Parkinson’s disease-like syndrome called Manganism. The study was carried out
through potentiometric and spectroscopic (UV-Vis, EPR, mono- and multidimensional NMR) techniques, to cast
light on the details of metal binding at different pH values and different ligand to metal molar ratios.
Keywords: Parkinson, Manganism, divalent cations
Introduction
Recently, a connection between genetic and environmental causes of Parkinson’s disease (PD) has been reported
[1,2]. It was known that environmental and occupational exposure to Mn(II) [3] can lead to symptoms that resemble
Parkinson’s disease, called Parkinsonism or Manganism [4], but two articles appearing almost at the same time in
the literature showed that a human PD gene, PARK9 (a member of the P5-type ATPase family, perhaps a metal
transporter, considering its richness in coordinating residues), and its homologue in yeast, YPK9, can prevent
manganese- induced PD and protect neurons and cells from manganese poisoning [1]. In fact, deletion of the
YPK9 gene, which is 58% similar and 38% identical in its amino acid sequence to human PARK9, confers
sensitivity to growth defects in the presence of cadmium, nickel, selenium, and manganese, suggesting that YPK9
protein may play a role in the sequestration of divalent heavy metal ions. In the same way, a mutation on PARK9
may expose humans to these cations, especially to manganese. We have thus decided to verify the possibility of
metal binding to some simplified portions of PARK9 and YPK9, by choosing promising sites for metal interaction
due to the presence of coordinating residues, far from the membrane-spanning and the alpha-helical rich domains
not easily accessible by metals, and taking into account highly conserved binding motifs on both PARK9 and
YPK9. We identified two interesting fragments and tested them for metal binding with manganese and zinc [5,6].
Materials and methods
Peptides were synthesized using solid-phase Fmoc chemistry in an Applied Biosystems Synthesizer. Peptides
were N-terminally acetylated and C-terminally amidated in order to mimic this region of YPK9 within the full-length
protein.
NMR experiments were performed on a Bruker AscendTM 400 MHz spectrometer equipped with a 5 mm automated
tuning and matching broad band probe (BBFO) with z-gradients. Samples were 2.5 or 5 mM in concentration and
dissolved in 90/10 (v/v) H2O/D2O solutions, at 298 K in 5 mm NMR tubes. NMR data were processed with TopSpin
(Bruker Instruments) software and analyzed by Sparky 3.11 MestRe Nova 6.0.2 (Mestrelab Research S.L.)
programs.
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Potentiometric titrations were performed with Orion EA 940 and Orion 720A pH-meters equipped with a combined
glass electrode (Metrohm EA125), Hamilton MicroLab M and MicroLab300 motor burettes, and Hamilton syringes.
The temperature was kept at 298.2 ± 0.1 K by use of a thermostat. All measurements were carried out under
nitrogen atmosphere.
EPR spectra were recorded with an X-band (9.4 GHz) Bruker EMX spectrometer, 100 kHz field modulation, at 120
K on neat aqueous solutions or mixed with few drops of ethylene glycol, at different ligand to metal molar ratios,
ranging from 1:0.2 to 1:1, pH 7 and 1 mM peptide concentration.
Results and discussion
Peptide 1, P1D2E3K4H5E6L7
Only mono-nuclear complexes have been potentiometrically detected in the systems containing Mn(II) and the
investigated peptides, and the speciation diagrams are in agreement with the results found with NMR, UV-vis and
EPR measurements.
Mn(II) is a paramagnetic ion, thus the effect of its addition to peptide 1 and 2 was monitored through the NMR line
broadening and/or disappearance it causes on the resonances of the free ligand (Fig.1).
1
13
Fig. 1 Selection of aliphatic regions in H- C HSQC NMR spectra for the Ac-P1D2E3K4H5E6L7- Am peptide, 2.5 mM,
pH 6.2, T 298 K in the absence (orange) and in the presence (blue) of 0.1 equivalents of Mn(II).
As expected whenever a histidine residue is present on a peptide fragment coordinating a metal, this was the
favoured binding site for both Mn(II) and Zn(II) ions with peptide 1.
From the different sets of experiments performed, it was clear that once His5 has anchored the peptide at slightly
acidic pH, other residues are involved in coordination as the pH is raised. Mn(II) progressively affected the
relaxation rates of the amidic HN protons from the backbone together with the complete disappearance of Asp2,
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Glu3 and Glu6 aliphatic protons signals in the 2D TOCSY spectra, and a decrease or total loss of their proton–
carbon correlations in the 2D 1H-13C HSQCs.
Moreover, the analysis of the aliphatic HSQC region revealed that Qγ protons of Glu3 and Glu6 vanished upon
Mn(II) addition, indicating that glutamic carboxylic groups are involved in the coordination, a fate shared also by the
aspartate residue. All the other signals remained unaffected upon manganese addition. From the analysis of the
data thus collected, the donor groups involved in metal coordination might be the Nε nitrogen from the imidazole
ring of His5, and the γ-O from three carboxylic moieties of Asp2, Glu3 and Asp6 side-chains (Fig 1).
EPR spectra of the Mn(II) complexes reflect the symmetry of the electronic environment of the ion which is imposed
by the ligands in the primary coordination sphere. Changes in the composition or geometric arrangement of the
ligands in the coordination sphere of the metal ion lead to changes in the position of the EPR spectral lines. The
Mn(II)–peptide 1 spectra are almost indistinguishable from those of free Mn(II). The spectral identity indicated that
the interaction, in aqueous solution, between the ligand and Mn(II) ions does not affect the electronic structure of
the metal ion itself. Thus, it is possible that the peptide does coordinate to Mn(II) by displacement of aqua ligands;
however, the six donor set and coordination geometry is almost maintained so that the zfs parameters remain
nearly unchanged. The spectra are in agreement with an octahedral or distorted octahedral geometry, probably
involving bidentate interaction of carboxyl groups carboxyl groups (Fig. 2b).
Fig. 2 a) The model of the YPK9 protein built with ESyPred3D using the 3D crystal structure of the sodiumpotassium pump (PDB 3B8E chain A) as the template, and the 3D structural models proposed for b) the Mn(II) ion
complexed with Ac-P1D2E3K4H5E6L7-Am and c) with the Ac- F1C2G3D4G5A6N7D8C9G10-Am peptide.
Diamagnetic zinc ion was studied in order to have more information about the coordination behaviour of both
investigated peptides. In fact, we were able to observe that the signals affected by diamagnetic shift under zinc
interaction were the same which experienced paramagnetic broadening under manganese interaction, suggesting
that the donor atoms involved in the coordination with peptide 1 are the same for both metal ions. In addition, a
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change in the resonances for HN and Hγ2 of Lys4, and for HN of Leu7 shows that these two residues, although not
directly involved in the complex formation, experience a new electronic environment after metal coordination.
Peptide 2, F1C2G3D4G5A6N7D8C9G10
Coordination of manganese to peptide 2 starts with the binding to a cysteine residue, as evidenced by the
broadening of its signals already at very low metal concentration. So, in the absence of a histidine residue, cysteine
acts as the anchoring site for the metal ion. In fact, Mn(II) progressively affected the relaxation rates in the
resonances of both Cys2 and Cys9 with a complete disappearance of their signals in the 2D TOCSY spectra and
1
13
the total loss of their proton–carbon correlations in the 2D H- C HSQCs (Fig. 3).
1
13
Fig. 3 Aliphatic regions of H- C HSQC NMR spectra of the Ac-F1C2G3D4G5A6N7D8C9G10-Am peptide, 2.5 mM, pH
6.3, T 298 K, in the absence (orange) and in the presence (blue) of 0.1 equivalents of Mn(II).
Asp4 and Asp8 residues experience the main difference in the observed Mn(II)-induced effects for the carbon and
proton frequencies, indicating that they take part, together with the cysteine residues, in the complex formation,
most likely through their carboxyl groups (Fig. 2c). The presence of the paramagnetic metal provides a signal
relaxation also for HN protons of Gly5, Ala6 and Asn7 residues. This behaviour could be due to an indirect
paramagnetic line broadening effect caused by a through-space relaxation of these residues along the peptide
chain which happen to be close to the Mn(II) centre.
The EPR spectra of Mn(II)–peptide 2 complex and of free Mn(II) ion in aqueous (glass) solution show that they are
qualitatively the same in the resolved sextet region, but the overall breadth of the pattern is a little different,
indicating that the axial zfs parameter is different for the Mn–peptide species. This can be explained by a decrease
in the number of water ligands around Mn(II) in the presence of the peptide fragment, suggesting their replacement
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12th International Symposium on Metal Ions in Biology and Medicine
by oxygen and sulphur donor atoms. Diamagnetic NMR with Zn(II) ions evidenced that the residues involved in
metal coordination are those experiencing the strongest chemical shift differences: Cys2 and Cys9. Other residues
appear to be indirectly interested in the complex formation by slight perturbations: Phe1, Gly3, Asp8 and Gly10.
Gathering the results from the NMR experiments we are able to infer that zinc coordination to peptide 2 involved
the sulphur donors from the two cysteine side chains, forming a macrochelate complex. It is interesting to point out
that the involvement of the two carboxylic groups from Asp4 and Asp8, in contrast to manganese coordination, has
not been evidenced for Zn(II) coordination, thus indicating that, in this case, the behaviour of peptide 2 is different
as a function of the different metal ion.
Conclusions
The preliminary results of the study on these two Park9 protein fragments show that both metals are able to bind
them in an effective way, although with different coordination features depending on the fragment chosen,
evidencing that portions of the same protein can interact in different ways with different ions. Although the
fragments used are small and only represent a part of the entire protein, nevertheless this kind of study may
provide insight in the divalent cations trafficking (transportation, detoxification) within the cell, at the base of the
connection between genetic and environmental Parkinson’s disease causes. Further investigations, involving
bigger domains of PARK9/YPK9 protein, are planned to shed light on the role of the entire protein in sequestering
manganese and in the involved detoxification mechanism.
References
1) Gitler AD, Chesi A, Geddie ML, Strathearn KE, Hamamichi S, Hill KJ, Caldwell KA, Caldwell GA, Cooper AA,
Rochet JC, Lindquist S. Alpha-synuclein is part of a diverse and highly conserved interaction network that includes
PARK9 and manganese toxicity. Nat Genet. 2009; 41; 308-15
2) Schmidt K, Wolfe DM, Stiller B, Pearce DA. Cd2+, Mn2+, Ni2+ and Se2+ toxicity to Saccharomyces cerevisiae
lacking YPK9p the orthologue of human ATP13A2. Biochem Biophys Res Commun. 2009 ; 383; 198-202
3) Covy JP, Giasson BI. α-Synuclein, leucine-rich repeat kinase-2, and manganese in the pathogenesis of
Parkinson disease. Neurotoxicology. 2011; 32; 622-9
4) Perl DP, Olanow CW. The neuropathology of manganese-induced Parkinsonism. J Neuropathol Exp Neurol.
2007; 66; 675-82
5) Medici S, Peana M, Delogu LG, Zoroddu MA. Mn(II) and Zn(II) interactions with peptide fragments from
Parkinson's disease genes. Dalton Trans. 2012 ;41; 4378-88
6) Remelli M, Peana M, Medici S, Delogu LG, Zoroddu MA. Interaction of divalent cations with peptide fragments
from Parkinson's disease genes. Dalton Trans. 2012; DOI: 10.1039/C2DT32222F
Acknowledgments
This work was also supported by the Regione Autonoma Sardegna FSE Sardegna 2007–2013 L.R.7/2007
“Promozione della ricerca scientifica e dell’innovazione tecnologica in Sardegna” programme, the L.R.7/2010
programme and the Fondazione Banco di Sardegna.
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12th International Symposium on Metal Ions in Biology and Medicine
Effect of zinc supplementation on the production of IL-17 in elderly individuals
Aguilar, A.E.1; Sanchez, M.T.C.1; Muñoz, B.1; Espejel, G.2; Saldivar, L.2; Pastelin, R.1
1
2
School of Chemistry, Biology Department, Analytical Chemistry Department, University of Mexico, UNAM, Mexico DF 04510,
Mexico
Abstract
The suboptimal contribution of micronutrients may damage the immune response. In the particular case of zinc, this
microelement has shown their participation in various immunological processes around perinatal stages. In the
elderly, eating lesser amounts of food can cause a negative balance in the concentration of zinc and its possible
effects on immunity. In this regard, it is interesting to know the impact of zinc may have on proinflammatory
cytokines such as IL-17 for its role in delayed hypersensitivity and its possible effect in the induction of
autoimmunity. This study aimed to evaluate the effects of zinc supplementation on the production of IL-17 over
elderly stages. For this, we used an experimental model of zinc supplemented mice (500 microgram/mL zinc
acetate, BALB/c inbred mice). We measured the microelement concentration in lymphoid organs by Atomic
Absorption Spectrometry and also the cytokine production in serum by double antibody enzyme-immuno assay.
Results showed that female group of supplemented mice diminished the cytokine production 34.35 % compared
with control group whereas male group of supplemented animals showed a noticeable reduction of 54.34 %.
Further, zinc concentration increased in the spleen of female supplemented mice (80%). We concluded that
supplementation of zinc in mice has the effect of causing decreased production of IL-17 by Th17 cells, which far to
produce a problem of autoimmunity this means a regulatory response to supplemented individuals.
Keywords: zinc, IL-17, elderly
Introduction
Zinc has various effects on the immune system, acting on the maturation of CD4+ and CD8+ T lymphocytes and
serum concentrations of this element are modified in autoimmune diseases. In case of deficiency causes an
imbalance in the function of the Th1 and Th2 cells in the periphery due to the reduced production of IFN-γ and IL-2
while the production of Th2 cytokines such as IL-4, IL-6 and IL-10, is not affected.[1]
Interleukins are a group of proteins that act as chemical messengers because these are the main intracellular
communication in a microbial attack. Cytokines serve to initiate the inflammatory response, and to define the extent
and nature of the specific immune response. Its main function is to regulate the events belonging to the functions of
the different populations of immune system cells, such as activation, differentiation or proliferation of various cell
types, antibody secretion, chemotaxis and regulation of other cytokines and factors. [2]
Some IL-17 studies have shown that IL-17/1L-17RA system is essential for host defense in various extracellular
bacterial infections. About the fungi infections, the Th cell activation occurs by 17 polysaccharides constituting the
cell wall. [3] Another important point is that in the absence of the signaling of IL-17, the influx and activation of
neutrophils in the infected organs undergoes serious delay, which means that there is not properly control of the
infections.[4] However, this cytokine overproduction can trigger autoimmune disorders like rheumatoid arthritis,
autoimmune encephalomyelitis, multiple sclerosis and psoriasis. [5]
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12th International Symposium on Metal Ions in Biology and Medicine
In the elderly, nutritional deficiency can cause a negative balance in the concentration of zinc and its possible
effects on immunity. In this regard, it is interesting to know the impact of zinc may have on proinflammatory
cytokines such as IL-17 for its role in delayed hypersensitivity and its possible effect in the induction of
autoimmunity. [6, 7]
This study aimed to evaluate the effects of zinc supplementation on the production of IL-17 over elderly stages.
Material and Methods
We used an experimental model in inbred mice. Balb/cAnN, male and female animals, received zinc acetate (500
microgram/mL, Mallinckrodt-Baker, Mexico, Cat 8740)) in drinking water for two weeks. Controls did not drink
supplement. The animals were divided into groups according to the received supplementation (Zn 0, Zn500), and
considering the gender of the animal. All mice were kept 7 months (28 weeks) in plastic boxes with stainless steel
covers. For water intake were used glass bottles and glass drinking tubes. The animals were fed ad libitum with
commercial diet 2018S HarlanTeklad Global Diets (USA). This study was performed according to the Guide for the
Care and Use of Laboratory Animals.
The Zn concentrations in thymus and spleen were obtained by AAS technique. In order to validated the method
and results we utilized the NIST (Standard Reference Material, 1577b, bovine liver).
To study the effect of zinc supplementation on the production of IL-17 and its detection in serum was assayed a
double antibody ELISA (PeproTech 900-M392), where antibodies anti-IL-17 were produced in rabbits and the
detection antibodies were biotinylated; as developing agent was employed the avidine-peroxidase system plus
ABTS (Sigma Cat A3219) and absorbance were measured to 405 nm in a Behring EL 301 microreader, with
correction absorbance at 630 nm.
Finally, statistical analysis was performed using GraphPad Prism software version 6.01
Results and Discussion
Aging is associated with damaged immune response which is linked with several immune related diseases in the
elderly. Nutritional intervention could be a promise for modulating immunity. [7]
Our studies showed that in vivo supplementation increases zinc concentration in the spleen and remains constant
the absorption in the thymus, and gender differences were observed because the trace element is localized mainly
in the spleen of elderly supplemented female group, with an increase of 80%, whereas in male individuals remains
mostly in the thymus, with an increase of 70%. Figure 1
This difference can be explained by the observation that the effect of zinc concentration is hormonal dependent
and the age of animal is critical to the analysis of results.
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We observed the typical proportional relationship between the absorbance and the concentration of murine IL-17,
in pg / mL. Figure 2
Figure 2. Murine IL-17 standard curve
The experimental groups were supplemented with zinc acetate (2H2O), in drinking water at 500 microgram/mL for
two weeks, showing a decline of IL-17 in serum (Figure 3A) being the diminish of 34.35% in the group of elderly
females and (Figure 3B) and 54.34% for elderly male mice. These data have some coincidence with the tendency
reported by Kitabayashi et al [1]. However our results indicated biological aspects which we need to investigate in
depth eg cytokine cellular concentration.
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12th International Symposium on Metal Ions in Biology and Medicine
Figure 3A, B. Murine IL 17 in pg/mL obtained in serum from supplemented groups compared with the controls
Conclusion
We concluded that supplementation of zinc in mice has the effect of causing decreased production of IL-17 by
Th17 cells, which far to produce a problem of autoimmunity this means a regulatory response to supplemented
individuals.
References
1. Kitabayashi Ch, Fukada T, Kanamoto M, Ohashi W, Hojyo Sh, Atsumi T, Ueda N, Azuma I, Hirota H, Murakami
M, Hirano T. Zinc suppresses Th17 development via inhibition of STAT3 activation. Int Immunol. 2010, 22: 375–386
2. Kindt TJ, Osborne BA, Goldsby RA, Kuby J. Immunology. Freedman WH Publisher, USA, 2007. pp 236-238
3. Louten J, Boniface K, de Waal Malefyt R. Development and function of TH17 cells in health and disease. J
Allergy Clin Immunol. 2009, 123:1004-1011
4. Korn T, Oukka M, Kuchroo V, Bettelli E. Th17 cells: Effector T cells with inflammatory properties. Seminars in
Immunology. 2007,19:362–371
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12th International Symposium on Metal Ions in Biology and Medicine
5. Hemdan NYA, Birkenmeier G, Wichmann G, Abu El-Saad AM, Krieger T, Conrad K, Sack U. Interleukin-17producing T helper cells in autoimmunity. Autoimmunity Rev. 2010, 9:785–792
6. Haase H and Rink L. The immune system and the impact of zinc during aging. Immunity and Ageing. 2009, 6:117
7. Pae M, Meydani S N, Wu D. The role of nutrition in enhancing immunity in aging. Aging and disease. 2012, 3:91119
8. National Research Council Guide for the Care and Use of Laboratory Animals. National Academy Press. Mexico
2002
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12th International Symposium on Metal Ions in Biology and Medicine
A novel Cu(II) ternary complex with iminodiacetate and dimethyl-bipyridine. Synthesis,
characterization and DNA interaction
a
c
a
b
a
Alvarez, N. ; Costa-Filho, A. ; Torre, M.H. ; Ellena, J. ; Facchin, G. .
a
c
Cátedra de Química Inorgánica, Facultad de Química, Universidad de la República, Montevideo, Uruguay.
b
Instituto de Física de São Carlos, Universidad de São Paulo, São Carlos, Brasil.
Faculdade de Filosofia Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, Brasil.
[email protected]
Abstract
The importance of metal complexes in medicine dates from the XVIth century with reports on the use of
metallodrugs in anticancer therapy. Inorganic Medicinal Chemistry has made great advances in search of new
metal compounds presenting antitumor activity, for instance, by use of essential metals. In this work, in the search
of new antitumor agents, the ternary complex [Cu(iminodiacetate)(4,4’-dimethyl-2,2’-bipyridine)]—4H2O was
prepared employing the reaction of copper acetate with iminodiacetic acid in presence of dimethyl-bipyridine. The
obtained complex was characterized by elemental analysis, single crystal X-ray diffraction, Fourier Transform
Infrared spectroscopy (FT-IR), Electron Paramagnetic Resonance (EPR) and UV-visible spectroscopy (UV-vis).
The complex crystallizes in a triclinic unit cell, in the centrosymmetric space group P-1. The Cu(II) center is bonded
to the bypiridine ligand -through its N atoms- and to the iminodiacetate ion -through the carboxylate O atoms and
the imine N atom-. The molecular structure was confirmed by FT-IR where bands involving the ligand and donor
atom-Copper bonds are observed. Knowing the atom array in solid state is considered fundamental for further
aqueous solution analysis and comprehension of the complex behavior in biological media. UV-vis absorption
bands in aqueous solution are consistent with a square pyramidal Cu(II) environment, which suggests the
maintenance of the coordination sphere observed in solid state. The ability of the complex to interact with
deoxyribonucleic acid (DNA) was studied by Circular Dichroism (CD) in constant concentration of DNA with
increasing concentration of complex. The profile observed in the CD spectra evidences complex-DNA interaction
which is consistent with an intercalative interaction, as observed in other complexes containing dimethyl-bipyridine.
Keywords: copper complex, crystal structure, DNA interaction.
Introduction
One of the target biomolecules for therapeutics with metallodrugs in cancer is the deoxyribonucleic acid (DNA). If
the complex binds to the DNA or participates in any form of interaction, it could alter its structure, impeding the
cellular system's ability to correctly read the genetic code, which can result in the interruption of protein synthesis or
synthesis of non-functional proteins. Any of these results leads eventually to cell death.
In the search of metallodrugs with fewer side effects, one of the strategies in Inorganic Medicinal Chemistry is to
synthesize coordination compounds with essential metals. The main advantage in the use of essential metals is
due to pre-existing biodistribution and excretion pathways. However, their therapeutic use should be controlled
since in excess they become toxic. In particular, different series of Cu(II) complexes have shown promising
antiproliferative activity in different tumor cell lines. Among them, the heteroleptic complexes with di-imine ligands
denominated Casiopeínas® have completed preclinical requirements and complexes CasII-gly [(Cu(II))(4,7dimethyl-1,10-phenanthroline)(glycinate)(NO3)(H2O)]
and
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CasI-II-ia
[(Cu(II))(4,4’-dimethyl-2,2’-
12th International Symposium on Metal Ions in Biology and Medicine
bipyridine)(acetilacetonate)(NO3)(H2O)] entered phase I clinical trials [1; 2]. The proposed mechanism for the latter
is DNA oxidative damage, as well as damage to other biomolecules and organelles, through production of reactive
oxygen species (ROS) in the presence of a reducing agent [3; 4].
The research group where the investigation takes place has experience in synthesis and characterization both in
solid state and aqueous solution of Cu(II) coordination complexes of pharmacological interest. The ability of these
complexes to interact with DNA has also been assessed. Antiproliferative studies of Cu-dipeptide and Cudipeptide-phenanthroline complexes in different tumor cell lines show promising results [5; 6].
In this work the study of a new ternary Cu(II) complex with iminodiacetic acid (ida) and dimethyl-bipyridine (dmb) is
presented.
Materials and Methods
Materials
All reactants, 4,4’-dimethyl-2,2’-bipyridine (Sigma, 99%), iminodiacetic acid (Sigma, 98%), Copper (II) acetate
(Sigma, 98%), as well as organic solvents and distilled water were used without further purification.
Synthesis and analytical characterization
To 10 mL of a 25 mM aqueous solution of Copper (II) acetate with constant stirring at 50°C, were added 10 mL of a
25 mM ethanolic solution of dimethyl-bipyridine and 10 mL of a 25 mM solution of iminodiacetic acid deprotonated
by NaOH (pH=7). Single crystals were grown by slow evaporation of solvent from the resulting solution at 4°C and
its crystal structure determined by X-ray diffraction. Anal. Calc. for C16H25CuN3O8: N: 9,32; C: 42,62; H: 5,59.
Found: N: 9,06; C: 42,93; H: 5,09.
Crystal structure determination
Data from suitable single crystals was collected at 298(2) K in a Enraf-Nonius FR590 Kappa-CCD diffractometer
using graphite monochromated MoKα radiation (0,71073 Å). The structure was solved by direct methods and the
model refined with SHELXL-97. Gaussian absorption correction was applied. All non-hydrogen atoms were refined
on F2 by full-matrix least-squares procedure using anisotropic displacement parameters. Hydrogen atoms for C-H
groups were stereochemically positioned, whereas imino hydrogen and water hydrogen atoms were found in the
difference Fourier map and positionally fixed. All hydrogen atoms were refined using the riding-model. The values
of the refinement indicators are: Goodness of fit: 1,070 (1 restrain), R1: 0,038 (I>2σ(I)) and wR2: 0,102 (I>2σ(I)).
Spectroscopic measurements
FT-IR spectra were measured on a Bomen MB 102 instrument in the range 4000-400 cm-1. Electronic absorption
spectra on aqueous solution were recorded on a Shimadzu UV-1800 in the range 200-900 nm using 1 cm quartz
cells. X-band (9.5 GHz) EPR measurements were carried out on polycrystalline samples and frozen aqueous
solution using a Bruker ELEXSYS E580 system with 100 kHz field modulation. Measurements were performed at
N2 liquid temperature (77 K).
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12th International Symposium on Metal Ions in Biology and Medicine
DNA-complex interaction
DNA-complex interaction was characterized by circular dichroism in a JASCO J-815 spectrometer in the 200-300
nm range. Dichroism spectra of 1.5 mM DNA aqueous solution with increasing concentration of complex were
measured at 20°C.
Results and discussion
Crystallographic data analysis
The coordination compound studied crystallizes in the spatial group P-1. Cell parameters are: a=8.0498(11),
b=10.8189(17), c=12.5674(14), α=67.215(7), β=88.141(5) and γ=84.128(8). The coordination sphere surrounding
the Cu(II) ion has in the equatorial positions three nitrogen atoms (two from the bipyridine ligand and one from the
iminodiacetate ion) and one oxygen (from the terminal carboxylate of the iminodiacetate ion), whereas the apical
position is occupied by the remaining oxygen from the iminodiacetate ion.
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12th International Symposium on Metal Ions in Biology and Medicine
a)
b)
Figure 1. (a) Representation of the complex [Cu(ida)(dmb)]—4H2O. (b) Crystal packing.
The crystalline arrangement is sustained by intermolecular hydrogen bonding (N-H•••O y H-O•••H) between water
molecules, carboxylate and imine groups as well as π-π interactions between the aromatic rings in the bypiridine
ligand (Figure 2).
Spectroscopic measurements
FT-IR spectroscopy confirms the coordination scheme observed in the crystal. Some of the most representative
stretching and rocking modes are: ν(C-H), ν(O-H) in 2900-3500 cm-1, ν(COO-)as in 1620 cm-1, ν(C-C), ν(C-N) in
-1
-1
-1
-1
1450, 1493 cm , δ(C-C-C), δ(C-C-N) in 921, 957 cm , aromatic δoop(C-H) in 839 cm , ν(Cu-N) in 514 cm and
-1
ν(Cu-N) in 425 cm .
Electronic spectrum of aqueous solution of the complex shows intraligand charge transferences in the ultraviolet
region and an asymmetric band at 656 nm (ε656=63 cm-1M-1) due to d-d electron transfer consistent with
pentacoordinated Cu(II) centers, suggesting that the coordination scheme observed in solid state is preserved in
solution.
EPR spectra both in solid state and frozen aqueous solution do not show any hyperfine structure. This is probably
a consequence of exchange interactions between the paramagnetic copper ions. In these cases, exchange
interaction averages out other interactions such as hyperfine.
DNA-complex interaction
The CD curves corresponding to DNA and DNA plus complex are shown in Figure 2. DNA concentration during the
whole experiment is fixed and the concentration of the complex is increased in each measurement. Even though
the general profile of the curve is similar it can be observed that after addition of the complex intensities around the
bands at 215, 244 and 270 nm vary due to an induced CD spectrum. In the case of the band at 244 nm the
intensities increase and have a shift to lower wavelengths, whereas in the case of the band at 270 there is no
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12th International Symposium on Metal Ions in Biology and Medicine
wavelength shift but the intensities decrease. This behavior is consistent with an intercalative mode of binding,
which has been previously observed for other complexes with dimethyl-bipyridine [7].
12
8
θ (mdeg)
4
0
-4
DNA
9 mM
25 mM
38 mM
49 mM
-8
-12
200
220
240
260
280
300
λ (nm)
Figure 2. Circular Dichroism spectra of DNA with increasing concentrations of complex.
Conclusions
The crystalline structure of a new heteroleptic complex [Cu(II)(ida)(dmb)]—4H2O with square-based pyramidal
geometry was determined. The molecular motif of the complex is maintained in aqueous solution. The complex
interacts with DNA, probably by intercalative binding.
Acknowledgements
The authors thank the Agencia Nacional de Investigación e Innovación (INI_X_2011_1_3940, N. Alvarez), Instituto
de Física de São Carlos (Winter internship, N. Alvarez) and PEDECIBA Química for financial support.
References
[1] J. Serment-Guerrero, P. Cano-Sanchez, E. Reyes-Perez, F. Velazquez-Garcia, M.E. Bravo-Gomez, L. RuizAzuara, Genotoxicity of the copper antineoplastic coordination complexes casiopeinas®. Toxicol In Vitro 25 (2011)
1376-1384.
[2] M.E. Bravo-Gómez, J.C. García-Ramos, I. Gracia-Mora, L. Ruiz-Azuara, Antiproliferative activity and QSAR
study of copper(II) mixed chelate [Cu(N–N)(acetylacetonato)]NO3 and [Cu(N–N)(glycinato)]NO3 complexes,
(Casiopeínas®). J Inorg Biochem 103 (2009) 299-309.
[3] A. Marı́n-Hernández, I. Gracia-Mora, L. Ruiz-Ramı́rez, R. Moreno-Sánchez, Toxic effects of copper-based
antineoplastic drugs (Casiopeinas®) on mitochondrial functions. Biochem Pharmacol 65 (2003) 1979-1989.
[4] X. Zhong, J. Yi, J. Sun, H.L. Wei, W.S. Liu, K.B. Yu, Synthesis and crystal structure of some transition metal
complexes with a novel bis-Schiff base ligand and their antitumor activities. Eur J Med Chem 41 (2006) 1090-1092.
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[5] G. Facchin, E. Kremer, D.A. Barrio, S.B. Etcheverry, A.J. Costa-Filho, M.H. Torre, Interaction of Cu-dipeptide
complexes with Calf Thymus DNA and antiproliferative activity of [Cu(ala-phe)] in osteosarcoma-derived cells.
Polyhedron 28 (2009) 2329-2334.
[6] N. Alvarez, S. Iglesias, R. Sapiro, M.H. Torre, G. Facchin, Antioxidant and pro-oxidant properties and
antiproliferative activity of homoleptic and heteroleptic copper complexes, potential antitumoral species, Badawi,
(Ed.), Metal Ions in Biology and Medicine, John Libbey Eurotext, Cambridge, United Kingdom, 2011, pp. 117-122.
[7] Y.S. Wu, K.R. Koch, V.R. Abratt, H.H. Klump, Intercalation into the DNA double helix and in vivo biological
activity of water-soluble planar [Pt(diimine)(N,N-dihydroxyethyl-N'-benzoylthioureato)]+Cl- complexes: a study of
their thermal stability, their CD spectra and their gel mobility. Arch Biochem Biophys 440 (2005) 28-37.
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Towards the development of new copper compounds for the treatment of cancer: Study
of the cytotoxic activity of [Cu(L-dipeptide)(1,10-phenanthroline)]] complexes
Iglesias, S.a; Noble, C.a; González, R.b; Torre, M.H.a; Kremer, E.a; Kramer, G.b; Facchin, G.a
a
Cátedra de Química Inorgánica, Facultad de Química, Universidad de la República, Montevideo, Uruguay.
Departamento de Desarrollo Biotecnológico, Instituto de Higiene, Facultad de Medicina, Universidad de la República,
Montevideo, Uruguay.
[email protected], [email protected]
b
Abstract
Coordination compounds of essential metals are being explored as new antitumor species, in the search of drugs
with expanded activity and/or reduced side effects. To date, several Cu(II) compounds have shown promising
antitumor activities in cellular lines, some of them are being studied in vivo. Their mechanisms of action are not
completely understood; however, it is well known that copper and its complexes, present the ability to participate in
redox reactions. This event generates reactive oxygen species (ROS) that are toxic to cancerous cells. In addition,
many of these complexes present the ability to intercalate DNA, interfering with DNA replication of dividing cells,
which may also be part of their mechanism of antitumor action. We have synthesized several new [Cu(Ldipeptide)(1,10-phenanthroline)] compounds. In solid state they present a pyramidal geometry, where the Cu(II) ion
is coordinated both to the phenanthroline, through its nitrogen atoms, and to the dipeptide, through the amine and
the amide nitrogen atoms and the carboxylic oxygen. These compounds are stable in aqueous solution, where its
coordination is maintained. In this work we have focused on the study of the lipophilicity and antitumor activity of
[Cu(L-dipeptide)(1,10-phenanthroline)] (were L-dipeptide= phe-phe, phe-val, phe-ala, ala-phe, gly-val, ala-gly). The
cancer cell lines utilized were HeLa (human cervical cancer), NMU (rat breast cancer) and 4T1 (mouse breast
cancer). The lipophilicity, depends on the dipeptide, being the [Cu(L-Phe-Phe)(1,10-phenanthroline)] complex the
most lipophilic. The estimated IC50 varied from 1 to 30 µM in these tumor cells, being the complexes containing the
dipeptides phe-val, phe-ala and ala-phe the ones that showed best cytotoxic activity in vitro. Therefore, complexes
[Cu(L-dipeptide)(1,10-phenanthroline)] with dipeptides phe-val, phe-ala and ala-phe, may be good candidates to
test its antitumor activity in animal models of cancer.
Keywords: copper phenanthroline dipeptide complex, lipophilicity, antiproliferative activity.
Introduction
Cisplatin, a coordination compound of Pt(II), is a widely used antitumor drug [1]. In search for new antitumor active
metal coordination complexes with expanded activity and/or reduced side effects, several copper (II) complexes
have been studied and reported to be promising as anticancer drugs. Copper is an essential metal therefore there
are specific metabolic routes of detoxification which may lead to complexes with less severe side effects. The
mechanisms of action of copper complexes are not completely understood; however, two early molecular events
are commonly accepted. One of them is the ability of copper and its complexes to participate in redox reactions
producing reactive oxygen species (ROS) that are toxic to cancerous cells, which are especially sensitive to
oxidative stress. The other one is the ability to intercalate or coordinate with the DNA, interfering with DNA
replication of dividing cells, which may also be part of their mechanism of antitumor action.
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12th International Symposium on Metal Ions in Biology and Medicine
Cu(I) complexes with the metal chelator bis-1,10-phenanthroline (phen) were synthesized and evaluated by
Sigman et al.. Especially, [Cu(phen)2]+ was reported to oxidatively degrade DNA and RNA by attacking the sugar
groups. The presence of large quantities of an external reductant, mediated the generation of freely diffusible OH
radical through the redox cycling between the Cu(II)/Cu(I) couple [2].
In spite of their large study, the biological application of bis phen copper complexes presents a restriction: their
stability is rather unfavorable under physiological conditions, because of the small association constant of the
second phen. One way to overcome this problem is to prepare mixed ligand complexes including only one phen
and additional ligands that form a stable complex [2]. An outstanding example of this approach are the complexes
denominated Casiopeinas, developed by L. Ruiz-Azuara et al., which are entering phase I human clinical studies
[3].
Our research group has developed different series of new mixed copper complexes, including phen and derivatives
as ligands. In particular, a series of new [Cu(L-dipeptide)(phen)] compounds has been synthesized and
characterized. They present a pyramidal geometry, where the Cu ion is coordinated both to one phen, through its
both nitrogen atoms, and to the dipeptide, through the amine and the amide nitrogen atoms and the carboxylic
oxygen. Its coordination is maintained in aqueous solution, where they are stable [4]. As the complexes present a
bulky lipophilic ligand, they may access lipophilic sites, such as solid tumors.
In this frame, the present work describes the study of the lipophilicity and evaluation of antitumor activity of [Cu(Ldipeptide)(1,10-phenanthroline)] (were L-dipeptide= phe-phe, phe-val, phe-ala, ala-phe, gly-val, ala-gly) in breast
cancer and cervical carcinoma cell lines.
Materials and Methods
Synthesis and analytical characterization
Copper complexes with the L-dipeptides (Sigma): phe-phe, phe-val, phe-ala, ala-phe, gly-val, ala-gly, and 1,10-ophenantroline (Sigma) were synthesized and characterized as reported previously [4]. The obtained stoichiometries
[Cu(L-dipeptide)(1,10-phenanthroline)] were determined by Elemental Analysis (Fig.1).
Figure 1. Representation of the solid state structure of [Cu(phe-phe)(1,10-phenanthroline)]
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12th International Symposium on Metal Ions in Biology and Medicine
Lipophilicity
Lipophilicity tests were performed determining the partition coefficient of the complexes in n-octanol/ physiological
solution. The copper concentration was measured by atomic absorption spectroscopy using a Perkin-Elmer 5000
instrument, with a Photron lamp for copper analysis.
Antiproliferative studies
Cell lines were obtained from the American Type Culture Collection (ATCC): HeLa (CCL-2™, human cervical
adenocarcinoma), NMU (CRL-1743™, mammary adenocarcinoma induced in Sprague Dawley rat), 4T1 (CRL2539™ mammary adenocarcinoma from BALB/c mouse). Cells were grown in Dulbecco's Modified Eagle Medium
(DMEM, Gibco) supplemented with 10% Fetal Bovine Serum (FBS) and removed enzymatically from T25 flasks
using 0.01% trypsin. Incubations of cultured cells were at 37°C on 5% CO2 atmosphere. Cell viability in response to
the Cu complexes was determined by colorimetric assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) and absorbance of converted dye was measured at 570 nm as specified by manufactures (SigmaAldrich).
Results and discussion
Lipophilicity
Table 1 presents the partition coefficients (Kp) between n-octanol and physiologic solution. The general trend
observed in the tabulated Kp-values for [Cu(dipeptide)(phen)] complexes suggests that lipophilicity increases with
ligand complexity. This trend follows a well known behavior, according to which in a homologous series the
partition coefficients increase by added CH2 or phenyl groups. In spite of this, [Cu(phe-ala)(phen)] is more lipophilic
than [Cu(ala-phe)(phen)], showing the importance of the overall arrangement of the ligands in the complex in
aqueous solution.
Table 1. Partition coefficients (Kp) between n-octanol and physiologic solution
Compound
Compound code
Kp
[Cu(phe-phe)(phen)]
1
4.12
[Cu(phe-val)(phen)]
2
0.59
[Cu(phe-ala)(phen)]
3
0.30
[Cu(ala-phe)(phen)]
4
0.18
[Cu(gly-val)(phen)]
5
ND
[Cu(ala-gly)(phen)]
6
ND
ND: not detected
Antiproliferative studies
Cell viability was determined by MTT Cell Proliferation Assay. MTT is a yellow substrate that can be reduced by
dehydrogenase enzymes in the mitochondria of living cells, giving a purple compound (MTT formazan).The amount
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of solubilized MTT formazan produced is directly proportional to the number of living cells [5]. An estimation of the
IC50 at 24 h was calculated using closer to 50% cell viability data and considering a linear correlation.
Table 2, 3 and 4 presents the mean percentage value of cell viability obtained for 4T1, NMU and HeLa cancer
cells, respectively, incubated for 24 and 48 hours in the presence of the indicated concentrations of the studied
complexes. Experiments were done in duplicate and standard deviation was less than 10 % of each value.
Table 2. Percentage of cell viability measured on 4T1 breast cancer cell line and IC50 estimated at 24 h.
5 µM
20 µM
100 µM
IC50 µM
Compound
24 h
48 h
24 h
48 h
24 h
48 h
24 h
1
40.13
2.08
0.64
0.44
0.73
0.73
4.2
2
46.26
2.53
1.23
0.82
0.36
0.36
4.7
3
28.81
1.45
5.00
0.19
0.45
0.45
3.5
4
17.50
1.45
0.68
0.63
0.55
0.55
3.0
5
105.63
82.23
30.68
2.31
1.17
1.17
14.4
6
59.32
8.10
1.75
0.84
0.68
0.68
6.1
7
113.4
124.31
108.93
106.13
23.37
23.37
65.3
Table 3. Percentage of cell viability measured on NMU breast cancer cell line and IC50 estimated at 24 h.
5 µM
20 µM
100 µM
IC50 µM
Compound
24 h
48 h
24 h
48 h
24 h
48 h
24 h
1
1.87
4.85
3.20
0.37
50.13
3.48
<5
2
2.40
0.87
2.67
0.50
57.07
0.99
<5
3
3.20
0.50
2.13
0.50
9.33
1.12
<5
4
4.00
1.37
3.20
0.37
20.00
1.49
<5
5
2.68
1.01
16.96
0.78
141.07
253.27
<5
6
2.38
0.89
3.27
0.34
100.89
15.86
<5
7
116.96
65.66
128.87
178.78
31.85
79.51
73.4
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12th International Symposium on Metal Ions in Biology and Medicine
Table 4. Percentage of cell viability measured on HeLa cervical cancer cell line and IC50 estimated at 24 h.
5 µM
20 µM
100 µM
IC50
Compound
24 h
48 h
24 h
48 h
24 h
48 h
24 h
1
4.49
4.47
4.33
3.35
13.98
6.70
<5
2
5.66
4.47
6.32
9.50
15.97
6.33
<5
3
5.49
4.47
6.99
6.70
7.32
9.31
<5
4
5.99
5.21
5.66
5.59
19.30
4.84
<5
5
7.16
2.70
12.08
2.43
94.41
101.08
<5
6
6.71
3.24
4.03
1.89
16.11
7.30
<5
7
13.42
10.81
33.56
27.84
9.40
4.05
-
We found that all Cu complexes present antitumor activity in the three studied cancer cell lines. Among them, the
complexes containing the dipeptides phe-val, phe-ala and ala-phe showed best antitumor activity in vitro. However,
we did not found a clear relationship between lipophilicity and antiproliferative activity. Possibly other factors as the
redox potential or the ability to interact with DNA influence the antiprolifferative activity.
Conclusions
The dipeptide present in the [Cu(L-dipeptide)(1,10-phenanthroline)] compounds determine lipophilicity, being
[Cu(L-Phe-Phe)(1,10-phenanthroline)] the most lipophilic. Among all the complexes studied, the ones containing
dipeptides phe-val, phe-ala and ala-phe, showed best antitumor activity in cancer cell lines and therefore, these
compounds may be good candidates to test its antitumor activity in vivo.
Acknowledgements
The authors thank PEDECIBA Química for financial support. We also thank members of both laboratories for
helpful technical advices.
References
1.
Florea, A.-M. and D. Büsselberg, Cisplatin as an Anti-Tumor Drug: Cellular Mechanisms of Activity, Drug Resistance
and Induced Side Effects. Cancers, 2011. 3: p. 1351-1371.
2.
Tisato, F., et al., Copper in Diseases and Treatments, and Copper-Based Anticancer Strategies. Medicinal Research
Reviews, 2010. 30(4): p. 708-749.
3.
Alemón-Medina, R., et al., Induction of oxidative damage by copper-based antineoplastic drugs (Casiopeínas). Cancer
Chemother Pharmacol, 2007. 60: p. 219-228.
4.
Alvarez, N., G. Facchin, and M. Torre, Antioxidant and pro-oxidant properties and antiproliferative activity of homoleptic
and heteroleptic copper complexes, potential antitumoral species. Metal Ions in Biology and Medicine, 2011: p. 117-122.
5.
Cole, S.P., Rapid chemosensitivity testing of human lung tumor cells using the MTT assay. Cancer Chemother
Pharmacol, 1986. 17: p. 259 -263.
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12th International Symposium on Metal Ions in Biology and Medicine
Preliminary study of superoxide dismutase activity in soybean seeds with good and poor
in vitro viability used in Uruguay
1
2
3
2
1
1*
Cardoso, J. ; Machado, I. ; Irigoyen, J.O. ; Pistón, M. ; Viera, I. ; Torre, M.H.
1
2
Química Inorgánica and Química Analítica, DEC, Facultad de Química, UdelaR, Gral. Flores 2124, CC1157, Montevideo,
Uruguay
3
Agropecuaria Valdense (J.J. Ferreira), Colonia, Uruguay
*e-mail: [email protected]
Abstract
Reactive oxygen species (ROS) such as superoxide and hydroxyl radicals are present in seeds, plants and animals
as a result of normal aerobic metabolism. If the organisms are not able to control the intracellular ROS level, they
may suffer damage in membrane lipids, proteins and nucleic acids leading to the death of the cells. For
detoxification of ROS, efficient antioxidant systems both enzymatic and non-enzymatic ones are present in all cells.
-
Superoxide dismutases (SOD) can react with O2 radical to prevent the damage. They are metalloproteins where
Cu, Mn, Zn or Ni are linked to the peptide residues. Therefore when the metal levels are low the activity of these
enzymes decrease and different disorders can appear.
The SOD extraction from seeds was optimized by means of multivariate experiments (best conditions: stirring time
60 min using 10 mL of phosphate buffer pH=7).
The SOD activity was determined by the method based on the inhibitory effect of SOD over the reduction of NBT
by the O2- generated by xanthine/xanthine oxidase system.
SOD activity of soybean seeds used in our country was in the range 20-63 %, similar in all the analyzed seeds. The
results showed that no significant difference was observed in seeds with viability less than 95%. In these
conditions, the poor viability of soybean seeds used in our country is not explained by oxidative stress.
I) Introduction
Loss of viability during plant seeds storage is an important problem in agriculture that produces great economic
losses. There are different factors producing changes in seed viability. One of them is the presence of reactive
-
-
oxygen species (ROS) including the superoxide radical (O2 ), hydroxyl radical (OH ) and hydrogen peroxide (H2O2),
in high levels [1]. These bioproducts are generated as a result of normal aerobic metabolism. If the organisms are
not able to control the intracellular ROS level, these species can produce damage in membrane lipids, proteins and
nucleic acids leading to cell death [2].
It is now known that the stress produced in seeds by drought, high temperatures, salinity, toxic metals, among
others, can cause molecular damage directly or indirectly through the formation of ROS [1,3-5].
To scavenge ROS and to avoid oxidative damage, efficient antioxidant systems both enzymatic and non-enzymatic
ones are present in all cells. One of them is the superoxide dismutases (SOD) that catalyze the reaction 2O2- +
+
2H → H2O2 + O2. These enzymes are Cu and Zn, Mn, Fe or Ni dependant and it is well known that if the metal
levels are low the activity of these enzymes decreases and different disorders can appear [2]. Besides there are
differences in the SOD levels depending on seed genotype, location, climatic and storage conditions and presence
of toxic metals, among others [4,5].
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12th International Symposium on Metal Ions in Biology and Medicine
With these antecedents, our work was to measure the SOD activity on soybean seeds with good and poor in vitro
viability previously determined with the aim to observe a possible correlation between SOD activity and viability and
provide new information about regional soybean seeds.
II) Experimental
Materials
Reagents and solvents were commercially available research-grade chemicals and were used without further
purification.
Seed samples
The soybean seeds were provided by Agropecuaria Valdense (Juan Jorge Ferreira), a seed importing company,
from Colonia (Uruguay).
Samples from 5 different batches of seed were analyzed: 3 of them with good viability and 2 presenting poor
viability. All tests were run in duplicate.
Enzyme extraction method
The SOD method of extraction from seeds was optimized by means of multivariate experiments (central composite
design) [7]. The variables were stirring time and volume of extraction and were analyzed in three levels. The effect
of Polyvinylpyrrolidone (PVP) concentration was also studied.
After the development of the method of extraction, the following technique was performed:
For each sample, 1 g of ground and dried (80ºC, 24 hours) soybean seeds was weighed and mixed with 0,2 g of
PVP. After that, 10 mL of extraction phosphate buffer, pH 7, was added. The homogenate was stirred for 1 hour,
centrifuged at 13,000 rpm for 15 min and finally extracted using a Sephadex G-25 column. The eluate was
collected in 10 tubes of two mL each one. These solutions were used to estimate protein concentration
(spectrophotometric method, A280) and initial superoxide anion level (as reduction of NBT, A
560)
(data not shown),
and to evaluate the SOD activity for the most concentrated tube.
SOD activity determination
The SOD activity was determined by the method based on the inhibitory effect of SOD over the reduction of NBT
-
by the O2 generated by xanthine/xanthine oxidase system, adapted from Beauchamp and Fridovich’s method [8].
The test tubes contained 0.1 mL of the sample, 0.2 mL of 4.6 mM xantine, 0.1 mL of 0.75 mM NBT, 0.1 mL of 3
-1
-1
mM EDTA, 0.1 mL of 1.50 mg mL bovine albumin, 0.1 mL of 10 mg prot. mL and buffer phosphate (pH=7.4) until
2 mL.
After the incubation at 28ºC, the reaction was stopped with 0.1 mL of 6 mM CuCl2 and the absorbance of the blank
solution and reaction solutions were measured with a spectrophotometer Thermo Scientific mEvolution60 at 560
nm.
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12th International Symposium on Metal Ions in Biology and Medicine
SOD activities were calculated with the equation: SOD activity (%) = (1 − A/B) × 100, being A= absorbance of
sample and B= absorbance of blank, taking into account the initial O2- level.
III) Results
III-1) Enzyme extraction method
The different experimental conditions of the extraction method and the result of the eluate in tube 4 are presented
in Table 1.
Table 1. Absorbance in different experimental conditions
Stirring Time (min)
Volume of extraction (mL)
Concentration (AU*) Tube 4
15
10
6,028
30
10
6,050
60
10
9,966
60
20
3,003
30
20
3,388
30
40
1,131
15
40
1,133
* AU = A280 x f(including dilution factor and absortivity)
As expected, more concentrated solutions are obtained when the volume is reduced and time of stirring is
increased.
The effect of changing the volume beyond 10 mL is more important than changing the time.
The analysis of the multivariate experiment is shown in Figure 1.
Figure 1. 3D Surface Plot, central composite design, two variables, three levels
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12th International Symposium on Metal Ions in Biology and Medicine
According to the 3D surface plot the conditions tested did not include the maxima of the curve for any variable. In
spite of this no more changes in experimental conditions were tried due to several reasons. First of all, any of the
protein concentrations obtained in these experiments proved adequate to perform both superoxide anion level and
SOD activity determinations by the method used. An increase in time would result in having to dilute the solution for
afore mentioned determinations.
On the other hand working below 10 mL, extraction mixture forms a semi-solid paste, very difficult to stir
homogenously.
Another variable was PVP quantity. The experiments showed that more than 1g meddled with the extraction
mixture and made it very difficult to separate from the solution in the column.
After the development of the technique, the elution profile was performed in all the experiments. Absorbance at 280
nm showed that the eluate in the fourth tube was the most concentrated in protein content for all samples.
III-2) SOD activity determinations
The results obtained in the SOD activity tests for seed sample 003 are presented in Figure 2.
Figure 2. Protein concentration and SOD activity for seed sample 003.
As the graph shows, tube 4 presented both the maximum protein concentration and maximum SOD activity. Similar
elution profile and SOD activity was observed in the other samples.
The preliminary SOD activity results for all the studied samples are shown in Table 3.
-
Table 3. Mean inhibition (%) of NBT reduction by the O2 generated by xanthine/xanthine oxidase system, in tube 4
Seed
001
002
003
V6,2
009
%
20
23
63
55
62
As Table 3 shows, the SOD activity of soybean seeds used in our country was in the range 20-63 %. To our
knowledge, there is no information about SOD activity in soybean seeds in the region but it is known that SOD
activity changes with latitude and genotype. For this reason, this preliminary study can be an interesting
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12th International Symposium on Metal Ions in Biology and Medicine
antecedent. Studies performed on other seeds than those used in Uruguay showed SOD activities in the range of
our results[5].
No differences were found between seeds with good or poor viability. Consequently, in these conditions, the poor
viability of soybean seeds used in our country is not explained by oxidative stress.
Further studies are necessary to investigate this aspect.
Biobliography
[1] Somone García J., Lupino Gratao P., Antunes Azevedo R., Zezzi Arruda M.A. Metal contamination effects on
sunflowers (Helianthus annuus L.) growth and protein expression in leaves during development. J Agric Food
Chem; 2006; 54; 8623-8630.
[2] Kaim W., Schwederski B. Bioinorganic Chemistry: Inorganic Elements in the Chemistry of Life. Chichester:
J.Wiley & Sons, 1994.
[3] Cabrini L., Barzanti V., Cipollone M., Fiorentini D., Grossi G., Tolomelli B., Zamboni L., Landi L. Antioxidants
and total peroxyl radical-trapping ability of olive and seed oils. J Agric Food Chem; 2001; 49; 6026-6032.
[4] Wang G., Huang J., Gao W., Lu J., Li J., Liao R., Jaleel C. A. The effect of high-voltage electrostatic field
(HVEF) on aged rice (Oryza sativa L.) seeds vigor and lipid peroxidation of seedlings. J Electrostatic; 2009; 67;
759-764.
[5] Bao X., Bing-chang Z., Qin-hua L., Jing-liang. Study on the superoxide dismutase of soybean seeds from
different species in subgenus soja. Acta Botanica Sinica; 1989; 31(7); 517-522.
[6] Kranner I., Colville L. Metals and seeds: Biochemical and molecular implications and their significance for seed
germination. Environ Exp Botany; 2011; 72; 93-105.
[7] Massart D. L., Vandeginste B. G. M., Buydens L. M. C., De Jong S., Lewi P.J., and Smeyers-Verbeke J.
Handbook of Chemometrics and Qualimetrics: Part A. Amsterdam: Elsevier Science, 1997.
[8] Beauchamp C., Fridovich J. Superoxide Dismutase: Improved assays and an assay applicable to acrylamide
gels. Anal Biochem; 1971; 44; 276-287.
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12th International Symposium on Metal Ions in Biology and Medicine
Metal ions and human diseases
Theophanides, T.1; Anastassopoulou, J.1; Dritsa, V.1; Pissaridi, K.2; Michalis, E.3
1
National Technical University of Athens, Chemical Engineering Department, Radiation Chemistry &Biospectroscopy, Zografou
Campus, 15780 Athens
2
Athens Institute for Education and Research, 8 Valaoritou Street, 10671 Athens
3
General Hospital, Messogion Av. Athens, Greece
Introduction
The metals are natural chemical elements and they were needed for life since the origin of life. Another aspect of
metals is the metals in everyday life.Without metals the computers, mobile phones and batteries could not exist.
The metals can be divided in three categories: 1) The life metals that are necessary for our health and life, for
example, the metal ions that are found in every human cell, i.e. Na+, K+,Mg2+ and Ca2+. In this category there are
also all the metal ions that are the life metals, e.g. iron, in haemoglobin, copper in copper-proteins,vanadium,
chrome, manganese, zinc, cobalt, molybdenum, etc. Iron makes our blood blue. 2) There are the very toxic metals,
such as arsenic, mercury, lead and cadmium and others and 3) There are the metals that have been used in
pharmaceuticals for several diseases, such as the antitumor coordination complex, cis-platinum, i.e. cisPt(NH2)2Cl2, discovered in 1965 by Barnett Rosenberg1 and second and third generation platinum complexes and
other metal complexes or metal salts used as pharmaceuticals such as the gold complexes for arthritis, etc.The
metals constitute almost 80% of all the elements of the Periodic Table.
4,5
The life metals and their usefulness in health and life are known for a long time . The metals sodium (Na),
potassium (K), magnesium (Mg) and calcium (Ca) are essential for all cells6,7. It is well known that the metal
sodium is very toxic and could kill a person immediately, if he tries to eat the metal, whereas when it is a cation,
+
Na
as in sodium chloride (NaCl) is not harmful at all. It is even very tasty. We use it in our food very often as
mineral nutrient, likewise for K, Mg and Ca. The metal, Na when is becoming an ion, is fundamental for our health,
both in cells and in our food (see equation [1]):
Na → Na + + e −
[1]
This sodium example is good evidence that a natural element can be toxic or good for our health, if it is
7
transformed chemically or energetically . The metals of the Periodical Table can be used in biology and medicine,
as nutrients or as pharmaceuticals or as toxic materials when they surpass the optimum concentration (Fig.1). In all
these categories the dose will determine their properties towards humans8.
Figure1. The double-bell function for the activity of all elements in our life.
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12th International Symposium on Metal Ions in Biology and Medicine
Patients and Methods
Materials: Blood serum and marrow were taken from 100 patients who had plasmacell dyscrasias (46-76 years).
They were characterized as IgG:4, IgA:1, IgM:1 and λ-light chains.The carotid and coronary arteries were taken
from 150 patients, who underwent endarterectomy, while 30 aortic valves were taken from patients, who underwent
surgical aortic valve substitution due to mineralization (45-85 years).
Methods: The spectra were recorded with a Nicolet 6700 thermoscientific spectrometer, equipped with an ATRFTIR technology. Each plot consisted of 120 co-added spectra at a spectral resolution of 4 cm−1 and the OMNIC
7.1 software was used for data analysis.
For the fine detection of aortic valve surfaces it was used the Scanning Electron Microscopy (SEM).The instrument
was from Fei Co, The Netherlands. It must be noted that there was not any coating of the samples with carbon or
gold. XRD was performed with a Simens D-500 X-Ray diffractometer based on an automatic adjustment and
analysis system. The diffraction interval was of 2θ 5o-80o and the scan rate was of 0.030o/s.
Results and Discussion
Metal ions found in cardiovascular systems
SEM-EDAX analyses showed that the atheromatic plaques were rich in foam cells, which were the preferential
sites of mineralization. In some atheromatic plaques were detected toxic metal ions, such as lead, silver, aluminum
and copper.Figures2A and C shows the morphology of atheromatic plaque of carotid artery as it was described by
SEM. From the electron density distribution, as it is plotted by pixel value (Fig. 2 B&D), in the mineral salts it
became clear that copper ions must bind to oxygen and nitrogen atoms of proteins most likely through chelation,
while calcium ions produced salts with the oxygen anions of phospholipids. This is indicated from the fact that in
the calcium salt the electron density is evenly distributed, as in an ionic salt, whereas in the case of copper there is
a hole in the middle showing a covalent bonding in the copper complex (see Fig.2, B and D).
The non-life metals, such as lead and silver, which were found at the arteries, were due to patient’s occupational
9,10
environment. It is well known from literature that copper induces oxidative stress, leading to atherosclerosis
. The
oxidized organic products that were formed during oxidative stress could be detected easily using the characteristic
absorption FT-IR bands and SEM analysis.
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12th International Symposium on Metal Ions in Biology and Medicine
Figure 2.A and C are SEM images of the carotid artery at different sites of the tissue. The squares show the
presence of Ca2+ and Cu2+ binding. B and D are the corresponding electron density environments at the square
surfaces using ImageJ software for the pixel analysis.
Figure 3.FT-IR spectra of 1: carotid artery, 2: coronary artery and 3: aortic valve
-1
The FT-IR spectra of the samples in the region 3000-2800 cm are shown in Fig. 3.The spectra showed significant
increase of the intensity of the bands,due to stretching vibrations of CH3 and CH2, suggesting that the permeability
of the membranes changed significantly (Fig.3). We propose that these changes could be related to free radical
reactions with lipid macromolecules, leading finally to a shortening of the chains of proteins. These findings are in
197
12th International Symposium on Metal Ions in Biology and Medicine
-1
accord with the appearance of the band at 3080 cm , assigned to C-H stretching vibration of the double bonds
related to LDL of patients. The band at 1735 cm-1 is assigned to aldehyde groups, which confirms the peroxidation
10
of LDL . The intensity of this band is also related to LDL of patients and could be used as a “marker band”.From
-1
the changes in the region of the infrared spectra, 1700-1500 cm , where the amide I and amide II of the proteins
absorb it was suggested that atherogenesis arises with breaking of intramolecular and intermolecular hydrogen
11
bonds of proteins and membranes .
-1
Finally, considerable changes were observed in the region 900-400 cm , which suggest that glutathioles (GRS), R-
S and R-SH were transformed into sulfones (S-S). In all patients’ spectra of aortic artery there were found bands in
the region, 1150-900 cm-1, which corresponded to the stretching vibrations of phosphate groups (vPO4), most likely
12
from hydroxyapatite . It was observed by XRD that in sites rich in magnesium concentration the hydroxyapatite
was amorphous. Magnesium cations (Mg2+) are known to regulate ATP metabolism and is cofactor in more than
300 intracellular enzymes13,14. It was described that Mg2+ inhibits the release of Ca2+ from the sarcoplasmic
2+
reticulum, blocks the influx of Ca
2+
competes with Ca
2+
into the cell by inactivating the Ca
channels in the cell membrane, and
at binding sites on troponin C and myosin, thereby inhibiting the ability of Ca2+cations to
15
stimulate myocardial tension .
Moreover, in the carotid and coronary arteries were detected molybdenum ions by the method of SEM-EDAX,
which indicates that the enzyme of xanthine (molybdenum-enzyme) is inert and that it does not reverse the
xanthine molecule into hypoxanthine16,17.
The influence of copper concentration in patients with plasma dyscrasias
Plasma dyscrasias is a malignant disease, which affects people usually after 50 years old. It refers in a series of
abnormalities of monoclonal immunoglobulin and includes the multiple myeloma and Waldenström’s
macroglobulinemia. In Figure 4 are shown the FT-IR spectra of a patient with plasma dyscrasias (1) and healthy
donor (2). It is important that the ratio of the intensities of Amide I/Amide II in the spectra of the patient is <1, due to
the influence of malignancy. In healthy people the ratio of the intensities Amide I/Amide II is found to be >1.
198
12th International Symposium on Metal Ions in Biology and Medicine
Figure 4.FT-IR spectra of blood plasma. 1: patient with plasma dyscrasias and 2: healthy donor.
Considerable changes were observed in the region, where the sugar moieties of DNA absorb. The intensity of the
band at 830 cm-1 increases in spectra of the patients and shifts to higher frequencies. These spectral changes in
the spectra of DNA are most likely due to DNA structural changes, maybe related to the high concentration of Cu2+
ions, which is known that strongly perturbs the sugar-phosphate group vibrations as well as the base vibrations
18,19
.
SEM-EDAX analyses showed that in the patients with plasma dyscrasias the relative concentration of copper
increases and varies between 7%-14%, while in the healthy donors the relative concentration was found to be
between 1% and 4,5%.
Summary
From all our research data it was shown that toxic metals could induce oxidative stress leading to atherogenesis.
The FT-IR spectra, in particular showed that the mechanism of plaque formation mustbe different in carotid and
coronary arteries from those of aortic valve mineral deposition.
In patients with blood diseases the influence of copper concentration compared to that of healthy subjects was
increased to double and even SEM-EDAX analysis showed that in the patients with plasma dyscrasias the copper
concentration varies between 7%-14%, while in the healthy donors the concentration is found between 1% and
4,5%, although no one of them had Wilson’s disease.
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200
Authors Index
Abbehausen, C., 82
Behar Cohen, F., 26
Adamek, D., 29
Bellanda, M., 63
Aguiar, R., 136, 138
Bellinger, D.C., 27
Aguilar Cardenas, A.E., 128
Benítez, J., 42, 104
Aguilar, A.E., 175
Beraldo, H. de O., 97
Aguilar, V.H., 46
Berchesi, A., 134
Alamiri, A., 36
Bergamini, F., 99
Alberto, R., 22
Bergamini, F.R.G., 82
Alemán, J., 107
Beyer, L., 84
Alemón, R., 15
Biestro, A., 55
Alonso, O., 60
Birriel, E., 104
Alvarez, C., 50
Bizzi, C.A., 32, 75
Alvarez, N., 41, 106, 180
Bollati- Fogolin, M., 76
Alvarez, S., 51, 52
Botta, B., 19
Álvarez-Valdés, A., 108
Botto, I.L., 142
Alves, F.R., 141
Bravo Gómez, M.E., 15
Anastassopoulou, J., 196
Brissos, R.F., 109
Arancibia, R., 35
Brodzka, R., 125
Arion, V.B., 36
Brown, E., 34
Aullón, G., 49
Büchel, G., 36
Azócar, M.I., 110
Cabello, C., 76
Baisch, P.R., 69, 144
Cabral, P., 62, 131, 132, 136, 138
Ballinas, G., 15
Cabrerizo, F., 100
Balter, H., 59, 61, 135
Calvo, C., 127, 165
Baran, E.J., 77, 88
Calzada, V., 132
Barbato, S., 73
Camacho, X., 132
Barg, G., 67, 143
Can, D., 22
Barh, G., 23
Cangelosi, V., 7
Bartesaghi, S., 64
Carballal, S., 65
Bastian, G., 13, 17
Cardoso, J., 126, 130, 191
Batinic-Haberle, I., 65
Carmo, A.M.L., 96
Batista, A., 83
Carnero, A., 108
Batista, A.A., 39, 111, 112
Carnizello, A., 83
Becco, L., 104, 109
Carvalho, F.A.O., 140
201
Carvalho, J.W., 141
Currier, J.M., 10
Carvalho, J.W.P., 80
Czyzycki, M., 29
Carver, P., 21
Da Costa Ferreira, A.M., 12
Castellano, E.E., 102
da Silva, E.G., 31
Castelli, R., 138
da Silva, S.A., 99
Castelli, S., 42
Dagher, J.M., 152
Castillo, I., 49
Dapueto, R., 136
Castro, W., 113
Dario, B.S., 12
Cebula-Obrzut, B., 105
De Almeida, R., 92
Cerecetto, H., 57, 58, 104, 134
de Araujo Fernandes, A.G., 47
Chabalgoity, J.A., 131, 132
de Azevedo Mello, P., 32, 75
Chammas, R., 136, 138
de Lima, A.P., 39
Chao, H., 93
de Paiva, R.E.F., 91, 94
Cipriani, M., 48
de Paula, F.C.S., 96
Clavijo, G., 116
de Paula, Q.A., 12
Colina-Vegas, L., 112, 113
Dědina, J., 10
Collery, P., 13
Deflon, V.M., 47
Collery, T., 13
Del Solar, V., 107
Cominetti, M.R., 111
Dematteis, S., 57
Comini, M., 103, 106
Demoro, B., 92
Comini, M.A., 63
Desideri, A., 42
Corbi, P.P., 82, 91, 94, 99
Desimone, M., 51
Corrêa, R., 83
Desmaele, D., 13
Correia, I., 104
Di Virgilio, A.L., 76
Cortés, F., 15
Diaz, L., 51
Cortes, S., 53
Dinamarca, N., 110
Costa Ferreira, A.M., 42
Dominguez, O., 129
Costa Pessoa, J., 92, 104
Dritsa, V., 196
Costa-Filho, A., 41, 86, 87, 180
Dupont-Soares, M., 69, 144
Courtois, Y., 26
Eberlin, M.N., 31
Cousillas, A., 18, 116, 117
Egusquiza, G., 76
Crócamo, N., 139
Ellena, J., 41, 83, 106, 180
Cruz, W.S., 97
Engler, H., 59, 135
Cubo, L., 108
Espejel, G., 128, 175
Cuin, A., 99
Espósito, B.P., 46
Curbelo, E., 48
Etcheverry, S., 88
202
Etcheverry, S.B., 76
Gomes, V. de C., 74
Evelson, P., 51, 52
Gómez, G., 110
Fabijanska, M., 105
González Baró, A.C., 89
Facchin, G., 41, 45, 55, 86, 87, 180, 186
González, L., 23
Farkas, E., 37
González, M., 23, 104, 134
Felice Guidugli, R.B., 31
González, M.J., 142
Fernadez, M., 131
González, R., 45, 186
Fernández, L., 56
Gonzatto, L., 119
Fernández, M., 104, 109, 132, 136, 138
Goso, C., 115
Fernández, S., 57, 58, 134
Graminha, A.E., 39, 111
Ferraesi Curotto, V., 89
Guidali, F., 106
Ferreira, J.C., 79
Guido, M., 124
Ferrer-Sueta, G., 65
Gumienna-Kontecka, E., 114
Fleitas, L., 63
Gutiérrez, A.G., 78
Flores, E.M.M., 32, 75
Gutiérrez, E., 132
Fontes, A.P.S., 96
Hardy, G., 55
Formiga, A.L.B., 91, 94
Hauser-Davis, R.A., 68, 74
Foster Mesko, M., 32, 75
Heinzen, H., 72
Franco, D.W., 102
Heller, T., 18
Freitag, L., 36
Hernández Gorritti, W.R., 84
Fuda, J., 133
Hernández-Lemus, E., 15, 78
Gagnon, Z.E., 66, 152
Höehr Nelci, F., 31
Galindo, R., 15
Holste, A., 30
Galusso, F., 55
Honorato, J., 111
Gambini, J.P., 131, 136, 138
Huertas, R., 73
Gambino, D., 15, 42, 48, 92, 103, 104, 109
Icimoto, M.Y., 79
Gamez, P., 109
Iglesias, S., 45, 87, 186
Garat, B., 104, 109
Irigoyen, J.O., 126, 130, 191
García Ramos, J.C., 15
Isaac, L., 66
Garcia, E., 69
Isaac; L.A.C., 152
Garcia, E.M., 144
Jansat, S., 120
Garcia, M.H., 103
Jeanny, J.C., 26
Gazzoli, D., 142
Juliano, C., 40
Gelpi, R.J., 52
Katkar, P., 42
Ghirga, F., 19
Katzin, A.M., 46
Giglio, J., 57, 58, 135
Klahn, H., 35
203
Knochen, M., 71, 72, 73, 160
Marcondes, M.Y., 79
Kordas, K., 20, 67, 124, 143
Marques, F., 92, 109, 136, 138
Kozlowski, H., 81, 114
Marti, M.A., 25
Kramer, G., 45, 186
Martínez Savio, G., 124
Kratzer, J., 10
Martínez-Alanis, P., 49
Kreimerman, I., 59
Martín-Santos, C., 107
Kremer, C., 54, 119
Marts, A.R., 36
Kremer, E., 45, 119, 186
Matoušek, T., 10
Krupp, E.M., 105
Maya, J.D., 35, 48
Lacerda, E de P.S., 39
Mebert, A., 51
Lafratta, A.E., 47
Medeiros, A., 103, 106
Lam, T., 152
Medici, S., 40, 81, 170
Lancellotti, M., 82, 94
Medrano, M.A., 108
Landeira-Fernandez, J., 74
Meini, M.R., 23
Lankosz, M., 29
Mejía, C., 15, 78
Lapier, M., 35
Melo Ruiz, V., 127
Lavaggi, M.L., 104, 134
Melo, V., 118, 129, 165
Leite, C.Q., 99
Mendoza, C., 120
León, I., 88
Menezes, A.C.S., 111
Leon, I.E., 76
Michalis, E., 196
Leonardi, N., 133
Mohsen, A., 13
Levin, P., 110
Molina, L., 53
Lima, B.A.V., 39
Molinari, M., 133
Lippert, B., 14
Mollo Filho, P.C., 31
Liu, H., 44
Mollo, A., 73
Llarrull, L., 23
Monahan, J.L., 152
Lyrio Tenorio Correia, C., 30
Mondelli, M., 83
Machado, I., 109, 126, 130, 191
Morais, T.S., 103
Macín, S., 127, 165
Morales, G., 71, 160
Magnani, N., 51, 52
Moreno, M., 131, 132
Mañay, N., 18, 50, 67, 115, 116, 117, 124, 143
Moreno, V., 15, 104
Manta, B., 63
Moreno-Esparza, R., 15
Manzanares,W., 55
Muccillo-Baisch, A.L., 69, 144
Manzur, J., 84
Muñiz, I., 127, 165
Mao, Z., 43
Muñoz, B., 128, 175
Marchini, T., 51, 52
Muñoz, H., 110
204
Murgida, D., 24
Peana, M., 40, 81, 170
Musil, S., 10
Pecoraro, V.L., 7
Nantes, I., 123
Pereira Testa, L., 18, 116, 117
Nantes, I.L., 79
Pereira, F. de C., 39
Nascimento, O., 123
Pereira, M.E.C., 89
Nascimento, O.R., 79
Pereira, N., 50
Navarro Marques, F.L., 47
Pereira-Maia, E.C., 95, 96
Navarro, M., 112, 113
Pericàs, M., 120
Navarro-Ranninger, C., 107
Pessôa, G. de S., 31
Navon, A., 114
Pessoa, J., 15
Neves, A., 16
Pianca Barroso, R., 86, 87
Noble, C., 45, 86, 87, 186
Picard, E., 26
Nóbrega, J. de A., 75
Pis Diez, R., 89
Ochocki, J., 105
Pissaridi, K., 196
Okamoto, A., 33, 146
Pistón, M., 71, 115, 126, 130, 160, 191
Olea Azar, C., 48
Pizzorno, P., 50
Oliveira Carvalho, F.A., 141
Plegaria, J., 7
Oliveira, V., 79
Porcal, W., 131, 136, 138
Oliver, P., 59, 135
Porro, V., 76
Osinaga, E., 135
Portela Luz, C., 47
Otero, L., 48, 92, 103
Potocki, S., 81
Paez, M.A., 110
Prieto, T., 123
Palacios, A., 23
Profirio, D.M., 94
Palacios, J., 129
Queirolo, E., 67, 124, 143
Palacios-Abrantes, J., 118
Quinn, T., 131
Palma-Tirado, L., 78
Quirino, T., 127, 129, 165
Parajón-Costa, B.S., 77, 89
Quiroga, A.G., 108
Parro, T., 108
Raab, A., 105
Pastelin, R., 128, 175
Raber, G., 105
Pastrana Alta, R.Y., 46
Radi, R., 8, 64, 65
Paula, F.C.S., 89
Radwanska, E., 29
Pavan, C., 63
Ragone, F., 100
Pavan, F., 99
Reina, M., 15
Pavel Sizemore, I.E., 152
Reisner, E., 36
Pavel-Sizemore, I., 66
Remelli, M., 40, 170
Paz Castillo, J., 84
Resende, J.A.L.C., 89, 97
205
Rey, A., 56, 57, 58, 134
Smolewski, P., 105
Rey, N.A., 89, 97
Soares, M.C.F., 69
Reyes, L., 56, 59, 135
Solinas, C., 40, 81, 170
Ribeiro, A. de S.B.B., 39
Soto, E.L., 142
Ribeiro, G.A., 12
Souza, M.L., 102
Riva, E., 132
Spitridonova, E., 84
Rocha, R., 74
Sterchook, R., 114
Rodrigues da Silva Júnior, F.M., 144
Sturlese, M., 63
Rogers, C., 34
Stýblo, M., 10
Roy, A., 67
Sucena, S., 91
Ruiz, G., 100
Suleiman, S., 22
Ruiz-Azuara, L., 15, 78
Svoboda, M., 10
Sabino, G.L., 12
Szabó, O., 37
Saldivar, L., 128, 175
Szczerbowska-Boruchowska, M., 29
Salgueiro, J., 133
Szebesczyk, A., 114
Sánchez Eguía, B., 49
Szenkier-Garcia, N., 114
Sanchez, M.T.C., 128, 175
Tabak, M., 80, 140, 141
Santi, E., 88
Tacchini, R., 55
Santi, L., 19
Tasat, D., 51, 52
Santoni, F., 13
Tavares, D., 83
Santos Júnior, J.C., 31
Tchounwou, P.B., 34
Sanz, I., 134
Tebo, A., 7
Sarniguet, C., 103
Tegoni, M., 7
Scarone, L., 106
Teixeira, V., 131
Schaumloffel, D., 30
Tejería, E., 58, 134
Schettino, B., 129
Telser, J., 36
Serra, G., 106
Terán, M., 56
Severo Fagundes Pereira, J., 32
Theophanides, T., 196
Shanzer, A., 114
Tholey, A., 30
Shen, Y., 22
Tierney, D.L., 36
Silva, I.M.P., 94
Tissot, F., 119
Silva, P.P., 95
Toloza, J., 48
Siqueira, S.A., 97
Tomaz, A.I., 103
Sixto, A., 72
Tomaz, I., 92
Smaili, S., 123
Torre, M.H., 41, 45, 55, 86, 87, 88, 106, 126, 130,
180, 186, 191
Smanioto Barin, J., 32, 75
206
Torres, J., 54, 119
Vilar, R., 120
Torti, H., 133
Vilche, M., 56
Trindade, V., 59, 135
Villarreal-Peña, W., 112
Trivedi, P., 66, 152
Wandzilak, A., 29
Trzcinka-Ochocka, M., 125
Wei, M., 13
Ugalde- Saldívar, V., 49
Wei, W., 122
Ulrich, A., 47
Wolcan, E., 100
Urbano, B., 118
Yañuk, J., 100
Vaisberg, A., 84
Yedjou, C.G., 34
Valderrama Negron, A.C., 46
Yefimova, M., 26
Valez, V., 65
Yu, F., 7
Varela, J., 104
Zabarska, N., 109
Vasilskis, E., 59, 135
Zanvettor, N.T., 82
Vázquez-Aguirre, A., 78
Zastrow, M., 7
Veiga, N., 54
Zezzi Arruda, M.A., 11, 31, 68, 74
Velluti, F., 106
Zhao, J., 122
Vieira, L.Q., 12
Ziolli, R., 74
Vieira, S., 42
Ziolli, R.L., 68
Viera, I., 88, 126, 130, 191
Zoroddu, M.A., 40, 81, 170
Viera, S., 73
Zubata, P., 133
Vila, A., 23
Zubillaga, M., 133
207