Kinetics of DNA methylation by the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in F344 rats

The carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) was injected intravenously (0.41 mmol/kg) into F344 rats. DNA from target organs (lung, liver) and a non-target organ (kidney) was extracted hydrolysed and analysed for methylated guanines by cation-exchange high-performance liquid chromotography-fluorimetry. Levels of O6-methylguanine, a promutagenic lesion, and 7-methylguanine were three to eight times higher in the liver than in the lung. Neither base could be detected in the kidneys. The extent of methylation of hepatic DNA by NNK was 35 times lower than observed with an equimolar dose of NDMA by Swann et al. (1983). The levels of the two methylated guanines in liver and lung DNA increased between 4 and 24 h following NNK injection. NNK is metabolized rapidly in F344 rats to 4-(methylnitrosamino)-1(3-pyridyl)-butan-1-ol (NNA1). The relatively slow methylation of hepatic DNA after injection of NNK could be due to a slow release of methylating species from the major circulating metabolite NNA1. This low but sustained level of O6-methylguanine induced by NNK could, in part, explain its carcinogenic potency.     

Dose-response study of DNA and hemoglobin adduct formation by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in F344 rats

Levels of hemoglobin adducts and DNA adducts were measured in F344 rats after 4 consecutive daily i.p. injections of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). The dose range was from 3 to 10,000 micrograms/kg/day. [5(-3)H]NNK and [C3H3]NNK were used to measure pyridyloxobutylation and methylation, in both globin and DNA, respectively. In globin, the level of binding increased linearly with dose. Total binding of [5(-3)H] NNK to globin was 3.2 to 8900 fmol/mg and total binding of [C3H3]NNK was 3.5 to 20,000 fmol/mg. The extents of pyridyloxobutylation of both DNA and globin were determined by measuring the amounts of 4-hydroxy-1-(3-pyridyl)-1-butanone released from each, over the dose range 15-5000 micrograms/kg/day. The levels of 4-hydroxy-1-(3-pyridyl)-1-butanone released were 3.2-650 fmol/mg globin, 18-3400 fmol/mg liver DNA, and 58-2180 fmol/mg lung DNA. The extents of DNA methylation in both lung and liver were greater than pyridyloxobutylation. When the dose range was 3-5000 micrograms/kg/day, the levels of 7-methylguanine were 0.22-246 pmol/mumol guanine (149-167,000 fmol/mg) in liver DNA and 0.23-78 pmol/mumol guanine (160-53,000 fmol/mg) in lung DNA. In the lung, the ratio of methylation to pyridyloxobutylation decreased as the dose decreased. In contrast to globin adduct formation, DNA adduct formation did not increase linearly with dose; adduct formation was greater at lower doses than would have been predicted by extrapolation from higher doses. Thus the results of this study demonstrate that there was not a linear relationship between globin adduct formation, neither pyridyloxobutylation nor methylation, and DNA adduct formation in the liver or the lung of rats treated with NNK.     

Effects of {alpha}-deuterium substitution on the tumorigenicity of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in F344 rats

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and itsanalogues substituted with deuterium at the methylene carbon,4,4-dideutero-4-(methylnitrosamino)-1-(3-pyndyl)-1-butanone[4,4-D₂)NNK], and the methyl carbon, 4-(trideuteromethylnitrosamino)-1-(3-pyridyl)-1-butanone[(CD₃NNK) adjacent to the N-nitroso group were tested for tumorigenlcityin F344 rats. Each compound was administered by 60 s.c. injectioiisover a 20-week period such that the total doses were either1.0 or 0.33 mmol/kg. The experiment was terminated after 104weeks. Survival of the rats treated with the higher dose of(4,4-D₂)NNK was significantly less than survival the groupstreated with the same doses of NNK or (CD₃)NNK Target tissueswere liver, lung and nasal cavity for all three compounds. Thehigher dose of (4,4-D₂)NNK induced higher numbers of nasal tumorsand malignant nasal tumors than did NNK. The lower dose of (4,4-D₂)NNKinduced a higher number of nasal tumors than did NNK. No othersignificant differences in tumor incidence were observed. Theresults suggest that 4-(3-pyridyl)-4-oxobutylation DNA mightbe important in induction of nasal cavity tumors by NNK.     

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.     

Evidence for 4-(3-pyridyl)-4-oxobutylation of DNA in F344 rats treated with the tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and N'-nitrosonornicotine

DNA was isolated from tissues of K344 rats 24 h after treatmentby s.c. injection with [5-³H]4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone([5-³H]NNK) or [5-³H]N'-nitrosonor-nicotine ([5-³H]NNN) It washydrolyzed with acid or at pH 7,100°C, and the hydrolysateswere analyzed by HPLC. The major product in each case was Identifiedas 4-hydroxy-1-(3-pyridyl)-1-butanone, formed by hydrolysisof a DNA adduct. It was detected in DNA from the livers of ratstreated with [5-³H]NNK or [5-³H]NNN, and in DNA from lungs ofrats treated with [5-³H]NNK. These results demonstrate that4-(3-pyridyl)-4-oxobutylation of DNA occurs in rats treatedwith NNK or NNN, and are consistent with the hypothesis thatthese nitrosamines are metabolically activated by -hydroxylation.     

Effect of snuff and nicotine on DNA methylation by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone

In this study we assayed the effects of snuff and nicotine onthe DNA methylation by 4-(methyhiitrosamino)-1-(3-pyridyl)-1-butanone(NNK), a powerful tobacco-specific N-nitrosa mine. Male F344rats were pretreated for 2 weeks with either a solution of asnuff extract or 0.002% nicotine in the drinking water. Subsequently,the rats were given a single dose of NNK and the effects ofsnuff and nicotine on the methylation of guanine by NNK in theDNA of target organs of this carcinogenic nitrosamine were determined.Formation of 7-methylguanine in the liver, nasal mucosae andoral cavity and of O⁶-methaylguanine in the liver and oral cavitywas much lower in the rats pretreated with snuff extract thanin those not pretreated. On the other hand, pretreatment ofthe rats with nicotine had no significant effect on the methylationof DNA by NNK nor on the elimination constants of NNK and itsmajor metabolite 4-(methylnitrosamino)-1-(3-pyridly)-1-butanol.     

Comparative metabolism of N'-nitrosonornicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone by cultured F344 rat oral tissue and esophagus

The metabolism and DNA binding of N'-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) by cultured F344 rat oral tissue and esophagus were investigated over a range of concentrations. The metabolites present in the culture media were separated by high performance liquid chromatography and were identified by comparison to standards. alpha-Hydroxylation of NNN, an esophageal carcinogen, was the major pathway for metabolism of this nitrosamine in both tissues. The metabolites formed from 2'-hydroxylation were between 3.0 and 3.9 times those formed from 5'-hydroxylation. 2'-Hydroxylation results in a pyridyloxobutylating species. DNA from esophagus cultured with [5-3H]NNN contained a pyridyloxobutylated adduct which upon acid hydrolysis released 3.8 pmol [5-3H]-4-hydroxy-1-(3-pyridyl)-1-butanone/mumol guanine. DNA from oral tissue cultured under the same conditions, where the extent of metabolism was the same, contained no measurable [5-3H]NNN DNA adduct. This suggests that factors, as yet unknown, cause the DNA of oral cavity tissue to be protected from pyridyloxobutylation by NNN. The metabolism of NNK by alpha-hydroxylation was as much as 10-fold less than the metabolism of NNN by this pathway in both tissues. alpha-Hydroxylation of NNK results in either a methylating species or a pyridyloxobutylating species. DNA from oral tissue cultured with [C3H3]NNK contained between 1.7 and 4.3 pmol 7-methylguanine/mumol guanine, respectively. No pyridyloxobutylated DNA (less than 0.2 pmol/mumol guanine) was detected in oral tissue incubated with [5-3H]NNK. The DNA from esophagi incubated with [C3H3]NNK contained no 7-methylguanine (less than 0.4 pmol/mumol guanine). The level of pyridyloxobutylation of DNA from esophagi incubated with [5-3H]NNK was 0.17 pmol/mumol guanine. The ability of the esophagus to metabolize NNN to a greater extent than NNK to a reactive species which pyridyloxobutylates DNA may be important in determining the carcinogenicity of NNN in the esophagus. In contrast, the metabolism of NNK to a methylating species by oral cavity tissue suggests that this tobacco-specific nitrosamine is important in tobacco-related oral cavity carcinogenesis.     

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.      

Effects of dietary sinigrin or indole-3-carbinol on O6-methylguanine-DNA-transmethylase activity and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced DNA methylation and tumorigenicity in F344 rats

The effects of dietary sinigrin and indole-3-carbinol(I3C) onDNA methylation and O⁶-methylguanine-DNA-trans-methylase activity,factors which may be of importance in the induction of tumorigenicityby the tobacco-specific nitrosamine 4-(methymitrosainino)-1-(3-pyridyD-1-butanone(NNK), were investigated. Additionally, the effects of dietarysinigrin on NNK tumorigenicity were assessed in a two-year bioassayin F344 rats. DNA methylation in target tissues of NNK tumorigenesiswas examined in F344 rats administered [³H-CH₃](NNK(0.6mg/kg,four doses)s.c. and fed control or experimental diets for twoweeks. Dietary sinigrin ata concentration of 3 µmol/gdiet decreased 7-methylguanine formation in hepatic DNA, buthad no effect on 7-methylguanine levels of lung or nasal mucosaDNA. Dietary 13C at a concentration of 30µmol/g diet increased7-methylguanine levels in hepatic DNA, but decreased DNA methylationin lung and nasal mucosa. No effects on O⁶-methylguanine-DNA-transmethylaseactivity were observed in tissue extracts derived from the livers,lungs and nasal mucosae of rats fed diets containing sinigrinor 13C. These results suggested that dietary sinigrin mightreduce the incidence of NNK-induced hepatic tumors with no effecton NNK tumorigenesis of the lung and nasal cavity, whereas 13Cmight increase hepatic tumor incidence and reduce NNK tumorigenesisof the lung and nasal cavity. The bioassay results showed thatdietary sinigrin had no effect on NNK tumorigenesis in thesetarget tissues. However, dietary sinigrin plus NNK resultedin a significant incidence of pancreatic tumors, a rare occurrencein F344 rats. While the results from DNA methylation studiesare in agreement with the bioassay data for lung and nasal cavity,the absence of any inhibitory effect of dietary sinigrin onNNK hepatic tumorigenesis indicates that factors other thanDNA methylation and (Amethylguanine repair should be consideredin assessing the effects of dietary compounds on NNK hepatictumorigenesis. The contrary effects on NNK-induced hepatic DNAmethylation by sinigrin and 13C, two major components of cruciferousvegetables, demonstrate the complexities of dietary modulationof carcinogenesis.     

Inhibition of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced DNA adduct formation and tumorigenicity in the lung of F344 rats by dietary phenethyl isothiocyanate

F344 rats fed diets containing phenethyl isothiocyanate (PEITC, 3 mumol/g diet), a cruciferous vegetable component, before and during treatment with the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), developed about 50% fewer lung tumors than NNK-treated rats fed control diets. NNK-induced liver and nasal cavity tumors in rats were, however, not affected by this dietary treatment. The effects of PEITC diets on the formation of DNA adducts by NNK were also investigated in these target tissues. DNA methylation and pyridyloxobutylation by NNK were both decreased by 50% in lung of rats fed PEITC diets compared to that of rats fed control diets, but the levels of DNA methylation were not affected in liver and nasal mucosa. These results correlated with those from the carcinogenicity bioassay, suggesting that DNA alkylations could be used as indicators for screening inhibitors of NNK tumorigenesis. A slight increase in the number of tumors of the exocrine pancreas was observed in PEITC-fed rats with or without NNK treatments. However, these incidences were not statistically significant when compared to the control groups. The potential toxicity of PEITC at concentrations ranging from 0.75 mumol to 6 mumol/g diet was evaluated in a 13-week study. The only toxicity caused by this treatment was minimal fatty metamorphosis in the liver. Considering the widespread human exposure to NNK through tobacco use, it is of practical importance to demonstrate inhibition of lung tumors induced by this carcinogen. These results provide a basis for studies designed to discover agents of better efficacy for the prevention of NNK-induced tumorigenesis.     

Effects of dietary indoles and isothiocyanates on N-nitrosodimethylamine and 4-(methylnitrosamino)-1-(3-pyridyl)- 1-butanone {alpha}-hydroxylation and DNA methylation in rat liver

Dietary-related indoles, isothiocyanates, and the allkyl isothiocyanateglucosinolate, sinigrin, were administered to F344 rats in thediet for 2 weeks (chronic protocol) or by gavage 2 h beforesacrifice (acute protocol) and the effects of these pretreatmentson the -hydroxylation of two carcinogenic nitrosamines, N-nitrosodimethylamine(NDMA) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK),were evaluated. -Hydroxylation was measured in vitro by quantitationof formaldehyde formation upon incubation of the nitrosamineswith liver microsomes, and in vivo by quantitation of levelsof 7-methylguanine and O⁶-methylguanine in hepatic DNA, 4 hafter nitrosamine treatment. Compounds shown to be inhibitoryin the in vitro assay were selected to be further evaluatedusing the in vivo assay. The results of the in vitro assaysshowed that indoles were inducers of the demethylation of bothnitrosamines. Indole, L-tryptophan and indole-3-carbinol werestrong inducers of NDMA and NNK demethylation, respectively.In contrast, isothiocyanates such as phenethyl isothiocyanateand phenyl isothiocyanate demonstrated a wide range of inhibitoryactivities toward demethylation of these nitrosamines in boththe acute and chronic studies. Chronic, but not acute, pretreatmentwith sinigrin also caused a significant decrease in the demethylationof NDMA and NNK. In view of their promising inhibitory activities,the effects of phenethyl isothiocyanate, phenyl isothiocyanateand sinigrin on the in vivo methylation of DNA by NDMA and NNKwere evaluated. The results were parallel to those obtainedin the in vitro assays. Phenethyl isothiocyanate, phenyl isothiocyanateand sinigrin generally inhibited the formation of 7-methylguanineand O⁶-methylguanine in rat hepatic DNA. The results of thisstudy suggest that these compounds could be anticarcinogenicto NDMA and NNK.     

Lack of correlation between DNA methylation and hepatocarcinogenesis in rats and hamsters treated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone.

Previous studies have demonstrated that the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) induced liver tumors in F344 rats but not in Syrian golden hamsters. The aim of this study was to determine whether there was a correlation between the persistence of O6-methylguanine (O6-mGua) adducts and the rate of recovery of O6-methylguanine-DNA methyltransferase (O6-mGuaT) after depletion in the liver and susceptibility to NNK in F344 rat and Syrian golden hamster injected s.c. with NNK (80 mg/kg). The levels of both 7-methylguanine and O6-mGua reached a maximum 24 h after NNK treatment. O6-mGua in NNK-treated rat liver was undetectable after 48 h. In the rat, the depletion of O6-mGuaT activity occurred within 4 h following NNK treatment. A subsequent rapid recovery of enzyme activity was observed 36 h after NNK exposure. In contrast, high levels of O6-mGua persisted in hamster liver DNA and no O6-mGuaT activity was detected up to 336 h after NNK injection. Thus, the persistence of O6-mGua in hamster liver is most likely related to a lack of recovery of the O6-mGuaT. These results suggested that factors other than O6-mGua may be determining NNK-induced hepatocarcinogenesis in rats. An aldehyde generated by alpha-hydroxylation of NNK, 4-oxo-4-(3-pyridyl)butanal, inhibited O6-mGuaT activity in rat hepatocytes, suggesting that this aldehyde contributes to the carcinogenicity of NNK by inhibiting this repair enzyme.     

Formation of hemoglobin adducts upon treatment of F344 rats with the tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and N'-nitrosonornicotine

[5-3H]4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone ([5-3H]NNK), [C3H3]NNK, and [5-3H]N'-nitrosonornicotine ([5-3H]NNN) were administered to F344 rats by i.p. injection. Levels of tritium present per milligram globin, 24 h after treatment were 720 fmol (0.1% of dose) for [5-3H]NNK, 640 fmol for [C3H3]NNK, and 370 fmol for [5-3H]NNN. Tritium was detectable in globin 7-8 weeks after treatment with [5-3H]NNK or [5-3H]NNN. Approximately 10-15% of the bound tritium in the globin of rats treated with [5-3H]NNK was released upon incubation of the globin with dilute NaOH or HCl. The released material was identified as 4-hydroxy-1-(3-pyridyl)-1-butanone; it was detectable in globin for 6 weeks (t1/2 = 9.1 days) after administration of [5-3H]NNK. 4-Hydroxy-1-(3-pyridyl)-1-butanone was also formed upon NaOH treatment of globin isolated from rats injected with [5-3H]NNN or [5-3H]4-(carbethoxynitrosamino)-1-(3-pyridyl)-1-butanone. The formation of 4-hydroxy-1-(3-pyridyl)-1-butanone under these conditions is consistent with a mechanism by which 4-(3-pyridyl)-4-oxobutyldiazohydroxide is produced upon metabolic alpha-hydroxylation of NNK or NNN and binds to globin of hemoglobin, yielding an adduct which is readily hydrolyzed by acid or base. Support for this mechanism was obtained by in vitro experiments. Levels of 4-hydroxy-1-(3-pyridyl)-1-butanone released upon base treatment of globin were 50 times greater after incubation of rat hemoglobin with [5-3H]4-(carbethoxynitrosamino)-1-(3-pyridyl)-1-butanone than with either [5-3H]NNK or [5-3H]4-hydroxy-1-(3-pyridyl)-1-butanone. The results of this study suggest methods that might be applicable for assessing the molecular dosimetry of NNK and NNN in individuals exposed to tobacco and tobacco smoke.     

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.     

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.     

Evaluation of the transplacental tumorigenicity of the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in mice

The transplacental tumorigenicity of the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) was assessed in three strains of mice: A/J; C3H/He x C57BL/6 F1 (hereafter called C3B6F1); and Swiss outbred [Cr:NIH(S)]. NNK (100 mg/kg) was administered i.p. on Days 14, 16, and 18 of gestation to A/J and C3H/He mice and on Days 15, 17 and 19 of gestation to the Swiss mice. The effects of postnatal treatment with tumor-promoting agents, including 0.05% sodium barbital in the drinking water until death or a single dose of Aroclor 1254 (a mixture of polychlorinated biphenyls, PCB) given on Postnatal Day 8 or 56, were also examined. Progeny were sacrificed at age 24 wk (A/J) or 72 wk (C3B6F1 and Swiss). Significant incidences of tumors occurred in the lungs of strain A/J progeny and in the livers of male C3B6F1 and Swiss progeny. Lung tumor incidence was 8 of 34 (24%) in the female offspring of the A/J mice treated with NNK, compared with 1 of 39 (3%) in controls (P less than 0.05). A 2-fold difference in lung tumor incidence in male offspring of NNK-treated (4 of 23, 13%) versus control (3 of 48, 6%) A/J mice was not of statistical significance. However, the incidence of lung tumors in NNK-exposed progeny A/J mice in both sexes combined (12 of 66, 18%) was also significantly greater than in controls (4 of 87, 5%). The incidence of liver tumors in the male C3B6F1 mice exposed transplacentally to NNK was 12 of 30 (40%) compared to 8 of 46 (17%) in controls (P less than 0.05). No effects of postnatal sodium barbital or PCB were observed on transplacental NNK tumorigenicity in C3B6F1 mice. The combined incidence of liver carcinoma in male mice in all NNK-treated groups (13 of 141, 9%) was significantly greater (P less than 0.05) than in controls (5 of 144, 3%). In male Swiss mice exposed transplacentally to NNK, the incidence of liver tumors was 3 of 57 (5%) compared to 0 of 35 controls, and postnatal treatment with PCB on Day 56 caused a significant increase (5 of 26, 19%) (P less than 0.05) in the incidence of NNK-induced liver tumors. The combined incidence of liver tumors in the male offspring of the Swiss mice treated with NNK, with or without PCB, was 8 of 83 (10%) which was significantly greater (P less than 0.05) than in controls (0 of 66).(ABSTRACT TRUNCATED AT 400 WORDS)     

Microautoradiographic localization of bound metabolites in the nasal cavities of F344 rats treated with the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), the tobacco-specific N-nitrosamine, labeled with 14C on the carbonyl group, was given iv or orally to F334 rats. The animals were killed 4 hours later, and the localization of bound radioactivity in the nasal cavities was studied by light microscopic autoradiography. The strongest labeling was observed in the following groups of glands situated in the lamina propria mucosae: 1) Bowman's glands in the olfactory region, 2) the group of serous glands situated in the anterior half of the nasal septum beneath the respiratory epithelium, 3) the group of serous glands in the middle portion of the lateral wall of the nasal cavity, and 4) the lateral nasal gland (Steno's gland) located lateroventrally to the maxillary sinus. The group of mucous glands in the septum in connection with Jacobson's organ and the group of glands in connection with the dorsal, medial, and lateral parts of the maxillary sinus were not labeled. The olfactory and respiratory surface epithelia in the various parts of the nasal cavity were only weakly labeled. These results indicate that Bowman's glands, some serous glands, and Steno's glands are the principal sites where NNK is activated to alkylating species.     

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     

Pharmacokinetics of N'-nitrosonornicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in laboratory animals

The pharmacokinetics of N'-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in the Syrian golden hamster, the CD-1 mouse, and the baboon were compared to the pharmacokinetics in the Fischer rat. The formation and biological half-life of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), the major metabolite of NNK, was also studied in these animal species. The biological half-life of NNN in these 4 animal species ranged from 0.24 h to 3.06 h, that of NNK from 0.21 h to 0.43 h and NNAL from 0.48 h to 2.9 h. The pharmacokinetic data obtained in the baboon suggest that treatment with NNN and NNK causes an enzyme induction which accelerates the rate of elimination of these compounds.