Animals and Treatments. Female ACI rats, 7-8 weeks of age, were treated with a single 20-mg pellet containing 3 mg of E2 + 17 mg of cholesterol, implanted sub-pannicularly in the shoulder region, as previously described (16). Control animals received 20-mg cholesterol pellets. Animals were killed after 6, 12, or 28 weeks and tissues were removed for analyses.
Preparation of Sub-cellular Fractions. Cytosol and microsomal fractions were prepared by differential centrifugation as described previously (17), and stored at-80°C until used. Protein concentration was determined by the BCATM protein assay kit.
Enzyme Assays. NAD(P)H quinone oxidoreductase (NQO1) was measured as reported by Jaiswal et al. (18). Cytosolic glutathione S-transferase (GST), glutathione peroxidase (GPx) and catalase (CAT) activities were measured as described earlier (19-21). Sulfotransferase (SULT) activity was determined as described previously (22) using PAP[35S] as cofactor. NADPH- dependent oxidation of E2 (CYP450 assay) was measured as described previously (23) using 5 jiM (MG) or 50 |-iM (liver) [3H]-E2 at 37 °C, for 60 and 30 min, respectively. Blanks, heat-inactivated microsomes, were analyzed with and without NADPH or ascorbic acid. Glucuronidation of E2 was assayed as described previously (24). ACO:E2 activity was determined with 25 nM (MG) or 50 fiM (liver) [3H]-E2 as described previously (25). Kinetic parameters were determined using six different concentrations of substrate.
Statistical Analysis. Differences among means were assessed by ANOVA followed by Bonferroni post hoc test for P values < 0.05. The kinetic parameters (Km and Vmax) ofthe enzymatic reactions were derived graphically by computer linear regression analysis using Khaleida Graph for Windows program.
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