This research proposal relates to the observed accumulation of the cytotoxic, mutagenic, and carcinogenic cholesterol epoxides in the human prostate gland detected just prior to and invariably with the development of the male diseases of benign prostatic hyperplasia and prostatic carcinoma. The additional observations that these cholesterol metabolites are also detected in canine prostatic hypertrophy and not in the rat prostate gland, which rarely develops either benign or malignant tumors, suggests that the epoxycholesterols might be important factors in the etiology and pathogenesis of human and canine prostatic diseases. The more recent additional observation that the in vivo administration of cholesterol alpha epoxide to the ventral prostate of the rat resulting in the significant enlargement of the gland within six weeks and associated with the development of hyperplasia and metaplasia makes it even more likely that the epoxycholesterols are important factors in the disease process. The overall purpose of this research proposal is to study the endogenous formation and metabolism of the cholesterol epoxides in the human prostate gland. Human prostate tissues characterized histopathologically as normal and those exhibiting benign hyperplasia and prostatic carcinoma will be examined for their ability to synthesize cholesterol epoxides from isotopically-labeled cholesterol. With cholesterol epoxide as a substrate the microsomal and cytosolic gluthathione S-epoxide transferases (EC 2.5.1.18) and epoxide hydrolases (EC 3.3.2.3) as isoenzymes will be examined in detail. This would include their isolation and purification to homogeneity employing tissue homogenization, centrifugation, affinity and fast protein liquid chromatography. Fractions will be analyzed for enzymatic activity and protein content. Enzyme homogeneity as well as pI values will be determined by isoelectric focusing (IEF). The enzymes will be characterized further as to substrate specificity, molecular weight, subunit molecular weight, kinetic properties, pH activity profile, specific activity, temperature stability, metal ion requirements, pI values, amino acid composition, peptide mapping, amino terminal sequencing, and immunological properties. The human prostate tissues will also be examined for their content of cholesterol, cholesterol epoxides, glutathione conjugates, and cholestane triol. Attempts will also be made to determine both qualitatively and quantitatively the glutathione S-transferase and epoxide hydrolase content of normal and diseased human prostate tissues. The in vivo effect of the epoxy- cholesterols on the ventral prostate gland of the rat will also b studied in detail.
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