Viswanath Gunda, Halder Sujata, Divya Gunda, Majumder Chandrajeet B, Roy Partha
Molecular Endocrinology Laboratory, Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee 247667, Uttarakhand, India.
Mol Cell Endocrinol. 2008 Nov 25;295(1-2):1-9. doi: 10.1016/j.mce.2008.08.021. Epub 2008 Aug 28.
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.
本研究描述了使用两步筛选系统鉴定（抗）孕激素内分泌干扰物（EDC）的方法。第一步，利用重组人孕激素受体（hPR）建立了竞争性结合试验。所测试的化学物质包括各种类型，如杀虫剂、其代谢物、工业化学品和污水处理厂（WWTP）的废水。所有测试的化学物质都显示出对hPR具有高亲和力结合。测试化学物质的平均IC50值在1-25微摩尔范围内。在筛选的第二步中，建立了基于哺乳动物细胞的hPR反式激活试验，其中HEK 293细胞与hPR和在孕激素反应元件控制下的荧光素酶报告基因共转染。用孕激素刺激细胞导致荧光素酶活性上调约25倍，EC50值为4纳摩尔。强效抗孕激素RU486显著抑制孕激素诱导的反式激活，而非孕激素类甾体在浓度达到1微摩尔时未能反式激活hPR。在竞争性结合试验中显示出高结合亲和力的化学物质随后在反式激活试验中进行测试，除了污水处理厂废水外，所有这些物质都被发现具有抗孕激素作用。使用从五个不同污水处理厂废水中提取的水样进行的反式激活试验表明，其中富含孕激素类化合物。这些废水引起的诱导水平在孕激素诱导水平的15-25%范围内，它们代表约6纳克/升等效孕激素活性。总之，我们证明了这种两步试验为检测各种样品中的（抗）孕激素EDC提供了一种有效的筛选工具。
