Dietary antioxidants such as t-butylated hydroxytoluene (BHT) and t-butylated hydroxyanisole (BHA) are known to retard chemical carcinogenesis induced by polycyclic aromatic hydrocarbons (PAH) such as benzo(a)pyrene (BP) in lung of mice. Recent studies provide strong evidence that this activity of the antioxidants is due to the induction of glutathione S-transferases (GST). GST isoenzymes, which arise from the different dimeric combinations of a number of distinct subunits, represent a family of multifunctional proteins. GST can detoxify various electrophilic xenobiotics and their metabolites (such as the highly carcinogenic epoxide diols of BP) by conjugating them to glutathione. Also, through non-catalytic binding, GST can remove the toxic PAH metabolites and other xenobiotics from circulation. We have shown that the different subunits of lung isoenzymes differ in their substrate specificities and non-catalytic binding properties, and in the lung and liver of rats and mice, these subunits are differentially induced by BHA and BHT. Also, only certain specific subunits of rat and mouse lung GST bind BP and its metabolites in vivo. In order to evaluate the effect of antioxidants on the detoxification functions, the isoenzymes of rat, mouse, and human lung and liver GST will be purified and studied for their functional and structural characteristics. The individual subunits of human, rat, and mouse lung isoenzymes will be isolated as active homodimers by in vivo hybridization of the subunits of purified isoenzymes. These subunits will be studied for their substrate specificities towards various xenobiotics, particularly with the PAH metabolites. In vitro binding of the human, rat, and mouse lung GST isoenzymes and their subunits with PAH and their metabolites will be studied. The antibodies specific to the various subunits of rats, and mouse lung and liver enzymes will be prepared, and using the techniques of ELISA, immunoprecipitation, and immunoaffinity chromatography, the effect of the antioxidants on the individual subunits will be quantitated. The in vivo bindind of radiolabelled BP and other PAH to lung GST subnits will be studied and quantitated in the control as well as BHA/BHT-treated rats and mice. The antibodies will also be used in immunofluorescence studies to localize various GST subunits in different cell types of lung. These studies on the effect of antioxidants on the detoxification functions of GST will not only help in elucidating the mechanism of antineoplastic activity of antioxidants, but may also, in the long term, provide a basis for developing inhibitors for chemical carcinogenesis.
Showing the most recent 10 out of 76 publications
