RESUMEN
Although several previous studies have demonstrated the presence of equine estrogens in the aquatic environment, limited data are currently available on the endocrine-disrupting potentials in fish and the risks they pose to aquatic organisms. To investigate the interactions of major equine estrogens equilin (Eq) and equilenin (Eqn), as well as their metabolites 17α-dihydroequilin, 17ß-dihydroequilin, 17α-dihydroequilenin and 17ß-dihydroequilenin, with the estrogen receptor α (ERα) of medaka (Oryzias latipes), a three-dimensional model of the ligand-binding domain (LBD) of ERα was built in silico, and docking simulations were performed. The docking simulation analysis indicated that the interaction of 17ß-dihydroequilenin with the ERα LBD is the most potent, followed by those of 17α-dihydroequilin and 17ß-dihydroequilin, whereas those of Eq and Eqn were least potent. We further analyzed gene expression profiles in the livers of male medaka exposed to Eq and Eqn. A DNA microarray representing 6000 genes revealed that 24-h exposure to Eq and Eqn (100 ng/L) upregulated the expression of 6 and 34 genes in the livers of males, respectively. Genes upregulated by Eq included the estrogenic biomarker genes vitellogenins and choriogenins, suggesting the estrogenic potential of Eq. In contrast, Eqn exposure upregulated several cancer-related genes, such as mediator complex subunit 16 and RAS oncogene family members, suggesting a carcinogenic potential for Eqn. These results suggest that equine estrogens may have not only endocrine-disrupting potentials via the ERα signaling pathway but also carcinogenic potency in male medaka.
Asunto(s)
Disruptores Endocrinos/toxicidad , Equilenina/toxicidad , Equilina/toxicidad , Hígado/efectos de los fármacos , Oryzias/metabolismo , Contaminantes Químicos del Agua/toxicidad , Animales , Disruptores Endocrinos/metabolismo , Equilenina/metabolismo , Equilina/metabolismo , Receptor alfa de Estrógeno/metabolismo , Receptor beta de Estrógeno/metabolismo , Ligandos , Hígado/metabolismo , Masculino , Simulación del Acoplamiento Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Unión Proteica , Transcriptoma/efectos de los fármacos , Contaminantes Químicos del Agua/metabolismoRESUMEN
Polycyclic aromatic hydrocarbons (PAHs) are suspect human lung carcinogens and can be metabolically activated to remote quinones, for example, benzo[a]pyrene-1,6-dione (B[a]P-1,6-dione) and B[a]P-3,6-dione by the action of either P450 monooxygenase or peroxidases, and to non-K region o-quinones, for example B[a]P-7,8-dione, by the action of aldo keto reductases (AKRs). B[a]P-7,8-dione also structurally resembles 4-hydroxyequilenin o-quinone. These three classes of quinones can redox cycle, generate reactive oxygen species (ROS), and produce the mutagenic lesion 8-oxo-dGuo and may contribute to PAH- and estrogen-induced carcinogenesis. We compared the ability of a complete panel of human recombinant AKRs to catalyze the reduction of PAH o-quinones in the phenanthrene, chrysene, pyrene, and anthracene series. The specific activities for NADPH-dependent quinone reduction were often 100-1000 times greater than the ability of the same AKR isoform to oxidize the cognate PAH-trans-dihydrodiol. However, the AKR with the highest quinone reductase activity for a particular PAH o-quinone was not always identical to the AKR isoform with the highest dihydrodiol dehydrogenase activity for the respective PAH-trans-dihydrodiol. Discrete AKRs also catalyzed the reduction of B[a]P-1,6-dione, B[a]P-3,6-dione, and 4-hydroxyequilenin o-quinone. Concurrent measurements of oxygen consumption, superoxide anion, and hydrogen peroxide formation established that ROS were produced as a result of the redox cycling. When compared with human recombinant NAD(P)H:quinone oxidoreductase (NQO1) and carbonyl reductases (CBR1 and CBR3), NQO1 was a superior catalyst of these reactions followed by AKRs and last CBR1 and CBR3. In A549 cells, two-electron reduction of PAH o-quinones causes intracellular ROS formation. ROS formation was unaffected by the addition of dicumarol, suggesting that NQO1 is not responsible for the two-electron reduction observed and does not offer protection against ROS formation from PAH o-quinones.
Asunto(s)
Oxidorreductasas de Alcohol/metabolismo , Equilenina/análogos & derivados , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , Hidrocarburos Policíclicos Aromáticos/metabolismo , Quinonas/metabolismo , Oxidorreductasas de Alcohol/genética , Aldehído Reductasa , Aldo-Ceto Reductasas , Benzopirenos/química , Benzopirenos/toxicidad , Biocatálisis , Línea Celular Tumoral , Equilenina/química , Equilenina/metabolismo , Equilenina/toxicidad , Humanos , Isomerismo , NAD(P)H Deshidrogenasa (Quinona)/genética , Oxidación-Reducción/efectos de los fármacos , Hidrocarburos Policíclicos Aromáticos/química , Hidrocarburos Policíclicos Aromáticos/toxicidad , Quinonas/química , Quinonas/toxicidad , Especies Reactivas de Oxígeno/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por SustratoAsunto(s)
Terapia de Reemplazo de Estrógeno , Estrógenos/análisis , Caballos , Aguas del Alcantarillado/efectos adversos , Aguas del Alcantarillado/química , Contaminantes Químicos del Agua/análisis , Animales , Aves/metabolismo , Carpas/metabolismo , Equilenina/análisis , Equilenina/metabolismo , Equilenina/toxicidad , Estrógenos/metabolismo , Estrógenos/toxicidad , Femenino , Humanos , Oncorhynchus mykiss/metabolismo , Reino Unido , Contaminantes Químicos del Agua/metabolismo , Contaminantes Químicos del Agua/toxicidadRESUMEN
Excessive exposure to synthetic and endogenous estrogens has been associated with the development of cancer in several tissues including the breast. 4-Hydroxyequilenin (4-OHEN), a major catechol metabolite of equine estrogens present in Premarin, an estrogen replacement formulation, has been shown to induce apoptosis and DNA damage in human breast cancer cells. It also has the potential to be a tumor initiator or promoter and complete carcinogen. To further understand the effects and mechanisms of equine catechol estrogen metabolite 4-OHEN action in vitro, human non-tumorigenic mammary epithelial MCF 10A cell line was used to study the toxic effects of 4-OHEN. In this study, we observed that 4-OHEN caused dose-dependent increases in apoptosis and DNA damage as measured by the DAPI nuclear screening assay and the Comet assay, respectively. Interestingly, cells treated with 100 nM 4-OHEN biweekly for 4 weeks became resistant to cisplatin-induced apoptosis. The resistance to apoptosis of the 100 nM 4-OHEN-treated cells was through multiple regulatory mechanisms. Compared to the DMSO-treated cells, the 100 nM 4-OHEN-treated cells had higher GSH levels and total SOD activity, and a stronger GSH response after cisplatin treatment. Expression levels of several genes involved in cell growth, DNA repair, and apoptosis were either up- or down-regulated. These data indicate that long-term low-level equine estrogen metabolite exposure could induce DNA damage and initiate cells to become resistant to apoptosis.
Asunto(s)
Apoptosis/efectos de los fármacos , Equilenina/análogos & derivados , Equilenina/toxicidad , Línea Celular , Cisplatino/toxicidad , Daño del ADN , ADN de Cadena Simple/efectos de los fármacos , Dimetilsulfóxido/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Glutatión/análisis , HumanosRESUMEN
Catechol-O-methyltransferase (COMT) plays an important role in the inactivation of biologically active and toxic catechols. It has been shown that COMT is genetically polymorphic with a wild-type and variant form where a valine has been substituted with a methionine. Several, but not all, epidemiological studies have shown that women, homozygous with the variant form, have an increased risk of developing breast cancer. Previously, we showed that 4-hydroxyequilenin (4-OHEN), a cytotoxic/genotoxic equine catechol estrogen metabolite, is both a substrate of COMT and an irreversible inhibitor of the methylation activity of COMT in vitro. To further understand the mechanism(s) of the association between the breast cancer risk and the COMT polymorphism, it was of interest to study the effect of the Val/Met polymorphism on COMT-catalyzed catechol estrogen methylation and 4-OHEN-mediated inhibition. In the present study, Michaelis-Menten analysis showed no difference between the relative ability of each form to methylate 4-OHEN. However, we found that the COMT variant form was more susceptible to 4-OHEN-mediated irreversible inactivation. Electrospray ionization mass spectrometry and SDS-gel analysis of COMT modified by 4-OHEN revealed that inhibition mechanisms include alkylation and/or oxidation of certain amino acids. In addition, site-directed mutagenesis experiments showed that Cys33 played a more important role in the variant form of COMT demonstrated by the fact that the C33A mutant of the variant form of COMT decreased its catalytic capability more dramatically as compared with that of wild type. Furthermore, thermotropic studies indicated that the variant form was more thermolabile, which suggested that the valine to methionine substitution may have changed the secondary/tertiary structure of the variant form of COMT, making it more susceptible to 4-OHEN and heat inactivation. These data suggest that 4-OHEN-mediated inhibition of the variant form of COMT in vivo might affect the detoxification efficiency of endogenous and/or exogenous catechol estrogens and play a role in the association between breast cancer risk and COMT polymorphism.
Asunto(s)
Neoplasias de la Mama/etiología , Neoplasias de la Mama/genética , Catecol O-Metiltransferasa/genética , Catecol O-Metiltransferasa/farmacología , Equilenina/análogos & derivados , Equilenina/farmacología , Equilenina/toxicidad , Inhibidores de Catecol O-Metiltransferasa , Congéneres del Estradiol , Femenino , Humanos , Metilación , Factores de Riesgo , TemperaturaRESUMEN
Estrogen replacement therapy has been correlated with an increased risk for developing breast and endometrial cancers. One potential mechanism of estrogen carcinogenesis involves metabolism of estrogens to 2- and 4-hydroxylated catechols, which are further oxidized to electrophilic/redox active o-quinones that have the potential to both initiate and promote the carcinogenic process. Previously, we showed that the equine estrogens, equilin and equilenin, which are major components of the estrogen replacement formulation Premarin (Wyeth-Ayerst), are primarily metabolized to the catechol, 4-hydroxyequilenin. This catechol was found to autoxidize to an o-quinone causing oxidation and alkylation of DNA in vitro and in vivo. To block catechol formation from equilenin, 4-halogenated equilenin derivatives were synthesized. These derivatives were tested for their ability to bind to the estrogen receptor, induce estrogen sensitive genes, and their potential to form catechol metabolites. We found that the 4-fluoro derivatives were more estrogenic than the 4-chloro and 4-bromo derivatives as demonstrated by a higher binding affinity for estrogen receptors alpha and beta, an enhanced induction of alkaline phosphatase activity in Ishikawa cells, pS2 expression in S30 cells, and PR expression in Ishikawa cells. Incubation of these compounds with tyrosinase in the presence of GSH showed that the halogenated equilenin compounds formed less catechol GSH conjugates than the parent compounds, equilenin and 17beta-hydroxyequilenin. In addition, these halogenated compounds showed less cytotoxicity in the presence of tyrosinase than the parent compounds in S30 cells. Also, as stated above, the 4-fluoro derivatives showed similar estrogenic effects as compared with parent compounds; however, they were less toxic in S30 cells as compared to equilenin and 17beta-equilenin. Because 17beta-hydroxy-4-halogenated equilenin derivatives showed higher estrogenic effects than the halogenated equilenin derivatives in vitro, we studied the relative ability of the 17beta-hydroxy-4-halogenated equilenin derivatives to induce estrogenic effects in the ovariectomized rat model. The 4-fluoro derivative showed higher activity than 4-chloro and 4-bromo derivatives as demonstrated by inducing higher vaginal cellular differentiation, uterine growth, and mammary gland branching. However, 17beta-hydroxy-4-fluoroequilenin showed a lower estrogenic activity than 17beta-hydroxyequilenin and estradiol, which could be due to alternative pharmacokinetic properties for these compounds. These data suggest that the 4-fluoroequilenin derivatives have promise as alternatives to traditional estrogen replacement therapy due to their similar estrogenic properties with less overall toxicity.
Asunto(s)
Equilenina/metabolismo , Equilenina/toxicidad , Halógenos/metabolismo , Halógenos/toxicidad , Adenocarcinoma/enzimología , Fosfatasa Alcalina/biosíntesis , Fosfatasa Alcalina/genética , Animales , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Equilenina/análogos & derivados , Ciclo Estral/efectos de los fármacos , Femenino , Halógenos/química , Glándulas Mamarias Animales/efectos de los fármacos , Glándulas Mamarias Animales/patología , Tamaño de los Órganos/efectos de los fármacos , Ovariectomía , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de Estrógenos/metabolismo , Relación Estructura-Actividad , Transfección , Útero/efectos de los fármacos , Útero/patología , Vagina/efectos de los fármacos , Vagina/patologíaRESUMEN
Excessive exposure to synthetic and endogenous estrogens has been associated with the development of cancer in several tissues. 4-Hydroxyequilenin (4-OHEN), a major metabolite of equine estrogens present in estrogen replacement formulations, has been shown to induce cytotoxic/carcinogenic effects. In the present study, we have found that 4-OHEN caused DNA damage in breast cancer cells, and cells that contain estrogen receptor alpha (S30) are more sensitive to 4-OHEN-mediated DNA damage as compared to estrogen receptor negative cells (MDA-MB-231). For example, concentration-dependent increases in 8-oxo-deoxyguanosine (8-oxo-dG), as measured by LC-MS-MS or by the Fpg comet assay, were only detected in the S30 cells, and the amount of this lesion could be enhanced by agents, which catalyze redox cycling (NADH) or deplete GSH (diethyl maleate). The role of the estrogen receptor in modulating DNA damage was further established in incubations with the ER antagonist tamoxifen, where decreases in 8-oxo-deoxyguanosine were observed. Another equine estrogen metabolite, 4,17 beta-hydroxyequilenin (4,17 beta-OHEN), was found to have the same cytotoxicity and a similar ability to induce reactive oxygen species (ROS), and caused the same oxidative DNA damage in S30 cells as compared to 4-OHEN. However, 4,17 beta-OHEN induced twice as much single strand DNA breaks in S30 cells compared to 4-OHEN. Also 4,17 beta-OHEN was more estrogenic than 4-OHEN as demonstrated by a higher binding affinity for ER alpha and an enhanced induction in activity of estrogen-dependent alkaline phosphatase in Ishikawa cells. These data suggest that the mechanism of DNA damage induced by equine estrogen metabolites could involve oxidative stress and that the estrogen receptor may play a role in this process.
Asunto(s)
Daño del ADN/efectos de los fármacos , Equilenina/análogos & derivados , Equilenina/toxicidad , Congéneres del Estradiol/toxicidad , Receptores de Estrógenos/metabolismo , Unión Competitiva , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Supervivencia Celular/efectos de los fármacos , Ensayo Cometa , ADN de Neoplasias/análisis , Relación Dosis-Respuesta a Droga , Receptor alfa de Estrógeno , Femenino , Humanos , Especies Reactivas de Oxígeno/metabolismo , Receptores de Estrógenos/antagonistas & inhibidores , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Tamoxifeno/farmacología , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/metabolismoRESUMEN
Recently, we investigated the inhibitory effects of 17 beta-estradiol and diethylstilbestrol on microtubule assembly, cytotoxicity, and aneuploidy in V79 cells. The present study analyzes the effects of equilin and equilenin (amongst the natural estrogens originally isolated from the urine of pregnant horses) and their related compounds, on the relative plating efficiency of Chinese hamster V79 cells. The results showed that a hydroxyl group on 17-C and a methoxyl group on 3-C of the estrogen skeleton were important for cytotoxicity. Of the various compounds analyzed, 2-methoxyestradiol had the strongest cytotoxicity, suggesting also the importance of a methoxyl group on 2-C.
Asunto(s)
Equilenina/toxicidad , Equilina/toxicidad , Estradiol/análogos & derivados , Estradiol/toxicidad , 2-Metoxiestradiol , Animales , División Celular/efectos de los fármacos , Línea Celular , Cricetinae , Relación Dosis-Respuesta a Droga , Equilenina/química , Equilina/química , Estradiol/química , Estrona/toxicidad , Relación Estructura-Actividad , Pruebas de ToxicidadRESUMEN
Epidemiological data strongly suggest that a woman's risk of developing breast cancer is directly related to her lifetime estrogen exposure. Estrogen replacement therapy in particular has been correlated with an increased cancer risk. Previously we showed that the equine estrogens equilin and equilenin, which are major components of the estrogen replacement formulation Premarin (Wyeth-Ayerst), are metabolized to the catechol, 4-hydroxyequilenin which autoxidizes to an o-quinone causing oxidation and alkylation of DNA in vitro [Bolton, J. L., Pisha, E., Zhang, F., and Qiu, S. (1998) Chem. Res. Toxicol. 11, 1113-1227]. In the present study, we injected 4-hydroxyequilenin into the mammary fat pads of Sprague-Dawley rats. Analysis of cells isolated from the mammary tissue for DNA single-strand breaks and oxidized bases using the comet assay showed a dose-dependent increase in both types of lesions. In addition, LC-MS-MS analysis of extracted mammary tissue showed the formation of an alkylated depurinating guanine adduct. Finally, extraction of mammary tissue DNA, hydrolysis to deoxynucleosides, and analysis by LC-MS-MS showed the formation of stable cyclic deoxyguanosine and deoxyadenosine adducts as well as oxidized bases. This is the first report showing that 4-hydroxyequilenin is capable of causing DNA damage in vivo. In addition, the data showed that 4-hydroxyequilenin induced four different types of DNA damage that must be repaired by different mechanisms. This is in contrast to the endogenous estrogen 4-hydroxyestrone where only depurinating guanine adducts have been detected in vivo. These results suggest that 4-hydroxyequilenin has the potential to be a potent carcinogen through the formation of variety of DNA lesions in vivo.
Asunto(s)
Mama/efectos de los fármacos , Aductos de ADN/efectos de los fármacos , Daño del ADN/efectos de los fármacos , ADN de Cadena Simple/efectos de los fármacos , Equilenina/análogos & derivados , Equilenina/toxicidad , Congéneres del Estradiol/toxicidad , Animales , Mama/química , Mama/metabolismo , Ensayo Cometa , Aductos de ADN/química , Aductos de ADN/metabolismo , Desoxiadenosinas/metabolismo , Desoxiguanosina/metabolismo , Relación Dosis-Respuesta a Droga , Equilenina/metabolismo , Congéneres del Estradiol/metabolismo , Femenino , Guanina/metabolismo , Oxidación-Reducción , Quinonas/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Estrogen replacement therapy has been correlated with an increased risk of developing hormone-dependent cancers. 4-Hydroxyequilenin (4-OHEN) is a catechol metabolite of equilenin and equilin which are components of the estrogen replacement formulation marketed under the name of Premarin (Wyeth-Ayerst). Previously, we showed that 4-OHEN autoxidizes to potent cytotoxic quinoids which can consume reducing equivalents and molecular oxygen, and cause a variety of DNA lesions, including formation of bulky stable adducts, apurinic sites, and oxidation of the phosphate-sugar backbone and purine/pyrimidine bases [Bolton, J. L., Pisha, E., Zhang, F., and Qiu, S. (1998) Chem. Res. Toxicol. 11, 1113-1127]. All of these deleterious effects could contribute to the cytotoxic/genotoxic effects of equine estrogens in vivo. In the study presented here, we studied the oxidative and carcinogenic potential of 4-OHEN and the catechol metabolite of the endogenous estrogen, 4-hydroxyestrone (4-OHE), in the JB6 clone 41 5a and C3H 10T(1/2) murine fibroblast cells. The relative ability of 4-OHEN and 4-OHE to induce oxidative stress was measured in these cells by oxidative cleavage of 2',7'-dichlorodiacylfluorosceindiacetate to dichlorofluoroscein. 4-OHEN (1 microM) displayed an increase in the level of reactive oxygen species comparable to that observed with 100 microM H(2)O(2). In contrast, 4-OHE demonstrated antioxidant capabilities in the 5-50 microM range. With both cell lines, we assessed single-strand DNA cleavage using the comet assay and the formation of oxidized DNA bases, such as 8-oxodeoxyguanosine, utilizing the Trevigen Fpg comet assay. 4-OHEN caused single-strand breaks and oxidized bases in a dose-dependent manner in both cell lines, whereas 4-OHE did not induce DNA damage. Since oxidative stress has been implicated in cellular transformation, we used the JB6 clone 41 5a anchorage independence assay to ascertain the relative ability of 4-OHEN and 4-OHE to act as tumor promoters. 4-OHEN caused a slight but significant increase in the extent of cellular transformation at the 100 nM dose; however, in the presence of NADH, which catalyzes redox cycling of 4-OHEN, the transformation ability of 4-OHEN was dramatically increased. 4-OHE did not induce transformation of the JB6 clone 41 5a in the 0.1-10 microM range. The initiation, promotion, and complete carcinogenic transformation potentials of both metabolites were measured in the C3H 10T(1/2) cells. 4-OHEN demonstrated activity in all stages of transformation at doses of 10 nM to 1 microM, whereas 4-OHE only demonstrated promotional capabilities at the 10 microM dose. These data suggest that oxidative stress could be partially responsible for the carcinogenic effects caused by 4-OHEN and that 4-OHEN is a more potent transforming agent than 4-OHE in vitro.
Asunto(s)
Transformación Celular Neoplásica/efectos de los fármacos , Equilenina/análogos & derivados , Equilenina/toxicidad , Congéneres del Estradiol/toxicidad , Animales , Carcinógenos/metabolismo , Carcinógenos/toxicidad , Línea Celular , Daño del ADN , Células Epidérmicas , Epidermis/efectos de los fármacos , Equilenina/metabolismo , Congéneres del Estradiol/metabolismo , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Caballos , Hidroxiestronas/metabolismo , Hidroxiestronas/toxicidad , Ratones , Ratones Endogámicos C3H , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Estrogen replacement therapy has been correlated with an increased risk of developing breast or endometrial cancer. 4-Hydroxyequilenin (4-OHEN) is a catechol metabolite of equilenin which is a minor component of the estrogen replacement formulation marketed under the name of Premarin (Wyeth-Ayerst). Previously, we showed that 4-OHEN autoxidizes to quinoids which can consume reducing equivalents and molecular oxygen, are potent cytotoxins, and cause a variety of damage to DNA, including formation of bulky stable adducts, apurinic sites, and oxidation of the phosphate-sugar backbone and purine/pyrimidine bases [Bolton, J. L., Pisha, E., Zhang, F., and Qiu, S. (1998) Chem. Res. Toxicol. 11, 1113-1127]. All of these deleterious effects could contribute to the cytotoxic and genotoxic effects of equilenin in vivo. In the study presented here, we examined the relative toxicity of 4-OHEN in estrogen receptor (ER) positive cells (MCF-7 and S30) compared to that in breast cancer cells without the estrogen receptor (MDA-MB-231). The data showed that 4-OHEN was 4-fold more toxic to MCF-7 cells (LC(50) = 6.0 +/- 0. 2 microM) and 6-fold more toxic to S30 cells (LC(50) = 4.0 +/- 0.1 microM) than to MDA-MB-231 cells (LC(50) = 24 +/- 0.3 microM). Using the single-cell gel electrophoresis assay (comet assay) to assess DNA damage, we found that 4-OHEN causes concentration-dependent DNA single-strand cleavage in all three cell lines, and this effect could be enhanced by agents which catalyze redox cycling (NADH) or deplete cellular GSH (diethyl maleate). In addition, the ER(+) cell lines (MCF-7 and S30) were considerably more sensitive to induction of DNA damage by 4-OHEN than the ER(-) cells (MDA-MB-231). 4-OHEN also caused a concentration-dependent increase in the amount of mutagenic lesion 8-oxo-dG in the S30 cells as determined by LC/MS-MS. Cell morphology assays showed that 4-OHEN induces apoptosis in these cell lines. As observed with the toxicity assay and the comet assay, the ER(+) cells were more sensitive to induction of apoptosis by 4-OHEN than MDA-MB-231 cells. Finally, the endogenous catechol estrogen metabolite 4-hydroxyestrone (4-OHE) was considerably less effective at inducing DNA damage and apoptosis in breast cancer cell lines than 4-OHEN. Our data suggest that the cytotoxic effects of 4-OHEN may be related to its ability to induce DNA damage and apoptosis in hormone sensitive cells in vivo, and these effects may be potentiated by the estrogen receptor.
Asunto(s)
Apoptosis/efectos de los fármacos , Neoplasias de la Mama/patología , Daño del ADN/efectos de los fármacos , ADN de Neoplasias/efectos de los fármacos , Equilenina/análogos & derivados , Congéneres del Estradiol/toxicidad , Neoplasias de la Mama/tratamiento farmacológico , Supervivencia Celular/efectos de los fármacos , Ensayo Cometa , Fragmentación del ADN , Relación Dosis-Respuesta a Droga , Células Epiteliales/efectos de los fármacos , Células Epiteliales/patología , Equilenina/metabolismo , Equilenina/toxicidad , Congéneres del Estradiol/metabolismo , Estrógenos de Catecol/toxicidad , Femenino , Humanos , Hidroxiestronas/toxicidad , Células Tumorales CultivadasRESUMEN
Premarin (Wyeth-Ayerst) is the estrogen replacement treatment of choice and continues to be one of the most widely dispensed prescriptions in North America. In addition to endogenous estrogens, Premarin contains unsaturated equine estrogens, including equilenin [1,3,5(10),6,8-estrapentaen-3-ol-17-one]. In previous work, we showed that the equilenin metabolite 4-hydroxyequilenin (4-OHEN) can be autoxidized to 4-OHEN-o-quinone which readily entered into a redox couple with the semiquinone radical catalyzed by NAD(P)H, P450 reductase, or quinone reductase, resulting in generation of reactive oxygen species [Shen, L., Pisha, E., Huang, Z., Pezzuto, J. M., Krol, E., Alam, Z., van Breemen, R. B., and Bolton, J. L. (1997) Carcinogenesis 18, 1093-1101]. As oxidative damage to DNA by reactive oxygen species generated by redox active compounds has been proposed to lead to tumor formation, we investigated whether 4-OHEN could cause DNA damage. We treated lambda phage DNA with 4-OHEN and found that extensive single-strand breaks could be obtained with increasing concentrations of 4-OHEN as well as increasing incubation times. If scavengers of reactive oxygen species are included in the incubations, DNA could be completely protected from 4-OHEN-mediated damage. In contrast, NADH and CuCl2 enhanced the ability of 4-OHEN to cause DNA single-strand breaks presumably due to redox cycling between 4-OHEN and the semiquinone radical generating hydrogen peroxide and ultimately copper peroxide complexes. We also confirmed that 4-OHEN could oxidize DNA bases since hydrolysis of 4-OHEN-treated calf thymus DNA and HPLC separation with electrospray MS detection revealed oxidized deoxynucleosides, including 8-oxodeoxyguanosine and 8-oxodeoxyadenosine. Our data suggest that DNA single-strand breaks and oxidation of DNA bases by 4-OHEN could contribute to the carcinogenic mechanism(s) of equine estrogens.