2-hydroxyestradiol and cumene-hydroperoxide

We demonstrated for the first time that nuclei were able to convert a stilbene estrogen (diethylstilbestrol) to reactive metabolites, which covalently bind to nuclear proteins and DNA. Depending on the cofactor used, nuclear enzymes catalyzed oxidation and/or reduction of stilbene and steroid estrogens. 2-Hydroxyestradiol (a major metabolite of steroidal estrogen, 17 beta-estradiol) and diethylstilbestrol (DES) were oxidized to 2,3-estradiol quinone and DES quinone, respectively, by peroxide-supported nuclear cytochromes P450. A Lineweaver-Burk plot of rate of formation of DES quinone at various substrate concentrations yielded a Km = 15 microM and Vmax = 10 nmol/mg protein/min. The oxidation of DES to DES quinone by nuclei was drastically decreased by known inhibitors of cytochromes P450. DES quinone was reduced back to DES by nuclei in the presence of NADPH, presumably through cytochrome P450 reductase. The reduction of DES quinone to DES by nuclei was significantly inhibited by antibodies and inhibitors of cytochrome P450 reductase. Under reaction conditions similar to oxidation of DES to DES quinone by nuclei, it was observed that nuclear metabolic products of DES were able to covalently bind to nuclear proteins and DNA. The data reported here establish that DES and a catechol estrogen can be oxidized to quinones and that the quinones may be reduced back to the hydroquinones by nuclear preparations when fortified with an appropriate cofactor and that reactive intermediates are involved based on observed covalent binding to macromolecules. The significance of these events, and their possible role in toxicity/cancer/teratogenicity, however, is not at all clear.

Topics: Animals; Benzene Derivatives; Cell Nucleus; Diethylstilbestrol; DNA; Estradiol; In Vitro Techniques; Male; NADP; Nuclear Proteins; Oxidation-Reduction; Quinones; Rats; Rats, Sprague-Dawley; Spectrum Analysis

Estrogen-2/4-hydroxylase (E-2/4-H) activity of rabbit hypothalamic tissue was previously found to be localized in the soluble subcellular fraction. In the present study, the enzymatic activity responsible for catechol estrogen formation in this subcellular fraction of the rabbit hypothalamus was purified by ammonium sulfate fractionation, ion exchange chromatography, and chromatofocusing. E-2/4-H activity was found to be associated with a group of hemoproteins with peroxidase activity. The characteristics of this hypothalamic E-2/4-H activity were reestablished in light of a peroxidatic mechanism for catechol estrogen formation. Organic hydroperoxides stimulated E-2/4-H activity, presumably by serving as oxidizing cosubstrate required for peroxidase-mediated reactions. E-2/4-H activity in a 17,500 X g supernatant of hypothalamic tissue was linear with time for at least 10 min and with protein concentration to at least 100 micrograms/150 microliter. It displayed a pH optimum of 6 and an apparent Michaelis-Menten constant (Km) of 32 microM with respect to estradiol. The amounts of 4-hydroxyestradiol formed were comparable to those of 2-hydroxyestradiol. The characteristics established in this study for the peroxidase-type E-2/4-H were distinct from those of the particulate, NADPH-dependent 2-hydroxylases found in rat liver and in porcine blastocyst and ovary. These differences provide a basis for differentiating between the two types of enzymatic activity that can lead to catechol estrogen formation in vitro.

Topics: Animals; Aryl Hydrocarbon Hydroxylases; Benzene Derivatives; Chromatography, Ion Exchange; Cytochrome P-450 CYP1A1; Cytochrome P-450 CYP1B1; Estradiol; Estrogens, Catechol; Hydrogen-Ion Concentration; Hypothalamus; Isoelectric Focusing; Polysorbates; Rabbits; Steroid Hydroxylases; Time Factors