邻苯二甲酸苄基酯暴露与男性生殖健康的相关性及其分子机理研究

Butyl benzyl phthalate (BBP) is one member of phthalates (PAEs), which are used as plasticizers, solvents and stabilizers in industry, medicine, building and consumer products. The widespread use of PAEs contributes to their omnipresent detection in the environment and human beings. PAEs can be transferred from maternal blood into the developing fetus via placenta and into neonates via breast milk. The possibility of PAEs entering into the biological system has generated concern about their reproductive and developmental toxic potential in humans. BBP and its metabolite (mono-benzyl phthalate (MBzP)) has been reported to induce a decrease in testis size, sperm production and mobility, morphological changes, embryolethality and malformations in mice, but its mechanism has not been clarified. Although preliminary, our study analyzed urinary phthalate metabolites from 511 mans with subfertile problems and found that the urinary MBzP levels were negatively correlated with the sperm concentration, sperm count, acrosomal enzyme and morphologically normal rate. This suggested that BBP and MBzP had a potential threat to the human reproductive health. Some reports showed that the urinary MBzP levels were significantly higher in children compared to adolescents and adults. The usage of BBP in baby products has been limited in US and other countries, but not yet limited in China. In our present program, firstly, we will collect 1200 urine samples from the males who were members of subfertile couples in China, analyze 14 phthalate metabolite concentrations using UPLC-MS/MS and determine serum hormon levels, the sperm production and qualities. The aim is to investigate the general concentrations of phthalate exposures in China and to evaluate correlations between phthalate metabolite concentrations with male reproductive parameters. This study will also help scientists plan and conduct research on exposure and health effects. In addition, we will establish a cross-foster animal model to study whether prenatal or postnatal exposure to MBzP alone can disturb the male reproductive function in mouse pups, and if so, which kind of exposure is a major cause. Besides, the mechanisms of action by which MBzP interferes with male reproductive system in mouse pups were also investigated. Moreover, we will establish a subchronic MBzP exposure model to adult male mouse, find the key proteins and pathways in mouse testis targeted by MBzP using proteomic technology. Furthermore, we will use in vitro exposure model through preparing primary Sertoli cells and Leydig cells, inhibiting PPARα expression by RNAi technology to further explore the mechanism of MBzP induced structural and functional damage to mouse testis and the roles of PPARα in MBzP induced male reproductive toxicity. We hope that this study will help scientist to evaluate the adverse effect of BBP to human health, and help government to draft related policy about BBP usage.

邻苯二甲酸丁基苄酯（BBP）作为增塑剂应用于化工、装修材料、食品包装等中，在环境介质及人体中广泛检出。我们的前期研究发现我国男性人群尿液中BBP的代谢物－邻苯二甲酸苄酯(MBzP)的水平与精子质量和数量呈负相关性。动物实验也发现BBP或MBzP具有明显的生殖毒性，但其机制尚未明确。本项目拟通过大样本量分析，揭示我国典型地区生育期男性尿液中PAEs暴露水平（特别是MBzP）与男性生殖健康的关联；进一步用MBzP暴露小鼠，通过交叉哺育实验，研究子宫暴露或母乳暴露对子代雄性生殖系统的影响，找到其敏感的暴露时期；通过成年小鼠体内、体外暴露实验，运用蛋白质组学、RNA干扰等技术手段，阐明MBzP对成年雄性生殖毒性效应及机制，明确PPAR?在MBzP诱导的生殖毒性中的作用；为正确评估BBP及其代谢物MBzP对男性生殖健康的影响提供科学依据。

邻苯二甲酸酯（Phthalates，PAEs）是一类常见的塑化剂，广泛存在于人类的生产和生活用品中，其作为一种内分泌干扰物，可能会男性生殖健康造成不利影响。因此，我们检测了1066个育龄男性尿液中14种PAEs水平，并与男性生殖健康指标（血清INSL3及激素水平，精子质量，顶体酶活性等）进行相关性分析。发现MEHP及%MEHP与INSL3 负相关；MBP、MiBP，MEHP和%MEHP与顶体酶活性负相关；MBP和MiBP与血清睾酮水平、游离雄激素指数，黄体生成素和精子形态存在显著负相关，而与精子DNA碎片化指数（DFI）正相关，暗示邻苯二甲酸酯在环境剂量下可能对生殖功能造成多方面的影响。另外，发现尿液中5mdC及5hmdC浓度与低分子量、高分子量及总PAESs浓度存在显著正相关，尿液中5mdC浓度与精子浓度、精子活力及精子浓度存在显著关联，暗示尿液中的5mdC可能是男性生殖健康潜在的生物标记物。.进一步利用交叉哺乳模型，用MiBP的前体DiBP（450 mg/kg/d）暴露孕鼠，F1代小鼠出生后继续母乳暴露21天（TT和TC），断奶后正常饮食（无DiBP暴露）至80天（TCC和TTC）。分别观察DiBP孕期及哺乳期暴露对21日龄和80日龄F1代子鼠的生殖毒性效应。生殖毒性指标包括脏器系数，精子数量和活力及睾酮水平等。研究结果发现：孕期哺乳期暴露可以显著降低21日龄（TC和TT组）和80日龄（TTC组）F1代仔鼠血清和睾丸睾酮水平，并且显著降低3b-HSD和CYP11A1基因的表达水平，同时，孕期及哺乳期暴露组（TTC）组F1代仔鼠精子浓度及活力均显著下降。以上结果说明，DiBP的早期暴露（孕期和出生后）可以通过降低睾酮水平或CYP11A1的表达对成年个体的雄性生殖功能造成损伤，而睾酮合成酶可能是这类物质的靶点。.另外，我们选择具有内分泌干扰效应的双酚AF（BPAF），对孕鼠进行暴露（100 mg/kg/d），F1代仔鼠进行交叉哺乳，发现BPAF可以通过脐带血在F1代仔鼠睾丸内积累；子宫和母乳双重暴露可以增加F1代雄性仔鼠睾丸睾酮水平，降低睾丸抑制素B水平，同时暴露后仔鼠睾丸中279个基因表达发生变化，这些基因参与细胞分化与分裂等，暗示BPAF子宫和母乳暴露可以损伤下一代雄性生殖功能。