Individual Difference in Drug Metabolish and Disposition : Toxicological Significance of Genotypes and Phenotypes of Human Polymorphic Arylamine N-acetyltransferase

药物代谢和处置的个体差异：人多态性芳胺 N-乙酰转移酶基因型和表型的毒理学意义

• 批准号: 08670487
• 负责人: IKEBUCHI Jun
• 金额: $ 1.41万
• 依托单位: Tottori University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08670487/
• 关键词: drug metabolizing enzyme; genetic polymorphism; genotype; phenotype; individual difference; interethnic difference; toxicogenetics; toxicology; チトクロームP-450遺伝子; N-アセチル転移酵素遺伝子; 遺伝的多型性

N-acetyltansferase (NAT) in human liver catalyzes N-acetylation of various arylamine-containing drugs and xenobiotics. Human NAT isoenzymes are encoded at two loci, and the NAT2 locus is polymorphic.Acentylation polymorphism has been studied by several methods with a test drug, such as isoniazid, and ethnic variation in N-acetylation capacity has been reported.In this study, we prepared the DNA samples from the blood of unrelated healthy Japanese, and developed a rapid and simple genotyping method using a polymerase chain reaction (PCR) based restriction fragment length polymorphism. NAT2 genotypes were determined based on the fact that four alleles in the NAT2 gene differe at single base substitutions which modify restriction enzyme cleavage sites. The sequence containing the polympophic sites was amplified, and the cleavage of the PCR product by the restriction enzymes was detected by polyacrylamide gel electrophoresis. Furthermore, NAT2 phenotypes were determined by measuring the molar ratio of caffeine urinary metabolites, 5-acetylamino-6-formylamino-3-methyluracil (AFMU) /1-methylxanthine (1X), taken at 4-5hr after caffeine ingestion.Consequently, the genotypes observed were NAT2^<**>1/^<**>1, NAT2^<**>1/^<**>2, NAT2^<**>1/^<**>3, NAT2^<**>1/^<**>4 and NAT2^<**>3/^<**>4. In vivo metabolic ratio (AFMU/1X) of wild-type was 1.55, whereas hetero-type was 0.87, and homo-mutated type 0.12. The genotype of NAT2 correlated with the phenotype.These results proved that genotyping assays of drug matabolizing enzymes would play more important role in assessing the severity and predicting the outcome of poisoning for forensic and clinical toxicology.

人体肝脏中的N-乙酰基转移酶(NAT)催化多种含芳胺类药物和外源化合物的N-乙酰化。人类NAT同工酶在两个基因座上编码，且NA2基因座具有多态特性。以异烟肼为例，用多种方法研究了乙酰化能力的种族差异。在本研究中，我们从无血缘关系的健康日本人的血液中制备了DNA样本，并建立了一种基于聚合酶链式反应(PCR)的限制性片段长度多态性的快速、简单的基因分型方法。根据Nat2基因中的4个等位基因在改变限制性内切酶切割位点的单碱基替换上存在差异，确定了Nat2的基因类型。扩增含多聚酶切位点的序列，用聚丙烯酰胺凝胶电泳法检测扩增产物被限制性内切酶切割的情况。此外，通过测量摄入咖啡因后4-5小时尿中咖啡因代谢产物5-乙酰氨基-6-甲氨基-3-甲基尿嘧啶(AFMU)/1-甲基黄嘌呤(1X)的摩尔比，确定了NAT2的表型。因此，观察到的基因型为NAT2^&lt；**&lt；**&gt；1，Nat2^&lt；**&gt；1/^&lt；**&gt；2，2^&lt；**&Gt；1/^&lt；**&gt；**&gt；野生型的体内代谢率(AFMU/1X)为1.55，杂合型为0.87，同变型为0.12。这些结果表明，药物代谢酶基因分型在法医学和临床毒理学中评估中毒的严重程度和预测结果方面有更重要的作用。

项目成果

山田 光子: "DNA多型(分担)" (DNA多型学会)東洋書店, 138-140 (1997)

山田光子：《DNA 多态性（共享）》（DNA 多态性学会）东洋书店，138-140（1997）

Mitsuko Yamada: "Ethnic Difference in Drug Metabolism" Proceedings of the International Meeting of the International Association of Forensic Toxicologists. 35(in press). (1998)

Mitsuko Yamada：“药物代谢的种族差异”国际法医毒理学家协会国际会议记录。