The broad objectives of the proposed research are to explore the chemical basis of the bioactivation and biotransformation of halogen- and sulfur-containing xenobiotics and environmental chemicals and their metabolites, the enzymology of bioactivation, and the development of in vitro expression systems to study haloalkene bioactivation. There are four Specific Aims:
Specific Aim 1 : The objective of this aim is to study the glutathione- and cysteine-conjugate beta-lyase-dependent bioactivation of haloalkenes. The goals include (a) the investigation of the activation of the microsomal glutathione transferase with haloalkenes as substrates, (b) investigation of the fate of 3H-labeled cysteine S-conjugates, of 13C-labeled cysteine S-conjugates by 13C-NMR spectroscopy, and of cysteine S-conjugates by Fourier-transform ion cyclotron resonance mass spectrometry, (c) the development of expression systems for studying the biosynthesis and bioactivation of glutathione- and cysteine S-conjugates of haloalkenes, and (d) the enzymology and molecular biology of aminoacylases that process haloalkene-derived mercapturic acids.
Specific Aim 2 : The objective of these studies is to map the tissue and organ distribution of mRNAs and enzymes of the beta-lyase pathway by immunohistochemistry, in situ hybridization, and in situ reverse-transcriptase polymerase chain reaction.
Specific Aim 3 : The objective of these studies is to investigate the bioactivation of dichloroacetic acid, which is a terminal product of the beta-lyase-dependent bioactivation of haloalkenes, as well as a drinking-water contaminant and a rodent carcinogen.
Specific Aim 4 : The objective of these studies is to investigate the bioactivation, biotransformation, and mutagenicity (Ames test) of the methionine-derived, food mutagen 2-chloro-4-methylthiobutanoic acid, which has been identified in processed fish. The proposed studies address an important human health issue: many of the halogen-and sulfur-containing compounds or classes of compounds are important environmental contaminants. Haloalkanes and haloalkenes, particularly chloroalkanes and chloroalkenes, are among the most common environmental chemicals, and several are carcinogenic in rodent bioassays. Because of widespread environmental contamination and occupational exposure, some, e.g., trichloroethene and tetrachloroethene, are listed as EPA Priority Chemicals. A thorough understanding of the bioactivation mechanisms of toxic halogen- and sulfur-containing compounds is important for assessing the hazards associated with human exposure and for the design of experiments to test the biological effects of such compounds in long-term animal studies.
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