The overall objective of this project is to define the role of a functionally unique alpha class murine glutathione (GSH) transferase (GST) (designated as ~mouse GST 9.5"""""""") and its human orthologue(s) in cellular protection against carcinogenic diol epoxides (Des) of polycyclic aromatic hydrocarbons, which are widespread environmental pollutants. Recent studies from our laboratory indicate that GST 9.5 is a heterodimer of A1 and A2 type murine GST subunits. The hypothesis of this project is that mouse GST 9.5 (mGSTA1-2), which, unlike other alpha class mammalian GSTs, is exceptionally active toward Des, and its human orthologue(s) may play an important role in the detoxication of Des. This hypothesis will be tested by: (I) determining the kinetic constants for tissue-isolated murine GSTs, including GST 9.5, as well as the recombinant mGSTA1-1 and m GSTA2-2 homodimers, which were recently produced by us, in the GSH conjugation of various bay-and fjord-region Des, (ii) identifying the critical amino acids(s) which may account for the relatively higher catalytic efficiency of the A1 type subunit of GST 9.5 toward Des as compared with the A2 type subunit (through site-directed mutagenesis and kinetic characterization of the mutants), (iii) determining the expression of GST 9.5 subunits in other A/J mouse tissues by GSH- affinity chromatography followed by reverse-phase HPLC, (iv) determine the effects of expression of GST 9.5 subunits in Hepa-1 cells, through stable transfection, on cytotoxicity of (+)-anti-BPDE (trans-N2dG-BPD adduct), (v) determining the correlation in vivo between chemopreventive efficacies of six anti-carcinogenic organosulfides from garlic against BP-induced cancer of forestomach and lung in A/J mice wit their effects on the levels of various GSTs, including GST 9.5, in the liver and target organs, (vi) purifying from stomach and/or liver of the human orthologue(s) of murine GST 9.5 for its/their structural, immunological and kinetic characterization, and (vii) determining the expression of human orthologue(s) of GST 9.5 in other human tissues. These studies will not only enhance our understanding of the protective mechanisms against carcinogenic Des but also shed light on the role of mouse GST 9.5 and its orthologue(s) in the detoxication of these carcinogens. In the long term, strategies to manipulate this group of GSTs to the advantage of cells/organ should be extremely important for cancer prevention.
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