University of Applied Sciences and Arts Northwestern Switzerland, School of Life Sciences, Hofackerstrasse 30, CH-4132 Muttenz, Switzerland.
University of Applied Sciences and Arts Northwestern Switzerland, School of Life Sciences, Hofackerstrasse 30, CH-4132 Muttenz, Switzerland; Swiss Federal Institute of Technology (ETH Zürich), Institute of Biogeochemistry and Pollution Dynamics, Department of Environmental Systems Science, CH-8092 Zürich, Switzerland.
Aquat Toxicol. 2020 Sep;226:105562. doi: 10.1016/j.aquatox.2020.105562. Epub 2020 Jul 3.
Fish are exposed to steroids of different classes in contaminated waters, but their effects are not sufficiently understood. Here we employed an anti-sense technique using morpholino oligonucleotides to knockdown the glucocorticoid receptors (GRs, GRα and GRβ) and androgen receptor (AR) to investigate their role in physiological and transcriptional responses. To this end, zebrafish embryos were exposed to clobetasol propionate (CLO), androstenedione (A4) and mixtures containing different classes of steroids. CLO caused a decrease of spontaneous muscle contraction and increase of heart rate, as well as transcriptional induction of pepck1, fkbp5, sult2st3 and vitellogenin (vtg1) at 24 and/or 48 h post fertilization (hpf). Knockdown of GRs eliminated these effects, while knockdown of AR decreased the ar transcript but caused no expressional changes, except induction of sult2st3 after exposure to A4 at 24 hpf. Exposure to a mixture of 6 steroids comprising progesterone (P4) and three progestins, cyproterone acetate, dienogest, drospirenone, 17β-estradiol (E2) and CLO caused a significant induction of pepck1, sult2st3, vtg1 and per1a. Knockdown of GRs eliminated the physiological effects and the up-regulation of vtg1, sult2st3, pepck1, fkbp5 and per1a. Thus, as with CLO, responses in mixtures were regulated by GRs independently from the presence of other steroids. Exposure to a mixture comprising A4, CLO, E2 and P4 caused induction of vtg1, cyp19b, sult2st3 and fkbp5. Knockdown of AR had no effect, indicating that regulation of these genes occurred by the GRs and estrogen receptor (ER). Our findings show that in early embryos GRs cause vtg1 and sult2st3 induction in addition to known glucocorticoid target genes. Each steroid receptor regulated its own target genes in steroid mixtures independently from other steroids. However, enhanced expressional induction occurred for vtg1 and fkbp5 in steroid mixtures, indicating an interaction/cross-talk between GRs and ER. These findings have importance for the understanding of molecular effects of steroid mixtures.
鱼类在受污染的水中会接触到不同类别的类固醇，但它们的作用尚未得到充分了解。在这里，我们使用反义技术，利用 morpholino 寡核苷酸敲低糖皮质激素受体（GRs，GRα 和 GRβ）和雄激素受体（AR），以研究它们在生理和转录反应中的作用。为此，我们将斑马鱼胚胎暴露于氯倍他索丙酸酯（CLO）、雄烯二酮（A4）和含有不同类固醇类别的混合物中。CLO 导致自发肌肉收缩减少和心率增加，以及受精后 24 和/或 48 小时（hpf）时 pepck1、fkbp5、sult2st3 和卵黄蛋白原（vtg1）的转录诱导。GRs 的敲低消除了这些作用，而 AR 的敲低降低了 ar 转录本，但除了暴露于 A4 后 24 hpf 时 sult2st3 的诱导外，没有引起表达变化。暴露于包含孕激素（P4）和三种孕激素（醋酸环丙孕酮、地诺孕素、屈螺酮）、17β-雌二醇（E2）和 CLO 的 6 种类固醇混合物导致 pepck1、sult2st3、vtg1 和 per1a 的显著诱导。GRs 的敲低消除了生理效应和 vtg1、sult2st3、pepck1、fkbp5 和 per1a 的上调。因此，与 CLO 一样，混合物中的反应受 GRs 调节，与其他类固醇的存在无关。暴露于包含 A4、CLO、E2 和 P4 的混合物中导致 vtg1、cyp19b、sult2st3 和 fkbp5 的诱导。AR 的敲低没有影响，表明这些基因的调节是由 GRs 和雌激素受体（ER）引起的。我们的研究结果表明，在早期胚胎中，GRs 除了已知的糖皮质激素靶基因外，还引起 vtg1 和 sult2st3 的诱导。每个类固醇受体在类固醇混合物中独立于其他类固醇调节其自身的靶基因。然而，在类固醇混合物中，vtg1 和 fkbp5 的表达诱导增强，表明 GRs 和 ER 之间存在相互作用/交叉对话。这些发现对于理解类固醇混合物的分子作用具有重要意义。
