Catechol Estrogens Research Articles

Modulation of ATM enhances DNA repair in G2/M phase of cell cycle and averts senescence in Fuchs endothelial corneal dystrophy

Fuchs Endothelial Corneal Dystrophy (FECD) is an aging disorder characterized by expedited loss of corneal endothelial cells (CEnCs) and heightened DNA damage compared to normal CEnCs. We previously established that ultraviolet-A (UVA) light causes DNA damage and leads to FECD phenotype in a non-genetic mouse model. Here, we demonstrate that acute treatment with chemical stressor, menadione, or physiological stressors, UVA, and catechol estrogen (4-OHE2), results in an early and increased activation of ATM-mediated DNA damage response in FECD compared to normal CEnCs. Acute stress with UVA and 4OHE2 causes (i) greater cell-cycle arrest and DNA repair in G2/M phase, and (ii) greater cytoprotective senescence in NQO1−/− compared to NQO1+/+ cells, which was reversed upon ATM inhibition. Chronic stress with UVA and 4OHE2 results in ATM-driven cell-cycle arrest in G0/G1 phase, reduced DNA repair, and cytotoxic senescence, due to sustained damage. Likewise, UVA-induced cell-cycle reentry, gamma-H2AX foci, and senescence-associated heterochromatin were reduced in Atm-null mice. Remarkably, inhibiting ATM activation with KU-55933 restored DNA repair in G2/M phase and attenuated senescence in chronic cellular model of FECD lacking NQO1. This study provides insights into understanding the pivotal role of ATM in regulating cell-cycle, DNA repair, and senescence, in oxidative-stress disorders like FECD.

Protection of HT22 neuronal cells against chemically-induced ferroptosis by catechol estrogens: protein disulfide isomerase as a mechanistic target

Ferroptosis is a form of regulated cell death, characterized by excessive iron-dependent lipid peroxidation. Biochemically, ferroptosis can be selectively induced by erastin through glutathione depletion or through inhibition of glutathione peroxidase 4 by RSL3, which leads to accumulation of cytotoxic lipid reactive oxygen species (ROS). Protein disulfide isomerase (PDI) was recently shown to mediate erastin/RSL3-induced ferroptosis and thus also become a new target for protection against chemically-induced ferroptosis. The present study aims to identify endogenous compounds that can protect against erastin/RSL3-induced ferroptotic cell death. We find that 2-hydroxyestrone, 2-hydroxyestradiol, 4-hydroxyestrone and 4-hydroxyestradiol, four major endogenous catechol estrogens, are effective inhibitors of PDI, and can strongly protect against chemically-induced ferroptotic cell death in cultured HT22 mouse hippocampal neuronal cells. The CETSA assay showed that these catechol estrogens can bind to PDI in live cells. PDI knockdown attenuates the protective effect of these catechol estrogens against chemically-induced ferroptosis. Mechanistically, inhibition of PDI’s catalytic activity by catechol estrogens abrogates erastin/RSL3-induced dimerization of nitric oxide synthase, thereby preventing the subsequent accumulation of cellular nitric oxide, ROS and lipid-ROS, and ultimately ferroptotic cell death. In addition, joint treatment of cells with catechol estrogens also abrogates erastin/RSL3-induced upregulation of nitric oxide synthase protein levels, which also contributes to the cytoprotective effect of the catechol estrogens. In conclusion, the present study demonstrates that the catechol estrogens are protectors of HT22 neuronal cells against chemically-induced ferroptosis, and inhibition of PDI’s catalytic activity by these estrogens contributes to a novel, estrogen receptor-independent mechanism of cytoprotection.

EFFECTS OF ARYL HYDROCARBON RECEPTOR LIGAND TCDD ON HUMAN TROPHOBLAST CELL DEVELOPMENT.

The primary interface between mother and fetus, the placenta, serves two critical functions: extraction of nutrients from the maternal compartment and facilitation of nutrient delivery to the developing fetus. This delivery system also serves as a barrier to environmental exposures. The aryl hydrocarbon receptor (AHR) is an important component of the barrier. AHR signaling is activated by environmental pollutants and toxicants that can potentially affect cellular and molecular processes, including those controlling trophoblast cell development and function. In this study, we investigated the impact of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), an effective AHR ligand, exposure on human trophoblast cells. Human trophoblast stem (TS) cells were used as in vitro model system for investigating the downstream consequences of AHR activation. The actions TCDD were investigated in human TS cells maintained in the stem state or in differentiating TS cells. TCDD exposure stimulated the expression of CYP1A1 and CYP1B1 in human TS cells. TCDD was effective in stimulating CYP1A1 and CYP1B1 expression and altering gene expression profiles in human TS cells maintained in the stem cell state or induced to differentiate into extravillous trophoblast cells (EVT) or syncytiotrophoblast (ST). These actions were dependent upon the presence of AHR. TCDD exposure did not adversely affect maintenance of the TS cell stem state or the ability of TS cells to differentiate into EVT cells or ST. However, TCDD exposure did promote the biosynthesis of 2 methoxy estradiol (2ME), a biologically active catechol estrogen, with the potential to modify the maternal-fetal interface. Human trophoblast cell responses to TCDD were dependent upon AHR signaling and possessed the potential to shape development and function of the human placentation site.

Microdroplet‐assisted protein adduct formation and characterization by mass spectrometry

AbstractThere have been increasing interests in developing proteomics‐based protein adduct detection for monitoring carcinogen exposure risk. Generating adducted protein standards and characterizing the adduction sites by bottom‐up proteomics, however, require lengthy adduction and enzymatic digestion. Here we demonstrated that microdroplet can greatly accelerate the catechol estrogen adduction on hemoglobin (Hb) as well as one‐pot reaction of adduction and enzyme digestion in millisecond for bottom‐up characterization. The adducted Hb generated by microdroplet reaction was characterized to contain one to two catechol estrogens with the β chain of Hb (Hb‐β) by online in situ intact measurement of mass spectrometry. The adduction sites were further identified to be C112‐Hb‐β or C93‐Hb‐β by microdroplet one‐pot or two‐step adduction and digestion. These results were consistent with the intact measurement and also same as the bulk or endogenous reaction. This method is expected to be applicable to prepare protein standards adducted by other reactive oxidizing species to greatly save time and sample amount.

Abstract LB296: Human CYP1B1 enhances cancer progression through induction of Sp1 via DNMT-mediated epigenetic regulation of miR-375

Abstract Human cytochrome P450 1B1 (CYP1B1) is a major catalytic enzyme for metabolism of 17β-estradiol (E2) to catechol estrogens, mainly carcinogenic metabolite 4-hydroxyestradiol (4-OHE2). To identify the unknown molecular mechanism for cancer progression induced by CYP1B1, we studied the role of CYP1B1 in human tumor cells, MCF-7, MDA-MB-231, and HeLa cells. Suppression of CYP1B1 by shRNA significantly reduced cancer cell viability and invasion. Clinical data from cancer patients showed that high level of CYP1B1 expression resulted in relatively lower survival rate. Interestingly, overexpression of CYP1B1 markedly activated Wnt/β-catenin signaling and EMT process, and suppression of specificity protein 1 (Sp1) by siRNA, an well-known transcription factor, demolished the promoting effects of CYP1B1 on cancer progression. To investigate the correlation between CYP1B1 and Sp1, we analyzed the clinical data from breast and cervical cancer patients and the results showed the positive correlation between two genes. Furthermore, TMS (tetramethoxystilbene), a specific CYP1B1 inhibitor, diminished the induction of Sp1 by CYP1B1, and 4-OHE2 exceedingly promoted the expression level of Sp1. Surprisingly, miR-375, known as an oncogenic microRNA, substantially decreased by 4-OHE2 and CYP1B1 overexpression in CYP1B1-transgenic (TG) mice. Methylation specific assay revealed that CYP1B1 suppressed miR-375 expression through induction of hyper-methylation on pre-miR-375. In addition, epigenetic regulation of miR-375 by CYP1B1 was executed via induction of DNA methyltransferases (DNMTs), DNMT1, 3a, and 3b. Enhanced expression level of Sp1 by 4-OHE2 was significantly deteriorated by 5-aza-dC, a DNMT inhibitor. Taken together, our data suggest that CYP1B1 enhances cancer progression through induction of cancer cell proliferation and invasion via DNMT-mediated epigenetic regulation of miR-375 and further promotion of Sp1 expression. Citation Format: Young-Jin Chun, Yeo-Jung Kwon, Tae-Uk Kwon. Human CYP1B1 enhances cancer progression through induction of Sp1 via DNMT-mediated epigenetic regulation of miR-375 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB296.

Menopause Hot Flashes and Molecular Mechanisms Modulated by Food-Derived Nutrients.

The causes of vasomotor symptoms, including hot flashes, are not fully understood, may be related to molecular factors, and have a polygenic architecture. Nutrients and bioactive molecules supplied to the body with food are metabolized using various enzymatic pathways. They can induce molecular cell signaling pathways and, consequently, activate effector proteins that modulate processes related to hot flashes in menopausal women. In this review, we analyzed the literature data from the last 5 years, especially regarding genome-wide association study (GWAS) analysis, and selected molecular factors and cell signaling pathways that may potentially be related to hot flashes in women. These are the kisspeptin-GnRH pathway, adipocyte-derived hormones, aryl hydrocarbon receptor signaling, catechol estrogens and estrogen sulfotransferase, inflammatory and oxidative stress biomarkers, and glucose availability. Then, single compounds or groups of food ingredients were selected that, according to experimental data, influence the course of the discussed molecular pathways and thus can be considered as potential natural therapeutic agents to effectively reduce the troublesome symptoms of menopause in women.

Hormonal and genotoxic estrogen-androgen carcinogenesis in the NBL rat prostate: A role for aromatase.

Androgens are generally thought to cause prostate cancer, but the data from animal studies suggest that they must be aromatized to estrogen and act in concert with genotoxic estrogen metabolites. The objective of this study was to determine whether treatment with testosterone (T) combined with a nonestrogenic estrogen metabolite and a nongenotoxic estrogenic compound would all be necessary and sufficient for the induction of a high incidence of prostate cancer in the susceptible NBL rat strain. NBL rats were treated with low-dose testosterone via slow-release Silastic implants and with the marginally estrogenic genotoxic catechol estrogen 4-hydroxyestradiol (4OH-E2) and the nongenotoxic estrogen 2-fluoroestradiol (2F-E2) and in one experiment the aromatase inhibitor letrozole via custom-made slow-release pellets. Animals were euthanized 52 weeks after implantation and their pituitaries and prostate complexes weighed and fixed in formalin. Hematoxylin and eosin (H&E)-stained step sections were prepared and examined microscopically for proliferative lesions. Animals treated with 2F-E2, with or without the other compounds, had enlarged pituitaries demonstrating its estrogenicity. Animals treated with T, with or without the other compounds, had enlarged prostates consistent with its androgenicity. Rats treated with T plus 2F-E2 and 4OH-E2 developed a high incidence of prostatic cancer (89%), while, surprisingly, rats treated with T plus only 2F-E2 also had a high incidence of prostate cancer (95%) contradicting our initial hypothesis. To test whether the formation of E2 from T by aromatase could lead to estrogen genotoxicity and prostate carcinogenesis we then rats treated with T and 2F-E2 also with letrozole and found that it reduced prostate cancer incidence by about 50%. These findings indicate that long-term treatment with a nongenotoxic estrogen (2F-E2) and T as well as uninhibited prostatic aromatase activity generating genotoxic E2 are all required for induction of a high incidence of prostatic adenocarcinomas in NBL rats. These and previous data indicate that androgen receptor-mediated action, estrogen receptor mediation, and estrogen genotoxicity are all required and sufficient for hormonal carcinogenesis in the NBL rat prostate. Interference with the estrogen genotoxicity is a potential approach to prostate cancer chemoprevention.

Bioconversion of 4-hydroxyestradiol by extradiol ring-cleavage dioxygenases from Novosphingobium sp. PP1Y

Livestock breeding activities and pharmaceutical wastes lead to considerable accumulation of steroid hormones and estrogens in wastewaters. Here estrogens act as pro-cancerogenic agents and endocrine disruptors interfering with the sexual development of aquatic animals and having toxic effects in humans. Environmental bacteria play a vital role in estrogens degradation. Their wide reservoir of enzymes, such as ring cleavage dioxygenases (RCDs), can degrade the steroid nucleus, catalyzing the meta-cleavage of A, B or D steroid rings. In this work, 4 extra-diol ring cleavage dioxygenases (ERCDs), PP28735, PP26077, PP00124 and PP00193, were isolated from the marine sphingomonad Novosphingobium sp. PP1Y and characterized. Enzymes kinetic parameters were determined on different synthetic catecholic substrates. Then, the bioconversion of catechol estrogens was evaluated. PP00124 showed to be an efficient catalyst for the degradation of 4-hydroxyestradiol (4-OHE2), a carcinogenic hydroxylated derivate of E2. 4-OHE2 complete cleavage was obtained using PP00124 both in soluble form and in whole recombinant E. coli cells. LC–MS/MS analyses confirmed the generation of a semialdehyde product, through A-ring meta cleavage. To the best of our knowledge, PP00124 is the first characterized enzyme able to directly degrade 4-OHE2 via meta cleavage. Moreover, the complete 4-OHE2 biodegradation using recombinant whole cells highlighted advantages for bioremediation purposes.

Catechol-O-methyl transferase suppresses cell invasion and interplays with MET signaling in estrogen dependent breast cancer

Catechol-O-methyl transferase (COMT) is involved in detoxification of catechol estrogens, playing cancer-protective role in cells producing or utilizing estrogen. Moreover, COMT suppressed migration potential of breast cancer (BC) cells. To delineate COMT role in metastasis of estrogen receptor (ER) dependent BC, we investigated the effect of COMT overexpression on invasion, transcriptome, proteome and interactome of MCF7 cells, a luminal A BC model, stably transduced with lentiviral vector carrying COMT gene (MCF7-COMT). 2D and 3D assays revealed that COMT overexpression associates with decreased cell invasion (p < 0.0001 for Transwell assay, p < 0.05 for spheroid formation). RNA-Seq and LC-DIA-MS/MS proteomics identified genes associated with invasion (FTO, PIR, TACSTD2, ANXA3, KRT80, S100P, PREX1, CLEC3A, LCP1) being downregulated in MCF7-COMT cells, while genes associated with less aggressive phenotype (RBPMS, ROBO2, SELENBP, EPB41L2) were upregulated both at transcript (|log2FC|> 1, adj. p < 0.05) and protein (|log2FC|> 0.58, q < 0.05) levels. Importantly, proteins driving MET signaling were less abundant in COMT overexpressing cells, and pull-down confirmed interaction between COMT and Kunitz-type protease inhibitor 2 (SPINT2), a negative regulator of MET (log2FC = 5.10, q = 1.04−7). In conclusion, COMT may act as tumor suppressor in ER dependent BC not only by detoxification of catechol estrogens but also by suppressing cell invasion and interplay with MET pathway.

A 'new' estrogen metabolite: an epoxide of estrone as a sulfated steroid.

Current heightened recognition of the importance of sulfated steroids led to the examination of conjugates in media from incubations of estrogens in tissues from the reproductive tract of stallions. Previously, we had reported a 'new' unidentified metabolite of estrone (E1) and [3H]-E1, located between 17β-estradiol (E2) and E1 reference standards on chromatography (HPLC) and identified tentatively as a stable 5α,6α-estrone epoxide. Stallion tissues were minced and incubated for 2 h with [3H]-E1 (1 × 106 cpm). Solid-phase extraction of unconjugated and conjugated steroids from media was followed by liquid scintillation counting (LSC), where radioactivity was mostly in the conjugate fractions (>80%). HPLC of conjugated steroids used an isocratic solvent system of acetonitrile/water (8:92) at 700 µL/min with detection by LSC. A radioactive peak between reference standards of E1 and E2 sulfates was examined, after solvolysis, in a second solvent system. Sulfated steroids yielded largely E1, whereas acid treatment of the unconjugated E1 epoxide had earlier formed 6α-OH-E1 almost exclusively. With sulfatase enzyme, at neutral pH, radioactivity was also seen mostly as E1 with trace amounts of polar material. Reduction with KBH4, however, led also to desulfation; radioactivity had alignment with E2 but even more had low retention times as for 6α/6β-OH-E2. These findings point to a different hydrolysis for desulfation; even more, they reveal an additional oxygen atom at C6 and are supportive of biological formation of 5α,6α-epoxides of E1 and E2. As possible alternatives to catechol estrogens, implicated in cancer, the 'new' estrogen metabolites and their sulfated forms may have special interest.

Abstract 2696: Catechol estrogen profiles of patients with EGFR- and ALK-positive non-small cell lung cancer (NSCLC)

Abstract Prior research from this group indicates that the human lung extensively metabolizes parent estrogens to reactive catechols. Estrone (E1) and estradiol (E2) are converted to the putative carcinogens, 4-hydroxyestrone (4-OHE1) and 4-hydroxyestradiol (4-OHE2), by cytochrome P450 1B1 (CYP1B1). In contrast, CYP1A1 metabolizes parent estrogens to 2-OHE1 and 2-OHE2, which may ultimately be converted to anti-proliferative derivatives. Our previous data strongly indicate that 4-OHEs contribute to lung tumorigenesis. However, their role in oncogene-driven NSCLC has not been investigated. The goal of this study was to compare the urinary profile of catechol estrogens in patients with EGFR- and ALK-positive NSCLC with those of cancer-free controls. The resulting levels of estrogen derivatives were correlated with genotypic variants in the estrogen metabolizing enzymes CYP1B1 and COMT. Patients with EGFR- and ALK-mutated NSCLC were recruited from the recently established Never-Smokers Lung Cancer Clinic at our institution. Study participants were 50 years of age or older to circumvent any confounding impact of pre-menopausal status on estrogen profiles. Mutation status was determined from tumor tissue biopsies. Cancer-free control subjects were Caucasian post-menopausal women with no prior smoking history. To date, 34 patients with tumors possessing EGFR mutations, 10 patients with ALK mutations, and 17 cancer-free control subjects have been enrolled. Urine specimens were collected from all participants and urinary estrogen species (E1, E2, E3, 4-OHE1, 4-OHE2, 2-OHE1, 2-OHE2, 16α-OHE1, 2-OME1, 4-OME1, OME2) were quantified using a newly established UPLC-MS/MS method. Blood lymphocytes, collected from NSCLC subjects, were genotyped for SNPs in CYP1B1 (L432V/rs1056836, R48G/rs10012, N453S/rs1800440) and COMT (V158M/rs4680) by real-time PCR. Preliminary investigation of the E1 metabolic pathway suggests a trend of higher ratios of 4-OHE1 to 2-OHE1 in patients with EGFR- and ALK-positive NSCLC as compared to cancer-free controls. Among NSCLC subjects, those possessing the high activity Leu432 variant of CYP1B1 (Leu/Leu + Leu/Val) tended to have higher ratios of 4-OHE1 to 2-OHE1 as compared to patients with the low activity Val/Val genotype. Early findings suggest enhanced production of C4 estrogen derivates may contribute to the development of oncogene-driven lung tumors. Targeting CYP1B1 may be of preventive and therapeutic interest. Recruitment of study participants is ongoing to validate these early findings in a larger cohort. (Supported by an In Vino Vita Award from Fox Chase Cancer Center) Citation Format: J. Nicholas Bodor, Joseph Treat, Daniel D. Krzizike, Carolyn L. Zawislak, Lisa Vanderveer, Sarah Eidbo, Eric A. Ross, Martin J. Edelman, Andrew J. Andrews, Margie L. Clapper. Catechol estrogen profiles of patients with EGFR- and ALK-positive non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2696.

Abstract 2345: Specificity of catechol-O-methyltransferase (COMT) for hydroxyestrogens favors 2-OHEs over 4-OHEs

Abstract The contribution of estrogen receptor-mediated signaling to cancer progression and metastasis has been well documented. However, much less attention has been given to the role of estrogen metabolism in carcinogenesis. Parent estrogens are metabolized to 2- and 4-hydroxyestrogens (2-OHEs and 4-OHEs) that can retain their estrogenic activity, form reactive quinones that cause DNA damage, or become inactivated primarily by the rate-limiting conjugation enzyme catechol-O-methyltransferase (COMT). The ability of 4-OHEs to induce genomic instability and the malignant transformation of human breast epithelial cells has been reported. Glucuronidation or sulfation of methylated hydroxyestrogens, produced by COMT, increases their solubility and facilitates their excretion via urine or feces. The extent to which catechol estrogens are methylated dictates, in part, their ability to initiate carcinogenesis verses protect against tumor formation. COMT is a ubiquitous polymorphic enzyme that is expressed in humans in both peripheral tissues (soluble, s-COMT) and the central nervous system (membrane-bound). The goal of the present study was to compare the rate at which human wild type (WT) s-COMT and its major polymorphic variant (V108M s-COMT) transfer the methyl group from S-adenosylmethionine to 2-OHE and 4-OHE. This was accomplished by expressing COMT in E. coli, followed by protein purification and steady state Michaelis-Menten kinetics. Production of methylated hydroxyestrogens (2-MeOEs and 4-MeOEs) was quantified using LC-MS/MS methodology. The resulting data indicate that WT s-COMT methylates catechol estrogens more efficiently than V108M s-COMT. In addition, the specificity constant (kcat/KM) of WT s-COMT for methylation of 4-OHE2 was 12-fold greater than that of V108M s-COMT. The kcat/KM of V108M s-COMT was 4-fold greater for 2-OHE2 vs. 4-OHE2. Based on the enhanced catalytic efficiency of WT s-COMT in converting 2-OHEs to 2-MeOEs, 4-OHEs may have greater potential to cause DNA damage. Additional studies are needed to determine if the V108M s-COMT variant contributes to increased risk for cancer. These data provide novel insight into the potential mechanism by which 4-OHEs promote hormone-induced carcinogenesis. This work was supported by an In Vino Vita Award from Fox Chase Cancer Center. Citation Format: Daniel D. Krzizike, Margie L. Clapper, Andrew J. Andrews. Specificity of catechol-O-methyltransferase (COMT) for hydroxyestrogens favors 2-OHEs over 4-OHEs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2345.

Quantification of the Endogenous Adduction Level on Hemoglobin and Correlation with Albumin Adduction via Proteomics: Multiple Exposure Markers of Catechol Estrogen.

Catechol estrogens (CEs) are genotoxic metabolites whose detection is challenging due to their low concentrations and high variability in the blood. By intact protein and free CE measurement of the spiked hemolysate, endogenous CEs were revealed to mainly (>99%) exist as hemoglobin (Hb) adducts in red blood cells. In order to detect endogenous CE-Hb adducts, we developed a two-step method that involved protein precipitation and solid phase extraction to purify Hb from red blood cells, and the method was coupled with proteomics using liquid chromatography-tandem mass spectrometry. Using bottom-up proteomics and standard additions, we identified C93 and C112 of Hb-β as the main adduction sites of Hb, and this accounted for CE-induced oxidization of adducted peptides by sample preparation. The non-adducted, adducted, and oxidized tryptic peptides that covered the same Hb-β sequences were targeted by parallel reaction monitoring to determine the adduction level in red blood cells. A quantification limit (S/N < 8) below the endogenous CE-Hb adduction level with relative standard errors that ranged from 5 to 22% was achieved and applied to clinical samples. The human serum albumin (HSA) adduction levels from the same patient were also determined using a previously developed method (Anal. Chem.2019,91, 15922-15931). A positive correlation (R2 = 0.673) between the CE-HSA and CE-Hb adduction level was obtained from all clinical samples, and both levels were significantly (p < 0.005) higher for patients with breast cancer compared to healthy controls. However, double indexes derived from the red blood cell and the serum, respectively, provide higher precision and confidence in predicting cancer risk than the single index. This study reported an efficient sample preparation for proteomics-based Hb adducts and revealed the potential of using multiple blood proteins for developing more reliable and specific markers based on protein adductomics.

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.

Investigation of Catechol-O-methyltransferase (COMT) gene Val158Met polymorphism in ovarian cancer

Objective:Catechol-O-methyltransferase (COMT), the product of the COMT gene, detoxifies the carcinogenic catechol estrogens. The aim of the present study was to examine the relationship between COMT Val158Met polymorphism and the risk of ovarian cancer.Material and Methods:The study groups consist of 94 individuals as a patients group with ovarian cancer (n=47) and control group (n=47). The allele and genotype frequencies were determined according to Hardy-Weinberg equilibrium (HWE). The allele and genotype frequencies. determined according to HWE. Genetic analysis were performed by real-time-polymerase chain reaction instrument, and the statistical analysis were performed by SPSS program.Results:Although no significant relationship was obtained among groups (p=0.413) regarding COMT gene Val158Met polymorphism, the genotype frequencies for COMT Val158Met (rs4860) polymorphism in groups was homozygote wild type GG genotype 25.5%, heterozygote GA genotype 46.8%, homozygote mutant AA genotype 27.7%.Conclusion:This study is the first to investigate the relationship between ovarian cancer and the Val158Met polymorphism in the COMT gene in a Turkish population. No statistically significant relationship was identified among genotypes belonging to the patient and control groups although sample sizes were relatively small and the analysis should be repeated in a larger cohort.

Discovery of breast cancer risk genes and establishment of a prediction model based on estrogen metabolism regulation

BackgroundMultiple common variants identified by genome-wide association studies have shown limited evidence of the risk of breast cancer in Chinese individuals. In this study, we aimed to uncover the relationship between estrogen levels and the genetic polymorphism of estrogen metabolism-related enzymes in breast cancer (BC) and establish a risk prediction model composed of estrogen-metabolizing enzyme genes and GWAS-identified breast cancer-related genes based on a polygenic risk score.MethodsUnrelated BC patients and healthy subjects were recruited for analysis of estrogen levels and single nucleotide polymorphisms (SNPs) in genes encoding estrogen metabolism-related enzymes. The polygenic risk score (PRS) was used to explore the combined effect of multiple genes, which was calculated using a Bayesian approach. An independent sample t-test was used to evaluate the differences between PRS scores of BC and healthy subjects. The discriminatory accuracy of the models was compared using the area under the receiver operating characteristic (ROC) curve.ResultsThe estrogen homeostasis profile was disturbed in BC patients, with parent estrogens (E1, E2) and carcinogenic catechol estrogens (2/4-OHE1, 2-OHE2, 4-OHE2) significantly accumulating in the serum of BC patients. We then established a PRS model to evaluate the role of SNPs in multiple genes. PRS model 1 (M1) was established from SNPs in 6 GWAS-identified high risk genes. On the basis of M1, we added SNPs from 7 estrogen metabolism enzyme genes to establish PRS model 2 (M2). The independent sample t-test results showed that there was no difference between BC and healthy subjects in M1 (P = 0.17); however, there was a significant difference between BC and healthy subjects in M2 (P = 4.9*10− 5). The ROC curve results showed that the accuracy of M2 (AUC = 62.18%) in breast cancer risk identification was better than that of M1 (AUC = 54.56%).ConclusionEstrogen and related metabolic enzyme gene polymorphisms are closely related to BC. The model constructed by adding estrogen metabolic enzyme gene SNPs has a good predictive ability for breast cancer risk, and the accuracy is greatly improved compared with that of the PRS model that only includes GWAS-identified gene SNPs.

Effects of the ovary and conceptus on uterine blood flow in the pig

Summary. Changes in uterine blood flow throughout pregnancy appear to be due to steroid-induced alterations in uterine arterial tone and contractility. Arterial contractility is a transient reduction in luminal diameter in response to nerve stimulation or to an alpha-1 adrenergic agonist, leading to short-term reduction in uterine blood flow. Tone is the pressure exerted by an arterial segment against an intraluminal flow (distensibility) and is considered to set the baseline rate of flow. These phenomena appear to be regulated individually, with tone changes predominating during pregnancy. In pregnancy, tone is markedly depressed as oestrogen concentrations rise, and the vessel is distended and flaccid. Arterial tone is a function of the amount of calcium available to the contractile proteins of the arterial smooth muscle, which is derived from extracellular sources. Calcium availability is regulated by the opening and closing of calcium channels in the surface membrane in response to changes in the membrane potential. The loss of uterine arterial tone associated with oestrogen results from a markedly depressed uptake of calcium by the vessels. A significant negative correlation (P < 0.001; r = – 0.93) is observed between uterine arterial uptake of calcium and the concentrations of oestrogens in systemic blood of pigs throughout gestation. Several lines of evidence suggest that the blockade of potential-sensitive calcium channels associated with uterine hyperaemia is produced by catechol oestrogens, short-lived metabolites of oestrogens that are found in the circulation when oestrogen levels are high. Synthesis of catechol oestrogens from the parent oestrogens has been shown to occur in the placenta, endometrium and uterine arteries of pigs. In addition, perfusion of uterine arteries from nonpregnant pigs with catechol oestrogens, but not free oestrogens, in vitro significantly reduces both the tone and the uptake of calcium by the vessels. Contractility of the arteries to nerve stimulation or phenylephrine, however, is not affected. These data suggest that the pig conceptus, through production of catechol oestrogens, locally increases arterial distensibility, resulting in increased baseline flow to each fetal—placental unit. The contractility (reactivity) of the uterine arterial vasculature to adrenergic agonists or other vasoactive agents, however, is not affected. In this way, the conceptus can maintain the locally elevated flows necessary for its survival while the maternal system can continue to respond to life-threatening stimuli by transiently rerouting blood flow away from the uterus and towards other vascular beds more necessary for maternal survival.

Risk of Breast Cancer Associated with Estrogen DNA Adduct Biomarker.

It is biologically plausible that genotoxic estrogens, namely estrogen DNA adducts (EDA), have a role in breast cancer development. Support comes from three prior studies that reported elevated concentrations of EDA relative to estrogen metabolites and conjugates (EDA:EMC) in women with breast cancer relative to control women. In postmenopausal women in the Women's Health Initiative (WHI), EDA:EMC in 191 controls was compared with findings in 194 prediagnosis urine samples from breast cancer cases. EDA:EMC determinations were by mass spectrometry as previously described, and logistic regression was employed to estimate ORs. EDA:EMC did not differ in breast cancer cases compared with controls overall [0.93 (95% confidence interval, 0.71-1.23)], with a mean (SD) of 2.3 (0.8) and 2.4 (1.1) in cases and controls, respectively. Similarly, the ratio did not differ when examined by estrogen receptor or recency of biospecimen collection prior to breast cancer. Despite the demonstrated genotoxic properties of certain catechol estrogens resulting in EDAs, this analysis did not provide evidence for an increased breast cancer risk in relation to an elevated EDA:EMC. This analysis, conducted prospectively within postmenopausal women in the WHI study, suggests that a strong association between EDA:EMC and breast cancer could be ruled out, as this study was powered to detect an OR of 2.2 or greater.

Identification of Cytosolic Protein Targets of Catechol Estrogens in Breast Cancer Cells Using a Click Chemistry-Based Workflow

Catechol estrogens (CEs) are known to be toxic metabolites and the initiators of the oncogenesis of breast cancers via forming covalent adducts with DNAs. CEs shall also react with proteins, but their cellular protein targets remain unexplored. Here, we reported the identification of protein targets of CEs in the soluble cytosol of estrogen-sensitive breast cancer cells by multiple comparative proteomics using liquid chromatography-tandem mass spectrometry (LC-MS/MS) coupled with an improved click chemistry-based workflow. Multiple comparative proteomics composed of an experimental pair (probe versus solvent) and two control pairs (solvent versus solvent and probe versus solvent without enrichment) were studied using stable isotope dimethyl labeling. The use of 4-hydroxyethynylestradiol (4OHEE2) probe with an amide-free linker coupled with on-bead digestion and redigestion of the proteins cleaved from the beads was shown to greatly improve the recovery and identification of CE-adducted peptides. A total of 310 protein targets and 40 adduction sites were repeatedly (n ≥ 2) identified with D/H (probe/solvent) ratio >4 versus only one identified with D/H >4 from the two control pairs, suggesting that our workflow imposes only a very low background. Meanwhile, multiple comparative D/H ratios revealed that CEs may downregulate many target proteins involved in the metabolism or detoxification, suggesting a negative correlation between CE-induced adduction and expression of proteins acting on the alleviation of stress-induced cellular damages. The reported method and data will provide opportunities to study the progression of estrogen metabolism-derived diseases and biomarkers.

Accurate and sensitive detection of Catechol-O-methyltransferase activity by liquid chromatography with fluorescence detection

Catechol-O-methyltransferase (COMT) is a major drug metabolizing enzyme in humans. COMT expression is directedly associated with various mental diseases and cancers due to its essential role in catalyzing metabolic inactivation of endogenous catecholamines and catechol estrogens. However, a practical method to precisely measure COMT activities in biological samples is lacking. In the current study, we established a liquid chromatography-fluorescence detection (LC-FD) method based on fluorometric detection of the methylated product of 3-BTD, a fluorescent probe for COMT, to sensitively quantify COMT activities in human erythrocytes and cell homogenates. Assay validation of the established LC-FD based method was conducted for selectivity and sensitivity, range of linearity, precision and accuracy, recovery, biological matrices effect and stability. The limit of quantification for 3-BTMD (the methylated product of 3-BTD by COMT) of this method was 0.0083 nM, which is nearly 10-fold lower than that for previously published methods. The method was precise with intra- and inter-day relative standard deviation (RSD) lower than 5%. In addition, this method showed an excellent anti-interference ability with no effects of the endogenous substances on the fluorometric detection of 3-BTMD. The practical use of this method was established by its successful application for the measurement of COMT activities in individual human erythrocytes (n = 13), and in cell homogenates generated from four different human cell lines. Our results suggest that this method will be of great value in accurately determining the native activity of COMT in biological samples, which is beneficial for a complete understand of the role of COMT both in physiological and pathological conditions.