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Assessment of Mutagenic and Carcinogenic Activity of Metabolites from Chemical Procarcinogen

化学原致癌物代谢物的致突变和致癌活性评估

基本信息

批准号：
05454222
负责人：
TOKUNAGA Rikio
金额：
$ 4.16万
依托单位：
KANSAI MEDICAL UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1994
项目状态：
已结题

项目摘要

The process of chemically induced carcinogenesis is generally divided into initiation and promotion. DNA adduct formation is target tissues is considered to be the first step of initiation ; therefor, detection of adducts would indicate the risks of cancer from exposure to compounds. ^<32>P-Postlabeling analysis, which can detect a very low level of adducts in DNA is a useful tool to detect this step in the initiation of cancer. Heterologous expression of human and rodent P450s in mammalian cells and yeast has been reported. Yeast contains intracellular organelles similar to mammalian cells and can be well used as a simple model of eukaryotic cells. We developed the method to measure P450-mediated metabolism of chemicals to DNA binding derivatives, using the yeast expression system, and examined the formation of DNA adducts derived from MBOCA in yeast expressing recombinant P450s (2B5 and 1A1) . These results show that recombinant rabbit P450 2B5 exhibits a potential activation of MBOCA and that rat P450 1A1 has some effect. The use of yeast expressing recombinant P450s and ^<32>P-Postlabeling facilitates a simple search for chemicals with carcinogenic potential.Ribonucleotide reductase (RNR) catalyzes the rate limiting step in the production of deoxyribonucleotides. RNR3 expression is inducible by DNA-damaging agents. We have constructed a fusion plasmid of 5'promoter region of RNR3 and lac Z,and measured beta-galactosidase activity in order to detect in-duction of RNR3 mRNA.We examined the induction of RNR3/lac Z in yeast upon exposure to various antitumor agents. We also examined RNR3 induction accompanied by P450 mediated activation of procarcinogen in yeast co-expressing rat CYP1A1 and RNR3/lacZ.

化学致癌过程一般分为启动和促进。靶组织中DNA加合物的形成被认为是起始的第一步;因此，加合物的检测将表明暴露于化合物的癌症风险。P<32>-后标记分析可以检测DNA中非常低水平的加合物，是检测癌症起始中这一步骤的有用工具。已经报道了人和啮齿动物P450在哺乳动物细胞和酵母中的异源表达。酵母含有类似于哺乳动物细胞的细胞器，可以很好地用作真核细胞的简单模型。我们开发了使用酵母表达系统测量P450介导的化学物质代谢为DNA结合衍生物的方法，并检查了在表达重组P450的酵母中源自MBOCA的DNA加合物的形成（2B 5和1A 1）。这些结果表明，重组兔P450 2B 5表现出潜在的激活MBOCA和大鼠P450 1A 1有一定的效果。使用表达重组P450和β-<32>P-后标记的酵母有助于简单地寻找具有致癌潜力的化学物质。核糖核苷酸还原酶（RNR）催化脱氧核糖核苷酸生产中的限速步骤。RNR 3的表达是由DNA损伤剂诱导的。我们构建了RNR 3 5 '启动子区与lac Z的融合质粒，测定了β-半乳糖苷酶活性，以检测RNR 3 mRNA的诱导，并研究了不同抗肿瘤药物对酵母中RNR 3/lac Z的诱导作用。我们还研究了在共表达大鼠CYP 1A 1和RNR 3/lacZ的酵母中RNR 3诱导伴随P450介导的前致癌原活化。