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.      

Carcinogenicity of 1,3-butadiene in C57BL/6 x C3H F1 mice at low exposure concentrations

The carcinogenicity of inhaled 1,3-butadiene was evaluated in C57BL/6 x C3H F1 mice exposed to concentrations of this gas ranging from 6.25 to 625 ppm. Butadiene is a high production volume chemical, used mainly in the manufacture of synthetic rubber. In these 2-yr inhalation studies, a potent multisite carcinogenic response was observed, including neoplasms of the lung at concentrations as low as 6.25 ppm. Early occurrence and extensive development of lethal lymphocytic lymphomas in mice exposed to 625 ppm of butadiene reduced the number of animals at risk for the expression of later developing neoplasms at other sites; at lower exposure concentrations, dose responses were demonstrated for hemangiosarcomas of the heart and neoplasms of the lung, forestomach, Harderian gland, preputial gland, liver, mammary gland, and ovary. So far, no long-term studies on butadiene have been conducted at exposure concentrations that have not shown a carcinogenic response. In separate experiments with reduced exposure durations, butadiene induced neoplastic responses at multiple organ sites even after only 13 wk of exposure. Because of the correspondence between these animal data and recent epidemiology findings, there is a worldwide public health need to reevaluate current workplace exposure standards for 1,3-butadiene.     

Molecular analysis of lacI mutants from transgenic fibroblasts exposed to 1,2-epoxybutene

1,3-Butadiene (BD) is a genotoxic carcinogen that is bioactivated to at least two mutagenic metabolites: 1,2-epoxybutene (EB) and 1,2:3,4- diepoxybutane (DEB). We reported previously that lacI transgenic mice exposed to BD had an increased frequency of specific base substitution mutations in the bone marrow and spleen relative to unexposed controls. In the experiments described here, we determined the mutagenicity and mutational spectrum of EB in Rat2 lacI transgenic fibroblasts as a means of assessing the contribution of this metabolite to the lacI mutational spectrum of BD. Rat2 cells were exposed to 0, 0.4, 0.6, 0.8 or 1.0 mM EB for 24 h, resulting in a range of cell survival from 100 to 15%, respectively. Mutagenicity was assessed at 0, 0.6 and 1.0 mM EB. Unexposed controls had a background mutant frequency of 6 +/- 1 +/- 10(-5), while the mutant frequency in cells exposed to 0.6 and 1.0 mM EB was increased 2- and 3-fold, respectively. DNA sequence analysis of 154 lacI mutants recovered in these experiments revealed an increase in the frequency of specific base substitution mutations in cells exposed to 1.0 mM EB compared with controls. These included G:C-->A:T transitions at non-CpG sites, G:C-->T:A transversions and A:T-->T:A transversions, which have all been observed in lacI mutants isolated from transgenic mice exposed to BD. These results suggest that EB causes mutation primarily by base substitution and that the spectrum of these mutations closely resembles that of BD. These data, along with previous findings from our laboratory, suggest that EB is more likely than DEB to be primarily responsible for the lacI mutational spectrum observed in lacI transgenic mice exposed to BD.      

Chromosome aberrations in mouse bone marrow cells following in vivo exposure to 1,3-butadiene

Chronic exposure to 1,3-butadiene (BD) results in a marked increasein the incidence of thymk lymphoma in male B6C3F1 relative toNIH Swiss mice whereas no demonstrable differences in bone marrow(target organ) toxicity exist. Repeated exposure to BD is knownto produce a macrocytic anemia and an increase in the frequencyof micronuclei in circulating erythrocytes in both strains.The present study was undertaken to determine if chromosomalbreakage, aneuploidy or both reflect differences in BD leukemogenicityobserved between B6C3F1 and NIH Swiss mice. Mice were exposedto a single concentration of BD (1250 p.p.m.) for 6 h. Bonemarrow cell preparations were made at 24, 48, 72 and 96 h aftercessation of exposure. In both strains comparable increasesin the frequency of chromosomal aberrations (of the chromatidtype) were observed following exposure to BD. Significant differencesin the number of chromosomes were not observed, although a patternof chromosomal loss in cells from treated animals was observed.These results indicate that BD-treatment in vivo produces significantincreases in chromatid aberrations but not aneuploidy in bothstrains. Therefore it is concluded that bone marrow toxidty,including cytogenetic abnormalities, is not predictive of leukemogenicityin these mice.     

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.      

Multiple organ carcinogenicity of inhaled chloroprene (2-chloro-1,3-butadiene) in F344/N rats and B6C3F1 mice and comparison of dose-response with 1,3-butadiene in mice.

Chloroprene (2-chloro-1,3-butadiene) is a high production chemical used almost exclusively in the production of polychloroprene (neoprene) elastomer. Because of its structural similarity to 1,3-butadiene, a trans-species carcinogen, inhalation studies were performed with chloroprene to evaluate its carcinogenic potential in rats and mice. Groups of 50 male and female F344/N rats and 50 male and female B6C3F1 mice were exposed to 0, 12.8, 32 or 80 p.p.m. chloroprene (6 h/day, 5 days/week) for 2 years. Under these conditions, chloroprene was carcinogenic to the oral cavity, thyroid gland, lung, kidney and mammary gland of rats, and to the lung, circulatory system (hemangiomas and hemangiosarcomas), Harderian gland, kidney, forestomach, liver, mammary gland, skin, mesentery and Zymbal's gland of mice. Survival adjusted tumor rates in mice were fit to a Weibull model for estimation of the shape of the dose-response curves, estimation of ED10 values (the estimated exposure concentration associated with an increased cancer risk of 10%) and comparison of these parameters with those for 1,3-butadiene. Butadiene has been identified as a potent carcinogen in mice and has been associated with increased risk of lymphatic and hematopoietic cancer in exposed workers. Shape parameter values for most of the neoplastic effects of chloroprene and 1,3-butadiene were consistent with linear or supralinear responses in the area near the lowest tested exposures. The most potent carcinogenic effect of 1,3-butadiene was the induction of lung neoplasms in female mice, which had an ED10 value of 0.3 p.p.m. Since the ED10 value for that same response in chloroprene exposed mice was also 0.3 p.p.m., we conclude that the carcinogenic potency of chloroprene in mice is similar to that of 1,3-butadiene. Cancer potency of chloroprene is greater in the mouse lung than in the rat lung, but greater in the rat kidney than in the mouse kidney and nearly equivalent in the mammary gland of each species.     

DNA adduct formation in the bone marrow of B6C3F1 mice treated with benzene

We used P₁-enhanced ³²P-postlabeling to investigate DNA adductformation in the bone marrow of B6C3F1 mice treated intraperitoneallywith benzene (BZ). No adducts were detected in the bone marrowof controls or mice treated with various doses of BZ once aday. After twice-daily treatment with BZ, 440 mg/kg, for 1 to7 days, one major and two minor DNA adducts were detected. Therelative adduct levels ranged from 0.06–1.46x10^–7.In vitro treatment of bone marrow from B6C3F1 mice with variousdoses of hydroquinone (HQ) for 24 h also produced three DNAadducts. These adducts were the same as those formed after invivo treatment of bone marrow with BZ. Co-chromatography experimentsindicated that the principal DNA adduct detected in the bonemarrow of B6C3F1 mice was the same as that detected in HL-60cells treated with HQ. This finding suggests that HQ may bethe principal metabolite of BZ leading to DNA adduct formationin vivo. DNA adduct 2 corresponds to the DNA adduct formed inHL-60 cells treated with 1,2,4-benzenetriol. DNA adduct 3 remainsunidentified. After a 7-day treatment with BZ, 440 mg/kg twicea day, the number of cells per femur decreased from 1.6x10⁷to 0.85xlO⁷, indicating myelotoxicity. In contrast, administrationof BZ once a day produced only a small decrease in bone marrowcellularity. These studies demonstrate that metabolic activationof BZ leads to the formation of DNA adducts in the bone marrow.Further investigation is required to determine the role of DNAadducts and other forms of DNA damage in the myelotoxic effectsof exposure to BZ.     

Life-span inhalation exposure to mainstream cigarette smoke induces lung cancer in B6C3F1 mice through genetic and epigenetic pathways

Although cigarette smoke has been epidemiologically associated with lung cancer in humans for many years, animal models of cigarette smoke-induced lung cancer have been lacking. This study demonstrated that life time whole body exposures of female B6C3F1 mice to mainstream cigarette smoke at 250 mg total particulate matter/m(3) for 6 h per day, 5 days a week induces marked increases in the incidence of focal alveolar hyperplasias, pulmonary adenomas, papillomas and adenocarcinomas. Cigarette smoke-exposed mice (n = 330) had a 10-fold increase in the incidence of hyperplastic lesions, and a 4.6-fold (adenomas and papillomas), 7.25-fold (adenocarcinomas) and 5-fold (metastatic pulmonary adenocarcinomas) increase in primary lung neoplasms compared with sham-exposed mice (n = 326). Activating point mutations in codon 12 of the K-ras gene were identified at a similar rate in tumors from sham-exposed mice (47%) and cigarette smoke-exposed mice (60%). The percentages of transversion and transition mutations were similar in both the groups. Hypermethylation of the death associated protein (DAP)-kinase and retinoic acid receptor (RAR)-beta gene promoters was detected in tumors from both sham- and cigarette smoke-exposed mice, with a tendency towards increased frequency of RAR-beta methylation in the tumors from the cigarette smoke-exposed mice. These results emphasize the importance of the activation of K-ras and silencing of DAP-kinase and RAR-beta in lung cancer development, and confirm the relevance of this mouse model for studying lung tumorigenesis.     

Activation of K-ras by codon 13 mutations in C57BL/6 X C3H F1 mouse tumors induced by exposure to 1,3-butadiene

1,3-Butadiene has been detected in urban air, gasoline vapors, and cigarette smoke. It has been estimated that 65,000 workers are exposed to this chemical in occupational settings in the United States. Lymphomas, lung, and liver tumors were induced in female and male C57BL/6 X C3H F1 (hereafter called B6C3F1) mice by inhalation of 6.25 to 625 ppm 1,3-butadiene for 1 to 2 years. The objective of this study was to examine these tumors for the presence of activated protooncogenes by the NIH 3T3 transfection and nude mouse tumorigenicity assays. Transfection of DNA isolated from 7 of 9 lung tumors and 7 of 12 liver tumors induced morphological transformation of NIH 3T3 cells. Southern blot analysis indicated that the transformation induced by 6 lung and 3 liver tumor DNA samples was due to transfer of a K-ras oncogene. Four of the 7 liver tumors that were positive upon transfection contained an activated H-ras gene. The identity of the transforming gene in one of the lung tumors has not been determined but was not a member of the ras family or a met or raf gene. Eleven 1,3-butadiene-induced lymphomas were examined for transforming genes using the nude mouse tumorigenicity assay. Activated K-ras genes were detected in 2 of the 11 lymphomas assayed. DNA sequencing of polymerase chain reaction-amplified ras gene exons revealed that 9 of 11 of the activating K-ras mutations were G to C transversions in codon 13. One liver tumor contained an activated K-ras gene with mutations in both codons 60 and 61. The activating mutation in one of the K-ras genes from a lymphoma was not identified but DNA sequence analysis of amplified regions in proximity to codons 12, 13, and 61 demonstrated that the mutation was not located in or near these codons. Activation of K-ras genes by codon 13 mutations has not been found in any lung or liver tumors or lymphomas from untreated B6C3F1 mice. Thus, the K-ras activation found in 1,3-butadiene-induced B6C3F1 mouse tumors probably occurred as a result of genotoxic effects of this chemical. The oncogenes most frequently detected in human pulmonary adenocarcinomas are K-ras genes. Activated K-ras genes have also been found in some human lymphomas. This suggest that activation of K-ras may be important in the induction of human pulmonary adenocarcinomas and lymphomas.(ABSTRACT TRUNCATED AT 400 WORDS)     

Analysis of diepoxide-specific cyclic N-terminal globin adducts in mice and rats after inhalation exposure to 1,3-butadiene

1,3-Butadiene is an important industrial chemical used in the production of synthetic rubber and is also found in gasoline and combustion products. It is a multispecies, multisite carcinogen in rodents, with mice being the most sensitive species. 1,3-Butadiene is metabolized to several epoxides that form DNA and protein adducts. Previous analysis of 1,2,3-trihydroxybutyl-valine globin adducts suggested that most adducts resulted from 3-butene-1,2-diol metabolism to 3,4-epoxy-1,2-butanediol, rather than from 1,2;3,4-diepoxybutane. To specifically examine metabolism of 1,3-butadiene to 1,2;3,4-diepoxybutane, the formation of the 1,2;3,4-diepoxybutane-specific adduct N,N-(2,3-dihydroxy-1,4-butadiyl)-valine was evaluated in mice treated with 3, 62.5, or 1250 ppm 1,3-butadiene for 10 days and rats exposed to 3 or 62.5 ppm 1,3-butadiene for 10 days, or to 1000 ppm 1,3-butadiene for 90 days, using a newly developed immunoaffinity liquid chromatography tandem mass spectrometry assay. In addition, 2-hydroxy-3-butenyl-valine and 1,2,3-trihydroxybutyl-valine adducts were determined. The analyses of several adducts derived from 1,3-butadiene metabolites provided new insight into species and exposure differences in 1,3-butadiene metabolism. Mice formed much higher amounts of N,N-(2,3-dihydroxy-1,4-butadiyl)-valine than rats. The formation of 2-hydroxy-3-butenyl-valine and N,N-(2,3-dihydroxy-1,4-butadiyl)-valine was similar in mice exposed to 3 or 62.5 ppm 1,3-butadiene, whereas 2-hydroxy-3-butenyl-valine was 3-fold higher at 1250 ppm. In both species, 1,2,3-trihydroxybutyl-valine adducts were much higher than 2-hydroxy-3-butenyl-valine and N,N-(2,3-dihydroxy-1,4-butadiyl)-valine. Together, these data show that 1,3-butadiene is primarily metabolized via the 3-butene-1,2-diol pathway, but that mice are much more efficient at forming 1,2;3,4-diepoxybutane than rats, particularly at low exposures. This assay should also be readily adaptable to molecular epidemiology studies on 1,3-butadiene-exposed workers.     

Disposition of butadiene monoepoxide and butadiene diepoxide in various tissues of rats and mice following a low-level inhalation exposure to 1,3-butadiene

1,3-Butadiene (BD), a chemical used extensively in the productionof styrene-butadiene rubber, is carcinogenic in Sprague-Dawleyrats and B6C3F₁ mice. Chronic inhalation studies revealed profoundspecies differences in the potency and organ-site specificityof BD carcinogenesis between rats and mice. BD is a potent carcinogenin mice and a weak carcinogen in rats. Previous studies fromour laboratory and others have shown marked differences betweenrats and mice in the metabolism of BD, which may account forspecies differences in carcinogenicity. The purpose of the presentstudy was to examine the production and disposition of two mutagenicBD metabolites, butadiene monoepoxide (BDO) and butadiene diepoxide(BDO₂), in blood and other tissues of rats and mice during andfollowing inhalation exposures to a target concentration of62.5 p.p.m. BD. BDO was increased above background in blood,bone marrow, heart, lung, fat, spleen and thymus tissues ofmice after 2 h and 4 h exposures to BD. In rats, levels of BDOwere increased in blood, fat, spleen and thymus tissues. Noincreases in BDO were observed in rat lungs. BDO₂, the moremutagenic of the two epoxides, was increased in the blood ofrats and mice at 2 and 4 h after initiation of exposure to BD.In mice, BDO₂ was detected in all tissues examined immediatelyfollowing the 4 h exposure. This metabolite was detected inheart, lung, fat, spleen and thymus of rats, but at levels 40-to 160-fold lower than those seen in mice. Immediately afterthe 4 h exposure, blood levels of BDO₂ were 204±15 pmol/gfor mice but were 41-fold lower for rats. In the sensitive mousetarget organs, heart and lungs, levels of BDO₂ exceeded BDOlevels immediately after the exposure. This study shows thatthe levels of BD epoxides are markedly greater in the mouseBD target organs. The high concentrations of BDO₂ in these organssuggest that this compound may be particularly important inBD-induced carcinogenesis. Thus, although BD is oxidativelymetabolized by similar metabolic pathways in rats and mice,the substantial quantitative differences in tissue levels ofmutagenic epoxides between species may be responsible for theincreased sensitivity of mice to BD-induced carcinogenicity.     

Comparison of blood concentrations of 1,3-butadiene and butadiene epoxides in mice and rats exposed to 1,3-butadiene by inhalation

1,3–Butadiene (BD), an important commodity chemical usedin the production of synthetic rubber, is carcinogenic in B6C3F1mice and Sprague-Dawley rats, raising concern for potentialcarcinogenicity in humans. Mice are more sensitive than ratsto the carcinogenic effects of BD. Metabolic activation of BDto form the putative DNAreactive metabolites, butadiene monoxide(BMO) and butadiene diepoxide (BDE), is mediated by cytochromeP450. Detoxication of the epoxides occurs by glutathione Stransferase-catalyzedconjugation with glutathione and hydrolysis by epoxide hydrolase.Species differences in metabolic activation and detoxicationmost likely contribute to the difference in carcinogenic potencyof BD by modulating the circulating blood levels of the epoxides.This study measured the in vivo concentrations of BD, BMO andBDE in the blood of male Sprague-Dawley rats and B6C3F1 miceduring and following 6 h nose-only exposure to inhaled BD at62.5, 625 or 1250 p.p.m. BD. Blood samples for BD and BMO (     

Frequency of Tk and Hprt lymphocyte mutants and bone marrow micronuclei in B6C3F(1)/Tk+/- mice treated neonatally with zidovudine and lamivudine

Mother-to-child transmission of the human immunodeficiency virus is substantially reduced by prenatal and postnatal treatment with anti-retroviral nucleoside analogues; however, the long-term consequences of these drug interventions are not known. The nucleoside analogue zidovudine (3'-azido-2',3'-dideoxythymidine; AZT) is carcinogenic in mice when administered transplacentally or neonatally, and this may be due to a genotoxic mechanism. Since single-drug treatment with AZT is being superseded by multidrug combinations, we have investigated the induction of mutations and micronuclei in mice treated neonatally with AZT, lamivudine (3'-thia-2',3'-dideoxycytidine; 3TC), or a combination of the two drugs. B6C3F(1)/Tk+/- mice were treated daily from days 1-8 of age with 200 mg AZT/kg/day, 200 mg 3TC/kg/day, or a mixture of 200 mg AZT + 200 mg 3TC/kg/day (AZT/3TC). One and 2 days after the last dose, bone marrow was collected to assess the induction of micronuclei in polychromatic erythrocytes; 3 weeks following treatment, the induction of mutants was determined in the hypoxanthine-guanine phosphoribosyltransferase (Hprt) and thymidine kinase (Tk) genes of spleen lymphocytes. AZT and AZT/3TC, but not 3TC, caused a significant increase in micronuclei, with the response being greatest one day after the last dose. None of the drugs induced mutations in the Hprt gene, while AZT and AZT/3TC, but not 3TC, caused a significant increase in the Tk mutant frequency. The increase in Tk mutants by AZT and AZT/3TC was associated with loss of the wild-type (Tk+) allele (loss of heterozygosity). These data suggest that AZT, but not 3TC, is genotoxic in neonatal mice, and that 3TC does not alter significantly the responses observed with AZT alone.     

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.     

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.     

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 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.     

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.     

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.     

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.