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".

由于R/D，生殖和发育（R/D）毒性的风险评估尤其具有挑战性 系统非常复杂，涉及在不同时间点的多个器官和系统之间的相互作用 和生活阶段。自从NAS（2007）的报告“ 21世纪的毒性测试”以来 体内动物测试最终可以用硅和体外方法的组合取代，需要 确定用于R/D的体外系统的毒性测试已增长。多年来，许多替代方法 作为用于评估R/D毒性的测试电池的一部分开发，但大多数集中于胚胎毒性 测试。需要开发针对诸如精子发生的过程的体外测定法。我们 已经开发了三维睾丸细胞共培养系统（3D-TC），包括胚芽，Sertoli和 leydig细胞。我们最初的验证研究表明，该3D-TC可以区分已知 发育有毒的邻苯二甲酸酯来自无毒的邻苯二甲酸酯。因此，进一步验证我们 已建立的3D-TCS系统对于允许该有前途的模型成为集成测试的一部分至关重要 用于R/D毒性评估的电池。在此提案中，我们将建立“黄金标准测试化合物”的列表 并在我们的3D-TCS系统中盲目测试这些测定的可预测性。我们建议整合基因组和 代谢组学方法开发基于途径的测定法，以使该3D-TCS更加有效，并且 基于机械的。这些测定不仅将直接链接到男性生殖的终点 毒性，但也与发育毒性有关，因为这些途径在 男性和女性的正常发育。具体目的（SAS）是（1）建立3D-TCS R/D毒性评估的模型； （2）定量比较毒素和代谢组的作用 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 R/D毒性的基因组和代谢组学“特征”； （3）开发基于综合途径的高 R/D毒性评估的3D-TCS模型中的内容（HCS）和高吞吐量（HTS）筛选分析。 SA1的里程碑是（1）优化和最终确定3D-TCS的标准操作程序； （2） 创建一个“黄金标准测试化合物”的数据库； （3）获取有关剂量和时间依赖的数据 70种化合物的细胞毒性和细胞活力； （4）计算预测性，精度和准确性 在此3D-TCS模型中测试的三类化合物。对于SA2，里程碑是（1）产生基因组 3D-SGC模型中40种化合物的曲线和（2）代谢组谱。对于SA3，里程碑是 （1）在3D-SGC模型中建立HCS/HTS分析； （2）生成和型号的R/D毒性用于评估 R/D有毒和非R/D有毒化合物； （3）设计指标用于接受或拒绝R/D有毒 基于剂量和基于时间依赖的途径的“ R/D毒性特征”，与非R/D有毒化合物相比。