MOLECULAR MECHANISMS OF CHEMICAL TOXICITY

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The long term objective of the proposed research is to elucidate the changes in DNA produced by representative environmental chemical toxins that react with DNA. It is well established that a component of toxicity of some chemicals is reaction with DNA and a great deal is known about the chemical nature of the adducts formed. Less is known about the consequences to the structure and function of DNA or about the molecular nature of alterations in DNA leading to mutagenesis and carcinogenesis. In the proposed work, genotoxicity in adult rat liver (ARL) epithelial cells will be studied because these lines are derived from a specific in vivo tissue and retain a substantial ability to bioactivate chemical genotoxins. In these proliferating lines, the DNA changes leading to mutation in the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus and the functional consequences of such mutations will be studied by a variety of means. These include the following: (a) Cloning of cDNA of the rat HGPRT gene and its characterization by DNA sequence analysis (b) In addition to the biochemical characterization of chemically induced ARL mutants, Southern analyses will be used to differentiate the nature of genomic alterations induced by genotoxic chemicals in ARL cells; (c) A highly precise technique, the polymerase chain reaction (PCR) will be adapted to detect single base substitutions in the rat HGPRT gene in chemically induced HGPRT mutants; (d) Northern blotting, RNase Protection analysis nuclear runoff transcription assay and in vitro translation assay will be used to explore the influence of mutation on the transcription, processing and half life of the HGPRT mRNA; (e) Elucidation of the overall organization of the rat HGPRT gene on the basis of hybridization studies and thus permit the distinction of functionally important sequences located on the X chromosome from related sequences that may occur on autosomal chromosomes. The proposed research, in addition to documenting the nature of chemically induced changes in DNA, will contribute to an understanding of the organization of the genome in the rat, an important species in experimental toxicology, and of changes in a gene involved in a human genetic disorder.