Körner W, Hanf V, Schuller W, Kempter C, Metzger J, Hagenmaier H
Institute of Organic Chemistry, University of Tübingen, Germany.
Sci Total Environ. 1999 Jan 12;225(1-2):33-48. doi: 10.1016/s0048-9697(99)80015-1.
A simplified proliferation test with human estrogen receptor-positive MCF-7 breast cancer cells (E-screen assay) was optimized and validated for the sensitive quantitative determination of total estrogenic activity in effluent samples from municipal sewage plants. After solid phase extraction of 1 l sewage on either 0.2 g polystyrene copolymer (ENV+) or 1 g RP-C18 material and removal of the solvent, analysis of the extracts in the E-screen assay could be performed without any clean-up step. This was even possible with untreated sewage. Parallel extraction of four sewage samples on both different solid phase materials gave comparable quantitative results in the E-screen. A blank sample did not induce cell proliferation. As additive behaviour of the estrogenic response of single compounds was proven for two different mixtures each containing three xenoestrogens, total estrogenic activity in the sewage samples, expressed as 17 beta-estradiol equivalent concentration (EEQ), could be calculated comparing the EC50 values of the samples with those of the positive control 17 beta-estradiol. The detection limit of the E-screen method was 0.05 pmol EEQ/l (0.014 ng EEQ/l), the limit of quantification 0.25-0.5 pmol EEQ/l (0.07-0.14 ng EEQ/l). In total, extracts of nine effluent and one influent sample from five different municipal sewage plants in South Germany were analyzed in the E-screen. All samples strongly induced cell proliferation in a dose-dependent manner which was completely inhibited by coincubation with 5 nM of the estrogen receptor-antagonist ICI 182,780. The proliferative effect relative to the positive control 17 beta-estradiol (RPE) was between 30 and 101%. 17 beta-Estradiol equivalent concentrations were between 2.5 and 25 ng/l indicating a significant input of estrogenic substances via sewage treatment plants into rivers.
对人雌激素受体阳性的MCF - 7乳腺癌细胞进行的简化增殖试验（E - 筛选试验）进行了优化和验证，用于灵敏定量测定城市污水处理厂出水样品中的总雌激素活性。在用0.2 g聚苯乙烯共聚物（ENV +）或1 g RP - C18材料对1升污水进行固相萃取并去除溶剂后，无需任何净化步骤即可在E - 筛选试验中对提取物进行分析。对于未经处理的污水也是如此。在两种不同的固相材料上对四个污水样品进行平行萃取，在E - 筛选试验中得到了可比的定量结果。空白样品未诱导细胞增殖。由于已证明两种不同混合物（每种混合物包含三种外源性雌激素）中单一化合物的雌激素反应具有加和性，因此通过比较样品与阳性对照17β - 雌二醇的EC50值，可以计算出污水样品中的总雌激素活性，以17β - 雌二醇当量浓度（EEQ）表示。E - 筛选方法的检测限为0.05 pmol EEQ / l（0.014 ng EEQ / l），定量限为0.25 - 0.5 pmol EEQ / l（0.07 - 0.14 ng EEQ / l）。总共对来自德国南部五个不同城市污水处理厂的九个出水样品和一个进水样品的提取物进行了E - 筛选分析。所有样品均以剂量依赖性方式强烈诱导细胞增殖，与5 nM雌激素受体拮抗剂ICI 182,780共同孵育可完全抑制这种增殖。相对于阳性对照17β - 雌二醇的增殖效应（RPE）在30%至101%之间。17β - 雌二醇当量浓度在2.5至25 ng / l之间，表明通过污水处理厂向河流中输入了大量雌激素物质。
