1. health and fitness
2. disease

MEDICAL UNIVERSITY − SOFIA
ACTA MEDICA
BULGARICA
2/2011
amb
vol. XXXVIII
This journal is indexed in Global Health Database,
in Bulgarian Medical Literature Database and
in Scopus
Central Medical Library
Editor in chief
Prof. V. Mitev, MD, Ph. D. DSc
Editorial board
Prof. K. Tsachev, MD, Ph. D., DSc
Prof. M. Marinov, MD, Ph. D., DSc
Prof. D. Ziya, MD, Ph. D., DSc
Prof. N. Lambov, Mag. Ph., Ph. D.
Prof. W. Bossnev, MD, Ph. D., DSc
L. Tacheva, MD
IOL DISLOCATION AND RETINAL DETACHMENT –
A STUDY BY ULTRASOUND BIOMICROSCOPY
B. Kutchoukov
Department of Ophthalmology
University Hospital “Alexandrovska” – Sofia, Bulgaria
Summary. A retrospective consecutive noncomparative case series of 5 eyes
(5 patients) with IOL dislocation and retinal detachment is reported. Following thorough ophthalmic exam, an ultrasound biomicroscopy (UBM) of the ciliary area was
performed in order to evaluate the position of the IOL haptics, the ciliary body area,
the vitreous base and far retinal periphery. In all eyes, the haptics were in various
positions – behind the iris, in the capsular bag, in the ciliary sulcus, in front of pars
plana. UBM proved to be a very useful imaging technique for assessment of IOL
haptic subluxation and the accompanying changes in the ciliary body and anterior
vitreous, which may play a role in the occurrence of retinal detachment in pseudophakic eyes and its unsuccessful scleral buckling surgery.
Key words: IOL, dislocation, retinal detachment, ultrasound biomicroscopy
C
INTRODUCTION
ataract surgery is the most common operation performed by ophthalmologists. Although it has a very high success rate, certain complications may occur. Intraocular lens (IOL) malpositions range from simple
decentration to luxation into the posterior segment. Subluxated IOLs involve such
extreme decentration that the IOL optic covers only a small fraction of the pupillary
opening. Luxation involves total dislocation of the IOL into the posterior segment.
Decentration of an IOL may be the result of the original surgical placement of the
lens, or it may develop in the postoperative period because of external (eg, trauma,
eye rubbing) or internal forces (eg, scarring, peripheral anterior synechiae, capsular contraction, size disparity) [2-5].
The IOL rarely dislocates completely onto the retinal surface. It usually lies
within the anterior vitreous with one haptic still adherent to the capsule or iris. It
Acta Medica Bulgarica, Vol. XXXVIII, 2011, № 2
3
may cause a vitreous hemorrhage by mechanical contact with ciliary body vessels.
The IOL may be related to retinal detachment or cystoid macular edema secondary
to vitreous changes. Rhegmatogenous retinal detachment (RD) develops in 0.5 to
1.0% of eyes even after modern cataract surgery [2-9] and is the most common
potentially blinding complication of this procedure [6-14].
The frequency of IOL dislocation has increased in the past few years because
phacoemulsification has a steep learning curve, and, as it becomes more popular,
more complications are occurring. Surgeons are more reluctant to implant anterior
chamber IOLs and aggressive placement of posterior chamber IOLs in the presence of capsular tears has become more common.
Ultrasound Biomicroscopy is a noninvasive real time diagnostic method [11]
allowing excellent resolution for anterior segment structures (incl. the IOLs) with
magnification similar to a low power light microscopy specimen. The high ultrasound frequency (50 MHz) leads to substantially higher axial and lateral resolution
(50 μm) however at the expense of decreased penetration (5-6 mm) [12]. It does
not depend on ocular media transparency.
Purpose: To study the relationship between IOL haptic position and ciliary body,
vitreous base and peripheral retina in pseudophakic eyes with retinal detachment.
Design: Retrospective consecutive noncomparative case series of 5 eyes (5
patients).
MATERIAL AND METHODS
For the period 01.05.2003 – 01.12.2003, five patients with pseudophakic
total rhegmatogenous retinal detachment were admitted to the Department of
ophthalmology at University Hospital “Alexandrovska”, Sofia, Bulgaria. Following a thorough ophthalmic exam, UBM was performed using a wire lid speculum
and an orbital cup (modified swimming goggles), thus improving the access to
the ciliary sulcus, ciliary body and peripheral retina [1]. A mixture of methylcellulose and sterile saline served as coupling medium. The IOL haptic position
and any changes in the ciliary body, vitreous base and far retinal periphery were
assessed.
RESULTS
In all patients, the IOL haptic position was revealed. The IOL haptics appeared
on UBM as highly reflective lesions with “comet-tail” like reverberations behind them.
Such a finding is typical for a foreign body (actually the IOL optic and haptics are a
foreign body within the eyeball). In patients № 1 (right eye) and № 2 (left eye), the
first IOL haptic was in the bag and the second was in the ciliary sulcus. In patient
№ 3, right eye, one haptic was with retroiridal position (Fig. 1) and the other was
4
IOL dislocation and retinal...
subluxated just below pars plicata (Fig. 2). The retinal detachment did not stop at
ora seratta rather than continued anteriorly as a ciliary epithelium detachment (Fig.
3). There were some opacities with intermediate acoustic reflectivity around the
ciliary body – a possible sign for an early anterior proliferative reaction. In patient
№ 4 (left eye), the first haptic was again behind the iris, almost in the ciliary sulcus
(Fig. 4), while the other was in front of pars plana (Fig. 5). Besides the detached
retina, we visualized with UBM a slit-like ciliary body detachment. Some opacities
with intermediate acoustic reflectivity were evident. Scleral buckling surgery failed
in last three eyes.
Fig. 1. The IOL haptic is pushing the iris and causes irido-corneal touch (arrow).
The detached retina continues in detached ciliary epithelium (arrow head)
Fig. 2. The IOL haptic is below pars plicata. Well seen are the “come-tail” like reverberations behind it
Acta Medica Bulgarica, Vol. XXXVIII, 2011, № 2
5
Fig. 3. The detached retina continues anteriorly
as a ciliary epithelium detachment. There are
some opacities with intermediate acoustic reflectivity – a possible sign for early anterior proliferative reaction (arrow head)
Fig. 4. The IOL haptic is below the iris, almost
in the sulcus (arrow head)
Fig. 5. The IOL haptic is in front pars plana (arrow head). Besides retinal detachment,
there is a slit-like ciliary body detachment (arrow)
CONCLUSIONS
UBM is a useful technique for assessment of IOL haptic dislocation and the
accompanying changes in the ciliary body and anterior vitreous, which may play a
role in the occurrence of retinal detachment in pseudophakic eyes and its unsuccessful scleral buckling surgery.
6
IOL dislocation and retinal...
REFERENCES:
1. K u t c h u k o v , B. et P. Yantchouleva-Gougoutchkova. [Ultrasound biomicroscopy and immersion
B-echography – comparative characteristics, advantages and limitations]. – Bulg. Ophthalmol.
Rev., 2003, № 1, 6-12. (in Bulgarian)
2. D a v i s , D. et al. Late in-the-bag spontaneous intraocular lens dislocation: evaluation of 86 consecutive cases. – Ophthalmology, 116, 2009, № 4, 664-670.
3. G i m b e l , H. et al. Late in-the-bag intraocular lens dislocation: incidence, prevention, and management. – J. Cataract. Refract. Surg., 31, 2005, № 11, 2193-2204.
4. G r o s s , J. G., G. T. Kokame et D. V. Weinberg. In-the-bag intraocular lens dislocation. – Am. J.
Ophthalmol., 137, 2004, № 4, 630-635.
5. J a v i t t , J. C., D. A. Street J. M. Tielseh. National outcomes of cataract extraction. Retinal detachment and endophthalmitis after outpatient cataract surgery. Cataract Patient Outcomes Research
Team. – Ophthalmology, 101, 1994, 100-105.
6. J a v i t t , J. C., S. Vitale et J. K. Canner. National outcomes of cataract extraction. I. Retinal detachment after inpatient surgery. – Ophthalmology, 98, 1991, 895-902.
7. N i e l s e n , N. E. et K. Naeser. Epidemiology of retina detachment following extra capsular cataract extraction: a follow-up study with an analysis of risk factors. – J. Cataract Refract. Surg., 19,
1993, 675-680.
8. N i n n – P e d e r s e n , K. et B. Bauer. Cataract patients in a defined Swedish population, 1986 to
1990. V. Postoperative retinal detachments. – Arch. Ophthalmol., 114, 1996, 382-386.
9. N o r r e g a a r d , J. C., H. Thoning et T. F. Andersen. Risk of retinal detachment following cataract
extraction: results from the International Cataract Surgery Outcomes Study. – Br. J. Ophthalmol.,
80, 1996, 689-693.
10. O l s e n , G. et R. J. Olson. Update on a long – term, prospective study of capsulotomy and retinal
detachment rates after cataract surgery. – J. Cataract Refract. Surg., 26, 2000, 1017-1021.
11. P a v l i n , C. J. et F. S. Foster: Ultrasound biomicroscopy of the eye. New York, Springer-Verlag; 1995.
12. P a v l i n , C. J. et K. Harasiewicz, Clinical use of ultrasound biomicroscopy. – Ophthalmology, 98,
1991, 287-295.
13. P o w e , N. R., O. D. Schein et S. C. Gieser. Synthesis of the literature on visual acuity and complications following cataract extraction with intraocular lens implantation. – Arch. Ophthalmol., 112,
1994, 239-252.
14. P o w e l l , S. K. et R. J. Olson. Incidence of retinal detachment after cataract surgery and neodymium: YAG laser capsulotomy. – J. Cataract Refract. Surg., 21, 1995, 132-135.
15. Ta p p i n , M. J. et D. F. Larkin. Factors leading to lens implant decentration and exchange. – Eye
(Lond), 14, 2000, Pt. 5, 773-776.
16. T i e l s e h , J. M., M. W. Legro et S. D. Cassard. Risk factors for retinal detachment after cataract
surgery. A population based case-control study. – Ophthalmology, 103, 1996, 1537-1545.
ª
Address for correspondence:
Borislav Kutchoukov, MD, PhD
Department of Ophthalmology
University Hospital “Alexandrovska”
Blvd. “Sv. Georgi Sofiiski” 1
Sofia 1431
+ 359 888556724
Acta Medica Bulgarica, Vol. XXXVIII, 2011, № 2
7
UNCOMMON CLINICAL PRESENTATION OF BALANITIS
CIRCUMSCRIPTA PLASMACELLULARIS ZOON
ASSOCIATED WITH MONOCLONAL GAMMOPATHY
OF IGG-TYPE AND ACUTE EXACERBATION
OF SEROPOSITIVE RHEUMATOID ARTHRITIS –
COMPLETE REMISSION AFTER SYSTEMIC
ADMINISTRATION OF DOXYCYCLINE
J. Ananiev1, G. Tchernev2, M. Penev3, M. Gulubova1, G. Kupcova4 and N. Sucha5
Department of General and Clinical Pathology, Medical Faculty, Trakia University –
Stara Zagora
2
Department of Dermatology and Venerelogy, Medical Faculty, Trakia University –
Stara Zagora
3
Department of II-nd Internal Clinic, Medical Faculty, Trakia University – Stara Zagora
4
Department of Dermatology and Venerelogy Kezmarok, Slovak Republic
5
Department of Dermatology and Venerelogy, Stara Lubovna Medical Faculty,
Slovak Republic
1
Summary. We present a case of a uncircumcised 82-year-old man with
clinical and histopathologic features of ulcerating balanitis circumscripta plasmacellularis (Zoon’s balanitis). Zoon’s balanitis is a benign condition of the
genital area that typically presents as a solitary, persistent plaque on the glans
penis of uncircumcised, middle-aged to older men. Its etiology and pathogenesis remain speculative. The eosin haematoxylin staining in our patient showed
lymphocytic infiltrates with some eosinophils. Immunohistochemically, there
was evidence of chronic histiocytic (macrophagocytic) and fibrosing plasmacellular balanitis. Histopathologic features of plasma cell balanitis in current
textbooks and publications vary considerably in the different stages of the disease. Some authors suggest that Zoon’s balanitis represents a non-specific
inflammatory response to a unknown exogenous agent. Therefore, it is often
associated with plasma cell infiltration by IgG producing cells, suggesting a
non specific polyclonal stimulation of B cells, as is common in chronic infections. We are presenting a patient with an acute form of rheumatoid arthritis
and monoclonal gammopathy of the IgG type, who has developed a rare ul8
Uncommon clinical presentation...
cerating type of Zoon’s balanitis. After a therapeutic course with doxycyline,
100 mg twice daily for a period of 3 weeks, and local application of octenidine
dihydrochloride solution (Octenisept) we were able to observe a complete
remission of the disease.
Key words: zoon’s, balanitis, rheumatoid arthritis, therapy
B
INTRODUCTION
alanitis is generally defined as inflammation of the glans penis,
which often involves the prepuce. It was first described by Zoon in
1952 [5]. There were thought to be a variety of predisposing factors. Balanitis is more common among uncircumcised men, possibly as a result
of poor hygiene and limited glans aeration or because of irritation by smegma
[1]. Underlying medical conditions may also predispose to ulcerating balanitis.
It has been reported as a source of fever and bacteremia in neutropenic men,
also in patients with chronic lymphocytic leukemia and acute promyelocytic leukemia [2-4].
Clinically, the lesions appear well circumscribed and orange-red in colour with
a characteristic glazed appearance and multiple pinpoint red macules-the so called
“cayenne pepper spots”. Symptoms of pain, irritation, and discharge can occur [6].
Histopathologic features of the balanitis plasmacellularis in current textbooks and
publications of dermatopathology vary considerably in the different stages of the
disease [6,7,8].
The etiology is unknown although chronic infection with Mycobacterium
smegmatis has been proposed as a cause [7]. In the most of the affected patients, the clinical course is chronic and poorly responsive to topical or systemic
therapy.
ANAMNESIS
An 82-year-old male had a two-month history of a progressive ulceration in the
genital area. The initial clinical suspicion was of a cutaneous neoplasm (figs.1a,1b).
His history was positive for rheumatoid arthritis with positive serology, diabetes
mellitus type II, glaucoma, heart failure (with bypass intervention), and hypertension. The patient’s systemic medications included: ISDN (isosorbide dinitrate) 120
mg 1-0-0, captopril 25/25 (1-0-1), Torasemide 10 mg 1-0-0, Kalinor (potassium
chloride) 1-0-0, and methotrexate 10 mg 1-0-0.
Clinical findings: Ulceration of the glans penis with fibrinous exudate
(figs.1a,1b).
Acta Medica Bulgarica, Vol. XXXVIII, 2011, № 2
9
1a), 1b) Ulceration in the area of the penis with fibrinous exudate in an 82-year-old patient
HISTOPATHOLOGIC FINDINGS
A bone marrow biopsy showed plasma cells and gammopathy of uncertain
significance. Skin biopsy showed lymphocytic infiltrates with some eosinophils:
Balanitis plasmacellularis circumscripta of Zoon (fig. 1c,1d). Immunohistochemically, there was evidence for a chronic histiocytic (macrophagic) and fibrosing balanitis plasmacellularis.
Direct immunofluorescence studies were negative.
1c), 1d) Lymphocytic infiltrates with some eosinophils. Immunohistochemically: chronic histiocytic
(macrophagоcytic) and fibrosing balanitis
Laboratory findings
− Erythrocyte Sedimentation Rate: 90/99, 76/88, 32/80.
− Ferritin: 1,225 (normal: 20-400 ng/ml).
10
Uncommon clinical presentation...
− Transferin : 1,52 (normal: 3-3,6 g/l).
− Hemoglobin 8,2 (normal: 8,7-11,2 mmol/l).
− Creatinine: 129 (normal: 53-133 μmol/l).
− Urea: 12,6 (normal:1,7-8.5 mmol/l).
− LDH: 4,1 (normal till 3,75 mmol/l).
− Immunofixation electrophoresis from the serum: paraproteinemia type kappa IgG could be observed.
− Immunofixation electrophoresis from the urine: no Bence Jones proteinuria.
− Serum protein electrophoresis: albumin- 54,5 (normal 60-71%) alpha 1 –
3,7 (normal 1,4-2,9%) gamma globulin – 20,9 (normal 9-16%).
Immunologic parameters:
− ANA: 1.640 with fine speckled muster.
− Autoantibodies against CCP: 8,2 (normal under 1).
− Rheumatoid factor: 92 (normal under 14 U/ml).
− pANCA, cANCA: negative.
− ENA: negative.
− Lymphocytic subpopulations: all data in normal issues.
− Quantitative Immunoglobulins: IgG und IgA- normal, IgM-5,88 (norm 0,4-2,3 g/l).
Serologies for infectious diseases
− TPHA test: negative.
− Chlamydia serology: IgA (Ratio 1,47), IgM positive (Ratio 1,08); IgG negative.
− Chlamydia smear, lesional tissue of the penis: negative.
− Herpes simplex virus serology (HSV1, HSV 2): IgG positive – 4,2, IgM negative.
− Fungal smear of the penis: negative.
− Bacterial smear of the penis: negative.
Other diagnostic studies
Roentgenography of thorax: right-sided lesion suspicious for tuberculoma.
Ultrasonography of lymph nodes and abdomen: steatosis hepatis, left band:
many enlarged, conflating lymph nodes up to 15 mm; right band: some solitary
painful lymph nodes.
Treatment and outcome
Administration of doxycycline 2 x 100 mg for 3 weeks and topical Octenisept®
(antiseptic solution) 2-3 times a day, with resultant complete remission (figs. 2a, 2b)
of skin lesions.
Acta Medica Bulgarica, Vol. XXXVIII, 2011, № 2
11
2a), 2b) Status after two and than after three weeks of therapy with doxycycline 2 x 100 mg.
Full remission during the recuperation process
DISCUSSION
Our patient was initially hospitalized due to the suspicion of a genital cancer.
The initial differential diagnosis included a non-typical presentation of pyoderma
gangrenosum within the framework of the existing kappa-type paraproteinemia. Infectious etiologies including fungal, bacterial, chlamydial, and viral (herpetic), were
ruled out. The data, obtained from the patient’s anamnesis, dod not contain information of any sexual contacts during the last 7-8 years.
At the time of presentation, the patient was also experiencing an acute,
serologically positive form of rheumatoid arthritis which was controlled as an outpatient. The slightly increased values of the chlamydia serology were interpreted
as erroneously positive because of the confounding arthritis serology. (The histopathology from the biopsy was surprising: lymphocytic infiltrates with some eosinophils, immunhistopathology: chronic histiocytic (macrophagic) and fibrosing
balanitis ).
Considering available literary data, we have concluded that the case represents a rare form of ulcerating balanitis plasmacellularis (Zoon’s balanitis).
The histopathologic findings of this disease do not represent a constant entity
but one that changes with the clinical course [7, 8]. These findings may include
epidermal atrophy, acanthosis, parakeratosis, superficial erosions, subepidermal clefts, superficial acantholysis, necrotic keratinocytes, and spongiosis [7,
8]. However these changes are likely clinical stage dependent. More advanced
cases show atrophy of the entire epidermis, superficial erosions, scattered neutrophils in the upper reaches of the epidermis, scant spongiosis, extravasation
of erythrocytes, and a much denser infiltrate with many plasma cells [7, 8]. At
even later stages, additional findings may include subepidermal clefts with loss
of the entire epidermis, marked fibrosis of the superficial dermis, and many si12
Uncommon clinical presentation...
derophages [7, 8]. Keratinocyte dysplasia and frank vesiculation are absent, as
in our case [7].
Dermal histopathologic findings include a dense band-like or lichenoid infiltrate
of plasma cells, neutrophils, eosinophils, lymphocytes and erythrocytes, prominent
blood vessels, siderophages, and fibrosis, as in our case [7]. Plasma cells are predominant in the infiltrate and usually exceed 50% of the cells present, although the
plasma cell population can demonstrate few plasma cells, or moderately dense to
dense infiltrate of plasma cells [5-7]. However, plasma cells are always detectable
[7]. An appreciable number of lymphocytes is constantly present. Polymorphonuclear leukocytes can be spotted in the dermis in most cases, but they are never
predominant [7, 8]. Eosinophils are only rarely seen and are mostly sparse, as in
our case [7, 8].
The histology of this case is heterogeneous but according to literary data
conforms to that of the advanced stages of balanitis circumscripta plasmacellularis.
CONCLUSIONS
We present a rare case of a ulcerating form of balanitis circumscripta
plasmacellularis, clinically manifested within the framework of an acute exacerbation of rheumatoid arthritis. Full remission was achieved after systemic
administration of doxycycline. In addition, a kappa-type paraproteinemia was
demonstrated.
ACKNOWLEDGEMENTS
No sources of funding were used to assist in the preparation of this manuscript.
REFERENCES
1 . V o h r a , S. et G. Badlani. Balanitis and and balanoposthitis. – Urol. Clin. North Am., 19, 1992,
143-147.
2 . M a n i a n , F. A. et R. H. Alford. Nosocomial infectious balanitis in neutropenic patients. – South
Med. J., 80, 1987, 909-911.
3 . G a t t o -W e i s , C. et al. Ulcerative balanoposthitis of the foreskin as a manifestation of chronic
lymphocytic leukemia: case report and review of the literature. – Urology, 56, 2000, № 4, 669.
4 . S t e i n b a c h , F. et al. Ulcerative balanoposthitis as the initial manifestation of acute promyelocytic
leukemia. – J. Urol., 160, 1998, № 4, 1430-1431.
5 . Z o o n , J. J. Balanoposthite chronique circonscrite benigne a plasmocytes. – Dermatologica, 105,
1952, № 1, 1-7.
Acta Medica Bulgarica, Vol. XXXVIII, 2011, № 2
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6 . S o u t e y r a n d , P., G. Wong et D. M. MacDonald. Zoon’s balanitis (balanitis circumscipta plasmacellularis). – Br. J. Dermatol., 105, 1981, 195-199.
7 . P a s t a r , Z. et al. Zoon plasma cell balanitis: an overview and role of histopathology. – Acta Dermatovenerol. Croat., 12, 2004, № 4, 268-273.
8 . W e y e r s , W. et al. Balanitis of Zoon: a clinicopathologic study of 45 cases. – Am. J. Dermatopathol., 24, 2002, № 6, 459-467.
14
Address for correspondence:
Assoc. Prof Georgi Tchernev, M.D. PhD
Department of Dermatology and Venerology
Trakia University of Stara Zagora
Medical Faculty
Armeiska Street № 11
6000 Stara Zagora, Bulgaria
00359 885 588424
e-mail: [email protected]
Uncommon clinical presentation...
EFFECT OF MENTAL WORK LOAD ON AUTONOMIC
CARDIOVASCULAR CONTROL
R. Nikolova1 and S. Danev2
National Center of Public Health Protection, Laboratory for Work Physiology and Psychology
2
Medeia, Sofia, Bulgaria
1
Summary. Psychophysiological and occupational medicine determination
of mental work load of air traffic controllers (ATC) reveal that ATC are exposed
to increased level of work load. The purpose of the study is to determine the
effect of mental work load on autonomic cardiovascular control assessed with
Heart Rate Variability (HRV) measures in air traffic controllers from Sofia airport.
Results of our study indicate that the effect of mental work load in ATC might
accelerate the ageing process and induces changes in autonomic cardiovascular control – decrease of parasympathetic activity and increase of sympathetic
activity. The level of work load of ATC working at the airport control might induce
change of the pattern of autonomic cardiovascular control with prevalence of
the sympathetic activity. The results of our study indicate that HRV measures
might be a useful tool for the assessment of the effect of work load on autonomic cardiovascular control.
Key words: mental work load, autonomic cardiovascular control, heart rate variability,
air traffic controllers
T
INTRODUCTION
he issue of measuring mental work load, mental effort and fatigue has
been an important topic in the field of psychophysiology and occupational medicine. Automation and industrialization have significant influence on work process and working conditions but in many cases mental work load
of operators and associated shift from physical to mental load is not associated with
real decrease of mental demands.
Mental work load induces changes in autonomic cardiovascular control measured with heart rate variability, and respiratory sinus arrhythmia which are sensiActa Medica Bulgarica, Vol. XXXVIII, 2011, № 2
15
tive indicators of cognitive activity [1]. Indices of cardiac activity, heart rate and
heart rate variability (HRV) are reliable occupational medicine and psychophysiological measures for assessing the effect of mental work load and stress on
operator’s functional state [2-6]. These measures reflect changes in the level of
operator’s mental work load and mental effort. HRV measures reveal the extent
of mental effort required of the operator to sustain the required level of cognitive
performance [7]. Performance of effortful mental tasks was related to a significant
reduction in HRV in laboratory and field conditions, and it was hypothesized to be
a result of the defence reaction which was associated with a decrease in baroreflex sensitivity [8].
Psychophysiological and occupational medicine determination of mental work
load of air traffic controller (ATC) reveal that ATC are exposed to increased level
of work load [9-12]. ATC perform several tasks at once: scanning radar and other
displays; giving instructions to pilots; annotating flight strips; calling down information onto tabular displays; planing ahead and anticipating future tasks; liaising; updating or removing out-of-date information, etc. There is considerable evidence in
traffic control that replacing routine functions might enable ATC to devote more time
to decision making and problem solving [13]. Mental tasks are associated with considerable cognitive and perceptive functions which might exert stress effect, and
induce mental strain and distress. Stress response might exert the influence of following factors: the working place; the working process – the intensity of work load,
information processing; the working task – scanning radars, planning, anticipating;
the association between tolerance to stress and mental strain; the operator-system
interaction. Stress response of ATC is dependent on the frequency and number of
planes to handle.
Air traffic control is considered a stressful occupation. Psychophysiological
studies of analysis of ATC tasks with measurements of heart rate, and heart rate
variability reveal that ATC task is emotionally stressing, and that ATC job is a stressful occupation [14-21]. Rose and Fogg, 1993 [15], Lille and Burnod, 1983 [22] found
increase of heart rate and systolic and diastolic blood pressure responses in ATC
with the increasing of the number of planes to handle and increase of mental load.
Henderson et al., 1990 [23] reveal that during exposure to mental load in ATC the
task demand induces increase of heart rate and decrease of the interval between
R wave and the pulse at the ear. Metzger and Parasuraman, 2001 [24] show the
sensitivity of heart rate variability for the study of mental work load in ATC. Health
analysis of ATC occupation shows high morbidity rate of diseases related to dysfunction of the autonomic nervous system – hypertension, heart rhythm disturbances, ulcer diseases [25].
16
Effect of mental work...
The purpose of the study is to investigate the effect of mental work load on
autonomic cardiovascular control assessed with heart rate variability measures in
air traffic controllers from Sofia airport.
METHOD
Fifty seven ATC participated in this study. ATC were divided into three groups:
25 ATC working at the airport control (AC); 27 ATC working at the flight control (FC),
and 5 ATC working at the airport tower main visual control point (TC).
1. Heart Rate Variability.
A computerized diagnostic system for the study of cardiovascular function was
applied [26]. Individuals were investigated for a period of 12 minutes between 9
a.m. and 11 a.m in sitting position after two hours monitoring of ATC radar task. Following indices were analyzed: 1. Time-domain HRV measures associated with the
time characteristics of consecutive series of cardiointervals (RR intervals): mean
cardiointerval (RR interval) (X) (msec); standard deviation (SD) (msec); mean difference of successive cardiointervals (V) (msec); total positive successive difference (S) (sec); number of the waves in the cardiotachogram (N) (number). 2. HRV
measures associated with RR intervals histogram distribution: mode (Mo) (msec);
amplitude of the mode (AMo) (%); homeostatic index (HI = AMo / Mo * SD) defined
as ratio of the AMo to the product of most frequent duration of RR intervals (Mo)
and standard deviation (SD) (s-2). 3. Frequency-domain measure of HRV (Index
of Autonomic Balance – IAB = PT / PRSA) (s/Hz2) – ratio of spectral power of RR
intervals in the temperature band (0.01-0.05 Hz) (PT) to the spectral power of RR
intervals in the respiratory sinus arrhythmia (RSA) band (0.15-0.50 Hz) (PRSA). 4.
Classification Index (CI). CI is the sume of main components of HRV multiplied by
their coefficients of weight) (arb. un.). CI value determines the functional state and
reflects the level of stress reaction. HRV measures are mediated by the activity of
two branches of the autonomic nervous system.
2. Mean values (X) and standard errors (Sx) of HRV measures were calculated. Student’s t-test for independent variables was used to determine the significance of differences between examined groups. A p value < 0.05 was considered
statistically significant.
RESULTS
Results might be summarized as follows:
1. There exists an age-related difference in comparing ATC divided into three
age groups: 20-29 (N = 21); 30-39 (N = 28); and 40-49 (N = 8). CI value in the first
age group was 31.3; in the second age group 5.5; and in third – 37.9 (Table 1).
Acta Medica Bulgarica, Vol. XXXVIII, 2011, № 2
17
Table 1. HRV indices in different age groups
HRV Indices
1. X (msec)
2. SD (msec)
3. V (msec)
4. S (sec)
5. N (number)
6. Mo (msec)
7. AMo (%)
8. HI (s-2)
9. IAB (s/Hz2)
10. CI (arb. un.)
GROUP 1
20-29 yr
N = 21
GROUP 2
30-39 yr
N = 28
GROUP 3
40-49yr
N=8
X±Sx
815.2±23.2
59.1±4.0
30.3±2.7
10.9±0.7
320.8±10.9
806.4±24.7
15.8±1.1
0.45±0.07
0.75±0.04
31.3±5.2
X±Sx
786.1±18.7
54.0±3.0
25.0±1.7
9.7±0.5
333.1±13.1
784.1±21.7
17.7±0.9
0.57±0.08
0.73±0.03
5.5±2.2
X±Sx
777.6±35.8
48.3±9.1
20.5±3.7
8.1±1.2
355.6±27.2
773.9±38.2
23.3±2.2
0.91±0.1
0.77±0.1
-37.9±8.6
P-value
1-2
0.67
0.3
0.08
0.5
0.5
0.5
0.1
0.2
0.6
0.1
1-3
0.5
0.2
0.05
0.04
0.1
0.5
0.002
0.005
0.7
0.01
2-3
0.8
0.5
0.2
0.1
0.5
0.8
0.01
0.06
0.5
0.08
2. As far as work-related distress may be evaluated through HRV parameters,
AC group seems to be exposed to more severe strain conditions in comparing with
FC and TC groups. ATC working in AC show lowest value of CI than ATC working
in FC and TC: CI_AC = 1.52; CI_FC = 12.7; CI_TC = 26.2) although the differences
are not statistically significant (Table 2).
Table 2. HRV indices in ATC groups
HRV Indices
1. X (msec)
2. SD (msec)
3. V (msec)
4. S (sec)
5. N (number)
6. Mo (msec)
7. AMo (%)
8. HI (s-2)
9. IAB (s/Hz2)
10. CI (arb.un.)
GROUP 1
AC
N = 25
X±Sx
796.6±19.7
51.8±3.2
24.0±1.7
9.1±0.4
325.3±11.0
796.9±23.2
18.1±0.98
0.58±0.07
0.74±0.07
1.52±1.3
GROUP 2
FC
N = 27
X±Sx
794.2±21.9
55.2±3.7
27.7±2.3
10.4±0.6
339.2±14.4
787.4±22.9
18.2±1.2
0.64±0.1
0.8±0.04
12.7±1.7
GROUP 3
TC
N=5
X±Sx
800.4±28.7
60.4±5.8
32.3±6.1
12.2±1.8
324.4±17.7
789.4±29.9
14.2±1.5
0.36±0.1
0.65±0.05
26.2±2.1
P-value
1-2
0.89
0.5
0.21
0.1
0.5
0.76
0.92
0.64
0.29
0.57
1-3
0.89
0.27
0.08
0.01
0.92
0.86
0.09
0.2
0.21
0.59
2-3
0.87
0.57
0.53
0.27
0.72
0.92
0.18
0.24
0.16
0.7
3. In comparing morning and afternoon shifts, it is not evident that the values
of HRV measures in afternoon shift show greater level of work-related strain than
HRV values in morning shift.
4. The results obtained were also compared with data from other occupational groups (operators from electrical power stations). This comparison indicated
that the air traffic control is rather difficult occupation.
18
Effect of mental work...
DISCUSSION
Changes in the values of HRV measures are valuable tool for comparing the
effect of work load on autonomic cardiovascular control. HRV indices may detect
changes in work load which do not result in any variation of the pulse. HRV indices
are more sensitive measure of mental work load than heart rate.
Results of our study reveal that age differences are better pronounced in comparing HRV measures based on pulse variability: V, S, AMo, HI, CI (see Table 1).
The effect of mental work load in ATC might accelerate the ageing process and induces changes in autonomic cardiovascular control – decrease of parasympathetic
activity and increase of sympathetic activity. We observed significant decrease
when comparing mean differences of successive cardiointervals (V), total positive
successive difference (S) and classification index (CI) between age groups: 20-29
and 40-49 yr. We observed also significant increase of mean values of amplitude
of the mode (AMo) and homeostatic index (HI) when compared 20-29 and 40-49
groups. The results of our study reveal that work load might accelerate aging process and change the pattern of autonomic cardiovascular control with sympathetic
predominance. The existence of real “functional” age difference was confirmed by
the results of psychosomatic complaints. Psychosomatic complaints increase with
the increasing of chronological age.
CI is an indicator of work-related stress. CI is mediated by the activity of the
two branches of autonomic nervous system. ATC working in AC show lowest value
of CI than ATC working in FC and TC although the difference is not statistically
significant. The level of work load of ATC working in AC might induce change of
the pattern of autonomic cardiovascular control with prevalence of the sympathetic
activity. ATC working in airport control might be exposed to a higher level of mental
work load and stress than FC and TC groups.
Changed autonomic cardiovascular control assessed with HRV indices indicate the influence of cognitive functions on cardiovascular functional state and the
extent of mental effort of the operator to sustain the required level of cognitive performance [7, 27]. Change of the pattern of autonomic cardiovascular control with
prevalence of the sympathetic activity reveal the invested level of mental effort and
the effect of mental work load on cardiovascular functional state. Similar results
are reported in the study of Backs et al., 2000 [18] who observed that autonomic
modes of control change as a function of the level of work load – high and medium workload elicited significant reciprocally-coupled sympathetic activation and
parasympathetic withdrawal, whereas low workload did not elicit significant change
from baseline.
The results of this study indicate that HRV measures might be a useful tool
for the assessment of the effect of work load on autonomic cardiovascular control.
Results of our study of analysis of mental tasks of ATC with measurements of heart
rate variability reveal that ATC job is a stressful occupation. Similar results are reported in the studies of [14, 18-21]. Stress does not only depend on the difficulties
Acta Medica Bulgarica, Vol. XXXVIII, 2011, № 2
19
of the task but also on the physical components of the working process, psychosocial stress, etc. Some of these parameters were carefully studied, and the results
obtained brought similar findings to the study of the effect of mental work load on
autonomic cardiovascular control assessed with heart rate variability measures.
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and models. – In: Handbook of Digital Human Modeling (Ed. V. Duffy). New York, CRC Press,
Taylor and Francis Group, 2009, 35-1–35-18.
2. F a h r e n b e r g , J. et C. Wientjes. Recording methods in applied environments. - In: Engineering
Psychophysiology. Issues and applications (Eds. R. Backs, W. Boucsein). Mahwah, N. J., Lawrence Erlbaum Associates, 2000, 111-136.
3. G a i l l a r d , A. et A. Kramer. Theoretical and methodological issues in psychophysiological research. – In: Engineering Psychophysiology. Issues and applications (Eds. R. Backs, W. Boucsein). Mahwah, N. J., Lawrence Erlbaum Associates, 2000, 31-58.
4. W i e n t j e s , C., H. Veltman et A. Gaillard. Cardiovascular and respiratory processes during a
complex decision-making task under prolonged isolation. – In: Advances in space biology and
medicine, JAI Press Inc., 1996, 5, 133-155.
5. V e l t m a n , J. et A. Gaillard. Physiological workload reactions to increasing levels of task difficulty.
– Ergonomics, 41, 1998, 656-669.
6. M i y a k e , S. Multivariate workload evaluation combining physiological and subjective measures.
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air traffic controllers. – Human Factors, 42, 2000, 349-366.
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of reported boredom and monotony while performing a simulated radar control task. – FAA Office
Aviation Med. Rep., 7, 1975, 75-89.
14. W i l s o n , G. et C. Russell. Operator functional state classification using multiple psychophysiological features in an air traffic control task. – Human Factors, 45, 2003, № 3, 381-389.
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325-338.
16. B r o o k i n g s , J., G. F. Wilson et C. Swain. Psychophysiological responses to changes in workload during simulated air traffic control. – Biol. Psychol., 42, 1996, № 3, 361-377.
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17. R o w e , D., J. Sibert et D. Irwin D. Heart rate variability: indicator of user state as an aid to humancomputer interaction. – Proc. of the SIGCHI conference on Human factors in computing systems,
New York, 1998.
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Address for corespondence:
Acta Medica Bulgarica, Vol. XXXVIII, 2011, № 2
21
EFFECT OF CHRONIC ALCOHOL CONSUMPTION
ON APOPTOSIS OF LYMPHOCYTES: ROLE
OF CASPASE-3 AND FAS LIGANDS
N. Boyadjieva and G. Bocheva
Department of Pharmacology and Toxicology, Medical Faculty,
Medical University – Sofia
Summary. Ethanol has been shown to acutely increase the apoptosis in various cells. But the effect of ethanol on FasL-mediated apoptosis of lymphocytes is
not well established. The aim of present study is to evaluate the mitogen-induced
proliferation in vitro and apoptosis of lymphocytes obtained from the spleens of
alcohol and nonalcohol fed rats. We also determined whether caspase-3 or soluble
FasL play a role in apoptotic effect of ethanol on lymphocytes. Here, we found that
chronic alcohol administration increased both apoptosis and caspase-3 activity and
decreased the number of T lymphocytes via depressing the proliferation and activating the apoptotic cell death in cultured splenocytes. Furthermore, we identified
that caspase-3 and soluble FasL play a role in apoptosis of lymphocytes.
Key words: apoptosis, Fas, Fas ligand (FasL), alcohol, caspase-3, T cells
A
INTRODUCTION
poptosis is a step of the cell cycle, important in the regulation of immune cell populations, which is essential mechanism used to remove
activated T cells. Ethanol (EtOH) intake affects both innate and adaptive
immune responses. Furthermore, several studies have shown that chronic ethanol
intake in both humans and mice is associated with changes of different subsets of
cytotoxic T-lymphocytes (e.g., CD8+ T cells) [15, 16, 24, 27, 31]. Excessive alcohol
consumption has a suppressive effect on the immune system’s ability to clear virusinfected cells and cells that have undergone neoplastic transformation. Previously,
it has been demonstrated that chronic alcohol consumption decreased the total
number of lymphocytes as well as the number of various subsets of T cells of male
22
Effect of chronic alcohol...
rats [3]. The mechanism of ethanol’s effect on lymphocytes is not well established.
Moreover, the role of apoptosis in lymphocyte pathways of death is not clear.
It is well documented that Fas is involved in both T-cell receptor- and membrane Ig-induced apoptosis [21, 25]. Binding of the Fas ligand (FasL) to the Fas
receptor (CD95), a member of TNF receptor family, may be an important element in
apoptosis of lymphocytes. FasL not only induces apoptosis in Fas receptor-bearing
target cells, but it is also able to transmit signals into the FasL-expressing cell via
its intracellular domain [17]. The effect of ethanol on FasL-mediated apoptosis of
lymphocytes is also not well established.
The aim of present study is to evaluate the mitogen-induced proliferation in
vitro and apoptosis of lymphocytes obtained from the spleens of alcohol and nonalcohol fed rats. We also determined whether caspase-3 or soluble FasL play a role
in apoptotic effect of ethanol on lymphocytes.
MATERIALS AND METHODS
Animals
Male rats (180-200 g) were used for acute and chronic experiments. Rats
were divided into 2 groups – controls and ethanol treated group (5 animals in each
group). Acute studies: Male rats were treated orally with alcohol (4 g/kg; 20%
solution; acute ethanol treatment-Ea) for a period of 6 h. Then rats were sacrificed,
spleens were taken and splenocytes were isolated. Splenocytes were cultured for a
period of 24 or 48 h, and studies on cell proliferation, apoptosis or Fas ligands were
performed. Chronic studies: Male rats were alcohol-fed with an alcohol diet for a
period of 3 weeks. Isolated splenocytes were cultured for further studies.
Study of Lymphocyte Proliferation
Lymphocytes were isolated from the spleens of male rats treated with 4 g/kg
20% solution of ethanol for a period of 6 h or 3 weeks. Splenocytes were isolated
in accordance with previously published method [2] and cultured (1 x10 (3) cells
per well) in duplicate in 96-well culture plates in complete growth medium containing RPMI1640, 1% penicillin/streptomycin, 2% glutamine, and 10% untreated fetal
bovine serum. Cells were treated with or without concanavalin A (Con A; 5 μg/ml)
or phytohemagglutinin (PHA; 5 μg/ml; all from Sigma Chemical Co., St. Louis, MO)
for 48 h. The number of cells was determined by a cell viability assay, which uses
a tetrazolium dye, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT; Sigma) [9]. Briefly, the cells were incubated with 1 mg/ml (100 μl per well) of
MTT for 4 h at 37°C. The MTT solution was removed, and 100 μl of 2-propanol was
added into each culture well for 30 min. The developing color was measured with
a spectrophotometer (HTS 7000, PerkinElmer, Boston, MA) with a 560-mm outer
diameter. The number of lymphocytes was determined by using a standard curve
of untreated spleenocytes (2.5, 10, 20, 40, 80, and 160 000) from ad libitum–fed
Acta Medica Bulgarica, Vol. XXXVIII, 2011, № 2
23
rats and the linear regression method. Although this cell viability assay is used to
determine the cell number, it does not indicate whether an increase of cell number
is due to increased cell proliferation or decreased cell death.
Apoptosis ELISA
The apoptosis in cultured splenocytes was measured by Nucleosome ELISA
assay in nucleosome units/ml by following the instructions of manufactures (Calbiochem, USA).
Caspase-3 activity
The Caspase-3 activity assay is a fluorometric, immunosorbent enzyme assay for the specific, quantitative in vitro determination of caspase-3 activity in microplates (Oncogene Research Product, Boston, USA). Caspase-3 activity was
determined in pmol/min by following the instructions of manufactures.
Fas Ligand ELISA
The Fas Ligand (FasL) was determined by FasL ELISA (Oncogene Research
Products, Boston, USA). It is a “sandwich” enzyme immunoassay employing mouse
monoclonal antibodies. It is achieved quantitative by the construction of a standard
curve using known concentrations of FasL. The FasL were determined in ng/ml by
following the instruction of manufactures.
Statistics
The mean and SE of the data were determined and are presented in the text
and figures. Data were analyzed with ANOVA. The differences between groups
were determined with the Student-Newman-Keuls test. A value of p < 0.05 was
considered a significant difference.
RESULTS
By using both splenocytes and polymorph nuclear cells in primary cultures
and performing MTT assay, we have previously shown that long-term exposure
to ethanol decreased mitogen-activated proliferations of lymphocytes [3]. In this
study, we used cells from the spleens of male rats, and found also that the chronic
ethanol exposure decreased the concanavalin A- and phytohemagglutinin-activated proliferation of cultured splenocytes (Fig. 1A and 1B; Fig. 2A and 2B). The results showed that alcohol decreased mitogen-induced proliferation of lymphocytes.
The acute exposure to ethanol did not affect the proliferation of cells. We also found
that chronic ethanol intake increased the amount of DNA damage in the cells as
determined by the nucleosome activity (a marker for DNA damage in the cells; Fig.
3) [1]. Taken together, the results demonstrate that ethanol depresses the proliferation of splenocytes and causes apoptotic cell death.
Studies were conducted to examine whether ethanol-induced cell apoptosis in splenocytes involves an activation of caspase-3. This enzyme is known
to increase endonuclease activity and cause cell apoptosis [6]. In our chronic
24
Effect of chronic alcohol...
study, ethanol increased the activity of caspase-3 in cells taken from spleens
of alcohol-fed male rats (Fig. 5). The data shown in Fig. 4 indicate that ethanol increases the levels of soluble FasL in cultured splenocytes in a dosedependent manner.
Number of lymphocytes in thousands
60
*
50
Number of lymphocytes in thousands
60
*
40
40
30
30
20
20
10
10
0
0
A.
C
Ea
*
50
C+Con Ea+Con
B.
a
*
*
C
Ec
C+Con Ec+Con
Fig. 1. Chronic alcohol consumption inhibits the Concanavalin A-activated (Con) proliferation of cultured
splenocytes. Male rats were treated with alcohol (4 g/kg; 20% solution; per os: acute ethanol treatmentEa; section A) or alcohol-fed for a period of 3 weeks (Ec; section B). The splenocytes were isolated,
cultured with or without Con (5 μg/ml) for a period of 48 h. The number of cells was determined by MTT
assay. Data are mean+ SE from 5 observations. *p< 0.001 as compared with controls without Con; a/p<
0.001 as compared with controls + Con
Number of lymphocytes in thousands
60
60
50
50
40
A
Number of lymphocytes in thousands
*
40
*
30
30
20
20
10
10
0
0
C
Ea
C+Ph Ea+Ph
*
a
*
*
C
Ec
C+Ph Ec+Ph
B.
Fig. 2. Effect of ethanol on phytohemagglutinin-stimulated (Ph) proliferation of cultured splenocytes.
Male rats were treated with alcohol (4 g/kg; 20% solution; per os: acute ethanol treatment-Ea; section
A) or alcohol-fed for a period of 3 weeks (Ec; section B). The splenocytes were isolated, cultured with
or without Ph (5 μg/ml) for a period of 48 h. The number of cells was determined by MTT assay. Data
are mean+ SE from 5 observations. *p < 0.001 as compared with controls without Ph; a/p < 0.001 as
compared with controls +Ph
Acta Medica Bulgarica, Vol. XXXVIII, 2011, № 2
25
*
3.5
*
3.0
FasL (ng/ml)
Nucleosome units/ml
3
2
1
*
2.5
2.0
1.5
1.0
0.5
0
C
Ea
0.0
Ec
Fig. 3. Chronic alcohol consumption stimulates
apoptosis in cultured splenocytes. Male rats
were treated with alcohol (4 g/kg; 20% solution; per os: acute ethanol treatment-Ea) or
alcohol-fed for a period of 3 weeks (Ec). The
splenocytes were isolated, cultured for a period
of 48 h. Then cells were lysed and apoptosis
was determined by nucleosome ELISA assay
in nucleosome units/ml. Data are mean+ SE
from 5 observations. *p < 0.001
C
E25
E100
Fig. 4. Acute effects of alcohol on Fas ligands
(FasL) in splenocytes. The splenocytes were isolated from control male rats, cultured for a period
of 48 h. Then cells were treated with various doses of ethanol (50 mM or 100 mM) for 48 h. Cells
were lysed and soluble FasL were determined by
ELISA assay in ng/ml. Data are mean+ SE from 5
observations. *p < 0.001
Caspase 3-activity
(% from controls)
200
*
*
100
0
C
Ea
Ec
Fig. 5. Acute and chronic effects of ethanol on caspase-3 activity in cultured splenocytes. Male rats were
treated with alcohol (4 g/kg; 20% solution; per os: acute ethanol treatment-Ea) or alcohol-fed for a period
of 3 weeks (Ec). The splenocytes were isolated, cultured for a period of 48 h. Then cells were lysed and
caspase-3 activity was determined by ELISA. The results are present as percentage of controls. Data
are mean+ SE from 5 observations. *p < 0.001
DISSCUSSION
The data presented here provide evidence that chronic ethanol administration decreases the concanavalin A- and phytohemagglutinin-activated proliferation
26
Effect of chronic alcohol...
of cultured splenocytes from male rats. We further show that ethanol exposure
increases apoptosis in cells. Furthermore, ethanol exposure activates caspase -3
and increases the cellular levels of FasL of cells.
Ethanol has been shown to acutely increase the apoptosis in various cells [7,
10]. Ewald SJ and Shao H (1993) demonstrated that ethanol increases apoptotic cell
death of thymocytes in vitro [11]. It was also published that ethanol promotes T cell
apoptosis through the mitochondrial pathway [12]. Various studies suggest that apoptosis plays a role in control of immune function [13, 14, 19, 20, 23]. Using fetal hypothalamic cells in primary cultures and DNA fragmentation assay, we have previously
shown that long-term exposure to ethanol induced apoptotic death of hypothalamic
cells during the developmental period [8]. Moreover, chronic alcohol consumption
decreased either, the total number of lymphocytes and the number of various subsets
of T cells [3], as well as both number and activity of NK cell in rats [3-5]. The results
presented here demonstrated that splenocytes died via apoptotic mechanism. Taken
together, the data suggest that alcohol decreased the number of T lymphocytes via
depressing the proliferation and activating the apoptotic cell death.
The Fas/FasL is one of the best-studied death systems. Fas-mediated apoptosis is known as one of mechanisms by which activated T cells undergo cell death
[14]. The sensitivity of T cells toward Fas-mediated apoptosis can be modulated
by the level of FasL expression upon activation. Like other TNF family members,
FasL is a homotrimetric molecule. Each FasL trimer binds three Fas molecules and
makes a complex which triggers a cascade of subcellular changes. This cascade
involves a number of proteins. It makes a “death domain” and contains enzymes
as caspases, which play an important role in apoptosis. We have previously shown
the role of caspase-3, 8 and 9 in apoptosis of lymphocytes. It is well established
that caspases have a central role in the regulation of most types of apoptotic pathways in various cells. Activation of the caspase cascade results in the cleavage of
cellular substrates that induces the biochemical as well as morphological features
of apoptosis.
Furthermore, the caspase-3 locates in the downstream of the caspase cascade and functions as an executor of cells. Because activated T cells express FasL,
and Fas-mediated signaling plays important parts in the induction of lymphocyte
apoptosis [18, 30], the contribution of FasL interaction in the apoptotic effect of alcohol was studied. The data demonstrated that ethanol in a dose dependent manner increases the levels of FasL in cultured splenocytes. There was not a significant
change in FasL in splenocytes of rats exposed chronically to ethanol. It remains
to be established whether soluble FasL could have systematically consequences
by increasing apoptosis in various tissue as well as in spleen. It was also demonstrated, that soluble FasL showed either agonist or antagonistic activity in various
activating mechanisms.
In the present study, exposure of male rats to chronic alcohol increased both
apoptosis and caspase-3 activity in cultured splenocytes. The increased activ-
Acta Medica Bulgarica, Vol. XXXVIII, 2011, № 2
27
ity of caspase-3 in cells suggestsing that alcohol facilitates apoptosis of splenocytes through promoting caspase activation with possible Fas-dependent pathway.
These data suggest the possibility that chronic ethanol exposure induces apoptosis
in lymphocytes by involvement of caspase-3 and FasL cascade. However, further
studies need to be conducted to determine the mechanism by which ethanol is affecting FasL in lymphocytes.
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Clin. Exp. Res., 17, 1993; 359-365.
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13. K a t z, G. G. et al. Signaling for ethanol-induced apoptosis and repair in vitro. – Clin. Biochem.,
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4, 672-676.
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of circulating lymphocytes that may be related with the development of liver injury. – Alcoholism:
Clin. Exp. Res., 34, 2010, 876–885.
17. L ü c k e r a t h, K. et al. Immune modulation by Fas ligand reverse signaling: lymphocyte proliferation is attenuated by the intracellular Fas ligand domain. – Blood, 117, 2011, № 2, 519-529.
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18. M c C o n k e y, D. J. et al. Calcium activated DNA fragmentation kills immature lymphocytes. –
FASEB J., 3, 1989, 1843-1849.
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CD4+ and CD8+ TCR thymocytes in vivo. – Science, 250, 1990, 1720-1722.
20. N e u m a n, M. G. Apoptosis in diseases of the liver. – Crit. Rev. Clin. Lab. Sci., 38, 2001, № 2,
109-166.
21. O r b a c h, A. et al. CD40·FasL and CTLA-4·FasL fusion proteins induce apoptosis in malignant
cell lines by dual signaling. – Am. J. Pathol., 177, 2010, № 6, 3159-3168.
22. P a n, H. N. et al. Chronic ethanol consumption inhibits hepatic natural killer cell activity and accelerates murine cytomegalovirus-induced hepatitis. – Alcohol Clin. Exp. Res., 30, 2006, 1615-1623.
23. P e r n e y, P. et al. Specific alteration of peripheral cytotoxic cell perforin expression in alcoholic patients: a possible role in alcohol-related diseases. – Alcohol Clin. Exp. Res., 27, 2003, 1825-1830.
24. S a n t o s - P e r e z, J. L. et al. T-cell activation, expression of adhesion molecules and response
to ethanol in alcoholic cirrhosis. – Immunol. Lett., 50, 1996, N 3, 179-183.
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162, 1999, 3031-3036.
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1070-1080.
Address for corespondence:
N. Boyadjieva
Department of Pharmacology and Toxicology
Medical Faculty
Medical University
2 Zdrave str.
1431 Sofia
91-72-61
Acta Medica Bulgarica, Vol. XXXVIII, 2011, № 2
29
FACTORS INFLUENCING ACUTE ALCOHOL POISONING
IN ADOLESCENTS IN BULGARIA
A. Loukova
Psychiatry Sector, Toxicology Clinic, Emergency Hospital Pirogov – Sofia, Bulgaria
Summary. The aim of the current study is to analyse the medical and social
dimensions of acute alcohol poisoning in children with regard to improve prevention of addictive habitis and develop effective preventive strategies for reducing
underage alcohol consumption. We studied the patients at the age up to 18 years
with acute alcohol poisoning hospitalized in the Children Toxicology Department
of Emergency Hospital Pirogov, Sofia, Bulgaria, from January 1, 2007 to June 31,
2008. All of them were at teen years – between 12 and 17 years old. Data on children were retrieved from hospital medical records (age, sex, level of consciousness, blood ethanol level). The initial blood ethanol level was measured on admission by thin-layer chromatography for each patient. We used the inquiry method
- specially created for the purpose of the survey questionnaire comprising 39 questions (location and the reason for drinking, type of alcoholic beverage, аge at first
drink, combination alcohol-illicit drug, type of family, education and employment
of parents, frequency of alcohol consumption by parents, consecutiveness of the
children in family, presence of siblings, presence of children in a single room, interests etc.). Psychiatric interview was used. We studied 137 adolescents with acute
alcohol poisoning. 77 were boys and 60 girls. The results demonstrated tendency
to increase of the poisonings in weekends and in late afternoon and evening. On
admission to hospital most of them had different levels of depressed consciousness. Blood ethanol level was over 2.00 mg/ml in 40.2%. No repeated hospitalization for acute alcohol poisoning in the study group for that period was registered.
Children most often used one type of alcoholic beverage. The most frequent alcoholic beverage leading to intoxication was vodka (63.1%). 64% of the children
came from complete families. Both parents had secondary education in 79.7% and
in 53.3% both parents were employed. 60% were the first born child in the family.
The most frequent reason for alcohol consumption was meeting with friends. The
students had approximately 1.20 euro daily. The research concerns one important
medico-social problem – alcohol consumption among children. The increasing alcohol consumption leads to increasing number of acute alcohol poisonings and
30
Factors influencing acute...
associated problems. The proposed preventive program may play an important role
in decreasing the consequences of alcohol consumption among young people. It
should be further developed and popularized among physicians.
Key words:
M
INTRODUCTION
edical and social problems associated with alcohol addiction have
raised great concern on the issue of the increasing prevalence of alcohol poisoning in adolescents. The overwhelming exhibit of alcoholic
beverages and the fact that they are easily available combined with the dominant
motivation of curiosity and aping in adolescents, the decreased preventive psychological protection, the low value thresholds in the majority of adolescents generate
an unfavourable psychosocial situation and have an influence on the prevalence
of alcohol intoxications among them. As a result, the society is faced a real lack of
protection of adolescents from the alcohol expansion [1, 2]. The preventive models
depend on the specific local conditions in individual countries and regions. The
preventive intervention is intended to reinforce the protective factors, identified to
have an influence on alcohol use in young people [3, 4].
In Bulgaria, there is a high social tolerability to alcohol consumption. The study
of Balabanova (1997) on alcohol consumption in Bulgaria found the pattern of drinking in Bulgaria. It used a survey of 1550 adults in Bulgaria in 1997, which found that
overall 50.7% of men and 13.6% of women drink at least weekly. In both sexes,
drinking is least common among the elderly and those living in villages. It is also
less common among those reporting their financial status as poor. Muslims are less
likely to drink than are orthodox Christians. Drinking is most common among those
living in cities, with higher education and high incomes. Heavy drinking, defined as
80 g/day or more, is rare among women, but is ascribed to 18.2% of men. Heavy
drinking is much more common among men living in Sofia and is less common
among those whose financial situation is poor. At the levels of drinking reported, it
can be expected that alcohol is making a substantial contribution to the burden of
disease and premature mortality in Bulgaria [5]. Alcohol is increasingly being recognized as a major cause of the greater burden of disease and premature death in
Eastern Europe than in the West [6].
The criteria for identifying the severity of the alcohol intoxication include: impairment of consciousness, breathing and hemodynamics, marked clinical signs
and syndromes from the organs and systems that are specifically targeted by the
poison [7]. Compared to adults, injuries due to alcohol intoxications in childhood
are manifested more rapidly and more severely due to the instability of enzyme
systems involved in alcohol metabolism in children. Hypokalemia is the most impor-
Acta Medica Bulgarica, Vol. XXXVIII, 2011, № 2
31
tant change in serum electrolytes. Alcohol intoxication causes metabolic acidosis
and respiratory depression in children [8]. Coma and vomiting are the commonest
symptoms in young teenagers intoxicated by alcohol. Severe toxicity, manifested
as coma, occurs at lower blood alcohol level in teenagers than in adults. The effect
of ethanol on the state of consciousness is directly proportional to blood alcohol
level [9].
PATIENTS AND METODS
Patients
We studied the patients at the age up to 18 years with acute alcohol poisoning
(diagnostic category T 51.0) [10], hospitalized at the Pediatric care unit, Toxicology Clinic of Emergency Hospital Pirogov, Sofia, Bulgaria, from January 1, 2007
to June 31, 2008. All of them were at teen years – between 12 and 17 years old.
Admission to the Pediatric care unit of Toxicology Clinic was made based on the
clinical presentation of acute alcohol poisoning. Pediatric care unit is the only specialized department nationwide scale on the for the treatment of acute poisoning
in children.
Data collection
Demographic and laboratory data on children were retrieved from hospital
medical records (age, sex, level of consciousness, blood ethanol level). The initial
blood ethanol level was measured on admission by thin-layer chromatography for
each patient. We used the inquiry method – specially created for the purpose of the
survey questionnaire comprising 39 questions (location and the reason for drinking,
type of alcoholic beverage, аge at first drink, combination alcohol-illicit drug, type of
family, education and employment of parents, frequency of alcohol consumption by
parents, consecutiveness of the children in family, presence of siblings, presence of
children in a single room, interests etc.). Psychiatric interview was used.
Statistical analysis
Data were analysed using the statistical package SPSS 16.0.1. Statistical
analysis was performed using Chi-squared test, Fisher’s exact test, KolmogorovSmirnov two-sample test, Mann-Whitney (U) test, Student’s t-test.
P value < 0.05 was considered as statistically significant.
Results
We studied 137 children with acute alcohol poisoning hospitalized from January 1, 2007 to June 31, 2008. 1022 children were hospitalized with intoxications
during the investigated period. Average age of children intoxicated by alcohol was
14.91±1.45 years. 77 (56.2%) were boys and 60 (43.8%) to girls. The results demonstrated interesting picture: tendency increase of the poisonings in weekends and
in late afternoon and evening. Twenty seven children (19.7%) were hospitalized on
Sunday, 21 (15.3%) on Fridays and 21 (15.3%) on Saturdays (Fig.1).
32
Factors influencing acute...
35
30
25
20
15
10
5
0
M
N
O
27
15
D
18
18
17
21
21
AY
AY
AY
AY
AY
AY
AY
D
D
D
D
D
ID
R
S
S
S
N
R
E
E
R
F
TU
N
U
SU
TU
SA
ED
TH
W
Fig. 1. Weekly distribution of acute alcohol poisonings in children
79 (57.7%) cases of intoxication were registred in the time frame from 7 p.m to
7 a.m, 37.2% from 1 p.m to 7 p.m and only about 5.0% between 7 a.m and 1 p.m. On
admission to hospital most of them had different levels of depressed consciousness:
84 (61.3%) patients were somnolent, 39 (28.5%) were soporous and 12 were comatose (5.1%). In 64 (46.7%) cases, blood ethanol level was 1.30 mg/ml-1.99 mg/ml
and in 55 (40.2%) it was 2.00 mg/ml-2.99 mg/ml. The highest estimated ethanol level
(3.80 mg/ml) was found in the blood of a 17-year-old boy. No repeated hospitalization
for acute alcohol poisoning in the study group for that period was registered. Combination alcohol-illicit drug was observed in 13 children (9.5%). The consumption implemented usually outdoors among friends and classmates. The location where children
got intoxicated was at park (56.2%), at home of friends (26.8%) and club (17.0%).
The mean consumed quantity of alcohol was higher among the girls (79.68 g of alcohol) compared to 55.98 g among the boys (P < 0.001). The largest average amount
of alcohol had been taken at the home of a friend (about 81 g of alcohol). Close to
this figure was the amount consumed at a pub (about 79 g of alcohol). Significantly
lesser was the average amount of alcohol known to have been consumed in the open
air (about 63 g of alcohol). To perform this analysis, different types of alcohol were
converted to an equivalent amount of ethanol, according to the following standards:
vodka, brandy, whiskey 40° – 31.2 g/100 ml; wine – 8.8 g/100 ml; beer – 3.5 g/100 ml
[11, 12, 13]. The reason for alcohol consumption was meeting with friends – 93 cases
(68.9%), birthdays (3.7%), emotional disorders (5.1%), family celebration (3.7%) and
other reason in 4 cases (2.9%). Children most often used one type of alcoholic beverage – 84 (61.0%). The most frequent alcoholic beverage leading to intoxication was
vodka – 63.1%. The majority of children was educated in families with more than one
child (68.6%). 64.% of the children came from complete (nuclear, with both parents)
families. Both parents has secondary education in 79.7% and in 53.3% both parents
were employed. The frequency of alcohol consummation by the parents was in connection with presence of alcohol at home (Table 1, Table 2)
Acta Medica Bulgarica, Vol. XXXVIII, 2011, № 2
33
Table 1. Availability of information self reported by a child on frequency of father’s alcohol
consumption and presence of alcohol at home*
Weekly distribution of
alcohol consumption
0 times a week
1 time a week
2 times a week
3 times a week
5 times a week
7 times a week
Total
Alcohol at home
N
24
4
0
0
0
2
30
No
%
80,00
13,33
0
0
0
6,67
100,00
Sp
7,30
6,21
4,55
N
12
13
23
8
3
23
82
Yes
%
14,63
15,85
28,05
9,76
3,66
28,05
100,00
P
Sp
3,90
4,03
4,96
3,28
2,07
4,96
< 0,05
n.s.
< 0,05
< 0,05
n.s.
< 0,05
* Abbreviations: Sp – standard error of a proportion;
† P – P value
‡ n.s – No statistically significant association
Table 2. Availability of information self reported by a child on frequency of mother`s alcohol
consumption and presence of alcohol at home*
Alcohol at home
No
N
%
Sp
N
0
40
93,02
3,88
60
1 time a week
3
6,98
3,88
7
2 times a week
0
0
7
3 times a week
0
0
1
5 times a week
0
0
1
7 times a week
0
0
7
Total
43
100,00
83
* Abbreviations: Sp – standard error of a proportion;
† P – P value
‡ n.s. – No statistically significant association
Weekly distribution of
alcohol consumption
Yes
%
72,29
8,43
8,43
1,20
1,20
8,43
100,00
P
Sp
4,91
3,05
3,05
1,20
1,20
3,05
< 0,05
n.s.
< 0,05
n.s.
n.s.
< 0,05
60.0% were the first born child in the family. Children who were a single child
in the family use alcohol less often than those who had brothers and sisters. No
significant difference in the self-stated frequency of alcohol consumption between
male and female children was observed. The gender of the children did not have a
significant effect on the age at which the first drink was consumed.
First alcohol consuption was at 12 years and 10 months for boys and at 13
years for girls. The intoxications were more frequent among the children with good
results in the school (55.0%), who studied at a college, who knew foreign languages (P = 0.001) and who possessed a computer (93.0%). From the data about the
after school interests among the children we can say that in their life predominated
the non institutional forms of communication (cinema, music, non formal contacts
34
Factors influencing acute...
etc.) at the expense of the institutional ones like school and family. Such factors as
the kind of family, and being a single child in the family were not associated with
alcohol consumption alone (P > 0.05). The majority of single children in the family
(n = 31) had a room of their own (72.1%). No statistically significant association between the availability of a child’s own room and the gender of the children, if more
than one in the family, had been detected (P = 0.818). In practical terms, children in
single parent families had no less pocket money than children in complete families.
The students had approximately 1.20 euro daily.
DISCUSSION
Our study represents the first systematic research of the alcohol intoxications
among children in Bulgaria. We analysed the data of the only Children Toxicology
Department in Sofia. 1022 children were hospitalized with intoxications during the
investigated period. We cannot exclude the possibility that children who manifested
minor signs of alcohol poisoning were treated at home but we believe that our study
shows the actual trend of alcohol poisoning among children in Sofia. The results
from our study coincide with data from other European countries studies.
A Slovak Republic study found that the average age of the patients with alcohol
intoxication presenting to hospital was 15.1±1.7 years. The proportion of children admitted with alcohol intoxication increased every year [14]. Our study showed the mean
age of alcohol poisoned patients (mean +/- standard deviation) 14,91 ± 1,45 years.
Croatian study by Bitunjac and Saraga [15] found that 73,2% of children were
hospitalized on weekends and 79% of children got intoxicated outseide their homes.
Our study showed that children usually drank outside their homes (56.20%), and
mostly on weekends (35%).
The results of study (Weinberg and Wyatt, 2006) confirm the heavy use of
alcohol by some young children. The most common types of alcohol consumed
were whisky, gin, vodka and tequila. No significant association was seen between
age and blood alcohol levels [16]. Our study showed that children most often used
one type of alcoholic beverage, and the most frequent alcoholic beverage leading
to intoxication was vodka.
Biochemical disturbances in young teenage alcohol intoxicants resemble
those previously found in adults. The severe toxicity by ethanol, manifesting in
coma, occurs in lower blood alcohol concentrations in children than in adults [17].
Our data have been compared to data from other studies. The most common are
intoxications with blood alcohol level between 1.30 mg/ml -1.99 mg/ml.
Lamminpää reported that motives leading to alcohol intoxication are a wish to
get drunk, experimenting and problems in human relations. The underlying problems are often family-related, such as divorce, an alcoholic parent and a lower
socioeconomic group. Underlying family problems were usual; in 45% of the cases
the family was broken and in 31% of the families one parent was an alcoholic. The
lower the mother’s social group was, the higher the frequency of alcohol intoxicaActa Medica Bulgarica, Vol. XXXVIII, 2011, № 2
35
tion. Previous intoxications were reported in 9% of the cases. Most of the children’s
intoxications were experimental (49%) [18, 19]. The most frequent reason for intoxication in our study was meeting with friends – 93 cases. Influence of ‘the group’
is the main cause for alcohol consumption in adolescence. Most of children are experiencing communication problems and the alcohol consumption `helps` them to
overcome this problem and make communications easier. The conveyance of the
center to socialization from the family towards the group leads to loss of emotional
attachments to parents and their replacement by relation with people in the group,
less influencing the personality as a whole, but forming certain behavioral models.
However, little is known about socio-economic differences in unhealthy lifestyles during adolescence. Parental socio-economic status is only of limited importance for episodes of drunkenness in adolescence [20]. Like social event the
alcohol intoxications depends on a lot of familiar, cultural, educational, psychological and behavioural factors. The parents with secondary education predominated.
The intoxications were more frequent among the children with good results in the
school. For majority of adolescents alcohol intake corresponds to attempt for building self-esteem, for integration in friend circle and in the world of grown-ups.
We developed a programme for active selective prevention of alcohol intoxication among children with aim to set limits of all health, social and economic damages due to alcohol by: limiting the demands for alcoholic beverages, the consumption of alcohol, increasing the quality of health education, upbringing, and value
formation guidance; creating a system for control at schools, in the family, in the
social group (among friends), and during leisure time. The model is based on psychological and social approach. The concept of autor is to develop different skills
toward a positive direction of children’s life. Very important elements are the ability
of resistance and the formation of habits for social frame of mind, communication
skills and desire for change of hasardous behavior by thinking, preparation and execution. The psychological aspect of prevention is directed toward the formation of
cognitive attitude to be successful. In the context of the health promotion, the focus
is to strengthen all aspects of the life [21].
ACKNOWLEDGMENTS
We are grateful to Prof. Elena Shipkovenska and Prof. Aneta Hubenova for
the critical reading of the manuscript.
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Croat. Med. J., 50, 2009, № 2, 151-156.
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Emerg. Med. J., 23, 2006, № 10, 774-776.
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Address for correspondence:
Anelia Loukova, MD, PhD
Toxicology Clinic – Psychiatry Sector
Emergency Hospital “N. I. Pirogov”
21 Totleben blvd.
1606 Sofia
Bulgaria
e-mail: [email protected]
Acta Medica Bulgarica, Vol. XXXVIII, 2011, № 2
37
DETERMINATION OF THE EFFECT OF LEAD-INDUCED
SUBCLINICAL TOXICITY ON AUTONOMIC
CARDIOVASCULAR FUNCTION
R. Nikolova1 and S. Danev2
National Center of Public Health Protection, Laboratory for Work Physiology and Psychology
2
Medeia, Sofia, Bulgaria
1
Summary. One of the main principles of physiological strategy for assessment of subclinical lead toxicity at concentrations below threshold limit value
is determination of the effect on the autonomic nervous system function. The
aim of the study is to determine the effect of lead-induced subclinical toxicity
on autonomic cardiovascular function assessed with heart rate variability (HRV).
Lead-exposed group showed significant increase of ratio of spectral power of
cardiointervals in the temperature band (0.01-0.05 Hz) (PT) to the spectral power
of cardiointervals in the respiratory sinus arrhythmia (RSA) band (0.15-0.50 Hz)
(PRSA) (PT/PRSA ratio), and reduction of classification index (CI) when compared with the control group. The results of our study reveal significant dependencies of PT/PRSA ratio and CI on work practice duration. Chronic lead toxicity
induced by prolonged exposure to lead may cause disturbed autonomic balance
with sympathetic predominance. The assessment of health risk associated with
chronic exposure to lead at concentrations below threshold limit value may be
performed with HRV measures which reflect quantitatively the association “exposure time – toxic concentrations”.
Key words: autonomic cardiovascular function, lead, Heart Rate Variability, subclinical
toxicity, health ris
C
INTRODUCTION
hronic lead toxicity is one of the major environmental health problems
in modern society. The presence of lead in bioavailable forms in the environment is due largely to the activities of humans [1-4]. Chronic lead
toxicity is also one of the major health issues because of the continuing use of lead in
industry [5-7] and in wide variety of consumer products including solder [8-9].
38
Determination of the effect...
Lead is considered today to cause adverse subclinical effects even at exposure levels below threshold limit value. These values were thought to be safe and
not producing specific and clinically detectable symptoms [4, 8]. Recognition of
subclinical toxicity arose from studies showing that asymptomatic exposure to lead
induce renal, male reproductive and neurologic dysfunctions. The underlying premise is that there exists a dose-related continuum of toxicity in which the clinically
apparent effects of lead have their asymptomatic functional analogues [10].
Renal subclinical toxicity affects glomerular and tubular functions with excretion of albumin and low-molecular weight proteins [11-12]. Subclinical lead toxicity
is expressed also in male fertility disturbances affecting sperm morphology and
count [4], and central, peripheral and autonomic nervous systems dysfunctions.
In the central nervous system, lead toxicity diminishes neuro-psychologic performance impairing visual intelligence and visual-motor coordination [13]. Murata et
al., 2009 [7] reveal that the critical organ of lead toxicity in workers is the nervous
system at critical blood lead level (PbB) between 107 and 175 μg/l. Lower levels
of lead exposure are associated with dysfunction of nerve conduction velocity [14].
Autonomic neurologic dysfunction in workers chronically exposed to lead is assessed using neurophysiological studies such as assessment of cardiointervals
(R-R interval) variability and heart rate variability (HRV) [15].
The assessment of different physiological aspects of subclinical lead toxicity
should be done by integrated strategy [16]. One of the main principles of that strategy is the determination of subclinical toxicity on the autonomic nervous system
(ANS) function by noninvasive assessment of autonomic cardiovascular control.
One of the methods for assessment of autonomic cardiovascular control is heart
rate variability (HRV). Indices of HRV are used to characterize ANS response patterns [17]. Subclinical lead toxicity might be examined with sensitive physiological
indicators showing dysfunction of ANS. HRV may be used for studying the effect
of lead on disturbances in autonomic cardiovascular control [4]. Examination of
R-R interval variability (CVRR) during deep breathing and the coefficient of variation of respiratory sinus arrhythmia (CVRSA) demonstrates potential autonomic
dysfunction – disturbance of parasympathetic activity following lead exposure [15,
18]. Parasympathetic autonomic dysfunction assessed with HRV is observed to be
affected also by exposure to environmental neurotoxic agent – lead [19]. Teruya
et al., 1991 [18] and Murata and Araki, 1991 [15] found decrease of HRV during
deep breathing in individuals with PbB above 340 μg/l (the biological threshold
limit is 400-600 μg/l) compared with controls. Böckelmann et al., 2002 (6) found
vagal depression (examined with HRV) and a delayed restoration of cardiac rhythm
parameters to the initial autonomic state caused by long-term chronic lead exposure. Teruya et al., 1991 [18] observed the statistical significant dose-response and
dose-effect relationships between PbB levels and HRV during deep breathing in
individuals with PbB level even above 200 μg/l. Murata et al. (1995) [5] suggested
that autonomic nervous function is more susceptible to lead than visual and audi-
Acta Medica Bulgarica, Vol. XXXVIII, 2011, № 2
39
tory nervous functions in glass workers and that lead affects sympathetic activity
more strongly than parasympathetic activity assessed with HRV. Gennart et al.,
1992 [9] found no correlation between the occupational exposure to lead and autonomic cardiovascular control evaluated with RR intervals variability, kidney and
endocrine function. Pfister et al., 1996 [20] studied the relationship between the
autonomic tone and exposure to lead and indicate that restoration of autonomic
tone is dependent on the duration of exposure to lead.
One of the main principles of physiological strategy for assessment of subclinical lead toxicity at concentrations below threshold limit value is determination of
the effect on the ANS function. Autonomic cardiovascular control is studied non-invasively with HRV analysis. Time- and frequency-domain HRV measures are used
to characterize ANS response patterns. Subclinical lead toxicity might be examined
with sensitive physiological HRV indicators showing dysfunction of ANS. The assessment of health risk associated with chronic exposure to lead at concentrations
below threshold limit value necessitates studies for determination the effect on the
ANS function.
AIM
The aim of the study is to determine the effect of lead-induced subclinical toxicity on autonomic cardiovascular function assessed with heart rate variability.
DESIGN AND METHODS
Subjects
Sixty-four (35 male and 29 female, mean age 36.35±3.27 years, age range
22-61 years) tin-lead solder workers from electronic production industry (mean
work practice duration 14.6±1.36 years) were examined. Control group consisted of 73 age/sex-matched healthy individuals who are selected from the working
country population (38 male and 35 female, mean age 35.43±2.53 years, age
range 21-61 years, mean work practice duration 13.4±1.03 years) who are not
exposed to toxic substances as being mainly employees in institutions, banks,
offices and business.
Criteria for exclusion from experimental and control group include: cigarette
smoking, alcohol consumption; body mass index > 25 kg/m2; and a history of cardiovascular, respiratory, renal, hepatic, gastrointestinal or systemic diseases.
Lead concentration on working place
Nevertheless the concentration of lead condensed aerosols in the breathing
zone was found to be under the threshold limit value (0.05 mg/m3) lead traces in
the same zone actually were observed at soldering of electronic components with
tin-lead alloys.
40
Determination of the effect...
Blood lead level and urinary excretion of δ-aminolaevulinic acid (δ-ALA)
The concentration of blood lead level (PbB) was found to be in the limit of the
biological threshold value = 400-600 μg/l for working environment. Dose-response
relationship between urinary excretion of δ-ALA and lead exposure was positively
correlated (increased excretion of δ-ALA was observed in 23.6% of the solders
at urinary excretion between 8160 to 10200 nmol/4h urine) when the biological
constant was 3930-7870 nmol/4h urine (data from the Hygiene-Epidemiological
Inspectorates).
Methods
1. Heart rate variability (HRV)
A computerized diagnostic system for the study of the autonomic cardiovascular function was applied [21]. Examination of the heart rate was conducted between
9-11 a.m. for a period of 10 minutes after 15 minutes relaxation period in supine
position.
Following indices were analyzed:
1.1. Time-domain HRV measure: classification index (CI) unifying time-domain
derived HRV measures multiplied by their coefficients of weight (arb. un.).
1.2. Frequency-domain measure:
PT / PRSA ratio – ratio of spectral power of R-R intervals in the Temperature
band (0.01-0.05 Hz) (PT) to the spectral power of R-R intervals in the Respiratory
Sinus Arrhythmia (RSA) band (0.15-0.50 Hz) (PRSA) (s/Hz2).
2. Blood pressure
Systolic and diastolic blood pressure (SBP; DBP) (mm/Hg) were measured
using the Riva-Rocchi method.
Data analysis
Mean values (X) and standard errors (Sx) of HRV measures, SBP, DBP, age
and work practice duration were calculated. Student’s t-test for independent variables was used to determine the significance of differences between the experimental and control groups. Correlation and linear regression analysis were applied
to determine the association and dependence of physiological measures on work
practice duration. A p value < 0.05 was considered statistically significant.
RESULTS
Groups mean values of HRV, SBP, DBP, work practice duration and age in
experimental and control group are presented in table 1. Time – and frequencydomain HRV measures were age-adjusted by multiplying every single value with
its age-related transfer coefficients [22]. Lead-exposed group showed significant
increase of PT/PRSA ratio and reduction of CI when compared with the control group.
Chronic lead toxicity induced by prolonged exposure to lead may cause disturbed
autonomic balance with sympathetic predominance. As a consequence of disturbed
Acta Medica Bulgarica, Vol. XXXVIII, 2011, № 2
41
autonomic function PT / PRSA ratio increased and CI decreased. SBP and DBP
although increased in the experimental group did not show significant differences.
Significant correlations were found to exist between: work practice duration
and CI (r = -0.616, p < 0.0001); work practice duration and PT/PRSA ratio (r = 0.533,
p < 0.0001). SBP and DBP did not show such a significant correlation.
Linear regression analysis revealed following dependences: dependence of
PT / PRSA ratio on work practice duration (WPD) – PT/PRSA ratio = 0.281 + 0.049 *
WPD (b = 0.049, p < 0.00001); dependence of CI on WPD – CI = 28.084 – 4.518 *
WPD (b = -4.518, p < 0.00001). Results obtained by regression analysis show that
chronic lead exposure time at lead concentration below threshold limit value can
significantly predict disturbed autonomic cardiovascular function.
Results of our study indicate that HRV measures are reliable health risk predictor for developing subclinical lead toxicity related to asymptomatic autonomic
response patterns [17] following prolonged lead exposure.
Table 1. Mean groups (X ± S x) and p-values of HRV measures, SBP, DBP, age and work
practice in lead-exposed and control groups.
Lead-exposed group
(N=64)
Control group
(N=73)
X±Sx
X±Sx
CI (arb.un.)
-50.9 ± 0.38
0.00 ± 0.17
< 0.05
Pt/Pr (s/Hz )
1.1 ± 0.13
0.2 ± 0.03
< 0.05
SBP (mm Hg)
146.46 ± 3.46
128.90 ± 2.73
n.s.
DBP (mm Hg)
91.70 ± 2.68
75.30 ± 1.36
n.s.
Work practice duration (yr)
14.6 ± 1.36
13.4 ± 1.03
n.s.
Age (yr)
36.35 ± 3.27
35.43 ± 2.53
n.s.
Variables
2
P – value
DISCUSSION
Recently, a substantial attention has been focused on increased health risk
associated with chronic exposure to lead at levels considered up to now as safe
[23-26]. Determining the ANS function by HRV promote for sensitive assessment
of subclinical toxicity induced by chronic exposure to lead as HRV detects subtle
changes in autonomic cardiovascular control patterns. Subtle changes in autonomic cardiovascular control precede or are early indicators of a pathological impairment as the effect of lead on the heart occurs via the autonomic cardiovascular
control. HRV analysis was used as a physiological marker reflecting the nonspecific
adverse drift in autonomic balance [4].
The increased chronic sympathetic tone which has been found in lead exposed group (revealed by increased Pt/Pr ratio and reduction of CI derived by HRV
42
Determination of the effect...
analysis) is positively correlated with the chronic lead exposure time although the
lead concentration does not exceed the threshold limit value. In fact it is difficult to
conclude that only the chronic lead exposure induces nonspecific autonomic disturbances but such a possibility cannot be rejected. Teruya et al. [18] and Murata and
Araki [15] demonstrate potential autonomic dysfunction affecting the parasympathetic activity (decreasing on the mean values of coefficient of variation of respiratory sinus arrhythmia during deep breathing) under PbB bellow biological threshold
limit: 360 μg/l and 340 μg/l. In addition Teruya et al. [18] showed significant doseresponse and dose-effect relationships during deep breathing between PbB levels
above 200 μg/l and R-R interval variation.
The main findings of this study are that subclinical asymptomatic lead toxicity
causes disturbed autonomic balance with sympathetic predominance (increasing
of Pt/Pr ratio and reduction of CI). Significant dependences exist between WPD
and CI; WPD and Pt/Pr ratio. Prolonged work practice which reflects dose-related
effect of chronic lead toxicity is a significant predictor of HRV measures. Subclinical toxicity associated with chronic lead exposure causes asymptomatic functional
autonomic response patterns.
The proposition inherent in this study is that dysfunction in autonomic cardiovascular control which is reflected in HRV components may determine subclinical lead toxicity. Therefore HRV may be used as an early nonspecific predictor of
subclinical lead toxicity when PbB concentration is within the limits of biological
threshold value. This conclusion is in agreement with previous studies showing
autonomic dysfunction assessed by HRV at PbB levels above 300 μg/l [15, 18].
Our results replicate the results WHO, 1995 [4], revealing the lack of causal
relationship between body burden of lead and blood pressure. The relation between
lead exposure and blood pressure necessitates further investigation as the results
are controversial. Contrary to the results of WHO, 1995 [4] and our findings, Metz
et al., 1997 [27] and Navas-Acien et al., 2007 [28] reported that cardiac toxicity
associated with lead exposure induces hypertension. Park et al., 2006 [29] found
that individuals with metabolic syndrome were more susceptible to autonomic dysfunction (examined with spectral powers in high-frequency (HF) and low-frequency
bands (LF), and LF/HF ratio, in association with chronic lead exposure.
Evaluation of subclinical toxicity needs specification in future studies of determining the toxic lead effect on the ANS response patterns based on HRV at different lead exposures at working places. The assessment of health risk associated
with chronic exposure to toxic substances at concentrations below threshold limit
value necessitates further studies for determining the extent to which HRV measures reflect quantitatively the association “exposure time – toxic concentrations”.
REFERENCES
1. S m i t h , M. Lead history. – In: Lead Toxicity: History and Environmental Impact. R. Landsdown,
W. Yule (Eds.) Baltimore, Johns Hopkins University Press, 1986, 7-24.
Acta Medica Bulgarica, Vol. XXXVIII, 2011, № 2
43
2. R i c e , D. Neurotoxicity of lead, methylmercury, and PCBs in relation to the Great Lakes. – Environ. Health Perspect., 103, 1995, 71-87.
3. B e r n i e r , J. et al. Immunotoxicity of heavy metals in relation to Great Lakes. – Environ. Health
Perspect., 103, 1993, 23-34.
4. W H O . International Program on Chemical Safety. Environmental Health Criteria 165. Inorganic
Lead. Geneva, WHO, 1995.
5. M u r a t a , K. et al. Autonomic and central nervous system effects of lead in female glass workers
in China. – Am. J. Industrial Med., 28, 1995, № 2, 233-244.
6. B ö c k e l m a n n , I. et al. Assessing the suitability of cross-sectional and longitudinal cardiac
rhythm tests with regard to identifying effects of occupational chronic lead exposure. – J. Occup.
Environ. Med., 44, 2002, 1, 59-65.
7. M u r a t a , K. et al. Lead toxicity: does the critical level of lead resulting in adverse effects differ
between adults and children. – J. Occup. Health, 51, 2009, № 1, 1-12.
8. A T S D R . Toxicological Profile for Lead. TP 92-12. Atlanta, GA, Agency for Toxic Substances and
Disease Registry, 1993.
9. G e n n a r t , J. P., A.Bernard et R. Lauweris. Assessment of thyroid, testes, kidney and autonomic
nervous system function in lead-exposed workers. – In Arch. Occup. Environ. Health., 64, 1992,
№ 1, 49-57.
10. L a n d r i g a n , P. The toxicity of lead at low dose. – Br. J. Ind. Med., 46, 1989, 593-596.
11. M u t t i , A. Detection of renal diseases in humans: developing markers and methods. – Toxicol.
Letters, 46, 1989, 177-191.
12. L a u w e r y s , R. et A. Bernard. Preclinical detection of nephrotoxicity: description of the tests and
appraisal of their health significance. – Toxicol. Letters, 46, 1989, 13-29.
13. S e p p a l a i n e n , A. et al. Early neurotoxic effects of lead exposure: a prospective study. – Neurotoxicology, 4, 1985, 181-192.
14. A r a k i , S. et al. Recovery of slowed nerve conduction velocity in lead-exposure workers. – Int.
Arch. Occup. Environ. Health, 46, 1980, 151-157.
15. M u r a t a , K. et S. Araki. Autonomic nervous system dysfunction in workers exposed to lead, zinc,
and copper in relation to peripheral nerve conduction: a study of R-R interval variability. – Am. J.
Ind. Med., 20, 1991, 663-671.
16. To d d , A. et al. Unraveling the chronic toxicity of lead: an essential priority for environmental
health. – Environ. Health Perspect., 104, 1996, 141-146.
17. Backs, R. Going beyond heart rate: Autonomic space and cardiovascular assessment of mental
workload. – Int. J. Aviat. Psychol., 5, 1995, 24-48.
18. Te r u y a , K. et al. Effect of lead on cardiac parasympathetic function. – Int. Arch. Occup. Environ.
Health Perspect., 62, 1991, 549-553.
19. v a n R a v e n s w a a i j - A r t s , C. et al. Heart rate variability. – Ann. Int. Med., 118, 1993, № 6,
436-447.
20. P f i s t e r , E., I. Böckelmann.et T. Ferl. Vegetative function diagnosis for early detection of lead
intoxication. – Int. Arch. Occup. Environ. Health, 69, 1996, № 1, 14-20.
21. D a n e v , S. [Informativeness of Heart Rhythm in Occupational Physiological Aspect.] (D. Sc. Thesis) Sofia, National Institute of Hygiene, Medical Ecology and Nutrition, 1989. (in Bulgarian)
22. N i k o l o v a , R. [Approbation of the method for analysis of heart rate variability under models
of neuro-psychic occupational strain and its methodological improvement.] (Ph.D. Thesis) Sofia,
National Institute of Hygiene, Medical Ecology and Nutrition, 1993. (in Bulgarian)
44
Determination of the effect...
23. N e e d l m a n , H. Human Lead Exposure. Boca Raton, FL, CRC Press, 1992.
24. M a h a f f e y , K. Dietary and Environmental Lead: Human Health Effects. New York, Elsevier, 1985.
25. L a n d s d o w n , R. et W. Yule. Lead Toxicity: History and Environmental Impact. Baltimore, Johns
Hopkins University Press, 1986.
26. R u t t e r , M. et J. Russel. Health: Sources and Effect of Low Level Exposure. Chichester, Wiley
& Sons, 1983.
27. M e t z , A. et al. Agents associated with the development of cardiac hypertrophy and/or congestive
heart failure. – In: Comprehensive Toxicology. I. Sipes, C. McQueen, A. Gandolfi (Eds.) Pergamon,
Cambridge, Cambridge University Press, 1997, 385-409.
28. N a v a s - A c i e n , A. et al. Lead exposure and cardiovascular disease – a systematic review. –
Environ. Health Perspect., 115, 2007, № 3, 472-482.
29. P a r k , S. et al. Low-level lead exposure, metabolic syndrome, and heart rate variability: The VA
Normative Aging Study. – Environ. Health Perspect., 114, 2006, № 11, 1718-1724.
Address for corespondence:
Dr. Rouja Nikolova, MD
Laboratory for Work Physiology and Psychology
National Center of Public Health Protection
1431 Bulgaria
Acta Medica Bulgarica, Vol. XXXVIII, 2011, № 2
45
CLINICAL ASSESSMENT AND MOLECULAR GENETICS
OF AN AUTOSOMAL DOMINANT RETINITIS PIGMENTOSA
IN A BULGARIAN ROMA FAMILY
K. Koev1, S. Cherninkova2, Ch. Chakarova3, R. Georgiev1, S. Dimitrova4, R. Kaneva5
and S. Bhatacharya3
1
Department of Ophthalmology, MU – Sofia, Bulgaria
Department of Neurology, University Hospital “Alexandrovska”, Sofia, Bulgaria
3
Department of Genetics, Institute of Ophthalmology, UCL, London, UK
4
Molecular Medicine Center, MU – Sofia, Bulgaria
5
Molecular Medicine Center and Department of Medical Chemistry and Biochemistry,
MU – Sofia, Bulgaria
2
Summary. Purpose was to make a clinical assessment and molecular genetic
analysis in patients with autosomal dominant form of retinitis pigmentosa (adRP)
in a Bulgarian Roma family. Clinical assessment and genealogical analysis in a
Bulgarian Roma family suggested the presence of RP with autosomal dominant
inheritance with at least 12 affected in 4 generations. Clinical results showed best
corrected visual acuity. Performed were kinetic Goldmann perimetry; direct and
indirect ophthalmoscopy; ERG; fluorescein angiography. The molecular genetic
analysis involved screening of 15 known adRP genes using microarray panel of
Asper Biotech in the index patient. T in exon 4 of the RP1 gene, leading to an
amino acid substitution T373I was found in heterozygous condition. Conclusion
shourd adRP is a severe and genetically heterogeneous retinal degeneration.
We present a Bulgarian Roma family with typical clinical symptoms of RP and
heterozygous change in the RP1 gene, which has previously been described
as a possible disease causing mutation in a Pakistani family with adRP and in
homozygous condition leading to a severe arRP in 2 consanguineous families
of Pakistani origin. The clinical and genetic analysis of additional affected and
unaffected family members is ongoing. This will allow better genotype-phenotype
correlations to be made.
Key words: autosomal dominant retinitis pigmentosa, molecular genetic analysis, tunnel visual field, ERG, fluorescein angiography
46
Clinical assessment and molecular genetics...
INTRODUCTION
R
etinitis pigmentosa (RP) is a type of progressive retinal dystrophy, a
group of inherited disorders in which abnormalities of the photoreceptors (rods and cones) or the retinal pigment epithelium (RPE) of the
retina lead to progressive visual loss [1].
Clinical variability and genetic heterogeneity have an important impact on genetic testing and counselling of affected families. RP is a group of inherited progressive retinal diseases affecting about 1 in 3500 people worldwide.
Linkage mapping in a large, seven-generation family with type 2 autosomal
dominant retinitis pigmentosa (ADRP) demonstrates linkage between the disease
locus (RP1) and DNA markers on the short arm of human chromosome 8 [2, 3, 4].
Purpose: To make a clinical assessment and molecular genetic analysis in
patients with autosomal dominant form of retinitis pigmentosa (adRP) in a Bulgarian Roma family.
MATERIAL AND METHODS
Clinical assessment and genealogical analysis in a Bulgarian Roma family
suggested the presence of RP with autosomal dominant inheritance with at least
12 affected in 4 generations.
Best corrected visual acuity; kinetic Goldmann perimetry; direct and indirect
ophthalmoscopy; ERG; fluorescein angiography.
The molecular genetic analysis involved screening of 15 known adRP genes
using microarray panel of Asper Biotech in the index patient.
CLINICAL RESULTS
We found the following symptoms in the 8 examined patients from the family:
1. The onset of the first visual complaints ranged from 7-8 years.
2. Bilateral symmetrically reduced visual acuity in the range between 0.01
till 0,3.
3. Bilateral tunnel visual field and central/paracentral scotomas.
4. Dustlike and granular pigmentation in the macular area and midperiphery.
5. RPE atrophy and optic disc pallor.
6. In all patients, the ERG are non detectable as well under red and blue
filter.
7. Fluorescein angiography showed an atrophy of the RPE in the macular
area and midperiphery.
Acta Medica Bulgarica, Vol. XXXVIII, 2011, № 2
47
Fluorescein angiography of the proband
Molecular genetic results
The mutation 1118C > T in exon 4 of the RP1 gene, leading to an amino acid
substitution T373I was found in heterozygous condition in the proband
RP1 EX4
48
1118C>T
T373I
CT/GA
Clinical assessment and molecular genetics...
DISCUSSION
Retinitis pigmentosa is a frequent retinal dystrophy characterized by a progressive loss of photoreceptors along with retinal degeneration [5]. RP1 gene mutations are the second most common cause of autosomal dominant retinitis pigmentosa [3].
As phenotypes do not always correlate with the respective genotypes, it is of utmost importance that clinicians, geneticists, counsellors, diagnostic laboratories and
basic researchers understand the relationships between phenotypic manifestations
and specific genes, as well as mutations and pathophysiologic mechanisms [6].
Dominant RP caused by mutations in the RP1 gene often shows late onset of
the disease phenotype, usually by the third decade of life. Pierce et al. [7] observed
that patients suffering from adRP who were heterozygous for the RP1 mutation had
classic, less severe adRP phenotype with late onset of disease.
The adRP is a severe and genetically heterogeneous retinal degeneration.
We present a Bulgarian Roma family with typical clinical symptoms of RP and pigmentation in the macular area.
The molecular genetic analysis of the known adRP genes led to the identification of heterozygous change in the RP1 gene (1118C>T,T373I), which has previously been described as a possible disease causing mutation in a Pakistani family
with adRP and in homozygous condition leading to a severe arRP in 2 consanguineous families of Pakistani origin [8].
The study [9] provides the first report of involvement of mutations in the RP1
gene in the autosomal recessive RP phenotype. All three recessive Pakistani families presented here showed the severe form of RP with early onset [9].
The clinical and genetic analysis of additional affected and unaffected family members is ongoing. This will allow better genotype-phenotype correlations
to be made.
REFERENCES:
1. H a r t o n g , D. T., E. L. Berson et T. P. Dryja. Retinitis pigmentosa. – Lancet, 368, 2006, № 9549,
1795-1809.
2. B l a n t o n , S. H. et al. Linkage mapping of autosomal dominant retinitis pigmentosa (RP1) to the
pericentric region of human chromosome 8. – Genomics, 11, 1991, № 4, 857-869.
3. P a y n e , A. et al. RP1 protein truncating mutations predominate at the RP1 adRP locus. – Invest.
Ophthalmol. Vis. Sci., 41, 2000, № 13, 4069-4073.
4. B e r s o n , E. L. et al. Clinical features and mutations in patients with dominant retinitis pigmentosa-1 (RP1). – Invest. Ophthalmol. Vis. Sci., 42, 2001, № 10, 2217-2224.
5. A d h i , M. R., Y. Jafri et S. Hussain. Molecular confirmation of the causes of inherited visual impairment in Northern Pakistan. – J. Coll. Physicians Surg. Pak., 19, 2009, № 12, 806-808.
Acta Medica Bulgarica, Vol. XXXVIII, 2011, № 2
49
6. B e l l , J. Predicting disease using genomics. – Nature, 429, 2004. 453-456.
7. P i e r c e , E. A. et al. Mutations in a gene encoding a new oxygen-regulated photoreceptor protein
cause dominant retinitis pigmentosa. – Nat. Genet., 22, 1999, 248-254.
8. R i a z u d d i n , S. A. et al. Autosomal recessive retinitis pigmentosa is associated with mutations
in RP1 in three consanguineous Pakistani families. – Invest. Ophthalmol. Vis. Sci., 46, 2005, №
7, 2264-2270.
9. K h a l i q , S. et al. Novel association of RP1 gene mutations with autosomal recessive retinitis
pigmentosa. – J. Med. Genet., 42, 2005, 436-438.
50
Adress for correspondence:
Assoc. Prof. Krassimir Koev, PhD
Medical University Sofia
2 Zdrave str.
Sofia
8365504
Clinical assessment and molecular genetics...
ACUTE INTERMITTENT PORPHYRIA DURING
PREGNANCY AND NORMAL DELIVERY: A CASE REPORT
A.Tsonev1, E. Kovachev1, I. Bakardzhiev2, S. Ivanov1, A. Abbud1 and M. Veseli1
1
Department of Obstetrics and Gynecology, Medical University of Varna, Bulgaria
2
Medical College, Varna, Bulgaria
Summary. We report a case of a 27-year-old woman, gravida 1, para 1 with
acute intermittent porphyria during pregnancy and normal delivery, who had a generalized seizure and hypertension during delivery of her 1st child at 37 weeks gestation. Proper treatment in collaboration with hematologist greatly improved the
outcome in this otherwise debilitating and potentially life-threatening disease.
Key words: porphyria, pregnancy, delivery
T
INTRODUCTION
he porphyrias comprise a heterogeneous group of rare, primarily hereditary, metabolic diseases caused by a partial deficiency in one of the
eight enzymes involved in the heme biosynthesis. These are classified
as hepatic or erythropoietic depending on the primary site of overproduction and
accumulation of the porphyrin. Acute intermittent porphyria, one of the hepatic porphyrias, is the most severe form of the disease, with gastrointestinal and neuropsychiatric manifestations, such as neuropathic abdominal pain, nausea, vomiting, peripheral neuropathy, hypertension, seizures, mental disturbances. Usually female
patients with porphyria cannot have normal pregnancies without experiencing any
of the above symptoms. However pregnancy is associated with increased levels of
hormones such as progesterone which potentially may aggravate porphyria.
CASE PRESENTATION
A 27-year-old woman, gravida 1, para 1, had a generalized seizure and hypertension during delivery of her 1st child at 37 weeks gestation. At admission the
patient was lethargic, drowsy, restless, irritable with BP=110/80 mmHg; PR=120/
Acta Medica Bulgarica, Vol. XXXVIII, 2011, № 2
51
min; RR=30/min. Her lethargic, drowsy, semiconscious behavior was attributed to
the disease. Her laboratory test results are shown in Table 1.
Table 1. Laboratory test results
Na+
K+
Mg2+
WBC
HGB
HCT
RBC
120
3.1
2.5
16.2 x10.9
98
32.0
4.2 x 10.12
mEq/L
mEq/L
mEq/L
L
g/L
%
L
MCV
MCH
PLT
Fibrinogen
ALAT
ASAT
79.8
26.6
173x10.9
4.1
34
84
fL
pg
L
g
U/L
U/L
Urine sample microscopy=5-6 Leu. Despite having her electrolyte abnormalities corrected on the next day, the patient remained lethargic. Her tendon reflexes
were markedly decreased, but her plantar reflex was normal. Later on a simple
bedside urine test was conducted and it came positive, as urine sample turned
to dark red color on exposure to open atmosphere. On further conformation urine
screening for porphobilinogen by the Window still test and Watson-Schwarz test
were also positive. Acute porphyria was confirmed. Penicillin was administered to
control the possible urinary tract infection. Initially i.v. Mannitol 20% was also given
prophylactically for 1st two days. She was given a high carbohydrate diet and high
iron diet. Her recovery of motor function was slow but definite.
DISCUSSION
The many precipitating factors that are known to induce acute porphyria attacks in susceptible persons include hormonal changes associated with pregnancy
and menstrual cycle, reduced caloric intake drugs, stress, and infections [2]. Barbiturates, sulfonamide, anticonvulsants, and oral contraceptives are by far the most
common drugs implicated in acute porphyria [1, 3]. These conditions or medications directly or indirectly induce the delta- aminolevulinic acid synthase activity,
which results in an increased biosynthesis of heme and the various intermediate
porphyrins (Figure 1).
We believe that the initial porphyria attack, which took the form of a generalized seizure and hypertension in our patient, occurred during the delivery and probably was related to low carbohydrate diet before delivery, and urinary tract infection.
Antibiotics like penicillin and streptomycin are considered to be safer in porphyrias
[4, 7]. The treatment of acute porphyria is high- caloric intake and heme arginate,
heme albumin or hematin 304 mg/day for 4 days [5, 6, 8]. Another important differential diagnosis of progressive weakness associated with pregnancy is GuillainBarre’ syndrome.
52
Acute intermittent porphyria during...
Fig. 1. Biosynthesis of heme
CONCLUSION
We emphasize that it is important to make the correct diagnosis of acute porphyria, because proper management will dramatically improve the outcome in this
debilitating and potentially life- threatening disease. A common error is the failure
to order urine porphyrins. Porphobilinogen, a porphyrin precursor, usually is not
included in a urine porphyrin screen and must be ordered specially. People experiencing severe attacks, especially those with severe neurologic symptoms, should
be treated with hematin. Most classic antiseizure medicines like Neurontin can lead
to acute porphyria attacks.
REFERENCES
1. A s h l e y , E. M. Anesthesia for porphyria. – Br. J. Hosp. Med., 36, 1996, 37-42.
2. C o x , T. M. et al. King George III and porphyria: an elemental hypothesis and investigation. – Lancet, 366, 2005, 332-335.
3. D u r m u s , M. et al. Remifentanil and acute intermittent porphyria. – Eur. J. Anaesthesiol., 19,
2002, 839-840.
4. J a m e s , M. F. M., et R. J. Hift. Porphyrias. – Br. J. Anaesth., 85, 2000, 143-153.
5. K a n t o r , G. et S. H. Rolbin. Acute intermittent porphyria and caesarean delivery. – Can. J. Anaesth., 39, 1992, 282-285.
6. K e r r , G. D. Acute intermittent porphyria and inappropriate secretion of antidiuretic hormone in
pregnancy. – Proc. R. Soc. Med., 66, 1973, 763-764.
7. K u l l e r , J. A. et al. Pregnancy complicated with Guillain-Barre’ syndrome. – South Med. J., 88,
1995, 987-989.
8. M i l o , R. et al. Acute intermittent porphyria in pregnancy. – Obstet. Gynecol., 73, 1989, № 3, 450-452.
ª
Address for correspondence:
Dr. Atanas Tsonev
150 Tzar Osvoboditel Str.
9000 Varna, Bulgaria
00359895551955, e-mail: [email protected]
Acta Medica Bulgarica, Vol. XXXVIII, 2011, № 2
53
LATE RADIATION RETINOPATHY IN A PATIENT TREATED
FOR RETINOBLASTOMA
B. A. Lafaut1, J.-J. De Laey1, F. M. Meire1 and B. M. Kutchoukov2
2
1
Department of Ophthalmology, Ghent University Hospital, Belgium.
Department of Ophthalmology, University Hospital “Alexandrovska” – Sofia, Bulgaria
Summary. We report a case of a young man who developed radiation
retinopathy 12 years after irradiation for retinoblastoma. With a history of bilateral retinoblastoma, our patient presented clustered retinal teleangiectasias in the
non-enucleated but treated with radiation eye. The lesions were associated with
hemorrhages and gradually increasing retinal exudation that deteriorated best
corrected visual acuity (BCVA) from 0.25 to 0.1. Detailed ophthalmic examination
was performed incl. fluorescein angiography (FA), indocyanine green angiography
(ICG) and optical coherence tomography (OCT). The ICG highlighted the vascular
anomalies that were clearly hyperfluorescent in the late phase. After 3 sessions
of focal ALC the outlined edema on Oct scans diminished and BCVA improved to
0.2. Some retinoschisis-like cystic changes remained on OCT retinal scans. The
manifested changes had common features with those found in diabetic retinopathy.
ICG staining of the retinal vascular anomalies was found. Radiation retinopathy
may occur more than a decade following radiation treatment for retinoblastoma.
Appropriate laser treatment may overcome the advancing process.
Key words: radiation retinopathy, OCT, ICG, retinoblastoma
R
INTRODUCTION
adiation retinopathy is a slowly progressive disease with a delayed
onset. The alterations in the structure and permeability of retinal
blood vessels usually occur months to years after exposure to radiation [3]. It was reported to develop in 12% [1] and 24% [4] and was first
detected 11-72 months (mean 37 months) after irradiation therapy [1] for retinoblastoma. However we present a patient whose initial clinical signs appeared 12
years (144 months) after ionizing radiation treatment.
54
Late radiation retinophaty....
CASE REPORT
A 16-year-old patient, previously irradiated for retinoblastoma, was referred for
vascular anomalies in the right eye. The latter were observed for about 3 years but
appeared to increase. At the age of 9 months, a bilateral retinoblastoma had been
diagnosed. The left eye with a large tumoral mass was enucleated and the pathologic
examination confirmed the diagnosis. The right eye presented two foci and was irradiated with 3650 cGy (3x/week 365 cGy; 8MeV). A supportive chemotherapy was
additionally given. Both lesions subsequently regressed, the small one as a sharply
demarcated zone of chorioretinal atrophy temporal of the fovea and the larger one as
an atrophic scar inferior to the optic disc with two foci of calcification.
His best corrected visual acuity was 20/80, the anterior segment was unremarkable. The retinal pigment epithelium was minimally pigmented and the choroidal vascular pattern could easily be seen. Two atrophic chorioretinal scars, corresponding
to the tumoral lesions, were present. Two clusters of retinal micro-aneurysms and/or
dot retinal hemorrhages were found, one temporally and another superotemporally
of the macular scar as well some scattered similar lesions. No retinal exudates or
larger retinal hemorrhages were seen. Fluorescein angiography (FA) revealed filling
of some of these lesions, in others fluorescence was blocked by associated intraretinal hemorrhages or the lesions were thrombosed (Fig 1A). Retinal ischemia was not
observed, neither in the macular area, nor in the periphery.
Two years later, his visual acuity did not changed, however larger retinal hemorrhages appeared as well as retinal exudation superior to the macular atrophic
scar (Fig.1B). A normal pattern of infrared autofluorescence was seen apart from
masking by the calcifications in the regressed tumor scar. Indocyanine green (ICG)
angiography highlighted better the vascular anomalies than FA, which became hyperfluorescent in the early venous phase (Fig. 2A). In the late phase, their walls remained hyperfluorescent (Fig. 2B). The atrophic scars were hypofluorescent while
the retinal exudates blocked choroidal fluorescence. The hemorrhages only caused
a minor masking phenomenon. An ill-defined area of hyperfluorescence was seen
superotemporally to the disc in the late phase.
Fig. 1. FA (venous phase) at the age of 16 years
showing two clusters of retinal vascular abnormalities (white arrowheads) and a perifoveal area of
well-demarcated chorioretinal atrophy at the place
of the regressed tumoral lesion
Acta Medica Bulgarica, Vol. XXXVIII, 2011, № 2
55
Fig. 2. Fundus photo at the age of 18. Note the occurred retinal exudation above the macular
scar (black arrowhead). The vascular abnormalities have increased
Fig. 3A. Early venous phase ICG angiogram at the age of 18 years showing hyperfluorescence
of the vascular abnormalities (black arrowheads)
Fig. 3B. Late phase ICG angiogram of the posterior pole – remark two obvious clusters of vascular lesions
(black arrowheads), the hypoflurescent chorioretinal scars and masking of exudates above the fovea
56
Late radiation retinophaty....
Fig. 4. A – OCT scan through the macula at the age of 19 years with severe macular edema; B – OCT
scan 9 months later, after 3 sessions of focal argon laser treatment. Note the substantially decreased
edema and some residual cystic changes
Almost a year later, his visual acuity decreased from 20/80 to 20/200. Optical
coherence tomography (OCT) revealed severe retinal edema in the macular area
(Fig 3). After three sessions of focal argon laser photocoagulation his best corrected visual acuity improved to 20/100 and the macular edema on OCT regressed.
On follow-up OCT sessions persisting intraretinal cystic changes were recorded in
the macula.
DISCUSSION
Alterations of retinal vessels, such as microaneurysms, dilated capillaries,
cotton-wool spots, perivascular sheathing, irregular loss of retinal capillaries,
hemorrhages and hard exudates become apparent months or several years after X-ray irradiation of the orbital region. The vascular changes are similar to
those found in diabetic retinopathy. Radiation retinopathy threshold depends on
the delivered total dose, irradiated retinal area and fractionation scheme. Retinal
damage usually develops after 3000 to 3500 cGy and may be enhanced by simultaneous chemotherapy.
Acta Medica Bulgarica, Vol. XXXVIII, 2011, № 2
57
This patient developed clustered retinal telangiectasias with intraretinal
hemorrhages 12 years after irradiation for retinoblastoma at the age of 9 months.
The number of dilated capillaries gradually increased. His visual acuity deteriorated because of intraretinal accumulation of fluid, documented by OCT and
formation of hard exudates. We consider these lesions to be a late radiation effect although FA failed to demonstrate major capillary dropout – the hallmark of
radiation-induced retinopathy [2]. The retinal vascular anomalies were strikingly
highlighted by ICG angiography: they appeared as highly fluorescent dots in the
early venous phase, while in the late phase their wall was stained. The regression of the macular edema after appropriate focal laser treatment, as confirmed
by OCT scans, was impressive.
REFERENCES:
1. A n t e b y , I. et al. Ocular and orbital complications following the treatment of retinoblastoma. –
Eur. J. Ophthalmol., 8, 1998, № 2,106-111.
2. B r o w n , G. C. et al. Radiation retinopathy. – Ophthalmology, 89, 1982, 1494-1501.
3. M u k a i , S., D. R. Guyer et E. S. Gragoudas. Radiation retinopathy. – In: Albert, D. M., Jakobiec, F. A., eds. Principles and practice of ophthalmology. Philadelphia, W. B. Saunders, 1994,
1038-1041.
4. P r a d h a n , D. G. et al. Radiation therapy for retinoblastoma: a retrospective review of 120 patients. – Int. J. Rad. Oncol. Biol. Phys., 39, 1997, № 1, 3-13.
ª
58
Address for correspondence:
Borislav Kutchoukov, MD, PhD
Department of Ophthalmology
University Hospital “Alexandrovska”
Blvd. “Sv. Georgi Sofiiski” 1
1431 Sofia
+ 359 888556724
Late radiation retinophaty....
SEVERE BACLOFEN SELF-POISONING – A CASE
REPORT
J. Radenkova-Saeva
Toxicology Clinic, MHATEM “N. I. Pirogov” – Sofia, Bulgaria
Summary. Baclofen is a lipophilic analogue of gamma-aminobutyric acid
(GABA), an inhibitory neurotransmitter in central nervous system. It is a muscle relaxant used in persons with neuromuscular disorders to control spasticity. Intentional or
accidental overdose of this drug can cause profound central nervous system depression, including coma, seizures, respiratory depression, and cardiovascular effects
such as bradycardia, hypotonia. A case of baclofen self-poisoning was presented in
a 21-year-old woman, hospitalized in the Toxicology Clinic, MHATEM “Pirogov” with
coma and respiratory depression. Primary importance in the treatment and management of such ingestions should focus on airway and respiratory support.
Key words: baclofen, self-poisoning, coma, seizures, respiratory depression
B
aclofen (brand names Kemstro and Lioresal) is a derivative of gammaaminobutyric acid (GABA). It is primarily used to treat spasticity and is
under investigation for the treatment of alcoholism. It is an agonist for the
GABAB receptors [4, 6]. The modulation of the GABAB receptor is what produces
baclofen’s range of therapeutic properties. Its beneficial effects in spasticity result
from actions at spinal and supraspinal sites. Baclofen can also be used to treat hiccups, and has been shown to prevent rises in body temperature induced by the drug
MDMA in rats [3]. In addition, research over the last five years has shown baclofen to
be effective in the treatment of alcohol dependence and withdrawal, by inhibiting both
withdrawal symptoms and cravings [1, 2]. Baclofen is widely used for the treatment
of spastic movement disorders, especially in instances of spinal cord injury, spastic
diplegia cerebral palsy, multiple sclerosis, amyotrophic lateral sclerosis (Lou Gehrig’s
disease), peripheral neuropathy and trigeminal and glossopharyngeal neuralgias.
Baclofen therapy is usually started with an initial low dose of about 10 mg daily
in divided doses and gradually titrated up in a stepwise fashion until symptomatic
relief occurs. The usual maximum dose is 80 mg per day.
Acta Medica Bulgarica, Vol. XXXVIII, 2011, № 2
59
Symptoms of a baclofen overdose include vomiting, weakness, drowsiness,
slow breathing, seizures, unusual pupil size, and coma.
CASE REPORT
A case of baclofen self-poisoning was presented in a 21-year-old woman, hospitalized in the Toxicology Clinic, MHATEM “Pirogov”. She was found unresponsive
with empty bottle and blister packs, containing baclofen, thioridazin and lizinopril.
She had ingested an unknown number of tablets in a suicidal attempt. In emergency toxicological department, initial vital signs were: blood pressure 110/ 70 mm
Hg; heart rate 68 beats (minute; respiratory rate 18 breaths) minute. Gastric lavage
was performed, nasal oxygen and activated charcoal were administered. Physical
exam revealed profoundly somnolent consciousness. She had dry skin and mucous
membranes. The pupils were normally broad. Lungs – clear vesicular breathing bilaterally. Cardiovascular system: heart – rhythmic activity without added noise. The
abdomen was soft, lacked defense. Not detectable hepatosplenomegaly. Extremities were without edema. In the next few hours, the patient condition was critical:
she was in deep coma, had muscle hypotonia, hyporeflexia and seizure activity
with apnoic breaks. Due to respiratory failure she received endotracheal tube insertion with mechanical ventilation. Toxicological analysis confirmed the presence
of baclofen together with small amounts of thioridazin and lizinopril. The treatment
with forced diuresis, nootropil, supportive therapy, including mechanical ventilation
for 2 days led to complete recovery.
DISCUSSION
Analysis of 37 cases cited in the literature suggests that there are two types
of baclofen intoxication syndrome. Patients with acute intoxication present with
four major clinical manifestations: encephalopathy (disturbance of consciousness
and/or seizure), respiratory depression, muscular hypotonia, and generalized hyporeflexia. Patients with chronic intoxication present with hallucinosis, impaired
memory, catatonia, or acute mania. The acute intoxication syndrome has a faster
onset, shorter duration, more severe clinical manifestations, and higher incidence
of seizures than the chronic intoxication syndrome. Baclofen intoxication, although
it may cause grave encephalopathic manifestations and electroencephalographic
findings, has a benign outcome if actively managed [5].
Severe toxicity of baclofen has been frequently reported in the literature in
patients with impaired kidney function after repeated ingestion of therapeutic doses.
Accumulation of baclofen due to diminished renal elimination has been postulated as
the main pathophysiologic mechanism. The patients presented with severe toxicity
immediately after ingesting the first therapeutic dose (25–50 mg) of baclofen [8].
A group of adolescents became symptomatic after ingesting 3 to 30, 20 mg
tablets of baclofen during a party at a suburban Boys’ Club. The most common
60
Severe baclofen self-poisoning...
clinical findings included coma, hypothermia, bradycardia, hypertension, and hyporeflexia. Mean length of mechanical ventilation was 40 hours. Three patients had
unifocal premature ventricular contractions. Two patients had tonic-clonic seizures.
A single dose of activated charcoal was given to all patients. All patients recovered
and were discharged home within 5 days of ingestion [7].
Our patient had many of the features of baclofen overdose described in the
literature, including coma, seizure activity, hypotonia, and respiratory depression.
Primary importance in the treatment of such ingestions should be placed on maintenance of airway and respiratory support [9].
CONCLUSION
Baclofen overdose may result in coma, apnea, autonomic disturbances, seizures. Primary importance in the treatment and management of such ingestions
should focus on airway and respiratory support.
REFERENCES
1. A d d o l o r a t o , G. et al. Baclofen in the treatment of alcohol withdrawal syndrome: a comparative
study vs diazepam. – Am. J. Med., 119, 2006, № 3, 276.e13-18.
2. A d d o l o r a t o , G. et al. Effectiveness and safety of baclofen for maintenance of alcohol abstinence in alcohol-dependent patients with liver cirrhosis: randomised, double-blind controlled
study. – Lancet, 370, 2007, 9603, 1915-1922.
3. B e x i s , S. et al. Baclofen prevents MDMA-induced rise in core body temperature in rats. – Drug
Alcohol Depend., 74, 2004, № 1, 89-96.
4. D z i t o y e v a , S., N. Dimitrijevic et H. Manev. Gamma-aminobutyric acid B receptor 1 mediates
behavior-impairing actions of alcohol in Drosophila: adult RNA interference and pharmacological
evidence. – Proc. Natl. Acad. Sci. USA, 100, 2003, № 9, 5485-5490.
5. L e e , T.-H. et al. Baclofen intoxication: report of four cases and review of the literature. – Clin.
Neuropharmacol., 15, 1992, № 1, 56-62.
6. M e z l e r , M., T. Müller et K. Raming. Cloning and functional expression of GABA(B) receptors
from Drosophila. – Eur. J. Neurosci., 13, 2001, № 3, 477-486.
7. P e r r y , H. E. et al. Baclofen overdose: drug experimentation in a group of adolescents. – Pediatrics, 101, 1998, 1045-1048.
8. S c h e n k - J a e g e r , K. M. et al. Severe toxicity of a single therapeutic dose of baclofen in patients with impaired renal function. – Clin. Toxicol., 48, 2010, № 3, 258.
9. V a n D i e r e n d o n k , D. R. et D. J. Dire. Baclofen and ethanol ingestion: a case report. – J.
Emerg. Med., 17, 1999, № 6, 989-993.
Address for correspondence:
Dr. Julia Radenkova – Saeva, MD, MSc, PhD,
Toxicology Clinic
Emergency Hospital “N. I. Pirogov” – Sofia
e-mail: [email protected]
Acta Medica Bulgarica, Vol. XXXVIII, 2011, № 2
61
MESANGIAL LIPOIDOSIS WITH CHOLESTEROL
DEPOSITION – A NEW CASE WITH NEPHROTIC
SYNDROME AND RAPIDLY PROGRESSING
RENAL FAILURE
R. Robeva1, T. Todorov2, V. Dimitrova1 M. Yordanov1 and D. Doychinov1
Clinic of Internal Disease Therapy, Nephrology, Dialysis and Clinical Pharmacology,
Univ. Hospital ”Sv . Ivan Rilski”
2
Department of Pathology, Medical University – Sofia
1
Summary. More than 140 years ago, first Virchow associated observed lipid
abnormalities with the concomitant renal disease. Despite a large body of clinical
and laboratory evidence, the precise role of the lipids in the development of the
renal disease remains unclear. In 1988, D. Doychinov described a new autosomal
dominant hereditary disease leading to cholesterol accumulation within the glomerular mesangium. We report the case of a young man (a member of the affected
family) with the same biopsy proven disease but with different clinical abnormalities
and course. This patient had an overt nephrotic syndrome from the onset: 5 g/24h
proteinuria, hypoproteinemia (52 g/l), hypoalbuminemia (28 g/l), hypercholesterolemia (8.5 mmol/l), edema as well as severe hypertension (180-200/110-120 mm
Hg). Five years later he developed renal failure and after another three he was put
on hemodialysis
Key words: mesangial lipoidosis, nephrotic syndrome, renal insufficiency
M
ore than 140 years ago first Virchow associated observed lipid abnormalities with the concomitant renal disease. Despite a
large body of clinical and laboratory evidence, the precise role
of the lipids in the development of the renal disease remains unclear. It has
been however demonstrated that lipid metabolism abnormalities may promote
glomerular injury by an increased intrarenal lipid deposition such as in Fabry`s,
Niemann – Pick and Gaucher` diseases, Alagille syndrome, the hereditary serum cholinesterase deficiency, type III hyperlipoproteinemia, lipoprotein glom62
Mesangial lipoidosis with...
erulopathy [1, 3, 4, 5]. In all of these, systemic signs and symptoms are also
present. Disorders of lipid deposition limited to the kidney are rare and are still
poorly understood.
In 1988, D. Doychinov described a new autosomal dominant hereditary disease leading to cholesterol accumulation within the glomerular mesangium [2]. This
injury was biopsy-proven in another four members of the same family (three men
and a woman) and it is probably present in another two older members of the
family (refusing biopsy) who developed renal failure at the age of 51and 38 years
respectively.
The onset of the disease was in the patient’s younger age. 10-20 years after
the typical onset with moderate proteinuria and hematuria, severe hypertension
and chronic renal failure developed progressively and hemodialysis was indicated.
Cholesterol crystal depositions were found in vitro in the glomerular mesangial matrix in all affected patients. No lipid storage in other renal structures or other organs
could be established. There were no abnormalities of serum cholesterol and triglycerides [2].
THE CASE REPORT
In 1998, we investigated another member of the affected family (4th generation) aged 17 years who is a son of single affected woman (see Fig. 1). The histological finding from his biopsy was no different from those of the other affected
family members (shown on Fig. 2). There were enlargement and lobulation of the
mesangial axes by cholesterol deposits, segmental sclerosis of the capillary wall
with fibrinoid deposits and peri-glomerular fibrosis in some glomeruli.
The extent of the injury was moderate – 10% sclerotic glomeruli, initial interstitial fibrosis and tubular atrophy were all documented (see Fig. 3).
Unlike his affected family members, this patients had an overt nephrotic syndrome just from the onset: proteinuria – 5 g/24 h, hypoproteinemia – 52 g/l, hypoalbuminemia-28 g/l, hypercholesterolemia – 8.5 mmol/l, edema as well as severe
hypertension- 180-200/110-120 mm Hg. His renal function at that time was normal.
The patient was treated with heparin, antihypertensive and lipid-lowering drugs.
The nephrotic syndrome however persisted; the hypertension was severe and very
difficult to treat. In 2003, the condition progressed to chronic renal failure (serum
creatinine 250 μmol/l). The follow-up renal biopsy manifested advanced sclerotic
changes in the glomeruli (38%), as well as interstitial fibrosis and focal tubular atrophy (see Fig. 4).
The patient was treated again with heparin, antihypertensives and symptomatic medications. Three years later he developed end-stage renal disease and was
referred to hemodialysis.
Acta Medica Bulgarica, Vol. XXXVIII, 2011, № 2
63
Fig. 1. Genealogy tree of the investigated family
Fig. 2. Histological finding in the first described
affected family member (renal biopsy)
64
Fig. 3. First biopsy finding of the reported
patient (Trichrom Masson x 100)
Mesangial lipoidosis with...
Fig. 4. Follow-up biopsy finding of the same patient
DISCUSSION
Essential lipoidosis with cholesterol deposition is an autosomal dominant hereditary disease. The onset of disease is in the younger age with moderate proteinuria and microscopic hematuria. Our patient however demonstrated that the
development of substantial protein losses with subsequent nephrotic syndrome is
also possible. In most affected members of this particular family, the hypertension
developed progressively, but in our patient the severe hypertension presented early
in the course of the disease.
Hypercholesterolemia is not a typical finding, and is probably related to the
nephrotic syndrome in our case. The onset of the disease with nephrotic syndrome
and hypertension was very abrupt. That is probably due to a faster progression of
the chronic renal failure (it takes usually 10- 20 years to develop; in this case – only
5 years).
The patient was treated with heparin as prevention of thrombotic complications of the nephrotic syndrome. There are some observations, that this drug may
exert an antisclerotic effect [5].
At the beginning, we were able to influence progression of the disease and
to bring the renal deterioration to a steady state. The patient however was non-
Acta Medica Bulgarica, Vol. XXXVIII, 2011, № 2
65
compliant and was lost for follow-up. That is why we did not evaluate the long term
effect of this treatment. To our knowledge, there is no evidence of an effective treatment of this disease.
REFERENCES:
1. D a v i s , J., R. Griffiths, D. Rozansky et al. Glomerular basement membrane lipoidosis in Alagille
syndrome. – Pediatr. Nephrol., 25, 2010, № 6,1181-1184.
2. D o i t c h i n o v , D. Lipoidosis of the glomerular mesangium with accumulation of cholesterol. A
novel hereditary disease. – Nephrologie, 9, 1988, № 6, 273-276.
3. H o n d a , K. et al. Hereditary serum cholinesterase deficiency associated with severe lipid deposition in the kidney. – Intern. Med., 32, 1993, № 2, 145-151.
4. P a s q u a r i e l l o , A. et al. Lipoprotein glomerulopathy: first report of 2 not consanguineous Italian
men from the same town. – J. Nephrol., 24, 2011, № 3, 381-385.
5. S a i t o , T. Abnormal lipid metabolism and renal disorders. – Tohoku J. Exp. Med., 181, 1997,
321-337.
6. X u , Q. et al. In vitro models of TGFβ- induced fibrosis suitable for high-throughput screening of
antifibrotic agents. – Am. J. Physiol. Renol. Physiol., 293, 2007, № 2, 631-640.
66
Address for correspondence:
Assoc. Prof. R. Robeva, MD
Univ. Hosp. ”St . Ivan Rilski”
Dialysis and Clinical Pharmacology
15, Blvd.” Acad. Ivan Geshov”
1431 Sofia
+35928510846
Mesangial lipoidosis with...
А CASE OF GIANT SQUAMOUS CELL CARCINOMA
OF THE TONGUE – A RARE FINDING WITHIN THE
FRAMEWORK OF DERMATOLOGICAL SCREENING?
G. Tchernev2, J. Ananiev1 and M. Gulubova1
1
Medical Faculty, University Hospital Lozenetz, Polyclinic for Dermatology and Venereology,
University Saint Kliment Ohridski – Sofia
2
Department of General and Clinical Pathology, Medical Faculty, Trakia University
Summary. Carcinomas of the mouth cavity, and in particular of the tongue, are
a comparatively rare form of pathology. For the correct establishing of the stages of
the tumor and for defining of the diagnosis and survival of the patients, a correct histological verification is needed as well as defining the stages as per the TNM criteria.
In this publication we present a rare case of a female patient with a tongue carcinoma,
diagnosed within the framework of a dermatological screening. The tumor has been
classified as a squamous carcinoma of the tongue with formed cornea and a moderate
degree of differentiation. Quite often, such an asymptomatic clinical finding creates diagnostic and from there prognostic difficulties, which appear as a result of both the late
discovery of the tumor as well as the impossibility for its complete surgical elimination.
The patient underwent a partial glossectomy of the right part of the tongue with a laser,
selective functional neck dissection on levels I – III under intubation anesthesia. Subsequently, a stabilisation of the clinical status followed and one year after the operation
no metastases and clinical complaints were registered. The careful examination of the
oral and genital mucosa, within the scope of the dermatological screening is of primary
importance for the timely diagnosis of the precancers and the tumors of the transitional
mucosa and in particular the mouth cavity.
Key words: squamous carcinoma, tongue, TNM classification
S
INTRODUCTION
quamous cell carcinoma (SCC) is a carcinomatous cancer occurring in
multiple organs. These include the skin, lips, mouth, esophagus, urinary
bladder, prostate, lungs, vagina, and cervix. It is a malignant tumor of
squamous epithelium (epithelium showing squamous cell differentiation). Despite
the common name, these are unique cancers with large differences in manifestation and prognosis.
Acta Medica Bulgarica, Vol. XXXVIII, 2011, № 2
67
Squamous carcinoma of the tongue is quite often a diagnosis which is placed
too late and in most cases does not presume a good forecast [1]. In spite of this,
this neoplasma continues to be the most frequently met neo-pathology of the mouth
cavity. During the last several years, the frequency of this type of carcinoma in Europe and the USA has grown, particularly among the younger patients not older
than 45 years of age [2, 3]. It turns out, that squamous carcinoma of the tongue
is exceptionally aggressive as clinical behaviour and is associated with frequent
occult metastases in the lymph glands of the neck. The prognosis is defined by
a number of histological and clinical factors, used as forecast markers, where of
greatest importance remains to be the tumor classification as per TNM [4].
We are here presenting the case of a patient with squamous carcinoma of the
tongue given the lack of data from the anamnesis for cigarette smoking, chronic
use of alcohol or infection from the human papilloma virus.
ANAMNESIS
The patient is a 65-years-old female with complaints of a non-painful mucous
lesion on the tongue (fig. 1a-b), who was examined within the framework of a screening program for examining skin and mucosa in the polyclinic of dermatology and venereology. The woman does not claim chronic use of alcohol and cigarette smoking
and there are no data of increased promiscuity. In the course of the anamnesis, no
data were obtained of infection with the human papilloma virus (HPV) in the sphere
of the oral or genital mucosa. Contradicting data were found for a recurring edema of
the larynx with an unclear genesis as well as accompanying diet-controlled diabetes.
The patient does not claim systematic use of medicinal products.
CLINICAL FINDING
In the course of the examination, a crater-shaped erosion plaque was found in
the area of the lateral right side of the tongue, dorsal, with no bleeding but with considerable induration of the tissue perilesional as well as loss of tongue elasticity.
Fig. 1a, b. Macroscopic view of the lesion – erosive form of a carcinoma of the tongue. This clinical case was
presented by Dr. Georgi TCHERNEV in 2004 at the Dessau dermatological conference held by the Department
of Dermatology, Venereology, Allergology and immunological centre in Dessau, the Academic Educational Hospital at the Martin Luther University in Halle-Wittenberg under the leadership of Professor H. D. Göring, former
long-standing chief of the Department of Dermatology, Venereology, Allergology and Immunology.
68
A case of giant squamous cell...
HISTOLOGICAL DATA
The biopsy of the tongue proved the availability of a moderately differentiated
squamous carcinoma with the formation of cornea. Tumor cell invasion deep in
the derma is observed together with the formation of solid nests, a well expressed
polymorphism and hyperchrome nucleus as well as the availability of partial keratinisation (fig. 2a-b).
Fig. 2a. Moderately differentiated squamous
carcinoma with formation of cornea and
sectors with partial keratinisation (x 40)
Fig. 2b. Polymorphism and hyperchrome
nucleus in a moderately differentiated
squamous carcinoma (x 200)
Paraclinic
• TPHA serology – negative
• Chlamydia serology IgG, IgA, IgM: negative
• Blood test, sediment, CRP, liver and kidney indicators: within norms
• HIV serology – negative
• Quantitative immunoglobulin and lymphocytic subpopulations – within the
norms.
Other examinations
• X-ray of the lung: no metastases
• X-ray of the neck vertebrae in two plains: lordosis, spondylarthrosis C5/C6
• ST of the neck: no metastases
• Ultrasound examination of the neck lymph glands, the area of the abdomen,
supraclavicular, axial: no metastases
TREATMENT
The patient underwent a partial glossectomy of the right part of the tongue
with a laser, selective functional neck dissection on levels I – III under intubation anesthesia. Subsequently, a stabilisation of the clinical status followed and one year
after the operation no metastases and clinical complaints were registered.
Acta Medica Bulgarica, Vol. XXXVIII, 2011, № 2
69
DISCUSSION
Twenty percent of the tongue carcinomas, as a part of the mouth cavity
carcinomas, possess squamous histogenesis [5]. From the clinical aspect, they
may be presented as formations having an ulcer, papillar or nodular type of form
as well as fibrosis, expressed to varying degrees. In most cases, this type of carcinoma develops on the basis of an already existing lesion (precancerosis), as,
for example: leucoplakia, eriythroplakia, chronic irritation, etc., but they may also
develop de novo [6]. The behaviour in the case of the squamous carcinoma of
the tongue is decisive for the prognosis and survival of the patients. In the cases,
where no metastasis of the lymph glands are suspected, there are two behavioural options: planned dissection with a subsequent histological verification and
observation. In order to solve this problem, it is usually necessary to identify a
reliable prognostic marker taking into consideration the histological type and the
TNM staging of the tumor.
In our case, we have a female patient, in the anamnesis of whom there were
no data of alcohol abuse or cigarette smoking as well as any existence of precancerous lesions. Examining the histological finding, we have placed the accent on
the following morphological features: degree of keratinisation, nucleus polymorphism, number of mitoses, invasive edge of the tumor as well as the infiltration with
lymphocytes. Its average diameter between its two most distant edges is 2 cm,
which practically places it in tumor stage T2. The lack of engaged lymph nodes as
well as the lack of distant metastasis form the end result, which is stage T2NxM0.
In spite of the basic importance, the prognosis of T1/T2 stage, with the availability of occult metastases in the locoregional nodes makes the assessment of
the tumor’s stage difficult and incorrect, all the more that the clinical examination
via palpation often leads to false negative data [7]. The histological description of
the tumor, together with the basal cell carcinoma and the verrucous carcinoma,
places it among the three most often met mouth cavity carcinomas, respectively
the tongue, while its moderate differentiation speaks for a large metastatic potential
at a later stage.
The examination of the mucosa is of primary importance in processing dermatological screening programs where quite often asymptomatic lesions in the area of
the tongue demonstrate an infiltrative and metastatic character and pose a serious
danger to the life of the patient. Often, however, the discovery of a primary tumor
comes too late while its initial histological verification does not give complete and
reliable information on the degree of development of the process which makes the
prognostication very difficult.
REFERENCES
1. A l v i , A. et J. Johnson. Development of distant metastasis after treatment of advanced-stage
head and neck cancer. – Head Neck, 19, 1997, 500-505.
70
A case of giant squamous cell...
2. S c h a n t z , S. et Y. Guo-Pei. Head and neck cancer incidence trends in young Americans,1973-1997, with a special analysis for tongue cancer. – Arch. Otolaryngol. Head Neck Surg.,
128, 2002, 268-274.
3. A n n e r t z , K. et al. Incidence and survival of squamous cell carcinoma of the tongue in Scandinavia, with special reference to young adults. – Int. J. Cancer, 101, 2002, 95-99.
4. B a g a n , J., G. Sarrion et Y. Jimenez. Oral cancer: clinical features. – Oral Oncol., 46, 2010,
№ 6, 414-417.
5. J i n , Y. et C. Jin. Oral squamous cell carcinoma. – Atlas Genet. Cytogenet. Oncol. Haematol.,
September 2006. http://AtlasGeneticsOncology.org/Tumors/OralSquamCellID5368.html
6. T i l a k a r a t n e , W. M. et al. Oral submucous fiberosis: A review of aetiology and patho-genesis.
– Oral Oncol., 42, 2006, 561.
7. S p a r a n o , A. et al. Multivariate predictors of occult neck metastasis in early oral tongue cancer.
– Otolaryngol. Head Neck Surg., 131, 2004, № 4, 472-476.
Address for correspondence:
Assoc. Prof Georgi Tchernev, M.D. PhD
Department of Dermatology and Venerology
Trakia University of Stara Zagora
Medical Faculty
Armeiska Street № 11
6000 Stara Zagora, Bulgaria
00359 885 588424
e-mail: [email protected]
Acta Medica Bulgarica, Vol. XXXVIII, 2011, № 2
71
Р53 – “THE GUARDIAN OF GENOME”
J. Ananiev1, G. Tchernev2, J. W. Patterson3, M. Gulubova1 and G. Ganchev4
Department of General And Clinical Pathology, Medical Faculty, Trakian University –
Stara Zagora, Bulgaria
2
Department of Dermatology and Venereology, Medical Faculty, Trakian University –
Stara Zagora, Bulgaria
3
Division of Surgical Pathology & Cytopathology, University of Virginia,
University of Virginia Hospital Health System, Charlottesville, VA, USA
4
Second Surgical Department, MHAT "Prof. Stoyan Kirkovich" – Stara Zagora, Bulgaria
1
Summary. p53 is a transcription-suppressing factor, which participates in control of cell cycle and apoptosis and in the regulation of cell genomic integrity. p53
is a tumor suppressor protein that in humans is encoded by the TP53 gene. p53
is crucial in multicellular organisms, where it regulates the cell cycle and, thus,
functions as a tumor suppressor that is involved in preventing cancer. The name
p53 is in reference to its apparent molecular mass: It runs as a 53-kilodalton (kDa)
protein on SDS-PAGE. But, based on calculations from its amino acid residues,
p53's mass is actually only 43.7 kDa. This difference is due to the high number of
proline residues in the protein, which slow its migration on SDS-PAGE, thus making
it appear heavier than it actually is. This effect is observed with p53 from a variety
of species, including humans, rodents, frogs, and fish. Mutations of p53 are observed in 50% of all human carcinomas and in 90% of melanomas being the most
frequently genome disturbance in carcinomas. Also, allele loss and mutation of the
p53 gene are detected in more than 60% of gastric cancers regardless of histological type. Different mechanisms of activation, physiological functions and the role of
p53 in cancerogenesis and in different neoplasms are viewed. Immunohistochemical recognition of p53 in normal cells and neoplastic altered cells gives information
about genesis, prognosis and sensitivity to therapy of different types of cancers.
Nevertheless, prospective studies need to address this issue definitely and this
marker need further investigation and confirmation.
Key words: p53, activation, biological function, immunohistochemical expression, malignant melanoma, gastric cancer
72
p53 – "The guardian of genome"
C
arcinomas form a very heterogeneous group of diseases. Although
their occurrence and development are influenced by various factors,
two of these factors – namely, the disturbed regulation of cellular proliferation and the inhibition of apoptosis – play a major role in the pathogenesis of
all tumors. The disturbed regulation of cellular proliferation in tumors is due to gene
mutations affecting normal cellular growth and division at various stages of cellular
cycle. Typical examples are the changes in pRb and p53 regulating cellular cycle
at different stages. On the other part, the survival of both normal somatic cells and
tumor cells is related to the regulation of apoptosis. The suppressed apoptosis
in tumor cells is a result of the combination of direct oncogene activity, genetic
mutations of regulating proteins, and the interaction of cellular proliferation and
apoptosis.
1.1. p53 – Introduction. The p53 protein is a transcription factor taking part in
the maintenance of human genomic integrity through regulation of cellular growth
within the cellular cycle as well as through regulation of cellular apoptosis. That is
why it is frequently named “the guardian of genome”. Mutations in the p53 gene
represent the most frequently occurring genetic damage observed in various types
of human cancer. These are found in more than 50% of all cancers and in more than
90% of cutaneous malignant diseases. Besides, malignancies or melanomas with
p53 mutations respond poorly to treatment and tend to have a poor prognosis [29].
1.2. Structure of p53. p53 is a protein with a short half-life of about 20 minutes that is found at a very low level in normal cells. Human p53 contains 393
amino acids arranged structurally and functionally in 4 domains: amino acidic (transcriptional domain); central nuclear domain (linking DNA); tetramerizing domain;
and c-terminal regulatory domain. The amino transcriptional domain reacts with
various proteins that positively regulate p53 transcription function in physiological
conditions. The central nuclear domain plays an important role in the linkage of
DNA sequences (fragments) as well as the various oncogenes and proteins that
negatively affect p53 transcription activity. The mutations affecting p53 suppressor activity emerge in this domain. The tetramerizing domain maintains p53 as a
tetramer in dissolved state at physiological conditions. The oligomerization of p53
is important for its suppressor activity. The c-terminal domain regulates the linkage
of DNA fragments by the central nuclear domain. The structural changes in the cterminal domain are required for p53 activation in view of linking DNA fragments to
the nuclear domain of p53.
1.3. Function and activation of p53. p53 was identified in 1979 by Lionel
Crawford, David P. Lane, Arnold Levine, and Lloyd Old, working at Imperial Cancer Research Fund (UK), Princeton University/UMDNJ (Cancer Institute of New
Jersey), and Sloan-Kettering Memorial Hospital, respectively. It had been hypothesized to exist before as the target of the SV40 virus, a strain that induced development of tumors. The TP53 gene from the mouse was first cloned by Peter Chumakov of the Russian Academy of Sciences in 1982, and independently in 1983 by
Acta Medica Bulgarica, Vol. XXXVIII, 2011, № 2
73
Moshe Oren in collaboration with David Givol (Weizmann Institute of Science). The
human TP53 gene was cloned in 1984, and the full length clone in 1985. The major
function of p53 is to respond to DNA damage as well as to protect from accumulation of oncogene mutations, thus preventing the occurrence of a state of genomic
instability. Several stress-related conditions lead to the emergence of signals for
p53 activation. Other p53-activating factors are vital or cellular oncogenes, hypoxia
and hypoglycemia [18]. Transcriptional activation of p53 is a major component of
its biological effects [4]. Activated p53 is bound to specific DNA fragments and activates the process of transcription [8, 9]. The exact mechanism through which p53 is
being activated in case of cellular stress is not completely clarified. Activation might
include increased p53 level as well as appear as a post-translation phenomenon –
due to DNA fragment linkage. The main question for p53 activation is how the cell
interprets DNA damage and transfers a signal for p53 stabilization. One possibility
is that cellular proteins recognize damaged DNA and bind to p53. Another possibility is that specific phosphorylation, dephosphorylation and acetylation lead to p53
activation [1, 29].
P14ARF
DNA damage
Mdm-2
Bax
Apoptosis
p53
P21
Celullar blockage
Fig. 1. Mdm-2 and ARF – their role in p53 regulation
The Mdm2 protein plays a major role in the control of p53 protein stability.
Mdm2 is a target of p53 transcriptional activation. At the same time, Mdm2 interacts
with and inhibits p53 transcriptional activity. This is done through rapid p53 disintegration, and this route is probably responsible for the low p53 level in normal cells.
In cancer cells with p53 mutations, it is believed that the low Mdm2 level contributes
to p53 stabilization [11]. The phosphorylation of p53 amino terminal occurring after
DNA damage reduces Mdm2 binding affinity and leads to p53 stabilization. Various
factors provoking alterations in cellular DNA specifically inhibit the transcription of
Mdm2 protein and induce an indirect response through p53. The reduced expression of Mdm2 in these cases is not dependent on p53, and the exact mechanism is
not known [41] (Fig. 2).
74
p53 – "The guardian of genome"
Oncogenes
damage
DNA damage
Е1А, c-Мyc
Anti-tumor drugs, γ-treatment, UV-treatment
ATM
DNS-PK
P19 ARF
Mdm-2
NF-kB
Bax
Cyt C
Release
Apaf-1
P53
Bcl-2
P21
CDKs
Bcl-2
Rb-E2F
Rb-PPP
CDK4
Caspases
P16
E2F
Apoptosis
damage
Fas/FasL
Ornithinedecarboxylase
Cellular blockage
damage
Fig. 2. Routes of regulating carcinogenesis
Meanwhile, it has been shown that several cellular oncogenes like E1A adenovirus, SV4OT antigens and Human papilloma virus 16E7 stabilize p53. Surprisingly, oncogenic viruses inducing DNA replication stabilize p53 that leads to growth
arrest or apoptosis. One plausible explanation is that p53 stabilization influenced
by viral oncogenes represents a defensive cellular response to viral infection. However, oncogenic viruses develop auxiliary mechanisms aimed at overcoming this
defensive cellular response. E1A inhibits p53 transcriptional activation that leads
to degradation of the protein and to inhibition of the negative growth signals from
p53. The mechanism of p53 stabilization in response to the expression of viral oncogenes was clarified following the discovery of p19ARF / p14ARF in humans. ARF
belongs to the INK4 proteins, p19ARF is a tumor suppressor-provoking blockage of
the cellular cycle like p53. ARF directly binds to p53 and/or Mdm2, altering p53 level
and activity. E2F1 activates 14ARF binding also the E1A oncogene to p14ARF and
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75
p53. Several other cellular oncogenes like c-myc and ras also stabilize p53 through
the route of ARF (Fig. 2). The damage (inactivation) of AFR in tumors complicates
the link between oncoproteins (mic, ras, E2F) and the subsequent p53 activation
which allows for cellular proliferation and survival in spite of the dysregulation of
oncogenes. The mutations specifically damaging AFR are not so frequent in human carcinomas. However, the disturbances of a series of regulatory mechanisms
of ARF activity frequently become the reason for its poor expression in a series of
tumors. Typical examples are adenomas and carcinoma of the colon, where ARF is
inactivated due to disturbed methylation of its promotor [7, 32].
1.4. Consequences of p53 activation. p53 activation leads to suppression
of cellular growth. p53 realizes these effects through individual or combined effects
on the cellular cycle (blockage of the cycle) or apoptosis. p53 also takes part in cellular differentiation, in the inhibition of angiogenesis and in the process of ageing of
tumorous cells [27, 29]. Though it is not quite clear how p53 exerts its effects on the
cells, now it is confirmed that p53 transcriptional activation is a major component of
its biological effects [7].
1.4.1. Regulation of the cellular cycle. p53 activation provokes cellular
blockage in G1 and G2/M which are the check-points of the cellular cycle (Fig. 3).
р53
р21
wafl/cyp1
Transcriptional
activation
Gadd45
Cyclin/CDK
Rb: E2F
14-3-3σ
Rbp + E2F
PCNA
Cdc2 Cdc25
Cdc2
Transcriptional
suppression of
Cyslin R1
G1 →S
G1 cellular blockage
DNA correction
G2 cellular blockage
Fig. 3. Cellular regulation through p53
76
p53 – "The guardian of genome"
A crucial role for the induction of the G1 cellular blockage plays p21 transcriptional activation. p21 binds and inhibits the activity of the cyclin-CDK complexes
that leads to increased expression of the phosphorylated (active) form of pRb and
of a subsequent G1 cellular blockage. p53 takes part in the induction of cellular
blockage during the G2 phase. p53 causes decreased transcriptional regulation
of cyclin B as well as cellular blockage at the margin of the G2/M phase. Cyclin B
is a major regulator required during the mitosis. GADD-45 and 14-3-3σ are other
transcriptionally activated proteins taking part in the induction of cellular blockage
in the G2 point of the cellular cycle. p21 and GADD-45 affect the activity of PCNA
that takes part in the process of DNA repair [7] (Fig. 2).
1.4.2. Induction of apoptosis by p53. p53 plays a major role in the regulation of apoptosis under physiological conditions. As in cellular arrest, here it is
also not quite clear how activated p53 affects apoptosis. It is admitted that p53
induces apoptosis in a transcriptionally dependent and independent manner [4].
The apoptosis is activated most frequently after DNA damage or tumor mutations.
The phosphorylation and activation of p53 are realized by DNA protein kinases
(DNA-PK) or ATM (ataxia teleangiectasia gene). Other activators of p53 are various antitumor drugs, gamma and ultraviolet radiation, and a series of oncogenes
(Fig. 3) [18]. Activated p53 relays the apoptotic signal to Bax that is translocated
from the cytoplasm to the mitochondria [25]. Bax is a major representative of the
Bcl-2 protein family and possesses proapoptotic activity. It is due to the opening of
a polyprotein transmission pore (PTP) in the mitochondrial wall caused by Bax activity. This is related to cytochrome C departure from the mitochondria. Cytochrome
C binds to Apaf 1 and procaspase 9. The subsequent activation of caspase 3 leads
to induction of the apoptosis. Bcl-2 inhibits apoptosis by blocking Bax function.
Thus described signal path is the target of many oncogenic mutations in malignant
melanoma [25].
Apoptosis might be activated by the oncogenes independently of p53. One
of these routes is NFKkB (the nuclear factor – kB). The myc oncogene possesses
a potent effect on the mitochondria. Myc strongly increases the sensitivity for signals of the Fas/FasL, CD95, TNF and TRAIL death receptors. E2F directly affects
apoptotic signals from death receptors [13]. Another common route through which
many of the various signals for proliferation affect the apoptosis programme is the
induction of ARF the function of which is to activate p53 using its inhibitory effect on
Mdm2 [28]. Oncogenes also lead to apoptosis by activation of Rb tumor suppressor
gene. The potential of Rb to take active part in the processes of proliferation and
apoptosis depends on its linkage to E2F (Fig. 3) [7].
1.4.3. Apoptosis in tumor cells. In tumor cells, apoptosis is suppressed below the critical threshold which gives these cells the chance to survive. In the majority of carcinomas, suppression of apoptosis and the chance of cellular survival
is due to inhibition of the paths through which p53 affects apoptosis [23, 35]. The
restoration of p53 function is enough to induce apoptosis in many tumor cells. SevActa Medica Bulgarica, Vol. XXXVIII, 2011, № 2
77
eral mechanisms exist for p53 reactivation in the treatment of tumors. In principle,
these mechanisms include the introduction of the wild type of p53 into the tumors
expressing a mutant protein or inhibiting the negative regulators of p53 (like Mdm2)
in the tumors including the wild type of p53 [7, 28].
Fig. 4. Immunohistochemical determination of p53 in melanoma cells of initial-stage melanoma (Antihuman p53 monoclonal antibody, own studies). Nuclear accumulation of strong intensity of staining.
80% of tumor cells show staining for the p53 marker
2. Significance of the immunohistochemical determination of p53 in malignant melanoma. The immunohistochemical determination of the markers characterizing the processes of proliferation and apoptosis, especially of p53, in the
case of various carcinomas is performed to allow subsequent pathogenetic conclusions, to predict the prognosis of the disease, and to determine the susceptibility of
the tumor to chemo- and/or radiotherapy. Immunohistochemically, p53 expression
is confirmed in the cell nucleus [34].
The evidence for increased expression of p53 in melanoma probably shows
better prognosis and therapeutic response. The data on the changes in the expression of p53 in malignant melanoma are controversial and should be subjected to critical consideration [9, 11, 12, 23, 37, 39]. The decreased expression
of p53 in melanoma cells is indicative of the impossibility for direct induction of
apoptosis through the route of Bax as well as probably decreased induction of
cellular blockage indirectly through the route of p21 (Fig. 5). Melanomas are often
positive when stained for mutant p53. As a result of these processes, tumor cells
proliferate uncontrollably and cannot be neutralized by the mechanisms of apoptosis. In these cases, melanoma advances very fast. Our recent studies show an
increase in the number of cases with loss of expression as well as a decrease
in the number of cases with strong p53 expression when melanoma progresses.
This is in support of the thesis that tumor progression is accompanied by decreased control of the cellular cycle and impossibility of inducing apoptosis [23,
30, 31, 32, 33, 38, 41].
78
p53 – "The guardian of genome"
Bax
p53
Release of cyt C
Apaf-1
p21
Cyclin/CDK complexes
G1 cellular blockage
Caspases
Apoptosis
Fig. 5. Major p53 functions in the induction of apoptosis and cellular blockage
It is important to stress that various factors playing a role in the occurrence
of a tumor are involved at various time points of its evolution, i.e. in the stage of
carcinogenesis. This means that they activate p53 at various time points of the progression of carcinogenesis. For example, the disturbed regulation of tumor markers
by oncogenes appears in the early stage of carcinogenesis while hypoxia plays a
role only after the tumor has become macroscopically apparent. Respectively, p53
exerts its tumor-suppressor role in various stages of the progression of carcinogenesis. This explains why p53 loss has such a profound effect on the development of
melanoma and of tumors in general [7, 21, 36].
3. The immunohistochemical determination of p53 in gastric cancer
p53 has been the subject of interests and research activity over the past 20
years because of its critical role in guarding against cancer development. The biological role and clinical importance of p53 alterations in gastric cancer are still unclear. This may be related to the very complicated and extensive p53 network and
to technical problems associated with surrogate markers to identify TP53 gene
defects, as most detection tests lack sensitivity and specificity.
Although mutations have been extensively discussed as a means of wild-type
p53 inactivation, there are also mutation-independent mechanisms to inactivate
p53 function. p53 lies within a pathway and perturbations occurring either upstream
or downstream negatively impact p53-dependent tumor suppression. Allelic loss
occurs in more than 60% of cases and mutations are identified in approximately
30%-50% of cases depending on the mutational screening method used and variable sample sizes [14]. Some mutations of p53 have even been identified in early
dysplastic and apparent intestinal metaplasia gastric lesions; however, most alteraActa Medica Bulgarica, Vol. XXXVIII, 2011, № 2
79
tions occurred in the advanced stages of neoplasia. The spectrum of mutations in
this gene within gastric tumors is not unusual with a predominance of base transitions. Many studies have used immunohistochemical analysis of tumors in an effort
to detect excessive expression of p53 as an indirect means to identify mutations
of this gene, but this assay does not seem to have consistent prognostic value in
patients with gastric cancers [10, 15].
Overexpression of p53 protein as the result of an altered gene has been demonstrated in 40–60% of primary gastric carcinomas [17, 19]. Also, few reports have
evaluated the relationship between p53 expression and progression in primary
gastric carcinoma [19]. For example, in a study from 2011, Ye et al. showed that the
prognosis of familial gastric cancer is closely related with p53 expression. A total of
162 patients had worse 5-year survival rates when they showed positive expression for p53 [42]. Many authors in early studies show the importance of this marker
in gastric carcinoma and describe it as a negative predictor in terms of survival and
prognosis [3, 20, 22].
Our observations also show that expression of this protein is associated with
expression of other protein molecules involved in the development of gastric cancer (such as: TGF-beta-1, Ki-67, HER2/neu), as well as survival and prognosis.
In our study from 2010, we examined immunohistochemically expression of p53
and found expression in all of gastric cancer specimens (Fig. 6a, b). The patients
with p53 expression had worse prognosis after surgical therapy compared to those
without, but 88.9% from patients with p53-positive status were in T1-T2 stage vs.
37.8% in T3-T4.
A
B
Fig. 6a, b. Positive immunohistochemical staining in gastric cancer specimens with monoclonal
antibody p53 (Magnification: a x 200; b x 800)
Despite own observations, there are many conflicting results related to the
importance of this protein, but the expansion of the groups, and targeted search for
meaning and mutations in various tumor types, is likely to lead to a clear conception
of the importance of p53.
80
p53 – "The guardian of genome"
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33. V o g t , T. et al. p53- protein and Ki- 67- antigen expression are both reliable biomarkers of prognosis in thick stage I nodular melanomas of the skin. – Histopathology, 30, 1997, 57-63.
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p53. – J. Immunol. Methods, 151, 1992, 237-244.
35. W a n g , X. Role of p53 and apoptosis in carcinogenesis. – Anticancer Res., 19, 1999, 4759-4771.
36. W e i n s t e i n , B. Disorders in cell circuity during multistage carcinogenesis: the role of homeostasis. – Carcinogenesis, 21, 2000, 857-864.
37. W e i s s , J. et al. Mutation and expression of TP53 in malignant melanomas. – Rec. Results Cancer Res., 139, 1995, 137-154.
38. W e i s s , J. et al. Expression of p53 protein in malignant melanoma: clinicopathological and prognostic implications. – Br. J. Dermatol., 113, 1995, 23-31.
39. W h i t e m a n , D., P. Parsons et A. Green. p53 expression and risk factors for cutaneous melanoma; a case-control study. – Int. J. Cancer, 77, 1998, 843-848.
40. W u , L. et A. Levine. Differential regulation of the p21/WAF- 1 and mdm2 genes after high dose
UV irradiation: p53-dependent and p53-independent regulation of the mdm2 gene. – Mol. Med.,
3, 1997, 441-451.
41. Y a m a m o t o , M. et al. Expression of the p53 protein in malignant melanomas as a prognostic
indicator. – Arch. Dermatol. Res., 287, 1995, 146-151.
42. Y e , Y. W. et al. Prognostic analysis of familial gastric cancer in Chinese population. – J. Surg.
Oncol., 104, 2011, № 1, 76-82.
43. Z i e g l e r , A. et al. Sunburn and p53 in the onset of skin cancer. – Nature, 372, 1994, 773-776.
Address for corespondence:
J. Ananiev
University "S. Kliment Ohridski"
Polyclinic for Dermatology and Venerelogy,
Universyti Hospital Lozenetz
Medical Faculty
1 Koziak str.
1407 Sofia, Bulgaria
82
p53 – "The guardian of genome"
AN IN VITRO STUDY ON THE SIZES OF PULP CHAMBERS
AND CLINICAL CROWNS OF MOLARS
E. Boteva
Department of Conservative Dentistry,
Faculty of Dental Medicine – Sofia, Bulgaria
Summary. The macromorphology of pulp chambers has been carefully studied in the last few decades. But there is a serious lack of knowledge on the sizes
in different dimensions of the pulp chambers of molars. Aims of the present study
are jo measure the range and mean dimensions of the pulp chambers of upper
and lower molars; to detect the relation between the sizes of clinical crowns and
pulp chambers for better endocavity preparation. 286 upper and lower molars, 161
first and second upper molars, 125 first and second lower molars – matured, fully
mineralized and sound are used for the study. Measurements of the clinical crownthree dimensions for each tooth in mm: mesio-distal, from the approximal marginal
ridge, bucco-lingual, from the top of buccal cusp to the top of the mesio-lingual
(palatal) cusp. The height of the crown is measured from the buccal side. Measurements of the pulp chambers: 40 randomly selected upper and lower molars, 20 in
each group without severe root canal curvatures are measured under the following
technology: the pulp chambers are opened with horizontal cuts, 1 mm apically from
the equator with diamond blend. After polishing the ridges the final size of the endocavity is 2 mm bellow the equator. Both buccolingual dimensions are measured
as L1 and L2, and the mean as L with endodontic file and endoblock in mm. It can
be useful for the lecture courses BL sizes less than ½ of the cusp distance to be
avoided for endocavity preparation. A careful approach to this sizes of crowns and
chambers can lead to safe hard dental tissues during endocavity preparation and
especially careful approach to the approximal walls of the teeth. This findings are
important for the prevention of crown fractures, of posts and pins and of crowns and
bridges in young age groups.
Key words: macromorphology, pulp chambers, sizes, crowus, molars
Acta Medica Bulgarica, Vol. XXXVIII, 2011, № 2
83
T
he macromorphology of pulp chambers has been carefully studied in
the last few decades. But there is a serious lack of knowledge on the
sizes in different dimensions of the pulp chambers of molars. This is an
important matter for the proper sizes of endodontic cavities [1, 2, 3, 4, 5, 6, 7, 9].
All parameters of the cavities are usually defined as distance between cusps or
mm from the buccal and lingual walls or from respective walls. Very little information other and completely out of date are some sizes of the crowns of molars in
one manual of Restorative Dentistry from 1976 [8] and in the translated booklet of
Wetzel (1947) [18]. This is the only available literature for our students at the moment. It has been established in the dental literature the relation between the size
and shape of the crown and the size and shape of the pulp chamber in youth age
and the age changes related to the reduction of the pulp chamber parameters [1].
Unfortunately, this fact is not always considered during endodontic cavity preparations, which often leads to iatrogenic errors. The literature review on the sizes of
clinical crowns in the last 40 years shows a significant reduction of the mesio-distal
and bucco-lingual dimension of the molars. An important matter is the preparation
of the pulp chamber on teeth with massive enamel and dentine loses, especially
complicated when the opposite or symmetrical teeth are destroyed too. From the
literature review and the on line cross cheque of the last 20 years only 8 articles can
be related to the macromorphology of the molars. Five of these studies are in vitro
with large variation in the number of cases from 5 up to 700 root canals. Only in two
of these studies, the sizes of the pulp chambers are measured. Only in one study,
differences are observed between “young” and “old” teeth (Bjorndal).
AIM
The aim of the present study is:
1. To measure the range and mean dimensions of the pulp chambers of upper and lower molars.
2. To detect the relation between the sizes of clinical crowns and pulp chambers for better endocavity preparation.
MATERIALS AND METHODS
Teeth: 286 upper and lower molars, 161 first and second upper molars, 125
first and second lower molars. No differences are made for left or right teeth.
All teeth are matured, fully mineralized and sound, used for the practical preclinical exam of endodontics.
Groups: two – upper and lower teeth.
Measurements of the clinical crown: three dimensions are measured for
each tooth in mm: mesiodistal, from the approximal marginal ridge, bucco-lingual,
from the top of buccal cusp to the top of the mesiolingual ( palatal) cusp. The height
84
An in vitro study on the sizes of...
of the crown is also measured from the buccal side (h) from the enamel border to
the middle part of the line between the cusps.
Measurements of the pulp chambers: 40 randomly selected upper and
lower molars, 20 in each group without severe root canal curvatures are measured
under the following technology:
1. The pulp chambers are opened with horizontal cuts, 1 mm apically from the
equator with diamond blend.
2. After polishing the ridges the final size of the endocavity is 2 mm bellow
the equator.
3. Both buccolingual dimensions are measured as L1 and L2, and the mean as
L and the mesiodistal sizes are measured in the widest part of the pulp chamber.
4. This measurements are performed with endodontic file and endoblock in mm.
Exclusion criteria: non vital teeth, massive tooth loses, teeth with root caries,
incisors and premolars, and not matured teeth.
RESULTS
The mean values of the sizes of the clinical crowns of all molars are closed
to the plastic Frasaco teeth for students preclinical teaching course in both dimensions MD and BL. The highest difference is in the height of the crown (h), where the
differences in the upper teeth are 1,6 mm and even higher in the lower teeth – 2,8
mm. For getting used to the future endodontic cavity preparation course, this fact in
the cariology course can be quite dangerous.
The mean values from these measurements are shown in table 1, 2 and 3:
Table1. Sizes of the clinical crowns of molars
Type of tooth
Upper
Teeth n = 161
Lower
teeth n = 125
Dimension
ВL
МD
H
ВL
МD
H
Mean
6,5
8,1
4,5
5,3
9,9
5,2
Range mm
5,8-7,8
7,9-9,4
4,0-6,2
4,0-8,0
8,0-13,0
3,5-7,0
Таble 2. Sizes of the pulp chambers of molars
Type of tooth
Upper teeth n = 20
Lower teeth n = 20
Dimension
Mean
Range mm
ВL 1
5,3
5,0-5,6
ВL 2
4,5
4,1-4,9
МD
2,9
2,6-3,2
ВL 1
4,6
4,4-4,8
ВL 2
3,9
3,6-4,2
МD
3,4
3,3-3,5
Acta Medica Bulgarica, Vol. XXXVIII, 2011, № 2
85
Table 3 shows that MD sizes of the crowns are twice bigger than the MD sizes
of the pulp chambers in both upper and lower molars. The BL sizes of the crowns
in upper and lower molars are with very little differences from the pulp chamber
sizes – 1,2 mm and 0,7 mm.
Таble 3. A comparison of both measurements
Type of the tooth
Upper teeth
Lower teeth
Dimension
ВL
МD
ВL
МD
Mean in mm
Crowns
6,5 mm
8,1 mm
5,3 mm
9,9 mm
Chambers
5,3 mm
4,5 mm
4,6 mm
3,9 mm
Fig. 1 and 2. Pulp chambers of lower and upper molars, measured in the study
DISCUSSION
The findings in this study are very different from the ones published by Ruskov
et al. (1976) [8], measured by Boianov. According to their criteria, MD of upper molars
are 9.3-10.1 mm, ВL 11.1-11.4 mm, аnd of lower MD 9.7-11.1, ВL 8.9-10.3 mm.
The comparison with the data from the literature shows that the differences
are up to twice, which is the case with the BL size of lower molars in Wenzel [18]. In
this book, for the upper molars the measurements are MD 9.8 mm, BL – 11.5 mm
and for the lower MD 10.7-11.5 mm and BL 9.8-10.4 mm.
The differences with table 1 in BL dimension are up to 4.5-6.5 mm.
Any data on the size of the pulp chambers of molars in the literature is non
existing. This is an explanation why after endodontic treatment the most common
mistakes are three:
1. Remaining pulp tissue in retentive lodges in the pulp chamber – sources of
infection and periapical lesions, which are 19.8% of all endodontic retreatments in
the Faculty of Dental Medicine in Sofia, shown in our previous study.
86
An in vitro study on the sizes of...
2. Not accurate exposure of pulp chambers, failures in working length estimation and bad preparation of root canals.
3. Overpreparation of cavity walls and crown fractures, mostly from overpreparation of medial and distal walls followed by use of posts and pins in 18.2% of
all endodontic treatments in the Faculty of Dental Medicine in Sofia, shown in our
previous study.
A simple algebra exercise shows that nearly in 50% of all endodontic treatments there are failures especially when we consider that with age all pulp chambers lower their sizes and orifices migrate up on cavity walls [12, 14, 16].
CONCLUSIONS:
1. It is essential to be included again in the lecture courses that endodontic
cavities cannot have BL sizes less than ½ of the cusp distance and that they need
at least parallel walls.
2. There is a need of knowledge not only on the pulp anatomy but on pulp
chamber sizes and crown sizes of the teeth.
3. A careful approach to these sizes can lead to safe hard dental tissues during endocavity preparation and especially careful approach to the approximal walls
of the teeth.
4. These findings are important for the prevention of crown fractures, the use
of posts and pins and the use of crowns and bridges in young age groups.
REFERENCES:
1. Б о т у ш а н о в , П. Кариесология и оперативно зъболечение, Пловдив, Автоспектър, 2000.
2. Б о т у ш а н о в , П. Ендодонтия – теория и практика, Пловдив, Автоспектър, 1998, 401-418.
3. Д а ч е в , Б. и кол. Ръководство за практически упражнения по пропедевтика на
терапевтичната стоматология. С., Мед. и физк., 1990, 47-48.
4. Е н д о д о н т и я 2002. Под ред. на Б. Инджов, С., Шаров, 2002.
5. И н д ж о в , Б. Основи на кавитетната препарация. С., Инджидент, 2006, 109-113.
6. Й о р д а н о в , Б. и И. Йончева. Ръководство за практически упражнения по пропедевтика на
протетичната стоматология. С., Мед. и физк., 2000.
7. М а с л и н к о в , Д. Ръководство за практически упражнения по пропедевтика на детската
стоматология. С., Мед. и физк., 1989.
8. Р у с к о в , Р., Ч. Ликов и Е. Евтимов. Ръководство за практически упражнения по пропедевтика
на ортопедичната стоматология. С., Мед. и физк., 1976, 6-26.
9. С т р а н с к и , Д. Детска стоматология. С., Мед. и физк., 1959, 85.
10. E l e f t h e r i a d i s , G. I. et T. P. Lambrianidis. Technical quality of root canal treatment and detection of iatrogenic errors in an undergraduate dental clinic. – Int. Endod. J., 38, 2005, 725-734.
11. G l i c k m a n , G. et al. The crisis in endodontic education: current perspectives and strategies for
change. – JOE, 31, 2005, № 4, 225-261.
12. H a y e s , S. J. et al. An audit of root canal treatment performed by undergraduate students. – Int.
Endod. J., 34, 2001, 501-505.
Acta Medica Bulgarica, Vol. XXXVIII, 2011, № 2
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13. M o n c a d a , G. et al. Increasing the longevity of restorations by minimal intervention: A two year
clinical trial. – Oper. Den., 33, 2008, № 3, 258-264.
14. Q u a l t r o u g h , A. J., J. M. Whitworth et P. M. Dummer. Preclinical endodontology: an international comparison. – Int. Endod. J., 32, 1999, 406-414.
15. S a n t o s , G. O. et al. Influence of C factor and light curring mode on gap formation in resin composite restorations. – Oper. Den., 34, 2009, № 5, 544-550.
16. S o n n t a g , D. et al. Pre-clinical endodontics: a survey amongst German dental schools. – Int.
Endod. J., 41, 2008, 863-868.
17. Ts i t r o u , E. et al. Fracture strength of minimally preparated resin bonded cerec inlays. – Oper.
Den., 34-5, 2009, 537-543.
18. W e t z e l , G. Анатомия хистология и ембриология на зъбите. Зъби и съзъбие. С., Алма
Матер, 1947, 13-51.
19. B j o r n d a l , L. et al. External and internal macromorphology in 3D- reconstructed maxillary molars using computerized X-ray microtomography. – Int. Endod. Y., 32, 1999, 3-9.
20. G u e l z o w , A. et al. Comparative study of six rotary NiTi systems and hand instrumentation for
root canal preparation. – Int. Endod. J., 10, 2005, № 38, 743-752.
21. H u l s m a n , M. et F. Stryga. Comparison of root canal preparation using different automated devices and hand instrumentation. – JOE, 19, 1993, № 3, 141-145.
22. I o d w a y , B. et M. Hulsmann. A comparative study of root canal preparation with NiTi – TEE and
K3 rotary NiTi instruments. – Int. Endod. Y., 39, 2006, № 1, 71-80.
23. S c h a f e r , E. et al. Roentgenographic investigation of frequency and degree of canal curvatures
in human permanent teeth. – JOE, 3, 2002, 211-216.
24. S c h a f e r , E. et R. Schlingemann R. Efficiency of rotary NiTi K3 instruments compared with stainless steel hand K- flexofile. Part 2. – Int. Endod. J., 36, 2003, № 3, 208-217.
25. Ta n , B. T. et H. H. Messer. The effect of instrument type and preflaring an apical file size determination. – Int. Endod. J., 35, 2002, № 9, 752-758.
26. W i l c o x , L. R. et M. L. Swift. Endodontic retreatment in small and large curved canals. – JOE,
17, 1991, № 7, 313-315.
88
Address for correspondence:
Assoc. Prof. E. Boteva
Faculty of Dentisty
Medical University – Sofia
3 Sv. G. Sofiiski
1431 Sofia
e-mail: [email protected]
An in vitro study on the sizes of...
ECONOMIC ANALYSIS OF THE COST SAVINGS AFTER
IMPLEMENTATION OF ANTIBIOTIC POLICY IN HOSPITAL
M. Manova1, A. Savova1, A. Stoimenova1, B. Angelovska2 and G. Petrova1
2
1
Department of Social Pharmacy, Faculty of Pharmacy, Medical University,
University in Shtip, Faculty of Medical Sciences, Department of Pharmacy, Shtip, FYROM
Summary. The antibiotic policy is an essential part of the hospital pharmacy
activities. It leads to decrease in the microbial resistance, optimisation of the medicine utilisation, and cost savings. The goal of the study is to analyse the results
of implementation of antibiotic policy on the medicine utilization and related cost
savings in a city hospital in Sofia. The study is a comparative retrospective and prospective analysis of the antibiotic prescribing and cost of therapy, before and after
the introduction of the antibiotic policy. There were gathered and systematized 2582
prescriptions for hospitalized patients in 3 hospital departments (surgical, urology,
and internal wards). For the evaluation of the most often prescribed combination,
the sample was made for 30 patients from each unit. The selection of patients was
made at random for the most common diagnoses in the studied wards. The antibiotic utilization was also measured in monetary terms and cost savings were calculated for the period after the antibiotic policy introduction. The comparison of the
cost of antibiotic therapy in urology, surgical and internal wards for 2004 and 2005,
after the introduction of AB policy showed that in all three studied wards significant
savings were realized. Reduction of costs in 2005 compared to 2004 was in total 26
832 BGN (28.48%). Total expenditures on medicines in the studied hospital were
447 810 BGN for 2004 and 396 659 BGN for 2005. Savings of 51 151 BGN were
realized, which is a significant value and a significant proportion of the total costs
(28.48%). Analysis of the results of the implementation of antibiotic drug policy confirmed that the optimization of drug administration leads to positive economic results. It also confirms the usefulness of measurement of utilization and cost saving,
especially in hospital wards with high cost services. The antibiotic policy should be
an obligatory part of drug policy in the hospitals with scarce resources. Measuring
the impact of antibiotic policy implementation is a useful tool for optimization of the
general hospital medicines policy and realization of cost savings. It gives possibility
Acta Medica Bulgarica, Vol. XXXVIII, 2011, № 2
89
for practical solutions on medicines utilization optimization. Introduction of antibiotic
policy in hospitals leads to the reduction of their usage, saves money and improves
therapeutic options.
Key words: antibiotic, DDD, cost reduction, hospital
I
IINTRODUCTION
n an era of growing concern about bacterial resistance and hospital costs,
limiting the use of broad spectrum antibiotic usage is important [16]. The current worldwide pandemic of antibiotic resistance shows no signs of abating.
It is clear that it is driven mainly by heavy and often inappropriate antibiotic use [5].
Lots of studies showed that multidisciplinary consultative approach reduced antibiotic
costs, contributed to infection control, and improved the quality of antibiotic prescription and led to costs savings [1, 4, 6, 10, 11, 14, 15, 17]. Antibiotic stewardship programs may take the form of management teams comprising infectious disease physicians and pharmacists [10, 12, 15]. A positive overall attitude to hospital antibiotic
policies is expressed by pharmacists and physicians in various studies [1].
The antibiotic (AB) policy is an essential part of the hospital pharmacist’s activities. It leads to decrease in the microbial resistance, optimisation of the medicines utilisation, and cost savings.
The goal of the study is to analyse the results of implementation of antibiotic
policy on the medicine utilization and related cost savings in a city hospital in Sofia.
MATERIALS AND METHODS
The study is a comparative retrospective and prospective analysis of the antibiotic prescribing and cost of therapy, before and after the introduction of the antibiotic policy in 3 hospital wards – internal, surgical and urology.
There were gathered and systematized 2582 prescriptions for hospitalized
patients and evaluated the share of the prescribed AB within every department. For
the evaluation of the most often prescribed combinations, the sample was made for
30 patients from each unit. The selection of patients was made at random for the
most common diagnoses in the studied wards.
The antibiotic utilization was also measured in monetary terms and cost savings were calculated for the period after the antibiotic policy introduction.
RESULTS AND DISCUSSION
Principles of antibiotic policy
The antibiotic policy comprised of three main principles:
90
Economic analysis of the cost...
1. Separation of antibiotic in three groups – for general usage, for limited usage, and with special permission;
2. Requirement of antibiogram for every permission;
3. Written rules for permission.
The antibiotic were separated in the three mentioned groups as presented on
Table 2.
Table 1. Separation of antibiotic according to groups of usage
Group of antibiotic
For general use
AB pharmacology group
penicillin’s
cephalosporins
cephalosporins 2nd
generation
With limited use
On approval regime
INN
penicillin, oxacillin, amoxicillin,
amoxacilling and clavulanic acid,
ampicillin, sulbactam
cefazolin, cefalotin,
cefamandol, cefaclor
aminoglycosides
macrolides
tetracyclines
sulfonamides
imidazoles
antimycotics
penicillin’s
cephalosporins 2nd
generation
gentamycin
azithromycin, claritromycin
doxacyclin
trimetoprim + sulfamethoxazol
metronizadol
nystatin
azlocillin, piperacillin
cefuroxim, cefoxitin
cephalosporins 3rd
generation
cefoperazon, cefotaxim, ceftriaxon,
cefixim
aminoglycosides
lincosamides
quinolones
antimycotics
penicillins and
carbapenemes
amicacin, tobramycin
clindamycin
ciprofloxacin per os
ketoconazol
pirepacillin + tazobactam, imipenem
cephalosporins 4rt
generation
ceftazidim, cefepim
glycopeptides
fluoriquinolones
antimycotics
vancomycin, teycoplanin
ciprofloxacin amp, fl
fluconasol inf., amphotericin B
The policy is oriented to regulation of the prescribing of second, third and
fourth generation antibiotic, as well as to the introduction of the written rules for
their usage and thus makes AB traceable and under control.
Changes in antibiotic’ prescribing and cost of therapy
Tables 1 and 2 present the most commonly prescribed antibiotic and combinations categorized according to the groups, spectrum, disease and the cost of
therapy, respectively for 2004 and 2005 in the urology ward.
Acta Medica Bulgarica, Vol. XXXVIII, 2011, № 2
91
A comparison of both tables revealed that before the introduction of the antibiotic policy rules in urology ward, the use of third-generation cephalosporins prevailed (Table 1), while after the introduction of the policy, first-generation cephalosporins became preferred and the shares of antibiotic combinations, and second
generation quinolones increased. In addition, transition from intravenous to oral
administration was recorded, which led to reduction in the cost of administration
of the antibiotic per patient. The change of antibiotic prescribing in urology department led to almost double decrease in the cost of therapeutic courses per patient
for the most commonly prescribed regimens.
Table 1. Prescribed AB and their cost in 2004 (urology ward)
Antibiotic
group
Ceftriaxone
Therapeutic group
Spectrum
Cephalosporin
3rd generation
Diagnosis
Metronidazole
Antiprotozoal
Bactericidal
Acute urethritis
Gram (-) aerobic
bacteria
Antiprotozoal
Ceftriaxone
Cephalosporin
3rd generation
Cephalosporin
3rd generation
Aminoglucosides
Gram (-) aerobic Prostatitis
bacteria
Gram (-) aerobic Orchiepididymitis
bacteria
Gram (-) aerobic
Cephalosporin
3rd generation
Gram (-) aerobic Acute
bacteria
Pyelonephritis
Fluoroquinolones
2nd generation
Bactericidal
Beta-lactamase
producing
organisms
Ceftriaxone
Gentamicin
Ceftriaxone
Ciproloxacin
Daily course cost
per patient (BGN)
19.20
Full course cost
per patient (BGN)
228
3.60
19.20
269
19.20
0.80
236
19.20
269
10.60
127
Acute cystitis
Table 2. Prescribed AB and their cost in 2005 (urology ward)
Antibiotic
group
Ciprofloxacin
Metronidazole
Ciprofloxacin
iv and реr os
Ciprofloxacin
Amoxicillin plus
clavulanic acid
4 days iv
6 days per os
Cephazoline
92
Therapeutic
group
Quinolones
Antiprotozoal
Quinolones
2nd generation
Fluoroquinolones
Spectrum
Diagnosis
Bactericidal
Antiprotozoal
Bactericidal
Acute urethritis
Gram (-)
aerobic
Semi-synthetic
Bactericidal
beta-lactamase
Gram (+),
resistant penicillins Gram (-)
and Betalactamase
producing
organisms
Cephalosporin
Bactericidal
1st generation
Gram (-)
Prostatitis
Orchiepididymitis
Acute pyelonephritis
Daily course cost
per patient (BGN)
10.40
3.60
10.40
0.80
10.40
Full course cost
per patient (BGN)
17.50
101
140
107
125
2.88
Acute cystitis
6.90
83
Economic analysis of the cost...
Table 3 and 4 show the prescribed antibiotic and their cost in the surgical ward for 2004 and 2005. In this ward, a reduction of costs for the two most
prescribed regimens was observed, for one regimen the costs remained almost
unchanged and the costs for the fourth regime remained the same. Compulsory
prophylactic dose and postoperative prophylaxis were introduced in 2005, a fact
that could be interpreted as real cost savings to therapy, because after the introduction of the antibiotic policy a twice daily double application of antibiotic at
lower costs was observed.
Table 5 and 6 present the most commonly prescribed combinations and their
cost in the internal ward before and after the introduction of AB policy. In internal
ward, the changes were most significant, both in terms of choice of antibiotic and
cost of therapy. Transition from 2nd to 3rd generation of cephalosporins and macrolides, quinolones and penicillinase-resistant penicillins was observed. The average cost for therapeutic regimen decreased almost twice although combinations
were preferred.
Table 3. Prescribed AB and their cost in 2004 (surgical ward)
Antibiotic/
combination
Therapeutic group Spectrum
Diagnosis
Cephazoline
Cephalosporin
1st generation
Antiprotozoal
Bactericidal
Gram (-)
Antiprotozoal
Mammal carcinoma
Cephalosporin
3rd generation
Antiprotozoal
Aminoglycosides
Gram (-)
Colon cancer
Metronidazole
Ceftriaxone
Metronidazole
Gentamicin
Daily course cost
per patient (BGN)
Full course cost
per patient (BGN)
6.90
64
3.60
Antiprotozoal
Bactericidal
Gram (-)
19.20
283
3.60
0.80
Amoxicillin plus
clavulanic acid
Semi-synthetic
beta-lactamase
resistant penicillins
Bactericidal
Gram (+),
Gram (-)
and Betalactamase
producing
organisms
Chole-cystectomy
17.50
123
Cefuroxime
Cephalosporin
2nd generation
Bactericidal
Gram (+) and
Gram (-)
Duodenal ulcer
complications
11.80
95
Acta Medica Bulgarica, Vol. XXXVIII, 2011, № 2
93
Table 4. Prescribed AB and their cost in 2005 (surgical ward)
Antibiotic/
combination
Cefuroxime
(prophylaxis)
Cephazoline
(after surgery)
Therapeutic group Spectrum
Diagnosis
Cephalosporin
2nd generation
Cephalosporin
1st generation
Bactericidal
Gram (+) and
Gram (-)
Mammal
carcinoma
Cefuroxime
(prophylaxis)
Metronidazole
Gentamicin
Ceftriaxone
(after surgery)
Cephalosporin
2nd generation
Atiprotozoal
Aminoglycosides
Cephalosporin
3rd generation
Bactericidal
Colon cancer
Cefuroxime and
Metronidazole
(prophylaxis)
Ciprofloxacin
(after surgery)
Cephalosporin
2nd generation
Antiprotozoal
Bactericidal
Antiprotozoal
Daily course cost Full course cost
per patient (BGN) per patient (BGN)
5.90
47
6.90
5.90
Antiprotozoal
Bactericidal
234
3.60
0.80
19.2
Chole-cystectomy
82
5.90
1.80
Bactericidal
10.4
Fluoroquinolones
Cefuroxime and
Metronidazole
(prophylaxis)
Cephazoline
(after surgery)
Cephalosporin
2nd generation
Antiprotozoal
Cephalosporin
1st generation
Bactericidal
Antiprotozoal
Duodenal ulcer
complications
79
5.90
3.60
Bactericidal
6.90
Table 5. Prescribed AB and their cost in 2004 (internal ward)
Antibiotic/
combination
Therapeutic
group
Spectrum
Diagnosis
Cephazoline
Cephalosporin
1st generation
Bactericidal
Gram (+/-)
Pneumonia
9.90
189
Ceftriaxone
Cephalosporin
3rd generation
Bactericidal
Gram (+/-)
Pneumonia
complications
19.20
384
Ceftriaxone
(14 days)
Azithromycin
Cephalosporin
3rd generation
Macrolides
Bactericidal
COPD
19.20
203
Cefuroxime
Cephalosporin
Bactericidal
2nd generation
Gram (-), E.coli.
Aminoglycos ides enterobacteria,
staphylococcus
Amikacin
94
Bacteriostatic
Daily course cost
per patient (BGN)
Full course cost per
patient (BGN)
10.85
11,80
196
Cholecystitis
7,80
Economic analysis of the cost...
Table 6. Prescribed AB and their cost in 2005 (internal ward)
Antibiotic/
combination
Azithromycin
З days iv. and
10 days per os
Therapeutic
group
Macrolides
Azithromycin
З days iv. and
10 days per os
Macrolides
Amoxicillin +
clavulanic acid
(10 days iv)
Ciprofloxacin (4
days per os)
Semi-synthetic
beta-lactamase
resistant
penicillins
fluoroquinolons
Spectrum
Diagnosis
Bacteriostatic
Gram (+) and
Gram (-) bacteria
incl. E. coli
Pneumonia
Bacteriostatic
Gram (+/-)incl.
E. coli
Pneumonia
complications
Bactericidal
Gram (+/-),Betalactamase
producing
organisms
COPD
Amoxicillin plus Semi-synthetic Bactericidal
clavulanic acid beta-lactamase
resistant
Metronidazole penicillins
Antiprotozoal
Antiprotozoal
Daily course cost
per patient (BGN)
Full course cost per
patient (BGN)
154
29.6
6.50
29.60
226
6.51
17.50
188
0.80
Cholecystitis
11.70
153
3.60
Based on the calculated cost of therapeutic course, the cost differences per
diseases which require AB administration were determined before and after implementation of AB policy. Table 7 presents cost savings realized for the most frequent
diagnoses. Total savings of the most often prescribed combinations were 537 BGN
in urology ward.
Table 7. Comparison of costs per therapeutic course in urology ward 2004-2005
Therapeutic regimen
costs in BGN/2004
Disease
Therapeutic regimen
costs in BGN/2005
Costs savings
Acute urethritis
228
140
88
Prostatitis
269
107
162
Orchiepididymitis
236
125
111
Acute pyelonephritis
230
98
132
Acute cystitis
127
83
Total
44
537
Surgical ward also reported a total decrease in the cost of commonly prescribed AB therapies of 123 BGN (Table 8). After the introduction of AB policy in
the whole surgical ward, from the beginning of 2005 the total costs for AB therapy
decreased by 6223 BGN (Table 8). The decrease in 2005 in comparison with 2004
was 43.23%.
Acta Medica Bulgarica, Vol. XXXVIII, 2011, № 2
95
Table 8. Comparison of costs per therapeutic course in surgical ward 2004-2005
Therapeutic regimen
costs in BGN/2004
Disease
Mammal carcinoma
Therapeutic regimen
costs in BGN/2005
Cost savings
54
47
17
Colon cancer
283
234
49
Cholecystectomy
123
82
41
95
79
16
Duodenal ulcer complications
Total
123
In the internal ward, the reduction of costs of the most commonly prescribed
AB regimens was 261 BGN and the most significant reduction was observed in the
treatment of complicated pneumonia (Table 9). For the whole internal ward, however, the decrease of the cost of AB therapy in 2005 in comparison with 2004 was
the most significant one (by value) and amounted to 12 752 BGN. Share of the cost
reduction for 2005 compared to 2004 was 20%.
Table 9. Comparison of costs per therapeutic course in internal ward 2004-2005
Therapeutic regimen
costs in BGN/2004
Disease
Therapeutic regimen
costs in BGN/2005
Costs savings
Pneumonia
189
154
35
Complicated pneumonia
384
226
158
COPD
203
188
25
Cholecystitis
196
153
43
Total
261
The comparison of the cost of antibiotic therapy in urology, surgical and internal wards for 2004 and 2005, after the introduction of AB policy showed that in all
three studied wards significant savings were realized. Total reduction of costs in
2005 compared to 2004 was 26 832 BGN (28.48%) (Table 10). Total expenditures
on medicines in the studied hospital were 447 810 BGN for 2004 and 396 659 BGN
for 2005. Savings of 51 151 BGN were realized, which is a significant value and a
significant proportion of the total costs (28.48%).
Table 10. Comparison of costs and savings in the studied wards 2004/2005
Ward
Costs (2004)
Costs (2005)
Savings (BGN)
Reduction (%)
Urological
15782
7925
7857
48,78
Surgical
14395
8172
6223
43,23
Internal
64029
51277
12752
20,00
94206
67374
26832
28,48
Total
96
Economic analysis of the cost...
CONCLUSIONS
Analysis of the results of the implementation of antibiotic drug policy confirmed that the optimization of drug administration leads to positive economic results. It also confirms the usefulness of measurement of utilization and cost saving,
especially in hospital wards with high cost services [2, 8, 9, 13].
The antibiotic policy should be an obligatory part of drug policy in the hospitals
with scarce resources. Measuring the impact of antibiotic policy implementation is useful tool for optimization of the general hospital drug policy and realization of cost savings. It gives possibility for practical solutions of drug utilization optimization [7, 3, 18].
Introduction of antibiotic policy in hospitals leads to the reduction of their usage, saves money and improves therapeutic options.
REFERENCES:
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¨
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Address for correspondence:
Manoela Manova, M. Pharm., Ph.D.
Department of Social Pharmacy
Faculty of Pharmacy
Medicla University
2 Dunav str.
1000 Sofia
(+3592 923 658 9)
(+3592 987 9874)
e-mail: [email protected]
Economic analysis of the cost...
CONTENTS
B. Kutchoukov. IOL dislocation and retinal detachment – a study by ultrasound biomicroscopy ...........3
J. Ananiev, G. Tchernev, M. Penev, M. Gulubova, G. Kupcova and N. Sucha.
Uncommon clinical presentation of balanitis circumscripta plasmacellularis zoon
associated with monoclonal gammopathy of igg-type and acute exacerbation of seropositive
rheumatoid arthritis – complete remission after systemic administration of doxycycline .................8
R. Nikolova and S. Danev. Effect of mental work load on autonomic cardiovascular control...............15
N. Boyadjieva and G. Bocheva. Effect of chronic alcohol consumption on apoptosis
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A. Loukova. Factors influencing acute alcohol poisoning in adolescents in Bulgaria ...........................30
R. Nikolova and S. Danev. Dеtermination of the effect of lead-induced subclinical toxicity
on autonomic cardiovascular function ............................................................................................38
K. Koev, S. Cherninkova, Ch. Chakarova, R. Georgiev, S. Dimitrova, R. Kaneva and
S. Bhatacharya. Clinical assessment and molecular genetics of an autosomal dominant
retinitis pigmentosa in a bulgarian roma family ..............................................................................46
A.Tsonev, E. Kovachev, I. Bakardzhiev, S. Ivanov, A. Abbud and M. Veseli. Acute intermittent
porphyria during pregnancy and normal delivery: a case report ....................................................51
B. A. Lafaut, J.-J. De Laey, F. M. Meire and B. M. Kutchoukov. Late radiation retinopathy
in a patient treated for retinoblastoma............................................................................................54
J. Radenkova-Saeva. Severe baclofen self-poisoning – a case report ................................................59
R. Robeva, T. Todorov, V. Dimitrova, M. Yordanov and D. Doychinov. Mesangial lipoidosis
with cholesterol deposition – a new case with nephrotic syndrome and rapidly progressing
renal failure ....................................................................................................................................62
G. Tchernev, J. Ananiev and M. Gulubova. А case of giant squamous cell carcinoma
of the tongue – a rare finding within the framework of dermatological screening? ........................67
J. Ananiev, G. Tchernev, J. W. Patterson, M. Gulubova and G. Ganchev. р53 – “The
guardian of genome” .....................................................................................................................72
E. Boteva. An in vitro study on the sizes of pulp chambers and clinical crowns of molars ...................83
M. Manova, A. Savova, A. Stoimenova, B. Angelovska and G. Petrova. Economic analysis
of the cost savings after implementation of antibiotic policy in hospital .........................................89
ACTA MEDICA BULGARICA 2/2011
Editor in Chief: Prof. V. Mitev, MD, Ph. D., DSc
Scientific editor: Prof. W. Bossnev, MD, Ph. D., DSc
Editor of the English text: B. Stancheva, MD
Art editor and computer design: D. Alexandrova
Organizing secretary: M. Dimitrova
Publisher’s sheets: 8.3
Printer’s sheets: 6.25
Format: 70 x 100/16
Issued by the Central Medical Library
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99