Investigations on the origin of tobacco-specific nitrosamines in mainstream smoke of cigarettes

The origin of tobacco-specific nitrosamines (TSNA) in mainstream smoke and the possible contribution of synthesis during the smoking procedure was investigated. Addition of the nitrosamine precursors nitrate and nicotine to the tobacco prior to smoking did not change the mainstream smoke concentrations of N'-nitrosonornicotine (NNN) and 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK), whereas the mainstream smoke concentration of N'-nitrosoanabasine (NAB) and N'-nitrosoanatabine (NAT) increased after spiking the cigarettes with nitrate. Data for TSNA in tobacco and in mainstream smoke and for nitrate in tobacco of commercial cigarettes of the West German market, taken from previous investigations, were used to calculate the mainstream smoke/tobacco ratios for NNN and NNK. These ratios were corrected for ventilation and cigarette length. It is shown that the ratios are constant and neither depend on the nicotine level nor on the nitrate level of the tobacco except for NNK in the nitrate rich dark tobacco type cigarettes. For nonfilter cigarettes the transfer rates of NNN and NNK which had been corrected for ventilation and cigarette length amounted to 23 or 34% respectively. For filter cigarettes a transfer rate of 13% for NNN and 23% for NNK was calculated. Furthermore it is shown that the mainstream smoke/tobacco ratios for NNN and NNK are constant over the whole length of the cigarettes except for NNK in dark tobacco type cigarettes. The results of this investigation indicate that pyrosynthesis of NNN does not occur and that it is very unlikely for NNK at least for lower nitrate levels. Thus with few exceptions the TSNA burden of smokers is predominantly influenced by the amount of preformed NNN and NNK in tobacco.     

Assessment of tobacco-specific nitrosamines in the tobacco and mainstream smoke of Bidi cigarettes

Bidi cigarettes, or bidis, are a tobacco product that originated in India and have been gaining popularity in the USA during the past few years, particularly with adolescents. As with conventional cigarettes, tobacco and smoke from bidis contain chemical constituents including carcinogenic chemicals such as the tobacco-specific nitrosamines (TSNAs). To help better assess the potential public health risk associated with bidi cigarettes, we developed modern high throughput methods to accurately quantify TSNA levels in tobacco and mainstream cigarette smoke particulate. We determined the TSNA levels in the tobacco filler and mainstream smoke from 14 bidi cigarette brands. In the bidi tobacco filler, the 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) levels ranged from 0.09 to 0.85 microg/g, while N'-nitrosonornicotine (NNN) levels ranged from 0.15 to 1.44 microg/g. These amounts are comparable with those in typical American blended cigarettes. The levels of NNK in mainstream smoke from bidis ranged from 2.13 to 25.9 ng/cigarette, and NNN levels ranged from 8.56 to 62.3 ng/cigarette. The wide variation in the TSNA levels most probably reflects the hand-rolled nature of the bidi cigarettes, resulting in a product with less homogenous tobacco amount and a wider variation in overall cigarette construction quality. TSNA levels of bidis were comparable with those of conventional cigarettes, and bidis should not be considered a lower-risk alternative tobacco product. Our analytical findings concur with the previous biologic and biochemical evidence supporting epidemiologic studies linking bidi use with various cancers, especially oral cavity and lung cancers.     

Tobacco specific N-nitrosamines: occurrence and bioassays

A new GC-TEA method for the analysis of tobacco-specific N-nitrosamines (TSNA) has been developed. Four TSNA have thus far been identified; these are N'-nitrosonornicotine (NNN), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), N'-nitrosoanatabine (NAT) and N'-nitrosoanabasine (NAB). The method is currently being applied to the development of cigarette filter-tips which will selectively remove these carcinogens from cigarette smoke. Since recent epidemiological studies have established a correlation between snuff dipping and oral cancer, we have analysed leading snuff brands for TSNA. Snuff products from Sweden, Denmark, Bavaria and the USA contained 5-106 mg/kg of the TSNA and the saliva of snuff dippers had TSNA levels of 20-890 micrograms/kg. NNN, NNK and NAB induce benign and malignant tumours of the respiratory tract of mice and rats. We have shown that NNN and NNK induce tumours in the upper respiratory tract of hamsters and that NNK is the most active carcinogen of the TSNA, also inducing adenoma and adenocarcinoma in the hamster lung. The reported chemical analyses and bioassay results support the epidemiological findings on the causal association of tobacco use and cancer in man.     

Preformed tobacco-specific nitrosamines in tobacco--role of nitrate and influence of tobacco type

Fifty-five types of con cigarettes on the open market in theFRG and several samples of pure tobacco types were analyzedfor preformed tobacco-specific nitrosamines (TSNA) and nitratein the tobacco. For the cigarette tobaccos the observed rangefor N'-nitrosonorrncotine (NNN) was 50–5316 ng/cigaretteand for 4-(methylnltrosamino)-1-(3-pyridyl)-1-butanone (NNK)from not detected (<50 ng/cigarette) to 1120 ng/cigarette.Nitrate levels ranged from 0.6 to 14.4 mg/cigarette. The highestTSNA values were obtained for cigarettes made of dark tobaccosand the lowest for Oriental type cigarettes. The results demonstratedthat there is a correlation between TSNA and nitrate levels.The tobacco type is another important influencing factor, especiallyfor NNK. In Virginia tobaccos unexpectedly high NNK values wereobserved. For the samples of the pure tobacco types, NNN levels rangedfrom 20 to 8850 p.p.b. and NNK levels from not detected (<50p.p.b.) to 1400 p.p.b. The observed range for nitrate was fromtraces (<0.005%) to 4.1%. Pure Oriental tobaccos which arelow in nitrate showed the lowest TSNA concentrations, hi thehigh nitrate Burley tobaccos the highest TSNA concentrationscould be determined. Virginia tobaccos which show a low nitratecontent are low in NNN but rather high NNK concentrations werefound. The results from these pure tobacco types demonstratethat the nitrate content of the tobacco has a great influenceon the TSNA level. However, Virginia tobaccos show higher NNKconcentrations than expected according to their low nitratecontent.     

Tobacco‐specific N‐nitrosamine exposures and cancer risk in the Shanghai cohort study: Remarkable coherence with rat tumor sites

The tobacco‐specific nitrosamines N′‐nitrosonornicotine (NNN) and 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone (NNK) are potent carcinogens for the rat esophagus and lung, respectively. Consistent with the animal carcinogenicity data, we previously reported a remarkably strong association between prospectively measured urinary total NNN, a biomarker of human NNN intake, and the risk of developing esophageal cancer among smokers in the Shanghai Cohort Study. We also demonstrated that urinary total 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanol (NNAL), a biomarker of exposure to the lung carcinogen NNK, is strongly associated with the risk of lung, but not esophageal cancer in smokers. In this study, we investigated the potential relationship between NNN intake and lung cancer risk in the same cohort. The prospectively collected urine samples from lung cancer cases and matching controls selected for this study, all current smokers, have been previously analyzed for total NNAL, cotinine (a biomarker of nicotine intake) and phenanthrene tetraol (PheT) (a biomarker of exposure to polycyclic aromatic hydrocarbons). Urinary levels of total NNN were not associated with the risk of lung cancer: odds ratios (95% confidence intervals) associated with the second and third tertiles of total NNN, relative to the lowest tertile, were 0.82 (0.36–1.88) and 1.02 (0.39–2.89), respectively (p for trend = 0.959), after adjustment for self‐reported smoking history, urinary cotinine and PheT. The results of this study reaffirm the previously reported specificity of urinary total NNN and total NNAL as predictors of esophageal and lung cancer risks, respectively, in smokers, and demonstrate remarkable coherence between rat target tissues of these carcinogens and susceptibility to cancer in smokers.     

On the formation of the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone during smoking

Tobacco-specific N-nitrosamines (TSNA) are the most abundant carcinogens identified in tobacco and its smoke. Reducing their levels in tobacco products and especially in cigarette smoke is, therefore, a primary goal towards minimizing the carcinogenic burden of the tobacco consumer. This study delineates the mechanisms of formation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), the most powerful of the carcinogenic TSNA during cigarette smoking. It demonstrates, by means of radiolabeled tracer compounds that 6.9-11.0% of the NNK formed in tobacco during the curing process transfers into the mainstream smoke. This constitutes 26-37% of the NNK present in the smoke. Addition of [methyl-14C]-nicotine to cigarettes, prior to smoking, led to the finding that 0.001% of nicotine in the cigarette column appears in the smoke as NNK. Thus, 63-74% of NNK in smoke is formed during smoking. NNK yield in the smoke was independent of nitrate content of the tobacco. These data serve to devise methods of reducing TSNA in smoke.     

Tobacco-specific nitrosamines, an important group of carcinogens in tobacco and tobacco smoke

Tobacco-specific nitrosamines are a group of carcinogens that are present in tobacco and tobacco smoke. They are formed from nicotine and related tobacco alkaloids. Two of the nicotine-derived nitrosamines, NNK and NNN, are strong carcinogens in laboratory animals. They can induce tumors both locally and systemically. The induction of oral cavity tumors by a mixture of NNK and NNN, and the organospecificity of NNK for the lung are particularly noteworthy. The amounts of NNK and NNN in tobacco and tobacco smoke are high enough that their total estimated doses to long-term snuff-dippers or smokers are similar in magnitude to the total doses required to produce cancer in laboratory animals. These exposures thus represent an unacceptable risk to tobacco consumers, and possibly to non-smokers exposed for years to environmental tobacco smoke. The permission of such high levels of carcinogens in consumer products used by millions of people represents a major legislative failure. Indeed, the levels of tobacco-specific nitrosamines in tobacco are thousands of times higher than the amounts of other nitrosamines in consumer products that are regulated by government authorities. Although the role of tobacco-specific nitrosamines as causative factors in tobacco-related human cancers cannot be assessed with certainty because of the complexity of tobacco and tobacco smoke, several lines of evidence strongly indicate that they have a major role, especially in the causation of oral cancer in snuff-dippers. Epidemiologic studies have demonstrated that snuff-dipping causes oral cancer. NNK and NNN are quantitatively the most prevalent known carcinogens in snuff, and they induce oral tumors when applied to the rat oral cavity. A role for NNK in the induction of lung cancer by tobacco smoke is likely because of its organospecificity for the lung. Tobacco-specific nitrosamines may also be involved in the etiology of tobacco-related cancers of the esophagus, nasal cavity, and pancreas. Because they are derived from nicotine, and therefore should be associated only with tobacco, tobacco smoke and other nicotine-containing products, tobacco-specific nitrosamines as well as their metabolites and macromolecular adducts should be ideal markers for assessing human exposure to, and metabolic activation of, tobacco smoke carcinogens. Ongoing research has demonstrated the formation of globin and DNA adducts of NNK and NNN in experimental animals. Sensitive methods for the detection and quantitation of these adducts in humans would provide an approach to assessing individual risk for tobacco-related cancers.(ABSTRACT TRUNCATED AT 400 WORDS)     

Transfer of the tobacco-specific carcinogens N'-nitrosonornicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and benzo [a] into the milk of lactating rats

The extent to which some of the more prevalent and potent carcinogensin cigarette smoke could be transferred from circulating bloodinto the milk of lactating rats was determined. One hour afteri.v. administration of benzo[a]pyrene (BaP), N'-nitrosonornicotine(NNN) and 4-(methylnitros-amino)-1-(3-pyridyl)-1-butanone (NNK)to the dams, the levels of these carcinogens were determinedin both blood and milk specimens. The average amount of radioactivitydetected 1 h after administration of ¹⁴C BaP was 0.21% of theadministered dose per ml of milk as compared with 0.17% perml of blood. The amount of NNN in milk ranged from 0.20 to 0.36%of the administered dose per ml which closely paralleled thelevels detected in blood. NNK is readily con verted in vivoto 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL). Thesum of NNK and NNAL was similar in the blood and milk of treateddams. There was, however, a major difference in the ratio ofNNAL/NNK as detected in milk and blood. The ratio of NNAL/NNKin blood ranged from 1.3:1 to 1.9:1 while the ratio in milkranged from 2.4:1 to 3.3:1. In a comparative study of the levelsof NNN in the blood and milk of lactating rats at <1.0, 20,60, 120 and 240 mm after administration, it was confirmed thatsimilar concentrations of NNN are present in blood and milk1 h after administration. These data indicate that these carcinogens,which are present in both cigarette smoke and tobacco, can betransferred into the milk of lactating rats.     

Analysis of tobacco-specific N-nitrosamines in indoor air.

A method was developed and applied for the assessment of tobacco-specific N-nitrosamines (TSNA) in indoor air polluted with tobacco smoke. Air samples were collected on Cambridge filters treated with 0.01 M potassium bisulfate, extracted with dichloromethane and enriched by column chromatography. The fraction containing the TSNA was concentrated and placed on a thermal desorption cartridge packed with Tenax GR. The sample was thermally desorbed and analyzed by capillary GC using a thermal energy analyzer. When the method was applied in a test laboratory in which one, two and four cigarettes were smoked during 30 min, linearity was observed. Field studies included sampling in bars, restaurants and trains. The concentration of N'-nitrosonornicotine (NNN) ranged from not detected to 23 pg/l, that of N'-nitrosoanatabine ranged from not detected to 9 pg/l, while 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) was detected in concentrations ranging from 1 to 29 pg/l. This means an exposure to NNN and NNK of 0.1-0.3 cigarette equivalents. Thus, non-smokers can be exposed to highly carcinogenic TSNA.     

Urinary levels of volatile organic carcinogen and toxicant biomarkers in relation to lung cancer development in smokers

Besides polycyclic aromatic hydrocarbons (PAH) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), which are established lung carcinogens, tobacco smoke also contains relatively large quantities of volatile organic carcinogens and toxicants, including 1,3-butadiene, ethylene oxide, benzene, acrolein and crotonaldehyde. Although animal experiments showed that some of these compounds can induce tumors in multiple organs including the lung, epidemiological studies of their relationship with lung cancer in smokers have not been reported. Therefore, in this study, we quantified urinary mercapturic acid metabolites of 1,3-butadiene, ethylene oxide, benzene, acrolein and crotonaldehyde in addition to urinary biomarkers for PAH, NNK and nicotine in 343 lung cancer cases and 392 matched controls among a cohort of 18,244 Chinese men in Shanghai, China, followed from 1986 to 2006. Compared with the lowest quartiles, highest quartiles of all measured mercapturic acids were associated with statistically significantly ~2-fold increased risk for lung cancer (all P's for trend <0.01) after adjustment for smoking intensity and duration. The positive associations between biomarkers of ethylene oxide, benzene or acrolein and lung cancer risk remained statistically significant after adjustment for biomarkers of PAH and NNK, whereas urinary total cotinine completely explained the mercapturic acid metabolites and lung cancer associations (all P's for trend ≥ 0.39). We conclude that mercapturic acid metabolites of 1,3-butadiene, ethylene oxide, benzene, acrolein and crotonaldehyde may not be independent risk predictors of lung cancer among Shanghai smokers, in contrast to biomarkers of PAH, NNK and nicotine exposure.     

Unusually high levels of carcinogenic tobacco-specific nitrosamines in Sudan snuff (toombak)

The aim of these studies was to determine the levels of carcinogenic tobacco specific nitrosamines (TSNA) in Sudanese oral snuff (toombak) as recent retrospective epidemiological studies suggested an association between the use of toombak and subsequent development of oral cancer. We have analyzed the TSNA levels in 20 samples of Sudanese toombak, of four different quality levels, collected from five different vendors. Using GC coupled with thermal energy analysis, four TSNA were quantified in snuff extracts: N'-nitrosonornicotine (NNN), N'-nitrosoanatabine (NAT), N'-nitrosoanabasine (NAB) and 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Unusually high levels of these TSNA (mean; range, mg/g snuff, dry wt) were detected; NNN (1.13; 0.50-3.08); NAT (0.08; 0.02-0.29); NAB (0.22; 0.02-2.37); and NNK (2.31; 0.62-7.87). Previously, the highest levels of NNN and NNK reported in any snuff were 0.154 and 0.014 mg/g dry wt respectively. In comparison, the levels in Sudanese toombak were up to 20 and 560 times higher respectively. As the public health implications of these findings are significant, attempts should be made to reduce exposure to TSNA in oral snuff users in Sudan.     

A study of snuff carcinogenesis

Dry snuff contains high levels of tobacco-specific N-nitrosamines (TSNA); their concentrations exceed by more than 100 times the quantities of nitrosamines found in any other consumer product. The concentrations of TSNA are similar in dry snuff and in the more popular moist snuff. In addition to the four TSNA identified earlier [N'-nitrosonornicotine (NNN), 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK), N'-nitrosoanatabine (NAT) and N'-nitrosoanabasine (NAB)], two new nitrosamines were detected in snuff, namely 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanol (NNAl; 0.07-0.15 ppm) and 4-(N-nitrosomethylamino)-4-(3-pyridyl)-1-butanol (iso-NNAl; 0.06-1.1 ppm). After oral swabbing with a mixture of NNN and NNK, rats developed tumours of the oral cavity and lung, showing that these TSNA are not only organ-specific carcinogens but can also induce local tumours. After swabbing an extract of snuff containing the same concentrations of NNN and NNK, significantly fewer tumours were induced in the oral cavity and lung, indicating inhibition of the tumorigenic activity of the TSNA by other snuff constituents.     

Carcinogenic agents in cigarette smoke and the influence of nitrate on their formation

The nitrate level of a nonfilter reference cigarette was elevatedfrom 0.52% to 1.2, 1.8, 2.4 and 3.05%, respectively, by additionof sodium nitrate. Data from the mainstream smoke analyses ofthese cigarettes were compared. Yields of carbon monoxide andcarbon dioxide were not significantly altered as a result ofnitrate elevation. Tar, nicotine, benzo [a]pyrene (BaP) andcatechol in mainstream smoke were reduced while yields of nitrogenoxides (NO_x), volatile N-nitrosamines (VNA) and tobacco-specificN-nitrosamines (TSNA) were significantly increased. On the basisof previous bioassays with smoke condensates from high-nitratecigarettes, it was expected that the cutaneous tumorigenicityof these tars would be reduced due to lower levels of Bap (andother carcinogenic PAH) and catechol. However, the total carcinogenicpotential of whole smoke from high nitrate cigarettes is consideredby us to be significantly increased due to the elevated yieldsof No_x, VNA and TSNA. The nitrosamines are regarded as a majorgroup of carcinogens in tobacco smoke; the nitrogen oxides arethe most important precursors for the endogenous formation ofN-nitrosamines upon smoke inhalation. The findings of this modelstudy support the recommendation that the nitrate content oftobacco products should be reduced.     

Tobacco-specific nitrosamines in mainstream smoke of West Gennan cigarettes-tar alone is not a sufficient index for the carcinogenic potential of cigarette smoke

Fifty-five types of commercial cigarettes on the West Germanmarket were analyzed for tobacco-specific nitrosamines (TSNA)in mainstream smoke. The cigarettes included filter and nonfiltercigarettes with very high, high, medium, low and ultra-low tarand nicotine yields. The observed range for N'-nitrosonornicotinewas from 5 to 625 ng/cigarette and for 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanonefrom not detected (<4 ng/cigarette) to 432 ng/cigarette.The highest TSNA values were obtained for nonfilter cigarettesmade of dark tobaccos and the lowest values for nonfilter Oriental-typecigarettes. Relatively high TSNA yields were also observed infilter cigarettes with moderate and lower tar deliveries. Theresults demonstrated that there is no correla tion between TSNAand tar deliveries in mainstrain smoke. The TSNA deliveriesIn mainstream smoke depended on the actual tobacco composition.According to these results the tar delivery, although crucial,is not a sufficient index for the biological activity and thecarcinogenic potential of cigarette smoke.     

Effect of nicotine and tobacco-specific nitrosamines on the metabolism of N'-nitrosonornicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone by rat oral tissue

The effect of N'-nitrosoanatabine (NAT) and nicotine on the metabolism of N'-nitrosonornicotine (NNN) and 4-(methyl-nitrosamino)-1-(3- pyridyl)-1-butanone (NNK) by cultured rat oral tissue was investigated. The effect of NNN on NNK metabolism and the effect of NNK on NNN metabolism was also determined. NNK inhibited NNN metabolism more than NNN inhibited NNK metabolism. NAT inhibited the metabolism of NNK but not of NNN. Nicotine, which is present at greater than 500 times the levels of NNN and NNK in smokeless tobacco, inhibited the metabolism of both nitrosamines. Inhibition of 1 microM NNN metabolism was greater than that of 1 microM NNK when the concentration of nicotine was 1, 10 or 100 microM. Nicotine at 100 microM inhibited the formation of all metabolites of NNN by 85-92%. These results suggest that NNN and nicotine may be metabolized by a common enzyme.     

Carcinogenic tobacco-specific nitrosamines are present at unusually high levels in the saliva of oral snuff users in Sudan.

Exposure to tobacco-specific nitrosamines (TSNA) has been measured in the saliva of 12 users of Sudanese oral snuff (toombak). Using GC coupled to thermal energy analysis, levels of N'-nitrosonornicotine (NNN), N'-nitrosoanatabine (NAT), N'-nitrosoanabasine (NAB) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were measured before, during and after snuff taking. In addition, two TSNA, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and 4-(methylnitrosamino)-4-(3-pyridyl)-1-butanol (iso-NNAL), were detected in the saliva of tobacco chewers for the first time and were confirmed by GC-MS. Nine out of 10 subjects had detectable saliva levels of total TSNA before chewing (0.01-1.0 micrograms/ml) and immediately following chewing (0.1-2.6 micrograms/ml). During dipping, TSNA concentrations reached microgram/ml levels; (range; number of subjects positive) NNN: (0.6-2.1; 12/12), NAT (0.06-0.5; 2/12), NAB (0.05-1.9; 12/12), NNK (0.06-6.7; 11/12), NNAL (0.05-3.3; 11/12) and iso-NNAL (0.07-0.4; 8/12). These saliva TSNA levels, which are 10-100 times the levels previously reported, are consistent with recent observations of unusually high TSNA levels in Sudanese toombak. As several of these TSNA have been shown to be carcinogenic in animals and epidemiological studies have associated human snuff use with tumours of the oral cavity, these findings draw attention to a significant potential public health hazard.     

Racial differences in exposure and glucuronidation of the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)

BACKGROUND: In the United States, Blacks who smoke cigarettes have a higher mean blood concentration of the nicotine metabolite cotinine than White smokers. It has not been determined whether there are racial differences in the exposure to the cigarette smoke carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and in the detoxification of NNK metabolites. METHODS: A community-based cross-sectional survey of 69 Black and 93 White smokers was conducted in lower Westchester County, New York. Information on smoking and lifestyle habits was collected and urinary concentrations of several tobacco smoke biomarkers were compared, including the NNK metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and its glucuronide (NNAL-Gluc). A frequency histogram and probit plot of NNAL-Gluc:NNAL ratios were constructed to determine slow and rapid glucuronidation phenotypes. RESULTS: The mean concentrations of total NNAL, urinary cotinine, plasma cotinine, and thiocyanate were significantly higher in Black men than in White men for each cigarette smoked. In women, the only biomarker that was significantly elevated in Blacks was plasma cotinine. A higher proportion of White versus Black women was categorized as "rapid" glucuronidators (two-tailed exact test, P = 0.03). In men, there were no significant differences in NNAL-Gluc:NNAL phenotypes. CONCLUSIONS: The higher rates of lung carcinoma in black men may be due in part to a higher level of exposure to tobacco smoke carcinogens.     

Mutagenic and cytogenetic studies of N'-nitrosonornicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone

The tobacco specific nitrosamines (TSNA) N'-nitrosonornicotine (NNN) and 4-(Methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK) were tested for mutagenic and clastogenic effects using a battery of short-term test systems. These test systems include the Ames test, micronucleus test (MNT), induction of chromosomal aberrations and sister chromatid exchange (SCEs). NNN and NNK were tested for their potency in inducing mutations in the Ames Salmonella/microsome assay and their clastogenic action were tested by the micronucleus inducing ability in vivo using Swiss mice. Studies on the induction of chromosomal aberrations and SCE exchange were carried out using human peripheral blood lymphocyte cultures. In the Ames test and MNT, NNN was positive but in comparisons with NNK, NNK was a more potent mutagen. Present studies clearly proves the genotoxic potential of both NNN and NNK and between the two NNK is more potent.     

Tobacco smoking and urinary levels of 2-amino-9H-pyrido[2,3-b]indole in men of Shanghai, China.

Carcinogenic heterocyclic aromatic amines (HAA) are formed in cooked meats, poultry, and fish and arise in tobacco smoke. We measured the concentrations of four prevalent HAAs in spot urine samples collected at baseline from 170 participants of the Shanghai Cohort study, a population-based cohort study of adult men recruited during 1986 to 1989 in Shanghai, China. Sixteen (18.6%) of 86 nonsmokers were positive for urinary 2-amino-9H-pyrido[2,3-b]indole (AalphaC) versus 41 (48.8%) of 84 cigarette smokers; the difference was statistically significant (P < 0.001). The number of cigarettes smoked per day was positively and significantly related to urinary levels of AalphaC in study subjects (P < 0.001); the mean level among nonsmokers was 2.54 ng/g creatinine, whereas the means for light (1-19 cigarettes per day) and heavy (20+ cigarettes per day) smokers were 7.50 and 11.92 ng/g creatinine, respectively. 2-Amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline was undetected in the urine of the 170 subjects. Only 5 (2.9%) and 6 (3.5%) subjects, respectively, showed detectable levels of urinary 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, and smoking status was unrelated to levels of either HAA. Quantitative measurements of HAAs in commonly eaten pork and chicken dishes in Shanghai showed low concentrations of HAAs (<1 ng/g meat). Our data indicate that AalphaC represents a major HAA exposure in adult men of Shanghai, China, and that tobacco smoke is an important point source of their AalphaC exposure.     

Approaches to the development of assays for interaction of tobacco-specific nitrosamines with haemoglobin and DNA

The tobacco-specific, nicotine-derived nitrosamines 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN) are among the most important carcinogens in tobacco and tobacco smoke. Treatment of Fischer 344 rats with these carcinogens resulted in alkylation of haemoglobin and DNA by the 4-(3-pyridyl)-4-oxobutyl group formed during their metabolism. This alkyl group can be detached from globin or DNA under mild hydrolytic conditions as 4-hydroxy-1-(3-pyridyl)-1-butanone, which appears to be a potentially useful dosimeter for human exposure to, and activation of, tobacco-specific nitrosamines.