Covalent binding to proteins of reactive intermediates resulting from prostaglandin H synthase-catalyzed oxidation of stilbene and steroid estrogens.

Prostaglandin H synthase (PSH) is known to metabolically activate a variety of xenobiotics in vitro by means of its peroxidase activity. Recently, stilbene and steroid estrogens have been found to be cooxidized by ram seminal vesical microsomes, a rich source of PHS, to nonextractable metabolites bound to microsomal protein. To investigate further the nature of this protein binding, different radiolabeled estrogens were incubated with purified PHS, holoenzyme in the presence of various amounts of albumin (BSA), and radioactivity bound to protein was determined after gel electrophoretic separation. Diethylstilbestrol (DES), its analog hexestrol, and the steroid estrogens estrone and 2-hydroxy-estrone were cooxidized by PHS in vitro to metabolites that bound covalently to PHS and to BSA. Although a preferential binding of DES to PHS was found in the presence of excess BSA, reactive intermediates derived from DES, or from the other estrogens, were sufficiently stable to react with the competing nucleophile BSA as well. With respect to the metabolic reactions catalyzed by PHS, in addition to one-electron oxidation of phenolic functions, PHS catalyzed the aromatic hydroxylation of synthetic and steroid estrogens as shown by 3H2O release from regiospecifically labeled compounds and confirmed by product identification. Although DES was extensively metabolized by PHS, its aromatic hydroxylation was minor by comparison to estradiol, a difference possibly related to the compounds' redox potentials. Thus, cooxidation of estrogens in vitro resulted in phenoxy radicals, semiquinones and quinones, reactive intermediates capable of protein binding that may contribute to the adverse effects of stilbene and steroid estrogen observed in vivo and in short-term assays.