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PURPOSE: To report age-specific serum estradiol concentration in nonsmoking, lean US men without comorbidities. We provide concentrations from 30 and 15 to 20 years ago given previously described declines in serum estradiol in US men over time. METHODS: We used data from the Third National Health and Nutrition Examination Survey (NHANES III; 1988 to 1991) and continuous NHANES (1999 to 2004). Serum estradiol and SHBG were previously measured by competitive electrochemiluminescence immunoassays. Free estradiol was estimated from estradiol, SHBG, and albumin. By age, we calculated median concentrations overall and for nonsmoking, lean (body mass index <25 kg/m2 and waist <102 cm) men without diabetes, cardiovascular disease, or cancer. RESULTS: Overall, respective total estradiol medians for men ages 20 to 39, 40 to 59, and ≥60 years old were 37.0, 33.9, and 33.5 pg/mL in NHANES III and 31.3, 30.5, and 27.0 pg/mL in continuous NHANES. In nonsmoking, lean men without comorbidities, respective total estradiol medians were 32.0, 32.1, and 32.0 pg/mL in NHANES III and 29.1, 22.7, and 26.1 pg/mL in continuous NHANES. Overall, respective free estradiol medians were 0.82, 0.72, and 0.64 pg/mL in NHANES III and 0.67, 0.61, and 0.47 pg/mL in continuous NHANES. In nonsmoking, lean men without comorbidities, respective free estradiol medians were 0.64, 0.67, and 0.62 pg/mL in NHANES III and 0.58, 0.42, and 0.40 pg/mL continuous NHANES. CONCLUSION: We report US nationally representative serum estradiol concentrations in healthy men, which could be used for targeting estradiol during testosterone supplementation and for general good health.
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Catechol-O-methyltransferase (COMT) acts as a 'gate-keeper' to prevent DNA damage during estrogen metabolism. Both experimental and epidemiological studies suggest the role of COMT in pathogenesis of human breast cancer (BCa). It was previously reported that inhibition of COMT enzyme activity in estradiol-treated human breast epithelial carcinoma-derived MCF-7 cells caused increased oxidative DNA damage and formation of mutagenic depurinating adducts. To improve our understanding of factors influencing estrogen metabolism in BCa, it requires a mechanistic study illustrating the regulation of this 'gate-keeper'. We investigated the epigenetic mechanisms underlying decreased COMT transcription in MCF-7 cells exposed to 17ß-estradiol (E2) and the phytoestrogen, genistein (GEN). CpG site-specific methylation at promoters for both soluble (S) and membrane-bound (MB) COMT transcripts were assessed. Both E2 and GEN induced CpG site-specific methylation within the distal promoter of MB-COMT. In addition, ChIP analysis showed that there was increased binding of DNMT3B, MBD2 and HDAC1 within this promoter. These epigenetic changes were associated with decreased COMT transcript levels. Interestingly, sulforaphane, an antioxidant commonly found in cruciferous vegetables, was able to reverse the estrogen-induced epigenetic changes and gene silencing of COMT. Our data provide a new insight in epigenetically targeting COMT transcription. Since reactive estrogen metabolites may contribute to breast cancer, our findings may help in developing prevention and/or intervention strategies for human BCa.
Assuntos
Neoplasias da Mama/genética , Catecol O-Metiltransferase/genética , Metilação de DNA/efeitos dos fármacos , Epigênese Genética/efeitos dos fármacos , Estradiol/toxicidade , Genisteína/toxicidade , Regiões Promotoras Genéticas , Transcrição Gênica/efeitos dos fármacos , Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Catecol O-Metiltransferase/metabolismo , Ilhas de CpG , Regulação para Baixo , Feminino , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Células MCF-7RESUMO
BACKGROUND: Given the recent findings from pooled studies about a potential inverse association between selenium levels and prostate cancer risk, this cross-sectional study aimed to investigate the association between serum selenium and serum concentrations of sex steroid hormones including estradiol in a nationally representative sample of U.S. men to investigate one mechanism by which selenium may influence prostate cancer risk. METHODS: The study included 1,420 men ages 20 years or older who participated in the Third National Health and Nutrition Examination Survey between 1988 and 1994. We calculated age/race-ethnicity-adjusted and multivariable-adjusted geometric mean serum concentrations of total and estimated free testosterone and estradiol, androstanediol glucuronide, and sex hormone binding globulin, and compared them across quartiles of serum selenium. RESULTS: Adjusting for age, race/ethnicity, smoking status, serum cotinine, household income, physical activity, alcohol consumption, and percent body fat, mean total estradiol [e.g., Q1, 38.00 pg/mL (95% confidence interval (CI), 36.03-40.08) vs. Q4, 35.29 pg/mL (95% CI, 33.53-37.14); P trend = 0.050] and free estradiol [e.g., Q1, 0.96 pg/mL (95% CI, 0.92-1.01) vs. Q4, 0.90 (95% CI, 0.85-0.95); P trend = 0.065] concentrations decreased over quartiles of selenium. Stratification by smoking and alcohol consumption, showed that the latter observation was stronger for never smokers (P interaction = 0.073) and those with limited alcohol intake (P interaction = 0.017). No associations were observed for the other sex steroid hormones studied. CONCLUSIONS: Our findings suggests that a possible mechanism by which selenium may be protective for prostate cancer is related to estrogen. IMPACT: Further studies of longitudinal measurements of serum and toenail selenium in relation to serum measurements of sex steroid hormones are needed.
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Carcinogênese/metabolismo , Estradiol/sangue , Estrogênios/sangue , Hormônios Esteroides Gonadais/sangue , Neoplasias da Próstata/sangue , Selênio/sangue , Adulto , Consumo de Bebidas Alcoólicas/efeitos adversos , Carcinogênese/patologia , Estudos Transversais , Seguimentos , Humanos , Masculino , Prognóstico , Neoplasias da Próstata/epidemiologia , Neoplasias da Próstata/prevenção & controle , Fumar/efeitos adversos , Estados Unidos/epidemiologia , Adulto JovemRESUMO
In the context of the Human Toxome project, mass spectroscopy-based metabolomics characterization of estrogen-stimulated MCF-7 cells was studied in order to support the untargeted deduction of pathways of toxicity. A targeted and untargeted approach using overrepresentation analysis (ORA), quantitative enrichment analysis (QEA) and pathway analysis (PA) and a metabolite network approach were compared. Any untargeted approach necessarily has some noise in the data owing to artifacts, outliers and misidentified metabolites. Depending on the chemical analytical choices (sample extraction, chromatography, instrument and settings, etc.), only a partial representation of all metabolites will be achieved, biased by both the analytical methods and the database used to identify the metabolites. Here, we show on the one hand that using a data analysis approach based exclusively on pathway annotations has the potential to miss much that is of interest and, in the case of misidentified metabolites, can produce perturbed pathways that are statistically significant yet uninformative for the biological sample at hand. On the other hand, a targeted approach, by narrowing its focus and minimizing (but not eliminating) misidentifications, renders the likelihood of a spurious pathway much smaller, but the limited number of metabolites also makes statistical significance harder to achieve. To avoid an analysis dependent on pathways, we built a de novo network using all metabolites that were different at 24 h with and without estrogen with a p value <0.01 (53) in the STITCH database, which links metabolites based on known reactions in the main metabolic network pathways but also based on experimental evidence and text mining. The resulting network contained a "connected component" of 43 metabolites and helped identify non-endogenous metabolites as well as pathways not visible by annotation-based approaches. Moreover, the most highly connected metabolites (energy metabolites such as pyruvate and alpha-ketoglutarate, as well as amino acids) showed only a modest change between proliferation with and without estrogen. Here, we demonstrate that estrogen has subtle but potentially phenotypically important alterations in the acyl-carnitine fatty acids, acetyl-putrescine and succinoadenosine, in addition to likely subtle changes in key energy metabolites that, however, could not be verified consistently given the technical limitations of this approach. Finally, we show that a network-based approach combined with text mining identifies pathways that would otherwise neither be considered statistically significant on their own nor be identified via ORA, QEA, or PA.
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Metabolismo Energético/efeitos dos fármacos , Estradiol/farmacologia , Estrogênios/farmacologia , Metaboloma/efeitos dos fármacos , Metabolômica/métodos , Modelos Biológicos , Metabolismo Secundário/efeitos dos fármacos , Toxicologia/métodos , Cromatografia Líquida de Alta Pressão , Biologia Computacional , Mineração de Dados , Bases de Dados Factuais , Disruptores Endócrinos/farmacologia , Humanos , Células MCF-7 , Reprodutibilidade dos Testes , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
The twenty-first century vision for toxicology involves a transition away from high-dose animal studies to in vitro and computational models (NRC in Toxicity testing in the 21st century: a vision and a strategy, The National Academies Press, Washington, DC, 2007). This transition requires mapping pathways of toxicity by understanding how in vitro systems respond to chemical perturbation. Uncovering transcription factors/signaling networks responsible for gene expression patterns is essential for defining pathways of toxicity, and ultimately, for determining the chemical modes of action through which a toxicant acts. Traditionally, transcription factor identification is achieved via chromatin immunoprecipitation studies and summarized by calculating which transcription factors are statistically associated with up- and downregulated genes. These lists are commonly determined via statistical or fold-change cutoffs, a procedure that is sensitive to statistical power and may not be as useful for determining transcription factor associations. To move away from an arbitrary statistical or fold-change-based cutoff, we developed, in the context of the Mapping the Human Toxome project, an enrichment paradigm called information-dependent enrichment analysis (IDEA) to guide identification of the transcription factor network. We used a test case of activation in MCF-7 cells by 17ß estradiol (E2). Using this new approach, we established a time course for transcriptional and functional responses to E2. ERα and ERß were associated with short-term transcriptional changes in response to E2. Sustained exposure led to recruitment of additional transcription factors and alteration of cell cycle machinery. TFAP2C and SOX2 were the transcription factors most highly correlated with dose. E2F7, E2F1, and Foxm1, which are involved in cell proliferation, were enriched only at 24 h. IDEA should be useful for identifying candidate pathways of toxicity. IDEA outperforms gene set enrichment analysis (GSEA) and provides similar results to weighted gene correlation network analysis, a platform that helps to identify genes not annotated to pathways.
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Estradiol/toxicidade , Receptor alfa de Estrogênio/efeitos dos fármacos , Receptor beta de Estrogênio/efeitos dos fármacos , Testes de Toxicidade/métodos , Animais , Proliferação de Células/efeitos dos fármacos , Estradiol/administração & dosagem , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Células MCF-7 , Fatores de Transcrição SOXB1/genética , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Fator de Transcrição AP-2/genética , Fatores de Transcrição/genéticaRESUMO
Common recommendations for cell line authentication, annotation and quality control fall short addressing genetic heterogeneity. Within the Human Toxome Project, we demonstrate that there can be marked cellular and phenotypic heterogeneity in a single batch of the human breast adenocarcinoma cell line MCF-7 obtained directly from a cell bank that are invisible with the usual cell authentication by short tandem repeat (STR) markers. STR profiling just fulfills the purpose of authentication testing, which is to detect significant cross-contamination and cell line misidentification. Heterogeneity needs to be examined using additional methods. This heterogeneity can have serious consequences for reproducibility of experiments as shown by morphology, estrogenic growth dose-response, whole genome gene expression and untargeted mass-spectroscopy metabolomics for MCF-7 cells. Using Comparative Genomic Hybridization (CGH), differences were traced back to genetic heterogeneity already in the cells from the original frozen vials from the same ATCC lot, however, STR markers did not differ from ATCC reference for any sample. These findings underscore the need for additional quality assurance in Good Cell Culture Practice and cell characterization, especially using other methods such as CGH to reveal possible genomic heterogeneity and genetic drifts within cell lines.
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Variação Genética/genética , Linhagem Celular Tumoral , Hibridização Genômica Comparativa/métodos , Perfilação da Expressão Gênica/métodos , Marcadores Genéticos/genética , Humanos , Células MCF-7 , Repetições de Microssatélites/genética , Reprodutibilidade dos TestesRESUMO
The Human Toxome Project, funded as an NIH Transformative Research grant 2011-2016, is focused on developing the concepts and the means for deducing, validating and sharing molecular pathways of toxicity (PoT). Using the test case of estrogenic endocrine disruption, the responses of MCF-7 human breast cancer cells are being phenotyped by transcriptomics and mass-spectroscopy-based metabolomics. The bioinformatics tools for PoT deduction represent a core deliverable. A number of challenges for quality and standardization of cell systems, omics technologies and bioinformatics are being addressed. In parallel, concepts for annotation, validation and sharing of PoT information, as well as their link to adverse outcomes, are being developed. A reasonably comprehensive public database of PoT, the Human Toxome Knowledge-base, could become a point of reference for toxicological research and regulatory test strategies.
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Toxicologia/métodos , Alternativas aos Testes com Animais , Animais , Bases de Dados Factuais , Disruptores Endócrinos , Humanos , Metabolômica , Camundongos , Testes de Toxicidade/métodos , TranscriptomaRESUMO
Studies in hamsters, mice and rats have demonstrated that estradiol (E2), its interconvertible metabolite estrone (E1) and their catechol metabolites, in particular 4-hydroxy E2/E1, are carcinogenic in the kidney, uterus and mammary gland. Observational studies and clinical trials consistently show that sustained exposure to E2/E1 is associated with the development of sporadic breast cancer. The weight of evidence supports the contribution of two complementary pathways in the initiation, promotion and progression of breast cancer. One pathway involves activation of nuclear and cytoplasmic signaling pathways through the binding of estrogen to nuclear and membrane-bound estrogen receptors leading to increased cell proliferation. The other pathway involves the oxidative metabolism of E2/E1 to catechols and then reactive quinones that can contribute to oxidative DNA damage and form specific, mutagenic depurinating adducts with adenine and guanine which then in turn can serve as biomarkers for the occurrence of these processes. Both pathways can serve as portals to preventive intervention. Antiestrogens are used clinically to block receptor-mediated signaling to block tumor growth. Various chemopreventive agents such as sulforaphane (SFN) and resveratrol have been shown in cell culture to block oxidative metabolism of E2/E1 and thus prevent DNA damage. Pretreatment of MCF-7 and MCF-10F cells with and inhibitor of catechol-O-methyltransferase (COMT) followed by treatment with E2 or 4-OH E2 caused increased oxidative DNA damage (8-oxo-dG) and depurinating DNA adducts showing the importance of E2-catechol O-methylation by COMT as a protective pathway. E2 treatment of MCF-10A cells with E2 or 4-OH E2 caused an increase in E2-adenine and guanine adducts. Treatment with sulforaphane increased NAD(P)H: quinone oxidoreductase 1 (NQO1) and glutathione-S-transferase A1 (GSTA1) expression without affecting expression of catechol-O-methyltransferase (COMT) or cytochrome P450 1B1. Pretreatment with SFN decreased depurinating DNA adducts while increasing levels of 4-OCH3E1/2 and 4-OHE1/2-glutathione conjugates. Treatment of MCF-10F cells with E2 or 4-OH-E2 also caused increased depurinating DNA adducts and neoplastic transformation while pretreatment with resveratrol caused a reduction in adduct levels and neoplastic transformation. Increased levels of estrogen-quinone conjugates and DNA adducts have also been detected in urine of women at increased risk for and with breast cancer. These observations support the notion that targeting the estrogen/estrone metabolism pathway may be another way to reduce breast cancer risk.
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Anticarcinógenos/farmacologia , Carcinógenos/metabolismo , Estrogênios/metabolismo , Estrogênios/toxicidade , Animais , Neoplasias da Mama/etiologia , Carcinógenos/toxicidade , Modelos Animais de Doenças , Estradiol/metabolismo , Estradiol/toxicidade , Estrona/metabolismo , Estrona/toxicidade , Feminino , Humanos , Isotiocianatos/farmacologia , Neoplasias Renais/etiologia , Camundongos , Ratos , Fatores de Risco , Sulfóxidos , Neoplasias Uterinas/etiologiaRESUMO
Sulforaphane (SFN) is a potent inducer of detoxication enzymes such as NAD(P)H:quinone oxidoreductase 1 (NQO1) and glutathione-S-transferase (GST) via the Kelch-like erythroid-derived protein with CNC homology-associated protein 1 (Keap1)-NF-E2-related factor 2 (Nrf2) signaling pathway. NQO1 reduces the carcinogenic estrogen metabolite, catechol estrogen-3,4-quinone, whereas GSTs detoxify it through conjugation with glutathione. These 3,4-quinones can react with DNA to form depurinating DNA adducts. Thus, SFN may alter estrogen metabolism and thus protect against estrogen-mediated DNA damage and carcinogenesis. Human breast epithelial MCF-10A cells were treated with either vehicle or SFN and either estradiol (E2) or its metabolite 4-hydroxyestradiol (4-OHE2). 4-Hydroxy-derived estrogen metabolites and depurinating DNA adducts formed from E2 and its interconvertable metabolite estrone (E1) were analyzed by mass spectrometry. Levels of the depurinated adducts, 4-OHE1/2-1-N3Adenine and 4-OHE1/2-1-N7Guanine, were reduced by 60% in SFN-treated cells, whereas levels of 4-OCH3E1/2 and 4-OHE1/2-glutathione conjugates increased. To constitutively enhance the expression of Nrf2-regulated genes, cells were treated with either scrambled or siKEAP1 RNA. Following E2 or 4-OHE2 treatments, levels of the adenine and guanine adducts dropped 60-70% in siKEAP1-treated cells, whereas 4-OHE1/2-glutathione conjugates increased. However, 4-OCH3E1/2 decreased 50% after siKEAP1 treatment. Thus, treatment with SFN or siKEAP1 has similar effects on reduction of depurinating estrogen-DNA adduct levels following estrogen challenge. However, these pharmacologic and genetic approaches have different effects on estrogen metabolism to O-methyl and glutathione conjugates. Activation of the Nrf2 pathway, especially elevated NQO1, may account for some but not all of the protective effects of SFN against estrogen-mediated DNA damage.
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Anticarcinógenos/farmacologia , Mama/efeitos dos fármacos , Adutos de DNA/efeitos dos fármacos , Estrogênios/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Isotiocianatos/farmacologia , Fator 2 Relacionado a NF-E2/metabolismo , Apoptose , Western Blotting , Mama/citologia , Mama/metabolismo , Proliferação de Células , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Feminino , Humanos , Técnicas Imunoenzimáticas , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteína 1 Associada a ECH Semelhante a Kelch , Fator 2 Relacionado a NF-E2/genética , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Sulfóxidos , Espectrometria de Massas em TandemRESUMO
Long term exposure to estrogens is associated with an increased risk of breast cancer. The precise mechanisms responsible for estrogen mediated carcinogenesis are not well understood. The most widely accepted theory holds that estradiol (E(2)), acting through estrogen receptor alpha (ERα), stimulates cell proliferation and initiates mutations arising from replicative errors occurring during pre-mitotic DNA synthesis. The promotional effects of E(2) then support the growth of cells harboring mutations. Over a period of time, sufficient numbers of mutations accumulate to induce neoplastic transformation. Laboratory and epidemiological data also suggest that non-receptor mediated mechanisms resulting from the genotoxic effects of estrogen metabolites are involved in breast cancer development. This manuscript critically reviews existing data implicating both ER-dependent and -independent mechanisms. The weight of evidence supports the possibility that both mechanisms are involved in the carcinogenic process. In addition, estrogen metabolites likely modulate stem cell functionality and cancer progression. The roles of ER dependent and independent actions in the carcinogenic process are pertinent to the consideration of breast cancer preventative agents as anti-estrogens block only receptor mediated pathways whereas the aromatase inhibitors block both.
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Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Receptores de Estrogênio/metabolismo , Neoplasias da Mama/epidemiologia , Estradiol/metabolismo , Feminino , Humanos , Redes e Vias Metabólicas/efeitos dos fármacos , Mutagênicos/toxicidadeRESUMO
Catechol estrogens are carcinogenic, probably because of their estrogenicity and potential for further oxidative metabolism to reactive quinones. Estrogenic quinones cause oxidative DNA damage as well as form mutagenic depurinating adenine and guanine adducts. O-Methylation by catechol-O-methyltransferase (COMT) blocks their estrogenicity and prevents their oxidation to quinones. A single gene encodes both membrane bound (MB) and soluble (S) forms of COMT. The COMT gene contains 34 single nucleotide polymorphisms (SNPs). The valine108 (S-COMT)/158 (MB-COMT) SNP encodes a low activity form of COMT and has been widely studied as a putative risk factor for breast cancer, with inconsistent results. Investigations of two other SNPs in the promoter of MB-COMT that may affect its expression have also provided mixed results. Future studies on the role of COMT in breast cancer should incorporate measurement of biomarkers that reflect COMT activity and its protective effects.
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The manganese superoxide dismutase (MnSOD) ala16val polymorphism has been associated with various diseases including breast cancer. In the present study, we investigated levels of MnSOD protein, enzymatic activity, and mRNA with respect to MnSOD genotype in several human breast carcinoma cell lines and in mouse embryonic fibroblasts (MEF), developed from the MnSOD knockout mouse, stably expressing human MnSOD-ala and MnSOD-val. In human breast cell lines, the MnSOD-ala allele was associated with increased levels of MnSOD protein and MnSOD protein per unit mRNA. In the MEF transformants, MnSOD activity correlated fairly well with MnSOD protein levels. MnSOD mRNA expression was significantly lower in MnSOD-ala versus MnSOD-val lines. MnSOD protein and activity levels were not related to MnSOD genotype in the transformed MEF, although, as observed in the human breast cell lines, the MEF human MnSOD-ala lines produced significantly more human MnSOD protein per unit mRNA than the human MnSOD-val lines. This suggests that there is more efficient production of MnSOD-ala protein compared to MnSOD-val protein. Examination of several indicators of reactive oxygen species levels, including superoxide and hydrogen peroxide, in wild-type MEF and in MEF expressing similar elevated amounts of MnSOD-ala or val activity did not show differences related to the levels of MnSOD protein expression. In conclusion, in both human breast carcinoma cell lines and MEF cell lines stably transfected with human MnSOD, the MnSOD-ala allele was associated with increased production of MnSOD protein per unit mRNA indicating a possible imbalance in MnSOD protein production from the MnSOD-val mRNA.
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Neoplasias da Mama/genética , Fibroblastos/enzimologia , Polimorfismo Genético , Superóxido Dismutase/genética , Alanina/genética , Alanina/metabolismo , Animais , Neoplasias da Mama/enzimologia , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos , Camundongos Knockout , Estresse Oxidativo , RNA Mensageiro/metabolismo , Superóxido Dismutase/metabolismo , Valina/genética , Valina/metabolismoRESUMO
There has been increasing evidence pointing to the mitochondrial respiratory chain (MRC) as a novel and important target for the actions of 17beta-estradiol (E(2)) and estrogen receptors (ER) in a number of cell types and tissues that have high demands for mitochondrial energy metabolism. This novel E(2)-mediated mitochondrial pathway involves the cooperation of both nuclear and mitochondrial ERalpha and ERbeta and their co-activators on the coordinate regulation of both nuclear DNA- and mitochondrial DNA-encoded genes for MRC proteins. In this paper, we have: 1) comprehensively reviewed studies that reveal a novel role of estrogens and ERs in the regulation of MRC biogenesis; 2) discussed their physiological, pathological and pharmacological implications in the control of cell proliferation and apoptosis in relation to estrogen-mediated carcinogenesis, anti-cancer drug resistance in human breast cancer cells, neuroprotection for Alzheimer's disease and Parkinson's disease in brain, cardiovascular protection in human heart and their beneficial effects in lens physiology related to cataract in the eye; and 3) pointed out new research directions to address the key questions in this important and newly emerging area. We also suggest a novel conceptual approach that will contribute to innovative regimens for the prevention or treatment of a wide variety of medical complications based on E(2)/ER-mediated MRC biogenesis pathway.
Assuntos
Transporte de Elétrons/efeitos dos fármacos , Transporte de Elétrons/fisiologia , Estrogênios/fisiologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/fisiologia , Receptores de Estrogênio/fisiologia , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/etiologia , Animais , Apoptose/genética , Apoptose/fisiologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/etiologia , Doenças Cardiovasculares/prevenção & controle , Proliferação de Células , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Resistencia a Medicamentos Antineoplásicos , Transporte de Elétrons/genética , Estradiol/farmacologia , Estradiol/fisiologia , Feminino , Genoma Mitocondrial , Humanos , Cristalino/efeitos dos fármacos , Cristalino/fisiologia , Masculino , Mitocôndrias/genética , Proteínas Mitocondriais/fisiologia , ATPases Mitocondriais Próton-Translocadoras/fisiologia , Modelos Biológicos , Neoplasias Hormônio-Dependentes/tratamento farmacológico , Neoplasias Hormônio-Dependentes/etiologia , Fármacos Neuroprotetores/farmacologia , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/etiologia , Biossíntese de Proteínas/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacosRESUMO
Cadmium (Cd) has been shown to bind to the human estrogen receptor (ER), yet studies on Cd's estrogenic effects have yielded inconsistent results. In this study, we investigated the effects of Cd on DNA synthesis and its simultaneous effects on both genomic (mediated by nuclear ER (nER)) and non-genomic (mediated by membrane-bound ER (mER)) signaling in human breast cancer derived T47D cells. No effects on DNA synthesis were observed for non-cytotoxic concentrations of CdCl(2) (0.1-1000 nM), and Cd did not increase progesterone receptor (PgR) or pS2 mRNA levels. However, Cd stimulated phosphorylation of ERK1/2 MAPK, detectable following 10 min and 18 h of treatment. The sustained Cd-induced ERK1/2 phosphorylation was inhibited by the ER antagonist ICI 182,780, suggesting the involvement of ER. In addition, Cd enhanced DNA synthesis and pS2 mRNA levels in estrogen (10 pM estradiol) treated T47D cells. The MEK1/2 specific inhibitor U0126 blocked DNA synthesis stimulated by estradiol (E2) and the E2-Cd mixtures. These findings indicate that the ERK1/2 signaling is critical in E2-related DNA synthesis. The sustained ERK1/2 phosphorylation may contribute to the Cd-induced enhancement of DNA synthesis and pS2 mRNA in mixture with low-concentration E2.
Assuntos
Neoplasias da Mama/metabolismo , Cloreto de Cádmio/toxicidade , Carcinógenos Ambientais/toxicidade , DNA de Neoplasias/biossíntese , Estradiol/farmacologia , Receptores de Estrogênio/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Humanos , Fosforilação , Receptores de Progesterona/metabolismo , Transdução de SinaisRESUMO
CumuIative and excessive exposure to estrogens is associated with increased breast cancer risk. The traditional mechanism explaining this association is that estrogens affect the rate of cell division and apoptosis and thus manifest their effect on the risk of breast cancer by affecting the growth of breast epithelial tissues. Highly proliferative cells are susceptible to genetic errors during DNA replication. The action of estrogen metabolites offers a complementary genotoxic pathway mediated by the generation of reactive estrogen quinone metabolites that can form adducts with DNA and generate reactive oxygen species through redox cycling. In this chapter, we discussed a novel mitochondrial pathway mediated by estrogens and their cognate estrogen receptors (ERs) and its potential implications in estrogen-dependent carcinogenesis. Several lines of evidence are presented to show: (1) mitochondrial localization of ERs in human breast cancer cells and other cell types; (2) a functional role for the mitochondrial ERs in regulation of the mitochondrial respiratory chain (MRC) proteins and (3) potential implications of the mitochondrial ER-mediated pathway in stimulation of cell proliferation, inhibition of apoptosis and oxidative damage to mitochondrial DNA. The possible involvement of estrogens and ERs in deregulation of mitochondrial bioenergetics, an important hallmark of cancer cells, is also described. An evolutionary view is presented to suggest that persistent stimulation by estrogens through ER signaling pathways of MRC proteins and energy metabolic pathways leads to the alterations in mitochondrial bioenergetics and contributes to the development of estrogen-related cancers.
Assuntos
Formação de Conceito , Hormônios/fisiologia , Mitocôndrias/fisiologia , Neoplasias/etiologia , Metabolismo Energético/fisiologia , Estrogênios/farmacologia , Humanos , Mitocôndrias/metabolismo , Modelos Biológicos , Neoplasias/induzido quimicamente , Neoplasias/metabolismo , Neoplasias Hormônio-Dependentes/metabolismo , Receptores de Estrogênio/fisiologia , Transdução de Sinais/fisiologiaRESUMO
Estrogen-DNA adducts are potential biomarkers for assessing the risk of developing of a number of hormonally modified cancers, including breast cancer. Formation of the 4-hydroxyestradiol-N(7)-guanine (4-OHE2-N(7)-guanine) adduct from the reaction of estradiol-3,4-quinone with DNA and its detection in vivo has been established. With the ultimate goal of exploring estrogen-DNA adducts as biomarkers in experimental and human investigations, the 4-OHE2-N(7)-guanine was synthesized, and preliminary studies demonstrated that this adduct was detectable in all 10 female human urine samples examined. Therefore, more extensive investigations were conducted to study this compound's chemical-physical properties and to examine the stability of 4-OHE2-N(7)-guanine under a range of pH conditions that might influence biomarker measurement. Under neutral to alkaline conditions, 4-OHE2-N(7)-guanine could completely oxidize to an 8-oxo-guanine derivative. This derivative was isolated by HPLC, and mass spectrometry confirmed the oxidized compound by demonstrating the formation of an m/ z 168 fragment, generated by oxygen addition to guanine. Furthermore, investigation of the 4-OHE2-N(7)-2'-deoxyguanosine nucleoside adduct showed that under alkaline conditions a formamidopyrimidine analogue was produced. The formamidopyrimidine derivative forms from ring opening of the guanine imidazole ring following C-8 oxidation in the N(7), N(9) disubstituted guanine. Formation of both of these oxidized estrogen-guanine DNA adducts has precedent with other chemical agents that covalently bind to the N(7) position in guanine. Therefore, the development and application of methods to measure estrogen-guanine adducts will need to also consider these new adducts, and the biological implications of these compounds will need to be explored to determine their contribution to estrogen toxicology.
Assuntos
Biomarcadores/urina , Adutos de DNA/metabolismo , Estradiol/análogos & derivados , Guanina/análogos & derivados , Biomarcadores/química , Cromatografia Líquida de Alta Pressão , Adutos de DNA/química , Estradiol/química , Estradiol/urina , Estrogênios de Catecol , Feminino , Guanina/química , Guanina/urina , Humanos , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
A single nucleotide polymorphism in the human COMT (catechol-O-methyltransferase) gene has been associated with increased risk for breast cancer and several CNS diseases and disorders. The G to A polymorphism causes a valine (val) to methionine (met) substitution at codon 108 soluble - (S)/158 membrane - (MB)-COMT, generating alleles encoding high and low-activity forms of the enzyme, COMT H and COMT L, respectively. Tissues and cells with a COMT LL genotype have decreased COMT activity compared to COMT HH cells. Previously, we reported that the decreased activity was due to decreased amounts of S-COMT L protein in human hepatocytes. In this study, we investigated the role of S-COMT protein synthesis and turnover as determinates of reduced COMT protein in COMT LL compared to COMT HH cells. No association between S-COMT protein synthesis and COMT genotype was detected. Using a pulse-chase protocol, the half-life of S-COMT H was determined to be 4.7 days, which was considerably longer than expected from the half-lives of other phase 2 enzyme proteins. The half-life of S-COMT L compared to S-COMT H protein was significantly shorter at 3.0 days, but the difference was affected by the medium used during the chase period. These results suggest that increased turnover may contribute to reduced COMT activity in cells and tissues from COMT LL individuals. Subtle differences appear to be able to affect the stability of the S-COMT L protein, and this may contribute to the differences observed in epidemiological studies on the association of this polymorphism with breast cancer risk.