4-hydroxyestradiol and 2-hydroxyestradiol

C2- and C4-position 17beta-estradiol metabolites play an important role in breast carcinogenesis. 2-Hydroxyestradiol and 4-hydroxyestradiol are implicated in tumorigenesis via two pathways. These pathways entail increased cell proliferation and the formation of reactive oxygen species that trigger an increase in the likelihood of deoxyribonucleic acid mutations. 2-Methoxyestradiol, a 17beta-estradiol metabolite, however, causes induction of apoptosis in transformed and tumor cells; thus exhibiting an antiproliferative effect on tumor growth. The 4-hydroxyestradiol:2-methoxyestradiol and 2-hydroxyestradiol:2-methoxyestradiol ratios therefore ought to be taken into account as possible indicators of carcinogenesis.

Topics: 2-Methoxyestradiol; Animals; Breast Neoplasms; Cell Proliferation; Estradiol; Estrogens, Catechol; Humans

Protein adducts covalently modified by catechol estrogens (CEs), referred to as estrogenized proteins, are potential biomarkers for estrogen homeostasis or exposure to environmental toxicants. However, serum proteins endogenously modified by CEs and the modification sites remain elusive. In this study, liquid chromatography-mass spectrometry (LC-MS)-based shotgun proteomics is applied to identify site-specific protein estrogenization in human blood via a systematic approach and stringent validation. We showed CEs, namely 2- and 4-hydroxyl estrogens which are regarded as biomarkers for estrogen homeostasis, form covalent bonds with proteins, mainly via side chain Cys, Lys, or His residue. Estrogenization of purified human serum albumin (HSA) and immunoglobulin G (IgG) at specific sites was achieved by co-incubation and used as the standards to confirm the identified estrogenization in serum proteins. Based on a database search, estrogenized peptides derived from serum proteins in patient blood were identified; endogenous estrogenization of HSA and IgG-1 at multiple sites were confirmed as compared to the standards. Based on a test using Ellman's reagent, estrogenization produced stable products and irreversibly abolished the reactivity of Cys34-HSA, which is the most important antioxidant and nitric oxide carrier in blood. Given the importance of estrogen metabolism in environmental toxicology, further exploration of estrogenized proteins is warranted for biomarker discovery and/or new mechanisms in disease process.

Topics: Binding Sites; Chromatography, Liquid; Databases, Protein; Estradiol; Estrogens, Catechol; Female; Humans; Immunoglobulin G; Middle Aged; Models, Molecular; Protein Binding; Protein Conformation; Protein Interaction Domains and Motifs; Protein Stability; Proteomics; Serum Albumin; Serum Albumin, Human; Tandem Mass Spectrometry

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

Germline mutations in BRCA1 predispose carriers to a high incidence of breast and ovarian cancers. BRCA1 functions to maintain genomic stability through critical roles in DNA repair, cell-cycle arrest, and transcriptional control. A major question has been why BRCA1 loss or mutation leads to tumors mainly in estrogen-regulated tissues, given that BRCA1 has essential functions in all cell types. Here, we report that estrogen and estrogen metabolites can cause DNA double-strand breaks (DSB) in estrogen receptor-α-negative breast cells and that BRCA1 is required to repair these DSBs to prevent metabolite-induced genomic instability. We found that BRCA1 also regulates estrogen metabolism and metabolite-mediated DNA damage by repressing the transcription of estrogen-metabolizing enzymes, such as CYP1A1, in breast cells. Finally, we used a knock-in human cell model with a heterozygous BRCA1 pathogenic mutation to show how BRCA1 haploinsufficiency affects these processes. Our findings provide pivotal new insights into why BRCA1 mutation drives the formation of tumors in estrogen-regulated tissues, despite the general role of BRCA1 in DNA repair in all cell types.

Topics: BRCA1 Protein; Breast; Breast Neoplasms; Cytochrome P-450 CYP1A1; DNA Breaks, Double-Stranded; DNA Repair; Estradiol; Estrogens; Estrogens, Catechol; Female; Genomic Instability; Humans; MCF-7 Cells

In the current study, the non-transformed prostatic epithelial cells (BPH-1) were exposed to the catechol estrogens (CE) 2-hydroxyestradiol (2-OHE2) or 4-hydroxyestradiol (4-OHE2), or the parent hormone 17-β-estradiol (E2) at an equimolar concentration (1μM) for a period of 6 weeks. It was found that both 2-OHE2 and 4-OHE2 have more potent proliferation-enhancing effect than E2. Exposure to 2-OHE2, 4-OHE2 or E2 resulted in a significant increase in the protein abundance of cyclin D1 and c-myc. The treated cells exhibited a shift toward the proliferative phase as indicated by FACScan. BPH-1 cells treated with 4-OHE2 showed increased abundance of estrogen receptor-α (ERα) and its downstream IGF-1R. Reduced abundance of estrogen receptor-β (ERβ) and its downstream tumor suppressor FOXO-1 were observed in cells exposed to E2, 2-OHE2 and, to a greater extent, 4-OHE2. Comet assay revealed that CE, especially 4-OHE2, elicited significant genotoxic effects as compared to E2. 4-OHE2 showed greater ability to neoplastically transform BPH-1 cells as indicated by increased colony forming capacity in soft agar and matrix invasion. In conclusion, in vitro exposure to CE could neoplastically transform human prostatic epithelial cells. Further, 4-OHE2 is more carcinogenic to prostate epithelial cells than the parent hormone E2.

Topics: Cell Cycle; Cell Line; Cell Proliferation; Cell Transformation, Neoplastic; Comet Assay; DNA Damage; Epithelial Cells; Estradiol; Estrogen Receptor alpha; Estrogen Receptor beta; Estrogens, Catechol; Flow Cytometry; Forkhead Box Protein O1; Forkhead Transcription Factors; Humans; Immunohistochemistry; Male; Prostate; Prostatic Neoplasms; Receptor, IGF Type 1

A diet high in linoleic acid (an ω-6 PUFA) increased the formation of miscoding etheno (ε)--DNA adducts in WBC-DNA of women, but not in men (Nair et al., Cancer Epidemiol Biomark Prev 1997;6:597-601). This gender specificity could result from an interaction between ω-6 PUFA intake and estrogen catabolism, via redox-cycling of 4-hydroxyestradiol (4-OH-E(2) ) and subsequent lipid peroxidation (LPO). In this study, we investigated whether in premenopausal women LPO-derived adducts in WBC-DNA are affected by serum concentration of 2- and 4-hydroxyestradiol metabolites and by fatty acid intake. DNA extracted from buffy coat and plasma samples, both blindly coded from healthy women (N = 124, median age 40 year) participating in the EPIC-Heidelberg cohort study were analyzed. Three LPO-derived exocyclic DNA adducts, εdA, εdC and M(1) dG were quantified by immuno-enriched (32) P-postlabelling and estradiol metabolites by ultra-sensitive GC-mass spectrometry. Mean M(1) dG levels in WBC-DNA were distinctly higher than those of εdA and εdC, and all were positively and significantly interrelated. Serum levels of 4-OH-E(2) , but not of 2-OH-E(2) , metabolites were positively related to etheno DNA adduct formation. Positive correlations existed between M(1) dG levels and linoleic acid intake or the ratios of dietary linoleic acid/oleic acid and PUFA/MUFA. Aerobic incubation of 4-OH-E(2) , arachidonic acid and calf thymus DNA yielded two to threefold higher etheno DNA adduct levels when compared with assays containing 2-OH-E(2) instead. In conclusion, this study is the first to compare M(1) dG and etheno-DNA adducts and serum estradiol metabolites in human samples in the same subjects. Our results support a novel mechanistic link between estradiol catabolism, dietary ω-6 fatty acid intake and LPO-induced DNA damage supported by an in vitro model. Similar studies in human breast epithelial tissue and on amplification of DNA-damage in breast cancer patients are in progress.

Topics: Adult; Cohort Studies; Dietary Fats; DNA Adducts; DNA Damage; Estradiol; Estrogens; Estrogens, Catechol; Fatty Acids, Omega-6; Female; Humans; Linoleic Acid; Lipid Peroxidation; Lymphocytes; Oleic Acid; Premenopause

The current study was undertaken to determine the involvement of cAMP/PKA and MAPK-mediated signalling pathways in the regulation of cell proliferation by hydroxylated metabolites of 17β-estradiol (E2).. MCF-7, human breast cancer cells, were cultured in phenol red-free DMEM and treated with 1 nM 2-OH-E2 or 4-OH-E2. E2 was used as a positive control. Cell proliferation was measured using the BrdU incorporation assay. Cellular levels of cAMP and PKA were determined using ELISA kits. ERK1/2 protein expression was evaluated by Western Blot analysis. To determine the involvement of different intracellular pathways in the regulation of cell proliferation appropriate activators or inhibitors were used.. Hydroxylated estrogens, as E2, exhibited no influence on cAMP accumulation and PKA activation. In concomitant treatments with forskolin, cell proliferation decreased to the amount noted under the influence of forskolin alone. A PKA inhibitor (PKI) had no statistically significant effect on proliferation stimulated by E2 and its hydroxylated metabolites. Phospho-ERK1/2 protein expression in cells stimulated with E2, 2-OH-E2 and 4-OH-E2 was not significantly different from the control. However, co-treatment with both PD98059 and E2 or its hydroxylated metabolites reversed the effect of tested compounds on cell proliferation.. We have shown that E2 hydroxylated metabolites do not activate cAMP/PKA in breast cancer cells and confirm previously published data, which showed a lack of ERK1/2 activation in a breast cancer cell line. The observed reversible action of PD98059 on cell proliferation can be explained by the fact that hydroxylated estrogens, as E2, stimulate secretion of a number of growth factors, which affect MAPK activity, suggested by Lobenhofer et al. (2000).

Topics: Breast Neoplasms; Carcinoma; Cell Line, Tumor; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Drug Evaluation, Preclinical; Enzyme Activation; Estradiol; Estrogens; Estrogens, Catechol; Female; Humans; Hydroxylation; MAP Kinase Signaling System; Signal Transduction

Glycogen is a uterine histotroph nutrient synthesized by endometrial glands in response to estradiol. The effects of estradiol may be mediated, in part, through the catecholestrogens, 2-hydroxycatecholestradiol (2-OHE2) and 4-hydroxycatecholestradiol (4-OHE2), produced by hydroxylation of estradiol within the endometrium. Using ovariectomized mink, our objectives were to determine the effects of estradiol, 4-OHE2, and 2-OHE2 on uterine: 1) glycogen concentrations and tissue localization; 2) gene expression levels for glycogen synthase, glycogen phosphorylase, and glycogen synthase kinase-3B; and 3) protein expression levels for glycogen synthase kinase-3B (total) and phospho-glycogen synthase kinase-3B (inactive). Whole uterine glycogen concentrations (mean ± SEM, mg/g dry wt) were increased by estradiol (43.79 ± 5.35), 4-OHE2 (48.64 ± 4.02), and 2-OHE2 (41.36 ± 3.23) compared to controls (4.58 ± 1.16; P ≤ 0.05). Percent glycogen content of the glandular epithelia was three-fold greater than the luminal epithelia in response to estradiol and 4-OHE2 (P ≤ 0.05). Expression of glycogen synthase mRNA, the rate limiting enzyme in glycogen synthesis, was increased by 4-OHE2 and 2-OHE2 (P ≤ 0.05), but interestingly, was unaffected by estradiol. Expression of glycogen phosphorylase and glycogen synthase kinase-3B mRNAs were reduced by estradiol, 2-OHE2, and 4-OHE2 (P ≤ 0.05). Uterine phospho-glycogen synthase kinase-3B protein was barely detectable in control mink, whereas all three steroids increased phosphorylation and inactivation of the enzyme (P ≤ 0.05). We concluded that the effects of estradiol on uterine glycogen metabolism were mediated in part through catecholestrogens; perhaps the combined actions of these hormones are required for optimal uterine glycogen synthesis in mink.

Topics: Animals; Estradiol; Estrogens, Catechol; Female; Gene Expression; Glycogen; Glycogen Phosphorylase; Glycogen Synthase; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Mink; Ovariectomy; RNA, Messenger; Uterus

Sex Hormone-Binding Globulin (SHBG) - specific carrier for sex steroids - regulates hormone bioavailable fraction and estrogen signaling system in breast cancer cells. This study was conducted to elucidate the effects of hydroxylated estrogen (E2) metabolites (2-OH-E2 and 4-OH-E2) on sex hormone binding globulin (SHBG) mRNA and protein expression as well as on intracellular and extracellular SHBG levels.. MCF-7 human breast cancer cells were cultured with 2-OH-E2 or 4-OH-E2 in concentration of 1, 10 and 100 nM for 24 h, 17β-estradiol being used as a positive control. SHBG levels were measured in medium and cells by ELISA, SHBG mRNA expression was evaluated by real-time-PCR and protein expression by Western blot analysis.. 4-OH-E2 in high doses and 2-OH-E2 in the highest dose, while 17β-estradiol in all doses used increased intracellular but not extracellular SHBG levels. Both metabolites increased SHBG mRNA expression, the rank order of potency being E2 > 4-OH-E2 > 2-OH-E2. Both E2 and its metabolites increased SHBG protein expression.. Although the metabolites showed a lower potency than 17β-estradiol, further studies are needed to clarify whether hydroxylated metabolites of E2 are potent ligands for SHBG.

Topics: Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Estradiol; Estrogens, Catechol; Female; Gene Expression; Humans; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sex Hormone-Binding Globulin

A 17beta-estradiol (E(2)) is hydrolyzed to 2-hydroxy-E(2) (2-OHE(2)) and 4-hydroxy-E(2) (4-OHE(2)) via cytochrome P450 (CYP) 1A1 and 1B1, respectively. In estrogen target tissues including the mammary gland, ovaries, and uterus, CYP1B1 is highly expressed, and 4-OHE(2) is predominantly formed in cancerous tissues. In this study, we investigated the inhibitory effects of chrysoeriol (luteorin-3'-methoxy ether), which is a natural methoxyflavonoid, against activity of CYP1A1 and 1B1 using in vitro and cultured cell techniques. Chrysoeriol selectively inhibited human recombinant CYP1B1-mediated 7-ethoxyresorufin-O-deethylation (EROD) activity 5-fold more than that of CYP1A1-mediated activity in a competitive manner. Additionally, chrysoeriol inhibited E(2) hydroxylation was catalyzed by CYP1B1, but not by CYP1A1. Methylation of 4-OHE(2), which is thought to be a detoxification process, was not affected by the presence of chrysoeriol. In human breast cancer MCF-7 cells, chrysoeriol did not affect the gene expression of CYP1A1 and 1B1, but significantly inhibited the formation of 4-methoxy E(2) without any effects on the formation of 2-methoxy E(2). In conclusion, we present the first report to show that chrysoeriol is a chemopreventive natural ingredient that can selectively inhibit CYP1B1 activity and prevent the formation of carcinogenic 4-OHE(2) from E(2.).

Topics: 2-Methoxyestradiol; Aryl Hydrocarbon Hydroxylases; Biocatalysis; Breast Neoplasms; Carcinogens; Catechol O-Methyltransferase; Cell Line, Tumor; Chemoprevention; Culture Media, Conditioned; Cytochrome P-450 CYP1A1; Cytochrome P-450 CYP1B1; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Cytosol; Enzyme Inhibitors; Estradiol; Estrogens, Catechol; Female; Flavones; Flavonoids; Gene Expression; Glucuronidase; Humans; Hydroxylation; Kinetics; Methylation; Oxazines; Recombinant Proteins; Sulfatases

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

The rapid, controlled, and cyclical nature of angiogenesis in the ovarian follicle suggests the potential for sex steroids to influence neovascularization. Angiogenesis is regulated by a local balance between the levels of endogenous stimulators and inhibitors. Multiple lines of evidence suggest that estrogens stimulate angiogenesis via effects on endothelial cells. However, it is of outstanding value to investigate the negative control of this process. Since the main estrogen metabolites, 2-hydroxyestradiol and 4-hydroxyestradiol (4-OHE2) have been demonstrated to function as anti-estrogen in several estrogen-dependent organs; the aim of this study was to investigate their potential involvement in the modulation of follicular angiogenesis. Hydroxyestrogens were quantified in swine follicular fluid and their effects were studied on granulosa cell vascular endothelial growth factor (VEGFA) production and tested in an angiogenesis bioassay. Our study documents that these molecules are physiologically present in swine follicular fluid and their concentrations significantly (P<0.001) increase during follicle development. Moreover, angiogenesis bioassay revealed that both hydroxyestrogens significantly (P<0.001) inhibited new vessel growth. We evidenced that the most potent negative effect is mediated by 4-OHE2. The anti-angiogenic potential of this molecule is also supported by its ability to inhibit (P<0.001) VEGFA production by granulosa cells. Increased knowledge in this area is of utmost importance for future therapeutic options to contrast infertility disorders associated with aberrant angiogenesis; this would be also very useful for the treatment of diseases characterized by disregulated angiogenesis and vascular regression.

Topics: Animals; Cell Line; Cells, Cultured; Estradiol; Estrogens, Catechol; Female; Gas Chromatography-Mass Spectrometry; Granulosa Cells; Microscopy, Phase-Contrast; Ovarian Follicle; Solid Phase Extraction; Swine; Vascular Endothelial Growth Factor A

Human catechol-O-methyltransferase (COMT, EC 2.1.1.6) catalyzes the transfer of the methyl group to a variety of endogenous and exogenous catechol substrates using S-adenosyl-L-methionine as the methyl donor. This enzymatic O-methylation plays an important role in the inactivation of biologically-active and toxic catechols. A number of studies in recent years have sought to characterize the polymorphism of human COMT, and also to determine the catalytic activity of polymorphic enzymes. We report here the identification of a new haplotype of the human COMT gene with triplet point mutations, which encodes the D51G/S60F/K162R mutant of the soluble COMT and the D101G/S110F/K212R mutant of the membrane-bound COMT. Kinetic analysis showed that these new COMT variants had essentially the same kinetic characteristics and catalytic activity as the wild-type COMTs for the O-methylation of 2-hydroxyestradiol and 4-hydroxyestradiol in vitro, but they have asignificantly reduced thermostability at 37 degrees C. In addition, the mutant enzymes have different binding affinities for S-adenosyl-L-methionine compared with the wild-type COMTs. In agreement with our biochemical observations, molecular modeling studies also showed that the variant human COMT proteins shared nearly the same overall structures as the wild-type proteins. The binding energy values of the mutant COMTs in complex with catechol estrogen substrates were similar to those of the wild-type COMTs bound with the same substrates.

Topics: Catechol O-Methyltransferase; Estradiol; Estrogens, Catechol; Haplotypes; Humans; Kinetics; Models, Molecular; Point Mutation; Polymorphism, Genetic; Protein Stability; Sequence Alignment

Animal studies demonstrated that females are more susceptible than males to benzo[a]pyrene (BaP)-induced toxicities, including lung carcinogenesis. Elevation of cyclooxygenase-2 (COX-2) expression has been shown to increase the risk of cancer development. BaP induces COX-2 expression, and an interaction between BaP and estrogen in relation to COX-2 expression is suspected. In the present study, 10 muM BaP alone only slightly increased COX-2 mRNA expression and 10 nM 17-beta estradiol (E(2)) alone slightly increased prostaglandin E2 (PGE2) secretion in human bronchial epithelial cells. However, co-treatment with BaP and E(2) potentiated COX-2 mRNA expression and significantly elevated PGE2 secretion. Utilizing specific inhibitors and reporter assays, we further investigated the potentiation mechanisms of E(2) on BaP-induced COX-2 expression. First, E(2) activated estrogen receptor to increase PGE2 secretion, which directly increased COX-2 expression. Second, E(2) potentiated BaP-induced nuclear factor-kappaB (NF-kappaB) activation, which regulates COX-2 expression. Third, although the aryl hydrocarbon receptor (AhR) did not play a role in BaP-induced COX-2 expression, the potentiation effect of E(2) itself was AhR dependent. We further demonstrated that BaP induced the production of genotoxic E(2) metabolites (2- and 4-hydroxyestradiols) via AhR-up-regulated cytochromes P450 1A1 and 1B1. These metabolites could directly activate NF-kappaB to further promote COX-2 mRNA expression in human lung epithelial cells. These findings were further supported by increased PGE2 secretion in rat lung slice cultures. Our findings that the BaP-E(2) interaction enhanced COX-2 expression and hydroxyestradiol accumulation in the media of cultivated lung cells and tissues provide the needed scientific basis for higher risk of BaP-associated lung cancer in females.

Topics: Air Pollutants; Animals; Aryl Hydrocarbon Hydroxylases; Benzo(a)pyrene; Bronchi; Carcinogens, Environmental; Cells, Cultured; Cyclooxygenase 2; Cytochrome P-450 CYP1A1; Cytochrome P-450 CYP1B1; Dinoprostone; Drug Synergism; Enzyme Activation; Epithelial Cells; Estradiol; Estrogens, Catechol; Fulvestrant; Humans; In Vitro Techniques; Lung; Male; Membrane Proteins; NF-kappa B; Rats; Rats, Sprague-Dawley; Receptors, Estradiol; Up-Regulation

Estradiol (E(2)) accelerates oviductal egg transport through intraoviductal nongenomic pathways in cyclic rats and through genomic pathways in pregnant rats. This shift in pathways, which we have provisionally designated as intracellular path shifting (IPS), is caused by mating-associated signals and represents a novel and hitherto unrecognized phenomenon. The mechanism underlying IPS is currently under investigation. Using microarray analysis, we identified several genes the expression levels of which changed in the rat oviduct within 6 hours of mating. Among these genes, the mRNA level for the enzyme catechol-O-methyltransferase (COMT), which produces methoxyestradiols from hydroxyestradiols, decreased 6-fold, as confirmed by real-time PCR. O-methylation of 2-hydroxyestradiol was up to 4-fold higher in oviductal protein extracts from cyclic rats than from pregnant rats and was blocked by OR486, which is a selective inhibitor of COMT. The levels in the rat oviduct of mRNA and protein for cytochrome P450 isoforms 1A1 and 1B1, which form hydroxyestradiols, were detected by RT-PCR and Western blotting. We explored whether methoxyestradiols participate in the pathways involved in E(2)-accelerated egg transport. Intrabursal application of OR486 prevented E(2) from accelerating egg transport in cyclic rats but not in pregnant rats, whereas 2-methoxyestradiol (2ME) and 4-methoxyestradiol mimicked the effect of E(2) on egg transport in cyclic rats but not in pregnant rats. The effect of 2ME on egg transport was blocked by intrabursal administration of the protein kinase inhibitor H-89 or the antiestrogen ICI 182780, but not by actinomycin D or OR486. We conclude that in the absence of mating, COMT-mediated formation of methoxyestradiols in the oviduct is essential for the nongenomic pathway through which E(2) accelerates egg transport in the rat oviduct. Yet unidentified mating-associated signals, which act directly on oviductal cells, shut down the E(2) nongenomic signaling pathway upstream and downstream of methoxyestradiols. These findings highlight a physiological role for methoxyestradiols in the female genital tract, thereby confirming the occurrence of and providing a partial explanation for the mechanism underlying IPS.

Topics: 2-Methoxyestradiol; Animals; Aryl Hydrocarbon Hydroxylases; Catechol O-Methyltransferase; Cytochrome P-450 CYP1A1; Cytochrome P-450 CYP1B1; Egg Proteins; Estradiol; Estrogens, Catechol; Female; Gene Expression Regulation; Menstrual Cycle; Methylation; Oviducts; Ovum; Phosphorylation; Pregnancy; Rats; Rats, Sprague-Dawley; Receptors, Estrogen; Sexual Behavior, Animal

Certain estradiol metabolites may play a pivotal role in breast carcinogenesis. Of special interest are the metabolites 2-hydroxyestradiol (2-OHE2), which can react anti-carcinogenically, and 4-hydroxyestradiol (4-OHE1) and 16a-hydroxyestrone (16-OHE1), which may have procarcinogenic potential. In the present study, we have compared for the first time the effect of these metabolites and their parent substance 17beta-estradiol (E2) on proliferation, apoptosis, apoptosis markers and markers of metastatic property of human breast cancer cells.. MCF-7 cells (human estrogen-receptor positive metastatic breast cancer cell line) were incubated with the estrogens at concentrations of 0.1-100 nM. Cell proliferation rate was measured by the ATP-assay. Apoptosis was measured by cell death assay and the apoptosis markers cytochrome C, Bcl-2, Fasl and p53 were determined in cell lysates by ELISAs. The markers of metastatic property of the cell line, VEGF and MCP-1 were measured in the cell supernatant by ELISAs.. The estrogens E2, 4-OHE2 and 16-OHE1 display a proliferative effect on MCF-7 cells which is accompanied by a down-regulation of apoptosis. Various markers of apoptosis such as Bcl-2, cytochrome C and p53 appear to be involved. No significant effect was found for the metabolite 2-OHE2. VEGF and MCP-1 were up-regulated by E2 and 16-OHE1, whereas 2-OHE2 and 4-OHE2 did not show any effect.. The most potent estrogen regarding proliferation, apoptosis and metastasis of breast cancer cells seems to be estradiol. However, the estradiol metabolites 4-OHE2 and 16-OHE1 elicit similar properties on cell proliferation, apoptosis and metastasis as compared to estradiol but only at higher concentrations. In contrast 2-OHE2 did not show any significant effect on these parameters. Thus, intracellular estradiol metabolism may determine an individual's risk for breast carcinogenesis.

Topics: Apoptosis; Biomarkers, Tumor; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Down-Regulation; Enzyme-Linked Immunosorbent Assay; Estradiol; Estriol; Estrogens; Estrogens, Catechol; Fas Ligand Protein; Female; Humans; Membrane Glycoproteins; Proto-Oncogene Proteins c-bcl-2; Tumor Necrosis Factors; Tumor Suppressor Protein p53; Up-Regulation

An elevated incidence of breast cancer in women has been associated with prolonged exposure to high levels of estrogens. Our laboratory demonstrated that treatment of the immortalized human breast epithelial cells MCF-10F with 17beta-estradiol (E2), 4-hydroxyestradiol (4-OHE2) or 2-hydroxyestradiol (2-OHE2) induces phenotypical changes indicative of neoplastic transformation. MCF-10F cells treated with E2, 4-OHE2 or 2-OHE2 formed colonies in agar methocel and lost their ductulogenic capacity in collagen, expressing phenotypes similar to those induced by the carcinogen benzo[a]pyrene. To investigate whether the transformation phenotypes were associated with genomic changes, cells treated with E2, 4-OHE2 or 2-OHE2 at different doses were analyzed using microsatellite markers. Since microsatellite instability (MSI) and loss of heterozygosity (LOH) in chromosomes 13 and 17 have been reported in human breast carcinomas, we tested these parameters in MCF-10F cells treated with E2, 2-OHE2, or 4-OHE2 alone or in combination with the antiestrogen ICI182780. MCF-10F cells treated with E2 or 4-OHE2, either alone or in combination with ICI182780, exhibited LOH in the region 13q12.3 with the marker D13S893 located at approximately 0.8 cM telomeric to BRCA2. Cells treated with E2 or 4-OHE2 at doses of 0.007 and 70 nM and 2-OHE2 only at a higher dose (3.6 microM) showed a complete loss of 1 allele with D13S893. For chromosome 17, differences were found using the marker TP53-Dint located in exon 4 of p53. Cells treated with E2 or 4-OHE2 at doses of 0.007 nM and 70 nM and 2-OHE2 only at a higher dose (3.6 microM) exhibited a 5 bp deletion in p53 exon 4. Our results show that E2 and its catechol estrogen metabolites are mutagenic in human breast epithelial cells. ICI182780 did not prevent these mutations, indicating that the carcinogenic effect of E2 is mainly through its reactive metabolites 4-OHE2 and 2-OHE2, with 4-OHE2 and E2 being mutagenic at lower doses than 2-OHE2.

Topics: Base Sequence; Breast; Breast Neoplasms; Cell Culture Techniques; Cell Transformation, Neoplastic; Epithelial Cells; Estradiol; Estrogens, Catechol; Female; Humans; Loss of Heterozygosity; Molecular Sequence Data; Mutagenesis; Phenotype

Strong evidence supports the idea that specific metabolites of estrogens, mainly catechol estrogen-3,4-quinones, can react with DNA to become endogenous initiators of breast, prostate, and other human cancers. Oxidation of the catechol estrogen metabolites 4-hydroxyestradiol (4-OHE2) and 2-OHE2 leads to the quinones, estradiol-3,4-quinone (E2-3,4-Q) and estradiol-2,3-quinone (E2-2,3-Q), respectively. The reaction of E2-3,4-Q with DNA affords predominantly the depurinating adducts 4-OHE2-1-N3Ade and 4-OHE2-1-N7Gua, whereas the reaction of E2-2,3-Q with DNA yields the newly synthesized depurinating adduct 2-OHE2-6-N3Ade. The N3Ade adducts are lost from DNA by rapid depurination, while the N7Gua adduct is lost from DNA with a half-life of approximately 3 h at 37 degrees C. To compare the relative reactivity of E2-3,4-Q and E2-2,3-Q, the compounds were reacted individually with DNA for 0.5-20 h at 37 degrees C, as well as in mixtures (3:1, 1:1, 1:3, and 5:95) for 10 h at 37 degrees C. Depurinating and stable adducts were analyzed. In similar experiments, the relative reactivity of 4-OHE2 and 2-OHE2 with DNA was determined after activation by lactoperoxidase, tyrosinase, prostaglandin H synthase (PHS), or 3-methylcholanthrene-induced rat liver microsomes. Starting with the quinones, the levels of depurinating adducts formed from E2-3,4-Q were much higher than that of the depurinating adduct from E2-2,3-Q. Similar results were obtained with lactoperoxidase or tyrosinase-catalyzed oxidation of 4-OHE2 and 2-OHE2, whereas with activation by PHS or microsomes, a relatively higher amount of the depurinating adduct from E2-2,3-Q was detected. These results demonstrate that the E2-3,4-Q is much more reactive with DNA than E2-2,3-Q. The relative reactivities of E2-3,4-Q and E2-2,3-Q to form depurinating adducts correlate with the carcinogenicity, mutagenicity, and cell-transforming activity of their precursors, the catechol estrogens 4-OHE2 and 2-OHE2. This is essential information for understanding the cancer risk posed by oxidation of the two catechol estrogens.

Topics: Adenine; Carcinogens; Deoxyadenosines; DNA; DNA Adducts; Estradiol; Estrogens, Catechol; Guanosine; In Vitro Techniques; Lactoperoxidase; Monophenol Monooxygenase; Mutagens; Oxidation-Reduction; Time Factors

Estrogens are hypothesized to contribute to breast cancer via estrogen receptor-mediated increases in cell proliferation and via genotoxic processes leading to mutations. In this latter process, estradiol (E(2)) is thought to be oxidized to 4-hydroxyestradiol and then to E(2)-3,4-quinone, which reacts with DNA leading to apurinic sites. These sites represent premutagenic lesions. Additionally, E(2)-3,4-quinone can undergo redox cycling with E(2)-3,4-hydroquinone, leading to the release of reactive oxygen species. Although there is evidence that estradiol and E(2)-3,4-quinone are carcinogenic or mutagenic in several systems, 4-hydroxyestradiol, a key intermediate in the proposed genotoxic pathway, has thus far been negative in mutagenesis assays. Another major metabolite of estradiol, 2-hydroxyestradiol, is essentially inactive in carcinogenicity or mutagenicity assays. Here, we report that when using multiple low-dose exposures 4-hydroxyestradiol is mutagenic in the cII assay in BB rat2 cells. Under similar conditions, 2-hydroxyestradiol is inactive. Furthermore, the mutational spectrum of 4-hydroxyestradiol contains a considerable proportion of mutations at A:T base pairs, consistent with the known ability of E(2)-3,4-quinone to form a significant fraction of DNA adducts at adenines. Thus, the results of this study support the proposal that estradiol can contribute to carcinogenesis via a genotoxic pathway.

Topics: Animals; Catechols; DNA; Dose-Response Relationship, Drug; Embryo, Mammalian; Estradiol; Estrogens, Catechol; Indicators and Reagents; Mutagenesis; Mutagens; Rats; Rats, Inbred BB; Reverse Transcriptase Polymerase Chain Reaction

Long-term exposure to steroidal estrogens is a key factor contributing to increases in the risk of developing breast cancer. Proposed mechanisms include receptor-activated increases in the rate of cell proliferation leading to the accumulation of genetic damage resulting from reading errors, and the production of DNA damage by species arising from metabolism of 17beta-estradiol (E2) resulting in mutations. In support of the second mechanism, catechol metabolites of E2 induce DNA damage in vitro. In the present study, utilizing the single-cell gel electrophoresis (Comet) assay, we observed increases in the number of single-strand breaks in estrogen receptor alpha-positive (MCF-7) and -negative (MDA-MB-231) breast cancer cells exposed to E2 (for 24 hr) or 4-hydroxy-17beta-estradiol (4-OH-E2; for 2 hr). The concentrations of 4-OH-E2 sufficient to induce these effects were approximately 100 nM, substantially lower than reported previously. The catechol 2-hydroxy-17beta-estradiol (2-OH-E2) also induced strand breaks. 2-OH-E2, often referred to as an improbable carcinogen in humans, is not a major metabolite of E2 in the breast; however, our findings show that it is as DNA-damaging as 4-OH-E2. Formamidopyrimidine glycosylase posttreatment of E2-, 4-OH-E2-, and 2-OH-E2-exposed MCF-7 cells led to an up to sixfold increase in mean tail moment, suggesting that oxidative DNA damage was formed. Comet formation could be partially attenuated by coincubation with dimethylsulfoxide, attributing a small DNA-damaging role to oxyradicals emanating from catechol redox cycling. Similar findings were obtained with MDA-MB-231 cells, indicating that estrogen receptor status is not relevant to these effects. Our observations show that exposure to E2 adds to the oxidative load of cells, and this may contribute to genomic instability.

Topics: Cell Line, Tumor; Comet Assay; DNA Damage; Estradiol; Estrogen Receptor alpha; Estrogens, Catechol; Humans; Oxidation-Reduction

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.

Topics: Animals; Binding, Competitive; Cytosol; Estradiol; Estrogens, Catechol; Female; Lung; Mice; Protein Binding; Proteins; Radioligand Assay; Uterus

The therapeutical beneficial effect of estrogen-derived metabolites or catecholestrogens is controversial. These molecules are produced during estrogen therapy based on 17-beta-estradiol treatment. The metabolization of 17-beta-estradiol is carried out in brain, kidney or liver, and triggers different products such as 2- and 4- hydroxyestradiol (2OH and 4OH). These products have shown antioxidant properties against oxidative stress (OS) in several experimental models. Different noxious side effects related to those metabolites have also been observed upon estrogen therapy. In this sense, catecholestrogens seem to be implicated in tumoral and mutagenic process after long treatment with estrogens substitutive therapy. In our study, we have verified that 2OH and 4OH have antioxidant and cardioprotective effects against adriamycin (AD)-induced cardiomyopathy in ovariectomized (OVX) rats. Catecholestrogens diminished the lipid peroxides and carbonyl protein (CO) content, and different enzymes related to cell injury (creatinine kinase, lactate dehydrogenase, aspartate aminotransferase, alanine aminotransferase) in cardiac tissue from OVX-, AD-, and OVX+AD-treated rats. All these changes were correlated to a recovery on reduced glutathione (GSH), glutathione peroxidase (GPx), superoxide dismutase (SOD) and catalase (CAT) in heart tissue. The present study showed that 2OH and 4OH reduced all the parameters related to OS, antioxidant depletion and cardiac injury in OVX rats treated or not with AD.

Topics: Alanine Transaminase; Animals; Cardiomyopathies; Catalase; Creatine Kinase; Doxorubicin; Estradiol; Estrogens, Catechol; Female; Glutathione Peroxidase; Hemodynamics; L-Lactate Dehydrogenase; Ovariectomy; Oxidative Stress; Rats; Rats, Wistar; Superoxide Dismutase

UDP-glucuronosyltransferase (UGT) 1A1 is involved in the inactivation of estradiol (E(2)) and its oxidized metabolites. These metabolites have been shown to contribute to the development of endometrial cancer in animal studies. Thus UGT1A1 represents a candidate gene in endometrial carcinogenesis. In this study, we established the substrate specificity of UGT1A1 for E(2) and its 2- and 4-hydroxylated metabolites. Intrinsic clearances indicated that UGT1A1 had a preference for the glucuronidation of 2-hydroxyestradiol, a metabolite associated with antiproliferative activity. Expression analysis demonstrated that UGT1A1 is present in the nonmalignant endometrium. Subsequently, we sought to determine whether the common UGT1A1 promoter allele, UGT1A1*28 [A(TA)(7)TAA], which decreases gene transcription, was associated with endometrial cancer risk in a case-control study nested within the Nurses' Health Study (222 cases, 666 matched controls). Conditional logistic regression demonstrated a significant inverse association with the UGT1A1*28 allele and endometrial cancer risk. Compared with women homozygous for the UGT1A1*1 [A(TA)(6)TAA] allele, the adjusted odds ratio (OR) was 0.81 [95% confidence interval (CI), 0.56-1.16] for the UGT1A1*1/*28 genotype and 0.40 (95% CI, 0.21-0.75) for the homozygous UGT1A1*28 genotype (P(trend) = 0.007). There was a suggestion of an interaction by menopausal status [OR = 0.39 (95% CI, 0.18-0.85) for premenopausal women and OR = 0.79 (95% CI, 0.55-1.13) for postmenopausal women who carry the UGT1A1*28 allele (P(interaction) = 0.05)]. These observations suggest that lower expression of UGT1A1 decreases the risk of endometrial cancer by reducing the excretion of 2-hydroxyestradiol, the antiproliferative metabolite of E(2), in the endometrium.

Topics: Adult; Alleles; Endometrial Neoplasms; Endometrium; Estradiol; Estrogens, Catechol; Female; Glucuronides; Glucuronosyltransferase; Humans; Middle Aged; Polymorphism, Genetic; Promoter Regions, Genetic; Substrate Specificity

Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric basic helix-loop-helix transcription factor composed of HIF-1alpha and HIF-1beta subunits. HIF-1 expression is induced by hypoxia, growth factors, and activation of oncogenes. HIF-1 activates downstream target genes such as vascular endothelial growth factor A (VEGF-A), which plays an important role in tumor progression and angiogenesis. Estrogen exposure is considered to be the major risk factor for ovarian cancer. Estradiol (E2) is usually metabolized by CYP1A1/1A2 and CYP3A4 to the 2-hydroxy estradiol (2-OHE2) and 4-hydroxy estradiol (4-OHE2) in human liver. Many reports have suggested that the formation of 4-OHE2 is important for mammary carcinogenesis. However, the formation of 2-OHE2 may play an important role in exhibiting anticarcinogenic effects. In the present study, we have demonstrated that one of the catechol estrogen metabolites of E2, 4-OHE2, induces HIF-1alpha and VEGF-A expression at protein level in two human ovarian cancer cell lines, OVCAR-3 and A2780-CP70 cells, in dose- and time-dependent manners, whereas the other catechol estrogen metabolite of E2, 2-OHE2, does not alter HIF-1alpha and VEGF-A expression. To explore the mechanism of 4-OHE2-induced HIF-1alpha and VEGF-A expression, we studied whether phosphatidylinositol 3-kinase (PI3K) or mitogen-activated protein kinase (MAPK) signaling pathways are involved in 4-OHE2-induced HIF-1alpha and VEGF-A expression. Our findings indicate that PI3K inhibitors, LY294002 and wortmannin, inhibited HIF-1alpha and VEGF-A expression, whereas MAPK inhibitor, PD98059, did not alter HIF-1alpha and VEGF-A expression induced by 4-OHE2. 4-OHE2, but not 2-OHE2, also induced Akt phosphorylation at Ser473 in dose- and time-dependent manners, and LY294002 and wortmannin inhibited Akt phosphorylation at Ser473 induced by 4-OHE2. Our results also indicated that the mTOR/FRAP inhibitor, rapamycin, inhibited 4-OHE2-induced HIF-1alpha and VEGF-A expression. These results suggest that the PI3K/Akt/FRAP signaling pathway is required for HIF-1alpha and VEGF-A expression induced by 4-OHE2, whereas the MAPK pathway is not required. The finding that induction of HIF-1alpha and VEGF-A expression occurs via the activation of the PI3K/Akt/FRAP signaling pathway could be an important mechanism of 4-OHE2-induced carcinogenesis.

Topics: Cell Line, Tumor; Enzyme Inhibitors; Estradiol; Estrogens, Catechol; Female; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Mitogen-Activated Protein Kinases; Ovarian Neoplasms; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Signal Transduction; Transcription Factors; Vascular Endothelial Growth Factor A

Estrogens and their oxidative metabolites, the catechol estrogens, have been implicated in the development of breast cancer; yet, relatively little is known about estrogen metabolism in the breast. To determine how the parent hormone, 17 beta-estradiol (E(2)), is metabolized, we used recombinant, purified phase I enzymes, cytochrome P450 (CYP) 1A1 and 1B1, with the phase II enzymes catechol-O-methyltransferase (COMT) and glutathione S-transferase P1 (GSTP1), all of which are expressed in breast tissue. We employed both gas and liquid chromatography with mass spectrometry to measure E(2), the catechol estrogens 2-hydroxyestradiol (2-OHE(2)) and 4-hydroxyestradiol (4-OHE(2)), as well as methoxyestrogens and estrogen-GSH conjugates. The oxidation of E(2) to 2-OHE(2) and 4-OHE(2) was exclusively regulated by CYP1A1 and 1B1, regardless of the presence or concentration of COMT and GSTP1. COMT generated two products, 2-methoxyestradiol and 2-hydroxy-3-methoxyestradiol, from 2-OHE(2) but only one product, 4-methoxyestradiol, from 4-OHE(2). Similarly, GSTP1 yielded two conjugates, 2-OHE(2)-1-SG and 2-OHE(2)-4-SG, from the corresponding quinone 2-hydroxyestradiol-quinone and one conjugate, 4-OHE(2)-2-SG, from 4-hydroxyestradiol-quinone. Using the experimental data, we developed a multicompartment kinetic model for the oxidative metabolism of the parent hormone E(2), which revealed significant differences in rate constants for its C-2 and C-4 metabolites. The results demonstrated a tightly regulated interaction of phase I and phase II enzymes, in which the latter decreased the concentration of catechol estrogens and estrogen quinones, thereby reducing the potential of these oxidative estrogen metabolites to induce DNA damage.

Topics: Animals; Aryl Hydrocarbon Hydroxylases; Catechol O-Methyltransferase; Cytochrome P-450 CYP1A1; Cytochrome P-450 CYP1B1; Estradiol; Estrogens, Catechol; Glutathione S-Transferase pi; Glutathione Transferase; Humans; Isoenzymes; Kinetics; Mammary Glands, Animal; Mammary Glands, Human; Models, Chemical; Models, Molecular; Oxidation-Reduction; Recombinant Proteins; Structure-Activity Relationship

Several investigators have suggested that certain hydroxylated metabolites of 17beta-estradiol (E2) are the proximate carcinogens that induce mammary carcinomas in estrogen-sensitive rodent models. The studies reported here were designed to examine the carcinogenic potential of different levels of E2 and the effects of genotoxic metabolites of E2 in an in vivo model sensitive to E2-induced mammary cancer. The potential induction of mammary tumors was determined in female ACI rats subcutaneously implanted with cholesterol pellets containing E2 (1, 2, or 3 mg), or 2-hydroxyestradiol (2-OH E2), 4-hydroxyestradiol (4-OH E2), 16alpha-hydroxyestradiol (16alpha-OH E2), or 4-hydoxyestrone (4-OH E1) (equimolar to 2 mg E2). Treatment with 1, 2, or 3 mg E2 resulted in the first appearance of a mammary tumor between 12 and 17 weeks, and a 50% incidence of mammary tumors was observed at 36, 19, and 18 weeks respectively. The final cumulative mammary tumor incidence in rats treated with 1, 2, or 3 mg E2 for 36 weeks was 50%, 73%, and 100% respectively. Treatment of rats with pellets containing 2-OH E2, 4-OH E2, 16alpha-OH E2, or 4-OH E1 did not induce any detectable mammary tumors. The serum levels of E2 in rats treated with a 1 or 3 mg E2 pellet for 12 weeks was increased 2- to 6-fold above control values (approximately 30 pg/ml). Treatment of rats with E2 enhanced the hepatic microsomal metabolism of E2 to E1, but did not influence the 2- or 4-hydroxylation of E2). In summary, we observed a dose-dependent induction of mammary tumors in female ACI rats treated continuously with E2; however, under these conditions 2-OH E2, 4-OH E2, 16alpha-OH E2, and 4-OH E1 were inactive in inducing mammary tumors.

Topics: Animals; Carcinoma in Situ; Carcinoma, Ductal, Breast; Dose-Response Relationship, Drug; Drug Implants; Estradiol; Estriol; Estrogens; Estrogens, Catechol; Female; Hydroxyestrones; Mammary Neoplasms, Experimental; Rats; Rats, Inbred ACI

A novel mechanistic hypothesis is proposed which suggests that hyperhomocysteinemia is a risk factor for the development of estrogen-induced hormonal cancer in humans. Mechanistically, hyperhomocysteinemia may exert its pathogenic effects largely through metabolic accumulation of intracellular S-adenosyl-L-homocysteine, a strong non-competitive inhibitor of the catechol-O-methyltransferase-mediated methylation metabolism of endogenous and exogenous catechol estrogens (mainly 2-hydroxyestradiol and 4-hydroxyestradiol). While a strong inhibition of the methylation metabolism of 2-hydroxyestradiol would decrease the formation of 2-methoxyestradiol (an antitumorigenic endogenous metabolite of 17beta-estradiol), an inhibition of the methylation of 4-hydroxyestradiol would lead to accumulation of this hormonally-active and strongly procarcinogenic catechol estrogen metabolite. Both of these effects resulting from inhibition of the methylation metabolism of catechol estrogens would facilitate the development of estrogen-induced hormonal cancer in the target organs. This hypothesis also predicts that adequate dietary intake of folate, vitamin B6, and vitamin B12 may reduce hyperhomocysteinemia-associated risk for hormonal cancer. Experimental studies are warranted to determine the relations of hyperhomocysteinemia with the altered circulating or tissue levels of 4-hydroxyestradiol and 2-methoxyestradiol and also with the altered risk for estrogen-induced hormonal cancer.

Topics: Anticarcinogenic Agents; Breast Neoplasms; Catechol O-Methyltransferase; Catechol O-Methyltransferase Inhibitors; Estradiol; Estrogens; Estrogens, Catechol; Female; Folic Acid; Humans; Hyperhomocysteinemia; Kinetics; Methylation; Models, Biological; Mutagenicity Tests; Neoplasms, Experimental; Neoplasms, Hormone-Dependent; Risk Factors; S-Adenosylhomocysteine; Uterine Neoplasms; Vitamin B 12; Vitamin B 6

The primary purpose of this research is to examine the hypothesis that reactive oxygen species generated by estrogen quinonoids are the main source for the formation of aldehydic DNA lesions (ADL) in genomic DNA. ADL induced by quinonoid metabolites of 17beta-estradiol (E2), e.g. 4-hydroxyestradiol (4-OH-E2), 2-hydroxyestradiol (2-OH-E2), estrogen-3,4-quinones (E2-3,4-Q) and estrogen- 2,3-quinone (E2-2,3-Q), were investigated in calf thymus DNA (CT-DNA) under physiological conditions. The abasic sites resulting from the spontaneous depurination-depyrimidination of the modified bases and the aldehydic base and sugar lesions resulting from the oxidative damage to deoxyribose moieties in the DNA molecules were measured by an aldehyde reactive probe and were estimated as the number of ADL per 106 nucleotides. With the addition of NADPH (100 micro M) and Cu(II) (20 micro M), nanomolar levels (100 nM) of 4-OH-E2 and 2-OH-E2 induced approximately 10-fold increases in the number of ADL over control (P<0.001). In parallel, increases in 8-oxoguanine were detected in DNA exposed to 4-OH-E2 and 2-OH-E2 (100 nM) plus Cu(II) and NADPH. Further investigation indicated that the ADL induced by estrogen catechols plus Cu(II) and NADPH were causally involved in the formation of hydrogen peroxide and Cu(I). Both E2-2,3-Q and E2-3,4-Q alone induced a 2-fold increase in the number of ADL over control (P<0.05) in CT-DNA at high concentrations (1 mM). Neither neutral thermal hydrolysis nor lower ionic strength of the reaction medium induced further increases in the number of ADL in E2-3,4-Q-modified CT-DNA. Conversely, with the inclusion of Cu(II) and NADPH, both E2-3,4-Q and E2-2,3-Q (1 micro M) induced parallel formation of DNA single strand breaks and approximately 20-fold increases in the number of ADL over control (P < 0.001). The data also demonstrated that the ADL induced by estrogen quinones with and without the presence of Cu(II) and NADPH contain 69 and 78% putrescine-excisable ADL in CT-DNA, respectively. Additionally, results of the ADL cleavage assay indicate that the ADL induced by estrogen quinones plus Cu(II) and NADPH in CT-DNA were predominantly T7 exonuclease-excisable (50%) and exonuclease III- excisable (20%) ADL, whereas the intact ADL, and other ADL accounted for 5 and 25%, respectively. These results suggest that the ADL induced by estrogen quinones in CT-DNA are derived from oxidative events rather than depurination/depyrimidination of labile estrogen qu

Topics: Aldehydes; Animals; Cattle; Copper; DNA Adducts; DNA Damage; Estradiol; Estrogens, Catechol; Guanine; NADP; Oxidative Stress; Reactive Oxygen Species

Catecholestrogens are endogenous metabolites that have been shown to modulate granulosa, theca, and luteal cell function in some species. The present study was aimed at determining the possible role of these steroids on oocyte maturation. Cumulus-enclosed bovine oocytes were matured for 24 h, fertilized, and then cultured for 8 days. Whereas estradiol was without effect, addition of catecholestrogens (2-hydroxyestradiol, 4-hydroxyestradiol, and 2-methoxyestradiol [2-MOE2]) to the maturation medium did not affect the cleavage rate but was associated with a decrease in blastocyst production on Day 8. Although 2-MOE2 was also able to inhibit blastocyst formation when added during embryo culture, the effects were less pronounced than those seen when the steroid was added only during maturation. In agreement with the known ability of 2-MOE2 to bind tubulin at the colchicine site, marked alterations were observed in the spindle assembly of oocytes exposed to 2-MOE2 during maturation, which lead to gross chromosomal aberrations after fertilization and consequent developmental arrest at the morula stage. Moreover, that the blastocyst rate was not affected when meiosis was blocked with roscovitine during 2-MOE2 exposure is consistent with the idea that altered nuclear maturation is the cause of the low developmental competence. Because 2-MOE2 could be increased in follicular fluid in response to aryl hydrocarbon-receptor ligands, such as some environmental contaminants, our results show that abnormally high intraovarian levels of catecholestrogens could have a deleterious effect on oocyte maturation and early embryonic development arising from the alterations in the meiotic spindle.

Topics: 2-Methoxyestradiol; Animals; Antioxidants; Apoptosis; Blastocyst; Cattle; Cells, Cultured; Colchicine; Cytogenetic Analysis; Dose-Response Relationship, Drug; Estradiol; Estrogens, Catechol; Female; Fertilization in Vitro; Male; Meiosis; Oocytes; Spindle Apparatus

Breast cancer patients frequently develop metastases. This process requires the degradation of extracellular matrix proteins which act as a barrier to tumour cell passage. These proteins can be degraded by proteases, mainly the matrix metalloproteinases (MMPs). MMP-2 and -9 which are frequently detected in breast cancer tissues. ProMMPs are released from cancer cells, and their activation is considered to be a crucial step in metastases development. In breast cancer, estrogen metabolism is altered favouring the accumulation of 2- and 4-hydroxyestradiol (2- and 4-OHE(2)). These estradiol metabolites can generate free radicals. Since reactive species are known activators of proMMPs, this study was designed to determine if the free radicals generated by 2- and 4-OHE(2) can activate proMMP-2 and -9. Activation of MMPs by hydroxyestradiol was determined by monitoring the cleavage of a fluorogenic peptide and by zymography analysis. Both estradiol metabolites activated the MMP-2 and -9. 4-OHE(2) was a more potent activator than 2-OHE(2), which reflects its higher capacity to generate free radicals. ProMMPs activation was mainly mediated through O(2)*-, although the free radical HO* also activated the proMMPs but to a lesser extent. ProMMPs activation was not observed with estrogens that cannot generate free radicals, i.e. estradiol, estrone, 2- and 4-methoxyestradiol, and 16alpha-hydroxyestrone. These results demonstrate that 2- and 4-OHE(2) at a concentration as low as 10(-8)M can activate the proMMP-2 and -9 and might play an important role in the invasion of breast cancer cells.

Topics: Copper; Enzyme Activation; Enzyme Precursors; Estradiol; Estrogens; Estrogens, Catechol; Fluorescent Dyes; Free Radicals; Kinetics; Mannitol; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Oxygen Consumption; Peptides; Phenylmercuric Acetate; Superoxide Dismutase

The main goal of our study was to assess estrogen contents of breast tumor tissues, having different estrogen receptor status, in relation to long-term follow-up of patients.. Twenty-one breast cancer cases, all collected from January 1986 to January 1988 at the M. Ascoli Cancer Hospital Centre in Palermo, were included in the study and compared with 6 healthy women as a control group. Average follow-up time of patients was 144 +/- 10 months. The estrogen receptor status of tissues was determined by both ligand binding and immunohistochemical assays. A high performance liquid chromatography-based approach, jointly with gas chromatography/mass spectrometry, was used to identify and measure main estrogens, various hydroxyestrogens, and their methoxy derivatives in both normal and tumor tissues.. Although variable concentrations of hydroxylated estrogens were detected, they consistently accounted for >80% of all of the estrogens. Significantly greater amounts of both 2- and 4-hydroxyestradiol, along with a marked increase of 16 alpha-hydroxyestrone (OHE(1)), were observed in cancer with respect to normal breast tissues. A significant positive association was observed with elevated 16 alpha OHE(1) (P = 0.015) in patients alive, leading to significantly lower (P = 0.043) 2OHE(1):16 alpha OHE(1) ratio values. Conversely, ratio values of 4:2 hydroxy+methoxy estrogens was significantly lower (P = 0.006) in deceased patients. Using cutoff values of 1.2 for 4:2 hydroxy+methoxy ratio and 150 fmol/mg tissue for 16 alpha OHE(1) we achieved a clear-cut separation of patients, with over-cutoff patients having 147 months and under cutoff patients showing only 47 months median survival time (P = 0.00008).. Our data imply that individual hydroxyestrogens may have a distinct role in the onset and the clinical progression of breast cancer, with greater 16 alpha OHE(1) levels being in turn associated to cancer with respect to normal tissues and to a prolonged survival of breast cancer patients.

Topics: Adult; Aged; Binding Sites; Breast Neoplasms; Chromatography, High Pressure Liquid; Estradiol; Estrogens; Estrogens, Catechol; Female; Follow-Up Studies; Humans; Hydroxyestrones; Immunoenzyme Techniques; Middle Aged; Receptors, Estrogen; Survival Rate

We examined by using 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) as a radical generator the ability of estrogens to scavenge carbon-centered and peroxyl radicals. Electron spin resonance signals of carbon-centered radicals from AAPH were diminished by catecholestrogens but not by phenolic estrogens, showing that catecholestrogens efficiently scavenged carbon-centered radicals. However, fluorescent decomposition of R-phycoerythrin by AAPH-derived peroxyl radicals was inhibited by catecholestrogens and phenolic estrogens. Evidently, peroxyl radicals were scavenged by catecholestrogens and by phenolic estrogens. However, the scavenging ability of 4-hydroxyestradiol was less than 2-hydroxyestradiol. Strand break of DNA induced by AAPH was inhibited by catecholestrogens, but not by phenolic estrogens under aerobic and anaerobic conditions. Inactivation of lysozyme induced by AAPH was completely blocked by 2-hydroxyestradiol under aerobic and anaerobic conditions, and by 4-hyroxyestradiol only under anaerobic conditions. Peroxidation of arachidonic acid by AAPH was strongly inhibited by catecholestrogens at low concentrations. Only large amounts of phenolic estrogens markedly inhibited lipid peroxidation. These results show that catecholestrogens were antioxidant against AAPH-induced damage to biological molecules through scavenging both carbon-centered and peroxyl radicals, but phenolic estrogens partially inhibited AAPH-induced damage because they scavenged only peroxyl radicals.

Topics: Amidines; Antioxidants; Arachidonic Acid; DNA Damage; Electron Spin Resonance Spectroscopy; Estradiol; Estrogens, Catechol; Free Radical Scavengers; Free Radicals; Lipid Peroxidation; Muramidase; Mutagens; Oxidation-Reduction; Oxygen; Peroxides; Phycoerythrin; Plasmids

A growing body of evidence supports the hypothesis that estrogens can be carcinogens as a result of their conversion to genotoxins after biotransformation to form the catecholestrogens (CEs) 2-hydroxyestrone (2-OHE1), 2-hydroxyestradiol (2-OHE2), 4-hydroxyestrone (4-OHE1) and 4-hydroxyestradiol (4-OHE2). CEs can then undergo further metabolism to form quinones that interact with DNA to form either stable or depurinating adducts. These events could potentially be interrupted by the sulfate conjugation of both the parent estrogens and/or the CEs. We set out to determine whether CEs can serve as substrates for sulfate conjugation, and-if so-which of the growing family of human sulfotransferase (SULT) isoforms are capable of catalyzing those reactions. We determined apparent K(m) values for 10 recombinant human SULT isoforms, as well as the three most common allozymes for SULT1A1 and SULT1A2, with 2-OHE1, 2-OHE2, 4-OHE1, and 4-OHE2, and with the endogenous estrogens, estrone (E1) and 17beta-estradiol (E2), as substrates. With the exception of SULT1B1, SULT1C1, and SULT4A1, all of the human SULTs studied catalyzed the sulfate conjugation of CEs. SULT1E1 had the lowest apparent K(m) values, 0.31, 0.18, 0.27, and 0.22 microM for 4-OHE1, 4-OHE2, 2-OHE1, and 2-OHE2, respectively. These results demonstrate that SULTs can catalyze the sulfate conjugation of CEs, and they raise the possibility that individual variation in this pathway for estrogen and CE metabolism as a result of common genetic polymorphisms could represent a risk factor for estrogen-dependent carcinogenesis.

Topics: Carcinogens; Dose-Response Relationship, Drug; Estradiol; Estrogens, Catechol; Humans; Hydroxyestrones; Isoenzymes; Kinetics; Models, Biological; Neoplasms; Risk Factors; Sulfates; Sulfotransferases

A method has been developed for the semiquantitative analysis of the catechol estrogens, 2- and 4-hydroxyestradiol, using tandem electrospray mass spectrometry in a quadrupole ion trap mass analyzer. The implication of catechol estrogens in the biogenesis of breast and prostate cancer makes these labile lipophilic compounds important analytical targets. Ferrocene boronic acid is reacted with 2- and 4-hydroxyestradiol to form their cyclic boronate esters. Sample ionization is accomplished during the electrospray process by a one-electron oxidation of the ferrocene functionality to form the radical cation. The analysis depends on a non-aqueous solvent system consisting of 90% acetonitrile and 10% dichloromethane with 100 microM lithium triflate as the supporting electrolyte. The sensitivity of the analysis is greatly increased by the use of a novel electrospray interface with a large surface area stainless steel electrode coupled to a pulled fused-silica needle. Collision-induced dissociation of the selected molecular ion within the ion trap produces fragment ion spectra that can be used to distinguish between the two isobaric isomers and ultimately determine the relative amounts in mixtures containing both components. The method is sensitive to analyte concentrations in the low nM range.

Topics: Esters; Estradiol; Estrogens, Catechol; Ferrous Compounds; Spectrometry, Mass, Electrospray Ionization

Estrogen metabolism is altered in most, if not all, breast cancer tumors. These alterations primarily lead to the formation of the catechol estrogen metabolites, 2- and 4-hydroxyestrogens, which can generate superoxide anion radicals (O(2)(*)(-)) through the redox cycling of semiquinone/quinone derivatives. In breast cancer cells, the activity of nitric oxide synthase is also frequently elevated, resulting in an increased level of exposure to nitric oxide ((*)NO). Since (*)NO rapidly reacts with O(2)(*)(-) to produce the peroxynitrite anion (ONOO(-)), this study was undertaken to determine whether ONOO(-) can be generated when 2- and 4-hydroxyestrogens are incubated in vitro with (*)NO donor compounds. Using dihydrorhodamine 123 as a specific probe for ONOO(-) formation, a ratio of 100 microM dipropylenetriamine NONOate (DPTA/NO) to 10 microM 4-hydroxyestradiol (4-OHE(2)) gave an optimal ONOO(-) production of 11.9 +/- 1.9 microM (mean +/- SD). Quantification of ONOO(-) was not modified by mannitol, supporting the idea that the hydroxyl radical was not involved. This production of ONOO(-) required the presence of the catechol structure of estrogen metabolites since all methoxyestrogens that were tested were inactive. Hydroxyestrogen metabolites derived from estradiol showed the same efficiency in producing ONOO(-) as those originating from estrone. With DPTA/NO, the 4-hydroxyestrogens generated 30-40% more ONOO(-) than the 2-hydroxyestrogens. Optimal production of ONOO(-) was assessed with DPTA/NO and diethylenetriamine NONOate (initial (*)NO generation rates of 0.76 and 0.08 microM min(-1), respectively). With faster (*)NO-releasing compounds, such as diethylamine NONOate and spermine NONOate, lower levels of ONOO(-) were detected. These data suggest that once the optimal concentration of (*)NO was obtained, the reaction between (*)NO and 4-OHE(2) was saturated. The excess of (*)NO would probably react with aqueous oxygen to form nitrite (NO(2)(-)). Since the third-order reaction rate for the reaction between 2(*)NO and O(2) is 2 x 10(6) M(-2) s(-1), it can therefore be suggested that the reaction between (*)NO and 4-OHE(2) occurs at a faster rate.

Topics: Chromatography, High Pressure Liquid; Estradiol; Estrogens, Catechol; Hydroxyestrones; Mass Spectrometry; Nitrates; Nitric Oxide

A sensitive and specific assay for the determination of the catecholestrogens 2-hydroxyestradiol (2-OHE2) and 4-hydroxyestradiol (4-OHE2) using gas chromatography with electron-capture detection (GC-ECD) is described. The formation of 2- and 4-OHE2 was assessed following activation of 17beta-estradiol in the microsomal fraction of female rat livers. The analytes were isolated by solid-phase extraction, derivatized to their heptafluorobutyryl esters with heptafluorobutyric acid anhydride, and subjected to solvent exchange prior to analysis; this resulted in minimal chromatographic interference, long column life, and stable derivatized analytes. Derivatized catechols were separated and confirmed with dual column chromatography (DB-5 and DB-608) and quantitated using GC-ECD. The DB-608 column was preferred for quantitation as it provided better 4-OHE2 resolution from interference. Key validation parameters for the assay include sensitivity, intra- and inter-assay precision, and accuracy. Instrument sensitivity and limits of detection (LOD) and quantitation (LOQ) were determined statistically from fortification data approaching expected limits. For 2-OHE2 and 4-OHE2, respective values for these parameters were; instrument sensitivities of 0.4 and 0.7 pg, LODs of 0.8 and 1.3 ng/mg, and LOQs of 2.6 and 4.3 ng/mg.

Topics: Animals; Chromatography, Gas; Estradiol; Estrogens, Catechol; Female; Microsomes, Liver; Rats; Rats, Sprague-Dawley; Reproducibility of Results; Sensitivity and Specificity

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 catechol metabolites of estradiol, 2- and 4-hydroxyestradiol (2-OHE(2) and 4-OHE(2), respectively) are potent signaling molecules and are hypothesized to be central to estrogen-linked carcinogenesis. Methylation by catechol-O-methyltransferase (COMT) is the principal means of catechol estrogen (CE) deactivation in the liver and other tissues. The present studies were conducted to determine the effects of PCBs and catechol metabolites of PCBs on the COMT-mediated catabolism of 4-OHE(2) and 2-OHE(2) in vitro and in vivo. Liver homogenates of female Sprague-Dawley rats treated with Aroclor 1254 for 21 days (5 mg/kg/day) showed a 30 and 40% reduction of COMT activity toward 2-OHE(2) and 4-OHE(2), respectively. Incubation of [(3)H]-beta-estradiol with these same liver homogenates, followed by HPLC analysis, demonstrated an elevation of CEs and a nearly complete reduction in levels of methylated catechol estrogens. In classical enzyme kinetics studies, COMT was demonstrated to have a high affinity for catechol PCBs, with K(m)'s approximately equivalent to those of CEs. Catechol PCBs were also potent inhibitors of CE O-methylation. These data suggest that PCBs may significantly alter the metabolism of catechol estrogens in vivo and that this effect may be mediated by catechol metabolites of PCBs. It is further speculated that methyltransferase inhibition by PCB catechols may contribute to PCB-mediated endocrine effects and liver carcinogenesis.

Topics: Animals; Catalysis; Catechol O-Methyltransferase; Catechol O-Methyltransferase Inhibitors; Catechols; Chlorodiphenyl (54% Chlorine); Chromatography, High Pressure Liquid; Enzyme Inhibitors; Estradiol; Estrogens, Catechol; Female; Kinetics; Liver; Methylation; Polychlorinated Biphenyls; Rats; Rats, Sprague-Dawley; Tritium

Phenolphthalein (PT), used in over-the-counter laxatives, has recently been identified as a multisite carcinogen in rodents, but the molecular species responsible for the carcinogenicity is not known. A catechol metabolite of PT, hydroxyphenolphthalein (PT-CAT), was recently identified and may be the molecular species responsible for at least part of the toxicity/carcinogenicity of PT. We hypothesize that PT-CAT inhibits the enzyme catechol-O-methyltransferase (COMT) and therefore potentiates genotoxicity by either PT-CAT itself or the endogenous catechol estrogens (CEs) in susceptible tissues. The present studies were conducted to determine the effects of PT treatment and PT-CAT itself on the COMT-mediated metabolism of 4- and 2-hydroxyestradiol both in vitro and in vivo. Female mice were treated with PT (50 mg/kg/d) for 21 days and then euthanized. PT-CAT concentration in urine reached plateau levels by 7 days of exposure. An O-methylated metabolite of PT-CAT was detected in feces. In vitro experiments demonstrated that PT treatment resulted in an increase in free CEs, which are normally cleared by COMT and a concurrent decrease in the capacity of hepatic catechol clearance by COMT. In vitro, PT-CAT was a substrate of COMT, with kinetic properties within the range measured with endogenous substrates. PT-CAT was an extremely potent mixed-type inhibitor of the O-methylation of the catechol estrogens, with 90-300 nM IC50s. The above data, when taken together, suggest that chronic administration of PT may enhance metabolic redox cycling of both PT-CAT and the catechol estrogens and this, in turn, may contribute to PT-induced tumorigenesis.

Topics: Animals; Carcinogens; Catechol O-Methyltransferase; Catechol O-Methyltransferase Inhibitors; Cathartics; Enzyme Inhibitors; Estradiol; Estrogens, Catechol; Female; Kinetics; Liver; Methylation; Mice; Mice, Inbred Strains; Phenolphthalein; Phenolphthaleins; Swine

Catechol estrogens may mediate estrogen-induced carcinogenesis because 4-hydroxyestradiol induces DNA damage and renal tumors in hamsters, and this metabolite is formed in the kidney and estrogen target tissues by a specific estrogen 4-hydroxylase. We examined the carcinogenic potential of catechol estrogen in an experimental model previously reported to result in a high incidence of uterine adenocarcinoma after neonatal exposure to diethylstilbestrol. Outbred female CD-1 mice were treated with 2- or 4-hydroxyestradiol, 17beta-estradiol, or 17alpha-ethinyl estradiol on days 1-5 of neonatal life (2 microg/pup/day) and sacrificed at 12 or 18 months of age. Mice treated with 17beta-estradiol or 17a-ethinyl estradiol had a total uterine tumor incidence of 7% or 43%, respectively. 2-Hydroxyestradiol induced tumors in 12% of the mice, but 4-hydroxyestradiol was the most carcinogenic estrogen, with a 66% incidence of uterine adenocarcinoma. Both 2- and 4-hydroxylated catechols were estrogenic and increased uterine wet weights in these neonates. These data demonstrate that both 2- and 4-hydroxyestradiol are carcinogenic metabolites. The high tumor incidence induced by 4-hydroxyestradiol supports the postulated role of this metabolite in hormone-associated cancers.

Topics: Adenocarcinoma; Animals; Animals, Newborn; Carcinogens; Cricetinae; Estradiol; Estrogens, Catechol; Ethinyl Estradiol; Female; Mice; Uterine Neoplasms; Uterus

To examine a direct involvement of genotoxic effects of estrogens in the initiation of hormonal carcinogenesis, the abilities of 17beta-estradiol (E2) and 8 of its metabolites to induce cellular transformation and genetic effects were studied using the Syrian hamster embryo (SHE) cell model. Treatment with E2, estrone (E1), 2-hydroxyestrone (2-OHE1), 4-hydroxyestrone (4-OHE1), 2-methoxyestrone (2-MeOE1), 16alpha-hydroxyestrone (16alpha-OHE1), 2-hydroxyestradiol (2-OHE2), 4-hydroxyestradiol (4-OHE2) or estriol (E3) for I to 3 days inhibited SHE cell growth in a concentration-dependent manner. Concentration-dependent increases in the frequency of morphological transformation in SHE cells were exhibited by treatment for 48 hr with each of all estrogens examined, except for E3. The transforming activities of the estrogens, determined by the induced transformation frequencies, were ranked as follows: 4-OHE1 > 2-OHE1 > 4-OHE2 > 2-OHE2 > or = E2 or E1 > 2-MeOE1 or 16alpha-OHE1 > E3. Somatic mutations in SHE cells at the Na+/K+ATPase and /or hprt loci were induced only when the cells were treated with 4-OHE1, 2-MeOE1 or 4-OHE2 for 48 hr. Some estrogen metabolites induced chromosome aberrations in SHE cells following treatment for 24 hr. The rank order of the clastogenic activities of the estrogens that induced chromosome aberrations was 4-OHE1 > 2-OHE1 or 4-OHE2 > 2-OHE2 > E1. Significant increases in the percentage of aneuploid cells in the near diploid range were exhibited in SHE cells treated for 48 hr or 72 hr with each of the estrogens, except for 4-OHE1 and E3. Our results indicate that the transforming activities of all estrogens tested correspond to at least one of the genotoxic effects by each estrogen, i.e., chromosome aberrations, aneuploidy or gene mutations, suggesting the possible involvement of genotoxicity in the initiation of estrogen-induced carcinogenesis.

Topics: Aneuploidy; Animals; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; Chromosome Aberrations; Cricetinae; Estradiol; Estrogens, Catechol; Fetus; Hydroxyestrones; Mesocricetus; Stem Cells

Chlorinated hydrocarbons (CHCs) are environmental contaminants that bioaccumulate and hence are detected in human tissues. Epidemiological evidence suggests that the increased incidence of a variety of human cancers, such as lymphoma, leukemia and liver and breast cancers, might be attributed to exposure to these agents. The ability of CHCs to disrupt estrogen homeostasis is hypothesized to be responsible for their biological effects. The present study examined the effect of CHCs on the expression of cytochrome P450 (CYP)1A1, CYP1A2 and CYP1B1 mRNAs and the consequent 2- and 4-hydroxylation of 17beta-estradiol (E(2)) in female Sprague-Dawley rats. Animals were administered a single dose of the LD(50) of 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) (25 microg/kg), 2, 4-dichlorophenoxyacetic acid (2,4-D) (375 mg/kg) and dieldrin (DED) (38 mg/kg) by gavage. Seventy-two hours after treatment, increased expression of CYP1A1, CYP1A2 and CYP1B1 was observed in the liver, kidney and mammary tissue. Since CYP1A and CYP1B1 are the major enzymes catalyzing 2- and 4-hydroxylation of E(2), respectively, the effect of these CHCs on the metabolism of E(2) was investigated in rat tissues. Formation of 2- and 4-catechol estrogens was increased in a tissue-specific manner in response to treatment. TCDD was the most potent inducer for CYP1 enzyme mRNA and for the 2- and 4-hydroxylation of E(2). 2,4-D and DED induced similar responses, but less than that of TCDD. These results suggest that induction of CYP1 family enzymes and consequent increases in estrogen metabolism by CHCs in target tissues may be factors contributing to the biological effects associated with exposure to these agents.

Topics: 2,4-Dichlorophenoxyacetic Acid; Animals; Aryl Hydrocarbon Hydroxylases; Cytochrome P-450 CYP1A1; Cytochrome P-450 CYP1A2; Cytochrome P-450 CYP1B1; Cytochrome P-450 Enzyme System; Dieldrin; Environmental Pollutants; Estradiol; Estrogens, Catechol; Female; Gene Expression; Hydrocarbons, Chlorinated; Hydroxylation; Isoenzymes; Kidney; Liver; Mammary Glands, Animal; Polychlorinated Dibenzodioxins; Rats; Rats, Sprague-Dawley; RNA, Messenger

DNA damage induced by estrogens dispersed in liposomes was investigated. 2-Hydroxyestradiol (2HOE(2)) and 4-hydroxyestradiol (4HOE(2)), but not estrone, estradiol-17beta or estriol, caused strand break of plasmid DNA damage in the presence of ADP-Fe(3+). The catechol structure may be necessary for DNA damage. When DNA was incubated with 2HOE(2) for a long time (24 h), DNA damage was induced even at very low concentrations. Adding hydrogen peroxide markedly enhanced the sensitivity of DNA to the attack by 2HOE(2). Hydroxyl radical (HO.) scavengers strongly inhibited the 2HOE(2)-induced DNA damage, and EDTA partially inhibited DNA damage. However, 2HOE(2) caused 8-hydroxyguanine formation from calf thymus DNA only in the presence of EDTA-Fe(3+), but not ADP-Fe(3+). In addition, deoxyribose, which is a detective molecule of HO(.), was not degraded by 2HOE(2) in the presence of ADP-Fe(3+). Upon adding EDTA 2HOE(2) rapidly degraded deoxyribose. These results suggest that DNA strand break caused by 2HOE(2) in the presence of ADP-Fe(3+) was due to ferryl ion rather than HO(.), whereas 8-hydroxyguanine (8HOG) induced by 2HOE(2) in the presence of EDTA-Fe(3+) was due to HO(.).

Topics: Adenosine Diphosphate; Animals; Cattle; Deoxyribose; DNA; DNA Damage; Dose-Response Relationship, Drug; Edetic Acid; Estradiol; Estrogen Antagonists; Estrogens, Catechol; Ferric Compounds; Ferritins; Free Radical Scavengers; Guanine; Hydrogen Peroxide; Iron; Iron Chelating Agents; Liposomes; Reactive Oxygen Species; Thiobarbituric Acid Reactive Substances; Time Factors

Certain estrogen metabolites are involved in carcinogenesis and the development of resistance to methotrexate (MTX). In this study, we determined whether these well-established biological effects correlate with the relative efficiency of several estrogen metabolites to induce DNA strand breaks in the presence of copper, and investigated the potential enhancing effect of reduced nicotinamide adenine dinucleotide (NADH). DNA strand breaks induced by estradiol metabolites were measured by the conversion of supercoiled phage phiX-174 RF1 DNA to open circular and linear forms. The most active catecholestrogens were the 4-hydroxy derivatives, which produced about 2.5 times more DNA double strand breaks than the 2-hydroxy derivatives, while estradiol and 16alpha-hydroxyestrone were inactive. In addition, our results show that 4-hydroxyestradiol (4-OHE2) at physiological concentrations was capable of exhibiting DNA cleaving activity. The formation of these catecholestrogen-induced DNA strand breaks was associated with the utilization of oxygen and the generation of H2O2, because catalase inhibited the DNA cleaving activity of 4-OHE2. Interestingly, we also observed that NADH enhanced the induction of DNA strands breaks by 4-OHE2/Cu(II), probably by perpetuating the redox cycle between the quinone and the semiquinone forms of the catecholestrogen. In conclusion, this study demonstrated that the relative efficiency of 2-, and 4-hydroxyestrogen in carcinogenesis and for the enhancement of MTX resistance correlates with their relative capability to induce DNA strand breaks. In order to inhibit these estrogen-mediated biological effects, it may be important to develop different strategies to block the production of reactive oxygen species by the catecholestrogen-redox cycle.

Topics: Bacteriophage phi X 174; Breast Neoplasms; Copper; DNA Damage; DNA, Superhelical; DNA, Viral; Electrophoresis, Agar Gel; Estradiol; Estrogens, Catechol; Humans; Hydrogen Peroxide; NAD; Oxidation-Reduction; Oxygen Consumption; Reactive Oxygen Species; Superoxides

Catechol estrogens are major estrogen metabolites in mammals and are the most potent naturally occurring inhibitors of catecholamine metabolism. These estrogen compounds have been implicated in carcinogenic activity and the 4/2-hydroxyestradiol concentration has been shown to be elevated in neoplastic human mammary tissue compared to normal human breast tissue. Three human liver UDP-glucuronosyltransferases, UGT2B7, UGT1A1, and UGT1A3, have been shown to catalyze the glucuronidation of catechol estrogens and lead to their enhanced elimination via urine or bile. The present study was designed to study the kinetic interaction of expressed human UGT2B7(Y) or (H), UGT1A1, and UGT1A3 toward 2- and 4-hydroxycatechol estrogens. cDNAs encoding UGT2B7(Y) or (H), UGT1A1, and UGT1A3 were expressed in HK293 cells, and cell homogenates or membrane preparations were used to determine their glucuronidation ability. UGT2B7(Y) reacted with higher efficiency toward 4-hydroxyestrogenic catechols, whereas UGT1A1 and UGT1A3 showed higher activities toward 2-hydroxyestrogens. UGT2B7(H) catalyzed estrogen catechol glucuronidation with efficiencies similar to UGT2B7(Y). Flunitrazepam (FNZ), a competitive inhibitor of morphine glucuronidation in hepatic microsomes, competitively inhibited catechol estrogen glucuronidation catalyzed by UGT2B7(Y), UGT1A1, and UGT1A3. Buprenorphine, an opioid substrate that reacts at high efficiency with each of these UGTs, was also studied. FNZ competitively inhibited buprenorphine glucuronidation with UGT1A1 and UGT2B7 but had no inhibitory activity toward UGT1A3. This suggests that buprenorphine and 2-hydroxycatechol estrogens react with separate active sites of UGT1A3. A catecholamine, norepinephrine, did not inhibit UGT2B7(Y)-, UGT1A1-, and UGT1A3-catalyzed glucuronidation of catechol estrogens. These results also suggest that drug-endobiotic interactions are possible in humans and may have implication in carcinogenesis.

Topics: Estradiol; Estrogens, Catechol; Flunitrazepam; Glucuronates; Glucuronosyltransferase; Humans; Hydroxyestrones; Isoenzymes; Norepinephrine

Catecholestrogens have been postulated to mediate the induction of kidney tumors by estradiol in male Syrian hamsters. In this study, we examined the mechanism of inhibition by quercetin of the catechol O-methyltransferase-catalyzed O-methylation of catecholestrogens as a basis for the previously reported enhancement of estradiol-induced tumorigenesis by this flavonoid. In hamsters treated with 50 micrograms of [6,7-3H]estradiol, quercetin increased concentrations of 2- and 4-hydroxyestradiol in kidney by 80 and 59%, respectively. In animals treated with two 10-mg estradiol implants, quercetin also decreased by 63-65% the urinary excretion of 2- and 4-hydroxyestradiol monomethyl ethers. Taken together, these results demonstrate the in vivo inhibition of the O-methylation of catecholestrogens by quercetin. S-Adenosyl-L-homocysteine, produced by the methylation of catecholestrogens, noncompetitively inhibited the O-methylation of 2- and 4-hydroxyestradiol by hamster kidney cytosolic catechol O-methyltransferase (IC50 approximately 10-14 microM). Due to the rapid O-methylation of quercetin itself, quercetin decreased renal concentrations of S-adenosyl-L-methionine by approximately 25% in control or estradiol-treated hamsters and increased concentrations of S-adenosyl-L-homocysteine by 5-15 nmol/g of wet tissue, which was estimated to cause a 30-70% inhibition of the enzymatic O-methylation of catecholestrogens. Quercetin or fisetin (a structural analog) inhibited the O-methylation of 2- and 4-hydroxyestradiol by a competitive plus noncompetitive mechanism (IC50 approximately 2-5 microM). These results suggest that the in vivo O-methylation of catecholestrogens is inhibited more by S-adenosyl-L-homocysteine than by quercetin. The accumulation of 2- and 4-hydroxyestradiol during co-administration of estradiol and quercetin may enhance metabolic redox cycling of catecholestrogens and thus estradiol-induced kidney tumorigenesis.

Topics: Animals; Catechol O-Methyltransferase Inhibitors; Cricetinae; Enzyme Inhibitors; Estradiol; Estrogens, Catechol; Flavonoids; Flavonols; Hesperidin; Kidney; Kidney Neoplasms; Kinetics; Male; Mesocricetus; Methylation; Oxidation-Reduction; Quercetin; S-Adenosylhomocysteine

Estrogen is a known risk factor in human breast cancer. In rodent models, estradiol has been shown to induce tumors in those tissues in which this hormone is predominantly converted to the catechol metabolite 4-hydroxyestradiol by a specific 4-hydroxylase enzyme, whereas tumors fail to develop in organs in which 2-hydroxylation predominates. We have now found that microsomes prepared from human mammary adenocarcinoma and fibroadenoma predominantly catalyze the metabolic 4-hydroxylation of estradiol (ratios of 4-hydroxyestradiol/2-hydroxyestradiol formation in adenocarcinoma and fibroadenoma, 3.8 and 3.7, respectively). In contrast, microsomes from normal tissue obtained either from breast cancer patients or from reduction mammoplasty operations expressed comparable estradiol 2- and 4-hydroxylase activities (corresponding ratios, 1.3 and 0.7, respectively). An elevated ratio of 4-/2-hydroxyestradiol formation in neoplastic mammary tissue may therefore provide a useful marker of benign or malignant breast tumors and may indicate a mechanistic role of 4-hydroxyestradiol in tumor development.

Topics: Adenocarcinoma; Animals; Biomarkers, Tumor; Breast; Breast Neoplasms; Cytochrome P-450 CYP1A1; Cytochrome P-450 Enzyme System; Estradiol; Estrogens; Estrogens, Catechol; Female; Fibroadenoma; Humans; Hydroxylation; In Vitro Techniques; Microsomes; Steroid Hydroxylases

Estradiol is converted to catechol estrogens via 2- and 4-hydroxylation by cytochrome P450 enzymes. 4-Hydroxyestradiol elicits biological activities distinct from estradiol, most notably an oxidant stress response induced by free radicals generated by metabolic redox cycling reactions. In this study, we have examined 2- and 4-hydroxylation of estradiol by microsomes of human uterine myometrium and of associated myomata. In all eight cases studied, estradiol 4-hydroxylation by myoma has been substantially elevated relative to surrounding myometrial tissue (minimum, 2-fold; mean, 5-fold). Estradiol 2-hydroxylation in myomata occurs at much lower rates than 4-hydroxylation (ratio of 4-hydroxyestradiol/2-hydroxyestradiol, 7.9 +/- 1.4) and does not significantly differ from rates in surrounding myometrial tissue. Rates of myometrial 2-hydroxylation of estradiol were also not significantly different from values in patients without myomata. We have used various inhibitors to establish that 4-hydroxylation is catalyzed by a completely different cytochrome P450 than 2-hydroxylation. In myoma, alpha-naphthoflavone and a set of ethynyl polycyclic hydrocarbon inhibitors (5 microM) each inhibited 4-hydroxylation more efficiently (up to 90%) than 2-hydroxylation (up to 40%), indicating > 10-fold differences in Ki (<0.5 microM vs. > 5 microM). These activities were clearly distinguished from the selective 2-hydroxylation of estradiol in placenta by aromatase reported previously (low Km, inhibition by Fadrozole hydrochloride or ICI D1033). 4-Hydroxylation was also selectively inhibited relative to 2-hydroxylation by antibodies raised against cytochrome P450 IB1 (rat) (53 vs. 17%). These data indicate that specific 4-hydroxylation of estradiol in human uterine tissues is catalyzed by a form(s) of cytochrome P450 related to P450 IB1, which contribute(s) little to 2-hydroxylation. This enzyme(s) is therefore a marker for uterine myomata and may play a role in the etiology of the tumor.

Topics: Benzoflavones; Cell Transformation, Neoplastic; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Enzyme Inhibitors; Estradiol; Estrogens, Catechol; Female; Humans; Hydroxylation; Leiomyoma; Microsomes; Myometrium; Naphthalenes; Phenanthrenes; Placenta; Pregnancy; Uterine Neoplasms

The preventive effects of estrogens on FeSO4-induced lipid peroxidation are closely correlated with their inhibition of Fe(II) oxidation during peroxidation. Estrogens affected the redox status of iron in aqueous solution with varying degrees of effectiveness. 2-Hydroxyestradiol substantially decreased the oxidation of Fe(II) and was the most potent Fe(III) reductant. Diethylstilbestrol and 4-hydroxyestradiol also exhibited reduction properties, whereas the phenolic estrogens 17 beta-estradiol, estrone, and 17 alpha-ethynylestradiol displayed slighter or no effects. Present results demonstrate that catecholestrogens and diethylstilbestrol directly alter the iron redox chemistry, this fact probably being involved in the antioxidant effects of these molecules.

Topics: Animals; Antioxidants; Diethylstilbestrol; Estradiol; Estrogens; Estrogens, Catechol; Iron; Lipid Peroxidation; Male; Microsomes, Liver; Models, Biological; Oxidation-Reduction; Rats; Rats, Sprague-Dawley; Solutions; Thiobarbituric Acid Reactive Substances; Time Factors

The catechol estrogens (CE), 2-hydroxyestradiol (2-OH-E2) and 4-hydroxyestradiol (4-OH-E2) were analyzed for their binding affinity to the estrogen receptor of MCF-7 cells. Applying a competitive binding assay to cytosols prepared from MCF-7 breast cancer cells, we measured a relative binding affinity of 23% (2-OH-E2) and 26% (4-OH-E2) compared to E2. Nuclear binding assays with the same cell line demonstrated a high specific binding with Kd's of 0.31 nM (2-OH-E2) and 0.21 nM (4-OH-E2). The relative binding affinity measured was 25% and 42% for 2-OH-E2 and 4-OH-E2, respectively. Based on this nuclear binding it can be concluded that the estrogen receptor occupied by CE is bound within the nucleus and might therefore be transcriptionally active.

Topics: Binding, Competitive; Breast Neoplasms; Cell Nucleus; Cytosol; Estradiol; Estrogens, Catechol; Humans; Prolactin; Receptors, Estrogen; Receptors, Progesterone; Tumor Cells, Cultured

The chronic administration of estradiol induces a high incidence (80-100%) of renal tumors in male Syrian hamsters. As part of our examination of a mechanism of carcinogenesis by free radicals generated during redox cycling of catecholestrogen metabolites, we assayed levels of 8-hydroxy-2'-deoxyguanosine (8-OHdGua), a marker product of hydroxy radical interaction with DNA, in livers and kidneys of hamsters treated with estradiol. Injections of 50 and 100 mg/kg estradiol doubled renal 8-OHdGua levels over controls [10.0 +/- 0.1 (SD) and 5.4 +/- 0.4 8-OHdGua/10(5) dGua, respectively] and raised hepatic 8-OHdGua levels almost 4-fold over control values, respectively. These changes were observed in kidney 4 h and in liver 1 or 2 h after treatment of hamsters with estradiol. Estradiol implants administered to hamsters for 3 days raised renal levels of 8-OHdGua by 50% over control values. Six days after 17 beta-estradiol implantation, 8-OHdGua levels returned to near-normal values. Liver DNA was not affected by estradiol implants. These data support a mechanism of estrogen-induced carcinogenesis by free radicals generated via redox cycling of catecholestrogen metabolites.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Cricetinae; Deoxyguanosine; Diethylstilbestrol; Drug Implants; Estradiol; Estrogens, Catechol; Free Radicals; Hydroxylation; Kidney; Liver; Male; Mesocricetus

The chronic administration of estradiol by subcutaneous (s.c.) implantation into male Syrian hamsters induces kidney tumors. Free radicals generated by redox cycling between catecholestrogens and their quinones have been proposed to damage DNA and to thus mediate renal hormone-induced carcinogenesis. As part of an examination of this postulate, we assayed by a filter elution technique DNA single-strand breaks in livers and kidneys of male hamsters treated with estrogen by single intraperitoneal (i.p.) injection, by s.c. implant or by continuous infusion and compared values to those in untreated controls. The DNAs of hamster liver and kidney were not affected by one i.p. injection of 5, 15 or 150 mg/kg estradiol. However, treatment of hamsters with one 25 mg estradiol implant/animal for 2 weeks elevated by 10% the levels of DNA single-strand breaks in kidney, but only to a minor extent in liver, which is not a target of estrogen-induced carcinogenesis. An infusion of 250 micrograms/day/animal of estradiol or 4-hydroxyestradiol for one week by osmotic pumps into hamsters resulted in a comparable increase of single-strand breaks in kidney DNA, whereas 2-hydroxyestradiol under these conditions had a negligible effect. It is concluded that the induction of DNA single-strand breakage by either estradiol or 4-hydroxyestradiol in hamster kidney supports a mechanism of estrogen-induced carcinogenesis by free radical generation via redox cycling between 4-hydroxyestradiol and its corresponding quinone.

Topics: Animals; Cricetinae; DNA; DNA Damage; DNA, Single-Stranded; Dose-Response Relationship, Drug; Estradiol; Estrogens; Estrogens, Catechol; Free Radicals; Kidney; Kidney Neoplasms; Male; Mesocricetus

Enzymatic O-methylation of catechol estrogens in red blood cells has been investigated with respect to species difference. In the presence of S-adenosylmethionine, 2- or 4-hydroxyestradiol (2-OHE2 or 4-OHE2) was incubated with blood lysate obtained from rats (five strains), guinea pigs, mice, rabbits, dogs, monkeys, and humans, respectively. The yielded guaiacols and unchanged substrate were determined by gas chromatography/mass spectrometry in a selected ion monitoring mode employing the corresponding 2H4-labeled compounds as internal standards. The total amounts of guaiacols formed from 2-OHE2 and 4-OHE2 were different, being the highest (79.6% and 38.1%) in monkeys and the lowest (5.1% and 1.9%) in humans. The ratios of isomeric guaiacols formed from 4-OHE2 (4Me/3Me) were 7.6-71, while those from 2-OHE2 (2Me/3Me) were 1.4-3.2. Thus, marked differences in O-methylation of catechol estrogens were observed among animal species, but no significant strain difference was detected in rats.

Topics: Adult; Animals; Dogs; Erythrocytes; Estradiol; Estrogens, Catechol; Guinea Pigs; Humans; Macaca fascicularis; Methylation; Mice; Mice, Inbred ICR; Rats; Rats, Inbred F344; Rats, Inbred SHR; Rats, Sprague-Dawley; Rats, Wistar; S-Adenosylmethionine; Species Specificity

Catecholestrogens are important metabolites of estradiol and estrone in the human. Considerable interest has focused on the catecholestrogens 2-hydroxy- and 4-hydroxyestradiol since they bind to the estrogen receptor with an affinity in the range of estradiol. Using the MCF-7 cell line, we analysed the capacity of purified catecholestrogens to transform the estrogen receptor into its high affinity nuclear binding form and to affect receptor-dependent processes such as proliferation and expression of the progesterone receptor (PR). Incubations with 2-hydroxy- and 4-hydroxyestradiol at 10(-8) M for 1 h resulted in tight nuclear binding of the estrogen receptor. During treatment of the cells with catecholestrogens we obtained a marked increase in proliferation rate of 36 and 76% for 2-hydroxy- and 4-hydroxyestradiol, respectively, relative to the inductive effect of estradiol (100%). The PR level, was slightly increased by treatment with 2-hydroxyestradiol (10%), whereas treatment with 4-hydroxyestradiol increased the PR level at 28%, compared to estradiol (100%). From these results we conclude that the 2- and 4-hydroxylated derivatives of estradiol are active hormones and are able to initiate estrogen receptor mediated processes in MCF-7 cells.

Topics: Breast Neoplasms; Cell Division; Cell Nucleus; Estradiol; Estrogens, Catechol; Female; Humans; Kinetics; Receptors, Estrogen; Receptors, Progesterone; Tamoxifen; Time Factors; Tumor Cells, Cultured

Catechol estrogens have been postulated to mediate estrogen-induced carcinogenesis. As part of our examination of this hypothesis, we studied the catechol-O-methyltransferase-catalyzed O-methylation of 2- and 4-hydroxyestradiol and the inhibition of this reaction by catecholamines. Epinephrine, norepinephrine, or dopamine (2 microM) inhibited the methylation of 2- and 4-hydroxyestradiol (20 mM) catalyzed by porcine liver catechol-O-methyltransferase by approximately 28-46% and 37-57%, respectively. One millimolar concentrations of catecholamines also inhibited the methylation of 5 microM 2- and 4-hydroxyestradiol by hamster kidney cytosol catechol-O-methyltransferase by approximately 27-31% and 19-33%, respectively. At a 15 microM 4-hydroxyestradiol concentration, the IC50 values for epinephrine and for dopamine were approximately 1200 and 3000 microM, respectively. Kinetic analyses of the methylation of 4-hydroxyestradiol in the presence of epinephrine, norepinephrine, or dopamine all revealed a competitive mechanism of inhibition. In contrast, the methylation of 160 microM 2-hydroxyestradiol was enhanced by approximately 75% in the presence of 1600 microM epinephrine or 2400 microM norepinephrine, likely due to a strong positive allosteric effect. An analysis of the substrate concentration dependence of O-methylation of 2-hydroxyestradiol revealed that at low concentrations (< 15 microM) this reaction was inhibited by epinephrine or norepinephrine, whereas it was significantly increased by approximately 50-100% at high substrate concentrations (50-200 microM). In contrast, dopamine competitively inhibited the methylation of all concentrations of 2-hydroxyestradiol (5-160 microM) tested. High levels of catecholamines were measured in hamster kidney or mouse uterus (1041 +/- 204 or 882 +/- 214 ng norepinephrine/g wet tissue, respectively) and in Fisher 344 rat pituitary (9.4 +/- 1.6 ng dopamine/mg protein), target organs of estrogen-induced carcinogenesis. Values were much lower in other organs of the same animals or in kidney, uterus, or pituitary of other rodent strains or species, which do not develop tumors under these conditions. High levels of catecholamines in target organs of hormonal cancer, the inhibition of O-methylation of 4-hydroxyestradiol, and the differential regulation of O-methylation of 2-hydroxyestradiol by catecholamines all support a role of 4-hydroxyestrogen metabolites in estrogen-induced carcinogenesis.

Topics: Animals; Catechol O-Methyltransferase; Catechol O-Methyltransferase Inhibitors; Catecholamines; Cricetinae; Estradiol; Estrogens, Catechol; Female; Kidney; Kinetics; Liver; Male; Mesocricetus; Methylation; Mice; Rats; Rats, Inbred F344; Rats, Sprague-Dawley; Rats, Wistar; Swine; Testis; Uterus

The pig conceptus and endometrium possess the ability to convert estrogens into catecholestrogens and catecholestrogens into methoxyestrogens. Experiments were carried out to evaluate the effect of catecholestrogens, methoxyestrogens and progesterone on the secretion of prostaglandin (PG) E and F2 alpha by porcine endometrial glandular and stromal cells in vitro. Both 2-hydroxyestradiol (2-OH-E2, 0-20 microM) and 4-hydroxyestradiol (4-OH-E2, 0-20 microM) increased (P less than .05) PGE and PGF2 alpha secretion by stromal cells in a dose response manner. Two-hydroxyestradiol tended (P less than .1) to decrease PGF2 alpha production by glandular cells. Two-methoxyestradiol (20 microM) suppressed (P less than .05) PGF2 alpha secretion by glandular and stromal cells. Four-methoxyestradiol (20 microM) stimulated (P less than .05) PGE production and PGE:PGF2 alpha ratio. Progesterone (.1 microM) suppressed (P less than .05) PG secretion in both cell types. We conclude that catecholestrogens, methoxyestrogens, and progesterone may participate in the establishment of pregnancy by modulating PG production in the endometrium.

Topics: 2-Methoxyestradiol; Animals; Endometrium; Estradiol; Estrogens, Catechol; Female; Pregnancy; Progesterone; Prostaglandins; Swine

The corpora lutea of several species contain estrogen receptors, but the role of estrogens in luteal function is unclear in most species. In this study, we investigated the direct effect of estradiol-17 beta (E2) and catecholestrogens (2-OHE2 or 4-OHE2) on rat and pig luteal steroidogenesis using in vitro cultures of small (SLC) and large (LLC) luteal cells prepared by elutriation. SLC and LLC were cultured at 37 degrees C for 36 h in serum-free media and treated with E2, 2-OHE2, or 4-OHE2; LH; forskolin (FORS); dibutyryl cAMP (dbcAMP); or combinations thereof. In the rat, E2 (2.5-10 micrograms/ml) inhibited progesterone (P4) production by both cell types dose-dependently. P4 production by rat SLC increased with increasing dose of 4-OHE2 up to the 2.5-microgram dose, then decreased to near control level at the 10-microgram dose. In LLC, P4 production in the presence of 4-OHE2 decreased initially (up to 2.5 micrograms/ml 4-OHE2), then increased at the 10-microgram dose. LH, FORS, and dbcAMP stimulated P4 production by SLC and LLC. For SLC, the stimulatory effects of LH and 4-OHE2 (2.5 micrograms) were comparable but lower than those of FORS and dbcAMP. For LLC, the effects of 4-OHE2 (10 micrograms), LH, and FORS were comparable but lower than those of dbcAMP. In time-course experiments, E2 inhibition of P4 production was observed at 36 and 72 h but not 6 h of culture for SLC and at all time points for LLC.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Bucladesine; Cells, Cultured; Colforsin; Estradiol; Estrogens, Catechol; Female; Kinetics; Luteal Cells; Luteinizing Hormone; Progesterone; Rats; Rats, Inbred Strains; Swine

A highly sensitive assay has been developed for measuring the rate of formation of 2-hydroxyestradiol and 4-hydroxyestradiol from estradiol by microsomal preparations. Catechol estrogens were converted to heptafluorobutyryl esters, which were separated by capillary column gas chromatography and quantified using electron-capture detection. 2-Hydroxyestradiol 17-acetate was used as an internal standard. The identity of catechol estrogen derivatives was verified by gas chromatography-mass spectrometry using negative-ion chemical ionization. Estrogens were identified by negative molecular ions and/or by characteristic fragments. This procedure permits quantification of catechol estrogens at the subpicogram level. The assay was validated by comparing estrogen 2- and 4-hydroxylase activities in microsomes from hamster and rat liver with values reported previously.

Topics: Animals; Aryl Hydrocarbon Hydroxylases; Chromatography, Gas; Cricetinae; Cytochrome P-450 CYP1A1; Cytochrome P-450 CYP1B1; Cytochrome P-450 Enzyme System; Electrochemistry; Estradiol; Estrogens, Catechol; Gas Chromatography-Mass Spectrometry; In Vitro Techniques; Male; Mesocricetus; Microsomes, Liver; Rats; Rats, Inbred Strains; Reference Standards; Regression Analysis; Steroid Hydroxylases

The 2-hydroxy and 4-hydroxyestradiols (2-/4-OHE2) caused marked cytotoxic effects, including vacuolation and nuclear changes, in rat epididymal epithelia, after exposure to very low levels (40 ng/rat/week) for 20 weeks. The effects of the 2-/4-OHE2 metabolites were more pronounced than that of estradiol-17 beta (E2).

Topics: Animals; Cell Nucleus; Dose-Response Relationship, Drug; Epididymis; Epithelium; Estradiol; Estrogens, Catechol; Male; Rats; Rats, Inbred Strains; Vacuoles

For identification of microsomal cytochrome P-450 (P-450) enzymes which catalyze 2- or 4-hydroxylations of estrogens in the rat liver, estradiol (E2) and estradiol 17-sulfate (E2-17-S) were selected as the substrates and incubated with various kinds of purified P-450 enzymes: PB-1, PB-2, PB-4 and PB-5 obtained from phenobarbital-treated male rats (Sprague-Dawley); MC-1 and MC-5 from 3-methylcholanthrene-treated male rats; and UT-1, UT-2, UT-4 and UT-5 from untreated animals. The reactions were carried out under the P-450-reconstructed system, and the resulting products were determined by HPLC using electrochemical detection. All the enzymes tested were shown to have varying degrees of catalytic activities for 2-hydroxylation of the two substrates; UT-1 and UT-2 had the highest activity. Of the induced P-450 enzymes, PB-2 and MC-1 showed fairly high catalytic activity for 4-hydroxylation of E2. The P-450 enzymes obtained from the untreated male rats, especially UT-4, showed the highest catalytic activity for 4-hydroxylation of the two substrates. From these results and also from kinetic experiments, the P-450 enzymes which catalyze 2- and 4-hydroxylations of estrogen were considered to be different species. A part of E2 was converted to such metabolites as estrone and those having a hydroxyl group at positions 6 beta, 15 alpha or 16 alpha, each production of which was estimated to be catalyzed by single or multiple P-450s.

Topics: Animals; Chromatography, High Pressure Liquid; Cytochrome P-450 Enzyme System; Estradiol; Estrogens, Catechol; Hydroxylation; Kinetics; Male; Methylcholanthrene; Microsomes, Liver; Phenobarbital; Rats; Rats, Inbred Strains

Various authors have demonstrated that estrogens stimulate both protein and DNA synthesis, as well as cell proliferation. This study was therefore undertaken to investigate the correlation between these physiological effects of estrogens and the total prostatic catecholestrogens. Either 17 beta-estradiol (5 ng), 2-hydroxyestradiol (40 ng), 4-hydroxyestradiol (17 ng), propane-1,2-diol or a saline solution was injected intraperitoneally to 22-day-old Sprague-Dawley rats daily. Regardless of the treatment, the prostate mass, the rate of DNA or protein synthesis correlates positively with the total prostatic catecholestrogen concentration. We therefore conclude that the effect of each estrogen per se should be regarded in terms of the total prostatic catecholestrogen concentration.

Topics: Animals; DNA; Estradiol; Estrogens, Catechol; Male; Propylene Glycol; Propylene Glycols; Prostate; Protein Biosynthesis; Rats; Rats, Inbred Strains

A role for eosinophils in the immune reaction has not been yet established. Considering that these leukocytes accumulate in lymphoid organs under glucocorticoid stimulation, we explored the possibility that they participate in the depression of immune reactions induced by these hormones and that they degranulate to exert this action. In this context, we investigated the dose effect of three estrogens on the number and degranulation of spleen red pulp eosinophils and on the percentage of spleen cross sectional area comprising white pulp. Estradiol-17 beta or 4 (OH) estradiol-17 beta increased red pulp eosinophils at low doses: 2 (OH) estradiol-17 beta increased them at a very high dose. The three estrogens degranulated the spleen eosinophils and decreased the lymphocyte containing spleen white pulp. We propose that the decrease in white pulp is a response mediated by agents released from degranulating eosinophils under the action of estrogen. Consequently, both estrogen-induced eosinophil degranulation and estrogen-induced increase in red pulp eosinophil numbers are conditions contributing to a decrease in white pulp volume. All above evidence supports the hypothesis that eosinophils are involved in immunoregulation by diminishing the number of lymphocytes contained in lymphoid organs.

Topics: Animals; Cytoplasmic Granules; Eosinophils; Estradiol; Estrogens; Estrogens, Catechol; Female; Rats; Rats, Inbred Strains; Spleen

Microsomal oestradiol-2/4-hydroxylase (OE-2/4-H) and cytosolic catechol-O-methyltransferase (COMT) (EC 2.1.1.6) activity in the uteri of pregnant and pseudopregnant rabbits during the periimplantation period were studied. The apparent Km for the 4-hydroxylation of oestradiol (3.18 microM) was considerably less than for the 2-hydroxylation reaction (13.36 microM), whereas the Vmax were almost equal. This suggests that 4-hydroxyoestradiol (4-OH-OE2) is the predominant product of OE-2/4-H in the rabbit uterus. These reactions were inhibited by SKF-525A, indicating the involvement of cytochrome P450 dependent monooxygenases. Uterine cytosolic COMT utilized 2-hydroxyestradiol (2-OH-OE2) as the preferred substrate as compared to 4-hydroxyoestradiol (4-OH-OE2). Since the rabbit uterus has a considerable capacity to synthesize 4-OH-OE2 and a lower capacity to metabolize it, it could be suggested that more 4-OH-OE2 than 2-OH-OE2 could be available to the uterus for its physiological activities. Furthermore, an increase in OE-2/4-H in Day 6 pseudopregnant and pregnant uteri with a concomitant decrease in COMT suggests the involvement of catecholoestrogens in the implantation process in the rabbit.

Topics: Animals; Aryl Hydrocarbon Hydroxylases; Catechol O-Methyltransferase; Cytochrome P-450 CYP1A1; Cytochrome P-450 CYP1B1; Cytosol; Embryo Implantation; Estradiol; Estrogens, Catechol; Female; Microsomes; Pregnancy; Pseudopregnancy; Rabbits; Steroid Hydroxylases; Substrate Specificity; Uterus

Twelve forms of human cytochrome P450 were synthesized in human hepatoma Hep G2 cells by means of cDNA-directed expression using vaccinia virus. The cDNA-expressed enzymes were tested for their ability to oxidize estradiol. Incubation of [14C]estradiol with cell lysates containing P450 IA2 resulted in the production of 2-hydroxy and 4-hydroxy metabolites with substrate turnovers of 2.74 and 0.27 min-1, respectively. P450s IIIA3 and IIIA4 yielded the same metabolites at about one third the rate of P450 IA2. Low levels of estradiol hydroxylation were also catalyzed by P450s IIC9, IIIA5, and IVB1. Six other P450 forms yielded no detectable metabolism. The roles of P450s IA2, IIA3, and IIIA4 were further established by immunoinhibition using antirat P450 antibodies. Antibody that specifically binds to P450 IIIA3 and IIIA4 inhibited 60-70% of estradiol hydroxylation, and antibody against P450 IA2 inhibited 20-40% of the estradiol hydroxylase activity in microsomes from two human liver specimens, suggesting that these enzymes constitute the major forms catalyzing estradiol oxidation in human liver. Immunoinhibition results also suggest that 2-hydroxy- and/or 4-hydroxycatechol estrogens are further metabolized to other yet uncharacterized metabolites by P450s IIIA3 and IIIA4.

Topics: Antibodies; Carcinoma, Hepatocellular; Cytochrome P-450 Enzyme System; DNA; Estradiol; Estrogens, Catechol; Gene Expression; Humans; Hydroxylation; Liver Neoplasms; Microsomes, Liver; Recombinant Proteins; Transfection; Tumor Cells, Cultured; Vaccinia virus

Formation of catecholestrogens (CE) by rat hepatic microsomes was re-examined because as recently shown; (1) CE formation can be catalyzed by an NADPH-dependent estrogen-4-hydroxylase (E-4-H(NADPH)) and by a peroxidatic, organic hydroperoxide-dependent estrogen-2/4-hydroxylase (E-2/4-H(OHP)), in addition to the established NADPH-dependent estrogen 2-hydroxylase (E-2-H(NADPH)); and (2) the indirect radiometric and the COMT-coupled radioenzymatic assays, used in many previous studies, may fail to provide an accurate measure, in particular, of 4-OH-CE. Using a direct product isolation assay, hepatic microsomes of both male and female rats were shown to express E-2/4-H(OHP) activity with properties similar to those of peroxidatic activity in other tissues. The activities of E-2/4-H(OHP) and E-2-H(NADPH) were affected differently by 5 out of 7 inducers of cytochromes P-450 administered in vivo. Phenobarbital and dexamethasone caused a 4- and 2-3-fold increase in E-2-H(NADPH) activity, respectively, but only a 38 and 20% increase in E-2/4-H(OHP) activity. Ketoconazol and beta-naphtoflavone caused a modest increase in E-2-H(NADPH) activity but a decrease in OHP-dependent activity. Clofibrate decreased peroxidatic activity by 50% and NADPH-dependent activity by approximately 20%. Both activities were increased by ethanol but decreased by isoniazide, an agent which induces the same form of cytochromes P-450 as ethanol. Polyclonal antibody against P-450p, a form of P-450 induced by glucocorticoids, inhibited E-2-H(NADPH) but not E-2/4-H(OHP) activity of untreated and of dexamethasone- and phenobarbital-treated rats. This study establishes that CE formation may occur in liver via the peroxidatic pathway and indicates that this pathway depends on forms of P-450 different from those mediating E-2-H(NADPH) activity. It also confirms and extends previous observations of the involvement of multiple, constitutive and induced forms of cytochrome P-450 in NADPH-dependent 2-hydroxylation in liver.

Topics: Animals; Antibodies; Benzoflavones; beta-Naphthoflavone; Clofibrate; Cytochrome P-450 Enzyme System; Dexamethasone; Enzyme Induction; Estradiol; Estrogens, Catechol; Female; Isoniazid; Kinetics; Male; Microsomes, Liver; NADP; Phenobarbital; Rats; Rats, Inbred Strains; Sex Factors

O-Methylation of catecholestrogens catalyzed by catechol-O-methyltransferase provides a major route for the rapid metabolic clearance of these steroids. However, the metabolic clearance rate of 4-hydroxyestradiol (4-OH-E2) is considerably lower than that of 2-hydroxyestradiol, although 2- and 4-hydroxycatecholestrogens (2- and 4-OH-CE) have similar apparent affinities for the enzyme. To determine the reason for this apparent paradox we have examined whether the efficiency of O-methylation of 4-OH-E2 could be affected by other catecholestrogens or their O-methyl ethers. The ratio of 4-methoxyestradiol:4-hydroxyestradiol 3-methyl ether was 2.6 at pH 8.5, the pH optimum for the reaction. The O-methylation of 4-OH-E2 (apparent Km 10 microM) was inhibited by 2-hydroxyestradiol (2-OH-E2) but not by 2- or 4-methyoxyestrogens. The values for Km, Vmax as well as the slope for the methylation of 4-OH-E2 were altered by 2-OH-E2 indicating a mixed inhibition. The inhibition constant for the intercept 1/V'max versus 2-OH-E2 concentrations and the inhibition constant for the slope versus 2-OH-E2 concentrations were 35 and 5.7 microM, respectively. The inhibition of O-methylation of 4-OH-E2 by 2-OH-E2 increased with the pH. In target tissues of the carcinogenic action of estrogens such as the rat pituitary, hamster kidney, or mouse uterus in which 2- and 4-OH-CE are both generated in almost equal amounts, the inactivation of 4-OH-CE by O-methylation may be impeded. Consequently, 4-OH-E2 would remain available as substrate for redox cycling, generation of active radicals and DNA damage.

Topics: Animals; Catechol O-Methyltransferase; Estradiol; Estrogens; Estrogens, Catechol; Hydrogen-Ion Concentration; Kinetics; Male; Methylation; Neoplasms; Rats; Rats, Inbred Strains

The binding of catechol estrogens, epoxyenones and methoxyestrogens was evaluated using estrogen receptors in cytosol prepared from human breast cancers. The relative affinity of 2-hydroxyestradiol, a metabolite formed in vitro from estradiol-17 beta by breast cancer cells, was indistinguishable from that of estradiol-17 beta. 4-Hydroxyestradiol, which is also a metabolite of estradiol-17 beta, associated with the estrogen receptor with a relative affinity approximately 1.5-fold greater than that of estradiol-17 beta. Epoxyenones and methoxyestrogens were weak competitors compared to the binding of estradiol-17 beta, exhibiting relative affinities 3% or less than the affinity of estradiol-17 beta. Sucrose density gradient centrifugation revealed that both 2- and 4-hydroxyestradiol inhibited the binding of estradiol-17 beta to both the 4S and 8S isoforms of the estrogen receptor in a competitive manner, with a Ki = 0.94 nM for 2-hydroxyestradiol and a Ki = 0.48 nM for 4-hydroxyestradiol. It can be concluded that these data demonstrate a specific receptor-mediated estrogenic action for both of these catechol estrogens.

Topics: Binding, Competitive; Breast Neoplasms; Centrifugation, Density Gradient; Cytosol; Estradiol; Estrogens, Catechol; Female; Humans; Kinetics; Receptors, Estrogen; Tumor Cells, Cultured

Reported values for the activity of enzymes mediating catechol estrogen formation by hamster kidney and liver, measured by catechol-O-methyltransferase-coupled radioenzymatic assay, have been uniformly low and there have been marked discrepancies in values reported from different laboratories. Therefore, we examined the validity of the radioenzymatic assay used in these studies. NADPH-dependent estrogen 2- and 4-hydroxylase activity of hamster liver microsomes measured by radioenzymatic assay was comparable to that reported in the literature but at least one order of magnitude lower than that obtained with a direct product isolation assay. Several features of the radioenzymatic assay were identified which, together, contribute to the underestimation of enzyme activity. They include, incomplete protection from oxidative degradation of both the catechol estrogens generated and of the catechol-O-methyltransferase and assay conditions which are suboptimal for O-methylation of the catechol estrogens. We conclude that results obtained using the catechol-O-methyltransferase-based radioenzymatic assay can only be considered valid if a consistent stoichiometric relationship can be demonstrated between the amounts of catechol estrogens and their O-methylated products.

Topics: Animals; Antioxidants; Buffers; Catechol O-Methyltransferase; Cricetinae; Estradiol; Estrogens, Catechol; In Vitro Techniques; Male; Mesocricetus; Microsomes, Liver; Predictive Value of Tests; Radiochemistry

Estrogen 2/4-hydroxylase (ESH) and catechol-O-methyltransferase (COMT) activities in mouse liver and uterus were studied. While 2-hydroxyestradiol (2-OHE2) was the predominant product in the liver, equal amounts of 2- and 4-hydroxyestradiol were produced in the uterus. Two-hydroxyestradiol was the preferred substrate for COMT in both tissues, but the level of this enzyme activity was much less in the mouse uterus (17-fold less). Thus, preferential production of 4-hydroxyestradiol (4-OHE2) in the presence of relatively less deactivation provides a mechanism for the local formation of a more chemically active form of estrogen by uterine tissue.

Topics: Animals; Aryl Hydrocarbon Hydroxylases; Catechol O-Methyltransferase; Cytochrome P-450 CYP1A1; Cytochrome P-450 CYP1B1; Estradiol; Estrogens, Catechol; Female; Mice; Steroid Hydroxylases; Time Factors; Uterus

A gas chromatographic/mass spectrometric assay for quantifying two catechol estrogens, 2-hydroxyestradiol and 4-hydroxyestradiol, in microsomal preparations is described. The assay employs deuterium-labeled analogs of the catechol estrogens as internal standards and permits quantification of catechol estrogens, in microsomal incubations, at low (1-2) microM concentrations. The compounds are analyzed as their trimethylsilyl derivatives following separation by capillary gas chromatography.

Topics: Animals; Deuterium; Estradiol; Estrogens, Catechol; Gas Chromatography-Mass Spectrometry; Male; Microsomes, Liver; Radioisotope Dilution Technique; Rats; Rats, Inbred Strains

The metabolic fate of the isomeric catecholestrogens 4-hydroxyestradiol (4-OHE2) and 2-hydroxyestradiol (2-OHE2) was studied to elucidate possible differences in their metabolism as an explanation for their different bioactivities. Healthy young men (n = 3 each) were infused (90 min) with 4-OHE2 (60 micrograms/h) or 2-OHE2 (100 micrograms/h). The main metabolites were determined in plasma and urine before, during and after infusion. Unconjugated and conjugated steroids, the latter after hot acid hydrolysis, were subjected to chromatography on LH-20 columns and measured by specific RIAs. During the infusion 4-OHE2 reached significant plasma concentrations whereas 2-OHE2 was so rapidly metabolised that its plasma levels remained virtually undetectable in spite of a higher infusion rate. The metabolism of 4-OHE2 was dominated by direct conjugation, that of 2-OHE2 by methyl ether formation. These findings were corroborated by the urinary excretion rates: during the infusion and the first hours afterwards, 4-OHE2 was mainly excreted as 4-OHE2 and 4-hydroxyestrone, while 2-OHE2 was predominantly excreted as 2-hydroxyestradiol 2-methyl ether and 2-hydroxyestrone 2-methyl ether.

Topics: Adult; Estradiol; Estrogens, Catechol; Humans; Kinetics; Male; Structure-Activity Relationship

The enzymatic metabolism of estradiol (E2) to the catecholestrogens, 2-hydroxyestradiol (2-OH-E2) and 4-hydroxyestradiol (4-OH-E2) in granulosa cells has been reported. Therefore, we evaluated the effects of these compounds and compared them to those of E2 on porcine granulosa cells cultured in serum-free medium. Cultures of granulosa cells were exposed to various treatments of E2, 2-OH-E2, 4-OH-E2 and(or) follicle-stimulating hormone (FSH) for 4 days and concentrations of progesterone in medium and cell numbers were determined. After 4 days of treatment, 2-OH-E2 and 4-OH-E2 stimulated basal progesterone production by granulosa cells, but 4-OH-E2 was less effective than 2-OH-E2. 2-OH-E2 (1 microgram/ml) stimulated progesterone production by 3.3 +/- 0.6-fold (n = 6 experiments), whereas E2 (1 microgram/ml) stimulated progesterone production 9.9 +/- 1.7-fold (n = 6 experiments). 2-OH-E2 at 4 micrograms/ml further stimulated progesterone production to 10.7 +/- 2.2-fold above controls (n = 9 experiments), whereas 4 micrograms/ml of E2 did not cause further stimulation of progesterone production. Thus, the average potency of 2-OH-E2 was less than E2. Concurrent treatment with 2-OH-E2 (4 micrograms/ml) and saturating concentrations of E2 resulted in further significant increases in progesterone production above the effects of either single treatment both in the absence and presence of FSH (200 ng/ml).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Cell Count; Cells, Cultured; Dose-Response Relationship, Drug; Drug Interactions; Estradiol; Estrogens, Catechol; Female; Follicle Stimulating Hormone; Granulosa Cells; Kinetics; Progesterone; Swine

This paper describes the induction of uterine eosinophilia as well as of deep endometrial edema and increase of uterine wet weight in the immature rat by the catecholestrogens 2-OH-estradiol and 4-OH-estradiol. These effects are thought to be mediated by eosinophils via a specific eosinophil receptor system. 4-OH-estradiol was equipotent with estradiol, whereas the effect of 2-OH-estradiol was significantly weaker.

Topics: Animals; DNA; Edema; Eosinophilia; Eosinophils; Estradiol; Estrogens, Catechol; Female; Organ Size; Rats; Rats, Inbred Strains; RNA; Uterine Diseases; Uterus

Catecholestrogens, formed locally, have been proposed to serve as local regulators of ovarian function. However, to date little or no activity of the critical biosynthetic enzyme, estrogen-2/4-hydroxylase, has been identified in the ovary. In the present study we have established the presence of this enzyme in porcine ovarian cells and characterized some of its biochemical features as well as its intraovarian distribution during the reproductive cycle. Homogenates of ovarian follicular tissue converted [3H]-17 beta-estradiol to the catecholestrogens 2-and 4-hydroxyestradiol (2-OH-E2 and 4-OH-E2) in a linear fashion for up to 60 min, with protein concentrations of equal to or less than 1 mg. The principal product, 2-OH-E2, was stable under the conditions of the assay. The reaction exhibited a dependence on nicotinamide cofactors, a pH optimum of 7.8, and an apparent Michaelis constant (Km) of approximately 10 microM for the production of 2-OH-E2 and 4-OH-E2. The activity of different ovarian preparations varied dramatically as a function of the reproductive cycle. Assayed at saturating substrate concentrations, immature follicular tissue and luteal tissue produced 50 or less pmol 2-OH-E2/mg protein X 40 min, while preovulatory follicles produced approximately 600 pmol 2-OH-E2/mg protein X 40 min. Even within the population of large presumptively preovulatory follicles, a variation in activity of more than 10-fold was encountered. Estrogen-2/4-hydroxylase activity of large preovulatory follicles correlated with the concentration of 17 beta-estradiol in the same follicles (r = +0.89, P less than 0.001). In large preovulatory follicles enzyme activity was present in both granulosa and theca layers. However, approximately 80% of the follicular activity was localized in the membrana granulosa. Under all conditions tested and in all ovarian compartments the formation of 2-OH-E2 was favored over that of 4-OH-E2. These studies show, for the first time, significant estrogen-2/4-hydroxylase enzyme activity within ovarian tissue. The striking increase in activity in the preovulatory follicle suggests physiological control of catecholestrogen synthesis. In conjunction with other data demonstrating stimulatory actions of catecholestrogens on ovarian cells, these observations are consistent with an intraovarian autocrine or paracrine regulatory function for these metabolites.

Topics: Animals; Cytochrome P-450 CYP1A1; Estradiol; Estrogens, Catechol; Female; Granulosa Cells; Kinetics; NAD; NADP; Ovarian Follicle; Ovary; Ovulation; Steroid Hydroxylases; Swine; Theca Cells

Estradiol and other estrogens induce renal carcinoma in male Syrian hamsters. The mechanism of carcinogenesis still remains unclear. Activation of estrogens to catechol metabolites has in the past been postulated to play a role in estrogen-induced carcinogenesis. Therefore, the carcinogenic activity of catechol estrogens was investigated. After 175 days of treatment, 4-hydroxyestradiol was found to be as carcinogenic as estradiol in male Syrian hamsters (4/5 and 4/5 animals with kidney tumors, respectively). Animals treated with 2-hydroxyestradiol (0/5) or 2-methoxyestradiol (0/6) did not develop renal carcinoma. The catechol estrogens failed to be mutagenic in the Ames test (reversions of his- S. typhimurium to histidine prototrophy in the TA 100 strain). The lack of carcinogenic activity of 2-hydroxyestradiol was not due to a failure to stimulate estrogen-dependent tumor growth. Growth of H-301 cells, an estrogen-dependent hamster kidney tumor cell line, was supported in vivo by estrogens in the following order: estradiol greater than 4-hydroxyestradiol greater than 2-hydroxyestradiol. Stimulation of tumor growth by 2-methoxyestradiol was not detected. It was concluded that the carcinogenic activity of 4-hydroxyestradiol was consistent with a role of catechol metabolites in estrogen-induced carcinogenesis. However, the intrinsic carcinogenic or hormonal activity of 2-hydroxyestradiol probably can not be assessed accurately in vivo because of its rapid methylation and metabolic clearance.

Topics: Animals; Carcinoma, Renal Cell; Cricetinae; Estradiol; Estrogens, Catechol; Kidney Neoplasms; Male; Mesocricetus; Mutagenicity Tests; Mutagens; Neoplasms, Hormone-Dependent; Salmonella typhimurium

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

Equimolar amounts (36.8 nmol) of estradiol and the two catecholestrogens (CE's) 4-hydroxyestradiol (4-OHE2) and 2-hydroxyestradiol (2-OHE2) were injected subcutaneously to ovariectomized adult rats. Serum levels of both LH and FSH were determined at short-term intervals. Moreover, serum levels of the administered steroids and their main free and conjugated metabolites were monitored. Serum levels of the injected steroids reached peak values at different time points: estradiol between 60 and 240 min, CE's between 30 and 60 min. Peak height also differed significantly: with estradiol highest (1500 pg/ml), followed by 4-OHE2 (540 pg/ml) and then 2-OHE2 (135 pg/ml) (ratio 11:4:1). This mirrored the different MCR's of the steroids: CE's, especially 2-OHE2, were rapidly and extensively methylated and/or - to a lesser degree - conjugated. Estradiol remained mainly unchanged. LH-serum levels in steroid treated animals showed - irrespective of the steroid used - a uniform reaction pattern: they were significantly depressed 60 to 240 min after injection and - with the exception of estradiol treated rats - reached pretreatment levels again 480 min after injection. Ultra-short (15 min) effects were not observed. FSH serum levels in CE treated animals were not significantly altered, only E2 application led to a significant but small decrease in FSH levels 240 and 480 min after its injection. In conclusion, neither the effect of 4-OHE2 nor that of 2-OHE2 corresponded to the different MCR's or the MCR-corrected affinities for the classical estrogen receptor. A non-genomic mechanism may be responsible for this impaired effect of CE's.

Topics: Animals; Castration; Estradiol; Estrogens, Catechol; Female; Follicle Stimulating Hormone; Luteinizing Hormone; Ovary; Rats; Rats, Inbred Strains; Time Factors

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

Microorganisms known to hydroxylate alkaloids, amino acids, and aromatic substrates were examined for their potential to hydroxylate 17 beta-estradiol and estrone. Thin-layer chromatography of fermentation extracts revealed a wide range of steroid products. Aspergillus alliaceus (UI 315) was the only culture capable of producing good yields of catechol estrogens with 17 beta-estradiol. The organism also transformed estrone but not to catechol products. Analytical experiments with high-performance liquid chromatography revealed that A. alliaceus formed 4- and 2-hydroxyestradiol with yields of 45 and 16%, respectively. A preparative-scale incubation was conducted in 2 liters of medium containing 1 g of 17 beta-estradiol as substrate. 4-Hydroxyestradiol was isolated and identified by proton nuclear magnetic resonance and high-resolution mass spectrometry. Ascorbic acid was added to microbial reaction mixtures as an antioxidant to prevent the decomposition of unstable catechol estrogen metabolites. The microbial transformation of 17 beta-estradiol by A. alliaceus provides an efficient one-step method for the preparation of catechol estrogens.

Topics: Aspergillus; Chromatography, High Pressure Liquid; Estradiol; Estrogens, Catechol; Hydroxylation

In the rat oestrogen is essential for induction of implantation in a progesterone primed uterus. The ability of the catechol oestrogens, 4-hydroxy-oestradiol (4-OH-E2) and 2-hydroxy-oestradiol (2-OH-E2) to initiate the implantation process (blue reaction, indicative of increased endometrial capillary permeability at the location of the blastocyst) in hypophysectomized delayed implanting rats was compared to that of oestradiol-17 beta (E2). Delayed implantation was maintained by daily administration of 2 mg of progesterone. A single sc injection of 100 ng of E2 or 400 ng of 4-OH-E2 consistently initiated implantation in all animals. When delivered sc via osmotic minipumps at a constant release rate of 10 ng/microliters/h, implantation of a full complement of embryos was obtained with E2 given for 6 or 24 h. This dose 4-OH-E2 was ineffective when administered for 24 h. However, 25 ng/microliters/h for 24 h was effective. When the dose was increased to 50 ng/microliters/h implantation was evident in the majority of animals exposed to the hormone for 6 or 8 h; all animals implanted when this dose was given for 24 h. In contrast, 2-OH-E2 given at 50 ng/microliters/h for 24 h was totally ineffective. When the dose of the latter steroid was raised to 200 ng/microliters/h implantation was achieved in half of the animals when exposure was 24 h. The results suggest that when administered systemically, 4-OH-E2 is less potent than E2, but more potent than 2-OH-E2, for initiating implantation.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Blastocyst; Capillary Permeability; Embryo Implantation; Embryo Implantation, Delayed; Endometrium; Estradiol; Estrogens, Catechol; Female; Hypophysectomy; Injections, Subcutaneous; Pregnancy; Rats

Electron spin resonance spectroscopy has been used to demonstrate production of semiquinone-free radicals from the oxidation of the catechol estrogens 2- and 4-hydroxyestradiol and 2,6- and 4,6-dihydroxyestradiol. Radicals were generated either enzymatically (using horseradish peroxidase-H2O2 or tyrosinase-O2) or by autoxidation, and were detected as their complexes with spin-stabilizing metal ions (Zn2+ and/or Mg2+). In the peroxidase system, radicals are produced by one-electron oxidation of the catechol estrogen and their decay is by a second-order pathway, consistent with their disproportionation to quinone and catechol products. With tyrosinase-O2, radical generation occurs indirectly. Initial hydroxylation of phenolic estrogen (at either the 2- or 4-position) gives a catechol estrogen in situ; subsequent two-electron oxidation of the catechol to the quinone, followed by reverse disproportionation, leads to the formation of radicals. A competing mechanism for radical production involves autoxidation of the catechol. Results obtained from the estrogen systems have been compared with those from the model compound 5,6,7,8-tetrahydro-2-naphthol.

Topics: Benzoquinones; Electron Spin Resonance Spectroscopy; Estradiol; Estrogens, Catechol; Magnesium; Monophenol Monooxygenase; Naphthols; Quinones; Zinc

In order to investigate the effects of 2-hydroxyoestradiol (2-OHE2) and 4-hydroxyoestradiol (4-OHE2) on the release of human pituitary hormones, each steroid (500 micrograms single bolus) was injected to young women during the follicular phase. Both steroids resulted in a transient suppression followed by a small rise of LH and FSH. 4-OHE2 appeared to be more potent than 2-OHE2. Injection of only the vehicle caused no change in gonadotrophin levels. The morning fall of plasma Prl was common in young women as an expression of circadian variation. Although the Prl fall tended to be greater in the 4-OHE2 group, analyses failed to reach statistical significance.

Topics: Adult; Estradiol; Estrogens, Catechol; Female; Follicle Stimulating Hormone; Humans; Luteinizing Hormone; Prolactin

Female rats were defeminized by neonatal treatment with estradiol-17beta benzoate, moxestrol (RU 2858), monohydroxytamoxifen ICI 79,280) or the dibenzoate esters of the catecholestrogens, 2-hydroxyestradiol-17beta and 4-hydroxyestradiol-17beta. When ovariectomized as adults and primed with estradiol-17beta benzoate all these rats demonstrated a deficient luteinizing hormone response to progesterone administration. However, estrogen responsiveness of progestin receptor induction was unimpaired in both the pituitary gland, the preoptic-hypothalamic brain and the uterus.

Topics: Animals; Animals, Newborn; Estradiol; Estrogen Antagonists; Estrogens, Catechol; Ethinyl Estradiol; Female; Hypothalamus; Male; Pituitary Gland; Preoptic Area; Rats; Receptors, Progesterone; Tamoxifen; Uterus

The effects of estradiol (E2) and its 2- and 4-hydroxylated metabolites on gonadotrophin regulation in the female rat brain were examined. Neonatal female rats were injected from day 1 through 5 with E2, 2-OHE2 and 4-OHE2, at doses of 0.1, 1 and 10 micrograms/day. At 2, 6 and 24 h after the last estrogen injection, some animals from each treatment group were killed and the concentration of estrogen receptors (ERn) in their brain cell nuclei determined. The remaining animals were allowed to mature. Their vaginal smear patterns were examined from 7 to 9 and from 15 to 17 weeks of age. They were then ovariectomized and tested for their capacity to exhibit a luteinizing hormone (LH) surge in response to estrogen and progesterone injections. In a parallel series of experiments, the affinities of the three test estrogens for alpha-fetoprotein (AFP) were determined from in vitro competition studies with fetal rat serum. All three estrogens increased brain cell nuclear ERn concentrations, measured at 2 h after the final injection. E2 was more potent in this respect than either 4-OHE2 or 2-OHE2. E2 and 4-OHE2 competed for binding to AFP to an approximately equal extent. 2-OHE2, however, was a much weaker competitor for AFP than either of the other two compounds. The neonatal E2 and 4-OHE2 treatments reduced the number of animals showing regular cyclic vaginal smears, at all three doses tested. In contrast, 2-OHE2 significantly affected vaginal cyclicity only at a dose of 10 micrograms/day.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: alpha-Fetoproteins; Animals; Animals, Newborn; Anovulation; Cell Nucleus; Estradiol; Estrogens, Catechol; Estrus; Female; Luteinizing Hormone; Pregnancy; Rats; Rats, Inbred Strains; Receptors, Estrogen; Time Factors; Vagina

Female rats were neonatally treated with estradiol-17 beta-benzoate or the long-acting dibenzoate esters of the isomeric catecholestrogens, 2-hydroxyestradiol-17 beta and 4-hydroxyestradiol-17 beta. Estrogen benzoates were administered subcutaneously from day 1 to 5 of life at doses of 0.05, 0.10, 0.50 and 1.00 micrograms/day. All rats were ovariectomized as adults and, 4 weeks later, the luteinizing hormone (LH) response to progesterone (2.5 mg) was tested after priming with estradiol-17 beta-benzoate (20 micrograms). At a dose of 0.5 micrograms/day, estradiol-17 beta-benzoate and 4-hydroxyestradiol-17 beta-dibenzoate were equally effective in neonatally defeminizing the LH surge mechanism. In contrast, up to a dose of 1.00 micrograms/day, 2-hydroxyestradiol-17 beta-dibenzoate did not interfere with the LH response in adult life. In the pituitary gland and uterus of the neonatally defeminized rats estrogen responsiveness of cytosolic progestin receptor induction was unimpaired. Moreover, in the uterus of these rats nuclear translocation of cytosolic progestin receptors was intact.

Topics: Animals; Animals, Newborn; Cell Nucleus; Cytosol; Estradiol; Estrogens, Catechol; Female; Luteinizing Hormone; Pituitary Gland; Rats; Rats, Inbred Strains; Receptors, Progesterone; Uterus

Using a competitive binding assay the effects of 2-hydroxyestradiol-17 beta, 4-hydroxyestradiol-17 beta, estradiol-17 beta and progesterone on the binding of tritiated catecholaminergic ligands to membrane preparations from rat brain and pituitary gland were studied. Up to a concentration of 10(-5) M none of the steroids tested was able to displace [3H]spiroperidol, [3H]dihydroergocryptine or [3H]dihydroalprenolol. The data suggest that the catecholestrogens do not interfere directly with the binding of catecholaminergic ligands to dopaminergic, alpha-adrenergic or beta-adrenergic receptors in the central nervous system. The view that a catechol structure is not essential for the interaction with dopaminergic receptors was further supported by the results obtained from additional studies on the competition of O-methylated and deaminated dopamine metabolites with [3H]spiroperidol binding.

Topics: Animals; Binding, Competitive; Brain; Dihydroalprenolol; Dihydroergotoxine; Estradiol; Estrogens, Catechol; Female; Humans; In Vitro Techniques; Pituitary Gland; Rats; Rats, Inbred Strains; Receptors, Adrenergic; Receptors, Catecholamine; Spiperone; Structure-Activity Relationship

We studied the action of catecholestrogens on prostaglandin and LH release by female rat pituitaries superfused in vitro. Their actions were compared to those of estradiol (E2) at the same concentration (10(-6) M). By comparison with E2 the action of 2-OHE2 was only the same on total PGE2 release. 4-OHE2-17 beta stimulated LH like E2 but the values for PGD2 and PGE2 were about 20-30% lower. However, the release of 6-keto-PGF1 alpha was about 35% higher. 4-OHE2-17 alpha had no comparable action on PG release but stimulated a significant LH release. There was no action on TxB2 in any case.

Topics: Animals; Estradiol; Estrogens, Catechol; Female; Luteinizing Hormone; Pituitary Gland; Prostaglandins; Prostaglandins D; Prostaglandins E; Prostaglandins F; Rats; Thromboxane B2

MCRs of the catechol estrogens 4-hydroxyestradiol (4-OHE2) and 2-hydroxyestradiol (2-OHE2) and of the parent estrogen 17 beta-estradiol (E2) were determined in rats. Long term ovariectomized Wistar rats were infused with the steroids at a constant rate for 3 days via a catheter placed in the abdominal aorta. Blood samples were drawn discontinually by retroorbital puncture, and the serum concentrations of E2, 4-OHE2, and 2-OHE2 were measured by RIA. Steady state was reached within 24 h of infusion. Mean serum MCRs were calculated to be 740 +/- 117 ml/h for E2, 2700 +/- 1000 ml/h for 4-OHE2, and 8300 +/- 1700 ml/h for 2-OHE2. Thus, the MCRs of the catechol estrogens were definitely higher than the MCR of E2 resulting in an apparent ratio of 1:4:11 (E2:4-OHE2:2-OHE2).

Topics: Animals; Castration; Estradiol; Estrogens, Catechol; Female; Metabolic Clearance Rate; Radioimmunoassay; Rats; Rats, Inbred Strains

The influence of estradiol-17 beta (E2) or catechol estradiols [4-hydroxyestradiol-17 beta (4-OH-E2) or 2-hydroxyestradiol-17 beta (2-OH-E2)] on prostaglandin (PG) production in the rabbit blastocyst and endometrial cell in vitro on Day 6 of pregnancy (144 h postcoitum) was studied. Blastocysts (4-6) were incubated in 1 ml of RPMI-1640 medium for a total period of 8 h. Media were changed every 2 h and stored at -80 degrees C. E2, 4-OH-E2 or 2-OH-E2 at various concentrations were added during the second and fourth periods, while the vehicle was added during the first and third periods. Single cell suspensions of endometrial tissues in 0.5 ml of RPMI-1640 were incubated for 2 h with and without estrogens. The media and cells were separated and stored at -80 degrees C. PGs were determined by radioimmunoassay. The results for blastocyst were expressed as the cumulative release of PGs over a period of two 2-h incubations of the same experimental conditions (pg/blastocyst per 4 h). The release of PGs from the blastocyst was not influenced by 8.8 microM of 4-OH-E2, whereas 44 microM of this steroid stimulated the release of PGE-A by about 25% and PGF by 59% (P less than 0.05) over the vehicle-treated controls. The release of PGE-A and PGF in the presence of 44 microM of 2-OH-E2 was stimulated by about 60% and 37%. On the other hand, E2 at any concentration, either showed none or inhibitory effects on the release of PGs from the blastocyst.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Blastocyst; Endometrium; Estradiol; Estrogens, Catechol; Female; In Vitro Techniques; Prostaglandins; Prostaglandins E; Prostaglandins F; Rabbits; Stimulation, Chemical

The ability of estradiol-17 alpha to serve as a substrate for estrogen-2/4-hydroxylase in rabbit hypothalamic tissue was determined and compared to that of estradiol-17 beta. Both 2- and 4-hydroxy metabolites of estradiol-17 alpha were formed by the hypothalamic tissue in vitro. The rates of formation of 2-hydroxyestradiol (2-OHE2)-17 alpha and -17 beta were similar as were their kinetic constants (Km and Vmax). In addition, 2-OHE2-17 alpha was shown to inhibit purified rat adrenal tyrosine hydroxylase with a potency comparable to that of 2-OHE2-17 beta, a finding similar to that reported by others with respect to catechol-o-methyltransferase. Since estradiol-17 alpha has a markedly reduced affinity for estrogen receptors compared with extradiol-17 beta, this steroid could be useful in studies designed to distinguish between receptor mediated effects of estrogens and effects that locally formed catechol estrogens may have through their direct interaction with catecholaminergic system in neural tissue.

Topics: Animals; Estradiol; Estrogens, Catechol; Hypothalamus; Kinetics; Rabbits; Tyrosine 3-Monooxygenase

Topics: Animals; Breast Neoplasms; Equilenin; Equilin; Estradiol; Estrogens; Estrogens, Catechol; Ethinyl Estradiol; Female; Humans; Hydroxyestrones; Hydroxylation; Microsomes, Liver; Papio

A high performance liquid chromatographic method is described for the rapid, non-destructive separation of a number of physiologically important steroidal estrogens, including the labile catechol estrogens. This procedures uses a "Diol" column and gradient elution to separate in a single run, estrogens ranging from 2-methoxy estrone, one of the least polar C18 steroids, to estriol, one of the most polar. Simpler, isocratic conditions, are provided for the separation of estrogens of similar polarity. A semi-preparative column of similar composition was used for the purification of samples containing 25 to 50 mg of individual steroids.

Topics: Chromatography, High Pressure Liquid; Estradiol; Estrogens; Estrogens, Catechol; Hydroxyestrones

The effects of primary and catechol oestrogens on the uterus of the immature rat were compared. Because differences between the in-vivo and in-vitro oestrogenic actions of catechol oestrogens on the secretion of LH had been observed, their effects on a peripheral target organ, the uterus, were examined under similar conditions. In-vivo effects were assessed by measurement of uterine weight, induction of uterine cytoplasmic progestogen receptors, and by histological examination. In-vitro actions were determined by measurement of oestrogen-specific induced protein. It was found that the uterotrophic effects in vivo of 4-hydroxyoestradiol were indistinguishable from those of oestradiol whereas 2-hydroxyoestradiol was only weakly oestrogenic and 2-hydroxyoestrone had no effect. However, in vitro, 2-hydroxyoestradiol was as effective as 4-hydroxyoestradiol or oestradiol in stimulating synthesis of uterine induced protein, and 2-hydroxyoestrone, although less potent than oestradiol, had a significant effect. These results were consistent with the observed effects on the secretion of LH. The differences between in-vivo and in-vitro uterotrophic properties of catechol oestrogens can be explained on the basis of known pharmacokinetic factors.

Topics: Animals; Estradiol; Estrogens; Estrogens, Catechol; Estrone; Female; Hydroxyestrones; Organ Size; Rats; Rats, Inbred Strains; Sexual Maturation; Uterus

Induction of implantation is among the most sensitive responses to estrogens. The ability of catechol estradiols, 4-hydroxy-estradiol-17 beta (4-OH-E2) and 2-hydroxy-estradiol-17 beta (2-OH-E2), to induce implantation in ovariectomized pregnant mice was compared to that of estradiol-17 beta. Delayed implantation was maintained by the daily administration of 2 mg of progesterone. A single injection of 3 ng of estradiol-17 beta, 50 ng of 4-OH-E2, or 2,000 ng of 2-OH-32 consistently induce a full complement of implantation sites in all animals. Before the estrogenicity of the latter steroid can be established the lack of contaminating estrogens must be proved.

Topics: Animals; Castration; Embryo Implantation; Estradiol; Estrogens, Catechol; Female; Mice; Ovary; Pregnancy; Progesterone

A direct product isolation assay for quantifying the formation of 2- and 4-hydroxyestradiol (2-OHE2 and 4-OHE2) from [6,7-3H]estradiol by rabbit hypothalami in vitro was developed, and the assay was used to characterize some properties of estrogen-2- and 4-hydroxylase activity in this tissue. The reaction was carried out under conditions that minimized further metabolism of enzymatically formed catechol estrogens. A simple two-step separation procedure, involving the use of a neutral alumina column, followed by thin layer chromatography, was developed to isolate the enzymatically formed catechol estrogens in a radiochemically homogeneous form. The detergent, Tween-80, was found to activate the enzyme and was used routinely at a concentration of 0.1% in the assay. The formation of 2-OHE2 was linear up to 10 min and with increasing protein concentrations up to 150 micrograms/incubation. Similar values were obtained for 4-OHE2. Maximum velocities (Vmax) for the formation of 2- and 4-OHE2 were 190 and 270 pmol/mg protein . 10 min, respectively. The apparent Km values with respect to estradiol for 2-OHE2 and 4-OHE2 were 125 and 150 microM, respectively. The highest specific activity for the enzyme was present in the 100,000 X g supernatant (S3), while the activity in the microsomal fraction (P3) was less than that in the original homogenate. Enzyme activity depended on the presence of NADPH and oxygen and was inhibited by CO as well as by high concentrations of SKF-525A. Estrogen-2- and 4-hydroxylase activity in rabbit hypothalamus differed from that in rat liver in two respects. In the liver, enzyme activity was localized in the microsomal fraction and was virtually abolished by Tween-80. In contrast, enzyme activity in rabbit hypothalamus was maximal in the soluble fraction (100,000 X g supernatant)and was stimulated by the detergent.

Topics: Animals; Carbon Radioisotopes; Cytochrome P-450 CYP1A1; Estradiol; Estrogens, Catechol; Estrone; Female; Hypothalamus; Kinetics; Rabbits; Radioisotope Dilution Technique; Steroid Hydroxylases; Tritium

Topics: Animals; Chromatography, High Pressure Liquid; Cytochrome P-450 CYP1A1; Electrochemistry; Estradiol; Estrogens; Estrogens, Catechol; Estrone; Hydrogen-Ion Concentration; Hydroxyestrones; Liver; Male; Rats; Rats, Inbred Strains; Steroid Hydroxylases

We have examined the effect of the catechol oestrogens 2-hydroxyoestradiol (2-OHE2), 4-hydroxyoestradiol (4-OHE2) and 2-hydroxyoestrone (2-OHE1) and their corresponding primary oestrogens on secretion of LH and FSH by enzymatically dispersed rat anterior pituitary cells in monolayer culture. Basal LH levels in the medium were significantly higher than in control wells when cells were exposed to 10(-8) M-oestradiol-17 beta for 40 h: oestrone and all three catechol oestrogens (in the same doses) also stimulated basal LH concentrations to levels quantitatively similar to those seem after oestradiol treatment. The same effects were observed when steroids were given at 10(-9) mol/l. Oestradiol, 2-OHE2, and 4-OHE2 but not 2-OHE1 increased pituitary responsiveness to LH releasing hormone (LH-RH) (given in a range of doses from 10(-11) to 10(-6) mol/l). The responses of cells treated with 2-OHE2 and 4-OHE2 were similar, though less than the response seen after treatment with oestradiol. This contrasts with the very different oestrogenic effects of 2- and 4-OHE2 previously observed in vivo. Neither oestradiol nor the catechol oestrogens had any effect on basal or LH-RH-stimulated FSH release.

Topics: Animals; Cells, Cultured; Estradiol; Estrogens, Catechol; Estrone; Female; Follicle Stimulating Hormone; Hydroxyestrones; Luteinizing Hormone; Pituitary Gland, Anterior; Rats; Secretory Rate; Stimulation, Chemical

Topics: 6-Ketoprostaglandin F1 alpha; Animals; Estradiol; Estrogens, Catechol; Estrone; Female; Humans; Hydroxyestrones; Prostaglandins; Prostaglandins E; Prostaglandins F; Rats; Uterus

Topics: Animals; Behavior, Animal; Brain; Cell Nucleus; Cytosol; Estradiol; Estrogens, Catechol; Estrone; Female; Glucosephosphate Dehydrogenase; Hydroxyestrones; Pituitary Gland; Posture; Rats; Receptors, Estrogen; Receptors, Progesterone

The ability of the catecholestrogens, 4-hydroxyestradiol (4-OHE2) and 2-hydroxyestradiol (2-OHE2) to induce lordosis in ovariectomized rats primed with progesterone was assessed following either short- or long-term exposure to the steroids. 4-OHE2 successfully induced lordosis whether administered as two bolus intrajugular injections separated by a 3-hour interval or continuously for 7 days via subcutaneously implanted osmotic minipumps. 2-OHE2, however, was ineffective under both conditions. When administered subcutaneously in conjunction with 3 days of estradiol benzoate (E2B) treatment, neither of the catecholestrogens exhibited any antiestrogenic effect as compared with the lordosis response to E2B alone. Likewise, simultaneous administration of 2-OHE2 did not significantly alter the response to intrajugular injections of estradiol (E2) or 4-OHE2. The results indicate that 4-OHE2 (but not 2-OHE2) mimicks the lordosis-inducing action of E2 and that neither catecholestrogen has any antiestrogenic effect in this behavioral test system.

Topics: Animals; Castration; Estradiol; Estrogens, Catechol; Female; Organ Size; Posture; Progesterone; Rats; Sexual Behavior, Animal

The pharmacological effect of 2-hydroxyoestradiol (2-OHE2) and 4-OHE2 on concentrations of LH in the chronically castrated rat have been compared with that of oestradiol in order to determine whether the in-vivo activity is altered by insertion of a hydroxyl group at position 2 or 4 of the aromatic A ring; these derivatives are naturally occurring oestrogen metabolites. Four groups of six adult male rats were used 4 weeks after bilateral orchidectomy. The right jugular vein was exposed under ether anaesthesia and a basal blood sample taken (10.00 h) immediately before an intravenous injection of vehicle alone (0.1 ml ethanol with 0.01% ascorbic acid), oestradiol, 2-OHE2 or 4-OHE2 (10 micrograms of each in 0.1 ml vehicle). Blood was taken from each animal at 2, 4, 6, 8 and 24 h after treatment and serum assayed for LH. Baseline LH levels were similar in the four groups. At 2 h there was no change in 2-OHE2-treated rats but there was a significant decrease of serum levels of LH in rats treated with oestradiol and 4-OHE2 compared with vehicle-treated controls. The decrease in LH was quantitatively similar in oestradiol- and 4-OHE2-treated groups and was sustained at 4, 6 and 8 h, returning to control values at 24 h. In subsequent experiments the effects of lower doses of these two steroids were compared and the potency of 4-OHE2 was estimated to be about 25% that of oestradiol. In a further experiment, 2-OHE2 (100 micrograms) had no effect when given alone, but when injected i.v. immediately before treatment with 1 microgram oestradiol, it was able to inhibit the suppression of LH by oestradiol. In conclusion, 4-OHE2 had a potent effect in lowering plasma LH levels whereas 2-OHE2, even at a high dose (100 micrograms), did not suppress LH but it was able to inhibit the effect of oestradiol. These differences in biological activity may reflect more rapid metabolism of 2-OHE2 or differences in binding properties of these catechol oestrogens to the oestrogen receptor.

Topics: Animals; Castration; Estradiol; Estrogens, Catechol; Luteinizing Hormone; Male; Rats

The 'positive feedback' effect of exogenous oestradiol-17 beta in advancing ovulation induced by pregnant mare serum gonadotrophin (PMSG) has been used in the present study as a model in which to test the possible oestrogenic or antioestrogenic effects of the catechol oestrogens, 2-hydroxyoestradiol (2-OHE2) and 4-OHE2. Sprague-Dawley rats of 26 days of age were injected with 20 i.u. PMSG together with either vehicle alone or test steroids. The animals were killed 72 h later and the Fallopian tubes were examined for the presence of ova. Advancement of induced ovulation by treatment with oestradiol was confirmed; 2-OHE2, in doses of up to 100 micrograms, influenced neither the time of ovulation nor the number of ova present but 4-OHE2 was equipotent with oestradiol in doses varying from 0.5 micrograms (the minimum effective dose for both steroids) to 10 micrograms. The possible antioestrogenic effect of 2-OHE2 was tested by giving a 100 micrograms dose either at the same time or 2 h before PMSG plus 2 micrograms oestradiol or 4-OHE2. The effects of oestradiol and 4-OHE2 were not altered by this treatment. These data show that, in this model of 'positive feedback', 2-OHE2 has neither an oestrogenic nor an antioestrogenic action but that 4-OHE2 has a potent oestrogenic action, thus raising the question of a physiological role for 4-OHE2 in the regulation of ovulation.

Topics: Animals; Catechols; Estradiol; Estrogens; Estrogens, Catechol; Female; Gonadotropins, Equine; Ovulation; Ovulation Induction; Rats; Sexual Maturation

2-Hydroxyest radiol-17 alpha and 4-hydroxyestradiol-17 alpha, the catechol derivatives of estradiol-17 alpha, have reduced affinity for hypothalamic, pituitary, and uterine estrogen receptors, but retain a potency for interaction with catechol-0-methyltransferase equal to that of the natural, 17 beta-hydroxy catechols. This dissociation of receptor binding and catecholamine interactions may allow the use of the 17 alpha catechols as a probe for the mechanism of action of the catechol estrogens.

Topics: Amygdala; Animals; Catechol O-Methyltransferase; Cytosol; Estradiol; Estrogens, Catechol; Female; Hypothalamus; Methylation; Norepinephrine; Pituitary Gland; Preoptic Area; Rats; Receptors, Estrogen

When [6,7-3H]estradiol was incubated with tissue homogenates of the brain, the pituitary, and the liver of two human female fetuses, a number of radioactive metabolites more "polar" than the incubated substrate were detected. Among these, the identification of two types of catecholestrogens, i.e. the 2- and 4-hydroxyestrogens, was of major interest. Compared on the basis of wet weight of tissues (250 mg), the conversion of estradiol to 2-hydroxyestrogens (2-hydroxyestradiol and 2-hydroxyestrone) was 0.8% in the frontal cortex, 1.0% in the hypothalamus, 2.1% in the pituitary, and 7.8% in the liver. For the first time, the formation of 4-hydroxyestrogens was demonstrated. The percentages of incubated estradiol hydroxylated at C-atom 4 (4-hydroxyestradiol and 4-hydroxyestrone) were 0.5 in the cortex, 0.4% in the hypothalamus, .1% in the pituitary, and 0.5% in the liver. The results show that fetal brain and pituitary tissue can hydroxylate estradiol in positions 2 and 4 to a similar extent, whereas in the liver, about 15 times more 2-hydroxy than 4-hydroxy compounds are formed. Moreover, the 2-hydroxylating capacity of the liver is definitely greater than that of the brain, whereas the 4-hydroxylating capacity is about the same as that of the brain.

Topics: Brain; Estradiol; Estrogens, Catechol; Female; Humans; Hydroxyestrones; Hydroxylation; Liver; Pituitary Gland

2-Hydroxyestrone, 2-hydroxyestradiol-17beta, 2-hydroxy-17alpha-ethynylestradiol, 2-hydroxyestriol, 4-hydroxyestrone, 4-hydroxyestradiol-17beta, 4-hydroxy-17alpha-ethynylestradiol and 4-hydroxyestriol are prepared on a preparative scale from the corresponding aminophenols using a new inverse oxidation procedure. By the synthesis described both the 2- and 4-hydroxylated estrogens are available in high yields.

Topics: Catechols; Chemical Phenomena; Chemistry; Estradiol; Estriol; Estrogens; Estrogens, Catechol; Ethinyl Estradiol; Hydroxyestrones; Methods