Mutagenicity of butadiene and its epoxide metabolites: I. Mutagenic potential of 1,2-epoxybutene, 1,2,3,4-diepoxybutane and 3,4-epoxy-1,2-butanediol in cultured human lymphoblasts

The mutagenic potential of the epoxide metabolites of butadiene(BD) was measured at the tk and hprt loci in TK6 human lymphoblastoidcells. TK6 cells were exposed for 24 h to 0–400 µM1,2-epoxybutene (EB), 0–800 µM 3,4-epoxy-1,2-butanediol(EBD), or 0–6 µM 1,2,3,4-diepoxybutane (DEB). Treatedcells were allowed to grow for several days and then seededin medium containing either 6-thioguanine or trifluorothymidineto select for hprt^– or tk^–/– mutants, respectively.All three metaboiltes were mutagenic at both loci, with DEBexhibiting activity at concentrations approximately 100-foldlower than EB or EBD. At the hprt locus, an induced mutationfrequency of 5 x 10^–6 (approximately twice backgroundhprt^– frequency) was produced by treatment with 3.5 µMDEB, 150 µM EB and 450 µM EBD. At the tk locus,a similar increase in mutation frequency (total tk^–/–frequency) was produced by treatment with 1.0 µM DEB,100 µM EB and 350 µM EBD. Each epoxide tested wascapable of inducing slow growth tk^–/– mutants. Thismutant phenotype, as shown previously by others, results fromlarge alterations in the tk region which completely remove theactive tk allele. In addition, Southern blot analysis revealedthat approximately half of DEB-induced hprt^– mutants displayedloss of wild-type hprt restriction fragments. No statisticallysignificant increase in the fraction of hprt deletions amongEB mutants was observed. The ability of DEB to induce deletionsmay be related to its ability to form DNA-DNA and DNA-proteincross-links.     

Comparison of the mutagenic potency of 1,3-butadiene at the hprt locus of T-lymphocytes following inhalation exposure of female B6C3F1 mice and F344 rats

1,3-Butadiene (BD) is an indirect alkylating agent that has greater cancer potency in the mouse than in the rat. The purpose of the present study was to compare the mutagenic potency of BD at the hprt locus of T- lymphocytes of exposed mice and rats and to determine whether mutations induced in this marker gene can be used as a quantitative indicator for species differences in susceptibility to cancer. To this end, experiments were conducted to define the effects of exposure duration and the time elapsed after exposures on the frequency of hprt mutations (Mf) in T-cells from female B6C3F1 mice and F344 rats of similar age (4- 5 weeks) when exposed to BD by inhalation. The accumulation of hprt mutations in T-cells from thymus was assessed in animals necropsied 2 weeks after exposure to 0 or 1250 ppm BD for 1 or 2 weeks, while the time course for the appearance of hprt mutant T-cells (i.e., the phenotypic expression and cell migration) in thymus and spleen was evaluated in animals necropsied at weekly/biweekly intervals up to 10 weeks after exposure for 2 weeks. At necropsy, T-cells were isolated from thymus and spleen and cultured in the presence of IL-2, concanavalin A, and 6-thioguanine (Walker and Skopek, Mutat. Res., 288, 151-162, 1993). BD exposures of 1 and 2 weeks led to mutagenic effects in mouse thymus, with the average Mfs being 3- and 5-fold greater than background values, respectively. In rat thymus, there was only a 1.7- fold increase in Mfs after 2 weeks of BD exposure. In the mutant expression experiment, hprt Mfs in thymus and spleen of both species increased for several weeks post-exposure and then declined. Hprt Mfs in thymus reached maximum levels at 2 weeks post-exposure in mice (Mfs = 11.3 +/- 2.4 x 10(-6)) and at 3 weeks post-exposure in rats (4.9 +/- 1.2 x 10(-6)), while hprt Mfs in spleen reached peak levels at 5 weeks post-exposure in mice (19.7 +/- 1.9 x 10(-6)) and 4 weeks post-exposure in rats (10.1 +/- 1.8 x 10(-6)). Background Mfs for mouse and rat thymus and spleen ranged from 1.6 +/- 0.3 x 10(-6) to 3.0 +/- 1.1 x 10(- 6). Statistical analyses of the hprt Mf data for spleen demonstrated that, under these exposure conditions, the mutagenic potency of BD (represented by the difference in the areas under the phenotypic expression curves of treated versus control animals) was 5-fold greater in mice than in rats. The magnitude of the species differences in mutagenic potency, observed after 2 weeks of BD exposure, resembles the species differences in metabolism more closely than the species differences in cancer potency.      

Forestomach neoplasm induction in F344/DuCrj rats and B6C3F1 mice exposed to sesamol.

Sesamol was administered at a dietary level of 2% to groups of 30 male and female F344/DuCrj rats and B6C3F1 mice for 104 and 96 weeks, respectively. Squamous cell carcinomas in the forestomach were induced in nine of 29 (31%) effective male rats, three of 30 (10%) female rats, eleven of 29 (38%) male mice and five of 30 (17%) female mice treated with sesamol. Papillomas developed in ten of 29 (34%) male rats and fourteen of 30 (47%) female rats, but not in any of the mice. Hyperplasias developed in almost all rats and mice of both sexes. Significant differences from control values were found for all three lesions in rats and for carcinoma and hyperplasia categories in mice. The incidences of other tumors in the 2% sesamol group were comparable with control values. In conclusion, sesamol induces squamous cell carcinomas in the forestomach of rats and mice, males being more susceptible than females.     

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.     

DNA adduct formation in B6C3F1 mice and Fischer-344 rats exposed to 1, 2, 3-trichloropropane

1, 2, 3-Trichloropropane (TCP) is a multispecies, multisitecarcinogen which has been found to be an environmental contaminantIn this study, we have characterized and measured DNA adductsformed in vivo following exposure to TCP. [¹⁴C]TCP was administeredto male B6C3F1 mice and Fischer-344 rats by gavage at dosesused in the NTP carcinogenesis bioassay. Both target and nontargetorgans were examined for the formation of DNA adducts. Adductswere hydrolyzed from DNA by neutral thermal or mild acid hydrolysis,isolated by HPLC, and detected and quanti-tated by measurementof radioactivity. The HPLC elution profile of radioactivitysuggested that one major DNA adduct was formed. To characterizethis adduct, larger yields were induced in rats by intraperitonealadministration of TCP (300 mg/kg). The DNA adduct was isolatedby HPLC based on coelution with the radiolabeled adduct, andcompared to previously identified adducts. The isolated adductcoeluted with S-[1-(hydroxymethyl)-2-(N⁷-guanyl)-ethyljglutathione,an adduct derived from the structurally related carcinogen 1,2-dibromo-3-chloropropane (DBCP). Analysis by electrospray massspectrometry suggested that the TCP-induced adduct and the DBCP-derivedadduct were identical. The ¹⁴C-labeled DNA adduct was distributedwidely among the organs examined. Adduct levels varied dependingon species, organ, and dose. In rat organs, adduct concentrationsfor the low dose ranged from 0.8 to 6.6 µmol per mol guanineand from 7.1 to 47.6 µmol per mol guanine for the highdose. In the mouse, adduct yields ranged from 0.32 to 28.1 µmolper mol guanine for the low dose and from 12.2 to 208.1 µmolper mol guanine for the high dose. The relationship betweenDNA adduct formation and organ-specific tumorigenesis was unclear.Although relatively high concentrations of DNA adducts weredetected in target organs, several nontarget sites also containedhigh adduct levels. Our data suggest that factors in additionto adduct formation may be important in TCP-induced carcinogenesis.     

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.     

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.     

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.     

Sulfuric acid esters as major ultimate electrophilic and hepatocarcinogenic metabolites of 4-aminoazobenzene and its N-methyl derivatives in infant male C57BL/6J x C3H/HeJ F1 (B6C3F1) mice

Liver cytosols from 12-day-old male C57BL/6 X C3H/HeJ F1 (B6C3F1) mice contain 3'-phosphoadenosine-5'-phosphosulfate (PAPS)-dependent sulfotransferase activity for N-hydroxy-4-aminoazobenzene and N-hydroxy-N-methyl-4-aminoazobenzene. No acetyl co-enzyme A-dependent transacetylase activity for these hydroxylamines was detected in the cytosols. Pentachlorophenol (PCP) and 2,6-dichloro-4-nitrophenol were only moderately active inhibitors of the sulfotransferase activity; at a 100-microM concentration each compound inhibited the activity by only 50-80%. A single dose of 0.04 mumol/g body weight of PCP administered to 12-day-old male B6C3F1 mice 45 min prior to a single dose of 0.1 mumol/g body weight of [3H]4-aminoazobenzene ([3H]AB) or [3H]N,N-dimethyl-4-aminoazobenzene ([3H]DAB) inhibited DNA adduct formation by approximately 50%. Under identical conditions, PCP also reduced the average number of hepatomas induced per mouse at 9 months by AB and N-methyl-4-aminoazobenzene (MAB) by 52 and 36%, respectively. PCP strongly inhibited the hepatocarcinogenicity of DAB or AB when this agent was administered in the diet with either dye to female CD-1 mice over a 10- month period. Single doses of 0.15 mumol/g body weight of [3H]AB and [3H]DAB bound to hepatic DNA of 12-day-old brachymorphic B6C3F2 mice, which are deficient in the synthesis of PAPS, at levels 15 and 20%, respectively, of those found in their phenotypically normal litter mates. Under identical conditions, the incidence of hepatomas in brachymorphic mice at 9 months were 11 and 29%, with averages of 0.2 and 0.8 hepatomas/mouse for AB and MAB, respectively. Incidences of 77 and 86%, with averages of 6.6 and 5.4 hepatomas/mouse, respectively, were found in their phenotypically normal litter mates. These data strongly indicate that N-sulfo?xy-AB is a major ultimate electrophilic and hepatocarcinogenic metabolite of AB in mice. Similarly, this ester and N-sulfo?xy-N-methyl-4-aminoazobenzene appear to be critical metabolites for these activities of DAB and MAB.     

Carcinogenesis studies of dichlorvos in Fischer rats and B6C3F1 mice

Dichlorvos (dichlorovinyl dimethyl phosphoric acid ester) is a cholinesterase inhibitor used widely as a contact and stomach insecticide for control of internal and external parasites. Carcinogenesis studies were conducted by administering dichlorvos in corn oil by gavage 5 times a week for 103 weeks to groups of 50 male and 50 female Fischer rats at 0, 4, or 8 mg/kg body weight, to groups of 50 male B6C3F1 mice at 0, 10, or 20 mg/kg, and to groups of 50 female B6C3F1 mice at 0, 20, or 40 mg/kg. During the course of the studies, body weights and survival rates of the male and female rats and mice were not different from those of their respective controls; females of both species appeared to gain more weight than controls. Neoplasms induced by dichlorvos included adenomas of the exocrine pancreas (male rats), mononuclear cell leukemia (male rats), and squamous cell papilloma of the forestomach (male and female mice; two other female mice had squamous cell carcinomas). Lesions observed in female rats that may have been due to dichlorvos administration included adenomas of the exocrine pancreas and fibroadenomas of the mammary gland. The results demonstrated that dichlorvos is carcinogenic for Fischer rats and B6C3F1 mice.     

ACCELERATED PAPER: Mutational spectra of the dietary carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) at the Chinese hamster hprt locus

The mutagenic ‘fingerprint’ of the cooked food carcinogen2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) was determinedin a Chinese hamster cell line genetically engineered to expresshuman CYP1A2 (XEMh1A2-MZ). The parental Chinese hamster V79and XEMh1A2-MZ cells were exposed to PhIP at various concentrationsfor 24 h. There was a dose-dependent increase in frequency ofmutations at the hypoxanthine-guanine phosphoribosyl-transferase(hprt)locus only in the metabolically competent XEMh1A2-MZ cells.The mutant frequency ranged from 25 to 90x10⁶ with final concentrationsof 2.5 to 100 µM PhIP compared to 8x10⁶ in the solventcontrols and the V79MZ cells. The molecular nature of PhlP-inducedmutations in XEMh1AZ-MZ cells was determined by examining DNAsequence modifications at the hprt locus in forty five 6-thioguanineresistant (6-TG¹) mutant clones. Single base substitutions,predominantly GC     

Development of hemangiosarcomas in B6C3F1 mice fed 2-methyl-1-nitroanthraquinone

Subcutaneous hemangiosarcomas developed in 97% of 172 B6C3F1 mice of both sexes fed either 0.03% or 0.36% 2-methyl-1-nitroanthraquinone in the diet. There was no significant relationship to dose or sex. In addition similar vascular tumors occurred in the mesentery of 14 mice. 2-Methyl-1-nitroanthraquinone is carcinogenic in B6C3F1 mice when given in food. One of 97 control mice had a splenic hemangiosarcoma.     

Mutagenicity of o-anisidine to the bladder of lacI- transgenic B6C3F1 mice: absence of 14C or 32P bladder DNA adduction

Earlier studies have established that the rodent bladder carcinogeno-anisidine (OA) gives negative results hi all of the standardrodent genetic toxicity assays. In the present study, a singleoral administration of the maximum tolerated dose level (750mg/kg) of OA to B6C3F1 mice yielded negative results in ³²P-post-labellingassays of bladder and liver DNA (24 h after dosing). Likewise,¹⁴C-ring-labelled OA administered orally to B6C3F1 mice gaveno evidence of DNA binding 6, 12 or 24 h later. Administrationof OA (750 mg/kg) to transgenic lacl{small tilde} mice (BigBlue^TM led to a small increase in mutation frequency (MF) inthe bladder, but not in the liver. Increased MFs were observedin the bladder following 1, 3 or 10 daily doses with samplingtimes of 1 or 2 weeks after the final dose. However, statisticalsignificance (P < 0.01) was only reached 2 weeks after either3 or 10 daily administrations of OA. The positive control chemical(dimethylnitrosamine) gave a positive result (P < 0.01) inthe liver, but not the bladder, 7 days after a single administrationof 10 mg/kg. The possibility that OA is mutagenic and carcinogenicto the rodent bladder via formation of radical species is suggested.     

Hepatocellular tumorigenicity of butylated hydroxytoluene administered orally to B6C3F1 mice

Butylated hydroxytoluene (BHT), a preservative widely found in food as a food additive, was orally administered at concentrations of 1% and 2% of the diet to B6C3F1 mice for 104 consecutive weeks. Treated animals underwent a 16-week recovery period prior to pathological examination. In male mice administered BHT, the incidence of mice with either a hepatocellular adenoma or a focus of cellular alteration in the liver was increased in a clear dose-response relationship. The incidences of male mice with other tumors and the incidences of female mice with any tumor were not significantly increased as a consequence of BHT administration. The results of this study indicate BHT to be tumorigenic to the liver of the B6C3F1 male mouse.     

Carcinogenicity of phenacetin: Long-term feeding study in B6C3F1 mice

Groups of 52 B6C3F₁ mice of each sex were maintained on a diet containing 1.25 or 0.6% phenacetin for 96 weeks and then fed a basal diet for 8 weeks. Control groups consisted of 50 mice of each sex and were fed a basal diet for 104 weeks. All animals were killed at the end of the experiment and all organs were examined histopathologically. Mice that died during the experiment were also autopsied and those that survived for more than 57 weeks, when the first tumor was observed, were also included in the effective number of mice. Tumors were found in the kidney, liver, lung, skin, hematopoietic system (leukemia or lymphoma) and occasionally in some other organs. The dose-related induction of renal cell tumors in the male mice fed phenacetin was clearly demonstrated in this experiment. Urinary bladder lesions that developed in the mice of either sex fed 1.25% phenacetin were also considered to be due to the tumorigenicity of phenacetin. Tumors of other organs in either the phenacetin-treated or the control group were regarded as strain-related spontaneous tumors of B6C3F₁ mice.     

Lack of tumorigenicity of aminopyrine orally administered to B6C3F1 mice

To test the tumorigenic potential of aminopyrine, an antipyretic analgesic, it was administered in drinking water at levels of 0 (control), 0.04 and 0.08% to 50 male and 50 female B6C3F1 mice for 100 weeks, and the mice were subsequently maintained without aminopyrine for a further 4 weeks. The most frequent types of tumor, in both treated and control groups, were hepatocellular tumor in male mice and malignant lymphoma/lymphoid leukemia in female mice. No statistically significant differences were observed in the incidences of these tumors between treated and control groups. The incidences of several other tumors in male and female mice also showed no statistically significant differences between treated and control groups. Therefore, no tumorigenic effect of orally administered aminopyrine in B6C3F1 mice was apparent in the present study.     

Phenobarbital promotion in diethylnitrosamine-initiated infant B6C3F1 mice: influence of gender

Phenobarbital (PB) promotes hepatic tumorigenesis when chronicallyadministered to male B6C3F1 mice after initiation with diethylnitrosamine(DENA) at 30 days of age. In contrast, when male B6C3F1 micewere initiated with DENA at 15 days of age, an inhibition ofhepatic tumorigenesis occurred. The present study was undertakento evaluate the Influence of gender on the inhibiting abilityof PB in the 15 day old DENA-initiated B6C3F1 mouse. Mice wereinjected with either DENA (5 µg/g) or saline at 15 daysof age. At weaning mice were given either PB (500 p.p.m.) containingdrinking water or deionized drinking water for 24 weeks. Malemice treated with DENA and PB demonstrated a significant decreasein the number of hepatocellular adenomas compared to males receivingDENA only. In contrast, females exposed to DENA and PB exhibitedan enhancement of hepatic adenoma number compared to those receivingonly DENA. In an additional experiment, individual preneoplastkfoci from male and female B6C3F1 mice initiated with DENA at15 days of age were examined for their responsiveness to theniitogenic stimuli of PB. Mice were exposed to either PB-containingor PB-free drinking water for 7 days. In non-PB treated malesand females, preneoplastk hepatocytes demonstrated higher ratesof DNA synthetic labelling compared to normal hepatocytes withno gender difference noted. Males exposed to PB exhibited increasedlevels of DNA synthesis in normal cells but not in preneoplastkfoci. Females treated with PB, however, demonstrated significantincreases in DNA synthesis in both preneoplastk and normal hepatocytescompared to non-PB treated females and PB-treated males. Thesefindings suggest that in male mice initiated with DENA at 15days of age, the preneoplastk foci are refractory to the proliferativeeffects of PB which may account for the observed inhibitionof hepatic tumorigenesis by PB in this mouse strain.     

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.     

Neoplastic and nonneoplastic lesions in aging (C57BL/6N x C3H/HeN)F1 (B6C3F1) mice.

Neoplastic and nonneoplastic lesions in untreated (C57BL/6N x C3H/HeN)F1 (B6C3F1) mice used as controls in carcinogenesis tests were tabulated and evaluated. The most common neoplasms in 2,543 male mice were hepatocellular adenomas and carcinomas. In 2,522 female mice, common tumors were lymphomas, leukemias, pulmonary adenomas and carcinomas, hepatocellular adenomas and carcinomas, and pituitary adenomas. The risk of developing most neoplasms increased with the age of the mouse. Hepatocellular carcinomas metastasized in 12% of the animals with these tumors. Other than lymphomas and leukemias, few other tumors metastasized. Nonneoplastic lesions included cystic hyperplasia of the uterus, nephritis, ovarian and uterine cysts, inflammatory lesions of the lung, mineralization in the brain, and focal hyperplasias in several tissues. The focal hyperplasias in lung and pituitary, adrenal, and thyroid glands were suggestive of the early stages of neoplasia. Comparative aspects of lesions in aging mice and their interpretation in carcinogenesis tests are discussed.