Koji Urano

Validation of transgenic mice harboring the human prototype c-Ha-ras gene as a bioassay model for rapid carcinogenicity testing

Studies were conducted to validate the transgenic (Tg) mice harboring human prototype c-Ha-ras gene, namely the rasH2 mice (CB6F1), as a model for rapid carcinogenicity testing. Short-term (26 weeks) carcinogenicity testing of 18 mutagenic (Salmonella) trans-species carcinogens, two mutagenic single-species (mouse-only) carcinogens, six non-mutagenic trans-species carcinogens, one non-mutagenic single-species (mouse-only) carcinogen, four mutagenic non-carcinogens and four non-mutagenic non-carcinogens were completed. The studies revealed that the Tg mice are able to detect various types of mutagenic carcinogens and may also detect various non-mutagenic carcinogens within 26 weeks. Dose-dependent tumor responses were observed with various carcinogens except for a few equivocal cases. The validation studies also revealed that the Tg mice are generally much more susceptible to both mutagenic and non-mutagenic carcinogens than control non-Tg mice. Most of the malignant tumors were observed in the carcinogen-treated Tg mice and only very few or none in the corresponding non-Tg mice. Most of the carcinogens tested induced some of the target organ tumors observed in B6C3F1 mice in a 2-year bioassay as well as certain types of tumors specific to the Tg mice, i.e. lung alveolar epithelial tumors, spleen hemangiosarcomas, forestomach squamous cell tumors. No significant tumor induction has been observed in the Tg mice either with mutagenic or non-mutagenic non-carcinogens. Although further validation studies are still required, the rasH2 mouse seems to be a promising candidate as an animal model for the development of a rapid carcinogenicity testing system.

Detection of the onset of ischemia and carcinogenesis by hypoxia-inducible transcription factor-based in vivo bioluminescence imaging.

An animal model for the early detection of common fatal diseases such as ischemic diseases and cancer is desirable for the development of new drugs and treatment strategies. Hypoxia-inducible factor 1 (HIF-1) is a transcription factor that regulates oxygen homeostasis and plays key roles in a number of diseases, including cancer. Here, we established transgenic (Tg) mice that carry HRE/ODD-luciferase (HOL) gene, which generates bioluminescence in an HIF-1-dependent manner and was successfully used in this study to monitor HIF-1 activity in ischemic tissues. To monitor carcinogenesis in vivo, we mated HOL mice with rasH2 Tg mice, which are highly sensitive to carcinogens and are used for short-term carcinogenicity assessments. After rasH2-HOL Tg mice were treated with N-methyl-N-nitrosourea, bioluminescence was detected noninvasively as early as 9 weeks in tissues that contained papillomas and malignant lesions. These results suggest that the Tg mouse lines we established hold significant potential for monitoring the early onset of both ischemia and carcinogenesis and that these lines will be useful for screening chemicals for carcinogenic potential.

Tumor Promotion by 12-O-Tetradecanoylphorbol-13-Acetate in an Ultra-Short-Term Skin Carcinogenesis Bioassay Using rasH2 Mice

Assessment of the skin tumor–promoting potential of 12-O-tetradecanoylphorbol-13-acetate (TPA) after initiation with 7,12-dimethylbenz[a]anthracene (DMBA) was conducted using rasH2 transgenic (Tg) mice and their nontransgenic (non-Tg) littermates. Mice were treated with DMBA (50 μg/100 μL acetone) on clipped back skin at the commencement of the study, and 1 week thereafter, TPA was applied at 8 μg/200 μL or 4 μg/200 μL acetone, once or twice weekly, for 7 weeks. Skin nodules were observed in the rasH2 Tg mice from week 4, and the incidence reached 100% at weeks 5 and 6. The number of skin nodules (multiplicity) in the 8-μg twice-weekly, 8-μg once-weekly, 4-μg twice-weekly, and 4-μg once-weekly groups was 62.4, 46.2, 62.6, and 36.9, respectively. The non-Tg mice also developed skin nodules, but the sensitivity to induction in the rasH2 Tg mice was higher. No nodules were observed in the acetone groups, but single nodules were apparent in the no-treatment rasH2 Tg and non-Tg groups. In conclusion, skin promotion effects could be detected within only 8 weeks in the rasH2 mice, and the concentration of 4 μg TPA once weekly was sufficient as a positive control. This short-term skin carcinogenesis bioassay using rasH2 mice could represent a useful tool for the assessment of drug and chemical safety with cutaneous treatment.