Several antioxidants, both natural and synthetic, are known to inhibit chemical carcinogenesis in animal models. Modulation of the electrophilic/oxidative stress on DNA through the induction of glutathione S-transferases (GSTs) is believed to be one of the major determinants for their anti-carcinogenic activity. During the funded years of this project, we have identified a novel group of GSTs in mammalian tissues which are distinct from the known alpha, mu, pi, and theta classes of GSTs. In humans, several isozymes of these GSTs (designated as hGSTs 5.8) are expressed in a tissue specifiC manner. We have shown that in experimental animals hGST 5.8 counterparts are inducible by antioxidants and by oxidative stress itself. Enzymes of this group show activities towards endogenously generated electrophiles, e.g. the genotoxic 4-hydroxynonenal (4- HNE), that are 50-100 fold higher than those of any other known GSTs, and also have GSH-peroxidase activity towards phospholipid hydroperoxides. We hypothesize that these GSTs may limit the electrophilic stress caused by lipid peroxidation, and that their induction by antioxidants should enhance the protection of DNA from electrophilic/mutagenic stress. This may be particularly relevant to the mechanisms of chemical carcinogenesis because it has been shown that omega-6 unsaturated fatty acids which are the major percursors of 4-HNE, potentiate carcinogenicity of various stwcturally unrelated carcinogens. To assess the physiological role of hGST 5.8, we propose: 1) to determine its localization by Northern and Western blot analyses, in situ hybridization, and immuno-electron microscopy. Since we have previously shown that specific hGST 5.8 isozymes are expressed in a gender-related manner, we will compare their expression in leukocytes and other tissues of men and women. 2) Isolate and charactenze cDNA and genomic hGSTs 5.8 clones. Detailed kinetic analyses of tissue isolated enzymes, enzymes obtained by expression in E. coli. and mutated enzymes (site directed mutagenesis) will be performed. The mechanisms of regulation of hGST 5.8 expression will be studied using chronic myeloid leukemia K-562 cells as the host. 3) Determine the physiological role of hGSTs 5.8 and related enzymes through in vitro and in vivo studies. Human lung cancer cell line H-69, and K-562 cells will be transfected with hGST 5.8 and mGSTA4-4, and the effect of their overexpression on the formation of 8-hydroxy-2-deoxyguanosine in response to oxidative stress will be determined. In vivo studies in rats will examine whether the induction of rat GST 8-8 (rat ortholog of hGST 5.8) by curcumin attenuates the oxidative stress on DNA, using 8-hydroxy-2-deoxyguanosine as the marker. In preliminary studies, we have shown that curcumin preferentialy induces rGST 8- 8 in rat liver. These studies will provide a better understanding of the role of GSTs in defense against chemical carcinogenesis.
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