多环芳烃致胎儿神经管缺陷作用及氧化应激机制研究

Neural tube defects (NTDs) are a group of common congenital malformations that often lead to perinatal deaths and life-long disabilities. The etiology and exact developmental mechanism of NTDs remain poorly understood. Our previous studies have shown that polycyclic aromatic hydrocarbons (PAHs), a group of ubiquitous environmental pollutants, may pay an important role in the development of NTDs. In the proposed research project, we intend to explore the role of PAHs in the development of NTDs and oxidative stress as an underlying mechanism of teratogenicity associated with PAH exposure, by using both human study and animal models. In the human study, a case-control design will be used. Cases are fetuses terminated due to prenatal diagnosis of an NTD and controls are fetuses terminated not for birth defects. Upon termination and obtaining consent from the mother, tissues of liver, brain and/or spinal cord of the terminated fetuses will be collected. PAH levels in liver tissue will be determined, and oxidant and antioxidant status, PAH-DNA adducts, markers of macromolecular (DNA, protein, lipid) oxidative damage , expression of PAX3 and p53 genes as well as proteins, and apoptosis will be assayed in the central nervous tissues. The association between PAHs exposure and the risk of NTDs, and the role of PAH-related oxidative stress in the development of NTDs will be analyzed. In the animal study, pregnant mice and whole mouse embryo culture system will be used to induce NTDs by administration of different doses of benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), with or without alpha- tocopherol (vitamin E). Oxidant and antioxidant status, DNA adducts, markers of macromolecule oxidative damage, Pax3 and p53 expression, and apoptosis will be determined in the mouse tissues of the central nervous system. The following potential molecular pathways in PAH-induced NTDs will be examined: a) electrophilic reactive intermediates, the products of PAHs bioactivation, covalently bind to DNA and thus induce neural crest cell apoptosis, resulting in closure failure of the neural tube; b) reactive oxygen species produced in the process of PAHs bioactivation cause macromolecular oxidative damage, which triggers neural crest cell apoptosis and results in neural tube defects, and c) reactive oxygen species inhibit Pax3 gene expression, which results in loss of control of p53 protein, which in turn leads to aberrant neural crest cell apoptosis, and finally neural tube defects. The information derived from the present study could provide the foundation for effective prevention strategies for NTDs.

神经管缺陷(NTDs)是一类常见致死、致残性先天缺陷，是影响出生人口素质的主要原因之一。NTDs的病因及发病机制尚未完全清楚。本课题组前期研究提示，多环芳烃(PAHs)可能与NTDs发病有关。本项目结合人体研究和动物模型，研究PAHs的致NTDs作用，检验氧化应激是否为PAHs的致畸机制。人体研究采用病例对照研究方法。病例为引产NTDs胎儿，对照为引产无缺陷胎儿。检测肝组织内PAHs水平；中枢神经组织内氧化-抗氧化状态、PAH-DNA加合物、生物大分子氧化损伤标志、PAX3和p53基因及蛋白表达水平，及细胞凋亡水平，分析PAHs与NTDs的关系及氧化应激所起作用。通过孕鼠体内染毒和体外全胚胎培养，构建PAHs致NTDs动物模型，检测胎鼠中枢神经组织内氧化-抗氧化状态、DNA加合物、生物大分子氧化损伤标志、Pax3和p53基因及蛋白表达水平，及细胞凋亡水平，揭示PAHs致NTDs分子机制。

神经管缺陷(NTDs)是一类常见致死、致残性先天缺陷。NTDs的病因及发病机制尚未完全清楚。本团队组前期研究提示，母亲多环芳烃(PAHs)暴露与胎儿NTDs风险存在关联性。本项目的目的是结合人体研究和动物模型，研究PAHs的致NTDs作用，检验氧化应激是否为PAHs的致畸机制。结果发现：①生育NTDs患儿风险随母亲血清PAHs水平升高而增加；②生育NTDs患儿母亲血液内DNA氧化损伤标志8-OHdG及蛋白氧化损伤标志蛋白羰基(PC)水平高与胎儿NTDs风险增加之间存在关联性；③胎儿脐带组织及脐带血PAH-DNA加合物水平高与胎儿NTDs风险增加之间存在关联性；④NTDs胎儿脊髓和脑组织内凋亡细胞增多，PAX3表达降低，但p53表达结果不一致。⑤BaP暴露可引起胎鼠神经管闭合缺陷；⑥小鼠胚胎暴露于BaP，体内ROS水平增加；⑦孕鼠BaP暴露，胎鼠氧化应激相关基因Cyp1a1, Sod1及Sod2表达增强；⑧BaP暴露，中枢神经组织凋亡细胞增多，裂解caspase-3蛋白表达增强；⑨BaP暴露，pax3 mRNA表达水平下降；与研究假设不同的是，p53 mRNA表达水平没有升高，反而降低；但p53蛋白表达水平升高；⑩BaP染毒孕鼠给于抗氧化物质维生素E，或体外全胚胎培养液内加入抗氧化剂N-乙酰-L-半胱氨酸，胎鼠神经管闭合缺陷率下降，氧化应激指标改善；给于维生素E后，p53蛋白表达下降，裂解caspase-3蛋白表达下降。综合以上结果，PAHs暴露通过引起氧化应激，进而导致Pax3基因表达下降，p53蛋白表达增强，中枢神经组织凋亡过度，最终引起神经管闭合障碍。本研究结果为NTDs的病因学和发育毒理学基础研究提供了新的方向，为环境污染的控制以及NTDs的预防提供了新的科学依据。