Distinct forms of hepatic androgen 6 beta-hydroxylase induced in the rat by indole-3-carbinol and pregnenolone carbonitrile.

The ability of indole-3-carbinol (IC), an anticarcinogen present in cruciferous vegetables, to induce CYP1A1, CYP1A2, CYP2B1/2, CYP2E1 and CYP3A1/2 in female rat liver was determined by Western analysis using monoclonal antibodies and compared to effects produced by pregnenolone carbonitrile in animals of both sexes. The ontogeny of induction of these cytochrome P450 isozymes in response to oral administration of IC was also investigated. An inverse correlation was observed between the 6 beta-hydroxylation of androsterone (A) and the induction by IC of CYP3A1/2, the P450 isozyme responsible for the bulk of hepatic 6 beta-hydroxylation of 4-androstenedione (AD). The effect of inhibitors on the formation of 6 beta-OHA from A or AD was also determined and shown to differ from their action on the P450 isozymes involved in the formation of the 6 beta-hydroxylated derivatives of AD or lithocholic acid. The results indicate that the enzyme induced by IC is distinct from the CYP3A1/2 which catalyzes hydroxylations at position 6 beta, allylic in AD but not in the fully saturated ring system of A. The increased hepatic conversion of A to its biologically less active 6 beta-OHA metabolite after treatment of female rats with IC could possibly contribute to the anticarcinogenic action of indole carbinols. It is also proposed that the action of multiple inducers present in cruciferous and other vegetables might produce androgen metabolic profiles very different from those produced by individual components isolated from them.

Antiandrogenic property of RU 486: enhancement of estrogen-induced uterine peroxidase activity in the rat.

The contragestational steroid RU 486 enhanced the increase in peroxidase activity produced by estradiol in estrogen-primed immature rat uteri and, like the antiandrogen flutamide, RU 486 reversed the inhibitory effect of testosterone on this estrogen-induced response. It antagonized the inhibition produced by progesterone but had no effect on peroxidase induction by itself or in unprimed immature animals. RU 486 also enhanced the effect of estradiol on the synthesis of complement component C3 in the rat uterus. The results confirm that RU 486 possesses antiandrogenic as well as antiprogestational properties. They also suggest that, in normal adult animals, the increase in peroxidase activity in the uterus in response to estrogen is not expressed fully but held in check by other endogenous steroids acting through their individual receptors.

Differential effect of Cu2+ and Zn2+ on the formation and further metabolism of catechol estrogen by rat liver microsomes.

The action of a number of different divalent metal ions on the rat liver microsomal release of 3H2O from estradiol and 2-hydroxyestradiol labeled with 3H at C-2 or C-4 was investigated. Cu2+ at low concentration (10 microM) produced a marked and specific inhibition of the 2-hydroxylation of estradiol with virtually no effect on the further oxidative activation of catechol estrogen. In contrast, Zn2+ inhibited the interaction of 2-hydroxyestradiol with microsomal protein as measured by the release of 3H from C-4 of the labeled steroids but did not influence 2-hydroxylation, except at high concentration. Other metal ions tested produced little or no change. Cu2+ inhibited the irreversible binding of estradiol to protein but activated this reaction with the catechol estrogen as substrate. The action of both Cu2+ and Zn2+ was reversed by glutathione. The differential effect of these metal ions on estrogen metabolism gives additional support for two different mechanisms in the cytochrome P-450-catalyzed formation of catechol estrogens and their further activation to form protein conjugates.

Inhibition of catechol estrogen formation in rat liver microsomes by hormonal steroids and related compounds.

The inhibitory action of a number of different hormonal steroids and related compounds on the 2-hydroxylation of estradiol by male rat liver microsomes was examined by a radiometric assay. Progesterone, Diethylstilbestrol, testosterone and 4-androstenedione were found to be the most potent of the compounds tested but inhibition was also observed with other steroids and a group of androgen analogs which are aromatization inhibitors. The kinetic constant Ki for those steroids which gave linear double reciprocal plots when added to [2-3H]estradiol was determined and the products from [14C]estradiol in the presence of the inhibitors were examined by TLC and autoradiography. The addition of steroids with a 17-hydroxyl group such as testosterone or dihydroequilin resulted in the formation of mainly 2-hydroxyestradiol with smaller amounts of other metabolites while those with a reducible ketonic group such as progesterone, 4-androstenedione, equilin or equilenin gave rise to considerable amounts of estrone in addition to the catechol estrogens. Further purification of the liver microsomes did not alter this effect. The possible role of progesterone and the catechol estrogens in the control of estrogen hydroxylation in liver as well as other aspects of steroid interaction are discussed.

Metabolism of estradiol by true and pseudoperoxidases.

The activation of 14C-labeled estradiol by "true" and "pseudo" peroxidases to form conjugates and other products was compared in four model systems using H2O2, glutathione, Mn2+ or irradiated riboflavin. Albumin was used as acceptor except in the glutathione system. The binding of estradiol to glutathione in the presence of the true peroxidases, lacto- or uterine peroxidase (no H2O2 added), was also examined and the conditions shown to differ from those required with the pseudoperoxidases, microperoxidase or trypsin-digested cytochrome c. The conjugates were purified by chromatography after elution from Amberlite XAD-2 and the relative amounts of these products assessed by autoradiography. The ratio of steroid to glutathione in the main water-soluble metabolite formed with lactoperoxidase was found to be approx 1:1 in a double label experiment with [14C]estradiol and [3H]glutathione. It was also shown, using estradiol labeled with 3H in different positions of the steroid molecule, that lactoperoxidase acts non-specifically in catalyzing the formation of glutathionyl conjugates as indicated by the release of 3H2O. The possible role of peroxidase and glutathione in the metabolism of estrogens and in the formation of artifactual products is discussed.

Androgen receptor-mediated inhibition of estrogen-induced uterine peroxidase.

Flutamide, an anti-androgen known to act through the androgen receptor, abolished the inhibitory action of testosterone on the induction of peroxidase in immature rat uteri without affecting inhibition produced by progesterone. The time course of the androgen effect on estrogen-induced uterine peroxidase, uterine weight and glucose 6-phosphate dehydrogenase activity was also determined together with the effect of flutamide on these steroid hormone-sensitive parameters. The possible mechanism of action of these compounds is discussed, particularly in the light of estrogen-induced eosinophilia. It is proposed that the observed interaction between testosterone and estradiol is mediated through their own specific receptors and not by illicit occupation of the estrogen receptor by the androgen. 5-Androstene-3 beta, 17 beta-diol (Adiol), an androgen known to exert estrogenic effects through the estrogen receptor, induced uterine peroxidase and was without significant effect on the action of estradiol, in contrast to testosterone.

Estrogen-androgen interaction: selective inhibition of estrogen-induced uterine peroxidase by testosterone and 5 alpha-dihydrotestosterone.

The effect of testosterone (T) and dihydrotestosterone (DHT) on the induction of peroxidase and glucose-6-phosphate dehydrogenase (G6PDH) in the immature rat uterus by estradiol (E2) was investigated. T (0.5-12.5 mg/rat) given on 3 successive days produced a large increase in uterine weight but, in contrast to E2, did not induce peroxidase or significantly augment uterine G6PDH. However, this androgen, even at a very low dose (50 micrograms/rat three times), inhibited the induction of peroxidase without a corresponding effect on G6PDH when given concurrently with E2 and was more effective than DHT. Inhibition by androgen was also observed when diethylstilbestrol was used to stimulate uterine growth. Combined treatment with E2 (3 micrograms/rat) and T (3 mg/rat) produced a cytosolic and nuclear estrogen receptor pattern in the uterus similar to that observed with E2 alone after various time intervals. The results speak against a direct inhibitory action or T on E2-induced uterine peroxidase via the estrogen receptor and confirm the lack of aromatization of T to E2 in the immature rat. Possible mechanisms for modifying the action of estrogens by androgens are discussed, particularly in the light of E2-induced eosinophilia. It is proposed that steroid hormones can interact in several ways and that uterine peroxidase provides a useful indicator to study steroid hormone action.

Modulation of estrogen-induced peroxidase activity in the rat uterus by thyroid hormones.

The effect of thyroid hormones, iodide, propylthiouracil and other thyroid-active agents on estrogen receptor concentration and on the induction of peroxidase in the immature rat uterus by estradiol (E2) was examined T3 (0.5 mg/Kg) given daily for 6 days produced a large decrease in E2-induced uterine peroxidase but lowered only slightly the concentration of cytosolic or nuclear receptors in this organ. T4 also decreased the effect of E2 on uterine peroxidase induction and the inhibitory action of T3 was observed in thyroidectomized rats. The administration of iodide (10 mM) in the drinking water for 6 days caused a 2-fold increase in estrogen-induced peroxidase in both normal and thyroidectomized immature animals without influencing the estrogen receptor concentration in the uterus. The effect of other thyroid-active agents on serum T3 and T4 levels was also determined. Treatment for 6 days with propylthiouracil (6 mM) increased the concentration of estrogen receptors in the uterus and decreased serum T3 and T4 levels without any effect on uterine peroxidase activity while bromide and perchlorate did not influence these parameters. Possible mechanisms for the inhibitory effects of thyroid hormones on uterine peroxidase induction by E2 are discussed.

Distribution of oestrogen-induced peroxidase in the rat uterus.

The activity of oestrogen-induced peroxidase in sections along the uterus of normal and mammary tumour-bearing adult rats was measured. Oestradiol increased the activity of this enzyme in the cervix as well as in other parts of the uterus in ovariectomized or immature rats. Peroxidase activity per mg protein was twice as high in the cervix as in the rest of the uterus where it was evenly distributed along both horns. The concentrations of oestrogen receptors in the cytosol and nucleus in each uterine horn and in the cervix was also determined and found to be lower in the cervix than in other sections of the uterus.

Inhibition of hepatic aryl hydrocarbon hydroxylase by 3-methylcholanthrene, 7,8-benzoflavone and other inducers added in vitro.

A close correlation has been observed between the ability of aromatic polycyclic hydrocarbons and 7,8-benzoflavone (7,8-BF) to induce hepatic aryl hydrocarbon hydroxylase (AHH) in vivo and to inhibit the induced enzyme system in vitro. The activity of this mono-oxygenase was measured by the conversion of 14C-labeled dimethylbenz(a)anthracene (DMBA) or benzo(a)pyrene (BP) to water-soluble products by rat liver preparations (8000 X g supernatant). DMBA as substrate had the advantage over BP in giving a wider range of ethyl acetate-soluble metabolites and allowing the observation of changes in the pattern of these products following injection or addition of the inducing agents. This property was used to detect low concentration (0.1 muM) of polycyclic hydrocarbons which are strong AHH inducers and which may also be carcinogenic. The liver preparation was active for several months when stored at --20 degrees. A possible mechanism of action for the in vitro behaviour of polycyclic hydrocarbons and 7,8-BF towards AHH is proposed.