CELLULAR & MOLECULAR TARGETS OF CHEMICAL CARCINOGENS

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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.