Catechol Estrogen Formation Research Articles

Catechol Estrogen 4-Hydroxyestradiol is an Ultimate Carcinogen in Breast Cancer

Excessive exposure to estrogens is the most important risk factor for the development of hormone-sensitive cancers, especially breast cancer. Estrogen stimulates the expression of genes and proteins involved in cell proliferation by binding to estrogen receptor (ER). Another possible mechanism of ER-independent carcinogenicity of estrogens is based on the hydroxylation of estradiol resulting in the formation of catechol estrogens. Catechol estrogen 4-hydroxyestradiol (4-OHE2) is further oxidized to catechol estrogen-3,4-quinones, the major carcinogenic metabolites of estrogens. Evidence increasingly supports the critical role of 4-OHE2 in hormonal carcinogenesis via DNA adduct formation or production of reactive oxygen species, which finally contribute to the transformation of normal mammary epithelial cells and the enhanced growth of breast cancer cells. It is also reported that the level of 4-OHE2 or its quinones is highly up-regulated in urine or tissues of breast cancer patients. Thus, we highlight the oncogenic roles of 4-OHE2 in catechol estrogen-induced breast carcinogenesis.

Abstract LB-64: Selective estrogen receptor modulators (SERMs) act as breast cancer chemopreventive agents by attenuating oxidative estrogen metabolism

Abstract Possibility of developing hormone dependent cancer in women will increase with long term exposure to endogenous estrogens. Estrogen induced, estrogen receptor (ER) mediated cell proliferation (hormonal pathway) and formation of reactive estrogen metabolites (chemical pathway) are believed to contribute to estrogen carcinogenesis. Estrogens are oxidized to the catechols, 2-hydroxyestradiol (2-OHE2) and 4-hydroxyestradiol (4-OHE2) by P450 1A1/1A2 and P450 1B1 respectively. Both catechols are further oxidized to form electrophilic o-quinones which can react with DNA and proteins, while 4 hydroxyestradiol O-quinone (4-OHE2-Q) is considered as the ultimate carcinogen. Selective Estrogen Receptor Modulators (SERMs) are used in cancer chemoprevention and in the treatment of post menopausal osteoporosis. The current study focuses on elucidating the influence of clinical and pre clinical benzothiophene SERMs on oxidative estrogen metabolism in ER negative non tumerigenic human breast epithelial cells (MCF-10A). Upon exposure to 17β-estradiol (E2), MCF-10A cells transformed into a malignant phenotype and showed anchorage independent colony growth in soft agar. After treatment of MCF-10A cells with E2 and SERMs, estrogen metabolites were analyzed using a sensitive LC-MS/MS method. Among the SERMs tested, raloxifene and desmethylarzoxifen (DMA) showed a significant reduction in catechol estrogen formation whereas 4′F-DMA had little effect. Furthermore, raloxifene and DMA significantly inhibited E2 induced malignant transformation and reactive oxygen species formation in MCF-10A cells. These data suggest that raloxifene and DMA possess chemopreventive activity through inhibition of genotoxic estrogen quinone formation, in addition to their action via estrogen receptors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-64. doi:1538-7445.AM2012-LB-64

Raloxifene and desmethylarzoxifene block estrogen-induced malignant transformation of human breast epithelial cells.

There is association between exposure to estrogens and the development and progression of hormone-dependent gynecological cancers. Chemical carcinogenesis by catechol estrogens derived from oxidative metabolism is thought to contribute to breast cancer, yet exact mechanisms remain elusive. Malignant transformation was studied in MCF-10A human mammary epithelial cells, since estrogens are not proliferative in this cell line. The human and equine estrogen components of estrogen replacement therapy (ERT) and their catechol metabolites were studied, along with the influence of co-administration of selective estrogen receptor modulators (SERMs), raloxifene and desmethyl-arzoxifene (DMA), and histone deacetylase inhibitors. Transformation was induced by human estrogens, and selectively by the 4-OH catechol metabolite, and to a lesser extent by an equine estrogen metabolite. The observed estrogen-induced upregulation of CYP450 1B1 in estrogen receptor negative MCF-10A cells, was compatible with a causal role for 4-OH catechol estrogens, as was attenuated transformation by CYP450 inhibitors. Estrogen-induced malignant transformation was blocked by SERMs correlating with a reduction in formation of nucleobase catechol estrogen (NCE) adducts and formation of 8-oxo-dG. NCE adducts can be formed consequent to DNA abasic site formation, but NCE adducts were also observed on incubation of estrogen quinones with free nucleotides. These results suggest that NCE adducts may be a biomarker for cellular electrophilic stress, which together with 8-oxo-dG as a biomarker of oxidative stress correlate with malignant transformation induced by estrogen oxidative metabolites. The observed attenuation of transformation by SERMs correlated with these biomarkers and may also be of clinical significance in breast cancer chemoprevention.

Abstract 5620: Polymorphisms in steroid hormone genes and endometrial cancer risk

Abstract The incidence of endometrial cancer is increased in the presence of elevated circulating levels of estrogens, especially when inadequately opposed by progestins. Estrogens are catabolized via catechol estrogen formation, oxidation, glucuronidation, and sulfation. Thus, it is possible that interindividual variation in these pathways might influence a woman's risk of endometrial cancer. To test this hypothesis, we examined whether the presence of the minor alleles in 26 SNPs in the genes CYP17, CYP1A1, CYP1B1, CYP3A4, CYP3A5, COMT, SULT1A1, UGT1A1, UGT1A8, UGT2B4, UGT2B7, and UGT2B15 was associated with risk of endometrial cancer in a population-based case-control study in Washington State, with 727 cases ages 50-74 years diagnosed between 1994-2005, and 736 controls frequency-matched to the cases by age, county and reference year. Analyses were restricted to non-Hispanic white women and adjusted for frequency matching variables. Only SNPs in CYP1A1 (involved in the formation of 2-hydroxyestrogens) and SULT1A1 (involved in estrogen sulfation) were associated with endometrial cancer risk. The CYP1A1 rs4646903 C allele was associated with a decreased risk of endometrial cancer (per C allele odds ratio (OR) 0.75, 95% confidence interval (CI) 0.58-0.95; ORs and 95% CIs for CT and CC vs TT were, respectively: 0.81, 0.61-1.07, and 0.38, 0.15-0.93). The SULT1A1 rs9282861 A allele was associated with increased risk of endometrial cancer (per A allele OR 1.22, 95% CI 1.04-1.42; ORs and 95% CIs for AG and AA vs GG were, respectively: 1.20, 0.96-1.50, and 1.50, 1.06-2.12). Both of these polymorphisms have been reported to be associated with alterations in the ability to metabolize estrogens; the CYP1A1 rs4646903 C allele may be associated with increased inducibility and increased endometrial expression of CYP1A1, and therefore could be associated with potentially higher levels of the 2-hydroxyestrogens in the target tissue. The SULT1A1 rs9282861 A allele is associated with reduced sulfotransferase activity, which could possibly allow the accumulation of estrogens. Thus, the observed associations with each SNP are consistent with their putative biologic function. However, several studies have examined variation in these and other genes in the steroid hormone metabolism pathways and endometrial cancer risk, and there are reports of both positive and negative findings for many of the SNPs studied. If a small effect size is expected, then additional studies and pooled analyses are needed to clarify possible associations with each of these polymorphisms. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5620. doi:10.1158/1538-7445.AM2011-5620

Estrogen metabolism and formation of estrogen-DNA adducts in estradiol-treated MCF-10F cells: The effects of 2,3,7,8-tetrachlorodibenzo- p-dioxin induction and catechol- O-methyltransferase inhibition

Formation of estrogen metabolites that react with DNA is thought to be a mechanism of cancer initiation by estrogens. The estrogens estrone (E 1) and estradiol (E 2) can form catechol estrogen (CE) metabolites, catechol estrogen quinones [E 1(E 2)-3,4-Q], which react with DNA to form predominantly depurinating adducts. This may lead to mutations that initiate cancer. Catechol- O-methyltransferase (COMT) catalyzes an inactivation (protective) pathway for CE. This study investigated the effect of inhibiting COMT activity on the levels of depurinating 4-OHE 1(E 2)-1-N3Ade and 4-OHE 1(E 2)-1-N7Gua adducts in human breast epithelial cells. MCF-10F cells were treated with TCDD, a cytochrome P450 inducer, then with E 2 and Ro41-0960, a COMT inhibitor. Estrogen metabolites and depurinating DNA adducts in culture medium were analyzed by HPLC with electrochemical detection. Pre-treatment of cells with TCDD increased E 2 metabolism to 4-OHE 1(E 2) and 4-OCH 3E 1(E 2). Inclusion of Ro41-0960 and E 2 in the medium blocked formation of methoxy CE, and depurinating adducts were observed. With Ro41-0960, more adducts were detected in MCF-10F cells exposed to 1 μM E 2, whereas without the inhibitor, no increases in adducts were detected with E 2 ≤ 10 μM. We conclude that low COMT activity and increased formation of depurinating adducts can be critical factors leading to initiation of breast cancer.

Expression of Cytochrome P450 1B1 and Catechol-O-Methyltransferase in Breast Tissue and Their Associations with Breast Cancer Risk

Cytochrome P450 1B1 (CYP1B1) and catechol-O-methyltransferase (COMT) are important estrogen-metabolizing enzymes that may affect breast cancer risk. Few studies have directly measured the expression of CYP1B1 and COMT genes in breast tissue samples. The subjects in this study were a subgroup of participants of the Shanghai Breast Cancer Study including 64 patients diagnosed with breast cancer and 68 patients diagnosed with benign breast diseases (BBD) who provided samples of tumor tissue and adjacent nontumor tissue to the study. We compared CYP1B1 and COMT mRNA expression in tumor tissue and adjacent nontumor tissue in both breast cancer patients and BBD patients. High levels of CYP1B1 expression and low levels of COMT expression in adjacent nontumor tissue were associated with a significantly increased breast cancer risk in a nonlinear manner. Odds ratios and 95% confidence intervals (in parentheses) for the midpoints of the first, second, fourth, and fifth quintiles of gene expression levels compared with the overall median levels in BBD subjects were 0.21 (0.07-0.67), 0.81 (0.69-0.95), 1.20 (1.05-1.38), and 1.55 (1.12-2.15) for CYP1B1 and 1.72 (1.17-2.55), 1.19 (1.05-1.35), 0.83 (0.73-0.95), and 0.78 (0.65-0.93) for COMT, respectively. These results support the hypothesis that the formation and accumulation of catechol estrogens in breast tissue through increased CYP1B1 expression and reduced COMT expression may play a significant role in breast cancer risk.

Genetic variation of Cytochrome P450 1B1 (CYP1B1) and risk of breast cancer among Polish women

Four single nucleotide polymorphisms (SNPs) in CYP1B1 (Ex2 + 143 C > G, Ex2 + 356 G > T, Ex3 + 251 G > C, Ex3 + 315 A > G) cause amino acid changes (R48G, A119S, L432V and N453S, respectively) and are associated with increased formation of catechol estrogens; however, epidemiologic evidence only weakly supports an association between these variants and breast cancer risk. Because genetic variability conferring increased susceptibility could exist beyond these putative functional variants, we comprehensively examined the common genetic variability within CYP1B1. A total of eight haplotype-tagging (ht)SNPs (including Ex3 + 315 A > G), in addition to two putatively functional SNPs (Ex2 + 143 C > G and Ex3 + 251 G > C), were selected and genotyped in a large case-control study of Polish women (1995 cases and 2296 controls). Haplotypes were estimated using the expectation-maximization algorithm, and overall differences in the haplotype distribution between cases and controls were assessed using a global score test. We also evaluated levels of tumor CYP1B1 protein expression in a subset of 841 cases by immunohistochemistry, and their association with genetic variants. In the Polish population, we observed two linkage disequilibrium (LD)-defined blocks. Neither haplotypes (global P-value of 0.99 and 0.67 for each block of LD, respectively), nor individual SNPs (including three putatively functional SNPs) were associated with breast cancer risk. CYP1B1 was expressed in most tumor tissues (98%), and the level of expression was not related to the studied genetic variants. We found little evidence for modification of the estimated effect of haplotypes or individual SNPs by age, family history of breast cancer, or tumor hormone receptor status. The present study provides strong evidence against the existence of a substantial overall association between common genetic variation in CYP1B1 and breast cancer risk.

Estrogen Bioactivation, Genetic Polymorphisms, and Ovarian Cancer

Recent experimental evidence has shown that catechol estrogens can be activated through metabolism to form depurinating DNA adducts and thereby initiate cancer. Limited data are available regarding this pathway in epithelial ovarian cancer. We conducted a case-control study of 503 incident epithelial ovarian cancer cases at the Mayo Clinic in Rochester, MN, and Jacksonville, FL, and a 48-county region in North Carolina. Six hundred nine cancer-free controls were frequency matched to the cases on age, race, and residence. After an interview to obtain data on risk factors, a sample of blood was collected for DNA isolation. Subjects were genotyped for seven common single nucleotide polymorphisms in four genes involved in catechol estrogen formation (CYP1A1 and CYP1B1) or conjugation (COMT and SULT1A1). Data were analyzed using logistic regression, stratified by race, and with adjustment for design factors and potential confounders. None of the individual genotypes were significantly associated with ovarian cancer risk. However, an oligogenic model that considered the joint effects of the four candidate genes provided evidence for an association between combinations of these genes and ovarian cancer status (P = 0.015). Although preliminary, this study provides some support for the hypothesis that low-penetrance susceptibility alleles may influence risk of epithelial ovarian cancer.

A Novel Mechanism for Adenylyl Cyclase Inhibition from the Crystal Structure of Its Complex with Catechol Estrogen

Catechol estrogens are steroid metabolites that elicit physiological responses through binding to a variety of cellular targets. We show here that catechol estrogens directly inhibit soluble adenylyl cyclases and the abundant trans-membrane adenylyl cyclases. Catechol estrogen inhibition is non-competitive with respect to the substrate ATP, and we solved the crystal structure of a catechol estrogen bound to a soluble adenylyl cyclase from Spirulina platensis in complex with a substrate analog. The catechol estrogen is bound to a newly identified, conserved hydrophobic patch near the active center but distinct from the ATP-binding cleft. Inhibitor binding leads to a chelating interaction between the catechol estrogen hydroxyl groups and the catalytic magnesium ion, distorting the active site and trapping the enzyme substrate complex in a non-productive conformation. This novel inhibition mechanism likely applies to other adenylyl cyclase inhibitors, and the identified ligand-binding site has important implications for the development of specific adenylyl cyclase inhibitors.

Impact of Metabolism on the Safety of Estrogen Therapy

Estrogen metabolism has received attention owing to the recognition that oxidative metabolism of these hormones could play a very important role in the initiation and evolution of estrogen-related cancerous processes. This review discusses principal metabolic pathways associated with the molecular constituents found in common hormone formulations. The focus is the formation of catechol estrogens that then oxidize to redox-active and electrophilic quinones, along with potential mechanisms by which these metabolites contribute to oxidative stress and induce damage to essential biological macromolecules. Recent investigations involving specific estrogen metabolites that may offer potential increase of therapeutic safety for estrogen therapy in the future have also been highlighted.

Resveratrol inhibits TCDD-induced expression of CYP1A1 and CYP1B1 and catechol estrogen-mediated oxidative DNA damage in cultured human mammary epithelial cells.

Resveratrol (3,5,4'-trihydroxystilbene), a naturally occurring phytoalexin present in grapes and other foods, has been reported to possess chemopreventive effects as revealed by its striking inhibition of diverse cellular events associated with tumor initiation, promotion and progression. In our present study, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), when treated with the cultured human mammary epithelial (MCF-10A) cells, induced the expression of cytochrome P450 1A1 (CYP1A1) and 1B1 (CYP1B1) that are responsible for the oxidation of 17beta-estradiol to produce catechol estrogens. Resveratrol strongly inhibited the TCDD-induced aryl hydrocarbon receptor (AhR) DNA binding activity, the expression of CYP1A1 and CYP1B1 and their catalytic activities in MCF-10A cells. It also reduced the formation of 2-hydroxyestradiol and 4-hydroxyestradiol from 17beta-estradiol by recombinant human CYP1A1 and CYP1B1, respectively. Furthermore, resveratrol significantly attenuated the intracellular reactive oxygen species (ROS) formation and oxidative DNA damage as well as the cytotoxicity induced by the catechol estrogens. Our data suggest that CYP1A1- and CYP1B1-catalyzed catechol estrogen formation might play a key role in TCDD-induced oxidative damage, and resveratrol can act as a potential chemopreventive against dioxin-induced human mammary carcinogenesis by blocking the metabolic formation of the catechol estrogens and scavenging the ROS generated during their redox cycling.

Polymorphism of estrogen metabolism genes and cataract

Cataract is the leading cause of visual impairment in older adults in the world. Age-related lens opacities are common and are frequent causes of loss of vision. The incidence of cataract increases significantly with increasing age in women only. The onset coincides with estrogen deficiency that occurs after menopause. Hormone replacement therapy has proven beneficial to selected postmenopausal women. Estrogen effects on biological system are modulated via the estrogen receptors (ER) and/or estrogen metabolites. Although ER have been detected in ocular tissue, whether ER polymorphism is related to cataract is not known at present. The polymorphisms of estrogen metabolizing enzymes are also related to the serum concentration and activity of estrogen. Polymorphism such as cytochrome P450c17 (A2/A2), cytochrome P450c1A (vt/vt) will result in increased formation of catechol estrogen, while people with catechol- O-methyltransferase (COMT) polymorphism COMT (L/L) will have decreased metabolism of catechol estrogen and decreased level of methoxyestradiol. COMT was also involved in tamoxifen metabolism which may further decrease the activity of COMT in breast cancer patients treated with tamoxifen. It is known that a 4–7% increase in cataract was found in tamoxifen-treated breast cancer patients than non-user. The 7.0% COMT (L/L) genotype in general population corresponded well with the 4–7% of cataract formation in tamoxifen-treated breast cancer patients. Our hypothesis is that breast cancer patients with COMT (L/L) genotype may be at increased risk of cataract formation after tamoxifen treatment.

Characterization of the Oxidative Metabolites of 17β-Estradiol and Estrone Formed by 15 Selectively Expressed Human Cytochrome P450 Isoforms

We systematically characterized the oxidative metabolites of 17beta-estradiol and estrone formed by 15 human cytochrome P450 (CYP) isoforms. CYP1A1 had high activity for 17beta-estradiol 2-hydroxylation, followed by 15alpha-, 6alpha-, 4-, and 7alpha-hydroxylation. However, when estrone was the substrate, CYP1A1 formed more 4-hydroxyestrone than 15alpha- or 6alpha-hydroxyestrone, with 2-hydroxyestrone as the major metabolite. CYP1A2 had the highest activity for the 2-hydroxylation of both 17beta-estradiol and estrone, although it also had considerable activity for their 4-hydroxylation (9-13% of 2-hydroxylation). CYP1B1 mainly catalyzed the formation of catechol estrogens, with 4-hydroxyestrogens predominant. CYP2A6, 2B6, 2C8, 2C9, 2C19, and 2D6 each showed a varying degree of low catalytic activity for estrogen 2-hydroxylation, whereas CYP2C18 and CYP2E1 did not show any detectable estrogen-hydroxylating activity. CYP3A4 had strong activity for the formation of 2-hydroxyestradiol, followed by 4-hydroxyestradiol and an unknown polar metabolite, and small amounts of 16alpha- and 16beta-hydroxyestrogens were also formed. The ratio of 4- to 2-hydroxylation of 17beta-estradiol or estrone with CYP3A4 was 0.22 or 0.51, respectively. CYP3A5 had similar catalytic activity for the formation of 2- and 4- hydroxyestrogens. Notably, CYP3A5 had an unusually high ratio of 4- to 2-hydroxylation of 17beta-estradiol or estrone (0.53 or 1.26, respectively). CYP3A4 and 3A5 also catalyzed the formation of nonpolar estrogen metabolite peaks (chromatographically less polar than estrone). CYP3A7 had a distinct catalytic activity for the 16alpha-hydroxylation of estrone, but not 17beta-estradiol. CYP4A11 had little catalytic activity for the metabolism of 17beta-estradiol and estrone. In conclusion, many human CYP isoforms are involved in the oxidative metabolism of 17beta-estradiol and estrone, with a varying degree of catalytic activity and distinct regioselectivity.

Catechol estrogen formation in liver microsomes from female ACI and Sprague-Dawley rats: comparison of 2- and 4-hydroxylation revisited.

Estradiol (E(2))-hydroxylation was studied in liver microsomes from ACI and Sprague-Dawley female rats, which differ markedly in their susceptibility to E(2)-induced formation of mammary tumors. NADPH-dependent oxidation of E(2) by liver microsomes from ACI and Sprague-Dawley rats produced several metabolites of which 2-hydroxyestradiol (2-OH-E(2)), estrone (E(1)), and 2-hydroxyestrone (2-OH-E(1)) were predominant. Incubations with either low (9 nM) or high (50 microM) concentrations of radiolabeled E(2) and with varying amounts of microsomal protein indicated the formation of only small amounts of 4-hydroxyestradiol (4-OH-E(2)). The ratio of 2-OH-E(2) to 4-OH-E(2) formed with the low concentration of E(2) was about 10:1 regardless of the amount of microsomal protein used, and about 20:1 using a high concentration of E(2). Thus, oxidation of E(2) by liver microsomes from female ACI and Sprague-Dawley rats occurs primarily via 2-hydroxylation, and 4-hydroxylation is only a minor pathway. These results are in disagreement with a recent report indicating substantial 4-hydroxylation of E(2) by liver microsomes from female ACI rats.

Urinary excretion pattern of catecholestrogens in preovulatory LH surge during the 4-day estrous cycle of rats

The formation of catecholestrogens represents a major pathway of estrogen metabolism and catecholestrogens are regarded as the main estrogen metabolite in non-pregnant state of various mammalian systems. In the present investigation, level of 2-hydroxyestrone, the major catecholestrogen excreted in rat urine, was measured by radioimmunoassay following acid hydrolysis and column chromatography of the 24-h urine samples of female Sprague Dawley non-pregnant rats during their 4-day estrous cycle. Urinary levels of estrone, estradiol and estriol were measured. Unlike the plasma level, urinary 2-hydroxyestrone showed a marked increase during the pre-ovulatory LH surge suggesting a plausible role of catecholestrogen in the mid-cycle elevation of the gonadotropin level in normal cycling rats.

NADPH- and hydroperoxide-supported 17β-estradiol hydroxylation catalyzed by a variant form (432L, 453S) of human cytochrome P450 1B1

Human cytochrome P450 1B1 (CYP1B1) catalyzes the hydroxylation of 17β-estradiol (E 2) at C-4, with a lesser activity at C-2. The E 2 4-hydroxylase activity of human CYP1B1 was first observed in studies of MCF-7 breast cancer cells. Sequencing of polymerase chain reaction products revealed that CYP1B1 expressed in MCF-7 cells was not the previously characterized enzyme but a polymorphic form with leucine substituted for valine at position 432 and serine substituted for asparagine at position 453. To investigate the NADPH- and organic hydroperoxide-supported E 2 hydroxylase activities of the 432L, 453S form of human CYP1B1, the MCF-7 CYP1B1 cDNA was cloned and the enzyme was expressed in Sf9 insect cells. In microsomal assays supplemented with human NADPH:cytochrome P450 oxidoreductase, the expressed 432L, 453S form catalyzed NADPH-supported E 2 hydroxylation with a similar preference for 4-hydroxylation as the 432V, 453N form, with maximal rates of 1.97 and 0.37 nmol (min) −1(nmol cytochrome P450) −1 for 4- and 2-hydroxylation, respectively. Cumeme hydroperoxide efficiently supported E 2 hydroxylation by both the 432V, 453N and 432L, 453S forms at several-fold higher rates than the NADPH-supported activities and with a lesser preference for E 2 4- versus 2-hydroxylation (2:1). The hydroperoxide-supported activities of both forms were potently inhibited by the CYP1B1 inhibitor, 3,3′,4,4′,5,5′-hexachlorobiphenyl. These results indicate that the 432V, 453N and 432L, 453S forms of CYP1B1 have similar catalytic properties for E 2 hydroxylation, and that human CYP1B1 is very efficient in catalyzing the hydroperoxide-dependent formation of catecholestrogens.

Influence of subchronic administration of catechol estrogens on the formation of reactive oxygen species in rat liver microsomes

Metabolic pathways of estrogens are the formation of catechol estrogens (CE; 2- and 4-hydroxy-estrogens), redox cycling of CE and free radical generation, mediated through cytochrome P450 (P450) oxidase/reductase activity. In previous investigations subchronic administration of estrogens showed prooxidative and antioxidative activities in rat liver microsomes (BARTH et al. 1999). To find out whether or not catechol metabolites are responsible for prooxidative activity, we checked 2- and 4-hydroxy-estradiol (2OH-E2 and 4OH-E2) and the non-catechol metabolite 6alpha-hydroxy-estradiol (6alpha-OH-E2) for formation of reactive oxygen species in liver microsomes of 30-day-old male Wistar rats after 5 days treatment (1, 10 mg/kg b. wt. orally, once a day). The results were compared with those after treatment of the rats with estradiol (E2), estradiol valerate (E2V) and ethinylestradiol (EE2). In liver homogenates glutathione and lipid peroxides were determined, in microsomes NADPH-Fe++-stimulated lipid peroxidation (LPO), H2O2 generation and lucigenin (LUC) and luminol (LUM) amplified chemiluminescence (CL) were investigated. In liver 9000 x g supernatants monooxygenase activities were measured. The two catechol estrogens did not show any antioxidative activity, whereas 6alpha-OH-E2 significantly diminished lipid peroxides in the liver as well as LPO and LUM-CL in liver microsomes. Among estrogens, only EE2 showed antioxidative activity. Both CE inhibited ethoxycoumarin O-deethylation. Peroxidative activity as enhanced LUC-CL was found after 2OH-E2 (1 mg/kg b.wt.) and E2, but 10 times higher doses of both CE did not change LUC-CL. Microsomal H2O2 generation was enhanced by E2, E2V and both CE, not by 6alpha-OH-E2. The lower level of H2O2 enhancement caused by CE in comparison to E2 and E2V together with unchanged LUC-CL after high CE doses did not unequivocally prove the CE to be mainly responsible for the prooxidative activities of E2 and E2V in liver microsomes, at least in 30-day-old male rats. Unchanged GSH in the liver after CE administration supports this hypothesis.

Estrogen metabolism by conjugation.

The involvement of estrogens in carcinogenic processes within estrogen-responsive tissues has been recognized for a number of years. Classically, mitogenicity associated with estrogen receptor-mediated cellular events was believed to be the mechanism by which estrogens contributed to carcinogenesis. Recently, the possibility that estrogens might contribute directly to mutagenesis resulting from DNA damage has been investigated. That damage is apparently a result of the formation of catechol estrogens that can be further oxidized to semiquinones and quinones. Those molecules represent reactive oxygen species and electrophilic molecules that can form depurinating DNA adducts, thus having the potential to result in permanent nucleotide mutation. Conjugation of parent estrogens to sulfate and glucuronide moieties; of catechol estrogens to methyl, sulfate, and glucuronide conjugates; and of catechol estrogen quinones to glutathione conjugates all represent potential "detoxification" reactions that may protect the cell from estrogen-mediated mitogenicity and mutagenesis. In this chapter, the biochemistry and molecular genetics of those conjugative reaction pathways are discussed. When applicable, the involvement of specific enzymatic isoforms is presented. Finally, the activity of many of these conjugative biotransformation reactions is subject to large interindividual variation--often due to the presence of common nucleotide polymorphisms within the genes encoding those enzymes. Functionally significant genetic polymorphisms that might contribute to variable conjugation of estrogens and catechol estrogens are also discussed.

Expression of dioxin-related transactivating factors and target genes in human eutopic endometrial and endometriotic tissues

Objective: Although an association between dioxin exposure and endometriosis has been proposed, the effects of this environmental toxin on human endometriosis are not known. To understand the potential underlying molecular mechanisms we studied the expressions of cytochrome P-450 genes ( CYP1A1, CYP1A2, and CYP1B1 ), which are induced by dioxin, and the expressions of cytosolic receptor for dioxin, aryl hydrocarbon receptor, and its nuclear translocator, aryl hydrocarbon receptor nuclear translocator protein, in endometriotic and eutopic endometrial tissues. Study Design: Levels of transcripts of CYP1A1, CYP1A2, CYP1B1, aryl hydrocarbon receptor, and aryl hydrocarbon receptor nuclear translocator protein were determined by a quantitative reverse transcriptase–polymerase chain reaction and Southern blot assay in total ribonucleic acid samples from endometriotic and eutopic endometrial tissues. Eutopic endometrial tissue samples (n = 33) and endometriotic tissue samples (n = 10) were obtained at the time of uterine curettage and laparoscopy from disease-free women and from patients with endometriosis. Portions of these eutopic endometrial and endometriotic tissues were obtained simultaneously from the same patients (n = 8 pairs of samples). Levels of transcripts of CYP1A1, CYP1A2, CYP1B1, aryl hydrocarbon receptor, and aryl hydrocarbon receptor nuclear translocator protein were determined in endometrial and endometriotic tissues during follicular and luteal phases of the cycle and in cultured endometriotic stromal cells treated with forskolin, phorbol diacetate, medroxyprogesterone acetate,and serum. Results: Transcripts of dioxin receptor, its nuclear translocator, and two dioxin-induced target genes (CYP1A2 and CYP1B1) were demonstrated during follicular and luteal phases of the cycle in both eutopic endometrial tissues and tissues affected by pelvic endometriosis, with no readily detectable differences between these tissues. On the other hand, levels of transcripts of another dioxin-induced gene, CYP1A1, were found to be strikingly higher in endometriotic tissues than in the eutopic endometrium. Mean levels in endometriotic tissues were 8.7 times those found in eutopic endometrium. Various hormonal treatments of endometriotic stromal cells did not significantly alter these levels. Conclusion: We demonstrated for the first time the expression of dioxin-related transcription factors aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator protein and target genes CYP1A1, CYP1A2, and CYP1B1 in endometriotic tissues and stromal cells. Strikingly elevated CYP1A1 transcripts in endometriosis may give rise to significantly increased P-4501A1 enzyme activity and thus promote the development and growth of endometriosis by either activating procarcinogens or inducing the formation of catechol estrogens or both. In fact, the proposed link between dioxin exposure and endometriosis may be explained in part by the up-regulation of the CYP1A1 gene expression in endometriotic tissues. (Am J Obstet Gynecol 2000;182:767-75.)

Responsiveness of plasma 2- and 4-hydroxycatecholestrogens to training and to graduate submaximal and maximal exercise in an untrained woman. Interuniversity Project on Reproductive Endocrinology in Women and Exercise.

A single-subject experimental design was used to obtain some preliminary findings on the plasma responses of catecholestrogens (CE) to acute exercise and brief, but exhaustive training on a cycle ergometer. One previously untrained eumenorrheic female (body fat: 26% VO2max: 43.3 ml x kg(-1) x min[-1]) participated in this study. Resting CE levels were for "total" (unconjugated + conjugated) 2-hydroxyestrogens (2-OHE) 162pg/ ml and 350 pg/ml in the follicular (FPh) and luteal phase (LPh), respectively. Plasma total 4-hydroxyestrogen (4-OHE) levels were 41 pg/ml in the FPh and 66 pg/ml in the LPh. For "total" 2-methoxyestrogens (2-MeOE), we found 257 pgl/ml in the FPh and 374 pg/ml in the LPh. Resting levels of 2-hydroxy CE following a period of brief, intensive training were decreased during the LPh (2-OHE: -38%; 2-MeOE: -19%), whereas 4-hydroxy CE were unaffected. After training, the formation of CE as expressed by the 2-OHE:E and 4-OHE:E ratios, was increased by 75% and 200% at rest, respectively. CE activity or O-methylation, as estimated from the 2-MeOE:2-OHE ratio, was higher following training (FPh: +22%; LPh: +30%). During acute exercise before training, we observed a small rise proportional to the exercise intensity in the plasma "total" primary estrogen concentrations (FPh: +28%; LPh: +16%), and no changes in either 2-OHE or 2 MeOE levels. Plasma concentrations of 4-OHE, however, doubled during maximal exercise intensity. The 2-OHE:E and 2-MeOE:2-OHE ratios did not alter during incremental exercise. Training effects on acute exercise responses were only noticed for 4-OHE, which contrary to pre-training conditions, now progressively decreased. The major findings of this study are that in response to training: a) during rest, a greater proportion of CE are formed from a lower amount of precursor hormone, b) the rate of O-methylation of CE increases.