Specific binding of 4-hydroxyestradiol to mouse uterine protein: evidence of a physiological role for 4-hydroxyestradiol.

There are several indications of a possible physiological role for 4-hydroxyestradiol (4-OHE(2)) in hormone-responsive tissues. To examine a hormonal activity of 4-OHE(2), we have studied the binding of (3)H-labeled 4-OHE(2) to mouse uterine cytosolic protein. In uteri of 3-week-old mice, total binding was 319.4 +/- 13.9 fmol/mg protein. Binding in the presence of excess unlabeled 4-OHE(2) dropped to 82.1 +/- 1.7 fmol/mg protein, whereas 214.6 +/- 9.4 fmol/mg protein bound while incubating in an excess of unlabeled 17beta-estradiol (E(2)). The difference between the two binding values in the presence of excess steroid (132.5 +/- 11.1 fmol/mg protein) is taken as selective binding of 4-OHE(2) to a specific protein. In mice older than 4 weeks, the specific 4-OHE(2) binding declined: 32.0 +/- 4.0 fmol/mg protein at 8 weeks, 54.8 +/- 6.3 fmol/mg protein at 12 weeks and 54.6 +/- 5.2 fmol/mg protein at 9 months. Of other organs tested (liver, kidney, lung and whole brain) only lung showed significant selective binding of 4-OHE(2). When E(2)-binding sites are blocked, binding follows first-order kinetics, yielding a dissociation constant (K(d)) value of 11.8 +/- 2.1 nM. The specific binding of 4-OHE(2) was not inhibited by any other steroids or estrogen metabolites that were tested, except for 2-hydroxyestradiol (2-OHE(2)), which displayed competitive inhibition of 4-OHE(2) binding with an inhibition constant (K(i)) value of 98.2 +/- 12.6 nM. These results lead us to conclude that 4-OHE(2) binds to a specific binding protein, distinct and different from binding to estrogen receptors (ERalpha and ERbeta). The physiological role of this binding remains to be elucidated.

Concentration dependence of prooxidant and antioxidant properties of catecholestrogens.

Estradiol is an established antioxidant in vitro and in vivo. In contrast, prooxidant effects such as 8-hydroxylation of guanine bases of DNA have been induced by various estrogens in hamsters and by 4-hydroxyestradiol or -estrone and a microsomal activating system in vitro. As part of an examination of these conflicting reports, we studied the enhancement or inhibition of lipid peroxidation (conjugated diene formation monitored at 240 nm) by catecholestrogens in human low-density lipoprotein (LDL) incubated with cupric sulfate in phosphate buffer. Addition of 2- or 4-hydroxyestradiol, 2- or 4-methoxyestradiol, or estradiol or estriol (0.5-50 microM) increased lag times for diene formation by 30 to <300% over control values in the absence of estrogens (lag time, 1.6 h). In contrast, low concentrations (5 pM-100 nM) of catecholestrogens decreased lag times by about 40-50%, demonstrating their prooxidant activities. The prooxidant capabilities of catecholestrogens were examined by assaying the reduction by estrogens of Cu(II) to Cu(I) and of Fe(III) to Fe(II). Both 2- and 4-hydroxyestradiol and 2- and 4-methoxyestradiol reduced Cu(II) and Fe(III) ions to their lower oxidation state. In conclusion, the reduction of Cu(II) to Cu(I) by catecholestrogens is proposed to initiate lipid peroxidation and thus oxidation of LDL. In contrast, at high concentrations of catecholestrogens, the scavenging of oxygen radicals may predominate over lipid peroxidation and free radical generation by analogy to the action of similar phenolic antioxidants. With estradiol, estriol, and the methoxyestrogen metabolites, only antioxidant effects were observed.