Genetic variability in the metabolism of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL)

Urinary metabolites of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and its glucuronides, termed total NNAL, have recently been shown to be good predictors of lung cancer risk, years before diagnosis. We sought to determine the contribution of several genetic polymorphisms to total NNAL output and inter-individual variability. The study subjects were derived from the Harvard/Massachusetts General Hospital Lung cancer case-control study. We analyzed 87 self-described smokers (35 lung cancer cases and 52 controls), with urine samples collected at time of diagnosis (1992-1996). We tested 82 tagging SNPs in 16 genes related to the metabolism of NNK to total NNAL. Using weighted case status least squares regression, we tested for the association of each SNP with square-root (sqrt) transformed total NNAL (pmol per mg creatinine), controlling for age, sex, sqrt packyears and sqrt nicotine (ng per mg creatinine). After a sqrt transformation, nicotine significantly predicted a 0.018 (0.014, 0.023) pmol/mg creatinine unit increase in total NNAL for every ng/mg creatinine increase in nicotine at p < 10E-16. Three HSD11B1 SNPs and AKR1C4 rs7083869 were significantly associated with decreasing total NNAL levels: HSD11B1 rs2235543 (p = 4.84E-08) and rs3753519 (p = 0.0017) passed multiple testing adjustment at FDR q = 1.13E-05 and 0.07 respectively, AKR1C4 rs7083869 (p = 0.019) did not, FDR q = 0.51. HSD11B1 and AKR1C4 enzymes are carbonyl reductases directly involved in the single step reduction of NNK to NNAL. The HSD11B1 SNPs may be correlated with the functional variant rs13306401 and the AKR1C4 SNP is correlated with the enzyme activity reducing variant rs17134592, L311V.     

Effects of deuterium substitution on the tumorigenicity of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol in A/J mice

Bioassays and DNA-binding studies of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its analogs with deuterium substitution at the positions alpha to the nitrosamino group ([4,4-D2]NNK and [CD3]NNK) were carried out in A/J mice in order to assess the potential importance of DNA methylation or pyridyloxobutylation in lung tumor induction. The tumorigenic activities of the major NNK metabolite, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and its analog with deuterium at the carbinol carbon ([1-D]NNAL) were also determined. Groups of A/J mice were given single i.p. injections of either 10 or 5 mumol of NNK, [4,4-D2]NNK, [CD3]NNK, NNAL and [1-D]NNAL, and were killed 16 weeks later. Lung tumor multiplicities were as follows in mice treated with 10 mumol: NNK, 7.3 +/- 3.5; [4,4-D2]NNK, 1.4 +/- 1.6; [CD3]NNK, 11.7 +/- 5.4; NNAL, 3.2 +/- 2.0; [1-D]NNAL, 3.2 +/- 2.0. Similar relative tumorigenic activities were observed in mice treated with 5 mumol of these compounds. These results demonstrated that [4,4-D2]NNK was less tumorigenic than NNK and [CD3]NNK was more tumorigenic than NNK. NNAL was less tumorigenic than NNK; substitution of deuterium at the carbinol carbon did not affect its activity. Levels of O6-methylguanine (O6-mG) were measured in pulmonary DNA of A/J mice treated with 10 mumol of NNK, [4,4-D2]NNK or [CD3]NNK, and killed 2 or 24 h later. O6-mG levels were lower in mice treated with [4,4-D2]NNK than in those treated with NNK; no difference in O6-mG levels was observed between those treated with NNK and [CD3]NNK. The results of this study support the hypothesis that O6-mG formation in pulmonary DNA is the key step in lung tumor induction by NNK in A/J mice.     

Identification and analysis of a new tobacco-specific N-nitrosamine, 4-(methylnitrosamino)-4-(3-pyridyl)-1-butanol

A new tobacco-specific N-nitrosamine, 4-(methylnitros-amino)-4-(3-pyridyl)-1-butanol(iso-NNAL) was isolated from snuff tobacco. Structural characterizationof this N-nitros-amine was confirmed by mass spectral analysis.Five popular US brands of moist snuff and three popular US brandsof dry snuff tobacco were analyzed for moisture, nicotine andtobacco-specific N-nitrosamines. The moisture content variedfrom 20 to 53% in moist snuff and from 4.7 to 5.6% in dry snuff.The nicotine levels in these samples varied from 0.6 to 3.2%.The newly identified iso-NNAL was present in con centrationsranging from 0.07 to 2.5 p.p.m. whereas other tobacco-specificN-nitrosamines, N-nitrosonornicotine, N-nitrosoanatabine, N-nitrosoanabasineand 4-(methylnitros-amino)-1-(3-pyridyl)-1-butanone were foundto range from 0.1 to 178 p.p.m. Iso-NNAL was not detected inmainstream and sidestream smoke of cigarettes. Iso-NNAL is genotoxicin primary rat hepatocytes; its tumorigenic properties are cur-rentlybeing tested in mice and rats.     

Effects of long term dietary phenethyl isothiocyanate on the microsomal metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and 4- (methylnitrosamino)-1-(3-pyridyl)-1-butanol in F344 rats

Phenethyl isothiocyanate (PEITC), a cruciferous vegetable component, inhibits lung tumor induction by the tobacco specific nitrosamine, 4- (methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). To gain insight into the mechanism of PEITC lung tumor inhibition, we examined, in male F344 rats, the effects of dietary PEITC (3 micromol/g NIH-07 diet) in combination with NNK treatment (1.76 mg/kg, s.c., three times a week) for 4, 12 and 20 weeks on liver and lung microsomal metabolism of NNK and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), a major metabolite of NNK and also a lung carcinogen. This was compared with rats fed NIH-07 diet, without PEITC, and treated with NNK alone or saline. The protocol was identical to that employed for inhibition of lung tumorigenesis by PEITC. We observed decreased rates of alpha- hydroxylation of NNK and NNAL in lung microsomes of 4-, 12- and 20-week PEITC + NNK treated rats compared with those treated with NNK or saline. NNK treatment alone also decreased lung alpha-methylene hydroxylation of NNK. Long-term NNK + PEITC administration did not significantly affect liver oxidative metabolism of NNK or NNAL, and did not affect the rate of glucuronidation of NNAL in liver microsomes when compared with rats treated with NNK or saline. Thus, PEITC selectively inhibited lung metabolic activation of NNK and NNAL. These results support the hypothesis that PEITC inhibits NNK-induced lung tumors by inhibiting metabolic activation of NNK in the lung. This study also demonstrated that PEITC inhibits lung alpha-hydroxylation of NNAL; this may play a role in PEITC inhibition of lung tumorigenesis by NNK.      

Disposition of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) in bile duct-cannulated rats: stereoselective metabolism and tissue distribution.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) is a chiral compound, and the primary metabolite of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a major carcinogen in tobacco smoke. The goal of the present work was to study the pharmacokinetics and stereoselective metabolism and tissue retention of NNK and NNAL in the rat. Groups of rats were dosed with [5-(3)H]NNK (n = 3) or racemic [5-(3)H]NNAL (n = 3) at a target dose of 8.45 micromol/kg and were killed at selected time points for tissue collection. Separate groups of rats (n =5 per group) received the same dose of either NNK or NNAL and serial sampling of blood, bile and urine was carried out over 24 h. All samples were analyzed by C(18) reversed-phase HPLC with gradient elution and radioflow detection. A gas chromatograph-thermal energy analyzer (GC-TEA) was used to separate the (R)-/(S)-NNAL enantiomers. Racemic NNAL and NNK had large volumes of distribution (321 +/- 137 ml for NNK and 2772 +/- 1423 ml for NNAL) and similar total body clearances (12.8 +/- 2.0 ml/min for NNK and 8.6 +/- 2.6 ml/min for NNAL). The results indicated that the enantiomers of NNAL are stereoselectively metabolized and excreted. The glucuronide of (R)-NNAL, ((R)-NNAL-Gluc) was identified as the major metabolite in the bile after administration of either NNK or NNAL. (R)-NNAL was the major NNAL enantiomer in the bile or urine samples. At 24 h after racemic NNAL administration, NNAL comprised an average of 75.4% of total radioactivity in the lung with an (S)-/(R)-ratio of >20. The stereoselective localization of (S)-NNAL to lung tissue may contribute to the lung selectivity of NNK carcinogenesis. The present studies suggest a need to look beyond metabolic activation as the sole mechanism for lung carcinogenesis.     

Tumorigenic activity of the tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), 4-(methylnitrosamino)-4-(3-pyridyl)-1-butanol (iso-NNAL) and N'-nitrosonornicotine (NNN) on topical application to Sencar mice

The tumor-initiating activities of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), 4-(methylnitrosamino)-4-(3-pyridyl)-1-butanol (iso-NNAL) and N'-nitrosonornicotine (NNN) were evaluated on the skin of female SENCAR mice. A total initiator dose of 28 mumol/mouse of each nitrosamine was applied in 10 subdoses administered every second day. Promotion commenced 10 days after the last initiator dose and consisted of twice weekly application of 2.0 micrograms of tetradecanoylphorbol acetate for 20 weeks. NNK induced a 79% incidence of skin tumors with an average of 1.6 tumors/mouse and a 59% incidence of lung adenomas. In contrast, iso-NNAL and NNN were not active as tumor initiators in either the skin or lung of mice. The tumorigenic activity of NNK on SENCAR mouse skin was evaluated at several doses. At a total initiator dose of 28 and 5.6 mumol/mouse, NNK exhibited significant activity (P less than 0.005) inducing a 59% and 24% incidence of skin tumors, respectively. In this dose response bioassay, NNK at a total initiator dose of 28 mumol induced a 63% incidence (P less than 0.005) of lung adenomas. The numbers of lung adenomas induced at the lower doses employed were not significant. NNK, at a total initiation dose of 1.4 mumol, did not exhibit significant tumorigenic activity (P greater than 0.05). Analysis of DNA from the skin of mice treated with NNK using HPLC with fluorescence detection failed to detect O6- and N-methylguanine (O6-MG and N7-MG) adducts. These data indicate that NNK can exert a contact carcinogenic effect and suggest that mechanisms other than DNA methylation may be involved in its activation to a tumorigenic agent in mouse skin.     

DNA and hemoglobin alkylation by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and its major metabolite 4-(methylnitros-amino)-1-(3-pyridyl)-1-butanol in F344 rats

Alkylation of DNA and hemoglobin was compared in male F344 ratsgiven a single s.c. injection of the tobacco-specific nitrosamine4-(methyInitrosamino)-1-(3-pyridyl)-1-butanone (NNK), or itsmajor metabolite formed by carbonyl reduction, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol(NNAL).In hepatic DNA, levels of 7-methylguanine and O⁶-methyl-guanineformed from NNK 1-48 h after treatment were similar to thoseformed from NNAL. In nasal mucosa and lung DNA, levels of 7-methylguanineand O⁶Amethylguanine were somewhat higher after treatment withNNK than with NNAL. Acid hydrolysis of hepatric DNA, isolatedfrom rats treated with either [5-³H]NNK or [5-³H]NNAL, gave180 ± 48 or 120 ± 23 µuno/mol guanine, respectively,of 4-hydroxy-1-(3-pyridyl)-1-butanone. Basic hydrolysis of globinisolated from rats treated with either [5-³H]NNK of 5-³H]NNALgave 4.1 ± 0.7 or 2.0 ± 0.1 pmol/mg, respectivelyof 4-hydroxy-1-(3-pyridyl)-1-butanone. These results indicatethat NNAL is not a detoxification product of NNK, since treatmentof rats with NNAL results in modifications of DNA which arequalitativerly and quantitatively similar to those observedupon treatment with NNK. Alkylation of DNA and globin by NNALmay result mainly from its metabolic reconversion to NNK.     

In vitro morphological changes induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-(butanone) in fetal hamster respiratory tract tissue

Previous studies conducted in our laboratories have shown that the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is metabolized by fetal hamster respiratory tissues to form DNA-alkylating and clastogenic intermediates. This study was conducted to explore if morphological changes compatible with such events could be detected. Explants of fetal hamster tracheas and lungs were exposed in vitro to various concentrations of NNK and investigated by light and electron microscopy. The explants demonstrated dose dependent preneoplastic lesions in the trachea, while the lung explants exhibited morphological changes compatible with disturbed production and/or release of surfactant.     

Stereoselective metabolism and tissue retention in rats of the individual enantiomers of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), metabolites of the tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is reduced to its main metabolite, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) in a reaction that is both stereoselective and reversible. (S)-NNAL has been shown to be equivalent to NNK in carcinogenic potency, and significantly more potent than (R)-NNAL. It was hypothesized that stereoselective differences in metabolism or tissue distribution contributed to the difference in carcinogenicity between the enantiomers. The individual NNAL enantiomers were therefore administered to bile duct-cannulated rats. Male Fisher F344 rats received i.v. doses of either (R)-NNAL (n = 10) or (S)-NNAL (n = 9) and bile, urine, blood and tissue samples were collected over 24 h. (R)/(S)-NNAL and metabolites were quantified by HPLC and radioflow detection. NNAL was also collected from the HPLC and silylated, and the two NNAL enantiomers were separated by chiral GC-TEA. (S)-NNAL had a much larger tissue distribution (Vss = 1792 +/- 570 ml) than did (R)-NNAL (Vss = 645 +/- 230 ml). Overall, (R)-NNAL tended to enter detoxification pathways, particularly glucuronidation, while reversible metabolism of (S)-NNAL to NNK was favored. For example, after (R)-NNAL administration, approximately 50% of the dose was excreted as (R)-NNAL-Gluc in bile and urine, and <5% was excreted as NNK or NNK metabolites. In contrast, only 10% of an (S)-NNAL dose was excreted as a glucuronide, while almost 20% of the (S)-NNAL dose was excreted as NNK or NNK metabolites. In tissues, particularly the lung, (S)-NNAL appeared to be stereoselectively retained. These findings suggest that the difference in carcinogenicity between the NNAL enantiomers may be attributed to stereoselective differences in tissue distribution and excretion.     

Mass spectrometric quantitation of nicotine, cotinine, and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol in human toenails.

Numerous studies have quantified total cotinine (the sum of cotinine and cotinine-N-glucuronide) and total 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol [NNAL; the sum of NNAL and its O- and N-glucuronides (NNAL-Glucs)] in the urine and blood of smokers, smokeless tobacco users, and nonsmokers exposed to environmental tobacco smoke. Analysis of hair and nails has several advantages over blood and urine testing, such as accumulation of xenobiotics during long-term exposure, ease of collection, and indefinite stability of samples. We developed sensitive methods for quantitation of nicotine, cotinine, and NNAL in human toenails. Nicotine and cotinine were analyzed by gas chromatography-mass spectrometry-selected ion monitoring. NNAL was assayed using liquid chromatography-electrospray ionization-tandem mass spectrometry-selected reaction monitoring. The detection limits of the methods were 0.01 ng/mg toenail for nicotine, 0.012 ng/mg toenail for cotinine, and 0.02 pg/mg toenail for NNAL. In 35 smokers, the mean nicotine level was 5.9 +/- 5.6 ng/mg toenail, mean cotinine was 1.6 +/- 1.3 ng/mg toenail, and mean NNAL was 0.41 +/- 0.67 pg/mg toenail. Samples collected from six nonsmokers were negative for NNAL. In smokers, NNAL correlated with cotinine (r = 0.77; P < 0.0001). The results of this study for the first time show the presence of cotinine and NNAL in human toenails. These sensitive and quantitative methods should be useful in epidemiologic studies of the role of chronic tobacco smoke exposure, including environmental tobacco smoke exposure, in human cancer.     

Tumorigenicity and metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol enantiomers and metabolites in the A/J mouse.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), a major metabolite of the tobacco-specific pulmonary carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), has a chiral center but the tumorigenicity of the NNAL enantiomers has not been previously examined. In this study, we assessed the relative tumorigenic activities in the A/J mouse of NNK, racemic NNAL, (R)-NNAL, (S)-NNAL and several NNAL metabolites, including [4-(methylnitrosamino)-1-(3-pyridyl)but-(S)-1-yl] beta-O-D-gluco-siduronic acid [(S)-NNAL-Gluc], 4-(methylnitrosamino)-1-(3-pyridyl N-oxide)-1-butanol, 5-(3-pyridyl)-2-hydroxytetrahydrofuran, 4-(3-pyridyl)butane-1,4-diol and 2-(3-pyridyl) tetrahydrofuran. We also quantified urinary metabolites of racemic NNAL and its enantiomers and investigated their metabolism with A/J mouse liver and lung microsomes. Groups of female A/J mice were given a single i.p. injection of 20 micromol of each compound and killed 16 weeks later. Based on lung tumor multiplicity, (R)-NNAL (25.6 +/- 7.5 lung tumors/mouse) was as tumorigenic as NNK (25.3 +/- 9.8) and significantly more tumorigenic than racemic NNAL (12.1 +/- 5.6) or (S)-NNAL (8.2 +/- 3.3) (P < 0. 0001). None of the NNAL metabolites was tumorigenic. The major urinary metabolites of racemic NNAL and the NNAL enantiomers were 4-hydroxy-4-(3-pyridyl)butanoic acid (hydroxy acid), NNAL-N-oxide and NNAL-Gluc, in addition to unchanged NNAL. Treatment with (R)-NNAL or (S)-NNAL gave predominantly (R)-hydroxy acid or (S)-hydroxy acid, respectively, as urinary metabolites. While treatment of mice with racemic or (S)-NNAL resulted in urinary excretion of (S)-NNAL-Gluc, treatment with (R)-NNAL gave both (R)-NNAL-Gluc and (S)-NNAL-Gluc in urine, apparently through the metabolic intermediacy of NNK. (S)-NNAL appeared to be a better substrate for glucuronidation than (R)-NNAL in the A/J mouse. Mouse liver and lung microsomes converted NNAL to products of alpha-hydroxylation, to NNAL-N-oxide, to adenosine dinucleotide phosphate adducts and to NNK. In lung microsomes, metabolic activation by alpha-hydroxylation of (R)-NNAL was significantly greater than that of (S)-NNAL. The results of this study provide a metabolic basis for the higher tumorigenicity of (R)-NNAL than (S)-NNAL in A/J mouse lung, namely preferential metabolic activation of (R)-NNAL in lung and preferential glucuronidation of (S)-NNAL.     

Formation and metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol enantiomers in vitro in mouse, rat and human tissues

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) is a major metabolite of the tobacco-specific lung carcino- gen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). NNAL has a chiral center at the 1-position, but little is known about the stereochemical aspects of its metabolic formation from NNK or its further metabolism. We investigated the metabolism of NNK to enantiomers of NNAL in microsomes and cytosol from male F-344 rat liver and lung, female A/J mouse liver and lung, and human liver, as well as in red blood cells from rats, mice and humans. In all systems, (S)-NNAL was the predominant enantiomer formed, ranging from 90 to 98% in the rodent tissues and averaging 64, 90 and >95% in human liver microsomes, liver cytosol and red blood cells, respectively. In rat liver microsomes, (R)- and (S)-NNAL were metabolized at similar rates by alpha-hydroxylation, considered to be the major metabolic activation pathway of NNAL. Pyridine-N-oxidation and adenosine dinucleotide phosphate adduct formation also occurred at similar rates from both enantiomers, while reoxidation to NNK was favored with (S)-NNAL as substrate. In rat lung microsomes, (S)-NNAL was more rapidly metabolized than (R)-NNAL by all oxidative pathways. In human liver microsomes, there were no significant differences in the rates of alpha-hydroxylation, pyridine-N-oxidation and reoxidation to NNK between the two enantiomers. The results of this study demonstrate that (S)-NNAL, the more tumorigenic enantiomer in mice, is preferentially formed from NNK in rodent and human tissues, and is a substrate for oxidative metabolism in rodent and human tissue microsomes.     

4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol and its glucuronides in the urine of infants exposed to environmental tobacco smoke.

Biomarkers of carcinogen uptake could provide important information pertinent to the question of exposure to environmental tobacco smoke (ETS) in childhood and cancer development later in life. Previous studies have focused on exposures before birth and during childhood, but carcinogen uptake from ETS in infants has not been reported. Exposures in infants could be higher than in children or adults because of their proximity to parents who smoke. Therefore, we quantified 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol and its glucuronides (total NNAL) in the urine of 144 infants, ages 3 to 12 months, who lived in homes with parents who smoked. Total NNAL is an accepted biomarker of uptake of the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. Cotinine and its glucuronide (total cotinine) and nicotine and its glucuronide (total nicotine) were also quantified. Total NNAL was detectable in 67 of 144 infants (46.5%). Mean levels of total NNAL in the 144 infants were 0.083 +/- 0.200 pmol/mL, whereas those of total cotinine and total nicotine were 0.133 +/- 0.190 and 0.069 +/- 0.102 nmol/mL, respectively. The number of cigarettes smoked per week in the home or car by any family member when the infant was present was significantly higher (P < 0.0001) when NNAL was detected than when it was not (76.0 +/- 88.1 versus 27.1 +/- 38.2). The mean level of NNAL detected in the urine of these infants was higher than in most other field studies of ETS exposure. The results of this study show substantial uptake of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in infants exposed to ETS and support the concept that persistent ETS exposure in childhood could be related to cancer later in life.     

Inhibition of 4-(methylnitrosamino)-1-(3-pyridyl)-1-1butanone tumorigenicity in mouse lung by the synthetic organoselenium compound, 1,4-phenylenebis(methylene)selenocyanate

The chemopreventive effect of 5, 10 and 15 p.p.m. (as selenium)of l, 4-phenylenebis(methylene)selenocyanate (p-XSC) on lungtumor induction by the tobacco-specific 4-(methylnitrosamino)-l-(3-pyridyI)-l-butanone(NNK) was examined in female A/J mice by administering p-XSCin the diet. Sodium selenite (5 p.p.m. selenium) was given inthe same manner for comparison with p-XSC. Mice were fed experimentaldiets containing the selenium compounds 1 week before i.p. injectionof 10 µunol NNK in 0.1 ml saline and throughout the experimentuntil termination, 16 weeks after carcinogen administration.Body weights of the mice in the different dietary groups didnot differ significantly. p-XSC significantly inhibited lungtumor multiplicity from 7.6 tumors per mouse in the controlgroup to 4.1, 3.3 and 1.8 tumors per mouse in animals given5, 10 and 15 p.p.m. of selenium respectively. In contrast, 5p.p.m. sodium selenite had no protective effect against lungtumor induction. The results of this study clearly indicatethat the structure of selenium-containing compounds is importantin determining their efficacy as chemopreventive agents.     

Combined analysis of r-1,t-2,3,c-4-tetrahydroxy-1,2,3,4-tetrahydrophenanthrene and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol in smokers' plasma.

Polycyclic aromatic hydrocarbons (PAH) and tobacco-specific nitrosamines, such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), are widely accepted to be two important types of lung carcinogens in cigarette smoke. In this study, we have developed a method to estimate individual uptake of these compounds by quantifying r-1,t-2,3,c-4-tetrahydroxy-1,2,3,4-tetrahydrophenanthrene (PheT) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) in 1 mL of smokers' plasma. PheT and NNAL are biomarkers of PAH and NNK uptake, respectively. [D10]PheT and [pyridine-D4]NNAL were added to plasma as internal standards. The plasma was treated with beta-glucuronidase to release any conjugated PheT and NNAL. The analytes were enriched by solid-phase extraction on a mixed mode cation exchange cartridge and the PheT fraction was further purified by high-performance liquid chromatography. The appropriate fractions were analyzed by gas chromatography-negative ion chemical ionization-mass spectrometry for PheT and liquid chromatography-electrospray ionization-mass spectrometry for NNAL. The method was sensitive (limits of quantitation: PheT, 13 fmol/mL; NNAL, 3 fmol/mL), accurate, and precise. Levels of PheT and NNAL in plasma from 16 smokers averaged 95 +/- 71 and 36 +/- 21 fmol/mL, respectively, which are approximately 1% to 2% of the amounts found in urine. This method should be useful in molecular epidemiology studies of carcinogen uptake and lung cancer in smokers.     

Biliary excretion of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in the rat

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone(NNK) is a potent pancreas carcinogen in rats. The biliary excretionof NNK was therefore studied in anesthetized female Sprague— Dawley rats following i.p. administration of 0.7 µmol/kg[carbonyl-¹⁴C]NNK. The concentration of radioactivity peakedwithin 30 min and decreased thereafter exponentially. Cumulativeexcretion of radioactivity reached a plateau at 6–9% ofthe total dose. HPLC analysis revealed the presence of 4-hydroxy-4-(3-pyridyl)butyricacid (hydroxy acid), 4-oxo-4-(3-pyridyl)-butyric acid (ketoacid), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butyl ß-D-glucopyranosiduronicacid (NNAL Glu), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol(NNAL) and NNK. NNAL Glu was the major metabolite contributing34 ± 4% of total radioactivity in bile at 30 min and58 ± 4% at 5 h. The percentage of acidic metabolitesremained constant at     

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

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and nitrosonornicotine (NNN) were administered to lacZ mice (MutaMouse) 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 approximately 6 to 40 mutants per 10(-5) plaque forming units This corresponds to approximately 2-13 times the background levels. A somewhat different pattern was observed with NNN, where the order was liver > esophagus oral tissue approximately 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 approximately 10-100% 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 approximately 15-50% in liver, lung, pooled oral tissue and esophagus.     

Comparison of DNA alkali-labile sites induced by 4-(methylnitrosamino)-1(3-pyridyl)-1-butanone and 4-oxo-4-(3-pyridyl)butanal in rat hepatocytes

4-Oxo-4-(3-pyridyl)butanal (OPB) is an aldehyde formed during the activation of the tobacco-specific N-nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Using the DNA alkaline elution technique, the properties of DNA alkali-labile sites induced in the isolated rat hepatocytes by NNK and OPB were compared. The DNA single-strand break (SSB) frequencies in vitro, as measured by the elution rate (ER), ranged from 0.015 to 0.479 and were proportional (r2 = 0.991) to the dose (0-2 mM) of OPB. These concentrations, however, were slightly cytotoxic. For example, the LC50 after 4 h of exposure was 2.8 mM. This suggests that OPB-induced DNA SSB result from additive effects of OPB-DNA interaction and the indirect DNA damage associated with OPB cytotoxicity. NNK induced a significant and dose-dependent increase of DNA fragmentation at concentrations ranging from 0.5 to 5.0 mM with ER values ranging from 0.012 to 0.274 (r2 = 0.951). Genotoxicity as measured by the DNA-damaging potency coefficient (DDP) was 810, 345, 131 and 75 for N-methyl-N-nitrosourea (MNU), N-nitrosodimethylamine (NDMA), OPB and NNK respectively. Both MNU- and NNK-induced DNA lesions showed increased lability with increased pH (from 12.1 to 12.5) of the eluting buffer (r2 = 0.979 and 0.967 respectively). In contrast, the number of OPB-induced labile sites were not affected by increases in the pH. These results indicate that OPB is not the metabolite contributing the majority of alkali-labile sites generated by NNK. The filter elution procedure was used to study the in vitro rejoining of SSB in DNA induced by NNK. The extent of DNA SSB rejoining after 18 h of culture of hepatocytes in NNK-free medium were dependent on the concentration of NNK (0.5, 2.0 and 5.0 mM) and ranged from 50 to 90%. Rats were injected s.c. with NNK (0.39 mmol/kg). SSB frequency in liver DNA increased rapidly and reached a maximum 12 h after injection. DNA SSB frequency declined during the next 2 weeks with biphasic kinetics. The fast phase (75% rejoining of DNA SSB between 12 h and 2 days) was followed by a slow one (25% of DNA SSB maintained during the next 5 days but not present after 2 weeks). The results of this study better define the role of OPB-induced DNA damage. The persistence of DNA SSB in the liver of NNK-treated rats reflects the inability of this tissue to repair all DNA lesions.     

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.