O6-methylguanine is a critical determinant of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone tumorigenesis in A/J mouse lung

The relative importance of the two alpha-hydroxylation pathways in the tumorigenicity of the tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), was examined in the A/J mouse lung. Methyl hydroxylation, which results in DNA pyridyloxobutylation, was investigated with 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKOAc) and N'-nitrosonornicotine. Methylene hydroxylation, which leads to DNA methylation, was studied by using acetoxymethyl-methylnitrosamine (AMMN). The tumorigenic activities of these compounds were compared to that of 10 mumol NNK at doses that yielded similar or greater adduct levels 24 h after exposure. The methylating agent AMMN was more tumorigenic than the pyridyloxobutylating agents, NNKOAc and N'-nitrosonornicotine. NNKOAc enhanced the tumorigenic activity of AMMN when the two compounds were given in combination. These results suggested that DNA methylation was more important than DNA pyridyloxobutylation in A/J mouse lung tumor induction by NNK and that pyridyloxobutylation enhanced the activity of the methylation pathway. However, the tumorigenicity of 10 mumol NNK could not be reproduced by AMMN +/- NNKOAc at doses that yielded similar levels of DNA adducts 24 h after exposure. Therefore, a second study was conducted in which the persistence of O6-methylguanine in lung DNA following various doses of NNK or AMMN +/- NNKOAc was compared to the tumorigenicity of these treatments. A strong correlation was observed between lung tumor yield and levels of O6-methylguanine at 96 h for NNK and AMMN +/- NNKOAc (r = 0.98). The ability of NNKOAc to increase the tumorigenic activity of AMMN was attributed to its ability to enhance the persistence of O6-methylguanine in lung DNA. These results demonstrate that the formation and persistence of O6-methylguanine are critical events in the initiation of A/J mouse lung tumors by NNK. They also suggest that DNA pyridyloxobutylation by NNK can increase the persistence of this promutagenic base in lung DNA.     

Ki-ras mutations in 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone—initiated and butylated hydroxytoluene—promoted lung tumors in A/J mice

The promotional effects of butylated hydroxytoluene (BHT) on lung tumorigenesis induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) was evaluated in a two-stage model of lung tumorigenesis in the A/J mouse. Mice were treated in two separate experiments with 1.54 mmol/kg (9.1 mg/mouse) NNK, which induced an average of 8.4 and 9.1 tumors/mouse in the experiments. Animals fed a diet that contained 1 g/kg BHT after administration of the carcinogen in these two experiments demonstrated an increase of the tumor multiplicity by 63% and 43%. Multiplicity of forestomach tumors was not effected by BHT in the diet. No differences in lung tumor morphology were seen as a result of the promoting effect of BHT. Mutations in the Ki-ras oncogene from lung tumors induced by NNK (19 tumors) or by NNK plus a diet containing BHT (34 tumors) were characterized by polymerase chain reaction, single-strand conformational polymorphism, and direct sequencing. All 19 NNK-induced tumors not promoted with BHT contained activated Ki-ras genes with GC→AT transitions at the second base of codon 12. Only 11 of 34 NNK-induced and BHT-promoted tumors (32%) had this characteristic Ki-ras alteration. These data suggest that the NNK-initiated mouse lung tumorigenesis pathway, which involves the specific mutation of the Ki-ras oncogene, is altered to a predominantly non-ras mechanism when these tumors are promoted by BHT in the diet. ©1994 Wiley-Liss, Inc.     

Dose-dependent ras mutation spectra in N-nitrosodiethylamine induced mouse liver tumors and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone induced mouse lung tumors

In a previous study, the spectrum of H-ras mutations detectedin B6C3F1 mouse liver tumors induced by 5, 50 or 150 µmol/kgbody wt of N-nitrosodiethylamine (NDEA) was similar to thatin spontaneous B6C3F1 mouse liver tumors, suggesting that activationof the H-ras gene in NDEA-Induced mouse liver tumors may notbe the direct result of the chemical interaction with the H-rasgene. In the present study, mutations in the H-ras oncogenefrom B6C3F1 mouse liver tumors induced by 5 or 50 µmol/kgbody wt of NDEA were characterized by DNA amplification withpolymerase chain reaction (PCR), single-strand conformationpolymorphism (SSCP) and direct sequence analysis. Twenty-oneof 66 NDEA-induced B6C3F1 mouse liver tumors contained activatedH-ras gene with 2 of 21 having a CG to AT transversion at thefirst base of codon 61, 17 of 21 having AT to GC transitionand 2 of 21 having an AT to TA transversion at the second baseof codon 61 in the H-ras gene. The predominant mutation, ATto GC transition (17/21, 81%) is consistent with the formationof O⁴-ethylthymine adduct, and is distinct from the predominantCG to AT transversion (50%) at the first base of codon 61 detectedin H-ras gene from NDEA-induced B6C3F1 mouse liver tumors ina previous study by Stowers et al. Mutations in the K-ras oncogenefrom 59 A/J mouse lung tumors induced by 0.53 mmol/kg body wtof 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) werealso characterized by using the above mentioned methods. Forty-sixof 59 NNK-induced A/J mouse lung tumors contained activatedK-ras genes. All 46 (100%) of the activated K-ras gene had GCto AT transitions at the second base of codon 12. The same mutationwas observed in 70% (7/10) of the K-ras oncogene from A/J lungtumors induced by 4.8 mmol/kg body wt (given in 21 doses) ofNNK. These data suggest that other factors in addition to genotoxiceffect might be involved in the induction of rodent tumors bysome carcinogens when given at higher doses. Therefore, furtherstudies to compare the dose-dependent differences in the profileof ras mutations induced by chemical carcinogens may help toassess human cancer risk. Mutation(s) in exons 5-8 of the p53gene was not found in these NDEA-induced mouse liver tumorsand NNK-induced mouse lung tumors.     

Interactions between methylating and pyridyloxobutylating agents in A/J mouse lungs: implications for 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced lung tumorigenesis

The tobacco-specific nitrosamine, 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone, is activated to lung DNA methylating and pyridyloxobutylating intermediates. It is likely that both pathways play a role in lung tumor initiation by this nitrosamine. Previous studies indicated that O(6)-methylguanine (O(6)-mG) persistence is critical for lung tumor formation in A/J mice. The model pyridyloxobutylating agent, 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKOAc), enhanced the tumorigenic activity of a model methylating agent, acetoxymethylmethylnitrosamine (AMMN), presumably by increasing O(6)-mG persistence in lung DNA. We have been testing the hypothesis that the pyridyloxobutylation pathway increases the mutagenic activity of the DNA methylation pathway by preventing the repair of O(6)-mG by O(6)-alkylguanine-DNA alkyltransferase (AGT). In this study, we report that NNKOAc depletes AGT in lungs but not livers of A/J mice. The consequences of AGT depletion by NNKOAc were then compared with those observed with a known AGT inhibitor, O(6)-benzylguanine (O(6)-bG). NNKOAc and O(6)-bG had similar effects on the levels of AMMN-derived O(6)-mG at 4 and 96 h postinjection. This increase in O(6)-mG levels correlated to increased lung tumor multiplicity in animals simultaneously treated with AMMN (0.75 or 1 micromol) and NNKOAc or O(6)-bG. Only NNKOAc significantly increased lung tumor multiplicity at doses of 0.25 or 0.5 micromol AMMN. The results from these studies indicate that the pyridyloxobutylating agent, NNKOAc, can influence the tumorigenic activity of methylating agents in two ways. At low AMMN doses, the increase in tumor multiplicity is dominated by the additive tumorigenic properties of AMMN and NNKOAc. At higher AMMN doses, NNKOAc appears to enhance the tumorigenic activity of AMMN through enhanced depletion of the repair protein, AGT, leading to increased O(6)-mG persistence. It is likely that similar interactions are important for the organospecific effects of 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone.     

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.     

Comparison of pulmonary O6-methylguanine dna adduct levels and Ki-ras activation in lung tumors from resistant and susceptible mouse strains

The role of O⁶-methylguanine (O⁶MG) DNA adduct formation and persistence in the formation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)—induced lung tumors from resistant C57BL/6 and susceptible A/J mice was investigated. In addition, the frequencies of pulmonary tumor formation and Ki-ras activation were defined in C57BL/6 mice treated with NNK or vinyl carbamate (VC), and the role of the p53 gene in pulmonary carcinogenesis in these resistant mice was examined. One day after treatment with 100 mg/kg NNK, O⁶MG adduct concentrations were twofold to eightfold higher in Clara cells and type II cells than in small cells or whole lungs from both mouse strains. The concentrations of O⁶MG in isolated cells decreased at a similar rate in the two strains of mice. Lung tumors were detected by 27 mo of age in 18% of the C57BL/6 mice after a single 100 mg/kg dose of NNK and in 46% of these mice after a single 60 mg/kg dose of VC. In contrast, the tumor incidence in untreated C57BL/6 mice was 4%. Only one of 22 lung tumors from C57BL/6 mice treated with NNK contained an activated Ki-ras gene that was associated with an O⁶MG DNA adduct, whereas previous studies detected activated Ki-ras oncogenes in most of the NNK-induced lung tumors analyzed from susceptible A/J and resistant C3H mice. The small differences in formation and persistence of the O⁶MG adduct in whole lung or isolated lung cells from A/J and C57BL/6 strains do not account for the differences in either susceptibility for tumor formation or activation of the Ki-ras gene between these strains. In contrast to the low number of NNK-induced tumors with Ki-ras mutations in the resistant mice, 11 of 20 lung tumors from VC-treated mice contained activated Ki-ras genes. Neither p53 tumor suppressor gene mutations nor overexpression of the p53 protein were detected in spontaneous or chemically induced lung tumors in C57BL/6 mice. Thus, although Ki-ras activation was detected in some tumors, pathways independent of ras activation and p53 inactivation also appear to be involved in lung tumorigenesis in this resistant mouse strain.     

Metabolism and macromolecular interaction of the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in cultured explants and epithelial cells of human buccal mucosa

Metabolism and macromolecular interaction of the tobacco-specificcarcinogen 4-(methylnitrosamino)-l-(3-pyridyl)-1-butanone (NNK)were studied in human buccal mucosa in vitro. Microautoradiographkanalysis of [5-³H]NNK-exposed explant cultures demonstrateda uniform distribution of bound radioactivity in the mucosalepithelium, without significant binding in the underlying connectivetissue. The metabolism of [5-³H)NNK at concentrations of both6 and 100 µM resulted in seven identified metabolitesin both explant and epithelial cell cultures. Formation of 4-(methylnitros-amino)-1-(3-pyridyI)butan-l-olby carbonyl reduction of NNK accounted for almost 95% of thetotal metabolism, whereas the proportions of other metabolitesobtained by     

Transformation of hamster pancreatic duct cells by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), in vitro

The tobacco specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone(NNK) is a potent carcinogen in laboratory animals. In the presentstudy, in vitro transformation of spontaneously immortal hamsterpancreatic duct cells following exposure to 20 mM NNK for 1,3,5and 7 days is described. NNK imparted a dose-dependent and time-dependenttoxicity to pancreatic duct cells in vitro. After NNK treatment,duct cells were grown either in complete duct medium (CDM) orin the absence of bovine pituitary extract, epidermal growthfactor and Nu-seruin (incomplete duct medium, 1DM). Additionof NNK to the culture for 1 and 3 days did not affect the growthof the cells, whereas exposure of the cells for 5 and 7 dayswas inhibitory. One and 3 day NNK-treated cells were able togrow in the absence of growth factors and serum immediatelyafter the treatment without any inhibition of growth. Untreatedcells grew as a monolayer consisting of tightly packed polygonalcells with single nuclei. NNK treated cells also grew as a monolayerwith numerous mitotic figures and multi-nucleated large cells.The doubling time between the untreated (16 h) and NNK-treatedcells (14 h) was not significantly different prior to injectioninto the nude mice. NNK treated cells grown in 1DM displayedanchorage independency in soft-agar. The tumorigenicity of theuntreated and NNK treated cells (5x10⁶) was determined in nudemice. One and 3 day NNK-treated cells grown in CDM producedwell-differentiated, mucinous tumors with a lower frequency(2/4 sites) and longer duration, but produced tumors at a higherfrequency (4/4 sites) and shorter duration when grown in IDM.Five and 7 day NNK-treated cells grown in CDM did not produceany tumors; however, they produced tumors when grown in CDMfollowed by IDM (5/8 and 6/8 sites) with a shorter durationin nude mice. Analysis of DNA for k-ras mutation at codons 12,13 and 61 showed G–A transition at codon 12 of the k-rasoncogene in tumor cells of 1 and 3 day NNK treatment. No mutationwas detected in tumor cells from 5 and 7 day treatment.     

Induction of hprt gene mutations in splenic T-lymphocytes from the rat exposed in vivo to DNA methylating agents is correlated with formation of O6-methylguanine in bone marrow and not in the spleen

The suitability of splenic T-lymphocytes as a substitute tissuefor detection of genotoxic effects induced in vivo by chemicalagents that are organ-specifically activated was tested in ratsexposed to single doses at the potent lung-carcinogen 4-(methylnitrosamino)-1-(3-pyri-dyl)-1-butanone(NNK), acetoxymethylmethylnitrosamine (AMMN) or N-methyl-N-nitrosourea(MNU). NNK, AMMN and MNU methylate DNA most likely via the formationof a methanediazohydroxide ion that decomposes to a methyl diazoniumion.For all three agents, an increase in the levels of O⁶-methylguanineand 7-methylguanine in DNA of rat liver and lung was detectedby reverse phase HPLC and electrochemical detection. Treatmentwith NNK did not result in the formation of O⁶-methylguanineand 7-methylguanine in DNA of bone marrow and spleen, correspondingwith the absence of metabolic activation pathways for this compoundin these tissues. For AMMN formation of both O⁶-methylguanineand 7-methylguanine was detectable in DNA of the spleen, whereasin DNA of bone marrow only very low frequencies of 7-methylguaninewere found at a toxic dose. MNU induced O⁶-methylguanine and7-methylguanine in both spleen and bone marrow. Using splenicT-lymphocytes from the rat no increase above control levelsof the hprt mutant frequencies was found for NNK and AMMN forall exposure levels tested, 32 days after chemical exposure.For MNU a dose-dependent increase in hprt mutant frequency wasfound at exposure levels of 0.097 mmol/kg up to 0.582 mmol/kg.DNA sequence analysis was performed on PCR products of hprtcDNA from 39 MNU-induced 6-thioguanine-resistant T-lymphocyteclones. Single base pair substitutions were found in 25 of thesemutants (64%), GC     

Comparison of transplacental and neonatal initiation of mouse lung and liver tumors by N-nitrosodimethylamine (NDMA) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and promotability by a polychlorinated biphenyls mixture (Aroclor 1254)

We have previously shown a positive tumor-promoting effect ofa single dose of Aroclor 1254 on lung and liver tumors initiatedneonatally in the mouse by N-nitrosodimethylamine (NDMA). Inthis study, we have confirmed and extended this observationwith NDMA and the tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyI)-1-butanone(NNK) given either transplacentally or postnatally, followedby a single dose of Aroclor 1254 on day 56. This polychlorinatedbiphenyl (PCB) mixture was an effective promoter of both lungand liver tumors; however, there were specific initiator andsex-related differences in this response. Aroclor administrationsignificantly increased the incidence of lung tumors initiatedtransplacentally by NDMA or NNK in male mice. Neither nitrosamineinitiated tumors transplacentally in females, but lung tumorsinitiated with NNK and liver tumors caused by NDMA in neonatalfemales were promoted by PCBs. Both liver and lung tumors initiatedneonatally by NDMA in male animals, but not NNK-initiated tumors,were promoted by PCBs. These data confirm that PCBs are ableto promote both NDMA- and NNK-initiated tumors, but with chemical-,sex- and age-dependent difference; this suggests influencesof both quantitative and qualitative factors in susceptibilityto tumor promotion.     

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.     

Mgmt deficiency alters the in vivo mutational spectrum of tissues exposed to the tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)

It has been proposed that O⁶-methylguanine DNA methyltransferase(MGMT) gene silencing in premalignant lesions and cancers ofthe lung might result in the acquisition of a ‘mutator’phenotype. Previously, however, we found that Mgmt^–/–mouse DNA failed to show an increase in spontaneous mutations.We thus hypothesized that only during exposure to specific environmentalcarcinogens would the consequences of MGMT deficiency becomeevident. Metabolism of the tobacco-derived nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone(NNK) generates alkylating species that can react with the O⁶position of deoxyguanine, thereby yielding substrates for MGMT-mediatedrepair. To investigate how MGMT might regulate the mutationaleffects of NNK, Mgmt^–/– mice were crossed with alacI-based transgenic reporter line (Big Blue^TM) thus enablingan assessment of the in vivo mutagenic effects of this agent.We observed the induction of a complex spectrum of NNK-dependentlacI mutations in both control and Mgmt^–/– tissues,but only a trend in the mutant frequency increases that couldbe attributed to MGMT deficiency. The mutational spectra ofNNK-treated Mgmt^–/– lungs revealed an increase inthe absolute number of G:C to A:T changes accompanied by a shiftin these from CpG to GpG sites, consistent with an S_N1 alkylationmechanism. In keeping with the high levels of MGMT expressedin the liver, more pronounced mutagenic effects and greaterdifferences in O⁶ position of deoxyguanosine adduct levels followingNNK were observed in Mgmt^–/– versus wild-type mice.Extrapolating to humans, MGMT-deficient cells would likely exhibitan increased mutational burden, but only following exposuresto specific environmental mutagens such as NNK. Abbreviations: dG, deoxyguanosine; dT, deoxythymidine; MCA, Monte Carlo estimation of the P-value of the hypergeometric test; Mf, mutation frequency; MGMT, O⁶-methylguanine DNA methyltransferase; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; O⁶-mG, O⁶-methylguanine; O⁶-pobG, O⁶-[4-oxo-4-(3-pyridyl)butyl]guanine; WT, wild-type Received March 21, 2007; revised December 28, 2007; accepted January 24, 2008.     

Effects of dietary 1,4-phenylenbis(methylene)selenocyanate on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced DNA adduct formation in lung and liver of A/J mice and F344 rats

1,4-Phenylenebis(methylene)selenocyanate (p-XSC) was testedfor its ability to inhibit DNA adduct formation induced by thetobacco-specific N-nitrosamine 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone(NNK) in the liver and lung of A/L mice and F344 rats. Dietaryp-XSC, providing a dose of 5 p.p.m. selenium, significantlyinhibited the formation of 7-methylguanine (7-mGua) inducedby a single i.p. injection of 10 µmol of NNK (12.8% inhibitionat 4 h and 19.9% at 96 h) and O⁶-methylguanine (O⁶-mGua) (16.5%at 4 h and 34.8% at 96 h) in the liver of A/J mice. Dietarysupplements of p-XSC providing 15 p.p.m. of selenium reducedthe levels of 7-mGua by 17.3% (4 h) and 33.6% (96 h). The formationof O⁶-mGua was inhibited by 69.5% (4 h) and 73.8% (96 h). InA/J mouse lung DNA the most significant reduction was observedin levels of O⁶-mGua. Dietary p-XSC at 5 p.p.m. as seleniuminhibited the formation of this adduct by 73.1% (4 h). Ninety-sixhours after NNK injection, and at both time points with p-XSCproviding 15 p.p.m. selenium,O⁶-mGua was not detected. Althoughlevels of 7-mGua in mouse lung DNA were also reduced, this wassignificant only 4 h after carcinogen administration. In general,selenite at 5 p.p.m. as selenium had no significant effect onthe levels of these lesions; however, it inhibited O⁶-mGua inthe liver only4 h after NNK administration. These effects mayexplain why there is chemopreventive activity for p-XSC, butnot for selenite, in NNK-induced lung carcinogenesis in A/Jmice. Moreover, these findings raised our interest in determiningthe potential chemopreventive activity of p-XSC against NNK-inducedlung adenocarcinomas in male F344 rats by first determiningits effects on NNK-induced DNA methylation in the lungs of rats.Diet supplemented with 10 p.p.m. selenium as p-XSC did indeedinhibit the formation of adducts in pulmonary DNA of F344 ratstreatedwith four consecutive injections of 81 mg/kg of NNK. Statisticallysignificant inhibition of O⁶-mGua formation was observed 4 hafter carcinogen treatment in both pulmonary (49.1% inhibition)and hepatic (39.8%) DNA. Statistically significant inhibitionof 7-mGua formation was also measured in lung DNA isolated 24h after the last NNK injection (45.0%) and in liver DNA 4 hafter carcinogen treatment (31.8%). Theseresults suggest thatp-XSC would also inhibit induction of lung adenocarcinoma inmale F344 rats by 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.     

Effects of {alpha}-deuterium substitution on the mutagenicity of 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK)1

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a carcinogenictobacco specific nitrosamine, can be converted to electrophilicdiazohydroxide intermediates by metabolic hydroxylalion of eitherthe methylene carbon (carbon 4) or the methyl carbon attachedto the nitrosamine group. To investigate the relative importanceof these two processes in NNK mutagenesis, we synthesized 4,4-dideutero-4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone([4,4,-D₂]NNK)and 4-(trideuteromethylnitrosamino)-1-(3-pyridyl)-1-butanone([CD₃] NNK), and evaluated their mutagenic activities in Salmonellatyphimurium tester strains. In the presence of Aroclor inducedrat liver 9000 g supernatant, NNK and [4,4-D₂]NNK had comparablemutagenic activities towards S. typhimurium TA 1535 and TA 100,but [CD₃]NNK was inactive in both strains. These results suggestthat hydroxylation of the methyl group of NNK is more importantthan hydroxylation of carbon 4 in its activation to a mutagen.To test the inherent mutagenicity of 4-oxo-4-(3-pyridyl)butyldiazohydroxideand methyldiazohydroxide which would be formed by methyl hydroxylationor carbon 4 hydroxylation, respectively, we compared the mutagenicities,without activation, of the corresponding model compounds, 4-(carbethoxynitrosamino)-1-(3-pyridyl)-1-butanoneand carbethoxynitrosaminomethane (methylnitrosourethane). Bothcompounds were highly mutagenic toward S. typhimurium TA 1535and TA 100, but at doses of 4 x 10^–3 to 4 x 10^–4µmol/plate,only 4-(carbethoxynitrosamino)-1-(3-pyridyl)-1-butanone wasmutagenic. These results are consistent with those obtainedwith the deuterium substituted compounds and indicate the importanceof 4-oxo-4-(3-pyridyl)butylation of DNA in NNK mutagenesis.     

Ki-ras oncogene mutations in tumors and DNA adducts formed by benz[j]aceanthrylene and benzo[a]pyrene in the lungs of strain A/J mice

Strain A/J mice received intraperitoneal injections of benz[j]aceanthrylene (B[j]A) or benzo[a]pyrene (B[a]P). At 24, 48, and 72 h, lung tissues were removed for analysis of B[a]P- or B[j]A-derived DNA adduct formation during the first 3 d of exposure. One group of mice exposed to these hydrocarbons was kept for 8 mo to determine lung tumor multiplicity, the occurrence of mutations in codons 12 and 61 of the Ki-ras gene in the tumors that arose, the relationship between Ki-ras oncogene mutations in tumors, and the presence and quantity of genomic DNA adducts. The major DNA adduct in the lungs of mice exposed to B[a]P was N²-(10β-[ + B,7α, 9α-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene]yl)-deoxyguanosine (BPDE-l-dGuo) arising from bay-region diolepoxide activation of B[a]P and was consistent with the occurrence of tumors with mutations GGT → TGT (56%), GGT → GTT (25%), and GGT → GAT (19%) in codon 12, all involving mutations of a guanine. B[j]A, a demethylated analogue of 3-methylcholanthrene (3-MCA) with an unsaturated cyclopenta ring, produced 16- to 60-fold more tumors at equivalent doses than did B[a]P; the mutations in tumors were GGT → TGT (4%), GGT → GTT (30%), and GGT → CGT (65%). Analysis of adduction patterns in DNA suggested that B[j]A was activated to form DNA-binding derivatives in A/J mouse lungs primarily at the cyclopenta ring even though B[j]A contains a bay region. As reported in the published literature, the mutation spectrum induced by 3-MCA in Ki-ras codon 12 of mouse cells is similar to that of B[a]P but not to that of its close relative B[j]A. In contrast to B[j]A, 3-MCA is activated mostly via a bay-region diol-epoxide since its cyclopenta ring is saturated and not easily epoxidated. Therefore, we propose that the GGT → CGT mutations produced by B[j]A in Ki-ras codon 12 were mostly the result of cyclopentaring-derived adducts.     

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.     

K-ras mutations in lung tumors from A/J and A/JxTSG-p53 F1 mice treated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and phenethyl isothiocyanate

The purpose of this study was to evaluate the effects of theloss of a p53 allele and phenethyl isothiocyanate (PEITC) pre-treatmenton the tumorigenicity of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone(NNK) and K-ras mutation frequency in a hybrid mouse model.Male TSG-p53 ‘knock-out’ mice were bred with A/Jfemale mice to produce (A/JxTSG-p53) F₁ mice either homozygous(p53+/+) or heterozygous (p53+/–) for p53 alleles. Thesemice, together with female A/J mice, were treated at 6–8weeks of age with NNK or dosed with PEITC prior to administrationof NNK. The A/J mice treated with NNK had a 100% incidence oflung tumors, with 9.7 ± 3.4 tumors/mouse. A/J mice pre-treatedwith PEITC prior to NNK administration had 3.5 ± 2.1lung tumors/animal, although the incidence remained at 100%.In (A/JxTSG-p53 F₁ mice with either the p53(+/–) or p53(+/+)genotype PEITC pre-treatment significantly decreased tumor incidence(100 to 40 and 36%, respectively) and multiplicity (2.0 ±0.5 to 0.5 ± 0.4 and 2.1 ± 0.5 to 0.5 ±0.4, respectively), indicating that PEITC is an effective chemopreventiveagent in both A/J mice and (A/JxTSG-p53) F₁ mice. Analysis oflung tumor DNA from A/J mice treated with NNK or NNK/PEITC indicatedthat 15 of 17 (88%) and 20 of 23 (87%) of the tumors, respectively,contained G     

Cytokine production by alveolar macrophages is down regulated by the alpha-methylhydroxylation pathway of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)

NNK, a nicotine-derived nitrosamine, is a potent lung carcinogen that generates electrophilic intermediates capable of damaging DNA. The effects of NNK on the immune response, which may facilitate lung carcinogenesis, are poorly understood. Alveolar macrophages (AM), a key cell in the maintenance of lung homeostasis, metabolize NNK via two major metabolic activation pathways: alpha-methylhydroxylation and alpha-methylenehydroxylation. We have shown previously that NNK inhibits the production of interleukin-12 (IL-12) and tumor necrosis factor (TNF), but stimulates the production of IL-10 and prostaglandin E(2) (PGE(2)) by AM. In the present study, we investigated the contribution of each activation pathway in the modulation of AM function. We used two precursors, 4-[(acetoxymethyl)-nitrosamino]-1-(3-pyridyl)-1-butanone (NNKOAc) and N-nitro(acetoxymethyl)methylamine (NDMAOAc), which generate the reactive electrophilic intermediates [4-(3-pyridyl)-4-oxo-butanediazohydroxide and methanediazohydroxide, respectively] in high yield and exclusively. Rat AM cell line, NR8383, was stimulated and treated with different concentrations of NNKOAc or NDMAOAc (12, 25 and 50 microM). Mediator release was measured in cell-free supernatants. NNKOAc significantly inhibited the production of IL-10, IL-12, TNF and nitric oxide but increased the release of PGE(2) and cyclooxygenase-2 expression suggesting that the alpha-methylhydroxylation pathway might be responsible for NNK modulation of AM cytokine release. In contrast, NDMAOAc did not modulate AM mediator production. However, none of these precursors, alone or in combination, could explain the stimulation of AM IL-10 production by NNK. Our results suggest that the alpha-methylhydroxylation of NNK leading to DNA pyridyloxobutylation also modulates cytokine production in NNK-treated AM.     

Activation of K-ras in aflatoxin B1-induced lung tumors from AC3F1 (A/JxC3H/HeJ) mice

In addition to being a potent hepatocarcinogen, aflatoxin B₁(AFB₁) is a pulmonary carcinogen in experimental animals andepidemiological studies have shown an association between AFB₁exposure and lung cancer in humans. Since point mutations atcodons 12, 13 and 61 of the K-ras protooncogene are often implicatedin chemically induced mouse lung tumors and in human lung adeno-carcinomas,we undertook an investigation of the role of K-ras activationin AFB₁-induced pulmonary carcino-genesis. Female AC3F1 (A/JxC3H/HeJ)mice were treated with AFB₁ (150 mg/kg i.p., divided into 24doses over 8 weeks), and 6–14 months after the completionof dosing mice were killed and pulmonary adenomas and carcinomasremoved. Of the 76 AFB₁-induced lung tumors analyzed by singlestrand conformation polymorphism (SSCP) and direct sequencing,75 possessed K-ras codon 12 mutations (46 GTT, 14 GAT, 13 TGTand 2 TTT; normal, GGT) and one had a GGC