RESUMEN
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.
Asunto(s)
Biodegradación Ambiental , Dioxigenasas , Estrógenos , Sphingomonadaceae , Humanos , Cromatografía Liquida , Dioxigenasas/genética , Dioxigenasas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Estrógenos/metabolismo , Estrógenos de Catecol , Sphingomonadaceae/genética , Sphingomonadaceae/metabolismo , Espectrometría de Masas en TándemRESUMEN
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.
Asunto(s)
Catecol O-Metiltransferasa , Neoplasias , Catecol O-Metiltransferasa/genética , Espectrometría de Masas en Tándem , Estrógenos/metabolismo , Catecoles , Receptores de Estrógenos/metabolismo , Estrógenos de CatecolRESUMEN
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-ß 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-ß 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.
Asunto(s)
Hemoglobinas , Proteómica , Cromatografía Liquida , Estrógenos de Catecol , Humanos , Albúmina Sérica HumanaRESUMEN
Nowadays, there have been attempts to use phytochemicals in fruits to reduce the risk of suffering a given sickness. In this work, we studied the potential effects of mango (cultivar "Nam Dok Mai") and banana (cultivar "Khai") to attenuate DNA oxidative damage in MCF-10A cells induced by 4-hydroxyestradiol (4-OHE2 ). The effects of mango extract (MNE) and banana extract (BKE) were comparable with three carotenoid compounds, ß-carotene, lycopene, and lutein. The oxidative-induced DNA damage was evaluated by 8-hydroxy-2-deoxyguanosine (8-OHdG) reduction. 4-OHE2 -induced DNA oxidative damage in MCF-10A cells showed a decrease in 8-OHdG formation when treated with MNE and BKE. Both fruit extracts also enabled the regaining production of Phase II detoxifying (GSTs and NQO1) and antioxidant (SOD, GPx, and CAT) enzymes during 4-OHE2 -induced DNA oxidative damage in the MCF-10A cells when compared with the untreated control. These results indicate that MNE and BKE can exert potential mitigating effects against 4-OHE2 -induced DNA oxidative damage in MCF-10A cells by enhancing the activities of detoxifying and antioxidant enzyme. PRACTICAL APPLICATIONS: Long-term exposure to estrogen increases the risk of sickness since oxidative stress via the estrogen pathway, leading to DNA damage. This study indicated that mango (cultivar "Nam Dok Mai") extract contains ß-carotene and lycopene, while banana (cultivar "Khai") extract contains ß-carotene and lutein, which act as natural antioxidants. Both fruit extracts have preventive properties against oxidative DNA damage and are potentially good supplements for women taking E2 between HRT.
Asunto(s)
Mangifera , Musa , Antioxidantes/farmacología , Estrógenos de Catecol , Estrés Oxidativo , Extractos Vegetales/farmacologíaRESUMEN
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.
Asunto(s)
Inhibidores de Catecol O-Metiltransferasa/química , Inhibidores de Catecol O-Metiltransferasa/farmacología , Catecol O-Metiltransferasa/metabolismo , Estrógenos de Catecol/metabolismo , Masoprocol/metabolismo , Masoprocol/farmacología , Mutágenos/toxicidad , Sitios de Unión , Muerte Celular/efectos de los fármacos , Daño del ADN , Estrógenos de Catecol/química , Estrógenos de Catecol/farmacología , Humanos , Células MCF-7 , Masoprocol/química , Metilación , Simulación del Acoplamiento Molecular , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato/efectos de los fármacosRESUMEN
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.
Asunto(s)
Neoplasias de la Mama , Estrógenos de Catecol , Neoplasias de la Mama/metabolismo , Cromatografía Liquida , Química Clic , Citosol , Humanos , Espectrometría de Masas en Tándem , Flujo de TrabajoRESUMEN
CONTEXT: Enhanced levels of catecholestradiols, 2-hydroxyestradiol (2-OHE2) or 4-hydroxyestradiol (4-OHE2), are reported in endometriosis. During gestation, catecholestradiol activation of adrenergic receptors (AR) elevates estrogen receptor (ER)-independent proliferation of uterine arterial endothelial cells. OBJECTIVE: To investigate ß-AR-mediated catecholestradiol effects on human endometrial stromal cell (HESC) and epithelial cell survival in endometriosis. DESIGN: ß-AR immunostaining of eutopic and ectopic endometria (nâ =â 9). Assays for cell viability, 5-bromo-2'-deoxyuridine proliferation, apoptosis, quantitative PCR, and estrogenicity (alkaline phosphatase activity), as well as siRNA ß-AR silencing and immunoblot analyses of cultured HESCs or Ishikawa cells treated with control or 2-OHE2 or 4-OHE2 ±ß-AR antagonist or ±p38 MAPK inhibitor. SETTING: University research institution. PATIENTS: Women with or without endometriosis. INTERVENTIONS: None. MAIN OUTCOME MEASURES: ß-AR expression in eutopic vs ectopic endometria and regulation of HESC survival by 2-OHE2 and 4-OHE2. RESULTS: Eutopic and ectopic endometrial stromal and epithelial cells displayed ß2-AR immunoreactivity with increased staining in the functionalis vs basalis layer (Pâ <â 0.05). Both 2-OHE2 and 4-OHE2 enhanced HESC and Ishikawa cell survival (Pâ <â 0.05), an effect abrogated by ß-AR antagonist propranolol, but not ER antagonist ICI182,780. 2-OHE2 or 4-OHE2 failed to induce cell survival and estrogenic activity in ADRB2-silenced HESCs and in Ishikawa cells, respectively. Although 2-OHE2 inhibited apoptosis and BAX mRNA expression, 4-OHE2 induced proliferation and decreased apoptosis (Pâ <â 0.05). Both catecholestradiols elevated phospho-p38 MAPK levels (Pâ <â 0.05), which was blocked by propranolol, and p38 MAPK inhibitor reversed catecholestradiol-enhanced HESC survival. CONCLUSIONS: Catecholestradiols increase endometrial cell survival by an ER-independent ß-AR-mediated p38 MAPK activation, suggesting that agents blocking ß-AR (e.g., propranolol) or inhibiting 2-OHE2- or 4-OHE2-generating enzymes (i.e., CYP1A1/B1) could treat endometriosis.
Asunto(s)
Endometriosis/tratamiento farmacológico , Endometrio/efectos de los fármacos , Estrógenos de Catecol/farmacología , Receptores Adrenérgicos/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Adulto , Estudios de Casos y Controles , Proliferación Celular , Supervivencia Celular , Endometriosis/metabolismo , Endometriosis/patología , Endometrio/metabolismo , Femenino , Estudios de Seguimiento , Humanos , Persona de Mediana Edad , Pronóstico , Transducción de Señal , Células del Estroma/metabolismoRESUMEN
Embryo implantation in the uterus is a critical step to achieve success following ART. Despite favorable uterine conditions, a great number of good quality embryos fail to implant, often for reasons that are unknown. Hence, improving the implantation potential of embryos is a subject of great interest. 4-Hydroxyestradiol (4-OH-E2), a metabolic product of estradiol produced by endometrial cells, plays a key role in endometrial-embryonic interactions that are necessary for implantation. Nonetheless, the effects of 4-OH-E2 on embryos obtained in vitro have not been yet described. This study was designed to determine whether culture media enriched in 4-OH-E2 could improve the quality and implantation rate of embryos obtained in vitro, using both in vitro and in vivo models. We also analyzed its effects on the epidermal growth factor (EGF)-binding capability of the embryos. Our results showed that the presence of 4-OH-E2 in the culture media of embryos during the morula to blastocyst transition increases embryo quality and attachment to endometrial cells in vitro. 4-OH-E2 can also improve viable pregnancy rates of mouse embryos produced in vitro, reaching success rates that are similar to those from embryos obtained directly from the uterus. 4-OH-E2 improved the embryos' ability to bind EGF, which could be responsible for the increased embryo implantation potential observed. Therefore, our results strongly suggest that 4-OH-E2 is a strong candidate molecule to supplement human IVF culture media in order to improve embryo implantation. However, further research is required before these findings can be translated with efficacy and safety to fertility clinics.
Asunto(s)
Blastocisto/efectos de los fármacos , Implantación del Embrión/efectos de los fármacos , Transferencia de Embrión , Factor de Crecimiento Epidérmico/metabolismo , Estrógenos de Catecol/farmacología , Fertilización In Vitro , Animales , Apoptosis/efectos de los fármacos , Blastocisto/metabolismo , Blastocisto/patología , Técnicas de Cultivo de Embriones , Femenino , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Embarazo , Índice de EmbarazoRESUMEN
Depression has been commonly associated with type 1 diabetes (T1D) and insulin covalently modified with catecholestrogens (CEs) was found in serum of these T1D patients. This study aimed to know whether depression link to higher antibodies against estrogenized insulin in T1D. ELISA (direct binding and competition) and quantitative precipitin titration were used to detect antibodies and their affinities against estrogenized insulin in the serum of 66 depressed T1D (DT1D) patients (out of 110 T1D) and 41 control subjects. Antibodies from DT1D patients showed high binding specificity to estrogenized insulin (2-hydroestradiol-insulin; 2-OHE2-Ins) in comparison to overall T1D patients (p < 0.05) or control subjects (p < 0.001). However, T1D sera demonstrate high recognition to 2-OHE2-Ins as compared to Ins (p < 0.05) or 2-OHE2 (p < 0.001). The affinity of antibodies from DT1D and T1D patients was 1.32 × 10-7 M and 1.43 × 10-7 M, respectively. Depression linked to higher antibodies production against estrogenized insulin in T1D. Furthermore, depression in T1D generates inflammatory conditions that further increased antibodies production in T1D patients.
Asunto(s)
Autoanticuerpos/biosíntesis , Autoanticuerpos/inmunología , Depresión/inmunología , Diabetes Mellitus Tipo 1/inmunología , Estrógenos de Catecol/inmunología , Animales , Autoanticuerpos/química , Autoanticuerpos/aislamiento & purificación , Depresión/sangre , Depresión/complicaciones , Diabetes Mellitus Tipo 1/sangre , Diabetes Mellitus Tipo 1/complicaciones , Ensayo de Inmunoadsorción Enzimática , Estrógenos de Catecol/sangre , Estrógenos de Catecol/química , Femenino , Humanos , Factores Inmunológicos/sangre , Resistencia a la Insulina/inmunología , Masculino , Persona de Mediana EdadRESUMEN
AIMS: Insulin (Ins) covalently modified by catecholestrogens (CEs) was commonly found in diabetic patients who have developed insulin resistance. Estrogenization of insulin altered its molecular function and effect carbohydrates metabolisms in these patients. Insulin resistance is a common phenomenon in diabetes but the exact mechanism remains unknown. In this study, binding specificity and affinity of autoantibodies against estrogenized insulin (4-hydroxyestradiol-insulin; 4-OHE2-Ins) were assayed in the serum of type 1 diabetes (T1D) patients in order to explain the phenomena behind insulin resistance. MATERIALS AND METHODS: Specificity and affinity of autoantibodies from the sera of 66 T1D patients and 41 controls were analyzed by direct binding, competition ELISA and quantitative precipitin titration. Insulin was also estimated in the serum of T1D patients by ELISA. KEY FINDING: Estrogenized insulin (4-OHE2-Ins) exhibited high affinity and specificity to T1D autoantibodies in comparison to Ins (p < .05) or 4-OHE2 (p < .001). Estrogenization of insulin alters its interaction with the insulin receptor (IR). The affinity constant of 4-OHE2-Ins with the T1D autoantibodies was found to be 1.41 × 10-7 M. SIGNIFICANCE: Estrogenization of insulin by catecholestrogen makes these molecules highly antigenic and produced high-affinity autoantibodies in T1D patients. As a result, patients develop insulin resistance and presented this molecule as a potential biomarker for T1D.
Asunto(s)
Autoanticuerpos/inmunología , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Estrógenos de Catecol/química , Hipoglucemiantes/química , Insulina/química , Adulto , Autoanticuerpos/metabolismo , Biomarcadores/metabolismo , Glucemia/análisis , Recolección de Muestras de Sangre , Propuestas de Licitación , Ensayo de Inmunoadsorción Enzimática , Estrógenos de Catecol/uso terapéutico , Femenino , Humanos , Hipoglucemiantes/uso terapéutico , Insulina/uso terapéutico , Resistencia a la Insulina , Masculino , Persona de Mediana Edad , Receptor de Insulina/inmunología , Receptor de Insulina/metabolismo , Sensibilidad y EspecificidadRESUMEN
Abundant blood proteins adducted by active electrophiles are excellent markers to predict the risk of electrophile-induced toxicity. However, detecting endogenously adducted proteins by bottom-up selective (or parallel) reaction monitoring (SRM/PRM) is challenging because of the high variability in sample preparation and detection as well as low adduction levels. Here, we reported a new approach in developing PRM methods by combining intact protein measurement with standard additions to target optimal conditions for detecting catechol estrogens (CEs)-adducted human serum albumin (HSA). Blood serum was added with multiple amounts of CEs to obtain serum standards. Intact protein measurement revealed two linear ranges of adduction levels (adducted-CE/HSA): 0.34-0.42 (R2 > 0.94) and 0.81-8.54 (R2 > 0.96) against the amount of added CEs, respectively. Six adduction sites were identified by trypsin (K20, C34, K73, K281, H338, K378) or chymotrypsin (K20, C34, K378) digestion. PRM methods targeting all adducted/nonadducted peptide pairs based on chymotrypsin or trypsin digestion were developed, and the data were compared with those obtained by intact protein measurement. Correlation plots indicated that chymotrypsin-PRM leads to poor sensitivity and largely underestimated protein adduction levels. Trypsin-PRM leads to sensitive and highly correlated (R2 > 0.91) protein adduction levels with a detection limit below the endogenous level and relative standard deviation <25%. As a proof of concept, clinical serum samples were examined by trypsin-PRM, and a slightly higher adduction level was observed for the obesity group when compared with the healthy group. This is the first report on determining adduction levels of blood proteins for long-term exposure to CEs. The standard addition approach can be generally applied to protein adductomics with resolvable mass increments by intact protein measurement to accelerate the development of bottom-up methods close to the inherent limit.
Asunto(s)
Estrógenos de Catecol/química , Espectrometría de Masas/métodos , Péptidos/análisis , Albúmina Sérica/química , Cromatografía Líquida de Alta Presión , Quimotripsina/metabolismo , Estrógenos de Catecol/metabolismo , Humanos , Espectrometría de Masas/normas , Nanotecnología , Péptidos/metabolismo , Péptidos/normas , Estándares de Referencia , Albúmina Sérica/metabolismo , Tripsina/metabolismoRESUMEN
Oncogenesis can be caused by an increase in the activity of genes responsible for initiating tumor growth in stem or progenitor cells, as well as a reduction in the functioning of suppressor genes. Endogenous estrogen exposure is associated with an increased risk of breast cancer in both pre- and postmenopausal women. The most important step in the understanding of the pathogenesis of breast cancer was the development of the theory of the switching of estrogen's effect from hormonal to genotoxic, in which the main culprits of carcinogenesis are not chemical metabolites of estrogens, but their derivatives, corresponding to chemical procarcinogens according to their damaging characteristics. The origin of these substances and the formation of estrogen genotoxicity lies in the disruption of the inactivation process of catechol estrogens in methylation reactions. The main epigenetic modification of the human genome is the methylation of cell DNA molecules. DNA methylation does not alter the primary sequence of nucleotides, but is necessary for the functional suppression of certain genes. The phenomenon of hypomethylation-hypermethylation underlies the long-term silencing of various genes, including tumor suppressor genes. Nutrition and a lifestyle associated with smoking and the consumption of excessive quantities of alcohol determine estrogen metabolism and the availability of methyl groups in the body, as well as epigenetic changes in the DNA of the genome. The assessment of individual risk of breast cancer on the basis of an assay for the expression and methylation of the COMT gene responsible for estrogen metabolism seems relevant.
Asunto(s)
Neoplasias de la Mama , Metilación de ADN , Estrógenos/metabolismo , Neoplasias de la Mama/genética , Catecol O-Metiltransferasa/genética , Estrógenos de Catecol , Femenino , HumanosRESUMEN
Life-long estrogen exposure is one of the major risk factors in the development and progression of breast cancer. However, little is known about the molecular mechanisms, by which chronic exposure to estrogen contributes to breast carcinogenesis. The aim of the present study was to investigate the effects of long-term exposure with 4-hydroxyestradiol (4-OHE2) on acquired cancer characteristics of human mammary epithelial MCF-10A cells. The possible regulators were further studied in chronic 4-OHE2-treated MCF-10A cells. We observed that MCF-10A cells long-term exposed to 4-OHE2 acquire the characteristics of cancer cells, such as enhanced cell growth, EMT properties, and increased migration and invasiveness. Moreover, the expression of CYP1B1 was significantly elevated in long-term 4-OHE2-treated MCF-10A cells. Block of CYP1B1 significantly reduced the cancer cell characteristics in long-term 4-OHE2-treated MCF-10A cells. Our results indicated that 4-OHE2 mediated enhanced cancer cell characteristics in mammary epithelial cells are an important key event for breast carcinogenesis process. CYP1B1 partially contributes to the 4-OHE2 induced cancer cell characteristics in MCF-10A cells. Targeting CYP1B1 might offer a new strategy for the treatment of estrogen-induced breast cancer.
Asunto(s)
Carcinogénesis/inducido químicamente , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Citocromo P-450 CYP1B1/biosíntesis , Transición Epitelial-Mesenquimal/efectos de los fármacos , Estrógenos de Catecol/toxicidad , Carcinogénesis/metabolismo , Carcinogénesis/patología , Técnicas de Cultivo de Célula , Línea Celular , Relación Dosis-Respuesta a Droga , Células Epiteliales/efectos de los fármacos , Células Epiteliales/enzimología , Células Epiteliales/patología , Femenino , Humanos , Glándulas Mamarias Humanas/citología , Glándulas Mamarias Humanas/enzimología , Regulación hacia ArribaRESUMEN
The cytochrome P450 (P450) 1 family is an important phase I enzyme involved in carcinogen activation. Nitidine chloride (NC) is a pharmacologically active alkaloid with polyaromatic hydrocarbon found in the roots of Zanthoxylum nitidum (Roxb.) DC, a traditional medicinal herb widely used in China. We examined the inhibitory effects of NC on CYP1A1, 1B1, and 1A2. NC significantly inhibited CYP1A1- and 1B1-catalyzed ethoxyresorufin O-deethylation activity (IC50 = 0.28 ± 0.06 and 0.32 ± 0.02 µM, respectively) in a concentration-dependent manner, but only showed slight inhibition of CYP1A2 activity (IC50 > 50 µM). Kinetic analysis revealed that NC competitively inhibited CYP1B1 with a K i value of 0.47 ± 0.05 µM, whereas NC caused a mixed type of inhibition on CYP1A1 with K i and K I values of 0.14 ± 0.04 and 0.19 ± 0.09 µM, respectively. The observed enzyme inhibition neither required NADPH nor revealed time dependency. Molecular docking manifested the generation of strong hydrogen-bonding interactions of Ser116 in CYP1A1 and Ser127 in CYP1B1 with methoxy moiety of NC. Additionally, NC-induced alteration of estradiol (E2) metabolism was also investigated in the present study. Hydroxyestradiols, including 2-hydroxyestradiol [(2-OHE2) nontoxic] and 4-hydroxyestradiol [(4-OHE2) genotoxic] generated in recombinant enzyme incubation systems and cultured MCF-7 cells were analyzed, and NC was found to preferentially inhibit the nontoxic 2-hydroxylation activity of E2 mediated by CYP1A1. In conclusion, NC was a mixed type inhibitor of CYP1A1 and a competitive inhibitor of CYP1B1. The remarkable inhibition on E2 2-hydroxylation might increase the risk of 4-OHE2-induced genotoxicity. SIGNIFICANCE STATEMENT: CYP1 enzymes catalyze oxidative metabolism of a variety of compounds and are known to play a crucial role in the development of cancer. CYP1A1 and CYP1A2 are responsible for hydroxylation of estradiol (E2) at the C-2 position, resulting in the formation of 2-OHE2, which is proposed to be a detoxification pathway. However, CYP1B1-mediated hydroxylation of E2 at the C-4 position has been suggested to be a tumor initiator. The present study found that nitidine chloride is a mixed type inhibitor of CYP1A1 and a competitive inhibitor of CYP1B1. NC preferentially inhibited the nontoxic E2 2-hydroxylation pathway mediated by CYP1A1, which might increase the risk of 4-OHE2-induced genotoxicity and cause severe drug-drug interactions.
Asunto(s)
Benzofenantridinas/farmacología , Citocromo P-450 CYP1A1/antagonistas & inhibidores , Citocromo P-450 CYP1B1/antagonistas & inhibidores , Estradiol/metabolismo , Redes y Vías Metabólicas/efectos de los fármacos , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP1B1/metabolismo , Interacciones Farmacológicas , Medicamentos Herbarios Chinos , Pruebas de Enzimas , Estrógenos de Catecol/metabolismo , Estrógenos de Catecol/toxicidad , Humanos , Concentración 50 Inhibidora , Células MCF-7 , Simulación del Acoplamiento Molecular , Pruebas de Mutagenicidad , Raíces de Plantas/química , Unión Proteica , Proteínas Recombinantes/metabolismo , Serina/metabolismo , Zanthoxylum/químicaRESUMEN
Androgens are thought to cause prostate cancer, but the underlying mechanisms are unclear. Data from animal studies suggest that for androgens to cause prostate cancer, they must be aromatized to estrogen and act in concert with estrogen metabolites. We tested the hypothesis that androgen-receptor and estrogen receptor-mediated effects of androgen and estrogen are necessary, as well as genotoxicity of estrogen metabolites. NBL rats were treated with androgenic and estrogenic compounds for 16-75 weeks through slow-release silastic implants or pellets. Testosterone alone induced cancer in the prostate of 37% of rats. 5α-Dihydrotestosterone, which cannot be converted to estradiol or testosterone, did not cause a significant prostate cancer incidence (4%). Addition of estradiol to 5α-dihydrotestosterone treatment did not markedly enhance prostate cancer incidence (14%), unlike adding estradiol to testosterone treatment which induced a 100% tumor incidence. Testosterone plus estradiol treatment induced a DNA adduct detectable by 32P-postlabeling, oxidative DNA damage (8-hydroxyguanosine), and lipid peroxidation at the site within the prostate where this treatment causes cancers, preceding later cancer formation. The non-estrogenic 4-hydroxy metabolite of estradiol, when combined with testosterone, induced prostatic dysplasia within 16 weeks and, after long-term treatment, a very low incidence of prostate cancer (21%). When an estrogen that cannot be hydroxylated (2-fluoroestradiol) was added to this combined treatment with testosterone and 4-hydroxyestradiol, dysplasia frequency after 16 weeks was doubled. These results strongly support the hypothesis, but additional definitive studies are needed which may identify new targets to interfere with these mechanisms that are clinically feasible in humans.
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Andrógenos/efectos adversos , Carcinogénesis , Estrógenos/efectos adversos , Neoplasias de la Próstata/inducido químicamente , Animales , Carcinoma , Aductos de ADN , Daño del ADN , Dihidrotestosterona/metabolismo , Estradiol/metabolismo , Estrógenos de Catecol/química , Guanosina/análogos & derivados , Guanosina/farmacología , Humanos , Incidencia , Masculino , Próstata , Ratas , Receptores de Estrógenos/metabolismo , Testosterona/metabolismoRESUMEN
Human cytochrome P450 1B1 (CYP1B1)-mediated formation of 4-hydroxyestradiol (4-OHE2) from 17ß-estradiol plays an important role in the progression of human breast cancer, while the biotransformation of 17ß-estradiol to 2-hydroxyestradiol mediated by cytochrome P450 1A1 (CYP1A1) is considered as a less harmful pathway. In this study, inhibitory effects of flavonoids baicalein and oroxylin A, a metabolite of baicalein in human body, on CYP1A1 and 1B1 activities were investigated in vitro. The inhibition intensities of baicalein and oroxylin A towards CYP1B1 were greater than towards CYP1A1 with a mixed mechanism. In addition, oroxylin A showed a stronger inhibitory effect than baicalein towards the CYP1B1-mediated 17ß-estradiol 4-hydroxylation, with the IC50 values of 0.0146 and 2.27 µM, respectively. Docking studies elucidated that oroxylin A had a stronger binding affinity than baicalein for CYP1B1. In MCF-7 cells, compared with baicalein-treated groups, oroxylin A with lower doses decreased and increased the formation of 4-OHE2 and 2-hydroxyestradiol, respectively, with a preferential induction of mRNA of CYP1A1 over CYP1B1. In conclusion, this study demonstrated that oroxylin A showed a stronger inhibitory effect than baicalein on CYP1B1-mediated 4-OHE2 formation in MCF-7 cells, providing crucial implications for their possibly preventive/therapeutic potential against breast cancer via inhibition of CYP1B1, particularly of oroxylin A.
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Carcinogénesis/efectos de los fármacos , Carcinogénesis/genética , Citocromo P-450 CYP1B1/genética , Estradiol/análogos & derivados , Estrógenos de Catecol/metabolismo , Estrógenos de Catecol/toxicidad , Flavanonas/farmacología , Flavonoides/farmacología , Neoplasias de la Mama/inducido químicamente , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Carcinogénesis/inducido químicamente , Carcinógenos/metabolismo , Carcinógenos/toxicidad , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Estradiol/metabolismo , Femenino , Flavanonas/metabolismo , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Células MCF-7RESUMEN
Estrogen hormones play an important role in controlling glucose homeostasis and pancreatic ß-cell function. Despite the significance of estrogen hormones for regulation of glucose metabolism, little is known about the roles of endogenous estrogen metabolites in modulating pancreatic ß-cell function. In this study, we evaluated the effects of major natural estrogen metabolites, catechol estrogens, on insulin secretion in pancreatic ß-cells. We show that catechol estrogens, hydroxylated at positions C2 and C4 of the steroid A ring, rapidly potentiated glucose-induced insulin secretion via a nongenomic mechanism. 2-Hydroxyestrone, the most abundant endogenous estrogen metabolite, was more efficacious in stimulating insulin secretion than any other tested catechol estrogens. In insulin-secreting cells, catechol estrogens produced rapid activation of calcium influx and elevation in cytosolic free calcium. Catechol estrogens also generated sustained elevations in cytosolic free calcium and evoked inward ion current in HEK293 cells expressing the transient receptor potential A1 (TRPA1) cation channel. Calcium influx and insulin secretion stimulated by estrogen metabolites were dependent on the TRPA1 activity and inhibited with the channel-specific pharmacological antagonists or the siRNA. Our results suggest the role of estrogen metabolism in a direct regulation of TRPA1 activity with potential implications for metabolic diseases.
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Estrógenos de Catecol/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Secreción de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Canal Catiónico TRPA1/metabolismo , Animales , Células Cultivadas , Glucosa/metabolismo , Humanos , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , RatasRESUMEN
Catechol estrogens (CEs) are metabolic electrophiles that actively undergo covalent interaction with cellular proteins, influencing molecular function. There is no feasible method to identify their binders in a living system. Herein, we developed a click chemistry-based approach using ethinylestradiol (EE2) as the precursor probe coupled with quantitative proteomics to identify protein targets of CEs and classify their binding strengths. Using in situ metabolic conversion and click reaction in liver microsomes, CEs-protein complex was captured by the probe, digested by trypsin, stable isotope labeled via reductive amination, and analyzed by liquid chromatography-mass spectrometry (LC-MS). A total of 334 liver proteins were repeatedly identified ( n ≥ 2); 274 identified proteins were classified as strong binders based on precursor mass mapping. The binding strength was further scaled by D/H ratio (activity probe/solvent): 259 strong binders had D/H > 5.25; 46 weak binders had 5.25 > D/H > 1; 5 nonspecific binders (keratins) had D/H < 1. These results were confirmed using spiked covalent control (strong binder) and noncovalent control (weak binder), as well as in vitro testing of cytochrome c (D/H = 5.9), which showed covalent conjugation with CEs. Many identified strong binders, such as glutathione transferase, catechol-O-methyl transferase, superoxide dismutase, catalase, glutathione peroxidase, and cytochrome c, are involved in cellular redox processes or detoxification activities. CE conjugation was shown to suppress the superoxide oxidase activity of cytochrome c, suggesting that CEs modification may alter the redox action of cellular proteins. Due to structural similarity and inert alkyne group, EE2 probe is very likely to capture protein targets of CEs in general. Thus, this strategy can be adopted to explore the biological impact of CEs modification in living systems.
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Estrógenos de Catecol/antagonistas & inhibidores , Proteínas/farmacología , Proteómica/métodos , Animales , Cromatografía Liquida , Química Clic/métodos , Etinilestradiol/química , Espectrometría de Masas , Microsomas Hepáticos/química , Sondas Moleculares , Unión Proteica , RatasRESUMEN
BACKGROUND: The development of hormone-dependent cancers, including endometrial carcinomas, in great part may be mediated by the genotoxic effects of estrogen metabolites, among which 4-hydroxyestradiol (4OHE2) is characterized by the most prominent DNA-damaging properties. It is assumed that the individual sensitivity to the 4OHE2 may determine the predisposition to endometrial cancer (EС). AIM: To analyze the sensitivity of peripheral blood lymphocytes (PBLs) of EC patients to the 4OHE2 and to evaluate the repair efficiency of 4OHE2-induced DNA damage. MATERIALS AND METHODS: The study was performed on the PBLs of 53 EC patients and 20 healthy women. The level of DNA damage was measured using the comet assay and was expressed as % tail DNA. The DNA repair efficiency (%) was evaluated by determining the ratio between the amount of repaired DNA damage and the level of 4OHE2-induced damage that appeared after incubation of PBLs with 4OHE2. RESULTS: In PBLs of EC patients, a higher level of 4OHE2-induced DNA damage (32.0 ± 2.2% tail DNA) and lower DNA repair efficiency (34.0 ± 4.5%) was observed compared to PBLs of healthy women (22.3 ± 2.3% tail DNA and 48.8 ± 4.5%, respectively). PBLs of EC patients with deep tumor invasion of myometrium were characterized by more prominent decrease of DNA repair than those with less invasive tumor (< ½ of myometrium) (20.9 ± 7.8 and 43.7 ± 6.7%, respectively). Furthermore, lower DNA repair efficiency was detected in the PBLs of EC patients with a family history of cancer compared to this parameter in patients with sporadic tumors (20.9±7.8 and 47.1 ± 5.5%, respectively). CONCLUSION: The PBLs of EC patients are characterized by increased sensitivity to the genotoxic effect of 4OHE2 and reduced repair efficiency regarding 4OHE2-induced DNA damage. A lower level of DNA repair is observed in EC patients with deep tumor myometrial invasion and a family history of cancer.
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Carcinoma Endometrioide/patología , Reparación del ADN/efectos de los fármacos , Neoplasias Endometriales/patología , Estrógenos de Catecol/toxicidad , Linfocitos/efectos de los fármacos , Adulto , Anciano , Daño del ADN/efectos de los fármacos , Femenino , Humanos , Persona de Mediana Edad , Mutágenos/toxicidadRESUMEN
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.