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Abstract：
Transgenic fish are powerful models that can provide mechanistic information regarding the endocrine activity of test chemicals. In this study, our objective was to use a newly developed transgenic zebrafish line expressing eGFP under the control of the cyp19a1a promoter in the OECD Fish Short Term Reproduction Assay (TG 229) to provide additional mechanistic information on tested substances. For this purpose, we exposed adult transgenic zebrafish to a reference substance of the TG 229, i.e. prochloraz (PCZ; 1.7, 17.2 and 172.6 μg/L). In addition to \"classical\" endpoints used in the TG 229 (reproductive outputs, vitellogenin), the fluorescence intensity of the ovaries was monitored at 4 different times of exposure using in vivo imaging. Our data revealed that 172.6 μg/L PCZ significantly decreased the number of eggs laid per female per day and the concentrations of vitellogenin in females, reflecting the decreasing E2 synthesis due to the inhibition of the ovarian aromatase activities. At 7 and 14 days, GFP intensities in ovaries were similar over the treatment groups but significantly increased after 21 days at 17.2 and 172.6 μg/L. A similar profile was observed for the endogenous cyp19a1a expression measured by qPCR thereby confirming the reliability of the GFP measurement for assessing aromatase gene expression. The overexpression of the cyp19a1a gene likely reflects a compensatory response to the inhibitory action of PCZ on aromatase enzymatic activities. Overall, this study illustrates the feasibility of using the cyp19a1a-eGFP transgenic line for assessing the effect of PCZ in an OECD test guideline while providing complementary information on the time- and concentration-dependent effects of the compound, without disturbing reproduction of fish. The acquisition of this additional mechanistic information on a key target gene through in vivo fluorescence imaging of the ovaries was realized without increasing the number of individuals.
摘要：
转基因鱼是强大的模型，可以提供有关测试化学品内分泌活动的机械信息。在这项研究中,我们的目标是在OECD鱼类短期繁殖试验（TG229）中使用在cyp19a1a启动子控制下表达eGFP的新开发的转基因斑马鱼系，以提供有关测试物质的其他机制信息。为此,我们将成年转基因斑马鱼暴露于TG229的参考物质，即丙氯胺（PCZ;1.7、17.2和172.6μg/L）。除了TG229中使用的“经典”端点(生殖输出，卵黄蛋白原),使用体内成像在4个不同的暴露时间监测卵巢的荧光强度。我们的数据显示，172.6μg/LPCZ显着降低了每个雌性每天产卵的数量和雌性卵黄蛋白原的浓度，反映了由于抑制卵巢芳香化酶活性而导致的E2合成减少。在7天和14天，卵巢中的GFP强度与治疗组相似，但在21天后以17.2和172.6μg/L显着增加。对于通过qPCR测量的内源性cyp19a1a表达观察到类似的概况,从而证实GFP测量用于评估芳香酶基因表达的可靠性。cyp19a1a基因的过表达可能反映了对PCZ对芳香酶酶活性的抑制作用的代偿反应。总的来说,这项研究说明了在OECD测试指南中使用cyp19a1a-eGFP转基因品系评估PCZ效应的可行性，同时提供了有关该化合物的时间和浓度依赖性效应的补充信息，不干扰鱼的繁殖。在不增加个体数量的情况下,通过卵巢的体内荧光成像实现了对关键靶基因的这种额外机制信息的获取。
