Programm MGU Mensch-Gesellschaft-Umwelt, Department of Environmental Sciences, University of Basel, Vesalgasse 1, Basel, Switzerland.
Aquat Toxicol. 2011 Feb;101(3-4):500-11. doi: 10.1016/j.aquatox.2010.12.008. Epub 2010 Dec 17.
A variety of natural and synthetic environmental substances have been shown to disrupt vertebrate reproduction through mimicking or modifying the regulation of the endocrine system. Tests to screen for any such chemicals that directly interact with the steroid hormone receptors are widely available; however, few tests have been developed to identify chemicals that affect endocrine function through non-receptor mediated mechanisms. The aim of this study was, therefore, to develop an assay for the identification of substances that disrupt the activity of enzymes involved in the sex steroid biosynthesis cascade, in particular the aromatase enzyme, CYP19, that catalyses the final conversion of androgens to estrogens. A gonad ex vivo assay was developed using gonad explants harvested from juvenile brown trout and cultured in a modified Leibovitz medium. Effects on sex steroid biosynthesis were quantified through measurement of 17β-estradiol (E2) and testosterone (T) concentrations in the medium after 2 days incubation. Exposure of ovary explants to 100 ng/mL 1,4,6-androstatriene-3,17-dione (ATD), a potent pharmaceutical aromatase inhibitor, reduced E2 concentrations and elevated T concentrations confirming that CYP19 activity could be inhibited in the assay. Exposure of ovary explants to 250 ng/mL prochloraz, an imidazole fungicide, also reduced E2 concentrations but did not affect T levels, consistent with reports that in addition to inhibiting CYP19 activity, prochloraz also inhibits enzymes in the steroidogenic pathway upstream of the CYP19 enzyme. Exposure to a third chemical, tributyltin (TBT), did not affect T or E2 concentrations, further supporting previous evidence that the CYP19 modulating effects of this chemical are not mediated through direct inhibition of CYP19 activity. These results demonstrate that the gonad ex vivo assay developed here can be successfully used to identify substances that disrupt sex steroid biosynthesis and further that it has the potential to inform on their specific mode of action.
各种天然和合成的环境物质已被证明通过模拟或改变内分泌系统的调节来干扰脊椎动物的繁殖。广泛提供了用于筛选任何与类固醇激素受体直接相互作用的此类化学物质的测试；然而，已经开发出的测试很少用于识别通过非受体介导的机制影响内分泌功能的化学物质。因此，本研究的目的是开发一种测定法，用于鉴定破坏参与性激素生物合成级联的酶的活性的物质，特别是芳香酶酶 CYP19，该酶催化雄激素向雌激素的最终转化。使用从幼年褐鳟鱼中收获的性腺外植体并在改良的 Leibovitz 培养基中培养，开发了一种性腺离体测定法。通过在孵育 2 天后测量培养基中 17β-雌二醇（E2）和睾酮（T）的浓度来量化对性激素生物合成的影响。暴露于 100ng/mL 1,4,6-雄甾三烯-3,17-二酮（ATD），一种有效的药物芳香酶抑制剂，可降低 E2 浓度并升高 T 浓度，证实 CYP19 活性可在测定中被抑制。暴露于 250ng/mL 咪鲜胺，一种咪唑类杀菌剂，也降低了 E2 浓度，但不影响 T 水平，这与咪鲜胺除了抑制 CYP19 活性外，还抑制 CYP19 酶上游的甾体生成途径中的酶的报道一致。暴露于第三种化学物质三丁基锡（TBT）不影响 T 或 E2 浓度，进一步支持了先前的证据，即该化学物质对 CYP19 的调节作用不是通过直接抑制 CYP19 活性介导的。这些结果表明，这里开发的性腺离体测定法可成功用于鉴定破坏性激素生物合成的物质，并且进一步表明它具有提供其特定作用模式的潜力。
