During the current funded period of this grant, we investigated the role of murine glutathione (GSH) transferases (GSTs) in detoxification of activated metabolites (diol epoxides) of polycyclic aromatic hydrocarbons (PAHs), which are suspected human carcinogens. Despite these advances, however, the role of individual human GSTs in defense against activated PAHs is poorly defined. In the present renewal application, we propose to shift emphasis from murine model to human GSTs and investigate their role in detoxification of diol epoxides. Recent studies from our laboratory and by others led us to hypothesize that the Alpha class GST isoenzymes play an important role in diol epoxide inactivation in human tissues. We propose to test this hypothesis by two different but complementary approaches. First, we will determine the kinetic constants and catalytic efficiencies for all four known Alpha class human GST isoenzymes (hGSTA1-1, hGSTA2-2, hGSTA3-3 and hGSTA4-4) toward a panel of structurally different bay- (chrysene and dibenz[a,h]anthracene) and fjord-region (benzo[c]phenanthrene and benzo[g]chrysene) type PAH diol epoxides (specific aim 1). The kinetic constants for Alpha class GSTs will be compared with those of other classes of human GSTs, including hGSTPI-1, hGSTMI-1 and hGSTTI-1, to substantiate the above hypothesis. Second, we will determine the relative contributions of human GSTs, including the Alpha class isoenzymes, for GSH conjugation of representative bay- and fjord-region diol epoxides using autopsied human liver (a major site for xenobiotic metabolism) and lung (a known target organ for PAH-induced tumorigenesis) (specific aim 2). The second objective of the present renewal application is to gain insights into the structural basis for catalytic differences in diol epoxide-GSH conjugation efficacy between human GSTs. This goal will be accomplished by two different approaches: First, we will determine the effects of mutations of key active site (H-site) residues on catalytic activity of Alpha class GSTs toward diol epoxides (specific aim 3). Second, X-ray crystallography studies are planned to elucidate the structural basis for differential substrate specificity pattern for human GSTs toward bay- and fjord-region diol epoxides (specific aim 4). In summary, the studies proposed in the present renewal application, which is a logical extension of our previous findings, will fill the gaps in our understanding of the mechanisms of diol epoxide inactivation in humans. In the long-term, this knowledge will guide us in developing strategies for prevention of PAH-induced cancers in humans.
Showing the most recent 10 out of 35 publications
