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.     

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.     

Molecular analysis of lacI mutants from bone marrow of B6C3F1 transgenic mice following inhalation exposure to 1,3-butadiene

Chronic exposure to 1,3-butadiene (BD) results in early occurrenceand high incidence of lethal lymphomas in male B6C3F1 mice.Male B6C3F1 lacI transgenic mice (BigBlue^TM) were exposed byinhalation to 0, 62.5, 625 or 1250 p.p.m. BD for 4 weeks (6h/day, 5 days/week). The lacI^- mutant frequency and mutationalspectrum were evaluated in DNA isolated from bone marrow cells.Two weeks after exposure the lacI^- mutant frequency in bonemarrow from BD-exposed mice had increased 2- to 3.5-fold overair control mice. DNA sequence analysis of 56 and 54 lacI^- mutantsfrom the air control and butadiene-exposed groups, respectively,demonstrated that there was a shift in the spectrum of basesubstitution mutations at A:T sites in BD-exposed mice (6/26)compared to the air control mice (2/45). A:T     

The role of hyperplastic nodules in dichloroacetic acid-induced hepatocarcinogenesis in B6C3F1 male mice

Dichloroacetic acid (DCA) has recently been shown to increase significantly the incidence of hepatic adenomas (HAs) and hepatocarcinomas (HCs) in male B6C3F1 mice. Although little is known about the mechanism of DCA carcinogenesis, chronic ingestion of the compound in drinking water induces primarily hyperplastic nodules (HNs) prior to the appearance of HAs and HCs. Given the putative preneoplastic potential of the HNs, we undertook this study to determine the role of the HNs in the progression of DCA-induced hepatocarcinogenesis. This role was assessed by detecting the expression of five different tumor markers: p21 ras, p39 c-jun, phosphotyrosine, tumor-associated aldehyde dehydrogenase and alpha-fetoprotein, all known from previous studies to be expressed more often in neoplastic liver lesions than in normal liver. Tumor marker expression was detected by immunohistochemical methods using formalin-fixed, paraffin-embedded sections of normal B6C3F1 mouse liver, and DCA-induced HNs, HAs and HCs. The results demonstrated that, except for the c-jun marker, HNs expressed the markers significantly less often than either HAs or HCs. Equal expression of c-jun occurred in any of the three lesion types. Although these results could be used to argue that no relationship existed between HNs and later-appearing HAs and HCs, those HNs that were marker positive contained small nests of marker-positive hepatocytes among a field of normally appearing unstained hepatocytes. No similar nests of marker-positive cells were detected in any area of normal liver outside the HNs. Also very few altered hepatic foci (AF) were detected with these markers or with hematoxylin and eosin, or with histochemical stains for ATPase or glucose-6-phosphatase deficiencies. These results suggested that these nests within some HNs were areas of transformed, or neoplastic hepatocytes. Phenotypic heterogeneity analysis, in which the number of tumor markers co-expressed by any given lesion was examined, confirmed a significantly greater percentage of HAs and HCs expressing multiple markers than HNs. Those HNs that expressed multiple markers, however, expressed at the same frequency as HAs and HCs and the expression was confined to the same nests of cells. Taken together, these data suggest that these nests of marker-positive cells within the HNs were neoplastic and could develop into later-appearing HAs and/or HCs. The absence of marker expression in normal liver and limited expression in the few AF indicates that the HNs may be the only significant preneoplastic lesion in DCA-induced 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.     

Evaluation of dichloromethane as an inducer of DNA synthesis in the B6C3F1 mouse liver

The potential of the mouse hepatocarcinogen dichloromethane(DCM) to induce hepatocellular division, as monitored by increasedDNA synthesis, has been evaluated using B₆C₃F₁ mice—thestrain in which it is carcinogenic but not apparently genotoxic.Male mice were exposed to DCM either by oral gavage in cornoil (1000 mg/kg) or by inhalation of an atmosphere containing4000 p.p.m. DCM for 2 h. Cells undergoing DNA synthesis (S-phase)were radiolabelled by means of four consecutive i.p. injectionsof tritiated thymidine at hourly intervals prior to killing.No evidence of S-phase activity was observed in the gavage studies.The inhalation studies resulted in some weak, but statisticallysignificant increases in S-phase incidence, but the biologicalsignificance was unclear due to similar increases being observedin some control groups. It is concluded that DCM does not sharethe mitogenic properties of such presumed non-genotoxic mouseliver carcinogens as trichloroethylene, polybrominated biphenylsand carbon tetrachloride, and as such its carcinogenicity tothe mouse liver remains mechanistically obscure.     

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.     

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.     

Loss of tumor-promoting activity of unleaded gasoline in N- nitrosodiethylamine-initiated ovariectomized B6C3F1 mouse liver

Unleaded gasoline (UG) vapor (2056 ppm) increased the incidence of liver tumors in a chronic bioassay and exhibited tumor-promoting activity in N-nitrosodiethylamine (DEN)-initiated female mouse liver. Estrogen inhibited mouse liver tumor development and the hepatocarcinogenic and tumor-promoting dose of UG produced uterine changes suggestive of estrogen antagonism. To directly test the hypothesis that UG-induced tumor-promoting ability is secondary to its interaction with the mouse liver tumor inhibitor, estrogen, we compared the tumor-promoting ability of UG in ovariectomized (Ovex) mice with the hepatic tumor-promoting ability of UG in intact mice. Ovaries were surgically removed at 4 weeks of age. Exposure to wholly vaporized UG (2018 ppm) under bioassay and tumor-promoting conditions began at 8 weeks of age. After 4 months of exposure, UG increased relative liver weight and hepatic microsomal cytochrome P450 pentoxyresourfin-O- dealkylase and ethoxyresorufin-O-deethylase activity to a similar extent in intact and Ovex mice. Non-focal hepatocyte proliferation, as measured by the incorporation of bromo-deoxyuridine, was not changed by UG exposure and was similar in all treatment groups. After 4 months of exposure to DEN-initiated mice, UG significantly increased the volume fraction of liver occupied by foci (three-fold) as compared to control intact mice. As expected, volume of foci was elevated in DEN/Ovex/control mice as compared to DEN/intact/control mice. In DEN/Ovex mice UG did not significantly increase the focal volume fraction. Thus, the tumor promoting activity of UG, as demonstrated by increased volume fraction of liver occupied by hepatic foci in intact mice, is greatly attenuated in Ovex mice. The volume fraction data in Ovex mice support the hypothesis that the tumor promoting activity of UG is dependent upon the interaction of UG with ovarian hormones. These data also indicate that hepatic microsomal cytochrome P450 PROD and EROD induction, hepatomegaly and non-focal hepatic LI are not specific markers of hepatic tumor promoting activity of UG.      

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.     

Dichloroacetic acid reduces Ha-ras codon 61 mutations in liver tumors from female B6C3F1 mice

Dichloroacetic acid (DCA), a disinfection by-product of chlorination found in drinking water, is a hepatocarcinogenic in the B6C3F1 mouse. Previous studies have shown that DCA does not significantly alter the incidence of Ha-ras codon 61 mutations in male mouse liver carcinomas from that observed in spontaneous tumors (approximately 50% have Ha-ras mutations) but it alters the proportions of mutations that occur in Ha- ras codon 61. Twenty-two tumors were produced in female B6C3F1 mice after treatment with 3.5 g DCA per liter of drinking water over a period of 104 weeks. To detect potential Ha-ras mutations in the liver tumor tissue of female B6C3F1 mice, genomic DNA was isolated from tumors that had been frozen. The polymerase chain reaction (PCR) and single-stranded conformational polymorphism (SSCP) was used to screen tumor DNA for mutations in Ha-ras exon 2. In DNA from liver tumors in female B6C3F1 mice induced by DCA-treatment we found only one mutation in exon 2 among the 22 tumors analyzed (4.5%). Direct-sequencing of exon 2 revealed a CAA to CTA transversion in Ha-ras codon 61. The result of this study indicates that tumor formation in DCA-treated female B6C3F1 mice is, therefore, not associated with a mutationally activated Ha-ras codon 61. This result differs from previous results obtained in male B6C3F1 mice.      

Promotion of preneoplastic lesions and induction of CYP2B by unleaded gasoline vapor in female B6C3F1 mouse liver

An initiation-promotion protocol was used to test the hypothesisthat unleaded gasoline (UG) vapor acts as a liver tumor promoterin female mice under exposure conditions in which UG was hepatocarcinogenicin a cancer bioassay. Twelve day old female B6C3F1 mice wereinjected with N-nitrosodiethylamine (DEN, 5 mg/kg, i.p.) orvehicle. Starting at 5–7 weeks of age, mice were exposedby inhalation 6 h/day, 5 days/week for 13 weeks to 0 or 2039p.p.m. of PS-6 blend UG, the same gasoline blend used in thecancer bioassay. Putative preneoplastic lesions in liver, characterizedmainly as basophilic foci in H&E-stained liver sections,were found exclusively in mice treated with DEN. While similarnumbers of altered hepatic foci were found in DEN-initiatedmice treated with 0 or 2039 p.p.m. UG, UG treatment significantlyincreased both the mean volume (3.2-fold) and the volume fraction(3.6-fold) of the foci. To determine if UG induced CYP2B, asubfamily of cytochrome P450 commonly induced by liver tumorpromoters in rodents, pentoxyresorufin-O-dealkylase (PROD) activitywas assayed in hepatic microsomes derived from the above livers.UG vapor increased hepatic PROD activity 8-fold, while increasingcytochrome P450 content only 30%. To ascertain if a more recentblend of UG, API 91-1, would have similar biological effectsas PS-6, female B6C3F1 mice were gavaged for 3 days with cornoil or 1800 mg/kg/day PS-6 or API 91–1 blend UG. PS-6and API 91-1 blend UG induced similar increases in relativeliver weight (25%), PROD activity (9-fold) and hepatocyte labelingindex (8-fold) relative to controls. These data demonstratethat PS-6 blend UG vapor promotes preneoplastic lesions andinduces CYP2B in female mouse liver under exposure conditionsin which it causes liver tumors, and suggest that a more recentblend of UG may have similar effects.     

Alterations in the methylation status and expression of the raf oncogene in phenobarbital-induced and spontaneous B6C3F1 mouse liver tumors

The liver tumor-prone B6C3F1 mouse (C57BL/6 ♂ × C3H/He♀), in conjunction with the more susceptible C3H/He paternal strain and the resistant C57BL/6 maternal strain, is an excellent model for studying the mechanisms involved in carcinogenesis. The study reported here indicated that the B6C3F1 mouse inherited a maternal raf allele containing a methylated site not present in the paternal allele. Seven days after partial hepatectomy or after administration of a promoting dose of phenobarbital (PB) for 14 d; raf in B6C3F1 mouse liver was hypomethylated. The additional methylated site in the allele inherited from C57BL/6 was not maintained. The methylation status of raf in the liver of the C57BL/6 mouse was not affected by PB treatment. This indicates that the B6C3F1 mouse is less capable of maintaining methylation of raf than the C57BL/6 strain is. In both PB-induced and spontaneous B6C3F1 liver tumors, raf was hypomethylated in a nonrandom fashion. The level of raf mRNA increased in seven of 10 PB-induced tumors but in only one of five spontaneous tumors, whereas the level of Ha-ras mRNA increased in nine of 10 PB-induced tumors and in four of five spontaneous tumors. The results of our investigation (a) support the hypothesis that hypomethylation of DNA is a nongenotoxic mechanism involved in tumorigenesis, (b) support the notion that PB promotes liver tumors that develop along a pathway different from that leading to spontaneous tumors, and (c) indicate that differences in DNA methylation between C57BL/6 and B6C3F1 mice could, in part, account for the unusually high tendency of the latter strain to develop liver tumors. © 1994 Wiley-Liss, Inc.     

Loss of heterozygosity in spontaneous and chemically induced tumors of the B6C3F1 mouse

The B6C3F1 mouse is used worldwide to gauge the carcinogenichazard posed by chemicals to humans. An assessment of the abilityof this rodent model to predict human neoplasia requires anevaluation of similarities and differences in the genetics oftumor formation between these two species. We examined 142 spontaneousand chemically-induced liver tumors isolated from the B6C3F1mouse for losses of heterozygosity (LOH) at 78 polymorphic lociand compared these results to genetic changes known to occurin human hepatocellular carcinoma. Approximately a third ofthe 142 mouse tumors exhibited LOH, suggesting that tumor suppressorgene inactivation may be involved in the formation of mouseliver tumors. Most of the LOH observed was restricted to sevenchromosome sites and most of the tumors that underwent LOH lostalleles from only one of those seven sites. The relatively fewlosses seen in these mouse tumors distinguished them from clinicalstage human tumors in that, in the mouse tumors, interstitialdeletions appeared more frequently than losses of whole chromosomes.Only four mouse tumors lost a whole chromosome. LOH occurredat loci of the mouse genome syntenic to areas of the human genomeknown to harbor the Wilms', retinoblastoma, APC, MCC and DCCtumor suppressor genes; these genes have never been associatedwith hepatocellular carcinomas. Losses observed on chromosomes5 and 8 (syntenic to human chromosomes 4 and 16) suggest tumorsuppressor genes that are common to hepatocellular carcinomasfrom both species, while losses on chromosome 9 suggest involvementof a previously unidentified tumor suppressor gene.     

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.     

Activation of H-ras oncogenes in male B6C3F1 mouse liver tumors induced by vinthionine or 2-chloroethyl methyl sulfide

Vinthionine (S-vinyl-DL-homocysteine) is hepatocarcino-genicin rats and mice. [Vinyl-¹⁴C]vinthionine binds cova-ently torat liver DNA, RNA and protein in vivo, but not in vitro. Thisamino acid is directly mutagenic in Salmonella typhimurium TA100and TA1535; the mechanism of its metabolic activation in vivoin bacteria and liver is under study. In the present study livertumors were induced in 12-day-old male B6C3F1 mice by singlei.p. injections of vinthionine or the alkylating agent 2-chloroethylmethylsulfide (CEMS).At 10 months the gross tumors were examinedfor the presence of activated H-ras oncogenes. DNA was isolatedfrom single tumors per mouse from 37mice treated with vinthionineand from 31 mice treated with CEMS. These DNAs were screenedfor codon 61 mutations by restriction fragment length polymorphismof PCR-amplified H-ras gene fragments. Thirty seven of 37 vinthionine-inducedhepatomas had H-ras mutations in this codon, which consistedof seven C     

H-ras oncogene mutation spectra in B6C3F1 and C57BL/6 mouse liver tumors provide evidence for TCDD promotion of spontaneous and vinyl carbamate-initiated liver cells

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent environmentaltoxin which has been found to be non-genotoxic in short termin vitro tests but strongly carcinogenic in two stage modelsof hepatocellular carcinogenesis in female rats. Many recentstudies have shown that after treatment of mice with variousgenotoxic or non-genotoxic compounds, the H-ras oncogene mutationalpatterns exhibited by hepatocellular tumors appear to vary specificallywith the chemical. To gain insight into the mechanism of TCDD-associatedcarcinogenesis, susceptible B6C3F1 mice and resistant C57BL/6mice were treated with a single dose of vinyl carbamate (VC)or vehicle, and TCDD was administered once every 2 weeks for1 year to half of the animals in each group. Liver tumor prevalencewas assessed and found to be highest in the VC+TCDD treatmentgroups, reaching nearly 100% at 600 days in both sexes and bothstrains of mice. DNA was isolated from 20 or more frozen livertumors (if available) from each exposure group and analyzedfor H-ras mutations in codon 61 by sequencing after PCR amplificationof exon 2. Fifty-one percent of tumors analyzed from B6C3F1mice treated with TCDD alone had H-ras codon 61 mutations witha pattern similar to that detected in spontaneous tumors. Seventy-eightpercent of tumors from B6C3F1 mice treated with both VC andTCDD had codon 61 mutations, and most mutations were A     

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.