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SAMPLE ABSTRACTS (from 2008)
CORESTA AP 2008 - ABSTRACT FORM
Please provide the following information.
Title:
Changes in nicotine to nornicotine conversion levels during the
production of Burley Foundation and Certified seed.
Author(s):
JACK A.M.; LI Xiaolong; FANNIN F.F.; BUSH L.P.
Principal author’s name first
Author(s) Affiliation(s):
University of Kentucky, Lexington, KY 40546, U.S.A.
Company name(s) and address(es)
Abstract Body (180-300 words):
Burley tobaccos have higher and more variable levels of nornicotine and NNN (N’-nitrosonornicotine)
than other tobacco types. Because of this, burley Foundation seed stocks are routinely rogued or
“screened” of plants which convert, or demethylate, nicotine to nornicotine. The subsequent
generation, Certified seed, is increased without screening, because screening on this scale is not
practical. However, it is known that conversion tends to increase with each cycle of unscreened seed
increase. The objective of this study was to quantify the decrease in conversion from the unscreened
Breeder seed to screened Foundation seed, and the subsequent increase from Foundation seed to
Certified seed. Three sequential seedlots (Breeder seed, Foundation seed and Certified seed) were
grown for each of three varieties (a high converter, VA509; a moderate-high converter, TN86; a
moderate converter, TN90). Individual plants were sampled accordingly to the LC protocol, and
analyzed for alkaloids. Conversion in the VA509 Breeder seed was 34.7%. It decreased to 14.3% in
the Foundation seed and increased to 25.1% in the Certified seed; these differences were significant.
In the TN86 and TN90, the trend was similar but not significant; the respective values for Breeder
seed, Foundation seed and Certified seed for TN86 were 11.2%, 6.7% and 9.1%, and for TN90 were
7.8%, 6.4% and 6.4%. It therefore appears that the lack of screening during Certified seed production
does not result in appreciable increases in conversion in most varieties. These data are consistent
with many of our other studies, where we have found that treatment differences in high converters are
generally significant, while those in more moderate converters follow the same trend but are not
significant. It should be noted that the LC method measures the maximum potential conversion, and
these conversion figures would be expected to be lower in a commercial crop.
Key Words: burley; seed; nornicotine; TSNA
CORESTA AP 2008 - ABSTRACT FORM
Please provide the following information.
Title:
Reducing nicotine conversion: stability of RNAi-mediated and
mutant down-regulation of nicotine demethylase.
Author(s): XU D.(1); SHEN Y.(1); LUSSO M.(1); BAILEY A.(2); NORMAN
Principal author’s name first D.(1); DAVIS G.(1);HART F.(1); CUI M.(1); NIELSEN M.(1)
1. U.S. Smokeless Tobacco Co., 4262 Colby Road, Winchester,
Author(s) Affiliation(s): KY 40391, USA
Company name(s) and address(es) 2. University of Kentucky, Research and Education Center, 1205
Hopkinsville Street, Princeton, KY 42445, USA
Abstract Body (180-300 words):
Nornicotine, a secondary alkaloid formed from the demethylation of nicotine in tobacco, is the
precursor of N’-nitrosonornicotine (NNN), a TSNA formed by nitrosation during tobacco curing. In
earlier studies, we reported that GMO (RNAi) and non-GMO (mutagenesis) strategies down-regulated
the nicotine demethylase (ND) gene and resulted in reduced nornicotine formation. Here, we report
on the stability of this down-regulated gene in advanced generation and male-sterile lines.
Experimental lines were grown in field trials at multiple locations for two years. Included in the tests
were four generations of RNAi transgenic lines and the male sterile counterparts of the R2 generation.
The lines were generated in both burley tobacco (TN90) and dark tobacco (NL Madole) backgrounds.
Also tested were four generations of mutant lines carrying a ND nonsense mutation in TN 90 and
mutant breeding lines that were back-crossed four times in two dark tobacco backgrounds, NL
Madole and KY171. Conversion levels (%nornicotine/(% nicotine + % nornicotine)) in the transgenic
lines were as low as 0.1% under field conditions and were stable throughout the four generations.
Mean nornicotine levels in the RNAi lines were reduced significantly when compared to the LC
control. No plants in the transgenic or mutant lines reverted to high nicotine conversion while some
plants in the control LC lines demonstrated this reversion. Molecular characterization of the
transgenic lines showed that RNAi down-regulation reduced the expression of the target ND gene as
well as its related P450 genes. Other, more recent data will also be discussed.
Key Words: nornicotine; TSNA; GMO
CORESTA SSPT 2008 - ABSTRACT FORM
Please provide the following information.
Title:
Market survey of the mutagenicity, cytotoxicity and genotoxicity
of smokeless tobacco products sold in Canada.
Author(s):
RICKERT W.S.; TRIVEDI A.H.; MOMIN R.A.; WAGSTAFF W.
Principal author’s name first
Author(s) Affiliation(s): Labstat International ULC, 262 Manitou Drive, Kitchener, ON,
Company name(s) and address(es) Canada N2C 1L3
Abstract Body (180-300 words):
This investigation was undertaken to characterize the range of toxicological responses for the majority
of smokeless tobacco (ST) products sold in Canada. A total of 31 ST products were extracted with
DMSO (1:9, w/v), in triplicate. These were tested for mutagenicity using the Ames assay using
Salmonella typhimurium strains TA98, TA100, TA102, TA1535 & TA1537 with S9 activation. In
addition, a subset of 12 DMSO extracts was tested for cytotoxicity (neutral red uptake (NRU) assay)
and genotoxicity (in vitro micronucleus (MN) assay). A subset of 4 ST products was also extracted
with (i) dichloromethane (DCM) and (ii) artificial saliva (AS). With one exception, all of the 31 DMSO
ST extracts were very similar in their mutagenic activity. The greatest response was observed from
strains TA100 and TA102 with slight TA98 activity. The maximum activity (TA100) was 30
revertants/mg extracted ST, which is about 3% of that observed for extracts of ‘tar’ prepared from
KR2R4F and greater than that observed from DCM and AS extracts. Cytotoxicity (IC50) of the 12
DMSO extracts ranged from 380 to 8850 µg ST/mL media in contrast to 54 µg/mL observed for
KR2R4F ‘tar’ extracts. When grouped by product type, the order of cytotoxicity was
Gutka>Snuff>Pouch>Plug>Long Cut. For most brands, DCM and AS extractants exhibited little or no
cytotoxic activity. There was some evidence for a difference in genotoxicity among the 12 ST
products; the genotoxicity of DMSO extracts of Gutka and Snuff (combined) being lower than the
combined result for Plug, Pouch and Long Cut (averages 0.282 vs. 0.587 % MN/mg ST/mL media).
However the maximum response was only 7% of that determined for KR2R4F ‘tar’ extracts. DMSO,
DCM and AS extracts were similar in genotoxic activity with the exception of Gutka where the order
was AS>DCM>DMSO.
Key Words: smokeless tobacco; toxicology
CORESTA SSPT 2008 - ABSTRACT FORM
Please provide the following information.
A study to estimate and correlate cigarette smoke exposure in
Title: smokers in Germany as determined by filter analysis and
biomarkers of exposure.
Author(s): SHEPPERD C.J.; ELDRIDGE A.C.; MARINER D.C.; McEWAN
Principal author’s name first M.
Author(s) Affiliation(s): British American Tobacco, Group R&D, Regents Park Road,
Company name(s) and address(es) Southampton, 5015 6TL, UK
Abstract Body (180-300 words):
A clinical study conducted in Germany compared two methods of estimating exposure to cigarette smoke.
Estimates of mouth level exposure (MLE) of nicotine, NNK, pyrene and acrolein were obtained by chemical
analysis of spent cigarette filters ('Filter Analysis'). Simultaneous estimates of smoke constituent uptake were
achieved by analysis of urinary biomarkers for nicotine (total nicotine equivalents), NNK (total NNAL), pyrene
(1-hydroxypyrene) and acrolein (3-hydroxylpropyl-mercapturic acid (3-HPMA)) plus the nicotine metabolite
cotinine in both plasma and saliva. The prime objective of this study was to establish the relationship
between the exposure estimates obtained by the two methods.
200 Volunteer subjects were recruited into the 19 day study; 50 smokers of each of 1-2 mg, 4-6 mg and 9-10
mg ISO tar yield cigarettes and 50 non-smokers. Smokers underwent two periods of home smoking, each
followed by confinement in a clinic. In the clinical setting, smoking was permitted ad libitum and spent
cigarette filters, cigarette consumption data, 24 hour urine, plasma and saliva samples were collected. Diet
was controlled to avoid potential interferences with biomarkers and ongoing health checks conducted.
Significant correlations between mouth level exposure and appropriate biomarker levels for all smoke
constituents were observed. The adjusted r values were 0.91 (nicotine), 0.85 (NNK), 0.88 (acrolein) and 0.75
(pyrene).
MLE estimates for nicotine, NNK and pyrene showed a dose response in line with ISO tar yield smoked, with
10 mg > 4 mg > 1 mg, and for acrolein 10 mg > 4 mg = 1 mg. The exposure estimates from biomarkers also
showed a dose response in line with ISO tar yield smoked with 10 mg > 4 mg > 1 mg > NS, and for pyrene
10 mg = 4 mg > 1 mg > NS
In conclusion, in this study, smokers of lower yield products were exposed to lower levels of the
smoke constituents as determined by filter analysis and by biomarkers of exposure. The estimates
from the two methods were well correlated.
Key Words: smoke; exposure; filter analysis; biomarkers