3D Testicular Cells Co-Culture Model for Reproductive and Developmental Toxicity

用于生殖和发育毒性的 3D 睾丸细胞共培养模型

• 批准号: 8333908
• 负责人: Elaine M Faustman
• 金额: $ 37.87万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-21 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8333908
• 关键词: 3D; Testicular; Cells; Co; Culture

Risk assessment for reproductive and developmental (R/D) toxicity is particularly challenging since the R/D system is highly complex, involving interactions between multiple organs and systems at different time points and life stages. Since the report "Toxicity Testing in the 21st Century" by the NAS (2007) envisioned that in vivo animal testing can eventually be replaced by a combination of in silico and in vitro approaches, the need to identify in vitro systems for R/D toxicity test has grown. Over the years, many alternative methods have been developed as a part of a test battery for assessing R/D toxicity, but the majority are focused on embryotoxicity testing. There is a great need to develop in vitro assays targeting processes such as spermatogenesis. We have developed a three-dimensional testicular cells co-culture system (3D-TCS) including germ, Sertoli and Leydig cells. Our initial validation studies demonstrated that this 3D-TCS can discriminate known developmentally toxic phthalate esters from non-toxic phthalate esters. Therefore, further validation of our established 3D-TCS system will be critical to allow this promising model to become part of an integrated testing battery for R/D toxicity assessment. In this proposal, we will build a list of "gold standard testing compounds" and blindly test the predictability of these assays in our 3D-TCS system. We propose to integrate genomic and metabolomic approaches to develop pathways-based assays to make this 3D-TCS more efficient and mechanistically based. These assays will not only be directly linked to the endpoint of male reproductive toxicity, but also linked to developmental toxicity since these pathways proposed also play critical roles during normal development for both males and females. The specific aims (SAs) are to (1) establish the 3D-TCS model for R/D toxicity evaluation; (2) to quantitatively compare toxicogenomic and metabolomic effects of potential R/D toxicants by using our established systems based GO-Quant analysis approach to identify genomic and metabolomic "signatures" for R/D toxicity; and (3) to develop an integrated pathway-based high content (HCS) and high throughput (HTS) screening assay in the 3D-TCS model for R/D toxicity evaluation. The milestones for SA1 are to (1) optimize and finalize a Standard Operating Procedure for the 3D-TCS; (2) create a database of "gold standard testing compounds;" (3) obtain dose and time dependent data on cytotoxicity and cell viability for 70 compounds; and (4) calculate the predictivity, precision, and accuracy for three classes of compounds tested in this 3D-TCS model. For SA2, the milestones are to (1) generate genomic profiles and (2) metabolomic profiles for the 40 compounds in the 3D-SGC model. For SA3, the milestones are to (1) establish the HCS/HTS assay in the 3D-SGC model; (2) generate and model R/D toxicity for evaluating the R/D toxic and non-R/D toxic compounds; and (3) design metrics for acceptance or rejection of R/D toxic versus non-R/D toxic compounds based on dose and time-dependent pathway-based "R/D toxicity signatures".

生殖和发育（R/D）毒性的风险评估特别具有挑战性，因为R/D 系统是高度复杂的，涉及多个器官和系统在不同时间点的相互作用 生命的各个阶段。由于NAS（2007年）的报告“21世纪世纪的毒性测试”设想， 体内动物试验最终可以被计算机和体外方法的组合所取代， 用于确定体外毒性测试系统的研发已经发展壮大。多年来，已经有许多替代方法， 作为评估研发毒性的测试组合的一部分开发，但大多数都集中在胚胎毒性上 试验.非常需要开发靶向过程如精子发生的体外测定。我们 已经开发了一种三维睾丸细胞共培养系统（3D-TCS），包括生殖细胞、Sertoli细胞和 睾丸间质细胞。我们的初步验证研究表明，这种3D-TCS可以区分已知的 从无毒的邻苯二甲酸酯中分离出具有发育毒性的邻苯二甲酸酯。因此，进一步验证我们的 建立3D-TCS系统将是至关重要的，使这个有前途的模型成为一个综合测试的一部分 用于研究/发展毒性评估的成套工具。在这份提案中，我们将建立一个“金标准测试化合物”清单。 并在我们的3D-TCS系统中盲测这些测定的可预测性。我们建议整合基因组和 代谢组学方法，以开发基于途径的测定，使这种3D-TCS更有效， 机械基础。这些检测不仅直接与男性生殖终点相关， 毒性，但也与发育毒性有关，因为这些途径也在发育过程中发挥关键作用。 男性和女性的正常发育。具体目标（SA）是（1）建立三维TCS R/D毒性评价模型;（2）定量比较 潜在的R/D有毒物质，通过使用我们建立的基于系统的GO-Quant分析方法来识别 基因组学和代谢组学“签名”的R/D毒性;和（3）开发一个综合的路径为基础的高 内容（HCS）和高通量（HTS）筛选试验中的3D-TCS模型的R/D毒性评价。 SA 1的里程碑是（1）优化并最终确定3D-TCS的标准操作规程;（2） 建立一个“金标准测试化合物”数据库;（3）获得剂量和时间依赖性数据， 70种化合物的细胞毒性和细胞活力;以及（4）计算70种化合物的预测性、精密度和准确度。 在该3D-TCS模型中测试的三类化合物。对于SA 2，里程碑是（1）生成基因组 3D-SGC模型中40种化合物的代谢组学特征。对于SA 3，里程碑包括 （1）在3D-SGC模型中建立HCS/HTS测定;（2）生成R/D毒性并建模以进行评估 R/D有毒和非R/D有毒化合物;（3）设计接受或拒绝R/D有毒化合物的指标 与非R/D毒性化合物相比，基于剂量和时间依赖性途径的“R/D毒性特征”。