Identification of cytochrome P450a (P450IIA1) as the principal testosterone 7α-hydroxylase in rat liver microsomes and its regulation by thyroid hormones

In the preceding paper, evidence was presented that rat liver microsomes contain two structurally related isozymes of cytochrome P450, namely cytochromes P450a and P450m, that can both catalyze the 7α-hydroxylation of testosterone. The aim of the present study was to determine the extent to which these two P450 isozymes are responsible for the 7α-hydroxylation of testosterone catalyzed by rat liver microsomes. Four monoclonal antibodies against cytochrome P450a, designated A2, A4, A5, and A7, were prepared in BALB/c mice. Monoclonal antibodies A2 (an IgM), A4 (an IgG2b), and A5 (an IgG1) were determined to be distinct immunoglobulins, whereas A7 could not be distinguished from A5. All of the antibodies were highly specific for cytochrome P450a; none cross-reacted with cytochrome P450m or with 10 other P450 isozymes purified from rat liver microsomes. Competition experiments between unlabeled and horseradish peroxidase-conjugated antibodies revealed that each of the monoclonal antibodies recognized the same epitope on cytochrome P450a. None of the monoclonal antibodies bound to denatured cytochrome P450a, suggesting that they each bound to a spatial epitope. A monospecific, polyclonal antibody against cytochrome P450a was also prepared, as described in the preceding paper. The levels of cytochrome P450a in liver microsomes were determined by single radial immunodiffusion, Western immunoblot (with polyclonal antibody), and enzyme-linked immunosorbent assay with monoclonal antibody. The levels of cytochrome P450a declined with age in male but not female rats, and were inducible up to 10-fold by treatment of rats with various xenobiotics. The levels of cytochrome P450a (but not cytochrome P450m) were also elevated (~3-fold) by thyroidectomy of mature male rats. Near normal levels of cytochrome P450a were restored by treatment of athyroid rats with triiodothyronine, whereas treatment with thyroxine was less effective in this regard. These changes in the levels of cytochrome P450a were highly correlated (r = 0.995) with changes in testosterone 7α-hydroxylase activity. None of the monoclonal antibodies inhibited the catalytic activity of cytochrome P450a when reconstituted with NADPH-cytochrome P450 reductase and lipid. In contrast, the polyclonal antibody not only inhibited the catalytic activity of purified cytochrome P450a, but also completely inhibited (>96%) the 7α-hydroxylation of testosterone catalyzed by liver microsomes from immature and mature rats of both sexes and by liver microsomes from male rats treated with a variety of cytochrome P450 inducers. Cytochrome P450m did not appear to contribute to the 7α-hydroxylation of testosterone catalyzed by rat liver microsomes. Although cytochrome P450m catalyzed the 15α-hydroxylation of testosterone in a reconstituted system, this reaction was a minor pathway of testosterone oxidation catalyzed by liver microsomes from mature male rats, which contain the highest levels of cytochrome P450m. In contrast to 7α-hydroxylation, the 6α-hydroxylation of testosterone was inhibited less than 40% by antibody against cytochrome P450a, except in liver microsomes from mature female rats (where the reaction was inhibited >95%). These results indicate that cytochrome P450a is solely responsible (>96%) for the 7α-hydroxylation of testosterone catalyzed by rat liver microsomes and suggest that enzymes other than cytochrome P450a can contribute significantly to the 6α-hydroxylation of testosterone.