1992 Fiscal Year Annual Research Report

生体内薬物濃度制御システムの関発研究・薬物代謝酵素の遺伝的多型性の解析を基礎として

Research Project

Project/Area Number	03557101
Research Institution	Hokkaido University
Principal Investigator	鎌滝哲也北海道大学, 薬学部, 教授 (00009177)
Co-Investigator(Kenkyū-buntansha)	澤田稔北海道大学, 薬学部, 助手 (30215917) 伊東進北海道大学, 薬学部, 助手 (70223154) 横井毅北海道大学, 薬学部, 助教授 (70135226)
Keywords	S-メフェニトイン / チトクロームP-450 / N-アセチル転移酵素 / カフェイン
Research Abstract	(1)ヒトS-メフェニトイン代謝型チトクロームP-450(P-450llC)のpoor metabolizer(PM)とextensive metabolizer(EM)について、遺伝的代謝能の差異を遺伝子レベルで検討を行った。第一にCYP2C8,2C9,2C18のcDNAを酵母で発現させ、S-メフェニトイン代謝活性を測定した。その結果いずれの分子種でも活性が認められたが、CYP2C18と2C9で比較的高い活性が得られた。第2にCYP2C9とCYP2C18について、cDNAレベルでの配列の差異を、比較検討した。その結果PM、EMで塩基配列に差は認められなかった。第3に、ヒト肝試料を用いて、in vitroでS-メフェニトイン代謝のPMとEMの判定を行なった。これらのヒト肝からRNAを調製し、CYP2C9とCYP2C18の発現量をRT-PCRで測定した。この値とS-メフェニトイン代謝能の相関を調べたところ、CYP2C18が高い相関を示した。第4に、CYP2C18に焦点を絞って、PMとEMからCYP2C18のゲノムDNAを単離し、その5'上流領域を-1500塩基まで検討した。しかし、PM特異的な配列は解読した範囲内では見い出されなかった。 (2)P-4501A2はフェナセチンやカフェインの代謝にかかわり、代謝能に多型性があることが示唆されている。第1に、カフェインを225人のボランティアに投与し代謝試験を行なった。その結果、約15%のヒトがPMと考えられるプロビットプロットの変曲点が得られた。第2に、10家族の2世代についてin vivo代謝試験を行い、PMが常染色体劣勢遺伝をすると、矛盾無く説明出来る結果が得られた。第3に、in vivo試験で典型的なPMとEMについて、ゲノムDNAの塩基配列を、PCR法を用いて検討した。その結果、エクソンまたは、エクソン-イントロンジャンクション部分に、PMとEMでの差異は認められなかった。 (3)N-アセチル転移酵素について、カフェインin vivo代謝試験の結果、2相性のプロビットプロットが得られた。同一人からゲノムDNAを抽出し、PCR-RFLP法で判定を行なった。In vivo代謝試験でのPMはhomo変異型であった。また、hetero変異型の群とwild型の群との代謝能に明らかな有為差を見いだした。

Research Products

(14 results)

All Other

All Publications (14 results)

[Publications] Sawada,M.et al.: "Stable expression of monkey cytochrome P-450lA1 cDNA in Chinese hamster CHL cells and its application for detection of mutagenicity of aflatoxin B_1." Mutation Research. 265. 23-29 (1992)
[Publications] Kitamura,R.et al.: "Stable expression of cytochrome P-450lllA7 cDAN in human breast cancer cell line MCF-7 and its applicationto cytotoxicity testing.Archs." Archs,Biochem.Biophys.292. 136-140 (1992)
[Publications] Itoh.S.et al.: "Genomic organization of human fetal specific P-450lllA7(cytochrome P-450HFLa)-related gene(s)and interaction of transcriptional regulatory factor with its DNA element in the 5'flanking region." Biochim.Biophys.Acta.1130. 133-138 (1992)
[Publications] Ubukata.K.et al.: "A new form of cytochrome P-450 responsible for the mutagenic activation of 2-amino-3-methy-limidazo[4,5-f] quinoline(IQ)in human livers." Cancer Res.52. 758-763 (1992)
[Publications] Yanagimoto,T.et al.: "Mouse liver cytochrome P-450(P-450lllAM1):its cDNA cloning and inducibility by dexamethasone." Biochim Biophys Acta.1130. 329-332 (1992)
[Publications] Komori,M.et al.: "Molecular cloning of monkey P-4501A1 cDNA and expression in yeast." Biochim Biophys Acta.1131. 23-29 (1992)
[Publications] Mori.T.et al.: "Examination for lipid peroxidation in liver microsomes of guinea pigs as a causal factor in the decrease in the content of cytochrome P-450 due to ascorbic acid deficiency." Res.Commun.Chem.Pathal.Pharmacal.75. 209-218 (1992)
[Publications] Nakura.H.et al.: "Decrease in the content of cytochrome P-450llE1 by fasting in liver microsomes of house musk shrew(suncus murinus)." Biochem.Pharmacal.43. 1907-1910 (1992)
[Publications] Fukuta.H.et al.: "Toxicological significance of dog liver cytochrome P-450:Examination with the enzyme expressed in Saccharomyces cerevisiae using recombinant expression plasmid." Mut.Res.269. 97-105 (1992)
[Publications] Ohgiya,S.et al.: "Six-base deletion occuring in messages of human cytochrome P-450 in the CYP2C subfamily results in reduction of tolbutamide hydroxylase activity." Biochem.Int.27. 1073-1081 (1992)
[Publications] Kitada,M.et.al.: "Immunochemical characterization and toxicological significance of P-450HFLb purified from human fetal livers." Biochim.Biophys.Acta.1117. 301-305 (1992)
[Publications] Sawada.M.et al.: "Stable expression of mouse NADPH-cytochrome P450 reductase and monkey P4501A1 cDNAs in Chinese hamster cells:Establishment of cell lines highly sensitive to aflatoxin B_1." Archs Biochem.Biophys.300. 164-168 (1993)
[Publications] Narita,M.et al.: "Inhibition of β-glucuronidase by natural glucuronides of Kampo medicines using glucuronide of SN-38(7-ethyl-10hydroxycamptothecin)as a substrate." Xenobiotica.,in press.
[Publications] Itoh.K.et al.: "Rat liver flavin-containing monooxygenase(FMO):cDNA cloning and expression in yeast." Biochim.Biophys.Acta.,in press.

1992 Fiscal Year Annual Research Report

生体内薬物濃度制御システムの関発研究・薬物代謝酵素の遺伝的多型性の解析を基礎として

Principal Investigator

鎌滝 哲也 北海道大学, 薬学部, 教授 (00009177)

Research Products

[Publications] Sawada,M.et al.: "Stable expression of monkey cytochrome P-450lA1 cDNA in Chinese hamster CHL cells and its application for detection of mutagenicity of aflatoxin B_1." Mutation Research. 265. 23-29 (1992)

[Publications] Kitamura,R.et al.: "Stable expression of cytochrome P-450lllA7 cDAN in human breast cancer cell line MCF-7 and its applicationto cytotoxicity testing.Archs." Archs,Biochem.Biophys.292. 136-140 (1992)

[Publications] Itoh.S.et al.: "Genomic organization of human fetal specific P-450lllA7(cytochrome P-450HFLa)-related gene(s)and interaction of transcriptional regulatory factor with its DNA element in the 5'flanking region." Biochim.Biophys.Acta.1130. 133-138 (1992)

[Publications] Ubukata.K.et al.: "A new form of cytochrome P-450 responsible for the mutagenic activation of 2-amino-3-methy-limidazo[4,5-f] quinoline(IQ)in human livers." Cancer Res.52. 758-763 (1992)

[Publications] Yanagimoto,T.et al.: "Mouse liver cytochrome P-450(P-450lllAM1):its cDNA cloning and inducibility by dexamethasone." Biochim Biophys Acta.1130. 329-332 (1992)

[Publications] Komori,M.et al.: "Molecular cloning of monkey P-4501A1 cDNA and expression in yeast." Biochim Biophys Acta.1131. 23-29 (1992)

[Publications] Mori.T.et al.: "Examination for lipid peroxidation in liver microsomes of guinea pigs as a causal factor in the decrease in the content of cytochrome P-450 due to ascorbic acid deficiency." Res.Commun.Chem.Pathal.Pharmacal.75. 209-218 (1992)

[Publications] Nakura.H.et al.: "Decrease in the content of cytochrome P-450llE1 by fasting in liver microsomes of house musk shrew(suncus murinus)." Biochem.Pharmacal.43. 1907-1910 (1992)

[Publications] Fukuta.H.et al.: "Toxicological significance of dog liver cytochrome P-450:Examination with the enzyme expressed in Saccharomyces cerevisiae using recombinant expression plasmid." Mut.Res.269. 97-105 (1992)

[Publications] Ohgiya,S.et al.: "Six-base deletion occuring in messages of human cytochrome P-450 in the CYP2C subfamily results in reduction of tolbutamide hydroxylase activity." Biochem.Int.27. 1073-1081 (1992)

[Publications] Kitada,M.et.al.: "Immunochemical characterization and toxicological significance of P-450HFLb purified from human fetal livers." Biochim.Biophys.Acta.1117. 301-305 (1992)

[Publications] Sawada.M.et al.: "Stable expression of mouse NADPH-cytochrome P450 reductase and monkey P4501A1 cDNAs in Chinese hamster cells:Establishment of cell lines highly sensitive to aflatoxin B_1." Archs Biochem.Biophys.300. 164-168 (1993)

[Publications] Narita,M.et al.: "Inhibition of β-glucuronidase by natural glucuronides of Kampo medicines using glucuronide of SN-38(7-ethyl-10hydroxycamptothecin)as a substrate." Xenobiotica.,in press.

[Publications] Itoh.K.et al.: "Rat liver flavin-containing monooxygenase(FMO):cDNA cloning and expression in yeast." Biochim.Biophys.Acta.,in press.

鎌滝哲也北海道大学, 薬学部, 教授 (00009177)