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     

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.     

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.     

Absence of gamma-glutamyltranspeptidase activity in lung metastasis in rats with hepatocellular carcinomas induced by ciprofibrate, a peroxisome proliferator

The incidence of lung metastasis in rats with hepatocellular carcinomas (HCC) induced by ciprofibrate, a peroxisome proliferator and the expression of gammaglutamyl transeptidase (GGT) in the metastatic lesions was studied. HCC were induced in 75 male F-334 rats by chronic dietary administration of ciprofibrate (0.025% w/w) for 16-22 months. The incidence of lung metastasis was 25% in rats killed between 14 and 16 months which increased to 56% in rats killed between 20 and 22 months. The lung metastases were multifocal and present in both the blood vessels and parenchyma. All the metastatic foci examined for the expression of GGT by histochemical stain were devoid of this enzyme. The results of this study clearly demonstrate the malignant behavior of ciprofibrate-induced liver tumors and the absence of GGT expression in metastatic lesions a phenotypic property that is peculiar to the primary HCC induced by several peroxisome proliferators.     

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.     

In vivo distribution of a carcinogenic hepatic peroxisome proliferator: whole-body autoradiography of [14C]ciprofibrate in the mouse

The distribution of radiolabel in male mice was studied by whole-bodyautoradiography at intervals after oral administration of [¹⁴C]ciprofibrate,a carcinogenic hepatic peroxisome proliferator. Radioactivitywas rapidly taken up by the liver and to a lesser extent bythe brown fat within 9 h after oral dosing of ciprofibrate.The radioactivity levels in blood, interstitial fluid and fatdecreased during the first 3 days after dosing, but the liverremained densely labeled. Between 3 and 27 days after dosing,liver exhibited a stippled pattern as a result of heavier labelingapparently around the central veins. The relatively low levelsof radiolabel in extra-hepatic tissues observed after oral dosing,together with the prolonged retention in this region of theliver, is consistent with the hepatotropic effects (i.e. hepaticperoxisome proliferation and development of liver tumors) exertedby this compound.     

Activation of protooncogenes in spontaneously occurring non-liver tumors from C57BL/6 x C3H F1 mice

The C57BL/6 x C3H F1 (hereafter called B6C3F1) mouse is an important animal model for long-term carcinogenesis studies. Maintained under normal laboratory conditions, these mice develop various types of spontaneous tumors during their lifetime. Activated Ha-ras genes have been detected in 66% of spontaneous hepatocellular tumors in the B6C3F1 mouse [Reynolds et al., Science (Washington DC), 237:1309, 1988]. In this study 49 spontaneous non-liver tumors were investigated for oncogene activation by DNA transfection techniques. Of the 49 tumor DNAs analyzed, only 5 yielded multiple foci in the NIH 3T3 focus assay: 2 of 10 pulmonary adenocarcinomas; 0 of 25 lymphomas; 2 of 2 Harderian gland adenomas; 0 of 1 adenocarcinoma of the small intestine; 1 of 6 malignant skin tumors; 0 of 4 hemangiosarcomas; and 0 of 1 lung metastasis of a hepatocellular carcinoma. DNA from six lymphomas which were negative in the NIH 3T3 focus assay were further analyzed for transforming genes by the nude mouse tumorigenicity assay. One of the five lymphomas tested positive with this assay. Southern blot analysis identified five activated ras genes: H-ras in two Harderian gland adenomas; K-ras in one pulmonary adenocarcinoma and in one s.c. adenocarcinoma; and N-ras in one lymphoma. The mutations involved were CG to AT and AT to TA in codon 61 of the Ha-ras genes, GC to AT or TA in codon 12 of the K-ras genes, and a GC to AT mutation in codon 12 of the N-ras gene. Transformant DNA from a pulmonary adenocarcinoma which yielded multiple foci in the transfection assay did not hybridize to DNA probes specific for the K-, H-, and N-ras, raf, neu, and met genes. Thirteen additional tumor DNAs yielded a single focus in the NIH 3T3 transfection assay. The transformant DNAs retransmitted in a second cycle transfection assay. Rearranged and/or amplified raf genes were detected in six of the transformant DNAs. At present we do not know whether these activated raf genes were present in the original tumor DNA. The other seven transformant DNAs did not hybridize with any of the above mentioned specific DNA probes utilized in Southern blot analysis. Unlike liver tumors, the activation of ras protooncogenes is not a frequent event in the development of spontaneous non-liver tumors of the B6C3F1 mouse. The results from this study should aid in understanding the neoplastic development associated with exposure to chemical carcinogens in the B6C3F1 mouse.     

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.     

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.     

Suppression of apoptosis in C3H mouse liver tumors by activated Ha-ras oncogene

Liver tumors were induced in male C3H mice by a single injection of N-nitrosodiethylamine and characterized with respect to the presence of base substitutions in the hot-spot position at codon 61 of the Ha-ras proto-oncogene. An increase in Ha-ras mutation prevalence was found with time after induction of tumors, suggesting that the activated ras gene provides a selective growth advantage. However, no significant differences in 5-bromodeoxyuridine labeling indices were evident between ras mutated and ras wild-type tumors, demonstrating that cell division rates in the two tumor populations were very similar. Apoptotic indices were determined by counting eosinophilic apoptotic bodies. The frequency of occurrence of apoptotic bodies was found to be approximately five times lower in tumors with Ha-ras mutations when compared with tumors not showing the mutation. This demonstrates that the activated p21(Ras) protein has anti-apoptotic activity in transformed mouse hepatocytes in vivo and suggests that the preferential outgrowth of Ha-ras-mutated hepatoma cells is mediated by suppression of apoptosis rather than by stimulation of cell division.     

Assessment of the mutagenicity of dichloroacetic acid in lacI transgenic B6C3F1 mouse liver

Dichloroacetic acid (DCA) is a chlorination byproduct found in finished drinking water. When administered in drinking water this chemical has been shown to produce hepatocellular adenomas and carcinomas in B6C3F1 mice over the animal's lifetime. In this study, we investigated whether mutant frequencies were increased in mouse liver using treatment protocols that yielded significant tumor induction. DCA was administered continuously at either 1.0 or 3.5 g/l in drinking water to male transgenic B6C3F1 mice harboring the bacterial lacI gene. Groups of five or six animals were killed at 4, 10 or 60 weeks and livers removed. At both 4 and 10 weeks of treatment, there was no significant difference in mutant frequency between the treated and control animals at either dose level. At 60 weeks, mice treated with 1.0 g/l DCA showed a 1.3-fold increase in mutant frequency over concurrent controls (P = 0.05). Mice treated with 3.5 g/l DCA for 60 weeks had a 2.3-fold increase in mutant frequency over the concurrent controls (P = 0.002). The mutation spectrum recovered from mice treated with 3.5 g/l DCA for 60 weeks contained G:C-->A:T transitions (32.79%) and G:C-->T:A transversions (21.31%). In contrast, G:C-->A:T transitions comprised 53.19% of the recovered mutants among control animals. Although only 19.15% of mutations among the controls were at T:A sites, 32.79% of the mutations from DCA-treated animals were at T:A sites. This is consistent with the previous observation that the proportion of mutations at T:A sites in codon 61 of the H-ras gene was increased in DCA-induced liver tumors in B6C3F1 mice. The present study demonstrates DCA-associated mutagenicity in the mouse liver under conditions in which DCA produces hepatic tumors.      

Inhibition by phenobarbital and lack of effect of amobarbital on the development of liver tumors induced by N-nitrosodiethylamine in juvenile B6C3F1 mice

The effects of phenobarbital (PB) and amobarbital (AB) on the rate of development of hepatocarcinogenesis induced by N-nitrosodiethylamine (DEN) were studied in mice. Groups of 40 B6C3F1 male mice were injected i.p. at 15 days of age with 5 micrograms DEN/g body wt. Beginning at 4 weeks of age, DEN treated groups were given either normal drinking water or water containing either 0.05% PB or AB for up to 36 weeks. DEN alone induced multiple focal hepatic lesions including hepatocellular foci, hepatocellular adenomas and trabecular carcinomas. Subsequent exposure to PB had a suppressing effect on DEN-induced hepatocarcinogenesis. Hepatocellular foci in PB-exposed mice were significantly smaller in size (area) and fewer in number throughout the study. Also, PB treatment either prolonged the latency period or significantly slowed the rate at which hepatocellular tumors developed in these mice. No such effects were seen in AB-exposed mice; AB neither inhibited nor promoted the development of focal hepatic lesions in DEN-pretreated mice. Possible mechanisms responsible for the inhibition of DEN-induced hepatocarcinogenesis include the feminizing effects of perinatal administration of PB.     

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.     

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.     

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.     

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     

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.      

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.