| Co-Investigator(Kenkyū-buntansha) |
NAKAYAMA Kazuo Hokkaido Univ., Fac.of Pharm.Sci., Instructor, 薬学部, 助手 (20261323)
YOKOI Tsuyoshi Hokkaido Univ., Fac.of Pharm, Sci., Associate Prof., 薬学部, 助教授 (70135226)
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| Research Abstract |
We have succeeded in establishing cell lines which stably expressed human NATI,NAT2, CYP1A2, CYP2E1, CYP3A4 and CYP3A7. We examined the cytotoxicity and mutagenicity of IQ and MeIQx in the cells expressing human CYP1A2, NAT1, and NAT2 alone or in combinations. The results led to the following conclusions : (a) no activation to produce cytotoxic and mutagenic metabolites from either compound was achieved by each enzyme alone ; (b) CYP1A2 in combination with NAT2 efficiently activated the compounds ; (c) expression of CYP1A2 togather with NAT1 was not effective for mutagemic activation. The cells expressing CYP2E1 showed 7-ethoxycoumarin OMICRON-deethylase activity. Moreover, these cells showed higher sensitivity to DMN in cytotoxicity assay as compared to parental cells. The cells expressing CYP3A4 and CYP3A7 were more sensitive to aflatoxin B_1 than parental cells. These cell lines provide a new valuable panel for studying the human metabolism of xenobiotics with an ability to directly
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relate to P450-catalyzed metabolism.
(2) We established several lines of transgenic mice harboring a CYP3A7 transgene.Of all the six transgenic lines established, M2 mice expressed the CYP3A7 gene in many tissues such as in the kidney, small intestine, and skin, etc, while M10 mice expressed the CYP3A7 transgene in the liver and testis, at both mRNA and protein levels. The potential application of this transgenic mice in drug metabolism was assessed using midazolam (MZ) as a substrate. Administration of this drug through intraperitoneal injection into adult mice of line M10 resulted in significantly higher levels of both 1'-OH-MZ and 4-OH-MZ in mouse serum. Kinetic study using liver microsomes from adult M10 mice indicated a higher Vmax with the same Km values in 1'-hydroxylation of MZ as compared with nontransgenic mice. These results indicate that CYP3A7 expressed in these transgenic mice possesses normal catalytic activities towards MZ with characteristics similar to those in the human livers. Less
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