Differences in the Genotoxic Responses of Two

Differences in the Genotoxic Responses of Two Tobacco Specific
Nitrosamines: NNK and NNN
Fiona H Cunningham1, Damien Breheny1, Sarah Mc Garry2, Mark Ballantyne2, Debbie Dillon1, Clive Meredith1
1British American Tobacco, Group Research and Development, Southampton, SO15 8TL, United Kingdom
2Covance Laboratories Ltd, Otley Road, Harrogate, HG3 1PY, United Kingdom
Correspondence: [email protected]
www.bat-science.com
Introduction
Tobacco smoke contains over 6,000 constituents, some with well-established
toxicological properties (1). To date approximately 150 tobacco smoke constituents
have been identified as ‘tobacco smoke toxicants’ (2). Tobacco-specific nitrosamines
(TSNAs) are related to nicotine and other tobacco alkaloids and some have been
shown to be carcinogenic in laboratory studies. The genotoxicity of two TSNAs (4(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N-Nitrosonornicotine (NNN))
was compared in a range of in vitro assays, in the presence and absence of rat liver
S9.
Methods
Mouse Lymphoma Assay
NNK and NNN were evaluated for the potential to induce forward mutation at the
thymidine kinase (tk) locus in mouse lymphoma L5178Y cells in the presence and
absence of S9. The procedures used in these studies complied with OECD Guideline
476 (1997).
Ames
NNK was tested for mutation (and toxicity) in five strains of Salmonella typhimurium
(TA98, TA100, TA1535, TA1537 and TA102), in two independent experiments, using
triplicate plates in the presence and absence of S9. NNN was tested in three strains
of Salmonella typhimurium (TA100, TA1535 and YG7108). The procedures used in
these studies complied with OECD Guideline 471 (1997), with the exception that only
three strains were used for the NNN study.
In Vitro Micronucleus
NNK and NNN were tested in an in vitro micronucleus assay using Chinese hamster
V79 cells, following 3 hours exposure (in the presence and absence of S9) and
following 24 hours exposure (in the absence of S9). The procedures used in these
studies complied with OECD Guideline 487 (2010).
Results
Mouse Lymphoma Assay
NNK did not induce mutations following 3 hour exposure in the absence of S9, but did
following 3 hour exposure in the presence of S9 when tested up to 2073 µg/ml
(equivalent of 10 mM) (see Figure 1) and following 24 hour exposure in the absence
of S9 (data not shown). NNN did not induce mutations following 3 hour exposure in
the presence or absence of S9 or following 24 hour exposure in the absence of S9
when tested up to 1772 µg/ml (equivalent of 10 mM) (see Figure 2).
Figure 1 – NNK Mouse Lymphoma Assay Data – 3 hour treatment in the
presence of S9
Figure 3 – NNK and NNN Ames data – TA100 in the presence of S9
Figure 4 – NNN Ames data – YG7108
Even following these additional supplements, there was no clear evidence for the
mutagenic potential of NNN. In these experiments, NNN induced some statistically
significant increases in revertants at the 1% level when the data were analysed using
Dunnett’s test, however these results were obtained only following testing at
concentrations higher than the maximum required regulatory concentration of
5000 µg/plate (8.82 mM^).
In Vitro Micronucleus
NNK treatments resulted in frequencies of micronucleated binucleate (MNBN) cells
that were significantly higher (p ≤ 0.001) than those observed in concurrent vehicle
controls at concentrations ranging from 30 to 2073 µg/mL (equivalent of 10 mM) (see
Figure 5). NNN did not induce biologically relevant increases in micronuclei
frequencies in V79 cells when tested for 3+21 hours with S9 when tested up to
1772 µg/mL (equivalent to 10 mM) (see Figure 6).
Figure 5 – NNK In Vitro Micronucleus data – 3 hour treatment, plus 21 hour
recovery in the presence of S9
Figure 2 – NNN Mouse Lymphoma Assay Data – 3 hour treatment in the
presence of S9
Figure 6 – NNN In Vitro Micronucleus data – 3 hour treatment, plus 21 hour
recovery in the presence of S9
Ames
NNK induced statistically significant increases in revertants when the data were
analysed at the 1% level using Dunnett’s test in strains TA100 and TA1535 with S9
when tested up to 5000 µg/plate (7.54 mM^) (see Figure 3 for exemplar data).
However, NNN gave no conclusive results in either strain. A further literature review
was conducted to investigate any other potentially responding strains. In addition to
this we believed that individual human Cytochrome P450 enzymes CYP1A1,
CYP1A2, CYP2A6 and CYP2A13 might be necessary for the metabolism of NNN.
Therefore, a series of additional experiments were designed to investigate the
additional strain YG7108 and the supplementation with these four individual human
Cytochrome P450 enzymes (see Figure 4).
^Approximation based on initial treatment volume of 3.2 mL
Conclusions
Both NNK and NNN are on the list of toxicants identified by the WHO Study Group on
Tobacco Product Regulation (TobReg) for mandatory lowering (3). Based on these
results, we conclude that there is a clear difference between NNK and NNN in terms
of their genotoxic responses.
References
1. Rodgman A, Perfetti TA. 2013. The chemical components of tobacco and tobacco smoke. 2nd edition. CRC Press. Pp. 2332.
2. Fowles, J. and Dybing, E., 2003. Application of toxicological risk assessment principles to the chemical constituents of cigarette smoke. Tobacco Control 12, 424-430.
3. Burns, D. M., Dybing, E., Gray, N., Hecht, S., Anderson, C., Sanner, T., O’Connor, R., Djordevic, M., Dresler, C., Hainaut, P., Jarvis, M., Opperhuizen, A. and Straif, K., 2008. Mandated Lowering of Toxicants in Cigarette Smoke: A Description of the World Health Organization TobReg Proposal. Tobacco Control
17, 132-141.
Covance Laboratories Ltd, Harrogate, UK, conducted all experimental work and were funded by British American Tobacco
The authors thank Study Directors Mel Lloyd and Gary Watters for the mouse lymphoma and micronucleus data assessment
Attendance at the meeting was supported by a bursary from UKEMS/IGG
GR&D Centre
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Differences in the Genotoxic Responses of Two
Tobacco Specific Nitrosamines: NNK and NNN
Cunningham, F.H.1, Breheny, D.1, Mc Garry, S.2, Ballantyne, M.2, Dillon, D.M.1,
Meredith, C.1
1British American Tobacco, Group Research & Development, Regents Park
Road, Southampton, SO15 8TL, United Kingdom
2Covance Laboratories Ltd., Otley Road, Harrogate, North Yorkshire HG3 1PY,
United Kingdom
Corresponding author: [email protected]
Presented at the
European Environmental Mutagenesis Society (EEMS) Annual Meeting hosted
by the United Kingdom Environmental Mutagen Society (UKEMS)
Lancaster University, UK
6th – 10th July 2014

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