Ryuji Kitamura

Stable expression of cytochrome P450IIIA7 cDNA in human breast cancer cell line MCF-7 and its application to cytotoxicity testing

A mammalian cell expression plasmid containing cytochrome P450IIIA7 complementary DNA was constructed. Breast cancer cells (MCF-7) were transfected with the plasmid and neomycin-resistant selection marker plasmid. We established three cell lines, termed M13, M21, and M27, which expressed the cytochrome P450IIIA7 as examined by RNA blot and immunoblot analyses. These cell lines showed 8- to 10-fold higher sensitivity against aflatoxin B1 compared to parental MCF-7 cells, suggesting that cytochromes P450IIIA7 expressed in the cells were responsible for the production of the cytotoxic metabolite of aflatoxin B1.

Occurrence of autoimmune antibodies to liver microsomal proteins in association with fulminant hepatitis in the LEC strain of rats

The Long Evans Cinnamon (LEC) rat, which has been established as a strain showing hereditary hepatitis and hepatic carcinoma, was found to possess autoimmune antibodies to liver microsomal proteins, particularly to a protein with the molecular weight of 56kD. The antibodies also recognized a protein(s) in liver microsomes from Long Evans Agouti and Sprague-Dawley rats. About 42 and 15 percent of respective female and male LEC rats died within a week after acute hepatitis; sera from all of the animals contained the antibodies. About 43 and 0 percent of the surviving female and male LEC rats possessed the antibodies, respectively. These results suggest that the autoantibodies occur in association with acute lethal hepatitis in the LEC rats.

Stable expression of monkey cytochrome P-405IA1 cDNA in Chinese hamster CHL cells and its application for detection of mutagenicity of aflatoxin B1

A monkey cytochrome P-450IA1 cDNA (MKah1) was transfected into Chinese hamster CHL cells using a vector containing the SR alpha promoter, and sublines stably expressing P-450IA1 were established. The cells showed 25-fold higher sensitivity to the cytotoxic effect of aflatoxin B1 than the parental CHL cells. This hypersensitivity was almost completely suppressed by alpha-naphthoflavone, which is a known specific inhibitor of P-450IA. The cells expressing P-450IA1, but not CHL cells, showed a positive response to aflatoxin B1 in an assay for mutagenicity at the HGPRT locus.