The Pi class GSH transferase isoenzyme hGSTPI-1 is polymorphic in human populations, and involves amino acid residues in positions 104 (isoleucine or valine) and/or 113 (alanine or valine). However, the toxicological relevance of hGSTPI-1 polymorphism is not well defined. During the current funded period of this project, we studied catalytic properties of the hGSTPI-1 variants for GSH conjugation (detoxification) of activated metabolites (diol epoxides) of PAH carcinogens. In the present renewal application, we propose to shift emphasis from in vitro biochemical characterization of the purified hGSTPI-1 variants to elucidation of their biological role in vivo. We hypothesize that the in vivo efficacy ranking of hGSTPI-1 variants in defense against carcinogenic effects of PAHs is dependent upon chemical structure of the diol epoxides, and that the expression of GSH-conjugate transporter MRP2 enhances the protection afforded by hGSTPI-I. These hypotheses will be tested by the following approaches, which extend our previous work in new directions. (a) We will determine the efficacy of hGSTPI-1 variants for protection against DNA damaging effects of PAH diol epoxides in a matched set of hGSTPI-1 variant-transfected cells (aim 1). (b) This work will be complemented by studies of the effect of MRP2 overexpression on hGSTPI-1 variant-mediated inactivation of PAH diol epoxides (aim 2). (c) We will generate 'knock-in' mice in which the endogenous murine GST Pi genes will be replaced by the allelic variants of human GSTPI-1. These mouse lines will enable us to determine the in vivo efficacy of the hGSTPI-1 variants for protection against diol epoxide-induced tumorigenesis in a physiological setting but under fully controlled conditions (aim 3). (d) Finally, in specific aim 4, we will determine the molecular mechanism of catalytic differences between hGSTPI-1 variants toward diol epoxides through X-ray crystallography and molecular modeling of their active sites. The planned studies, which are a logical extension of our previous work, will enhance our understanding of the significance of hGSTPI-1 polymorphism in relation to metabolism of chemical carcinogens that are highly relevant to human health. In the long term, these studies may be valuable in formulating strategies for cancer prevention in humans. Moreover, the 'knock-in' mice generated through support of this project will be valuable reagents for future toxicological studies (beyond the scope of the present proposal) to define significance of hGSTPI-1 polymorphism. ? ? ? ?
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