Mutations at codon 61 of the Ha-ras proto-oncogene in precancerous liver lesions of the B6C3F1 mouse

Liver tumors of the B6C3F1 mouse frequently contain mutations at specific sites of codon 61 of the Ha-ras proto-oncogene. To address whether these mutations occur early or late during carcinogenesis, we analyzed mutations in the Ha-ras gene in small precancerous liver lesions of the B6C3F1 mouse. For this purpose, 10-microns frozen liver sections were prepared and stained for glucose-6-phosphatase activity. Using punching cannuli, we then took small tissue samples of approximately 5-30 micrograms from enzyme-deficient liver lesions and from normal parts of the liver. These tissue samples were analyzed for mutations in the Ha-ras gene by in vitro amplification of DNA via the polymerase chain reaction combined with selective oligonucleotide hybridization. By this approach we were able to analyze mutations in the Ha-ras gene within lesions with diameters of less than 0.5 mm. Our results demonstrate that approximately 15% of the glucose-6-phosphatase-negative lesions that occurred 24-28 wk after a single injection of diethylnitrosamine contain either C----A transversions at the first base or A----G transitions and A----T transversions at the second base of codon 61 of the Ha-ras gene. The same types of mutations, although with a somewhat higher frequency (33%), were found in liver tumors taken 68 wk after diethylnitrosamine treatment. These findings demonstrate that Ha-ras mutations can be detected even in very small precancerous liver lesions, suggesting that these mutations may be an early, perhaps even the first, critical event during murine hepatocarcinogenesis.     

Characterization of p53 mutations in methylene chloride-induced lung tumors from B6C3F1 mice

Mutations of the p53 tumor suppressor gene are the most commondefined genetic alterations seen in a wide variety of humancancers. In contrast, little is known about the importance ofthe p53 gene in chemically induced tumors of rodents, whichare widely used as models for the evaluation of human healthrisks. In this study we examined 54 methylene chloride-inducedand seven spontaneously arising lung tumors from female B6C3F1mice for losses of heterozygosity (LOH) at markers near thep53 gene on chromosome 11. LOH was detected in seven methylenechloride-induced lung carcinomas by Southern analysis of a restrictionfragment length polymorphism and PCR analysis of five simplesequence length polymorphisms. In each case allele loss wasobserved at all six markers; thus, these chromosomal alterationswere likely to have resulted from mitotic nondisjunction. Incontrast, LOH was not detected in 20 liver tumors from methylenechloride-treated mice at the Acrb locus, which is tightly linkedto the p53 gene on chromosome 11. In addition single strandconformation polymorphism analysis was performed to screen formutations in the most conserved regions of the p53 gene (exons5 to 8). Consequently, potential mutations identified by directsequencing, were only detected in four of the seven tumor sampleswith LOH, but not in any of the remaining lung tumors. Overexpressionof the p53 protein by immuno-histochemical staining was detectedonly in the four tumors that contained p53 point mutations andin a focal area of another tumor. Finally, using a simple sequencelength polymorphism within the retinoblastoma tumor suppressorgene, LOH on mouse chromosome 14 was also detected in threelung carcinomas and one liver tumor. Inactivation of p53 andpossibly the retinoblastoma tumor suppressor gene appear tobe infrequent events in lung and liver tumors from methylenechloride treated mice.     

Incidence of mutations at codon 61 of the Ha-ras gene in liver tumors of mice genetically susceptible and resistant to hepatocarcinogenesis

By selective oligonucleotide hybridization of polymerase chain reaction (PCR) amplified tumor DNAs we have analysed the incidence of mutations at codon 61 of the Ha-ras gene in 42 liver tumors spontaneously developed in Balb/c, C3Hf and B6C3 male mice, and in 79 liver tumors induced by the chemical carcinogens diethylnitrosamine (NDEA) and urethan in B6C3 and B6C male and female mice. The incidence of Ha-ras gene mutations in both spontaneously developed and urethan-induced liver tumors was 50%-63% in mice genetically susceptible to hepatocarcinogenesis (C3Hf, B6C3) and 7%-9% in mice genetically resistant (Balb/c, B6C). Urethan-induced tumors showed about the same incidence of ras mutations in male and in female B6C3 mice. NDEA-induced tumors showed a low incidence of Ha-ras mutations in both the hybrid mice (3/18 and 1/13 in B6C3 and B6C male mice, respectively). The most frequently found mutations were a C----A transversion at the 1st base of codon 61 in spontaneous tumors, and an A----T transversion at the 2nd base in urethan-induced tumors. Our results indicate that liver tumors induced by NDEA or urethan or spontaneously arisen have a different pattern of Ha-ras mutations at codon 61 and that these mutations constitute a rare molecular alteration in the pathogenesis of liver tumors in genetically resistant mice.     

Effect of varying exposure regimens on methylene chloride-induced lung and liver tumors in female B6C3F1 mice

Methylene chloride is a high production chemical used in a varietyof applications resulting in estimated occupational and consumerexposures of at least one million people per day. Results ofpreviously reported chronic evaluations of inhaled methylenechloride indicated that it caused mammary tumors in Fischer344 rats and neoplasia in the lungs and liver of B6C3F1 mice.Mechanism(s) for methylene chloride-induced carcinogenesis havenot been adequately elucidated. In this paper we describe thehistologic evaluation of animals at a number of intermittenttimes for the purposes of assessing the progressive developmentof liver and lung neoplasia. Additionally, a series of stop-exposuretreatments was conducted to evaluate the role of different methylenechloride exposure durations on the induction of hepatic andpulmonary neoplasia in female mice. Inhalation exposure to 2000p.p.m. methylene chloride for 6 h per day, 5 days per week,for 104 weeks resulted in an 8-fold increase in the incidenceof exposed animals having a lung adenoma or carcinoma (63 versus7.5%; P < 0.01) and a 13-fold increase in the total numberof pulmonary adenomas and carcinomas per animal at risk (0.97versus 0.075; P < 0.01). This exposure also caused a 2.5-foldincrease in the incidence of mice having liver tumors (69 versus27%; P < 0.01) and a 3-fold increase in the total numberof hepatic adenomas and carcinomas per animal at risk (1.34versus 0.46; P < 0.01). Methylene chloride exposure hastenedthe first appearance of lung tumors (by 1 year) compared tothat observed in control animals; chemical-induced and spontaneousliver tumors first occurred simultaneously. A shorter exposureduration was sufficient to attain maximal numbers of lung tumorsthan that needed for a maximal liver tumor burden. Lung tumormultiplicity was substantially increased by having additionaltime after cessation of the chemical treatment. This contrastswith the findings in liver, where additional post-exposure latencytime did not effect tumor multiplicity compared to that of miceevaluated immediately after cessation of exposure. The incidenceof lung alveolar hyperplasia in methylene chloride exposed animalswas very low, even in tumor-bearing animals and the hyperplasiaswere not seen until at least 13 weeks after appearance of adenomasand carcinomas. Thus, the genesis of methylene chloride inducedlung tumors in B6C3F1 mice is not preceeded by overt cytotoxicity,enhanced cell proliferation nor observed hyperplasia. The differentialbehavior of the target organs to varying exposure regimens,suggests to us that the mechanisms responsible for its tumorigenicityare different in the lung and the liver.     

H-ras 61st codon activation in archival proliferative hepatic lesions isolated from female B6C3F1 mice exposed to the leukotriene D4-antagonist, LY171883

LY171883, a peroxisome proliferator and leukotriene D_4–antagonist,induced a statistically significant increase in the number ofhepatic lesions in B6C3F1 female mice in a 2 year oncogenicitystudy at dietary doses of 0.0225% and 0.075%. The mutation frequencyand spectrum of the 61st codon of H-ras was determined for 64independent, archived lesions from the LY171883 2 year oncogenicitystudy using the polymerase chain reaction (PCR), allele specificoligo hybridization (ASO) and DNA sequencing. Results showed41 (64%) of these lesions had mutations at the 61st codon (16/21hepatocellular carcinomas, 4/10 hepatocellular adenomas, 19/26focal hepatocellular hyperplasias and 2/7 focal hepatocellularatypia). These mutations consisted of 18 C-A transversions,16 A-G transitions and seven A-T transversions. Compared tothe mutation frequency for spontaneously occurring archivalB6C3F1 hepatic lesions (41%), the frequency of LY171883 lesions(64%) was significantly higher (P < 0.01). Hie frequenciesof H-ras 61st codon mutations among the LY171883 lesion types(hepatocellular carcinomas 76%, hepatocellular adenomas 40%,focal hepatocellular hyperplasias 73% and hepatocellular atypia29%) were also significantly different (P = 0.035). In contrast,spontaneous lesions showed no statistical difference in thefrequencies of mutation among lesion types (P > 0.5). Themutation spectrum of the LY171883 lesions was not significantlydifferent from the spontaneous spectra. It may be concludedthat based on the similarity in mutation spectrum and the increasein mutation frequency, LY171883 may selectively promote spontaneoushepatic lesions containing H-ras 61st codon mutations. In addition,the difference in mutation frequency among lesion types doesnot support a linear progression of all LY171883 lesions throughfocal atypia, focal hepatocellular hyperplasias, hepatocellularadenomas and hepatocellular carcinomas.     

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.     

Tumorigenicity of nitropolycyclic aromatic hydrocarbons in the neonatal B6C3F1 mouse bioassay and characterization of ras mutations in liver tumors from treated mice

Tryptamine is an endogenous neuroactive metabolite of tryptophan. Interpretation of the function of this bioamine, however, is restricted to manipulation with tryptamine synthetic pathways. Meanwhile, tryptamine is a potent inhibitor of protein biosynthesis, via the competitive inhibition of tryptophanyl-tRNA synthetase (TrpRS). The influence of the persistent tryptamine inhibition on the half-life and cellular content of TrpRS was examined by chase labeling of HeLa cells and the tryptamine-resistant subline with [35S]methionine. The results indicate that long-term tryptamine treatment of HeLa cells led to a significant increase in the half-life of TrpRS while the content, in vivo phosphorylation and gene dose of TrpRS were unchanged. These findings suggest that survival of drug-resistant cells may not be due to TrpRS gene amplification, but to stabilization of TrpRS. It was shown that tryptamine is an effective inhibitor of HeLa cell growth. In contrast to the well-characterized antineoplastic compounds, conferring a many hundred-fold elevated drug resistance to tumor cells, resistance to tryptamine at very low levels was difficult to achieve, i.e. the 2-fold resistant subline was selected after 19 months of treatment of HeLa cells with gradually increasing concentrations of tryptamine. The tryptamine-resistant HeLa subline exhibited a slower growth rate than the original HeLa line when similar concentrations of both cell populations were seeded on the plates. A low tryptamine resistance and a lack of TrpRS gene amplification were observed in two tryptamine-resistant HeLa sublines and three Chinese hamster sublines. The role of TrpRS in oncogenesis and the perspective for tryptamine as a potential anti-cancer drug are discussed.     

ras proto-oncogene activation in dichloroacetic add-, trichloroethylene- and tetrachloroethylene-induced liver tumors in B6C3F1 mice

The frequency and mutation spectra of proto-oncogene activationin hepatocellular neoplasms induced by tetrachloroethylene,trichloroethylene and dichloroacetic acid were examined to helpdefine the molecular basis for their carcinogenicity. H-rascodon 61 activation was not significantly different among dichloroaceticacid- and trichloroethylene-induced and combined historicaland concurrent control hepatocellular tumors (62%, 51% and 69%respectively). The mutation spectra of H-ras codon 61 mutationsshowed a significant decrease in AAA and increase in CTA mutationsfor dichloroacetic acid- and trichloroethylene-induced tumorswhen compared to combined controls. The H-ras codon 61 mutationfrequency for tetrachloroethylene-induced tumors was significantlylower (24%) than that of combined controls and also that ofthe two other chemicals. Mutations at codons 13 and 117 plusa second exon insert contributed 4% to the total H-ras frequenciesfor trichloroethylene and tetrachloroethylene. There was alsoa higher incidence of K-ras activation (13%) in tetrachloroethylene-inducedtumors than in the other chemically induced or control tumors.Four liver tumors were found to contain insertions of additionalbases within the second exon of K- or H-ras. These findingssuggest that exposure to dichloroacetic acid, trichloroethyleneand tetrachloroethylene provides a selective growth advantageto spontaneously occurring mutations in codon 61 of H-ras and,at the same time, is responsible for a small number of uniquemolecular lesions suggestive of either a random genotoxic modeof action or a non-specific result of secondary DNA damage.However, the absence of ras activation in many of the liverneoplasms suggests that alternative mechanisms are also importantin B6C3F1 mouse hepatocarcinogenesis.     

Ras mutations in methyiclofenapate-induced B6C3F1 and C57BL/1OJ mouse liver tumours

The majority of genotoxic carcinogen-induced liver tumours ofthe sensitive B6C3F1 mouse contain activated H-ras oncogenes.Such mutations also occur in hepatocarcinogenesis resistantstrains. In order to determine whether this is true of non-genotoxiccarcinogen-induced tumours, liver tumours induced in B6C3F1and C57BL/10J mice by methylclofena pate (MCP) were compared.Polymerase chain reaction (PCR) analysis revealed H-ras codon61 mutations in 11/46 B6C3F1 and 4/31 C57BL/10J liver tumours.The nude mouse tumorigenicity (NMT) assay was used to analysetumours without codon 61 mutations. Of the 12 B6C3F1 liver tumourDNAs subjected to this assay, one contained a H-ras codon 117mutation. Further PCR analysis on frozen tumour samples (46B6C3F1 and 15 C57BL/10J) revealed no codon 12 mutations; oneadditional codon 117 mutation was identified in a B6C3F1 tumour.Overall, then, H-ras codon 61 mutations were detected in MCP-inducedB6C3F1 tumours less frequently than in genotoxin-induced tumours.Two B6C3F1 tumours contained codon 117 mutations similar tothose previously found in tumours induced by ciprofibrate, furanand furfural, and in at least one spontaneous tumour. Ras mutationswere also detected in some C57BL/10J tumours, providing furtherevidence that ras oncogenes can participate in hepatocarcinogenesisin resistant mice.     

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     

Search for Ha-ras codon 61 mutations in liver tumours caused by hexachlorobenzene and Aroclor 1254 in C57BL/10ScSn mice with iron overload.

C57BL/10ScSn mice administered iron--dextran and fed the environmental pollutants hexachlorobenzene (HCB) and polychlorinated biphenyls (PCBs) develop hepatic nodules and carcinomas within 18 months. A range of lesions from the livers were analysed for the presence of mutations in the Ha-ras proto-oncogene at codon 61 using the polymerase chain reaction to amplify DNA from formalin-fixed sections, followed by oligonucleotide hybridization. Only two mutations from 23 preneoplastic and neoplastic lesions induced by HCB were detected (a focus of altered cells and a trabecular cell carcinoma). With Aroclor 1254 no mutations were detected in 28 areas at various stages of carcinogenesis analysed. Sequencing of the two mutations generated by HCB showed a C-->T transversion at the first base of codon 61 (carcinoma) and an A-->T transversion at the second base (proliferative focus). Thus, in marked contrast to some other systems of mouse liver tumour induction, hepatocarcinogenesis caused by HCB and PCBs in C57BL/10ScSn mice is an example of carcinogenesis which does not involve a high frequency of Ha-ras gene mutation at codon 61.     

Methyl tertiary butyl ether lacks tumor-promoting activity in N-nitrosodiethylamine-initiated B6C3F1 female mouse liver

Methyl tertiary butyl ether (MTBE) is an additive in some formulationsof unleaded gasoline (UG) that enhances octane and reduces carbonmonoxide emissions from motor vehicles. MTBE in CD-I mice andUG in B6C3F1 mice increased the incidence of liver tumors selectivelyin female mice in their chronic bioassays. Both agents werenegative in in vitro tests of genotoxicity, and exhibit similarhepatic microsomal cytochrome P450 activity and hepatocyte proliferationafter short-term exposure. We previously demonstrated that UGhas hepatic tumor-promoting activity in DEN-initiated femaleB6C3F1 mice. Thus, we hypothesized that MTBE would have hepatictumor-promoting activity in the same initiation-promotion modelsystem in which UG was a hepatic tumor promoter. Twelve-day-oldfemale B6C3F1 mice were initiated with a single i.p. injectionof the mutagen N-nitrosodiethylamine (DEN) (5 mg DEN/kg, 7.1ml/kg body weight) or saline. Beginning at 8 weeks of age, micewere exposed to 0 ppm or the hepatocarcinogenic dose of approximately8000 ppm MTBE. After subchronic exposure, MTBE significantlyincreased liver weight and hepatic microsomal cytochrome P450activity without hepa-totoxicity or an increase in non-focalhepatocyte DNA synthesis. These are subchronic effects similarto those produced by UG. However, MTBE did not significantlyincrease the mean size of hepatic foci and volume fraction ofthe liver occupied by foci as compared to DEN-initiated controlsat either 16 or 32 weeks. The lack of tumor-promoting abilityof MTBE in DEN-initiated female mouse liver was unexpected andsuggests that MTBE does not produce liver tumors through a tumor-promotingmechanism similar to that of UG.     

Comparison of effect of tumor promoter treatments on DNA methylation status and gene expression in B6C3F1 and C57BL/6 mouse liver and in B6C3F1 mouse liver tumors

The effects of different liver tumor-promoting treatments (i.e., a choline-devoid, methionine-deficient (CMD) diet, phenobarbital (PB), or both) on Ha-ras and raf methylation status and expression were determined in mouse strains with different susceptibilities to liver tumor formation: the relatively sensitive B6C3F1 and the relatively resistant C57BL/6. Additionally, B6C3F1 mouse liver tumors, spontaneous or PB induced, were assessed for alterations in global DNA methylation status and expression of Ha-ras and raf. The CMD diet led to hypomethylation of Ha-ras and raf after 12 wk of administration in B6C3F1 and C57BL/6 mice. At this early phase of tumor promotion, the frequency of increased expression of both Ha-ras and raf mRNAs was higher in the B6C3F1 but not the C57BL/6 mice. This is a mechanism that may, in part, underlie the heightened sensitivity of the B6C3F1 mouse to liver tumorigenesis. Subpopulations of B6C3F1 mouse liver tumors displayed altered global methylation status, with both hypomethylation and hypermethylation evident. Carcinomas were significantly more hypomethylated than adenomas. The level of raf mRNA was not changed in spontaneous or PB-induced B6C3F1 mouse liver tumors. Increased expression of Ha-ras was evident in some spontaneous B6C3F1 liver tumors and in most of the PB-induced liver tumors. These experiments support the concept that altered DNA methylation plays a key role in tumorigenesis and indicate that the high propensity of the B6C3F1 mice to liver tumorigenesis may be due, in part, to a decreased ability to maintain normal methylation status. Mol. Carcinog. 18:97–106, 1997. © 1997 Wiley-Liss, Inc.     

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.     

Spontaneous and urethan-induced tumor incidence in B6C3F1 versus B6CF1 mice

The incidences of spontaneous tumors of the murine hybrids (C57BL/6J X C3Hf)F1 (B6C3F1) and (C57BL/6J X BALB/c)F1 (B6CF1) were compared in untreated mice kept until 110 weeks of age. Male B6C3F1 and B6CF1 mice had respectively 16% and 20% incidence of lymphomas, 26% and 4% of liver tumors and 12% and 22% of lung tumors. Among B6C3F1 and B6CF1 females, a 36% and 12% incidence of lymphomas, a 6% and zero incidence of liver tumors, and a 4% and 16% of lung tumors were observed. A few other tumors were seen in both hybrids. Groups of male and female mice of the 2 hybrids received 5 i.p. injections of 1000 mg/kg urethan once every other day starting at 10 days of age, and were kept under observation until 65-80 weeks of age. Treated B6C3F1 mice had an earlier mortality than B6CF1 mice due to tumor development. The statistical analysis, allowing for survival, showed a significantly higher lymphoma incidence in male and female B6C3F1 than B6CF1 mice, which had instead a higher incidence of lung tumors. Hepatocellular tumors were seen in both sexes of the 2 hybrids, with a higher frequency in B6C3F1 mice. Male mice of both hybrids had a higher incidence of liver tumors than females.     

Carcinogenicity of catechol in F344 rats and B6C3F1 mice

Carcinogenicity of catechol, a naturally occurring and industrialchemical which has been shown to have strong cell proliferatingpotential on rat glandular stomach epithelium, was investigatedin male and female F344 rats and B6C3F₁ mice. Groups of 30 maleand female F344 rats and B6C3F₁ mice were treated with 0.8%catechol in powdered diet continuously for 104 weeks (rats)or 96 weeks (mice). At necropsy, neoplastic lesions were observedmainly in the glandular stomach of both species. Adenomas werefound in all rats and in the majority of mice: 29 out of 30(97%) in males and 21 out of 29 (72%) females. In addition 15out of 28 (54%) and 12 out of 28 (43%) of the male and femalerats respectively, had well differentiated adenocarcinomas.No adenocarcinomas were found in mice of either sex. In theforestomach epithelium, although significant increase in papillomadevelopment was not evident, incidences of squamous cell hyperplasiawere significantly increased in rats and mice of both sexes.In other organs examined, incidence and numbers of liver hyperplasticfoci per cm² liver section were significantly lower in malerats. Although the incidence was not different, the numbersof hyperplastic foci were also significantly reduced in femalerats. Thus the present experiment clearly demonstrated thatcatechol exerts carcinogenic activity in rodent glandular stomachepithelium.     

Detection of activated proto-oncogenes in N-nitrosodiethylamine-induced liver tumors: a comparison between B6C3F1 mice and Fischer 344 rats

DNA from B6C3F₁ mouse and Fischer 344 rat liver tumors inducedby N-nitrosodiethylamine (DEN) were examined for the abilityto induce morphological transformation of NIH3T3 cells. DNAsfrom 14 of 33 of the mouse liver tumors induced by a singleinjection of DEN at 12 or 15 days of age were positive in thisassay while DNA from only one of 28 DEN-induced rat liver tumorswas active. Southern blot analysis of the NIH3T3 transformantsderived from the mouse liver tumors revealed amplified and/orrearranged restriction fragments homologous to the H-ras proto-oncogene.DNA from two independent foci induced by the rat tumor DNA didnot hybridize to probes for members of the ras gene family orc-raf. Activating mutations in the H-ras genes from the DEN-inducedmouse liver tumors were characterized by selective oligonucleotidehybridization and the detection of a new XbaI restriction siteby Southern blot analysis. In activated H-ras genes from theDEN-induced mouse liver tumor DNA, seven of 14 had a CGAT transversionat the first base of the 61st codon, three of 14 had an ATGCtransition and four of 14 had the ATTA transversion at the secondbase of codon 61. This spectrum of mutations is very similarto that recently observed in activated H-ras genes found inspontaneously occurring B6C3F₁ mouse liver tumors. Taken together,the data suggest that the DEN-induced rat and mouse liver carcinogenesismay involve genetic targets other than or in addition to theH-ras gene.     

Beta-catenin mutations and protein accumulation in all hepatoblastomas examined from B6C3F1 mice treated with anthraquinone or oxazepam

The molecular pathogenesis of hepatoblastomas in the B6C3F1 mouse is unclear but may involve alterations in the beta-catenin/Wnt signaling pathway as was recently described for chemically induced hepatocellular neoplasms and human liver cancers. The objective of this study was to characterize the mutation frequency and spectrum of beta-catenin mutations and the intracellular localization of beta-catenin protein accumulation in chemically induced hepatoblastomas. In this study, beta-catenin mutations were identified in all 19 anthraquinone-induced hepatoblastomas and all 8 oxazepam-induced hepatoblastomas examined. Although several hepatoblastomas had multiple deletion and/or point mutations, the pattern of mutations in the hepatoblastomas did not differ from that identified in hepatocellular neoplasms. In a majority of the hepatoblastomas (six of seven) examined by immunohistochemical methods, both nuclear and cytoplasmic localization of beta-catenin protein were detected, whereas in hepatocellular adenomas, carcinomas, and normal liver only membrane staining was observed. Our data suggest that beta-catenin mutations and the subsequent translocation of beta-catenin protein from the cell membrane to the cytoplasm and nucleus may be critical steps in providing hepatocellular proliferative lesions with the growth advantage to progress to hepatoblastoma.     

Altered gene expression in spontaneous hepatocellular carcinomas from male B6C3F1 mice

In this study, we analyzed spontaneous hepatocellular carcinomas (HCCs) from male B6C3F1 mice for alterations in the expression of the genes for c-myc, insulin-like growth factor II (IGF-II), cyclin D1, transforming growth factor-α (TGF-α), and the epidermal growth factor receptor (EGFR). These genes are all important in growth control in the rodent liver, and therefore, alterations in these genes or their products may result in unregulated growth. Northern blot analysis demonstrated an increase in expression of c-myc mRNA in five of 21 (24%) spontaneous HCCs compared with nontumor tissue. Tumors that had an increase in c-myc mRNA did not have an amplified c-myc gene. Of the HCCs analyzed, 18 of 29 (62%) showed reexpression of IGF-II RNA when compared with controls. Cyclin D1 mRNA was overexpressed in seven of 27 (26%) of the tumors analyzed relative to controls. Tumors with an increase in cyclin D1 mRNA also overexpressed the cyclin D1 protein. RNA encoding for the EGFR was decreased in 21 of 23 (91%) HCCs when compared with controls. None of the 29 liver tumors analyzed for alterations in expression of TGF-α mRNA differed from controls. Also, each individual tumor had a unique set of molecular alterations even when different tumors from the same animal were analyzed. These novel findings suggest that IGF-II, cyclin D1, c-myc, and EGFR are important mediators of carcinogenesis in spontaneous mouse liver tumor formation. Mol. Carcinog. 19:31–38, 1997. © 1997 Wiley-Liss, Inc.     

Effect of phenobarbital on diethylnitrosamine and dimethylnitrosamine induced hepatocellular tumors in male B6C3F1 mice

The effect of the type of carcinogen initiator on the ability of phenobarbital (PB) to promote hepatic tumor formation in 15-day-old initiated male B6C3F1 mice was evaluated. Fifteen-day-old male B6C3F1 mice were divided into 6 groups of 10 mice each. Groups 1 and 2 received a single intraperitoneal (i.p.) injection of diethylnitrosamine (DENA) (5 micrograms/body wt). Groups 3 and 4 received a single i.p. injection of diethylnitrosamine (DENA) (5 micrograms/g body wt). Groups 3 and 4 received a single i.p. injection of dimethylnitrosamine (DMNA) (5 micrograms/g body wt). Groups 5 and 6 received a single i.p. injection of saline. At weaning (28 days of age), mice in groups 2, 4 and 6 received PB (500 mg/ml) in their drinking water. Mice in groups 1, 3 and 5 received deionized drinking water. Drinking water treatment continued for 24 weeks at which time mice were sampled. At sampling, mice were examined for hepatic tumors by histology. Mice in groups 5 (no treatment) and 6 (PB only) did not exhibit hepatic tumors. Groups 2 (DENA + PB) displayed a decrease in hepatic adenomas from that of group 1 (DENA only), confirming previous observations. Treatment with DMNA and PB (group 4), however, resulted in a significant increase in both hepatic adenoma incidence and number over that of DMNA only (group 3) treated mice. The promoted adenomas appeared to be predominantly eosinophilic in appearance. The type of initiator therefore appears important in determining if 15-day-old initiated male B6C3F1 mice respond to the promotion effects of PB.