High frequency of K-ras mutations in spontaneous and vinyl carbamate-induced lung tumors of relatively resistant B6CF1 (C57BL/6JxBALB/cJ) mice

The murine K-ras proto-oncogene is hypothesized to be a pulmonaryadenoma susceptibility gene. This postulate is supported bythe previous demonstration of a preference for mutation of theK-ras allele from the susceptible parent in lung tumors of A/JxC3HF1 mice. We have examined K-ras activation in control and vinylcarbamate (VC) (single dose 0.03 µmol/g i.p.) treatedB6CF1 mice, the progeny of resistant C57BL/6J and intermediatelysensitive BALB/cJ parents. Thirty-four of 37 tumors from VC-treatedmice and 17 of 23 from controls contained activating K-ras weresimilar for the two groups, except that 7 tumors from VC-treatedmice had A     

Tumor multiplicity, DNA adducts and K-ras mutation pattern of 5-methylchrysene in strain A/J mouse lung

This study was undertaken to evaluate the carcinogenic potenlialof 5-methylchrysene (5-MeC) in strain A/J mouse lung and tocorrelate the 5-MeC-DNA adduct profile in lung tissue with themutation spectrum in the K-ras gene of lung tumors. Strain A/Jmice received a single i.p. injection of 5-MeC at doses of 10,50, 100 and 200 mg/kg and after 24, 48 and 72 h their lungswere collected for DNA adduct analysis. Eight months later,lungs from the remaining mice were harvested and the lung tumorscounted and collected for subsequent mutational analysis ofthe K-ras gene. 5-MeC was found to be a potent lung carcinogenin strain A/J mice, inducing more than 100 tumors/mouse at aconcentration of 200 mg/kg. Six 5-MeC-DNA adducts were observed;one adduct comigrated with the standard N²-deoxyguanosine adductof 5-MeC-diol-epoxide I [1R,2S,3S-trihydroxy-4R-(N²-deoxyguanosyl-3'-phosphate)-1,2,3,4-tetrahydro-5-methyl-chrysene],derived from the bay-region diol-epoxide of 5-MeC. DNAs isolatedfrom 5-MeC-induced lung tumors were evaluated for activatingmutations in the K-ras gene by polymerase chain reaction-singlestrand conformation polymorphism and direct DNA sequencing analysis.Mutations were detected in 44 of 49 (90%) 5-MeC-induced tumorsand the mutations were GGT     

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.     

Carcinogenicity, DNA adduct formation and K-ras activation by 7H-dibenzo[c, g]carbazole in strain A/J mouse lung

N-Heterocyclic polynuclear aromatic hydrocarbons (NHA) are environmentalpollutants formed during the combustion of organic materials.7H-Dibenzo[c,g]carbazole (DBC) is a potent carcinogen in lung,liver and skin. We undertook these studies to determine whethertissue specificity for DBC lung carcinogenicity inthe strainA/J mouse is mirrored by formation of DBC-DNA adducts in lungtissue and whether these adducts are consistent with mutationpatterns in the K-ras gene. Strain A/J mice were given a singlei.p. injectionof DBC at doses of 0, 5, 10, 20 or 40 mg/kg andlevels of DNA adducts in the lung were monitored by ³²P-postlabelingon days 1,3,5,7,14 and 21. The remaining animals were sacrificed8 months after DBC treatment and lung tumor multiplicity andK-ras mutation patterns in the tumors were determined. The lungtumor response to DBC was dose related, with an average of 4.7± 1.2 tumors/mouse at 5 mg/kg and 48.1 ± 5.5 tumors/mouseat 40 mg/kg. As many as seven DBC-DNA adducts were observedin the lung. DNA binding levels in the lung were highest at40 mg/kg, with maximum binding at 5-7 days. At lower dose levelsthe maximum binding to DNA decreased and shifted to earliertime points. The DBC-DNA adduct in the lung with the highestlevel of binding at all dose levels was DBC-DNA adduct 3. Themajority of DBC-induced mutations in the K-ras gene in the lungwere A     

Ras proto-oncogene activation in liver and lung tumors from B6C3F1 mice exposed chronically to methylene chloride

Methylene chloride has been the subject of recent toxicologicaland carcinogenesis studies because of significant human exposureand widespread use in industrial processing, food preparationand agriculture. In this study, liver and lung tumors, inducedin female B6C3F1 mice by inhalation of 2000 p.p.m. methylenechloride (6 h/day, 5 days/week continuous exposure), were examinedfor the presence of activated rasproto-oncogenes. DNA was isolatedfrom 49 spontaneous and 50 methylene chloride-induced livertumors and screened by oligonucleotide hybridization of PCRamplified H-ras gene fragments for codon 61 mutations. In thechemically induced tumors, 38 mutations were detected, 16 Cto A transversions in base 1, 16 A to G transitions in base2 and 6 A to T transversions in base 2. This mutation profilewas similar to that identified for the H-ras gene in the spontaneousliver tumors and suggests that methylene chloride acts in liverby promoting cells with spontaneous lesions. Tumors in whichH-ras codon 61 mutations were not detected were examined forthe presence of transforming genes by the nude mouse tumorigenicityassay. Except for activated K-ras genes detected in DNA fromtwo methylene chloride induced tumors and one spontaneous tumor,no other transforming genes were identified. DNA from 54 lungtumors was screened by direct sequencing of PCR amplified DNAfragments of the K-ras gene for first and second exon mutations,and 12 mutations were identified, 5 in exon one and 7 in exon2. The low number of spontaneous tumors available in this studylimits the interpretation of the data, and thus the frequencyand spectrum of K-ras activation in the methylene chloride inducedtumors was not significantly different from that in the sevenspontaneous tumors analyzed. Since K-ras activation was notdetected in 80% of the tumors, the nude mouse tumorigenicityassay was used to examine the lung tumors for the presence ofother transforming genes. At present no transforming genes otherthan ras genes were identified in either liver or lung tumors.     

Increased frequency of K-ras mutations in lung neoplasms from female B6C3F1 mice exposed to ozone for 24 or 30 months

The National Toxicology Program recently completed long-termozone inhalation studies in B6C3F1 mice and F344/N rats. Miceand rats were exposed to 0, 0.5 or 1.0 p.p.m. ozone by inhalationfor 24 or 30 months. There was an increased incidence of lungneoplasms in B6C3F1 mice. However, there was no evidence ofcarcinogenicity in F344/N rats. The objectives of this studywere to (i) evaluate benign and malignant lung neoplasms fromB6C3F1 mice for mutations in the K-ras gene at codons 12, 13and 61, (ii) determine if the frequency and spectra of K-rasmutations were unique for ozone-induced lung neoplasms, (iii)determine if specific K-ras mutations were associated with thesize and morphological patterns of lung neoplasms or ozone exposureconcentrations and (iv) screen lung neoplasms by immunohistochemicalmethods for the p53 protein. K-ras mutations were detected bysingle-strand conformation analysis and identified by directsequencing of polymerase chain reaction-amplified DNA isolatedfrom formalin-fixed, paraffin-embedded neoplasms. K-ras mutationswere detected in 73% of ozone-induced neoplasms, as comparedwith 33% of lung neoplasms from controls. The predominant mutationsconsisted of A     

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.     

Benzo[b]fluoranthene: tumorigenicity in strain A/J mouse lungs, DNA adducts and mutations in the Ki-ras oncogene

The polycyclic aromatic hydrocarbon benzo[b]fluoranthene (B[b]F)is a pervasive constituent of environmental combustion products.We sought to examine the lung tumorigenic activity of B[b]Fin strain A/J mice, to study the relationship between formationand decay of B[b]F-DNA adducts and to examine mutations in theKi-ras proto-oncogene in DNA from B[b]F-induced tumors. Micewere given i.p. injections of 0, 10, 50, 100 or 200 mg/kg bodywt and lung adenomas were scored after 8 months. B[b]F inducedsignificant numbers of mouse lung adenomas in a dose-relatedfashion, with the highest dose (200 mg/kg) yielding 6.95 adenomas/mouse, with 100% of the mice exhibiting an adenoma. In micegiven tricaprylin, the vehicle control, there were 0.60 adenomas/mouse,with 55% of the mice exhibiting an adenoma. Based on dose, B[b]Fwas less active than benzo[     

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     

Glutathione S-transferase {micro}1 null genotype is associated with K-ras gene mutation in lung adenocarcinoma among smokers

Glutathione S-transferase µ1 (GSTM1) plays a role in thedetoxification of benzo[a]pyrene (BP) diol epoxide in tobaccosmoke. Individuals who genetically lack the GSTM1 gene are likelyto have an increased risk of smoking-related lung cancers, however,the target oncogenes for mutation are unknown. To investigatethe relation between GSTM1 genotype and K-ras gene mutationwe examined 193 adenocarcinomas and 119 squamous cell carcinomasof lung. The GSTM1 genotype was determined by PCR and K-rasgene mutations at codons 12 and 13 were detected by dot-blothybridization analysis using sequence-specific oligonucleotideprobes. K-ras gene mutations were found in 29 of 312 (9.3%)tumors. All of them arose in patients who were habitual smokers.Mutations of the K-ras gene were detected in 6 of 100 (6%) and15 of 93 (16.1%) adenocarcinoma cases with the GSTM1(+) andGSTM1(–) genotypes, respectively, and the difference wasstatistically significant. These findings suggest that the causeof K-ras gene mutation in smokers with lung adenocarcinoma maybe in part an accumulation of BP diol epoxide which is not welldetoxified in individuals with the GSTM1 null genotype.     

Relationships of DNA adduct formation, K-ras activating mutations and tumorigenic activities of 6-nitrochrysene and its metabolites in the lungs of CD-1 mice

6-Nitrochrysene (6-NC), an environmental pollutant and a potentmouse lung carcinogen, is activated by two major metabolic pathwaysto yield DNA adducts derived from either trans-1,2-dihydro-1,2-dihydroxy-6-aminochrysene(1,2-DHD-6-AC) or N-hydroxy-6-aminochrysene (N-OH-6-AC). Whilethe former pathway has been shown to be the major activationpathway leading to DNA adducts in mice treated with 6-NC, thepotential contribution of the minor nitroreduction pathway totumorigenicity in this system is not clear. To evaluate theroles of these activation pathways and the resulting DNA adductsin mouse lung tumorigen-esis, we studied DNA adduct formation,the induction of tumors and tumor K-ras mutational spectra inthe lungs of male CD-1 mice treated with 6-NC and its metabolites.6-NC, 6-AC and 1,2-DHD-6-AC produced predominantly a singlechromatographically identical dG adduct, and 6-nitrosochrysene(6-NOC) gave a single major adduct that was most likely derivedfrom reaction at the C8 position of deoxyadenosine. 6-NC-, 1,2-DHD-6-AC-and 6-NOC-treated mice developed both adenomas and adenocarcin-omasin the lung, whereas only lung adenomas were observed in 6-AC-treatedanimals. K-ras mutations in adenomas resulting from 6-NC andits metaboliteswere primarily at G: C basepairs in codons 12and 13, while adenocarcinomas had K-ras mutations distributedbetween codons 12, 13 and 61, and involved both G:C and A:Tbasepairs. The K-ras mutational spectra in codons 12 and 13were similar in both adenomas and adenocarcinomas, whereas ahigher percentage of mutations at A:T incodon 61 was found inadenocarcinomas. These results support the conclusion that the1,2-DHD-6-AC-derived adduct is associated with both adenomaand adenocarcinoma formation and is the primary lesion involvedin the induction of mouse lung tumors by 6-NC. The major adductdetected after 6-NOC treatment, which is derived from N-OH-6-AC,is apparently less efficient as an inducer of mouse lung tumorsand is associated more specifically with adenocarcinoma formation.     

SHORT COMMUNICATION: K-ras mutations in benzotrichloride-induced lung tumors of A/J mice

Benzotrichloride (BTC) is used extensively as a chemical intermediatein the synthesis of benzoyl chloride and benzoyl peroxide. Epidemiologkaldata suggest that BTC is a human lung carcinogen. BTC is alsoa carcinogen in the A/J mouse lung tumor bioassay. ActivatedK-ras protooncogenes were detected in BTC-induced lung tumorsfrom A/J mice. The polymerase chain reaction was used to amplifyspecific DNA segments likely to contain activating mutations,and the amplified DNAs were sequenced to identify the mutation.The activating mutation present in the K-ras gene from all BTC-inducedlung tumors (24/24) was a GC     

Analysis of activated protooncogenes in B6C3F1 mouse liver tumors induced by ciprofibrate, a potent peroxisome proliferator

Liver tumors from B6C3F1 mice induced by the potent peroxisomeproliferator ciprofibrate, a hypolipidemic drug, were evaluatedfor the presence of transforming genes by the nude mouse tumorigenicityassay. As reported earlier, the tumors were not activated bya point mutation in codon 61 of H-ras. Two of the eight tumorsexamined contained a mutation in codon 13 or an H-ras gene mutatedin codon 117. Screening of another 23 ciprofibrate-induced livertumors by oligonucleotide hybridization analysis and directDNA sequencing resulted in the identification of three tumorDNA samples with point mutations in codon 117 of the H-ras gene.In addition, another tumor sample contained a K-ras gene witha mutation in codon 61. Mutations in these codons have beenseen only rarely in chemically induced liver tumors from thismouse strain. Of 15 spontaneous B6C3F1 liver tumors screenedin the same manner, one exhibited a K-ras gene activated bya mutation in codon 13 and a second contained an H-ras geneactivated by a mutation in codon 117. These ras gene mutationshave not been reported previously from spontaneous liver tumors.The frequency and spectrum of ras oncogene mutations characterizedin ciprofibrate-induced liver tumors differ significantly fromthe frequency and pattern identified in spontaneously occurringliver tumors. The results of this study with a limited numberof samples suggest that ras protooncogene activation or activationof other protooncogenes that can be detected by the nude mousetumorigenicity assay are not frequent events in the mechanismof carcinogenicity of the peroxisome proliferator ciprofibrate.However, the lower frequency and distinct pattern of H-ras mutationsobserved in these tumors disprove the assumption of promotionof spontaneous hepatocarcinogenesis by ciprofibrate.     

Hepatocarcinogenicity of chlordane in B6C3F1 and B6D2F1 male mice: evidence for regression in B6C3F1 mice and carcinogenesis independent of ras proto-oncogene activation

Logistic regression analysis of age-specific prevalences forneoplastic and non-neoplastic liver lesions was used to examinetreatment responses for B6C3F1 and B6D2F1 male mice continuouslyexposed to chlordane (55 p.p.m.) and to determine whether neoplasmswere dependent on continuous exposure in the B6C3F1 mice. Inorder to determine if ras oncogene activation plays a role inthe carcinogenicity of chlordane and whether the activationis dependent on genetic background, liver tumors from chlordane-treatedB6C3F1 and B6D2F1 mice were analyzed for the presence of activatingmutations in the ras oncogene. The overall liver tumor prevalenceat terminal killing was nearly 100% for both strains; however,the age-specific prevalence increased more rapidly in B6C3F1mice than in B6D2F1 mice. Tumor-bearing B6C3F1 mice had an averageof two more tumors per liver than B6D2F1 mice at their respectiveterminal killings (5.4 versus 3.3). When chlordane exposurewas discontinued for a group of B6C3F1 mice (‘stop’group) at 491 days of age, overall tumor multiplicity significantlydecreased by 30% from an average of 4.4 per tumor-bearing-animalat 525 days to 3.1 at terminal killing (568 days). Over thesame time period the prevalence of hepatocellular carcinomassignificantly decreased from 80 to 54% and adenomas from 100to 93% by terminal killing in B6C3F1 ‘stop-group’mice. Chlordane induced diffuse hepatocellular centrilobularhypertrophy, frequent multinucleate hepatocytes, toxic changeand hepatoproliferative lesions composed predominantly of acidophilichepatocytes in nearly 100% of both the B6C3F1 and B6D2F1 mice.The development of histological evidence of toxicity closelyparalleled the temporal development of hepatocellular neoplasiaand decreased in severity when the tumor burden was maximal.No H- or K-ras mutations were detected in the chlordane-inducedhepatocellular tumors in B6C3F1 mice (15 adenomas and 15 carcinomas)or B6D2F1 mice (10 adenomas and 10 carcinomas). In conclusion,chlordane induced liver tumors in both B6C3F1 and B6D2F1 malemice by mechanisms independent of ras oncogene activation and30% of both benign and malignant liver tumors in the B6C3F1mice regressed after exposure was discontinued.     

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     

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.     

Variant mutational activation of the K-ras oncogene in renal mesenchymal tumors induced in newborn F344 rats by methyl(methoxymethyl)nitrosamine

Renal mesenchymal tumors were induced at high incidence in F344rats by a single intraperitoneal injection of methyl(methoxymethyl)nitrosamine(DMN-OMe) within 48 h after birth. DNAs from 18 of 35 mesenchymaltumors contained transforming ras sequences in NIH3T3 transfectionassays: K-ras (17/18) or N-ras (1/18). Single-stranded conformationalpolymorphism analysis or dideoxy sequencing of polymerase chainreaction-amplified K-ras gene fragments revealed that theseneoplasms contained a variety of activating mutations in theK-ras oncogene. Alterations in codon 12 predominated and includedGGT GAT transitions, GGT GTT or TGT transversions, and previouslyreported insertion mutations, although some tumors expressedmore than one mutation and the pattern of mutations even variedwithin tumors. Mutations were also found in exons 2 and 3. Inaddition, tumor trans-plantability into syngeneic hosts correlatedpositively and significantly with K-ras activation. Renal mesenchymaltumors with transforming mutations in exon 1 were often successfullypassaged (10/12) while tumors which lacked mutations in exon1 were infrequently transplantable (2/14). While the observedbase substitutions in K-ras are consistent with adduct formation,the presence of insertion mutations and intratumor heterogeneityof alterations suggest that ras activation in DMN-OMe-inducedtumors is not necessarily an early event in tumorigenesis.     

DNA adduct formation and repair in hamster and rat tracheas exposed to benzo[a]pyrene in organ culture

Syrian golden hamsters are much more susceptible than Wistarrats to the induction of tracheal tumors by benzo[a]pyrene (B[a]P).To investigate whether this difference is reflected in the patternof DNA adduct induction and removal, tracheas from either specieswere isolated and exposed to B [a]P (5 µg/ml) in organculture. At various time- points B[a]P-DNA adducts were quantifiedby ³²P-postlabeling; unscheduled DNA synthesis (UDS) and cellproliferation were determined by [³H]thymidine incorporationduring the 18 h before sampling. In an induction-repair experimenttracheas were exposed to B[a]P for 2 days, and cultured foranother 4 days without B[a]P After 2 days of exposure totalB[a]P-DNA adduct levels were 10 times higher in hamster comparedto rat tracheas. In hamster tracheas one major adduct was formed(95%), namely the adduct between (+)-anti-benzo[a]pyrene diolepoxideand deoxy guanosine (BPDE-N²dG). In rat tracheas BPDE-N²dG comprised60% of the total B[a]P-DNA adduct level. The other major adductfound in rat tracheas is probably derived from interaction ofsyn-BPDE and deoxyadenosine. During exposure to B[a]P in hamstersthe adduct level increased to 36 ± 19 adducts/10⁶ nucleotides(add/10⁶n) on day 2. Two days after removal of B[a]P-the B[a]P-DNAadduct level had decreased to 60% of that on day 2; there wasno further decrease in the B[a]P-DNA adduct level, despite considerablecell proliferation at the end of the 6 day culture period. UDSincreased during exposure to B[a]P and decreased after removalof B[a]P. In rats removal of B[a]P did not lead to a decreasein the B[a]P adduct level, which agreed with the observed absenceof UDS. In a second experiment tracheas were exposed to B[a]Pcontinuously for 15 days. In hamster tracheas the total B[a]P-DNAadduct level increased from 11 ± 0.7 add/10⁶n after 1day of exposure to 105 ± 2 add/10 after 15 days; alsoUDS increased with increasing exposure until day 11. Cell proliferationwas low at the end of the culture period. In rat tracheas noprogressive increase in the B[a]P-DNA adduct level was seen,UDS was not increased and cell proliferation had increased significantlyat the end of the exposure period. The extent of adduct inductionin the trachea of the two species corresponded with the differentsusceptibilities to B[a]P-induced tumor formation.     

Ras oncogene activation during hepatocarcinogenesis in B6C3F1 male mice by dichloroacetic and trichloroacetic acids

Dichloroacetic (DCA) and trichloroacetic (TCA) acids, two majorby-products formed during chlorine disinfection of drinkingwater, increase the incidence of tumors in B6C3F1 mice by 6-and 3-fold respectively. In order to understand better the mechanismby which these two compounds induce liver tumors, the incidenceand spectrum of mutations in the K- and H-ras proto-oncogenesin these tumors were analyzed. DNA from spontaneous, DCA- andTCA-induced liver tumors from B6C3F1 male mice was evaluatedfor point mutations in exons 1, 2 and 3 of the two genes bysingle-stranded conformation polymorphism. Results demonstrateda similar incidence of mutations for exon 2 of H-ras in spontaneouscarcinomas (58%), and in carcinomas induced by DCA 3.5 g/l (50%),1.0 g/1(48%) and TCA 4.5 g/l (45%). Only four samples showedmutations in the other exons of H-ras or in K-ras. Sequenceanalysis of spontaneous tumor samples with second exon H-rasmutations revealed a change in codon 61 from CAA to AAA in 80%and CAA to CGA in 20% of tumors. In contrast, tumors with H-rasmutations from DCA-treated mice revealed a H-61 change fromCAA to AAA in 21 % at 3.5 g/l and 16% at 1.0 g/l. CAA to CGAwas observed in 50% of tumors from mice given DCA 3.5 or 1.0g/l, and CAA to CTA was present in 29% and 34% of the two dosagegroups respectively. Interestingly, TCA showed the same mutationalspectrum as the spontaneous liver tumors. The data indicatesthat induction of liver carcinoma by DCA and TCA involves activationof the H-ras proto-oncogene at a frequency similar to that observedin spontaneous tumors. However, the mechanism(s) for inducinghepatocellular carcinoma does not appear to be identical forDCA and TCA.     

Characterization of benzo[a]pyrene-initiated mouse skin papillomas for Ha-ras mutations and protein kinase C levels

The frequency and spectrum of Ha-ras mutations in benzo[a]pyrene(B[a]P)-initiated/12-O-tetradecnoylphorbol-13-acetate (TPA)-promotedCD-1mouse skin papillomas were characterized by amplifying highmolecular weight papilloma DNA using the polymerase chain reaction(PCR) followed by direct DNA sequencing. Analysis of 10 individualB[a]P-initiated early emergence papillomas indicated that 90%contained a Ha-ras mutation. Twenty percent of these papillomascontained a GGA