Toshimi Usui

The Tg rasH2 mouse in cancer hazard identification.

The Tg rasH2 transgenic mouse has been developed as an alternative to the lifetime mouse bioassay to predict the carcinogenic potential of chemicals. Unlike the p53+/- mouse, the Tg rasH2 mouse is sensitive to both genotoxic and nongenotoxic carcinogens. The Tg rasH2 mouse, officially designated CB6F1-TgN (RasH2), contains multiple copies of the human c-Ha-ras oncogene and promoter within its genome. These mice develop spontaneous and chemically induced neoplasms earlier in life and in greater numbers than wild-type mice, reflecting their enhanced sensitivity to neoplasia. The most common spontaneous neoplasms in control Tg rasH2 mice 8 to 9 months of age are lung adenomas and carcinomas (7.4% incidence), splenic hemangiomas and hemangiosarcoma s (5.4%), forestomach squamous cellpapillomas and carcinomas (2.4%), and skin neoplasms (1.2%). Simulations have demonstrated that 20 to 25 mice/sex/treatment group are required to provide the assay with adequate statistical power. Four of 6 known or suspected human carcinogens tested in Tg rasH2 mice were positive in this assay. For 19 nonmutagenic agents testing positive in conventional rodent bioassays, 7 chemicals were positive, 10 chemicals were negative, and 2 were equivocal. None of the 10 nonmutagenic rodent carcinogens that were negative in the Tg rasH2 mouse model are considered to be human carcinogens. All nonmutagenic chemicals that were negative in the conventional rodent bioassays were also negative in the Tg rasH2 model. Results for 15 of 18 mutagenic chemicals tested in Tg rasH2 mice agreed with the results of conventional rodent bioassays, and 3 results were equivocal. The Tg rasH2 mouse model appears to predict known or suspected human carcinogens as well as the traditional mouse bioassay, but with fewer positive results for nongenotoxic compounds that are not considered human carcinogens. The Tg rasH2 mouse model is the most thoroughly tested in vivo alternative to the lifetime mouse bioassay for nongenotoxic compounds administered by oral or parenteral routes. The U.S. FDA Carcinogenicity Assessment Committee has determined that the Tg rasH2 model has been adequately evaluated for consideration for carcinogenicity testing of pharmaceutical candidates and its use could contribute to the weight of evidence for carcinogenicity assessment. The FDA will consider proposals to replace lifetime mouse carcinogenicity studies with 6-month Tg rasH2 mouse studies to support pharmaceutical registration on a case-by-case basis.

Interlaboratory Comparison of Short-Term Carcinogenicity Studies Using CB6F1-rasH2 Transgenic Mice

In order to evaluate a short-term carcinogenicity testing system using CB6F1-Tg rasH2 ( rasH2-Tg) mice carrying a human prototype c-Ha-ras gene, 26-week studies were conducted in 12 different facilities as a part of an International Life Science Institute Health and Environmental Science Institute (ILSI HESI) international collaborative project. In each study N-methyl-N-nitrosourea (MNU) was administered to a separate group of rasH2-Tg mice by single intraperitoneal injection (75 mg/kg) as a positive control. We herein have summarized the mortality, body weight change, and neoplastic and nonneoplastic lesions detected in these positive control groups as representative historical positive control data. Also, we performed an interlaboratory comparison of the response of rasH2-Tg mice to MNU based on the data of 11 positive control groups from these studies. Although the body weight of rasH2-Tg mice showed lower values than that of non-Tg mice during the experimental period, body weight gain in the rasH2-Tg mice was similar to that in non-Tg mice. The mortality of rasH2-Tg mice during the study period was very low, the same as for the non-Tg mice. Incidences of spontaneous alveolar/bronchiolar adenomas and splenic hemangiomas/hemangiosarcomas were also low in the rasH2-Tg mice. Nonneoplastic lesions detected in the rasH2-Tg mice were similar to those in non-Tg mice, excluding the incidence of myopathy. There were interlaboratory differences in mortality and incidence of some lesions in the MNU-treated groups. However, the causes of death were common among the 11 laboratories and almost all the MNU-treated rasH2-Tg mice developed forestomach squamous cell papillomas/carcinomas or malignant lymphomas. This suggests that there is no appreciable difference in the response of the rasH2-Tg mouse to MNU used as a positive control. Therefore, it is concluded that MNU would be an adequate positive control compound in this testing system.

Mutation analysis of vinyl carbamate or urethane induced lung tumors in rasH2 transgenic mice

Previous studies showed that significant differences in mutation frequency of the human c-Ha-ras transgene between vinyl carbamate (VC)- and ethyl carbamate (urethane)-induced lung tumors were observed in rasH2 mice. It remains unclear why the point mutation frequency is extremely low in VC-induced lung tumors, although this compound is much more carcinogenic than urethane. In this study, we examined the somatic point mutations of the transgene at the RNA level in VC- and urethane-induced lung tumors of rasH2 mice. We did not find any mutation at codon 12 of the transgene in any of these lung tumors, but codon 61 showed frequent mutations in not only urethane-induced lung tumors (15 out of 16) but also VC-induced lung tumors (11 out of 11) in rasH2 mice. These results suggested that point mutations at codon 61 of the transgene play an important role in the carcinogenesis of VC- and urethane- induced lung tumors in rasH2 mice.

Interlaboratory comparison of the CB6F1-Tg rasH2 rapid carcinogenicity testing model

Several genetically engineered mouse models are currently being examined for potential use in cancer hazard identification. We have undertaken an interlaboratory comparison of the performance of the CB6F1-Tg rasH2 transgenic mouse in cancer bioassays concurrently conducted in the United States and Japan. Chemicals selected for study included known human carcinogens (melphalan and cyclosporin A) and known rodent carcinogens (p-cresidine and vinyl carbamate) tested at carcinogenic doses, and non-carcinogens (p-anisidine and resorcinol) tested at appropriate high doses. Because of abdominal adhesions caused by the intraperitoneal dosing vehicle, melphalan was excluded from the study results. The remaining five studies showed similar results between the two laboratories conducting each study. Vinyl carbamate gave the strongest positive response inducing lung adenomas and carcinomas and splenic hemangiosarcomas. p-Cresidine was considered positive for urinary bladder transitional neoplasia. Cyclosporin A, p-anisidine, and resorcinol were negative in all studies. Although only five chemicals were successfully tested in this interlaboratory comparison, there was good concordance in outcome for the strong carcinogens and for the non-carcinogens. Successful testing of chemicals with less carcinogenic potential may require modifications in study design to include more animals and longer study duration.

Lack of susceptibility of transgenic mice carrying the human c-Ha-ras proto-oncogene (rasH2 mice) to phenolphthalein in a 6-month carcinogenicity study.

Phenolphthalein has carcinogenic activity, causing malignant lymphomas in B6C3F1 mice at a dietary dose of 3000 ppm in a 2-year carcinogenicity study and in heterozygous p53-deficient female mice at the same dose in a 6-month study. To examine whether phenolphthalein carcinogenic potential can be detected in male and female transgenic (Tg) mice carrying the human c-Ha-ras gene (rasH2 mice) and their wild-type littermates (non-Tg mice), a diet containing 3000, 6000 or 12000 ppm was given for 6 months. Unequivocal induction of neoplastic lesions was not apparent, suggesting that rasH2 mice are resistant to the induction of malignant lymphomas by the treatment of phenolphthalein.

Skeletal Myopathy in Transgenic Mice Carrying Human Prototype c-Ha-ras Gene

Skeletal myopathy was found in almost all-transgenic mice carrying the human prototype c-Ha-ras gene (rasH2 mouse). Microscopically, variation of the muscle fiber size, centrally placed nuclei, regenerating fibers, and interstitial fibrosis were evident; hyalinization and necrosis were sometimes observed in the skeletal muscle (femoralis and pectoralis) of the rasH2 mice. Inflammatory changes in the skeletal muscle or abnormality of adjacent peripheral nerve were not observed. The features were essentially similar to those of muscular dystrophy. Although the severity was relatively mild compared to 34-week-old rasH2 mice, the skeletal myopathy was also observed in younger male (10 weeks of age) rasH2 mice. In nontransgenic littermates, skeletal myopathy was not observed. The mRNA of human c-Ha-ras product was detected in femoral muscle from the rasH2 mice by RT-PCR. In conclusion, these data suggest that skeletal myopathy is occurring in almost all rasH2 mice. Integration of c-Ha- ras gene is thought to be crucial to pathogenesis of skeletal myopathy in the rasH2 mice. Further characterization of the muscular lesion and its pathogenesis are needed to explore the possibility of rasH2 mouse becoming a new model for muscular dystrophy.

Lack of susceptibility of heterozygous p53-knockout CBA and CIEA mice to phenolphthalein in a 6-month carcinogenicity study.

Phenolphthalein has carcinogenic activity, causing malignant lymphomas in B6C3F1 mice at a dietary dose of 3000 ppm in a 2-year carcinogenicity study and in female heterozygous p53-knockout TSG mice (C57BL/6 background) at the same dose in a 6-month study. To examine whether carcinogenic potential of phenolphthalein can be detected in other p53-knockout mouse [p53 (+/-)] strains such as p53 (+/-) CBA mice or p53 (+/-) CIEA mice (C57BL/6 background) and their littermates, they were given a diet containing 0, 6000 or 12000 ppm for 6 months. Unequivocal induction of neoplastic lesions was not apparent, suggesting that p53 (+/-) CBA mice and p53 (+/-) CIEA mice are resistant to the induction of malignant lymphomas by the treatment of phenolphthalein.