双酚A干扰稀有鮈鲫性腺类固醇激素合成和效应的DNA甲基化调控及其机制研究

Bisphenol A (BPA) is an ubiquitous compound in the environment. The previous studies and our preliminary experiments suggest that the reported properties of BPA can not completely underlie the toxicological mechanism of BPA. We speculate that the effects of BPA on DNA methylation could comprehensively and deeply elucidate the toxicological mechanism of BPA. In the present work, the rare minnow Gibiocypris rarus will be exposed to BPA at different concentrations for diverse durations. The methods of molecuar biology, epigenetics, biochemistry, analytical chemistry and histology will be used in the present study. The DNA methylation involved in BPA disturbance to steroid hormone biogenesis and the actions will be analyzed. How BPA affects activities and gene expressions of DNA methyltransferase will be investigated. The regulation of BPA exposure on production of methyl donor S-adenosyl methionine (SAM) in gonads via changes of glutathione (GSH) level will be analyzed. How BPA modulates the production of critical intermediates of the citric acid cycle involved in activity of methyl cytosine hydroxylase TET and finally the DNA methylation will be investigated. In the present study, the disturbance of BPA to biogenesis of steroid hormones and their actions will be investigated in epigenetic level for the first time. The present study will throw a new light on the toxicological mechanism for BPA. This study will provide sensitive epigenetic endpoints for BPA detection. The present findings will provide novel ideas to elucidate the relationship between the BPA's epigenetic toxicities and oxidative stress, and the final metabolic pathways.

BPA在环境中普遍存在，文献及我们的预实验均表明，目前公认的BPA的毒性作用机制无法完全解释其干扰效应。我们认为BPA对DNA甲基化的影响可以更全面深入地解释BPA的效应。本项目用BPA暴露稀有鮈鲫，采用分子生物学、表观遗传学、生物化学、分析化学和组织学方法，分析DNA甲基化的调控如何参与BPA干扰性腺类固醇激素的合成及其作用，BPA对DNA甲基转移酶活性及表达的影响，BPA如何改变GSH合成而调节甲基供体SAM水平，BPA如何影响三羧酸循环的关键中间产物而调控甲基胞嘧啶羟化酶TET的活性，最终改变DNA甲基化水平。本项目首次在表观遗传层次探讨BPA对稀有鮈鲫性腺类固醇激素的合成及其作用的干扰，本项目有助于从新的角度更全面地阐明BPA的毒理机制，为BPA这种环境中普遍存在的内分泌干扰物提供敏感的表观遗传测试终点，为揭示BPA的表观遗传毒性与氧化应激及营养物质的最终代谢通路的关系提供新思路。

哺乳动物的研究显示，BPA可以通过表观遗传改变而导致生殖毒性，而在鱼类，BPA所引起的表观遗传影响还未有深入研究，目前几乎没有鱼类生殖毒性的表观遗传机制的研究。本项目以稀有鮈鲫为试验动物，检测BPA对性腺类固醇合成基因5’侧翼区DNA甲基化的影响, 研究BPA干扰鱼类精巢DNA甲基化的机制及BPA引起的GSH库的扰动和DNA甲基化改变之间的关系，研究抗氧化剂N-乙酰半胱氨酸（NAC）添加对BPA所引起毒性的保护作用，研究BPA对稀有鮈鲫亲代及亲代暴露2个月的子代性腺的组蛋白甲基化和DNA甲基化干扰机制。获得如下结果：BPA可通过干扰类固醇合成酶基因的表达影响卵巢的类固醇激素的合成；BPA暴露可引起cyp19a1a、star、cyp11a1、cyp17a1、hsd11b2和hsd3b 5’侧翼的DNA甲基化的变化与其对应基因的表达变化有关；BPA能够影响star、hsd11b2和hsd3b基因5’侧翼ERE与ER的相互作用而干扰基因的表达；BPA短期和延长暴露可通过影响DNMTs介导的DNA甲基化过程或TETs介导的去甲基化过程最终引起稀有鮈鲫精巢DNA高甲基化，而不同浓度BPA及暴露时间均不会对卵巢全基因组甲基化水平产生明显影响；高浓度BPA短期暴露时精巢组织中GSH从头合成过程推动了DNA的甲基化；NAC添加能增加半胱氨酸的含量以抑制BPA导致的精巢DNA高甲基化；稀有鮈鲫精巢抗氧化系统的积极响应和NAC本身的抗氧化活性防止了BPA和（或）NAC处理可能导致的精子氧化损伤；BPA可改变亲鱼性腺和亲代BPA暴露后的子代雌鱼卵巢中的DNA甲基化和组蛋白甲基化水平；BPA可以通过影响类固醇合成酶类的DNA和组蛋白甲基化修饰进而影响亲代及F1代卵巢类固醇激素合成；BPA暴露雌雄亲鱼可以影响F1代胚胎和性腺发育，由BPA产生的遗传毒性效应可逆转恢复。