CELLULAR & MOLECULAR TARGETS OF CHEMICAL CARCINOGENS

实验方法是基于我们实验室最近的观察 细胞色素P-450型单加氧酶系统的存在 在大鼠肝线粒体中激活肝致癌物黄曲霉毒素 B1，二甲基亚硝胺和2-乙酰氨基芴; 这些致癌物质对线粒体DNA的影响 转录-翻译过程。 本提案的目标是， 因此，为了将DNA上的损伤与改变的基因表达相关联， 并确定这些细胞质基因在癌变中的作用。 它 提出了定位线粒体DNA上的黄曲霉毒素B1结合区域， 使用限制性内切核酸酶进行物理作图。 同样，线粒体 在变性条件下通过电泳分析转录物。 转录模式的变化将与转录因子的面积相关。 通过DNA-RNA杂交使用Southern和“北方”印迹检测mt基因组 方法的变化，并最终与翻译模式的变化相关联。 除此之外，我们还打算纯化大鼠肝线粒体细胞色素 P-450的同质性，并研究其激活各种肝脏和 非肝致癌物。 最后，线粒体基因的作用， 将使用体细胞杂交来确定肿瘤过程 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.