Mutagenesis induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-4- (methylnitrosamino)-1-(3-pyridyl)-1-butanone and N-nitrosonornicotine in lacZ upper aerodigestive tissue and liver and inhibition by green tea
Marcia d.M.von Pressentin1,
Michael Chen1 and
Joseph B. Guttenplan1,,2,,3
1 Division of Basic Sciences/Biochemistry, New York University Dental Center, 345 East 24th Street, New York, NY 10100, USA and
2 Department of Environmental Medicine, New York University Medical Center, New York, NY, USA
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Abstract
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4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and nitrosonornicotine (NNN) were administered to lacZ mice (MutaTMMouse) at equal concentrations in drinking water (2 weeks at 0.1 followed by 2 weeks at 0.2 mg/ml) over a 4 week period, for a total estimated dose of 615 mg/kg) and mutagenesis in a number of organs was measured. For mutagenesis induced by NNK the potency order was: liver > lung> pooled oral tissues kidney > esophagus > tongue. The mutant fraction varied from ~6 to 40 mutants per 105 plaque forming units This corresponds to ~213 times the background levels. A somewhat different pattern was observed with NNN, where the order was: liver > esophagus oral tissue
tongue > lung > kidney. The potency of NNK was about twice that of NNN in liver and lung, but somewhat less in aerodigestive tract tissue. When compared with results previously obtained for a similar administered dose of benzo[a]pyrene, NNK was ~10100% as mutagenic in the corresponding organs. Reported target organs for carcinogenesis by NNN and NNK in rodents were targets for mutagenesis, but mutagenesis was also observed at other sites, suggesting that these sites are initiated. The effect of green tea consumption on mutagenesis by NNK was also investigated. Green tea reduced mutagenesis by ~1550% in liver, lung, pooled oral tissue and esophagus.
Abbreviations: BaP, benzo[a]pyrene; MF, mutant fraction; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; NNN, nitrosonornicotine; TSN, tobacco-specific nitrosamines.
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Introduction
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4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and nitrosonornicotine (NNN) are potent carcinogenic tobacco-specific nitrosamines (TSN) (1,2). Both are present in tobacco and cigarette smoke, along with many other carcinogens (1,2). They are also the major identified carcinogens in smokeless tobacco products (1,2). Thus, there is reason to suspect that they are involved in carcinogenesis of the lung, mouth and possibly other sites (1,2). NNK is reported to be predominantly a lung and liver carcinogen in rodents and NNN a carcinogen for several aerodigestive sites, such as lung, esophagus and nasal cavity (1,2). Tumors of the oral cavity have also been induced in rats upon oral administration of TSN (3,4). Metabolites of NNK have been detected in the urine of smokers (5) and adducted to hemoglobin in the blood of tobacco chewers (6), demonstrating that NNK can be absorbed and metabolized by humans.
The potential contribution to human cancer posed by exposure to NNN and NNK in tobacco smoke is difficult to evaluate using experimental animal models because NNN and NNK are usually present in small amounts in complex mixtures containing many carcinogens. In smokeless tobacco NNK and NNN are the major potent identified carcinogens, but which of these is potentially more important in oral carcinogenesis is not known.
It would be difficult to compare the carcinogenic potencies of TSN with the other major tobacco smoke carcinogens under relevant conditions in experimental animals because of the time and expense of conducting long-term assays on a large number of compounds. However, a potential shorter term in vivo assay is the lacZ mouse assay (7). In this system (and the related lacI system; ref. 8) mutations can be measured in any organ of the animal, in a reporter gene present in genomic DNA (79). As the assay can be completed in weeks rather than months or years and it utilizes small numbers of mice per group, it may prove to be a practical model system for comparing the relative initiating abilities of tobacco smoke components. In addition, effects of potential inhibitors can also be efficiently examined in this system.
Among the agents that have been investigated as possible chemopreventives against the carcinogenic effects of nitrosamines is green tea (10). Several epidemiological studies have shown inverse associations between the consumption of green tea and incidences of certain cancers (11). Green tea contains a number of phenolic antioxidants (10) which may play protective roles in the replication-dependent steps of carcinogenesis, as certain of these steps appear to involve reactive oxygen species (12). Since the fixation of mutations is dependent on replication, inhibitors of replication may also inhibit mutagenesis.
In this study we have compared the mutagenic effects of NNK and NNN in a number of organs of the lacZ mouse (MutaTMMouse). We have also investigated the effects of green tea on mutagenesis induced by NNK and found significant inhibition in certain organs.
Female mice (MutaTMMouse, 6 weeks old) were purchased from Covance Research Products (Denver, PA) and acclimated for 1 week before the start of treatments. This strain is derived from a CD-2 (BALB/cxDBA/2) F1 mouse. The mice were maintained on the AIN-76 diet (ICN Biomedicals, Costa Mesa, CA) and were allowed food and water ad libitum. The mice were weighed once per week and the mean weekly weight was 28.7 g and the mean daily water consumption was 4.2 ml per mouse. No significant weight differences between groups were noted.
Two weeks after the acclimation period NNK and NNN (Chemsyn Science Laboratories, Lexena, KS) were given in the drinking water at a concentration of 0.1 mg/ml for 2 weeks, then 0.2 mg/ml for 2 weeks, with 1 week separating the treatment periods and a 2 week expression period after the last treatment. Drinking water containing NNK and NNN was changed twice weekly. In the green tea study green tea was administered starting 2 weeks before and continued during the treatment and expression periods.
Green tea extract was prepared by adding 500 ml of boiling water to 25 g of tea leaves [purchased as Chinese green tea (Foo Joy) in teabags from an Asian market], allowing the mixture to cool to room temperature, decanting the supernatant, repeating the process with fresh boiling water and combining the supernatants. When NNK was co-administered with green tea a 10 mg/ml solution of NNK was added to the cooled tea to bring the tea to the desired concentration of NNK. To ascertain that NNK was stable in the green tea solution the concentration of NNK in a green tea solution was compared with that of the same concentration of NNK left in water for 1 week at room temperature. After extraction with methylene chloride both solutions were analyzed by HPLC (4.6x100 mm, 3 µm Microsorb MV column; Rainin/Varian, Walnut Creek, CA) with UV254 nm detection and the concentrations of NNK in the two solutions were, within experimental error (<5%), identical.
After treatment of the animals DNA was extracted from the organs of interest by homogenization, lysis and digestion with proteinase K (Sigma Chemical Co., St Louis, MO), followed by treatment with RNase A (Sigma), precipitation of proteins with ammonium acetate and, finally, precipitation of DNA with isopropyl alcohol. Details of the procedure have been previously described (13). Samples designated `pooled oral tissue' consisted of a mix of sublingual, pharyngeal and gingival tissue.
Phage packaging was carried out using a Transpack packaging mix (Stratagene, La Jolla, CA) and the positive selection (galE) mutation assay was performed according to published techniques (14). The necessary bacterial strain, Escherichia coli C lac galE, was obtained from Ingeny (Leiden, The Netherlands). At least 25 mutants and 5x105 plaques were assayed for each value in the figures.
Both NNK and NNN were mutagenic in most of the tissues examined (Figure 1
). Upon administration in drinking water NNK was most mutagenic in liver, followed by lung. In mice NNK has been assayed for carcinogenicity predominantly in the A/J strain, where it is mainly a lung carcinogen (1,2). This strain is particularly susceptible to lung carcinogenesis (15). However, when given orally to other strains of mice it is also a liver carcinogen (16,17). NNK was about half as mutagenic in pooled oral tissues as in lung and showed weaker, but significant, activity in the other tissues examined (Figure 1
). Thus, the major target organs for carcinogenesis by NNK in mice are among the targets for mutagenesis. In rats NNK is also carcinogenic in liver, lung and in other sites (1,2). Also, in contrast to the current study, many previous studies with NNK were carried out using i.p. or s.c. injection; so that `first pass' absorption by the liver would not occur and metabolism by other organs would be favored relative to the oral route. NNK also exhibited some activity in kidney, although this is not a major target organ for experimental carcinogenesis by NNK. It has been established, however, that cigarette smoking increases the risk of neoplasia of the kidney (18).

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Fig. 1. Mean MF in several organs of female lacZ mice (MutaTMMouse) administered 0.1 mg/ml NNK or NNN for 2 weeks followed by 0.2 mg/ml for 2 weeks, with 1 week separating the two treatment periods and a 2 week expression period. Results are expressed as group means ± SD (n = 5 for the NNK and NNN groups and n = 6 for the control). A single asterisk (*) denotes P < 0.05 in a MannWhitney U-test versus the corresponding control and a double asterisk (**) denotes P < 0.01. Values for NNK and NNN in oral tissue and tongue are from von Pressentin et al. (13). At least 25 mutants and 5x105 plaques were assayed for each value. O.T., oral tissue; esoph., esophagus; cont., control (untreated).
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NNN was also most mutagenic in the liver, followed by the aerodigestive tissues. Among these it was similarly active in all tissues examined. It was not appreciably mutagenic in kidney. Compared with NNK it was about half as mutagenic in liver and lung, similar in pooled oral tissues and more active in tongue and esophagus. Thus, a major difference between NNK and NNN is a stronger specificity of NNK for lung and relatively higher activity of NNN in other aerodigestive tissues. The higher activity of NNN than NNK in esophagus is consistent with carcinogenicity data in rodents (1,2). NNN has also been reported to be carcinogenic in the liver of Swiss and Balb/c mice (17). TSN are activated by cytochrome P450 isozymes (1,2,19) and it seems likely that the sites of greatest mutagenic activity to some extent reflect sites with the highest levels of the specific forms of P450 that activate NNN and NNK. In a report that long-term administration of NNN + NNK induced tumors of the oral cavity the mixture consisted of a large excess of NNN, to approximate the ratio in smokeless tobacco (3). In the current study, carried out at equal concentrations, NNN was more potent than NNK in tongue and similar in pooled oral tissue. This result can be extrapolated to suggest that due to the large excess of NNN over NNK in that carcinogenesis study, NNN was the major oral carcinogen. It is noteworthy, however, that for both NNN and NNK the mutant fraction (MF) in aerodigestive tissues varied by much less than an order of magnitude. This observation suggests that these tissues have similar potential susceptibilities to the genotoxic effects of TSN; the actual targets for carcinogenicity may be dependent, in part, on their tissue disposition.
As NNK and NNN in smokeless tobacco products can directly interact with the tongue, esophagus and oral tissues, NNK and NNN were administered in drinking water to allow topical exposure of these tissues, as well as systemic exposure of these and other tissues. We have previously reported on the mutagenic activity of benzo[a]pyrene (BaP) in female lacZ mice when BaP was administered by gavage over a 12 day period (13). In our previous study the total administered dose of BaP was 625 mg/kg (2.48 mmol/kg) body wt and in this study a similar total administered dose of NNK, 615 mg/kg (2.40 mol/kg), was estimated. Although an exact comparison of potencies cannot be made, due to the different mode and duration of administration, an approximate comparison of the results of these studies indicates that BaP is ~110 times more potent in the overlapping tissues examined. For instance, in lung and pooled oral tissues the MF induced by BaP was several times higher than observed here for NNK. A direct comparison between BaP and NNK is subject to certain caveats. For instance, in contrast to NNK and NNN, BaP was administered as a bolus via gavage. Because of the rapid deposition of BaP in the digestive tract the concentrations of BaP absorbed into the liver may have significantly exceeded the Km values, resulting in relatively more metabolism at peripheral sites. Other differences can certainly be envisaged. However, the mutagenic potencies of NNK and BaP are consistent with comparisons of their carcinogenic potencies in lung (20).
In terms of the relationship between exposures here and in the human situation, it has been estimated that the total dose of TSN over 40 years of snuff dipping is ~130 mg/kg (2). This is several times less than the dose of NNK or NNN here and that dose increased the MF ~2to 10-fold over background levels in most tissues. It also appears that spontaneous, but not induced, mutant frequencies are ~10 times greater in lac transgenes than in endogenous genes (21). This suggests that the doses of NNN and NNK used here increased mutagenesis relative to background in endogenous genes ~20to 100-fold. There are, of course, many uncertainties in a linear extrapolation from high dose, body weight-based, short-term exposures in experimental animals to low dose long-term exposures in humans, but in view of the large population exposed to TSNs, the results observed here are consistent with potential genotoxic effects of TSNs in humans. It should also be noted that the above discussion considers mutations detectable in the lacZ in vivo mutagenesis system, which distinguishes mainly base pair substitutions and frameshift mutations. It would not detect certain other types of genetic and epigenetic damage, such as very large deletions, chromosome alterations and altered methylation patterns, and NNK has been reported to lead to these types of events (2224).
One pilot study on inhibition of NNK-induced mutagenesis by green tea was carried out. Reductions in the MF were observed in most of the tissues studied, with levels of induced mutagenesis reduced by ~1550% in most of the tissues (Figure 2
). The effect is somewhat greater if the MF attributable to spontaneous mutagenesis is subtracted. Both green and black tea have demonstrated protective effects against carcinogenesis and other markers of genotoxicity in experimental animals (10,25). Among the possible protective agents, the antioxidant polyhydroxy phenolics have often been proposed (10), although green tea is a complex mixture; its effects may result from multiple mechanisms. With respect to in vivo mutagenesis, the antioxidant effects of green tea may be important, as administration of NNK has been reported to increase the levels of the reactive oxygen species marker 8-hydroxydeoxyguanosine in rat liver (26). Also, the tea polyphenol epigallocatechin gallate has been reported to inhibit BaP-induced mutagenesis in the lung of rpsL transgenic mice (27).

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Fig. 2. Effect of green tea on mean mutant frequencies in several organs of lacZ mice treated with NNK as above. Mice in the green tea group received a 2.5% (w/v) solution of green tea in their drink starting 2 weeks before administration of NNK and continuing during NNK administration and for 2 weeks after. A single asterisk (*) denotes P < 0.05 in a MannWhitney U-test of MF of NNK versus the corresponding tissue from mice receiving NNK + green tea and a double asterisk (**) denotes P < 0.01 (n = 5 for the NNK group and n = 6 for the NNK + green tea group). At least 25 mutants and 5x105 plaques were assayed for each value. T, tea; O.T., oral tissue; esoph., esophagus.
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This study extends the range of carcinogens that are mutagenic in target organs in an in vivo rodent system. It is also consistent with studies on tumorigenesis showing inhibition of NNK-induced carcinogenesis by green tea. However, it also extends a pattern of detecting mutagenic effects in organs which thus far are not reported targets for tumorigenesis. This observation suggests that such latter sites are initiated and could give rise to tumors under appropriate promoting conditions.
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Notes
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3 To whom correspondence should be addressed Email: joseph.guttenplan{at}nyu.edu 
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Acknowledgments
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This work was supported by grant no. 95B 104 from the American Institute of Cancer Research and grant no. 0727 From the Smokeless Tobacco Research Council. Some of these results were presented at the 1999 Annual Meeting of the American Association of Cancer Research.
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Received May 24, 2000;
revised September 5, 2000;
accepted September 25, 2000.