CELLULAR & MOLECULAR TARGETS OF CHEMICAL CARCINOGENS

实验方法是基于我们实验室最近的观察结果。 一种独特的细胞色素P-450型单加氧酶系统的存在 大鼠肝线粒体对致癌物质黄曲霉毒素的激活作用 B1、二甲基亚硝胺和2-乙酰氨基荧烯； 线粒体DNA上的这些致癌物影响线粒体 转录-翻译过程。这项建议的目标是， 因此，要将DNA上的损伤与基因表达的变化联系起来 并确定这些细胞质基因在癌症发生中的作用。它 建议通过以下方法定位线粒体DNA上的黄曲霉毒素B1结合区 使用限制性内切酶的物理作图。同样，线粒体 转录本将在变性条件下进行电泳法分析。 转录模式的变化将与细胞的面积相关 DNA-RNA杂交结合Southern和Northern杂交检测MT基因组 方法，并最终与翻译模式的变化相关联。 除此之外，我们还打算提纯大鼠肝脏线粒体细胞色素 P-450均一化，并研究其激活多种肝脏和 非肝脏致癌物质。最后，线粒体基因在人类进化中的作用 肿瘤过程将通过体细胞杂交来确定 程序。

The experimental approach is based on the recent observations in our laboratory on the existence of a unique cytochrome P-450 type monooxygenase enzyme system in rat liver mitochondria for the activation of hepatic-carcinogens Aflatoxin B1, Dimethyl nitrosamine and 2-Acetyl aminofluorene; and on the direct attack of these carcinogens on mitochondrial-DNA affecting mitochondrial transcription-translation processes. The objectives of this proposal are, therefore, to correlate the lesions on the DNA with the altered gene-expression and also to determine the role of these cytoplasmic genes in carcinogenesis. It is proposed to localize the Aflatoxin B1 binding regions on mitochondrial DNA by physical mapping using restriction-endonucleases. Similarly, mitochondrial transcripts will be analyzed by electrophoresis under denaturing conditions. The changes in the transcription pattern will be correlated with the area of the mt genome by DNA-RNA hybridization using both the Southern and "Northern" blot methods, and finally be correlated with the changes in the translation patterns. In addition to this, we intend to purify the rat liver mitochondrial cytochrome P-450 to homogeneity and study its ability to activate varied hepatic and nonhepatic carcinogens. Finally, the role of mitochondrial genes in the neoplastic process will be determined using somatic cell hybridization procedures.