Evaluation and Application of Genotoxicity by Determining Response of DNA-damage Responsive

我们通过将表达哺乳动物细胞色素P-450同工酶的酵母暴露于化学致癌物中，检测了DNA加合物的形成或DNA损伤反应基因的诱导。这些系统的实现将有利于对化学致癌物进行简单的评价。显示大鼠和人细胞色素P-450 1A1、1A4和2B5的酵母菌与致癌物质阿尔法曲霉毒素B1，2-氨基荧烯和2-乙酰氨基荧烯(AAF)接触时，在酵母染色体上检测到DNA加合物，但这些结构在不同的化学物质中是不同的。RNR3是核糖核苷酸还原酶的一个小亚基，是DNA损伤反应基因之一。DNA损伤化学物质4-硝基喹啉-1-氧化物、环孢菌素和博莱霉素可显著诱导酵母中RNR3mRNA的表达。然后将RNR3的5‘-启动子区域连接到β-半乳糖苷酶的结构基因上，并进行报告实验。酵母暴露于黄曲霉毒素和AAF可以诱导报告基因的活性，而这种诱导依赖于哺乳动物P-450的表达。因此，构建表达不同P-450的酵母将有助于检测其致癌能力。为了阐明RNR3基因在DNA损伤中的顺式作用调控，我们对其启动子区域进行了缺失分析。然后我们发现，至少有三个潜在的顺式元件是诱导表达RNR3响应DNA损伤所必需的。最后，我们研究了哺乳动物DNA损伤反应基因在培养细胞中的诱导，并显示了几个基因，如gadd152和gadd74，都是通过接触致癌物而诱导的。然而，诱导的机制很复杂，或者我们无法弄清楚哺乳动物基因表达对DNA损伤的反应规律。

We examined formation of DNA adducts or induction of DNA-damage responsive gene by exposure of yeast expressing mammalian cytochrome P-450 isozymes to chemical carcinogens. The achievement of these systems will facilitate to simply evaluate the chemical carcinogens. When yeast exhibiting rat and human cytochrome P-450 1A1,1A4 and 2B5 was exposed to carcinogens such as alfatoxin B1,2-aminofluorene and 2-acethylaminofluorene (AAF), DNA-adducts in yeast chromosomes were detected, but these structures were different among chemicals. RNR3, a small subunit of ribonucleotide reductase, is one of potent DNA-damage responsive genes. RNR3mRNA in yeast was markely induced by the treatment with DNA damage chemicals, including 4-nitroquinoline-1-oxide, cysplatin and bleomycin. Then the 5'-promoter region of RNR3 was connected to the structural gene of beta-galactosidase, and the reporter assay was made. Exposure of yeast to aflatoxin and AAF resulted in the induction of reporter activity, and these inductions depend on the expression of mammalian P-450. Therefore, construction of yeast expressing various P-450s will be useful to detect carcinogenic potency.To clarify cis-acting regulation of RNR3 gene by DNA damage, deletion analysis of the promoter region was performed. Then we found that at least three potential cis-elements are neccessary for the inducible expression of RNR3 in response to DNA damage. Finally we examined induction of mammalian DNA-damage responsive genes in cultured cells, and showed that several genes such as gadd152 and gadd74 were induced by exposure to carcinogens. However, the mechanisms involved in the inductions are complicate or we cannot figure out the regulations of mammalian gene expression in response to DNA damage.