Dietary antioxidants such as BHA and BHT are known to inhibit chemical carcinogenesis induced by polycyclic aromatic hydrocarbons (PAH) in lung of mice. We have demonstrated that these antioxidants induce specific glutathione S-transferase (GST) isoenzymes of rodent lung. Our studies strongly suggest that the enhanced detoxification of the carcinogenic metabolites of benzo(a)pyrene (B(a)P) due to induction of GSTs is responsible for the anticarcinogenic activity of these antioxidants and emphasize the protective role of GST against chemical carcinogenesis in lung. GSTs represent a family of multifunctional enzymes and in mammals. GSTs are encoded by at least three distinct genetic loci corresponding to the three major classes of isoenzymes designated as alpha, mu, and II. Each class of human GST has a number of tissue specific isoenzymes. In human lung, we have demonstrated that (a) all classes of GST are expressed, (b) each class of GST is heterogenous and polymorphic and, (c) in lung tumors and cancer cell lines expression of GSTs is differentially altered. Therefore, in order to elucidate the protective role of GSTs against chemical carcinogenesis we will characterize GSTs of human lung belonging to all three classes. These isoenzymes will be isolated by affinity chromatography, IEF, chromatofocusing, and HPLC, and studies for their catalytic properties, non-catalytic binding, and structural properties as detailed in the Specific Aims Section. We have polyclonal antibodies against the alpha, mu, and II classes of GSTs and are developing the monoclonal antibodies against different classes of GTSs, Western blotting, ELISA, and immunotitration studies will be performed using these antibodies to compare the expression of GST in normal lung, lung tumors, and cancer cell lines. The antibodies will also be used for immunocytochemical localization of GST in lung. In parallel studies, the effect of antioxidants on the detoxification of B(a)P metabolites and on each class of GST isoenzymes of mouse and rat lung, and rat hepatocytes in culture will be determined. We have shown that in rat hepatocytes, GST can be induced by BHT within a short time. We will use the hepatocyte model study the mechanism of GST induction by antioxidants. These studies will help in evaluating whether or not the results of rodent studies on the anticarcinogenic effect of BHA/BHT could be applied to humans and will help in achieving the long term objective of developing strategies for chemo-prevention of carcinogenesis.
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