We have previously shown that the protective effect of antioxidants BHA and BHT against chemical carcinogenesis in lung is manifested through induction of glutathione (GSH) S-transferase (GST) isozymes. The objectives of this proposed project are to characterize various GST isozymes, define their role in the detoxification of carcinogens and elucidate the mechanisms by which antioxidants differentially induce GST isozymes and provide protection against chemically induced carcinogenesis. Or preliminary studies indicate that in humans, several closely related GSTs having as little as one amino acid residue difference in their sequences nd having distinct kinetic characteristics are present within each of the three major classes alpha, mu and pi of GSTs. In addition our studies show that at least two types of mu-class subunits which have high activity towards toxic epoxides such as the carcinogenic B(a)P epoxides are expressed differentially in human lung. Therefore, in the proposed studies we will determine the primary structures, substrate specificities, kinetic characteristics and binding characteristics of mu and alpha class GSTs of lung, and compare their expression in normal lung and lung tumor tissue and in cell lines derived from tumors. We will also characterize the novel human GST isozymes zeta and omega, an mouse lung GST 5.7 for their primary structures and kinetic and binding properties, and study their expression in lung and lung tumors. We will quantitate the effect of BHT on individual alpha, mu and pi class GSTs as well as the analogs of GST zeta and omega in mouse lung by isolating individual isozymes and also by Western and Northern blot analyses. We will compare the expression and characteristics of individual lung GST isozymes of male and female mice and humans because our preliminary studies show sex-related differences in the expression and characteristics of GSTs of mouse liver and human colon. The effect of BHT on the corresponding GST isozymes of male and female mouse lung will also be compared. Since our preliminary studies show lower levels of GSH and GST in female mouse lung and liver as compared to the male, we will investigate whether or not carcinogenic effect of B(a)P in mouse lung and the protective effect of BHA differs in male and female mice. We will clone and sequence CDNA for the mu class GSTs of human lung (GST 6.25 and GST 6.45), the alpha class GST 9.4, and novel human GSTs omega and zeta, as well as novel mouse lung GST 5.7, using specific antibodies and/or synthetic oligonucleotide probes generated from the AA sequences, and compare the expression of these isozymes in human lung and lung tumors by Northern blot analysis using the CDNA probes. These studies will help in understanding the mechanism(s) of the anti-carcinogenic effect of antioxidants and define the roles of GSTs in defense mechanisms against chemical carcinogenesis. Overall, studies proposed in this project will help immensely in developing strategies for the prevention of cancer.
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