ascorbic-acid and 2-hydroxyestradiol

Estradiol 17β (E2β) and ascorbic acid (AA) have been implicated in cancer progression. However, little is known about the actions of biologically active metabolites of E2β, 2-hydroxyestradiol (2OHE2), 4-hydroxyestradiol (4OHE2), 2-methoxyestradiol (2ME2), and 4-methoxyestradiol (4ME2) synthesized sequentially by cytochrome P450, family 1, subfamily A (CYP1A1) and B (CYP1B1), polypeptide 1, and catechol-O-methyltransferase (COMT) on ovarian cancer. Herein, we examined the expression of CYP1A1, CYP1B1, COMT, and estrogen receptor α (ERα) and β (ERβ) in human ovarian surface epithelial (IOSE-385) and cancer cell lines (OVCAR-3, SKOV-3, and OVCA-432). We also investigated the roles of E2β, 2OHE2, 4OHE2, 2ME2, and 4ME2 in cell proliferation, and their interactive effects with AA on ovarian cells. We found the expression of CYP1A1, CYP1B1, COMT, ERα, and ERβ in most cell lines tested. Treating cells with physiological concentrations of E2β and its metabolites promoted (13%-42% of the control) IOSE-385 and OVCAR-3 proliferation. The ER blockade inhibited IOSE-385 (∼76%) and OVCAR-3 (∼87%) proliferative response to E2β but not to its metabolites. The ERα blockade inhibited (∼85%) E2β-stimulated OVCAR-3 proliferation, whereas ERβ blockade attenuated (∼83%) E2β-stimulated IOSE-385 proliferation. The AA at ≥250 μmol/L completely inhibited serum-stimulated cell proliferation in all cell lines tested; however, such inhibition in IOSE-385, OVCAR-3, and OVCA-432 was partially (∼10%-20%) countered by E2β and its metabolites. Thus, our findings indicate that E2β and its metabolites promote cell proliferation and antagonize the AA-suppressed cell proliferation in a subset of ovarian cancer cells, suggesting that blocking the actions of E2β and its metabolites may enhance AA's antiovarian cancer activity.

Topics: 2-Methoxyestradiol; Aryl Hydrocarbon Hydroxylases; Ascorbic Acid; Catechol O-Methyltransferase; Cell Line, Tumor; Cell Proliferation; Cytochrome P-450 CYP1A1; Cytochrome P-450 CYP1B1; Dose-Response Relationship, Drug; Drug Interactions; Estradiol; Estrogen Antagonists; Estrogen Receptor alpha; Estrogen Receptor beta; Estrogens, Catechol; Female; Humans; Ovarian Neoplasms; Time Factors

The mechanisms underlying the pathogenesis of estrogen-induced breast carcinogenesis remain unclear. The present study investigated the roles of estrogen metabolism and oxidative stress in estrogen-mediated mammary carcinogenesis in vivo. Female August Copenhagen Irish (ACI) rats were treated with 17beta-estradiol (E(2)), the antioxidant vitamin C, the estrogen metabolic inhibitor alpha-naphthoflavone (ANF), or cotreated with E(2) + vitamin C or E(2) + ANF for up to 8 months. E(2) (3 mg) was administered as an subcutaneous implant, ANF was given via diet (0.2%) and vitamin C (1%) was added to drinking water. At necropsy, breast tumor incidence in the E(2), E(2) + vitamin C and E(2) + ANF groups was 82, 29 and 0%, respectively. Vitamin C and ANF attenuated E(2)-induced alterations in oxidative stress markers in breast tissue, including 8-iso-prostane F(2alpha) formation and changes in the activities of antioxidant enzymes superoxide dismutase and glutathione peroxidase. Quantification of 2-hydroxyestradiol (2-OHE(2)) and 4-hydroxyestradiol (4-OHE(2)) formation in breast tissue confirmed that ANF inhibited 4-hydroxylation of E(2) and decreased formation of the highly carcinogenic 4-OHE(2). These results demonstrate that antioxidant vitamin C reduces the incidence of estrogen-induced mammary tumors, increases tumor latency and decreases oxidative stress in vivo. Further, our data indicate that ANF completely abrogates breast cancer development in ACI rats. The present study is the first to demonstrate the inhibition of breast carcinogenesis by antioxidant vitamin C or the estrogen metabolic inhibitor ANF in an animal model of estrogen-induced mammary carcinogenesis. Taken together, these results suggest that E(2) metabolism and oxidant stress are critically involved in estrogen-induced breast carcinogenesis.

Topics: Animals; Antioxidants; Ascorbic Acid; Benzoflavones; Cell Transformation, Neoplastic; Dinoprost; Estradiol; Estrogens, Catechol; Female; Mammary Neoplasms, Experimental; Neoplasms, Hormone-Dependent; Oxidative Stress; Rats; Rats, Inbred ACI

Catechol estrogens are considered critical intermediates in estrogen-induced carcinogenesis. We demonstrated previously that 17beta-estradiol (E(2)), estrone (E(1)) and four of their catechol estrogens, 2- and 4-hydroxyestradiols (2- and 4-OHE(2)), and 2- and 4-hydroxyestrones (2- and 4-OHE(1)) induce morphological transformation in Syrian hamster embryo (SHE) fibroblasts, and the transforming abilities vary as follows: 4-OHE(1) > 2-OHE(1) > 4-OHE(2) > 2-OHE(2) vertical line E(2), E(1). To examine the involvement of catechol estrogens in the initiation of hormonal carcinogenesis, we studied the ability of E(2), E(1) and their catechol estrogens to induce DNA adducts in SHE cells by using a (32)P-post-labeling assay. DNA adducts were detected in cells treated with each of all the catechol estrogens at concentrations of 10 microg/ml for 1 h and more. 2- or 4-OHE(2) formed a single DNA adduct, which was chromatographically distinct from each other. In contrast, 2- or 4-OHE(1) produced one major and one minor adduct, and the two adducts formed by each catechol estrogen exhibited identical mobilities on the chromatograms. Neither E(2) nor E(1) at concentrations up to 30 microg/ml induced DNA adducts. The abilities of the estrogens to induce DNA adducts were ranked as follows: 4-OHE(1) > 2-OHE(1) > 4-OHE(2) > 2-OHE(2) > > E(2), E(1), which corresponds well to the transforming and carcinogenic abilities of the estrogens. In addition, the level of DNA adducts induced by the catechol estrogens was markedly decreased by co-treatment of cells with the antioxidant L-ascorbic acid. The results indicate the possible involvement of oxidative metabolites of catechol estrogens of E(2) and E(1) in the initiation of endogenous estrogen-induced carcinogenesis.

Topics: Animals; Antioxidants; Ascorbic Acid; Cell Survival; Cell Transformation, Neoplastic; Cricetinae; DNA Adducts; Estradiol; Estrogens, Catechol; Fibroblasts; Hydroxyestrones; Mesocricetus

The effect of estrogens, including estrone (E1), estradiol-17beta (E2), estriol (E3) and 2-hydroxyestradiol (2-OH-E2), on the oxidative damage induced by ferrylmyoglobin (ferrylMb) was investigated. These estrogens inhibited lipid peroxidation induced by ferrylMb. The ability of 2-OH-E2 to inhibit lipid peroxidation was much greater than the other estrogens. Furthermore, 2-OH-E2 trapped 2,2'-azobis-(2-amidinopropane)-dihydrochloride peroxyl radicals more rapidly, and among these estrogens only 2-OH-E2 reacted with 2,2-diphenyl-1-picrylhydrazyl. These results suggest that the ability of 2-OH-E2 to inhibit lipid peroxidation is because it scavenges lipid peroxyl and carbon-centered radicals. Estrogens, except for 2-OH-E2, partially prevented the inactivation of alcohol dehydrogenase (ADH) induced by ferrylMb. Of interest, however, the exposure of sulfhydryl (SH) enzymes to ferrylMb in the presence of 2-OH-E2 dramatically increased the inhibition of the enzyme activity. Ascorbic acid (ASA) and reduced glutathione (GSH) strongly inhibited the inactivation of ADH induced by ferrylMb in the presence of 2-OH-E2. During the reaction of ferrylMb with ASA or GSH in the presence of 2-OH-E2, large amounts of oxymyoglobin were formed, suggesting the involvement of the semiquinone from 2-OH-E2 in the reduction of metmyoglobin. Presumably, the semiquinone formed from 2-OH-E2 oxidizes the SH group of enzymes to facilitate the rapid inactivation of the SH enzymes induced by ferrylMb.

Topics: Alcohol Dehydrogenase; Animals; Ascorbic Acid; Estradiol; Estriol; Estrogens; Estrone; Free Radical Scavengers; Glutathione; Kinetics; Lipid Peroxidation; Metmyoglobin; Microsomes, Liver; Rats; Thiobarbituric Acid Reactive Substances

We have shown that the metabolism of 17 beta-estradiol in hamster liver microsomes is concentration-dependent. At low (< 25 microM) concentrations of 17 beta-estriol, 16 alpha-hydroxylase activity predominated, and estriol was the major metabolite. At higher concentrations (25-75 microM), 16 alpha-hydroxylation and aromatic hydroxylation at C2 contributed equally to 17 beta-estradiol metabolism. Aromatic C4-hydroxylation was maximal at 75 microM of 17 beta-estradiol and was always less than C2-hydroxylation. Dehydrogenation of the 17 beta-hydroxyl group to the ketone (estrone) was also observed, but both estrone and 2-hydroxyestrone were minor (approximately 3%) metabolites of 17 beta-estradiol, only detectable at concentrations of 50 microM and above. Catechol-O-methyl transferase (COMT) effectively converted both 2- and 4-hydroxyl-17 beta-estradiol to their corresponding monomethoxy metabolites. Effective reducing conditions are required for COMT activity, because catechol estrogens are readily oxidized to their corresponding ortho-quinones, and ascorbic acid is routinely added to assays of COMT activity. Interestingly, although ascorbic acid (1 mM) increased the recovery of 2- and 4-hydroxy-17 beta-estradiol from microsomal incubations, it decreased the recovery of the methoxy metabolites (approximately 40%). Since the enediol function of ascorbate resembles that of a catechol group, ascorbate is a substrate for COMT and probably competes with the catechol estrogens for methylation. Because previous studies describing the ability of COMT to inhibit the covalent binding of electrophilic reactive metabolites of [4-(14)C]17 beta-estradiol to microsomal protein were performed in the presence of high (100 mM) Mg2+ concentrations, we also investigated the effects of Mg2+ on 17 beta-estradiol metabolism. Concentrations of Mg2+ > 10 mM inhibited the metabolism of 17 beta-estradiol, as evidenced by i) the increased recovery of substrate; ii) a decrease in the formation of estriol, estrone, and 2-, and 4-hydroxy-17 beta-estradiol; iii) a decrease in the recovery of water-soluble metabolites when incubations were performed in the presence of glutathione (GSH) to trap the reactive electrophilic metabolites; and iv) a decrease in the amount of reactive electrophilic metabolites bound to microsomal protein. GSH also decreased the covalent binding of electrophilic metabolites of [4-(14)C]17 beta-estradiol to microsomal protein, with the concomitant formation of water

Topics: Animals; Ascorbic Acid; Catechol O-Methyltransferase; Cricetinae; Estradiol; Estrogens, Catechol; Glutathione; Inactivation, Metabolic; Magnesium; Male; Mesocricetus; Methylation; Microsomes, Liver; S-Adenosylmethionine; Steroid 16-alpha-Hydroxylase

We investigated the inhibition mechanism of lipid peroxidation by estrogens. Estradiol and 2-hydroxyestradiol showed strong inhibitory activities toward NADPH and ADP-Fe(3+)-dependent lipid peroxidations in the microsomes from rat livers only when the steroids were added to the reaction system before the start of the peroxidation reaction. These steroids also strongly inhibited oxygen uptake only when added before the start of the reaction. These results suggest that estradiol and 2-hydroxyestradiol inhibit the initial stage of microsomal lipid peroxidation. Lipid peroxidation of erythrocyte membranes induced by the systems of xanthine oxidase-hypoxanthine and ascorbate was strongly inhibited by 2-hydroxyestradiol, but not by estradiol. Lipid peroxidation of erythrocyte membranes induced by 2.2'-azobis- (amidinopropane) dihydrochloride was not markedly inhibited by estradiol and 2-hydroxyestradiol, suggesting that the steroids have low reactivity with lipid peroxyl radicals. However, lipid peroxidation induced by t-butyl hydroperoxide-Fe3+ was strongly inhibited only by 2-hydroxyestradiol. It seems that 2-hydroxyestradiol may interact with alkoxyl rather than with peroxyl radicals during lipid peroxidation.

Topics: Adenosine Diphosphate; Amidines; Animals; Ascorbic Acid; Butylated Hydroxytoluene; Dose-Response Relationship, Drug; Erythrocyte Membrane; Estradiol; Free Radical Scavengers; Hypoxanthine; Iron; Lipid Peroxidation; Microsomes, Liver; NADP; Rats; Rats, Wistar; Thiobarbituric Acid Reactive Substances; Xanthine Oxidase

Compounds like indole-3-carbinol (I3C) have been shown to increase catechol estrogen formation and reduce mammary tumor incidence in mice. These compounds may exert a protective effect for breast cancer development by decreasing the overall estrogen pool available for the formation of 16 alpha-hydroxyestrone (16 alpha-OHE1), a metabolite that retains significant estrogenic activity, may be mutagenic and could represent a potential carcinogenic intermediate of estradiol degradation. I3C and ascorbigen originate from the breakdown of glucobrassicin. We have compared the inductive effects of I3C with ascorbigen and beta-naphthaflavone (Bnf) in microsomes from rats pretreated with these compounds using isotope dilution GC-MS and a radiometric method. Incubated microsomes from rats pretreated with I3C and ascorbigen yielded high levels of 2-hydroxyestradiol (2-OHE2) that were comparable to levels induced by Bnf and were significantly above control group levels (p < 0.005). Absolute values determined by the radiometric method were approximately 40% lower than 2-OHE2 concentrations determined by GC-MS, although the relative changes in each group were the same. These differences may be attributed to the radiolabel becoming trapped in microsomal intermediates in the sequence leading to tritium entering the aqueous compartment. Both ascorbigen- and Bnf-treated animals exhibited significant increases in 2-hydroxyestrone (2-OHE1) (p < 0.05). The ability of ascorbigen to induce estradiol C-2 hydroxylation has not been previously reported. Based on these data, we speculate that ascorbigen will act as an anticarcinogenic agent and will inhibit or reduce the incidence of mammary tumor formation.

Topics: Animals; Ascorbic Acid; Benzoflavones; beta-Naphthoflavone; Estradiol; Estrogens, Catechol; Female; Gas Chromatography-Mass Spectrometry; Hydroxyestrones; Hydroxylation; Indoles; Microsomes, Liver; Radiometry; Rats; Rats, Sprague-Dawley

Porcine granulosa cells synthesize and respond to catecholestrogens, but the stimulatory potency of catecholestrogens on progesterone production is much less than that of estradiol (E2). Therefore, to determine if metabolism of catecholestrogens by granulosa cells could account for the reduced potency of 2-hydroxyestradiol (2-OH-E2) observed in vitro, porcine granulosa cells were cultured with [3H]2-OH-E2 and medium collected at 0, 0.5, 1, 2, 4, 6, or 12 h in the presence or absence of 1 microgram/ml 2-OH-E2, 0.5 mM L-ascorbate or 10 microM U-0521 (a specific catechol-O-methyltransferase inhibitor). Metabolism of [3H]2-OH-E2 was very rapid with only 16% of the original [3H]2-OH-E2 remaining after 4 h exposure to cells. The main metabolite comigrated with 2-methoxyestradiol (2-MeO-E2) on thin-layer chromatography. Although appreciable degradation of [3H]2-OH-E2 occurred with time in the absence of cells, formation of the O-methyl derivative was minimal. Rather, formation of polar metabolites occurred in the absence of cells. Ascorbate dramatically reduced this noncellular degradation. Ascorbate added to cell cultures had no effect on the rate of formation of O-methyl products but slowed the formation of polar compounds as well as the overall rate of degradation of [3H]2-OH-E2 by nearly 2-fold. U-0521 completely blocked the formation of O-methyl products, slowed the overall rate of degradation of [3H]2-OH-E2 by half and resulted in an increase in polar metabolites. The effects of U-0521 and ascorbate on 2-OH-E2-stimulated progesterone production in vitro was also examined. Ascorbate (0.5 mM) enhanced the effect of 2-OH-E2 (but not E2) on progesterone production by 2-fold (p less than 0.05). The addition of 10 microM U-0521 in the presence of 0.5 mM ascorbate had no effect on 1 microgram/ml 2-OH-E2-stimulated progesterone production, but it increased (p less than 0.05) the response to 4 micrograms/ml 2-OH-E2. The effects of 2-MeO-E2, 2-OH-E2, and E2 on progesterone production by cultured granulosa cells were then compared. The ED50 of E2 was 6- to 8-fold lower than that of 2-OH-E2 and 2-MeO-E2, whereas the ED50 of 2-OH-E2 was 15% lower than that of 2-MeO-E2. In the presence of ascorbate (0.5 mM), the maximal effect of E2 and 2-OH-E2 was approximately equal, whereas 2-OH-E2 was nearly 2-fold more efficacious than 2-MeO-E2.(ABSTRACT TRUNCATED AT 400 WORDS)

Topics: 2-Methoxyestradiol; Animals; Ascorbic Acid; Catechol O-Methyltransferase; Catechol O-Methyltransferase Inhibitors; Cell Count; Cells, Cultured; Dose-Response Relationship, Drug; Estradiol; Female; Granulosa Cells; Progesterone; Propiophenones; Swine

Formation of the catechol estrogens 2- and 4-hydroxyestradiol (2-OHE2 and 4-OHE2) from estradiol by pig blastocysts was studied using a direct product isolation assay for estrogen-2/4-hydroxylase (E-2/4-H). Blastocyst E-2/4-H activity was characterized biochemically using homogenates of blastocysts obtained on day 12 of pregnancy. This information was used to establish appropriate incubation conditions for the assay of E-2/4-H activity in blastocysts during the preimplantation period. Catechol estrogen formation was linear with time for up to 30 min and with blastocyst protein concentrations of up to 100 micrograms in a reaction volume of 150 microliters. The E-2/4-H activity of pig blastocysts was maximal at pH 7.9 and was not affected by the nonionic detergent Tween-80. The E-2/4-H activity was dependent on nicotinamide cofactor, with NADPH preferred over NADH for 2-OHE2 formation. The predominant catechol estrogen formed was 2-OHE2: maximum velocities (Vmax) for the formation of 2- and 4-OHE2 were 1570 and 174 pmol/mg protein . 30 min, respectively. The apparent Km values with respect to estradiol for 2- and 4-OHE2 were similar, 4.39 and 4.27 microM, respectively. Blastocyst E-2/4-H activity was detectable in one of two samples of blastocysts from day 10 of pregnancy (4.4 pmol 2-OHE2/mg protein . 30 min), increased to a maximum on days 12 and 13 (628 +/- 153 and 516 +/- 227 pmol 2-OHE2/mg protein . 30 min, respectively), and declined by day 14 (63.2 +/- 32.9 pmol 2-OHE2/mg protein . 30 min). The activity of E-2/4-H was positively correlated with aromatase activity assayed in the same tissue samples from days 10-14 of pregnancy. The surge in E-2/4-H activity coincides with several of the critical events that occur near the time of implantation. Our findings are consistent with the hypothesis that catechol estrogens mediate some of the actions of estrogens in early pregnancy in the pig.

Topics: Animals; Aromatase; Ascorbic Acid; Blastocyst; Cytochrome P-450 CYP1A1; Embryonic Development; Estradiol; Estrogens, Catechol; Female; Hydrogen-Ion Concentration; Kinetics; NAD; Polysorbates; Pregnancy; Proteins; Steroid Hydroxylases; Swine

In the course of establishing an assay for estrogen-2-hydroxylase activity, a detailed comparison was made between the formation of tritiated water (3H2O) and [6,7-3H]2-hydroxyestradiol (2-OHE2) by rabbit hypothalami in vitro from 2-3H- and 6,7-3H-labeled estradiol, respectively. The amounts of both 3H2O and [6,7-3H]2-OHE2 formed were stimulated several-fold by the nonionic detergent Tween-80. Maximum activity for both functions was associated with the soluble fractions (S2, 17,500 X g supernatant, for tritium release; S3, 100,000 X g supernatant, for 2-OHE2 formation). In contrast, maximal 3H2O formation by rat liver was associated with the microsomal (P3, 100,000 X g pellet) fraction and was virtually abolished by Tween-80. The amount of 3H2O formed exceeded, up to severalfold, the amount of 2-OHE2 produced under all conditions examined and in all subcellular fractions. The bulk of the excess 3H2O formation, unrelated to the production of 2-OHE2, could be eliminated by adding ascorbic acid (10 mM) to the incubation medium. However, a second, smaller component of spurious 3H2O release could not be suppressed. This component was responsible for a persistent lack of stoichiometry between the formation of 3H2O and 2-OHE2, with the former exceeding the latter by up to 2-fold. This discrepancy was unaffected by ascorbic acid (up to 20 mM), unlabeled 2-OHE2 (up to 10 microM), and reducing the temperature of incubation from 37 to 30 C, measures that prolonged the t1/2 of 2-OHE2 during incubation with hypothalamic tissue from under 3 min to over 100 min. These findings 1) raise doubts about the validity of using 3H2O formation from [2-3H]estradiol as a quantitative index of estrogen-2-hydroxylase activity, and 2) establish conditions under which further metabolism of 2-OHE2 is inhibited, thereby making it practical to quantify enzyme activity on the basis of the amount of catechol estrogen formed. Evidence is also presented that the release of 3H2O from [2-3H]estradiol by hypothalamic tissue, unrelated to 2-OHE2 formation, may be enzymatically mediated.

Topics: Animals; Ascorbic Acid; Cytochrome P-450 CYP1A1; Estradiol; Estrogens, Catechol; Female; Hypothalamus; Kinetics; Rabbits; Radioisotope Dilution Technique; Steroid Hydroxylases; Subcellular Fractions; Tritium