青春期砷暴露干扰雄性大鼠HPT轴内分泌/代谢通路的生殖毒性研究

Recently, arsenic (As) exposure has been tightly associated with increased risk of reproductive problems. Exposure to arsenic in drinking water represents a significant health problem for people around the world. Besides, dietary arsenic and inhaled particulates may be other important sources for general population exposures. For a typical Chinese adult living in an urban area where safe drinking water is supplied, the daily dietary intake of iAs is estimated to be 42.81 μg/day. This exposure source in combination with aggregate exposures from other pathways may result in exposure levels that may pose a health risk to the general population. However, few data has been reported for the adverse effect of low dose arsenic exposure on reproduction health. Puberty is the process of physical changes through which a child's body matures into an adult body capable of sexual reproduction to enable fertilization. Puberty is a sensitive period of reproductive development. Pubertal exposure to arsenic may affect male reproductive health. Currently, only a few reproductive toxicity studies have been reported for early exposure to arsenic, and most of them just focus on the adverse effects. The specific molecular mechanism underlying the effects remained unclear. Hypothalamic - pituitary -testicular axis (HPT axis) is the most important factor in male reproduction. Arsenic exposure not only disrupts hormone secretion, but also affects a large number of male reproduction - related metabolic pathways. Based on previous reports and our preliminary studies, we assume that arsenic exposure may disturb global endocrine and metabolism regulatory networks of HPT axis (especially the hormone and neurotransmitter, sugar and lipid pathways), eventually lead to reproductive toxicity. Based on this hypothesis, we will expose young rats to arsenic during puberty (PND23-PND53) to mimic pubertal arsenic exposure of human being. The used doses cover environmental dose of general population exposure and the relatively high dose found in arsenic endemic area. Previous studies focused on a single organ and limited numbers of indexes. Advances in omics technologies have enabled simultaneous and profiling of thousands of genes, proteins and metabolites in complex biological matrices. These non-hypothesis-driven omics approaches have been used to identify exposure-specific biomarkers and related pathways. In this project, through combining conventional toxicological methods and high-throughput omics technologies, we aim to investigate the adverse effects of different doses of chronic arsenic exposure on global endocrine and metabolism regulatory networks (including target and non-target analysis) of HPT axis, and elucidate the male reproductive toxicity mechanism of arsenic from systems biology angle. In this project, we investigate the influence of arsenic exposure on serum hormone levels and sperm quality; screen effect biomarkers of exposure by integrated conventional toxicology methods and proteomics, targeted/non-targeted metabolomics technology; combine the data from different molecular levels by using IPA software, discovery the important molecular events involved in arsenic-induced endocrine/metabolism disruption (focusing on the hormone and neurotransmitter, sugar and lipid pathway); on the basis of animal model, conduct in vitro experiment, discover the important toxicity target of arsenic through intervening the expression of the key gene in the pathways found in in above vivo experiments; consequently elucidate the disruption effects of pubertal exposure to arsenic on endocrine/metabolism pathways and related mechanism of reproductive toxicity. This project will improve our understanding of the reproductive health impact of arsenic exposure for different populations (e.g. general population and the population from arsenic endemic area), and provide new knowledge to toxicity mechanism and environmental risks of arsenic exposure.

青春期砷暴露会影响远期的男性生殖健康，然而具体的分子机制尚不明确。下丘脑-垂体-睾丸轴(HPT轴)是雄性生殖的决定因素，我们推测：青春期砷暴露影响HPT轴各器官内分泌/代谢调控网络(尤其是激素和神经递质、糖和脂质通路)，最终导致生殖毒性。本项目以青春期大鼠为模型开展长期暴露(覆盖普通环境剂量-高砷地区典型剂量)，探讨砷对血清激素水平和精子质量的影响；将常规毒理学方法与蛋白质组、靶向/全代谢组学技术相结合，兼顾已知和未知通路，筛选暴露效应标志物，利用IPA软件整合各分子层面的数据，发现砷扰乱HPT 轴内分泌/代谢调控网络中重要的分子通路；在此基础上，开展细胞暴露实验，对重要通路中标志性基因进行干预，发现关键的毒性靶分子；综合体内、体外实验数据，阐明砷的内分泌/代谢干扰效应及相关生殖毒性机理。该项目有助于我们更好的理解砷对不同人群的生殖健康的影响，为砷的毒性效应以及环境风险提供新的认识。

流行病学研究表明砷会显著影响男性生殖健康，但是我们尚不清楚砷通过哪些重要途径损害雄（男）性生殖功能，所产生的生殖健康影响是否可逆。本项目通过对雄性幼鼠进行为期30天的饮水砷暴露来模拟人类青春期暴露情况，聚焦HPT轴和肠-脑轴研究砷的雄（男）性生殖毒性作用，并且通过额外的恢复组研究损伤效应的恢复情况。我们发现，砷对体重和睾丸脏体比无明显影响。肛门生殖器间距显著减小，但部分剂量组在恢复期后得到恢复。砷暴露导致肝功（CHE, TBA, TP）和心肌（LDH, α-HBDH, CK-MB）指标水平增加，并显著影响了HPT轴激素相关重要基因、性激素和单胺类物质水平，暴露停止后，部分指标可逐渐恢复至正常水平。但是，睾酮水平在恢复期后仍然显著升高，表明了睾酮对砷响应的滞后效应，可能需要更长的时间才能恢复到正常水平。相反，睾丸组织中的甲状腺激素水平几乎未受到砷的影响。相关性分析表明砷可能通过血清单胺类物质影响睾丸组织的激素合成和分泌通路。组学分析表明砷暴露明显扰乱了睾丸代谢网络，对脂质代谢的影响最为显著，甘油磷脂代谢和亚油酸代谢通路是砷生殖毒性的关键通路。我们还从睾丸中筛选出四种潜在的暴露效应标志物（Glu-Phe, DAG, LysoPE 18:3, LysoPC 20:4）。砷的生殖毒性效应可能是通过改变神经递质和HPT轴激素合成通路基因的表达水平，进而影响激素的分泌和相关代谢物的合成。除此以外，我们还发现，砷暴露影响了肠道微生物组及代谢组的稳态，乳酸菌菌株可能作为抵抗砷诱导健康损害的保护剂。砷暴露后，特异性OTUs并未与性激素存在强相关关系，但恢复期某些OTUs与睾酮水平呈现强相关。以上这些发现加深了我们对青春期砷暴露生殖毒性的理解，为砷的毒性分子机理以及环境风险提供新的认识。