Nordihydroguaiaretic Acid as a Novel Substrate and Inhibitor of Catechol O-Methyltransferase Modulates 4-Hydroxyestradiol-Induced Cyto- and Genotoxicity in MCF-7 Cells.

Nordihydroguaiaretic acid (NDGA) is a major lignan metabolite found in Larrea spp., which are widely used in South America to treat various diseases. In breast tissue, estradiol is metabolized to the catechol estrogens such as 4-hydroxyestradiol (4-OHE2), which have been proposed to be cancer initiators potentially involved in mammary carcinogenesis. Catechol-O-methyltransferase (COMT) catalyzes the O-methylation of catechol estrogens to their less toxic methoxy derivatives, such as 4-O-methylestradiol (4-MeOE2). The present study investigated the novel biological activities of NDGA in relation to COMT and the effects of COMT inhibition by NDGA on 4-OHE2-induced cyto- and genotoxicity in MCF-7 human breast cancer cells. Two methoxylated metabolites of NDGA, 3-O-methylNDGA (3-MNDGA) and 4-O-methyl NDGA (4-MNDGA), were identified in the reaction mixture containing human recombinant COMT, NDGA, and cofactors. Km values for the COMT-catalyzed metabolism of NDGA were 2.6 µM and 2.2 µM for 3-MNDGA and 4-MNDGA, respectively. The COMT-catalyzed methylation of 4-OHE2 was inhibited by NDGA at an IC50 of 22.4 µM in a mixed-type mode of inhibition by double reciprocal plot analysis. Molecular docking studies predicted that NDGA would adopt a stable conformation at the COMT active site, mainly owing to the hydrogen bond network. NDGA is likely both a substrate for and an inhibitor of COMT. Comet and apurinic/apyrimidinic site quantitation assays, cell death, and apoptosis in MCF-7 cells showed that NDGA decreased COMT-mediated formation of 4-MeOE2 and increased 4-OHE2-induced DNA damage and cytotoxicity. Thus, NDGA has the potential to reduce COMT activity in mammary tissues and prevent the inactivation of mutagenic estradiol metabolites, thereby increasing catechol estrogen-induced genotoxicities.

Targeting Endogenous Adduction Level of Serum Albumin by Parallel Reaction Monitoring via Standard Additions and Intact Protein Measurement: Biological Dosimetry of Catechol Estrogens.

Abundant blood proteins adducted by active electrophiles are excellent markers to predict the risk of electrophile-induced toxicity. However, detecting endogenously adducted proteins by bottom-up selective (or parallel) reaction monitoring (SRM/PRM) is challenging because of the high variability in sample preparation and detection as well as low adduction levels. Here, we reported a new approach in developing PRM methods by combining intact protein measurement with standard additions to target optimal conditions for detecting catechol estrogens (CEs)-adducted human serum albumin (HSA). Blood serum was added with multiple amounts of CEs to obtain serum standards. Intact protein measurement revealed two linear ranges of adduction levels (adducted-CE/HSA): 0.34-0.42 (R2 > 0.94) and 0.81-8.54 (R2 > 0.96) against the amount of added CEs, respectively. Six adduction sites were identified by trypsin (K20, C34, K73, K281, H338, K378) or chymotrypsin (K20, C34, K378) digestion. PRM methods targeting all adducted/nonadducted peptide pairs based on chymotrypsin or trypsin digestion were developed, and the data were compared with those obtained by intact protein measurement. Correlation plots indicated that chymotrypsin-PRM leads to poor sensitivity and largely underestimated protein adduction levels. Trypsin-PRM leads to sensitive and highly correlated (R2 > 0.91) protein adduction levels with a detection limit below the endogenous level and relative standard deviation <25%. As a proof of concept, clinical serum samples were examined by trypsin-PRM, and a slightly higher adduction level was observed for the obesity group when compared with the healthy group. This is the first report on determining adduction levels of blood proteins for long-term exposure to CEs. The standard addition approach can be generally applied to protein adductomics with resolvable mass increments by intact protein measurement to accelerate the development of bottom-up methods close to the inherent limit.

Nitidine Chloride-Induced CYP1 Enzyme Inhibition and Alteration of Estradiol Metabolism.

The cytochrome P450 (P450) 1 family is an important phase I enzyme involved in carcinogen activation. Nitidine chloride (NC) is a pharmacologically active alkaloid with polyaromatic hydrocarbon found in the roots of Zanthoxylum nitidum (Roxb.) DC, a traditional medicinal herb widely used in China. We examined the inhibitory effects of NC on CYP1A1, 1B1, and 1A2. NC significantly inhibited CYP1A1- and 1B1-catalyzed ethoxyresorufin O-deethylation activity (IC50 = 0.28 ± 0.06 and 0.32 ± 0.02 µM, respectively) in a concentration-dependent manner, but only showed slight inhibition of CYP1A2 activity (IC50 > 50 µM). Kinetic analysis revealed that NC competitively inhibited CYP1B1 with a K i value of 0.47 ± 0.05 µM, whereas NC caused a mixed type of inhibition on CYP1A1 with K i and K I values of 0.14 ± 0.04 and 0.19 ± 0.09 µM, respectively. The observed enzyme inhibition neither required NADPH nor revealed time dependency. Molecular docking manifested the generation of strong hydrogen-bonding interactions of Ser116 in CYP1A1 and Ser127 in CYP1B1 with methoxy moiety of NC. Additionally, NC-induced alteration of estradiol (E2) metabolism was also investigated in the present study. Hydroxyestradiols, including 2-hydroxyestradiol [(2-OHE2) nontoxic] and 4-hydroxyestradiol [(4-OHE2) genotoxic] generated in recombinant enzyme incubation systems and cultured MCF-7 cells were analyzed, and NC was found to preferentially inhibit the nontoxic 2-hydroxylation activity of E2 mediated by CYP1A1. In conclusion, NC was a mixed type inhibitor of CYP1A1 and a competitive inhibitor of CYP1B1. The remarkable inhibition on E2 2-hydroxylation might increase the risk of 4-OHE2-induced genotoxicity. SIGNIFICANCE STATEMENT: CYP1 enzymes catalyze oxidative metabolism of a variety of compounds and are known to play a crucial role in the development of cancer. CYP1A1 and CYP1A2 are responsible for hydroxylation of estradiol (E2) at the C-2 position, resulting in the formation of 2-OHE2, which is proposed to be a detoxification pathway. However, CYP1B1-mediated hydroxylation of E2 at the C-4 position has been suggested to be a tumor initiator. The present study found that nitidine chloride is a mixed type inhibitor of CYP1A1 and a competitive inhibitor of CYP1B1. NC preferentially inhibited the nontoxic E2 2-hydroxylation pathway mediated by CYP1A1, which might increase the risk of 4-OHE2-induced genotoxicity and cause severe drug-drug interactions.

Oroxylin A, a methylated metabolite of baicalein, exhibits a stronger inhibitory effect than baicalein on the CYP1B1-mediated carcinogenic estradiol metabolite formation.

Human cytochrome P450 1B1 (CYP1B1)-mediated formation of 4-hydroxyestradiol (4-OHE2) from 17ß-estradiol plays an important role in the progression of human breast cancer, while the biotransformation of 17ß-estradiol to 2-hydroxyestradiol mediated by cytochrome P450 1A1 (CYP1A1) is considered as a less harmful pathway. In this study, inhibitory effects of flavonoids baicalein and oroxylin A, a metabolite of baicalein in human body, on CYP1A1 and 1B1 activities were investigated in vitro. The inhibition intensities of baicalein and oroxylin A towards CYP1B1 were greater than towards CYP1A1 with a mixed mechanism. In addition, oroxylin A showed a stronger inhibitory effect than baicalein towards the CYP1B1-mediated 17ß-estradiol 4-hydroxylation, with the IC50 values of 0.0146 and 2.27 µM, respectively. Docking studies elucidated that oroxylin A had a stronger binding affinity than baicalein for CYP1B1. In MCF-7 cells, compared with baicalein-treated groups, oroxylin A with lower doses decreased and increased the formation of 4-OHE2 and 2-hydroxyestradiol, respectively, with a preferential induction of mRNA of CYP1A1 over CYP1B1. In conclusion, this study demonstrated that oroxylin A showed a stronger inhibitory effect than baicalein on CYP1B1-mediated 4-OHE2 formation in MCF-7 cells, providing crucial implications for their possibly preventive/therapeutic potential against breast cancer via inhibition of CYP1B1, particularly of oroxylin A.

The role of 17ß-estradiol metabolites in chromium-induced oxidative stress.

BACKGROUND: The increasing incidence of estrogen-dependent breast cancer and the presence in the environment of a large number of factors that interact with estrogen receptors have sparked interest in chemical influences on estrogen-dependent processes. In a previous work, the authors examined the interaction of estradiol with chromium. In the present article the importance of estradiol biotransformation in these interactions is investigated. There is no information in the available literature about the role of metabolites in exposure to chromium. It seems important because estradiol metabolites have various carcinogenic abilities and their formation during biotransformation could be increased or decreased by environmental enzyme inducers or inhibitors. The metabolites could play a detoxifying role or create a toxic synergism in free radical processes induced by chromium VI (CrVI). OBJECTIVES: The aim of this study was to evaluate the influence of 2 17ß-estradiol metabolites - 4-hydroxyestradiol (4-OHE2) and 16α-hydroxyestrone (16α-OHE1) - in conditions of oxidative stress caused by CrVI. MATERIAL AND METHODS: Human blood, erythrocytes or mitochondria isolated from human placentas after natural deliveries were used in the experiments. The influence of CrVI, 4-OHE2 and 16-OHE1 on thiobarbituric acid reactive substances (TBARS), the hydroxyl radical (•OH), superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione-S-transferase (GST), and the interactions of the metabolites exposed to chromium expressed by these factors were examined. RESULTS: 4-OHE2 reduced the level of TBARS induced by CrVI in mitochondria (p < 0.05) and in erythrocytes (p < 0.05), and increased SOD activity (p < 0.05). 16α-OHE1 increased the activity of GST in erythrocytes exposed to CrVI (p < 0.05). CONCLUSIONS: The metabolites do not have toxic interactions with CrVI. On the contrary, they exhibited a protective effect. The mechanism of protection varied: 4-OHE2 decreased TBARS and increased SOD activity, while 16α-OHE1 increased GST activity.

Determination of urinary 15α-hydroxyestrogen levels via immunoaffinity extraction.

15α-Hydroxyestrogens (15α-OHEs) are metabolites of the female hormone estradiol. In this study, to discover physiological markers that can be utilized for monitoring fetal conditions and estrogen-induced cancers, we established a method for quantifying 15α-OHEs in rat urine via immunoaffinity column extraction and HPLC-electrochemical detection, and detected 15α-OHEs in urine obtained male rats treated with estradiol. Notably, the standard curves for quantification obtained using the column were linear over a range of 0.5-50ng 15α-OHEs. The accuracy of the analytical method with cleanup was 97-109% for the three kinds of 15α-OHEs examined, and the intra-assay precision of the measured values had a coefficient of variation of ≤20.6%. Therefore, the theoretical limit of quantification was 0.5ng. However, the actual measured values obtained from the urine of male rats indicated that the detection limits were 0.425, 0.103, and 0.047ng for estetrol, 15α-hydroxyestradiol, and 15α-hydroxyestrone, respectively. Our newly established method for measuring 15α-OHE concentrations in urine could facilitate characterization of the in vivo metabolic profile of 15α-OHEs in mammals under various physiological conditions, which could comprise clinical markers for monitoring human fetal health conditions in mammals.

[The biological and clinical relevance of estrogen metabolome]. / Az ösztrogénmetabolom biológiai és klinikai jelentosége lokális folyamatokban.

Considerable knowledge has been gathered on the physiological role of estrogens. However, fairly little information is available on the role of compounds produced in the breakdown process of estrone and estradiol wich may play a role in various diseases associated with estrogen impact. To date, approximately 15 extragonadal estrogen-related compounds have been identified. These metabolites may exert protective, or, instead, pro-inflammatory and/or pro-oncogenic activity in a tissue-specific manner. Systemic and local estrogen metabolite levels are not necesserily correlated, which may promote the diagnostic significance of the locally produced estrogen metabolites in the future. The aim of the present study is a bibliographic review of the extragonadal metabolome in peripheral tissues, and to highlight the role of the peripheral tissue homeostasis of estrogens as well as the non-hormonal biological activity and clinical significance of the estrogen metabolome. Orv Hetil. 2017; 158(24): 929-937.

Unique effect of 4-hydroxyestradiol and its methylation metabolites on lipid and cholesterol profiles in ovariectomized female rats.

Animal studies have shown that endogenous estrogens such as 17ß-estradiol (E2) can modulate lipid profiles in vivo, and this effect is generally thought to be mediated by the estrogen receptors (ERs). The present study sought to test a hypothesis that some of the endogenous estrogen metabolites that have very weak estrogenic activity may exert some of their modulating effects on lipid metabolism in an ER-independent manner. Using ovariectomized female rats as an in vivo model, we found that 4-hydroxyestradiol (4-OH-E2) has a markedly stronger effect in reducing the adipocyte size and serum cholesterol level in rats compared to E2, despite the weaker estrogenic activity of 4-OH-E2. Moreover, when E2 or 4-OH-E2 is used in combination with ICI-182,780 (an ER antagonist), some of their lipid-modulating effects are not blocked by this antiestrogen. Interestingly, two of the O-methylation metabolites of 4-OH-E2, namely, 4-methoxyestradiol and 4-methoxyestrone, which have much weaker estrogenic activity, were also found to have similar lipid-modulating effects compared to 4-OH-E2. Mechanistically, up-regulation of the expression of leptin, cytochrome P450 7A1 and LXRα genes is observed in the liver of animals treated with E2 or 4-OH-E2, and the up-regulation is essentially not inhibited by co-treatment with ICI-182,780. These results demonstrate that some of the endogenous E2 metabolites are functionally important modulators of lipid metabolic profiles in vivo. In addition, our findings indicate that an ER-independent pathway likely mediates some of the lipid-modulating effects of endogenous estrogens and their metabolic derivatives.

The Val158Met polymorphism in COMT gene and cancer risk: role of endogenous and exogenous catechols.

Catechol-O-methyltransferase, COMT, is an important phase II enzyme catalyzing the transfer of a methyl-group from S-adenosylmethionine to a catechol-containing substrate molecule. A genetic variant Val158Met in the COMT gene leads to a several-fold decrease in the enzymatic activity giving rise to the accumulation of potentially carcinogenic endogenous catechol estrogens and their reactive intermediates and increasing thus the risk of tumorigenesis. However, numerous association studies between the COMT genotype and susceptibility to various malignancies have shown inconsistent and controversial findings indicating that additional gene-gene and gene-environment interactions might be crucial in modulating the physiological role of the COMT. In this review article, the important contribution of dietary catechol-containing flavonoids to modification of the relationships between the COMT genotype and cancer risk is discussed. Whereas, the diverse anticancer activities of common phytochemicals, such as green tea polyphenols, quercetin, fisetin or luteolin, can be markedly changed (both decreased or increased) by the COMT-mediated O-methylation of these exogenous substrates, flavonoids can also behave as potent inhibitors of the COMT enzyme slowing detoxification of endogenous catechol estrogens. Such a many-featured functioning of the COMT and its complex regulation by several different genetic and environmental factors, including plant-based food ingredients, emphasizes the necessity to further stratify the association studies between the COMT genotype and tumor risk by consumption of catechol-containing dietary flavonoids. Currently, it can be only speculated that some of the possible associations might be masked by the regular intake of specific food polyphenols, taking effect in certain communities or populations.

CYP1B1 Enhances Cell Proliferation and Metastasis through Induction of EMT and Activation of Wnt/ß-Catenin Signaling via Sp1 Upregulation.

Cytochrome P450 1B1 (CYP1B1) is a major E2 hydroxylase involved in the metabolism of potential carcinogens. CYP1B1 expression has been reported to be higher in tumors compared to normal tissues, especially in hormone-related cancers including breast, ovary, and prostate tumors. To explore the role of CYP1B1 in cancer progression, we investigated the action of CYP1B1 in cells with increased CYP1B1 via the inducer 7,12-dimethylbenz[α]anthracene (DMBA) or an overexpression vector, in addition to decreased CYP1B1 via the inhibitor tetramethoxystilbene (TMS) or siRNA knockdown. We observed that CYP1B1 promoted cell proliferation, migration, and invasion in MCF-7 and MCF-10A cells. To understand its molecular mechanism, we measured key oncogenic proteins including ß-catenin, c-Myc, ZEB2, and matrix metalloproteinases following CYP1B1 modulation. CYP1B1 induced epithelial-mesenchymal transition (EMT) and activated Wnt/ß-catenin signaling via upregulation of CTNNB1, ZEB2, SNAI1, and TWIST1. Sp1, a transcription factor involved in cell growth and metastasis, was positively regulated by CYP1B1, and suppression of Sp1 expression by siRNA or DNA binding activity using mithramycin A blocked oncogenic transformation by CYP1B1. Therefore, we suggest that Sp1 acts as a key mediator for CYP1B1 action. Treatment with 4-hydroxyestradiol (4-OHE2), a major metabolite generated by CYP1B1, showed similar effects as CYP1B1 overexpression, indicating that CYP1B1 activity mediated various oncogenic events in cells. In conclusion, our data suggests that CYP1B1 promotes cell proliferation and metastasis by inducing EMT and Wnt/ß-catenin signaling via Sp1 induction.

Potent Chemopreventive/Antioxidant Activity Detected in Common Spices of the Apiaceae Family.

Spices are used worldwide, particularly in the Asian and Middle Eastern countries, and considered protective against degenerative diseases, including cancer. Here, we report the efficacy of aqueous and non-aqueous extracts of 11 Apiaceae spices for free radical-scavenging activity and to inhibit cytochrome P450s in two separate reactions involving: 1) 4-hydroxy-17ß-estradiol (4E2), DNA, and CuCl2 and 2) 17ß-estradiol, rat liver microsomes, cofactors, DNA and CuCl2. Oxidative DNA adducts resulting from redox cycling of 4E2 were analyzed by (32)P-postlabeling. Aqueous (5 mg/ml) and non-aqueous extracts (6 mg/ml) substantially inhibited (83-98%) formation of DNA adducts in the microsomal reaction. However, in nonmicrosomal reaction, only aqueous extracts showed the inhibitory activity (83-96%). Adduct inhibition was also observed at five-fold lower concentrations of aqueous extracts of cumin (60%) and caraway (90%), and 10-fold lower concentrations of carrot seeds (76%) and ajowan (90%). These results suggests the presence of 2 groups of phytochemicals: polar compounds that have free radical-scavenging activity and lipophilic compounds that selectively inhibit P450 activity associated with estrogen metabolism. Because most of these Apiaceae spices are used widely with no known toxicity, the phytochemicals from the Apiaceae spices used in foods may be potentially protective against estrogen-mediated breast cancer.

Differences in 4-hydroxyestradiol levels in leukocytes are related to CYP1A1(∗)2C, CYP1B1(∗)3 and COMT Val158Met allelic variants.

Exposure to estrogen and its metabolites, including catechol estrogens (CEs) and catechol estrogen quinones (CE-Qs) is closely related to breast cancer. Polymorphisms of the genes involved in the catechol estrogens metabolism pathway (CEMP) have been shown to affect the production of CEs and CE-Qs. In this study, we measured the induction of CYP1A1, CYP1B1, COMT, and GSTP1 by 17ß-estradiol (17ß-E2) in leukocytes with CYP1A1(∗)2C, CYP1B1(∗)3, COMT Val158Met and GSTP1 Ile105Val polymorphisms by semi quantitative RT-PCR and compared the values to those of leukocytes with wild type alleles; we also compared the differences in formation of 4- hydroxyestradiol (4-OHE2) and DNA-adducts. The data show that in the leukocytes with mutant alleles treatment with 17ß-E2 up-regulates CYP1A1 and CYP1B1 and down-regulates COMT mRNA levels, resulting in major increments in 4-OHE2 levels compared to leukocytes with wild-type alleles. Therefore, we propose induction levels of gene expression and intracellular 4-OHE2 concentrations associated with allelic variants in response to exposure of 17ß-E2 as a noninvasive biomarker that can help determine the risk of developing non-hereditary breast cancer in women.

FSH-induced p38-MAPK-mediated dephosphorylation at serine 727 of the signal transducer and activator of transcription 1 decreases Cyp1b1 expression in mouse granulosa cells.

Most mammalian follicles undergo atresia at various stages before ovulation, and granulosa cell apoptosis is a major cause of antral follicular atresia. Estradiol is an essential mitogen for granulosa cell proliferation in vivo and inhibition of apoptosis. The estradiol-producing capacity and metabolism levels are important for follicle health, and sufficient estradiol is necessary for follicle development and ovulation. Cyp1b1, a member of the cytochrome P450 1 subfamily, is responsible for the metabolism of a wide variety of halogenated and polycyclic aromatic hydrocarbons in diverse tissues. In mouse follicles, Cyp1b1 converts estradiol to 4-hydroxyestradiol. We investigated mouse granulosa cells (MGCs) in vivo and in vitro and found that Cyp1b1 played a crucial role in estradiol metabolism in dominant follicles. Follicle-stimulating hormone (FSH) decreased estrogen metabolism by reducing Cyp1b1 mRNA and protein levels in MGCs. Furthermore, FSH regulated signal transducer and activator of transcription 1 (STAT1), a significant transcription factor of Cyp1b1, by mediating the dephosphorylation of STAT1 on serine 727 (Ser(727)) in MGCs. p38 mitogen-activated protein kinase (MAPK) may be involved in the FSH-induced dephosphorylation of STAT1 on Ser(727) in MGCs. These results suggested that FSH functions via p38 MAPK-induced dephosphorylation at Ser(727) of STAT1 to downregulate Cyp1b1 expression and maintain the estradiol levels in mouse dominant follicles.

The molecular etiology and prevention of estrogen-initiated cancers: Ockham's Razor: Pluralitas non est ponenda sine necessitate. Plurality should not be posited without necessity.

Elucidation of estrogen carcinogenesis required a few fundamental discoveries made by studying the mechanism of carcinogenesis of polycyclic aromatic hydrocarbons (PAH). The two major mechanisms of metabolic activation of PAH involve formation of radical cations and diol epoxides as ultimate carcinogenic metabolites. These intermediates react with DNA to yield two types of adducts: stable adducts that remain in DNA unless removed by repair and depurinating adducts that are lost from DNA by cleavage of the glycosyl bond between the purine base and deoxyribose. The potent carcinogenic PAH benzo[a]pyrene, dibenzo[a,l]pyrene, 7,12-dimethylbenz[a]anthracene and 3-methylcholanthrene predominantly form depurinating DNA adducts, leaving apurinic sites in the DNA that generate cancer-initiating mutations. This was discovered by correlation between the depurinating adducts formed in mouse skin by treatment with benzo[a]pyrene, dibenzo[a,l]pyrene or 7,12-dimethylbenz[a]anthracene and the site of mutations in the Harvey-ras oncogene in mouse skin papillomas initiated by one of these PAH. By applying some of these fundamental discoveries in PAH studies to estrogen carcinogenesis, the natural estrogens estrone (E1) and estradiol (E2) were found to be mutagenic and carcinogenic through formation of the depurinating estrogen-DNA adducts 4-OHE1(E2)-1-N3Ade and 4-OHE1(E2)-1-N7Gua. These adducts are generated by reaction of catechol estrogen quinones with DNA, analogously to the DNA adducts obtained from the catechol quinones of benzene, naphthalene, and the synthetic estrogens diethylstilbestrol and hexestrol. This is a weak mechanism of cancer initiation. Normally, estrogen metabolism is balanced and few estrogen-DNA adducts are formed. When estrogen metabolism becomes unbalanced, more catechol estrogen quinones are generated, resulting in higher levels of estrogen-DNA adducts, which can be used as biomarkers of unbalanced estrogen metabolism and, thus, cancer risk. The ratio of estrogen-DNA adducts to estrogen metabolites and conjugates has repeatedly been found to be significantly higher in women at high risk for breast cancer, compared to women at normal risk. These results indicate that formation of estrogen-DNA adducts is a critical factor in the etiology of breast cancer. Significantly higher adduct ratios have been observed in women with breast, thyroid or ovarian cancer. In the women with ovarian cancer, single nucleotide polymorphisms in the genes for two enzymes involved in estrogen metabolism indicate risk for ovarian cancer. When polymorphisms produce high activity cytochrome P450 1B1, an activating enzyme, and low activity catechol-O-methyltransferase, a protective enzyme, in the same woman, she is almost six times more likely to have ovarian cancer. These results indicate that formation of estrogen-DNA adducts is a critical factor in the etiology of ovarian cancer. Significantly higher ratios of estrogen-DNA adducts to estrogen metabolites and conjugates have also been observed in men with prostate cancer or non-Hodgkin lymphoma, compared to healthy men without cancer. These results also support a critical role of estrogen-DNA adducts in the initiation of cancer. Starting from the perspective that unbalanced estrogen metabolism can lead to increased formation of catechol estrogen quinones, their reaction with DNA to form adducts, and generation of cancer-initiating mutations, inhibition of estrogen-DNA adduct formation would be an effective approach to preventing a variety of human cancers. The dietary supplements resveratrol and N-acetylcysteine can act as preventing cancer agents by keeping estrogen metabolism balanced. These two compounds can reduce the formation of catechol estrogen quinones and/or their reaction with DNA. Therefore, resveratrol and N-acetylcysteine provide a widely applicable, inexpensive approach to preventing many of the prevalent types of human cancer.

Estradiol 17ß and its metabolites stimulate cell proliferation and antagonize ascorbic acid-suppressed cell proliferation in human ovarian cancer cells.

Estradiol 17ß (E2ß) and ascorbic acid (AA) have been implicated in cancer progression. However, little is known about the actions of biologically active metabolites of E2ß, 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 ß (ERß) in human ovarian surface epithelial (IOSE-385) and cancer cell lines (OVCAR-3, SKOV-3, and OVCA-432). We also investigated the roles of E2ß, 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 ERß in most cell lines tested. Treating cells with physiological concentrations of E2ß 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 E2ß but not to its metabolites. The ERα blockade inhibited (∼85%) E2ß-stimulated OVCAR-3 proliferation, whereas ERß blockade attenuated (∼83%) E2ß-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 E2ß and its metabolites. Thus, our findings indicate that E2ß 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 E2ß and its metabolites may enhance AA's antiovarian cancer activity.

Cancer morbidity in rheumatoid arthritis: role of estrogen metabolites.

Estrogen metabolites have been implicated in rheumatoid arthritis (RA) and cancer, although the mechanism remains unestablished. Some estrogen metabolites, which are used for the assessment of cancer risk, play an important role in RA. The pathways by which malignancies associated with RA remain elusive. Possible mechanism involves enzymatic or nonenzymatic oxidation of estrogen into catecholestrogen metabolites through semiquinone and quinone redox cycle to produce free radicals that can cause DNA modifications. Modifications of DNA alter its immunogenicity and trigger various immune responses leading to elevated levels of cancer and RA antibodies. However, the role of different estrogen metabolites as a mediator of immune response cannot be ruled out in various immune-related diseases.

Common genetic variation in CYP1B1 is associated with concentrations of T4, FT3 and FT4 in the sera of polycystic ovary syndrome patients.

CYP1B1 encodes an estrogen enzyme that oxidizes 17ß-estradiol to 4-hydroxyestradiol. The evidence demonstrates there may be a relationship between CYP1B1 and thyroid function. To date, no study has evaluated if genetic polymorphisms that regulate concentrations of serum FT3 and FT4 contribute to Polycyctic Ovary Syndrome (PCOS). To identify polymorphisms in the CYP1B1 locus associated with PCOS, we genotyped three common polymorphisms across the CYP1B1 locus in 226 patients. A test for association of common variants with susceptibility to PCOS was conducted in a large cohort of 609 subjects. The functional polymorphism CYP1B1 L432V (rs1056836) is associated with serum T4 (P = 0.003), serum FT3 (P < 0.001) and serum FT4 concentrations (P < 0.001). Our study provides the first evidence that genetic variants in CYP1B1 can be associated with serum T4, FT4 and FT3 levels in PCOS. These findings imply novel pathophysiological links between the CYP1B1 locus and thyroid function in PCOS.

On methods for the detection of reactive oxygen species generation by human spermatozoa: analysis of the cellular responses to catechol oestrogen, lipid aldehyde, menadione and arachidonic acid.

Oxidative stress is known to have a major impact on human sperm function and, as a result, there is a need to develop sensitive methods for measuring reactive oxygen species (ROS) generation by these cells. A variety of techniques have been developed for this purpose including chemiluminescence (luminol and lucigenin), flow cytometry (MitoSOX Red, dihydroethidium, 4,5-diaminofluorescein diacetate and 2',7'-dichlorodihydrofluorescein diacetate) and spectrophotometry (nitroblue tetrazolium). The relative sensitivity of these assays and their comparative ability to detect ROS generated in different subcellular compartments of human spermatozoa, have not previously been investigated. To address this issue, we have compared the performance of these assays when ROS generation was triggered with a variety of reagents including 2-hydroxyestradiol, menadione, 4-hydroxynonenal and arachidonic acid. The results revealed that menadione predominantly induced release of ROS into the extracellular space where these metabolites could be readily detected by luminol-peroxidase and, to a lesser extent, 2',7'-dichlorodihydrofluorescein. However, such sensitivity to extracellular ROS meant that these assays were particularly vulnerable to interference by leucocytes. The remaining reagents predominantly elicited ROS generation by the sperm mitochondria and could be optimally detected by MitoSOX Red and DHE. Examination of spontaneous ROS generation by defective human spermatozoa revealed that MitoSOX Red was the most effective indicator of oxidative stress, thereby emphasizing the general importance of mitochondrial dysregulation in the aetiology of defective sperm function.

Polymorphisms of catechol estrogens metabolism pathway genes and breast cancer risk in Mexican women.

Breast cancer is associated to estrogen exposure. Allelic variants involved in estrogen metabolism might change the risk of developing this neoplasia. We examined the potential association of breast cancer risk in Mexican women with the polymorphisms CYP1A1 rs1048943, CYP1B1 rs1056836, COMT rs4680, GSTP1 rs1695, GSTT1 null and GSTM1 null which are involved in estrogen metabolism pathway. This study included 150 cases and 150 controls. A significant association was observed between, CYP1A1 rs1048943 (OR = 1.95, C.I. 1.13-3.36) and GSTP1 rs1695 (OR = 2.39, C.I. 1.24-4.24) polymorphisms with the risk of breast cancer. This risk was increased when the women were stratified according to their menopausal status. The results show that breast cancer risk significantly increases in women with 3-6 risk polymorphisms (OR = 3.75, C.I. 1.44-9.74).

Role of catecholestrogens on ovarian prostaglandin secretion in vitro in the catfish Heteropneustes fossilis and possible mechanism of regulation.

Seasonal, periovulatory and 2-hydroxyestradiol-17ß (2-OHE(2))-induced changes on ovarian prostaglandin (PG) E(2) and F(2α) were investigated under in vivo or in vitro in the female catfish Heteropneustes fossilis. Both PGE(2) and PGF(2α) increased significantly during ovarian recrudescence with the peak levels in spawning phase. The PGs showed periovulatory changes with the peak levels at 16 h after the hCG treatment. Incubation of postvitellogenic ovary fragments with estradiol-17ß (E(2)), 2-OHE(2) or 2-methoxyE(2) produced concentration-dependent increases in PG levels; 2-OHE(2) was more effective. In order to identify the receptor mechanism involved in the 2-OHE(2)-induced PG stimulation, the ovarian pieces were incubated with phentolamine (an α-adrenergic antagonist), propranolol (a ß-adrenergic antagonist) or tamoxifen (an estrogen receptor blocker) alone or in combination with 2-OHE(2). The incubation of the tissues with the receptor blockers alone did not produce any significant effect on basal PG levels. However, co- and pre-incubation of the tissues with the blockers resulted in inhibition of the stimulatory effect of 2-OHE(2) on the PGs. Phentolamine was more effective than propranolol. The signal transduction pathway(s) involved in the 2-OHE(2)-induced PG secretion was investigated. The incubation of the ovarian pieces with 3-isobutyl-1-methylxanthine (IBMX, a phosphodiesterase inhibitor), chelerythrine (a protein kinase C inhibitor) and PD098059 (a mitogen-activated protein kinase inhibitor) significantly lowered the basal secretion of PGF(2α) and PGE(2). In contrast, H89 (a protein kinase A inhibitor) increased the basal secretion of PGs at 1 and 5 µM concentration and decreased it at 10 µM concentration. The co- or pre-incubation with IBMX, H89, chelerythrine and PD098059 significantly inhibited the stimulatory effect of 2-OHE(2) on PGF(2α) and PGE(2) levels. The inhibition was higher in the pre-incubation groups. Chelerythrine was the most effective followed by PD098059, IBMX and H89. The results suggest that 2-OHE(2) may employ both adrenergic and estrogen receptors, or a novel receptor mechanism having properties of both adrenergic and estrogen receptors.