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

生殖和发育(R/D)毒性的风险评估特别具有挑战性，因为 系统高度复杂，涉及多个器官和系统在不同时间点的相互作用 和生命阶段。由于美国国家科学院在《21世纪的毒性测试》报告(2007)中预计， 活体动物试验最终可以被体内和体外相结合的方法所取代，需要 用于R/D毒性试验的体外鉴定系统已经发展起来。多年来，已经有了许多替代方法 作为测试电池的一部分，用于评估R/D毒性，但大多数侧重于胚胎毒性 测试。非常需要开发针对精子发生等过程的体外检测方法。我们 开发了三维睾丸细胞共培养系统(3D-TCS)，包括生殖细胞、Sertoli和 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提取的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系统将是使这一前景光明的模型成为集成测试的一部分的关键 用于研发毒性评估的电池。在这份提案中，我们将建立一个“黄金标准测试化合物”的清单 并在我们的3D-TCS系统中盲目测试这些分析的可预测性。我们建议将基因组和 代谢学方法，以开发基于途径的分析，使这种3D-TCS更有效和 以机械为基础。这些化验结果不仅直接与男性生殖终点有关 毒性，但也与发育毒性有关，因为所提出的这些途径在 男性和女性都有正常的发育。具体目标是：(1)建立3D-TCS R/D毒性评价模型；(2)定量比较青蒿素的毒理基因组效应和代谢效应 使用我们已建立的基于GO-QUANT分析方法的系统来识别潜在的研发毒物 R/D毒性的基因组和代谢“信号”；以及(3)开发基于整合途径的高 3D-TCS模型中含量(HCS)和高通量(HTS)筛选试验用于R/D毒性评估。 SA1的里程碑是：(1)优化和最后确定3D-TCS的标准操作程序；(2) 建立“黄金标准测试化合物”数据库；(3)获得与剂量和时间相关的数据 70种化合物的细胞毒性和细胞活力；以及(4)计算其预测性、精密度和准确性 在这个3D-TCS模型中测试了三类化合物。对于SA2，里程碑是(1)产生基因组 和(2)3D-SGC模型中40种化合物的代谢谱。对于SA3，里程碑是 (1)在3D-SGC模型中建立HCS/HTS检测方法；(2)建立用于评价R/D毒性的模型 R/D有毒和非R/D有毒化合物；(3)接受或拒绝R/D有毒的设计指标 与基于剂量和时间依赖途径的“R/D毒性特征”的非R/D有毒化合物进行比较。

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".