低剂量双酚A生殖毒性的分子和表观遗传作用机制

The plastic monomer and plasticizer bisphenol A (BPA) is an endocrine disruptor chemicals (EDCs) that is ubiquitous in environment and human body. The structure similarity between BPA and the 17β-estradiol (E2) has prompted researchers to suspect that BPA is one of the main causative factors for the continuous decline of human reproductive health. Previous research has been driven by the assumption that BPA acts as an estrogen disruptor through binding with classical nuclear estrogen receptors (ERs). However, BPA was normally detected at low doses in human body, and it has also been shown to have weak binding affinity with ERs. It thus seems impossible for BPA to compete with E2 for binding sites on ERs and initiate gene expression changes in vivo. More recent studies indicate that BPA exhibits very strong binding affinity for estrogen related receptor gamma (ERRγ) while ERRγ is an orphan nuclear receptor that has no known endogenous ligand, suggesting BPA may affect reproduction through ERRγ. Currently, it is unknown whether BPA exerts its reproductive toxicity through ERs or ERRγ. To bridge this gap, our proposal thus hypothesize that low dose BPA-induced reproductive toxicity is mediated through ERRγ. This proposal will use zebrafish as the model animal and take the advantage of the modern molecular and epigenetic techniques in combination with classic toxicology methods to reveal the underlying mechanism for BPA-induced reproductive toxicity. The specific objectives are to 1) identify the morphological/physiological and genetic signature phenotypes that are specific to low-dose BPA induced reproductive toxicity; 2) elucidate the in vivo receptor target of low-dose BPA induced reproductive toxicity when exposure during the sensitive developmental window; and 3) explore the potential role of epigenetic regulation in BPA-mediated reproductive toxicity. The findings of our research will not only reveal the underlying genetic and epigenetic mechanisms of BPA or other similar EDCs induced reproductive toxicity, but also provide strong scientific evidence for reproductive health risk assessment of EDCs in general.

内分沁干扰物双酚A（BPA）因在环境和人体内普遍存在而被认为是现代生殖健康问题的主要因素之一。其结构与雌激素相似，因而多数研究均假设BPA是通过与雌激素受体（ERs）结合而作用于靶基因的。然而BPA在人体内的低含量及与ERs的弱亲和力让人对此产生质疑。最近研究雌激素相关受体ERRγ（与BPA的结合力更强且缺乏内源性配体）更有可能是BPA的作用受体。针对这一研究空白，本项目以斑马鱼为模式动物，采用传统毒理学和现代分子生物学研究手段，通过低剂量BPA于敏感窗口的慢性暴毒：1）识别低剂量（环境浓度值）BPA生殖毒性的形态/生理表型及特征基因表型；2）阐释低剂量BPA在敏感窗口期暴毒所引发的生殖毒性的分子作用受体；3）探索BPA生殖毒性是否存在表观遗传作用机制。研究结果能诠释以BPA为代表的一些典型内分沁干扰物的生殖毒性作用机制，而且可为这些化合物对生物和人类生殖健康的风险评价提供重要的科学依据。

BPA因在环境和人体内普遍存在而被认为是现代生殖健康问题的主要因素之一，但是其作用机制不清楚，故本课题采用斑马鱼模式生物系统地研究了BPA的生殖毒性及其作用机理。研究发现，斑马鱼在不同发育期慢性暴露于环境相对存在BPA浓度下，对斑马鱼精巢重量、精子总数、精子密度有着不同程度的负面效应，亲代的产卵量少，子一代的受精率与孵化率下降，而畸形率和死亡率升高，而且这些负面效应均在最低浓度1nM组的表现最为显著。且0-21dpf暴露窗口为BPA敏感的暴露窗口，qPCR分析发现21dpf原始生殖细胞标记基因vasa上调，其调控基因Nanos3下调，提示它们在该雌性化过程中具有作用。.进一步研究了1 nM BPA多世代暴露对斑马鱼的生殖毒性及其作用机制。发现BPA可引起斑马鱼亲代和子一代雌性化趋势不断加剧，并使雄性精子密度下降、精子质量下降（活力和运动分析）、ATP含量下降，MDA增加。且整体分析发现BPA对雄鱼的生殖毒性明显大于对雌性的生殖毒性。通过对子一代成体期睾丸组织的RNA-Seq分析发现有1320个基因发生了显著变化，信号通路分析发现BPA激活了线粒体生物合成、和非典型性Wnt/平面细胞极化及Wnt/钙离子信号通路。qPCR确认了Wnt信号通路中差异表达的基因多数为上调，Wnt信号在生殖毒性和性别分化过程中具有作用，推测BPA暴露通过激活Wnt信号和线粒体生物合成而引起雌性化发生和生殖毒性效应。此外对子三代胚胎期的表观遗传相关基因的表达分析发现它们均被显著抑制，推测其在BPA生殖毒性过程中也具有作用。为进一步探究BPA生殖毒性机制，本研究还利用CRISPR-cas9技术进行关键基因fzd8b基因敲除构建突变体和该基因MO下调与BPA联合作用研究，以阐明BPA生殖毒性机制。目前已成功构建突变体。.此外，我们还研究了BPA急性高剂量暴露对斑马鱼发育毒性和神经毒性的影响，结果表明，BPA在μM级别暴露不引起发育毒性，但能显著影响各个神经行为毒性指标。对神经毒性作用机制研究发现BPA暴露中DNA不稳定和ROS的浓度上升导致了神经细胞凋亡和肌肉肌丝松弛，最终导致运动神经元发育延迟和行为运动改变。.综上所述，本研究结果能诠释以BPA为代表的一些典型内分沁干扰物的生殖毒性作用机制，而且为这些化合物对生物和人类生殖健康的风险评价提供重要的科学依据。