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

通过干扰激素的功能和作用，内分泌干扰化学品（EDCs），如增塑剂邻苯二甲酸二（2-乙基己基）酯（DEHP），可对发育和生殖产生负面影响。在伊丽莎白城州立大学担任教职时，Traore博士获得了一项研究促进奖（SC 2），他发现妊娠期间体内接触DEHP对成年人的睾酮（T）产生有负面影响，并且早期接触会加剧后续接触的影响。现在在坎贝尔大学医学院，Traore博士建议进行体外机制研究，研究DEHP对Leydig细胞产生T细胞的急性和慢性影响。直到最近，通过可用的方法分离和培养的Leydig细胞已经能够产生并维持高水平的T产生仅3-5天。因此，它还没有可行的体外使用原代细胞检查环境相关浓度的内分泌干扰物的直接，长期的影响，或严格评估其作用于Leydig细胞的机制。然而，兹金博士在约翰霍普金斯的实验室 彭博公共卫生学院（JHSPH）最近开发了一种方法，通过该方法，来自Leydig干细胞分化的Leydig细胞能够产生并维持更长时间（数月）的高水平T细胞生产。Zirkin博士曾担任SC2奖的导师职位，他将继续与Traore博士和他的学生合作。初级Leydig细胞能够维持高水平的T生产的可用性将是非常有价值的，我们提出的研究环境相关的邻苯二甲酸酯浓度在高度受控的条件下的慢性效应，以及它们的功能机制。随着这些细胞的可用性，我们将进行体外研究MEHP对干细胞间质细胞形成的直接影响，MEHP暴露对成年间质细胞的影响，以及早期暴露对后续暴露后果的影响。我们还将对邻苯二甲酸酯诱导的氧化应激对T细胞的急性和长期影响进行机制研究。 生产坎贝尔大学拥有拟议研究所需的设备和设施，以及 优秀的医学生、研究生和本科生将参与本研究项目。有了这笔REAP赠款的资金，Traore博士将能够支持和扩大他在坎贝尔大学实验室正在进行的研究，该大学位于北卡罗来纳州的一个农村，服务不足的地区。约翰霍普金斯公共卫生学院和坎贝尔大学之间的广泛合作不仅有助于激发坎贝尔大学的学生，而且还增加了他们考虑从事基础科学或临床研究的兴趣。此外，该项目无疑将吸引其他坎贝尔大学的教师寻求资金来发展他们的研究，并有助于加强校园的研究环境。

By interfering with hormone function and action, endocrine-disrupting chemicals (EDCs) such as the plasticizer di-(2-ethylhexyl) phthalate (DEHP) can have negative effects on development and reproduction. With the support of a Research Enhancement (SC2) Award, when he was a faculty member at Elizabeth City State University, Dr. Traore found that in vivo exposure to DEHP during gestation had negative effects on testosterone (T) production in the adult, and that early exposure exacerbated the effects of subsequent exposures. Now at Campbell University School of Medicine, Dr. Traore is proposing to conduct in vitro mechanistic studies of both acute and chronic DEHP effects on T production by Leydig cells. Until recently, Leydig cells isolated and cultured by available methods have been able to produce and maintain high levels of T production for only 3-5 days. Therefore, it has not yet been feasible to use primary cells in vitro to examine the direct, long-term effects of environmentally relevant concentrations of EDCs or to critically evaluate mechanisms involved in their actions on Leydig cells. However, Dr. Zirkin’s lab at the Johns Hopkins Bloomberg School of Public Health (JHSPH) has recently developed a method by which Leydig cells derived from the differentiation of Leydig stem cells are able to produce and sustain high levels of T production for longer periods, several months. Dr. Zirkin, who served in a mentorship position for the SC2 Award, is continuing to collaborate with Dr. Traore and his students. The availability of primary Leydig cells able to sustain high levels of T production will be of great value for our proposed studies of the chronic effects of environmentally relevant phthalate concentrations under highly controlled conditions, and the mechanisms by which they function. With the availability of these cells, we will conduct in vitro studies of the direct effects of MEHP on Leydig cell formation from stem cells, the effects of MEHP exposure on the adult Leydig cells, and the effects of early exposures on the consequences of subsequent exposures. We also will conduct mechanistic studies of acute and long-term effects of phthalate-induced oxidative stress on T production. Campbell University has the equipment and facilities necessary for the proposed studies, as well as outstanding medical students, graduate students and undergraduates who will be involved in this research project. With funds of this REAP grant, Dr. Traore will be able to support and expand ongoing research in his lab at Campbell University, an institution located in a rural, underserved area of North Carolina. The extensive collaboration between the Johns Hopkins School of Public Health and Campbell University will not only help excite Campbell University students but also increase their interest in considering careers in basic science or clinical research. Moreover, the project undoubtedly will attract other Campbell University faculty to seek funding to develop their research, and help strengthen the research environment on campus.