Many halogenated hydrocarbons are of interest because of their high production volume and demonstrated ability to cause cancers in experimental animals. The vinyl halide trichloroethylene (TCE) can be activated via oxidation or conjugation; the focus in this proposal will be on oxidation and the key product TCE oxide. The mechanism of TCE oxide hydrolysis will be established using kinetics and isotope labeling patterns. Reaction products formed with proteins and DNA will be characterized with the working hypothesis that acyl halides are the major electrophiles that react. Dihalomethanes (CH2X2) are activated by enzymatic glutathione (GSH) conjugation. The characterization of CH2X2-derived GSH-DNA adducts will be done using mass spectrometry, with work extended to in vitro settings with CH2Cl2. Rat and human GSH transferases will be compared (in terms of DNA adduction) with a bacterial analog (DM11) that allows for growth of Methylophilus on CH2Cl2. This work and structure-activity relationships among CH2X2 compounds will be done to help establish the conjugation mechanism and to determine if GSCH2X or HCHO is the genotoxic product (of CH2X2). Other work will involve a search for GSH-containing DNA adducts derived from 4-carbon bifunctional electrophiles (e.g. butadiene diepoxide) as an explanation for enhancement of bacterial mutation by GSH transferase expression. Work with ethylene dibromide (EDB) will involve (i) completion of bacterial site-directed mutagenesis work with guanyl N2-, N7-, and O6-CH2CH2SG adducts to determine the relative contributions of each and (ii) analysis of the basis of O6-alkylguanine tranferase-increased mutagenicity of EDB (and CH2Br2). These experiments involving enzymatic oxidation and conjugation reactions are intended to help define important concepts that can be applied to other halogenated hydrocarbons and to other cancer suspect chemicals as well.
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