Endocrine disrupting chemicals, 4-nonylphenol, bisphenol A and butyl benzyl phthalate, impair metabolism of estradiol in male and female rats as assessed by levels of 15α-hydroxyestrogens and catechol estrogens in urine.

Bisphenol A (BPA), 4-nonylphenol (NP) and butyl benzyl phthalate (BBP), termed endocrine-disrupting chemicals, are known to mimic estrogen activity. The effects of these chemicals on 17ß-estradiol (E2 ) metabolism in vivo in rats were examined. Male and female rats were given NP (250 mg kg-1  day-1 ), BPA (250 µg kg-1  day-1 ) or BBP (500 mg kg-1  day-1 ) by gavage for 14 days, followed by a single intraperitoneal injection of E2 (5 mg kg-1 ) on the final day. The urinary excretion over 72 hours of 2-hydroxyestrone 1-N-acetylcysteine thioether, 2-hydroxyestrone 4-N-acetylcysteine thioether, 4-hydroxyestrone 2-N-acetylcysteine thioether, 2-hydroxy-17ß-estradiol (2-OHE2 ), 2-hydroxyestrone (2-OHE1 ), 4-hydroxy-17ß-estradiol, 4-hydroxyestrone, 15α-hydroxyestriol (E4 ), 15α-hydroxy-17ß-estradiol and 15α-hydroxyestrone was measured. Increases in urinary excretion of 2-OHE1 and decreases in E4 were observed in males treated with NP or BBP. Decreases in urinary excretion of 2-OHE2 and E4 were observed in males treated with BPA. Decreases in urinary excretion of 2-OHE1 and 2-OHE2 were observed in females treated with BBP. Normalized liver and weights were increased in both sexes treated with NP or BBP. Histologic observations revealed marked changes in the distal tubules and collecting ducts in the kidneys of rats exposed to NP and BBP, and hypertrophy in the hepatocytes of the centrilobular zone of the liver. No BPA-related effects on organ weight and on liver or kidney histopathology were found. These results suggest that the 14 day oral dosing of NP and BBP disrupted E2 metabolism, resulting from marked morphological and functional alterations in the liver and kidneys. In addition, BPA could induce metabolic and endocrine disruption.

Critical depurinating DNA adducts: Estrogen adducts in the etiology and prevention of cancer and dopamine adducts in the etiology and prevention of Parkinson's disease.

Endogenous estrogens become carcinogens when dangerous metabolites, the catechol estrogen quinones, are formed. In particular, the catechol estrogen-3,4-quinones can react with DNA to produce an excess of specific depurinating estrogen-DNA adducts. Loss of these adducts leaves apurinic sites in the DNA, generating subsequent cancer-initiating mutations. Unbalanced estrogen metabolism yields excessive catechol estrogen-3,4-quinones, increasing formation of depurinating estrogen-DNA adducts and the risk of initiating cancer. Evidence for this mechanism of cancer initiation comes from various types of studies. High levels of depurinating estrogen-DNA adducts have been observed in women with breast, ovarian or thyroid cancer, as well as in men with prostate cancer or non-Hodgkin lymphoma. Observation of high levels of depurinating estrogen-DNA adducts in high risk women before the presence of breast cancer indicates that adduct formation is a critical factor in breast cancer initiation. Formation of analogous depurinating dopamine-DNA adducts is hypothesized to initiate Parkinson's disease by affecting dopaminergic neurons. Two dietary supplements, N-acetylcysteine and resveratrol complement each other in reducing formation of catechol estrogen-3,4-quinones and inhibiting formation of estrogen-DNA adducts in cultured human and mouse breast epithelial cells. They also inhibit malignant transformation of these cells. In addition, formation of adducts was reduced in women who followed a Healthy Breast Protocol that includes N-acetylcysteine and resveratrol. When initiation of cancer is blocked, promotion, progression and development of the disease cannot occur. These results suggest that reducing formation of depurinating estrogen-DNA adducts can reduce the risk of developing a variety of types of human cancer.

Estrogen genotoxicity causes preferential development of Fuchs endothelial corneal dystrophy in females.

Fuchs endothelial corneal dystrophy (FECD) is a genetically complex, age-related, female-predominant disorder characterized by loss of post-mitotic corneal endothelial cells (CEnCs). Ultraviolet-A (UVA) light has been shown to recapitulate the morphological and molecular changes seen in FECD to a greater extent in females than males, by triggering CYP1B1 upregulation in females. Herein, we investigated the mechanism of greater CEnC susceptibility to UVA in females by studying estrogen metabolism in response to UVA in the cornea. Loss of NAD(P)H quinone oxidoreductase 1 (NQO1) resulted in increased production of estrogen metabolites and mitochondrial-DNA adducts, with a higher CEnC loss in Nqo1-/- female compared to wild-type male and female mice. The CYP1B1 inhibitors, trans-2,3',4,5'-tetramethoxystilbene (TMS) and berberine, rescued CEnC loss. Injection of wild-type male mice with estrogen (E2; 17ß-estradiol) increased CEnC loss, followed by increased production of estrogen metabolites and mitochondrial DNA (mtDNA) damage, not seen in E2-treated Cyp1b1-/-male mice. This study demonstrates that the endo-degenerative phenotype is driven by estrogen metabolite-dependent CEnC loss that is exacerbated in the absence of NQO1; thus, explaining the mechanism accounting for the higher incidence of FECD in females. The mitigation of estrogen-adduct production by CYP1B1 inhibitors could serve as a novel therapeutic strategy for FECD.

Label-free and de-conjugation-free workflow to simultaneously quantify trace amount of free/conjugated and protein-bound estrogen metabolites in human serum.

Different chemical forms of sex hormones including free/conjugated metabolites as well as their protein/DNA adducts in human serum are a panel of important indicators of health conditions. It is, however, hard to quantify all species simultaneously due to the lack of general extraction, derivatization, and de-conjugation methods. Here we developed a label-free and de-conjugation-free workflow to quantify 11 free/conjugated estrogen metabolites including depurinating DNA and protein adduct forms of 4-hydroxyestradiol (4OHE2) in human serum. Acetonitrile acts as an excellent solvent to purify adducted and non-adducted human serum albumin (HSA) by precipitation as well as to extract free/conjugated metabolites and depurinating DNA adducts from the supernatant by salting-out effect. The adduction level of 4OHE2 on HSA was determined by proteomics; free/conjugated metabolites were quantified by a newly developed microflow liquid chromatography (microflow LC)-nanoelectrospray ionization (nanoESI)-multiple reaction monitoring (MRM) method with high reproducibility (7-22% RSD, n > 3) and sub-picogram levels (0.6-20 pg/mL) of quantification limits (S/N = 8) by using non-pulled capillary as nano-ESI emitter. This workflow was demonstrated to reveal endogenous adduction level of 4OHE2 on HSA as well as circulation levels of free/conjugated metabolites in clinical samples. 4OHE2 in human serum were solely detected as protein-bound form, indicating the merit of such integrated platform covering unstable or active metabolites. Compared to traditional methods using labeling or de-conjugation reaction, this workflow is much simplier, more sensitive, and more specific. Moreover, it can be widely applied in omics to concurrently access various bio-transformed known and un-known markers or drugs.

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.

Loss of NQO1 generates genotoxic estrogen-DNA adducts in Fuchs Endothelial Corneal Dystrophy.

Fuchs Endothelial Corneal Dystrophy (FECD) is an age-related genetically complex disease characterized by increased oxidative DNA damage and progressive degeneration of corneal endothelial cells (HCEnCs). FECD has a greater incidence and advanced phenotype in women, suggesting a possible role of hormones in the sex-driven differences seen in the disease pathogenesis. In this study, catechol estrogen (4-OHE2), the byproduct of estrogen metabolism, induced genotoxic estrogen-DNA adducts formation, macromolecular DNA damage, and apoptotic cell death in HCEnCs; these findings were potentiated by menadione (MN)-mediated reactive oxygen species (ROS). Expression of NQO1, a key enzyme that neutralizes reactive estrogen metabolites, was downregulated in FECD, indicating HCEnC susceptibility to reactive estrogen metabolism in FECD. NQO1 deficiency in vitro exacerbated the estrogen-DNA adduct formation and loss of cell viability, which was rescued by the supplementation of N-acetylcysteine, a ROS scavenger. Notably, overexpression of NQO1 in HCEnCs treated with MN and 4-OHE2 quenched the ROS formation, thereby reducing the DNA damage and endothelial cell loss. This study signifies a pivotal role for NQO1 in mitigating the macromolecular oxidative DNA damage arising from the interplay between intracellular ROS and impaired endogenous estrogen metabolism in post-mitotic ocular tissue cells. A dysfunctional Nrf2-NQO1 axis in FECD renders HCEnCs susceptible to catechol estrogens and estrogen-DNA adducts formation. This novel study highlights the potential role of NQO1-mediated estrogen metabolite genotoxicity in explaining the higher incidence of FECD in females.

Phytoestrogens and breast cancer prevention: possible mechanisms of action.

OBJECTIVE: Phytoestrogens display an array of pharmacologic properties, and in recent years investigation of their potential as anticancer agents has increased dramatically. In this article we review the published literature related to phytoestrogens and breast cancer as well as suggest the possible mechanisms that may underlie the relationship between phytoestrogens and breast cancer. DATA SOURCES: Electronic searches on phytoestrogens and breast cancer were performed on MEDLINE and EMBASE in June 2007. No date restriction was placed on the electronic search. DATA EXTRACTION: We focused on experimental data from published studies that examined the characteristics of phytoestrogens using in vivo or in vitro models. We also include human intervention studies in this review. DATA SYNTHESIS: We evaluated evidence regarding the possible mechanisms of phytoestrogen action. Discussions of these mechanisms were organized into those activities related to the estrogen receptor, cell growth and proliferation, tumor development, signaling pathways, and estrogen-metabolizing enzymes. CONCLUSIONS: We suggest that despite numerous investigations, the mechanisms of phytoestrogen action in breast cancer have yet to be elucidated. It remains uncertain whether these plant compounds are chemoprotective or whether they may produce adverse outcomes related to breast carcinogenesis.

4-Hydroxyestradiol induces mammary epithelial cell transformation through Nrf2-mediated heme oxygenase-1 overexpression.

Estrogen (17ß-estradiol, E2) undergoes oxidative metabolism by CYP1B1 to form 4-hydroxyestradiol (4-OHE2), a putative carcinogenic metabolite of estrogen. Our previous study showed that 4-OHE2-induced production of reactive oxygen species contributed to neoplastic transformation of human breast epithelial (MCF-10A) cells. In this study, 4-OHE2, but not E2, increased the expression of heme oxygenase-1 (HO-1), a sensor and regulator of oxidative stress, in MCF-10A cells. Silencing the HO-1 gene in MCF-10A cells suppressed 4-OHE2-induced cell proliferation and transformation. In addition, subcutaneous administration of 4-OHE2 markedly enhanced the growth of the MDA-MB-231 human breast cancer xenografts, which was retarded by zinc protoporphyrin, a pharmacological inhibitor of HO-1. 4-OHE2-induced HO-1 expression was mediated by NF-E2-related factor 2 (Nrf2). We speculate that an electrophilic quinone formed as a consequence of oxidation of 4-OHE2 binds directly to Kelch-like ECH-associated protein 1 (Keap1), an inhibitory protein that sequesters Nrf2 in the cytoplasm. This will diminish association between Nrf2 and Keap1. 4-OHE2 failed to interrupt the interaction between Keap1 and Nrf2 and to induce HO-1 expression in Keap1-C273S or C288S mutant cells. Lano-LC-ESI-MS/MS analysis in MCF-10A-Keap1-WT cells which were treated with 4-OHE2 revealed that the peptide fragment containing Cys288 gained a molecular mass of 287.15 Da, equivalent to the addition of a single molecule of 4-OHE2-derived ortho-quinones.

Etiology and prevention of prevalent types of cancer.

Endogenous estrogens become carcinogens when excessive catechol estrogen quinone metabolites are formed. Specifically, the catechol estrogen-3,4-quinones can react with DNA to produce a large amount of specific depurinating estrogen-DNA adducts, formed at the N-3 of Ade and N-7 of Gua. Loss of these adducts leaves apurinic sites in the DNA, which can generate subsequent cancer-initiating mutations. Unbalanced estrogen metabolism yields excessive catechol estrogen-3,4-quinones, increasing formation of the depurinating estrogen-DNA adducts and the risk of initiating cancer. Evidence for this mechanism of cancer initiation comes from studies in vitro, in cell culture, in animal models and in human subjects. High levels of estrogen-DNA adducts have been observed in women with breast, ovarian or thyroid cancer, and in men with prostate cancer or non-Hodgkin lymphoma. Observation of high levels of depurinating estrogen-DNA adducts in high risk women before the presence of breast cancer indicates that adduct formation is a critical factor in breast cancer initiation. Two dietary supplements, N-acetylcysteine and resveratrol, complement each other in reducing formation of catechol estrogen-3,4-quinones and inhibiting formation of estrogen-DNA adducts in cultured human and mouse breast epithelial cells. They also inhibit malignant transformation of these epithelial cells. In addition, formation of adducts was reduced in women who followed a Healthy Breast Protocol that includes N-acetylcysteine and resveratrol. Blocking initiation of cancer prevents promotion, progression and development of the disease. These results suggest that reducing formation of depurinating estrogen-DNA adducts can reduce the risk of developing a variety of types of human cancer.

Depurinating estrogen-DNA adducts, generators of cancer initiation: their minimization leads to cancer prevention.

Estrogens can initiate cancer by reacting with DNA. Specific metabolites of endogenous estrogens, the catechol estrogen-3,4-quinones, react with DNA to form depurinating estrogen-DNA adducts. Loss of these adducts leaves apurinic sites in the DNA, generating mutations that can lead to the initiation of cancer. A variety of endogenous and exogenous factors can disrupt estrogen homeostasis, which is the normal balance between estrogen activating and protective enzymes. In fact, if estrogen metabolism becomes unbalanced and generates excessive catechol estrogen 3,4-quinones, formation of depurinating estrogen-DNA adducts increases and the risk of initiating cancer is greater. The levels of depurinating estrogen-DNA adducts are high in women diagnosed with breast cancer and those at high risk for the disease. High levels of depurinating estrogen-DNA adducts before the presence of breast cancer indicates that adduct formation is a critical factor in breast cancer initiation. Women with thyroid or ovarian cancer also have high levels of estrogen-DNA adducts, as do men with prostate cancer or non-Hodgkin lymphoma. Depurinating estrogen-DNA adducts are initiators of many prevalent types of human cancer. These findings and other discoveries led to the recognition that reducing the levels of estrogen-DNA adducts could prevent the initiation of human cancer. The dietary supplements N-acetylcysteine and resveratrol inhibit formation of estrogen-DNA adducts in cultured human breast cells and in women. These results suggest that the two supplements offer an approach to reducing the risk of developing various prevalent types of human cancer. Graphical abstract Major metabolic pathway in cancer initiation by estrogens.

Estrogen-like metabolites and DNA-adducts in urogenital schistosomiasis-associated bladder cancer.

An estrogen-DNA adduct mediated pathway may be involved in the pathogenesis of the squamous cell carcinoma of the bladder associated with infection with the blood fluke Schistosoma haematobium. Extracts from developmental stages of S. haematobium, including eggs, induce tumor-like phenotypes in cultured cells. In addition, estrogen-derived, reactive metabolites occur in this pathogen and in sera of infected persons. Liquid chromatography-mass spectrometry analysis was performed on urine from 40 Angolans diagnosed with urogenital schistosomiasis (UGS), half of who also presented UGS-associated squamous cell carcinoma and/or urothelial cell carcinoma. The analysis revealed numerous estrogen-like metabolites, including seven specifically identified in UGS cases, but not reported in the database of metabolites in urine of healthy humans. These schistosome infection-associated metabolites included catechol estrogen quinones (CEQ) and CEQ-DNA-adducts, two of which had been identified previously in S. haematobium. In addition, novel metabolites derived directly from 8-oxo-7, 8-dihydro-2'-deoxyguanosine (8-oxodG) were identified in urine of all 40 cases of UGS. These metabolites can be expected to provide deeper insights into the carcinogenesis UGS-induced bladder cancer, and as biomarkers for diagnosis and/or prognosis of this neglected tropical disease-linked cancer.

Identification of Endogenous Site-specific Covalent Binding of Catechol Estrogens to Serum Proteins in Human Blood.

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

Why does infection with some helminths cause cancer?

Infections with Opisthorchis viverrini, Clonorchis sinensis and Schistosoma haematobium are classified as Group 1 biological carcinogens: definitive causes of cancer. These worms are metazoan eukaryotes, unlike the other Group 1 carcinogens including human papilloma virus, hepatitis C virus, and Helicobacter pylori. By contrast, infections with phylogenetic relatives of these helminths, also trematodes of the phylum Platyhelminthes and major human pathogens, are not carcinogenic. These inconsistencies prompt several questions, including how might these infections cause cancer? And why is infection with only a few helminth species carcinogenic? Here we present an interpretation of mechanisms contributing to the carcinogenicity of these helminth infections, including roles for catechol estrogen- and oxysterol-metabolites of parasite origin as initiators of carcinogenesis.

Regioselective deuterium labeling of estrone and catechol estrogen metabolites.

Increased exposure to estrogens and estrogen metabolites is linked with increased rates of breast, ovarian and other human cancers. Metabolism of estrogen can led to formation of electrophilic o-quinones capable of binding to DNA. In order to gain insight into the mechanism of estrogen-induced DNA damage, estrone and catechol estrogens derived from estrone, have been regioselectively labeled with deuterium at the 1-position. Estrone-1-d, estrone-1,2,4-d3, 4-hydroxyestrone-1-d and 2-hydroxyestrone-1-d have been synthesized with or without deuteriums at the 16-position. The key labeling step involves deuterated trifluoroacetic acid exchange catalyzed by t-butyl alcohol. This economical, straightforward labeling technique makes available a range of estrone compounds containing deuterium at the 1-position.