RESUMEN
The present work describes the identification of (anti)progestin endocrine disrupting chemicals (EDC) using a two step screening system. In the first step a competitive binding assay was developed using recombinant human progesterone receptor (hPR). The tested chemicals were of various classes like insecticides, their metabolites, industrial chemicals and waste water treatment plant (WWTP) effluents. All the tested chemicals demonstrated a high affinity binding for hPR. The average IC50 values of the test chemicals were within the range of 1-25microM. In the second step of screening, a mammalian cell-based hPR transactivation assay was developed where HEK 293 cells were co-transfected with hPR and luciferase reporter gene under the control of progesterone-response element. Stimulation of the cells with progesterone resulted in about 25-fold up regulation of luciferase activity, with EC50 value of 4nM. Potent anti-progesterone, RU486, significantly inhibited progesterone-induced transactivation and non-progestagenic steroids failed to transactivate hPR till 1microM concentrations. The chemicals showing high binding affinities in competitive binding assays were then tested in transactivation assay and all of them were found to be anti-progestative except WWTP effluents. Transactivation assays using extracted water samples from five different WWTP effluents showed that it was rich in progestative compounds. The levels of induction caused by these effluents were in the range of 15-25% of induction by progesterone and they represented about 6ng/l equivalent progesterone activities. In conclusion, we demonstrated that this two step assay provides an efficient screening tool for the detection of (anti)progestative EDC in various samples.
Asunto(s)
Bioensayo , Disruptores Endocrinos/farmacología , Antagonistas de Hormonas/farmacología , Progestinas/farmacología , Receptores de Progesterona/efectos de los fármacos , Elementos de Respuesta/efectos de los fármacos , Activación Transcripcional/efectos de los fármacos , Contaminantes Químicos del Agua/farmacología , Unión Competitiva , Línea Celular , Relación Dosis-Respuesta a Droga , Disruptores Endocrinos/metabolismo , Monitoreo del Ambiente , Genes Reporteros , Antagonistas de Hormonas/metabolismo , Humanos , Progesterona/metabolismo , Progestinas/metabolismo , Receptores de Progesterona/genética , Receptores de Progesterona/metabolismo , Proteínas Recombinantes/metabolismo , Transfección , Contaminantes Químicos del Agua/metabolismoRESUMEN
The present study was aimed to develop a sensitive, fast and user friendly progesterone receptor transactivation assay using recombinant yeast cells, Saccharomyces cerevisiae, modified to express human progesterone receptor (PR) and progesterone response element (PRE) driving the expression of green fluorescent protein. Stimulation of cells with increasing concentrations of progesterone resulted in significant elevation in fluorescence activity, with the minimum effective dose of progesterone being 0.1 nM. RU486, significantly inhibited progesterone induced transactivation and non-progesterogenic steroids failed to transactivate PR till 10 microM concentrations. About 7 different chemicals (mostly pesticides or their metabolites) like DDT and its metabolites, nonylphenol, endosulfan were screened in this assay system for their role in transactivation and they were all found to be anti-progestative and IC50 values within the range of 3-20 microM. Further, the assay was used to analyze the endocrine disrupting activity of extracted water samples from leather industries known for their high content of various chemicals and it was found to be rich in anti-progestative compounds. It resulted in about 30% reduction in transactivation. In conclusion, we demonstrated that this yeast based bioassay provides a rapid and robust assay for high throughput screening of (anti)progestative compounds from various sources.
Asunto(s)
Disruptores Endocrinos/farmacología , Progestinas/antagonistas & inhibidores , Saccharomyces cerevisiae/efectos de los fármacos , Bioensayo , Western Blotting , Evaluación Preclínica de Medicamentos , Disruptores Endocrinos/química , Disruptores Endocrinos/aislamiento & purificación , Genes Reporteros/genética , Proteínas Fluorescentes Verdes/genética , Antagonistas de Hormonas/farmacología , Residuos Industriales/análisis , Ligandos , Mifepristona/farmacología , Plásmidos/genética , Progesterona/agonistas , Progesterona/antagonistas & inhibidores , ARN/aislamiento & purificación , Receptores de Progesterona/efectos de los fármacos , Receptores de Progesterona/genética , Elementos de Respuesta , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Saccharomyces cerevisiae/genética , Activación Transcripcional/efectos de los fármacosRESUMEN
We have constructed an efficient and reliable yeast-based detection system to evaluate the androgenic activity of endocrine disruptors from pulp and paper mill effluents (PPME). This system consists of human androgen receptor and androgen response elements driven ß-galactosidase genes transformed in yeast, Saccharomyces cerevisiae. The transcriptional activation by known androgens, correlated with androgenic activities as measured by other assay systems. This assay system when applied to evaluate anti-androgenic activities, the known anti-androgens effectively inhibited reporter gene induction by testosterone. The specificity of the assay was tested by incubating the transformed cells with supraphysiological concentrations of non-androgenic steroids and none of them gave a significant response. The extracted PPME from five different mills demonstrated strong androgenic activities (about five- to eight-folds over control). These results suggest that PPME are rich in androgenic chemicals and the employed detection system could be applicable to primary screening for effectors on androgen receptor functions.