Title: In Vitro Analysis of the Effects of Acute and Chronic Phthalate Exposures on Leydig Cell Testosterone Production, and the Molecular Mechanisms Involved

项目摘要 通过干扰正常的荷尔蒙功能和作用，内分泌干扰物(EDCs)，如 增塑剂邻苯二甲酸二(2-乙基己基)酯(DEHP)会对男性生殖系统产生负面影响。在……里面 在之前的研究中，Traore博士发现，在子宫内暴露于DEHP会导致血清睾酮水平下降 在成人体内的水平。在体外进行的研究中，他发现急性(短期)暴露于单-2-乙基己基 邻苯二甲酸酯(MEHP)是DEHP的代谢物，可抑制分离的间质细胞产生睾酮。 MA-10小鼠肿瘤间质细胞孕酮的产生。此外，他发现MEHP会对人体产生负面影响 影响MA-10细胞的线粒体功能和胆固醇移位。直到最近，这还不可行。 使用从睾丸分离的间质细胞进行长期研究，因为尽管这些细胞能够 产生高水平的睾丸素，它们的作用时间不会超过1-2天。因此，这是不可行的 使用分离的间质细胞来检查环境相关浓度的直接、长期影响 或批判性地评估邻苯二甲酸酯类作用的机制。Zirkin博士在约翰·霍普金斯大学的实验室 彭博公共卫生学院(JHSPH)最近开发了一种方法，可以用来提取间质细胞 通过与生精小管相关的干细胞的体外分化，以及衍生的间质 细胞能够产生并维持高水平的睾酮产生数月。可得性 这些细胞对我们提出的对睾酮产生的影响的研究将具有重要的价值 间质细胞对环境相关邻苯二甲酸盐的急性、慢性和重复暴露 浓度，特别是为了解决邻苯二甲酸盐的分子机制- 导致睾丸素的减少。我们在此R15续订申请中提出的研究将： (1)加深对环境应激源调节抑制的机制的理解 睾丸激素的产生；以及(2)提供关于间质细胞多次暴露于 邻苯二甲酸盐，我们一生中接触到的化学物质；以及3)解决早期接触是否 在以后的生活中易受随后的无毒剂量暴露的影响。坎贝尔大学有 将参与建议的优秀医学生、研究生和本科生 研究。用当前申请寻求续签的REAP赠款的资金，Traore博士支持和 他在坎贝尔大学的实验室里培训了许多学生，这是一所位于农村地区的机构，服务不足 北卡罗来纳州。延长拨款将允许Traore博士继续并极大地扩展正在进行的研究 同时提高学生对基础科学和/或临床研究的兴趣。此外，他的 研究的成功无疑将影响吸引其他坎贝尔大学教职员工寻求资金发展 他们实验室里的研究项目。

Project Summary By interfering with normal hormonal function and action, endocrine-disrupting chemicals (EDCs) such as the plasticizer di-(2-ethylhexyl) phthalate (DEHP) can have negative effects on the male reproductive system. In previous studies, Dr. Traore found that in utero exposure to DEHP can result in reduced serum testosterone levels in the adult. In studies conducted in vitro, he found that acute (short-term) exposure to mono-2-ethylhexyl phthalate (MEHP), a metabolite of DEHP, can inhibit testosterone production by isolated Leydig cells and progesterone production by MA-10 mouse tumor Leydig cells. Additionally, he found that MEHP can negatively affect mitochondrial function and cholesterol translocation in MA-10 cells. Until recently, it has not been feasible to conduct long-term studies using Leydig cells isolated from the testis because although these cells are able to produce high levels of testosterone, they do not do so for longer than 1-2 days. Therefore, it has not been feasible to use isolated Leydig cells to examine the direct, long-term effects of environmentally relevant concentrations of EDCs, or to critically evaluate mechanisms involved in phthalate actions. Dr. Zirkin’s lab at the Johns Hopkins Bloomberg School of Public Health (JHSPH) recently developed a method by which Leydig cells can be derived from the in vitro differentiation of stem cells associated with the seminiferous tubules, and that the derived Leydig cells are able to produce and sustain high levels of testosterone production for several months. The availability of these cells will be of great value for our proposed studies of the effects on testosterone production of acute, chronic, and repeat exposures of Leydig cells to environmentally relevant phthalate concentrations, and particularly so for addressing the molecular mechanisms involved in phthalate- induced reductions in testosterone. The studies that we propose in this R15 renewal application will: (1) increase our understanding of the mechanism by which environmental stressors mediate the inhibition of testosterone hormone production; and (2) provide new insights regarding multiple exposure of Leydig cells to phthalates, chemicals to which we are exposed throughout life; and 3) address whether early exposures predispose susceptibility to subsequent non-toxic dose exposure later in life. Campbell University has outstanding medical students, graduate students and undergraduates who will be involved in the proposed research. With funds from the REAP grant that the current application seeks to renew, Dr. Traore supported and trained many students in his lab at Campbell University, an institution located in a rural, underserved area of North Carolina. Renewal of the grant will allow Dr. Traore to continue and greatly extend ongoing research while increasing the interest in basic science and/or clinical research of the involved students. Moreover, his research success undoubtedly will influence attract other Campbell University faculty to seek funding to develop research programs in their labs.