Van den Belt K, Berckmans P, Vangenechten C, Verheyen R, Witters H
VITO-Flemish Institute for Technological Research, Expertisecenter Environmental Toxicology, Boeretang 200 B-2400 MOL, Belgium.
Aquat Toxicol. 2004 Feb 10;66(2):183-95. doi: 10.1016/j.aquatox.2003.09.004.
The estrogenic activity of compounds was evaluated in a comparative approach both with in vitro and in vivo assays. By comparing simultaneously obtained experimental data, we evaluated the differences in response sensitivity (by EC10) and concentration-response relationships (including EC50) in order to get an idea about the predictive value of in vitro assays for in vivo estrogenic potencies or effects in fish. Two human estrogen receptor-based assays, the MVLN-assay (transformed MCF-7 human breast cancer cell line) and the yeast estrogen screen (YES-screen) were used for the in vitro evaluation of the estrogenic potencies. An in vivo model with the female zebrafish (Danio rerio) with plasma vitellogenin (VTG) as a biomarker for exposure and the ovarian somatic index (OSI) as an effect endpoint was used for the in vivo work. Compounds tested were 17beta-estradiol (E2), estrone (E1), 17alpha-ethynylestradiol (EE2) and the alkylphenolic compound nonylphenol (NP). All compounds were found to be estrogenic in both in vitro assays and were able to induce VTG and to reduce the ovarian somatic index in female zebrafish. The MVLN-assay appeared up to 15 times more sensitive than the YES-screen. Concentration-response relationships, determined by EC10 and EC50 (concentration of test compound causing 10% or 50% effect compared to control) for VTG and OSI were of the same order of magnitude, indicating that VTG induction as an exposure biomarker can be predictive for effects on ovaries in females. We further demonstrated that for E1 and NP, the in vitro observed estrogenic potencies, based on EC50 values, were of the same order of magnitude as the in vivo estrogenic potencies. For EE2, a difference between in vitro and in vivo relative estrogenic potency was observed, being about 25 times more potent in vivo than could be expected based on the in vitro results. These experimental results showed the suitability of in vitro assays for screening purposes with qualitative assessment of estrogenicity, but they meanwhile point to the need of in vivo tests for an accurate hazard assessment for wildlife.
采用比较法，通过体外和体内试验评估化合物的雌激素活性。通过同时比较获得的实验数据，我们评估了反应敏感性（以EC10表示）和浓度-反应关系（包括EC50）的差异，以便了解体外试验对鱼类体内雌激素效力或效应的预测价值。两种基于人雌激素受体的试验，即MVLN试验（转化的MCF-7人乳腺癌细胞系）和酵母雌激素筛选试验（YES筛选试验），用于体外评估雌激素效力。体内试验采用雌性斑马鱼（Danio rerio）作为模型，以血浆卵黄蛋白原（VTG）作为暴露生物标志物，以卵巢体指数（OSI）作为效应终点。所测试的化合物为17β-雌二醇（E2）、雌酮（E1）、17α-乙炔基雌二醇（EE2）和烷基酚类化合物壬基酚（NP）。在两种体外试验中均发现所有化合物具有雌激素活性，并且能够诱导雌性斑马鱼体内的VTG并降低卵巢体指数。MVLN试验的敏感性比YES筛选试验高15倍。由VTG和OSI的EC10和EC50（与对照相比引起10%或50%效应的测试化合物浓度）确定的浓度-反应关系处于相同数量级，表明作为暴露生物标志物的VTG诱导可预测对雌性卵巢的影响。我们进一步证明，对于E1和NP，基于EC50值在体外观察到的雌激素效力与体内雌激素效力处于相同数量级。对于EE2，观察到体外和体内相对雌激素效力存在差异，体内效力比基于体外结果预期的效力高约25倍。这些实验结果表明体外试验适用于雌激素性的定性评估筛选目的，但同时也指出需要进行体内试验以对野生动物进行准确的危害评估。
