Risk assessment for reproductive and developmental (R/D) toxicity is particularly challenging since the R/Dsystem is highly complex, involving interactions between multiple organs and systems at different time pointsand life stages. Since the report 'Toxicity Testing in the 21st Century' by the NAS (2007) envisioned that invivo animal testing can eventually be replaced by a combination of in silico and in vitro approaches, the needto identify in vitro systems for R/D toxicity test has grown. Over the years, many alternative methods have beendeveloped as a part of a test battery for assessing R/D toxicity, but the majority are focused on embryotoxicitytesting. There is a great need to develop in vitro assays targeting processes such as spermatogenesis. Wehave developed a three-dimensional testicular cells co-culture system (3D-TCS) including germ, Sertoli andLeydig cells. Our initial validation studies demonstrated that this 3D-TCS can discriminate knowndevelopmentally toxic phthalate esters from non-toxic phthalate esters. Therefore, further validation of ourestablished 3D-TCS system will be critical to allow this promising model to become part of an integrated testingbattery 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 andmetabolomic approaches to develop pathways-based assays to make this 3D-TCS more efficient andmechanistically based. These assays will not only be directly linked to the endpoint of male reproductivetoxicity, but also linked to developmental toxicity since these pathways proposed also play critical roles duringnormal development for both males and females.
The specific aims (SAs) are to (1) establish the 3D-TCSmodel for R/D toxicity evaluation; (2) to quantitatively compare toxicogenomic and metabolomic effects ofpotential R/D toxicants by using our established systems based GO-Quant analysis approach to identifygenomic and metabolomic 'signatures' for R/D toxicity; and (3) to develop an integrated pathway-based highcontent (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 oncytotoxicity and cell viability for 70 compounds; and (4) calculate the predictivity, precision, and accuracy forthree classes of compounds tested in this 3D-TCS model. For SA2, the milestones are to (1) generate genomicprofiles and (2) metabolomic profiles for the 40 compounds in the 3D-SGC model. For SA3, the milestones areto (1) establish the HCS/HTS assay in the 3D-SGC model; (2) generate and model R/D toxicity for evaluatingthe R/D toxic and non-R/D toxic compounds; and (3) design metrics for acceptance or rejection of R/D toxicversus non-R/D toxic compounds based on dose and time-dependent pathway-based 'R/D toxicity signatures'.
challenge for toxicologists has been the need to identify chemicals for their potential to cause reproductive and developmental (R/D) toxicity. Through the milestone-driven research proposed, we will show that the 3D-TCS provides a stable HCS/HTS assay system, makes accurate prediction of potential for adverse effects of R/D toxicity, and paves the way for both accelerated and mechanistically based testing of compounds while significantly reducing use of animals. Thus, this proposal is of great significance to public health.
| Weldon, Brittany A; Griffith, William C; Workman, Tomomi et al. (2018) In vitro to in vivo benchmark dose comparisons to inform risk assessment of quantum dot nanomaterials. Wiley Interdiscip Rev Nanomed Nanobiotechnol 10:e1507 |
| Harris, Sean; Wegner, Susanna; Hong, Sung Woo et al. (2016) Phthalate metabolism and kinetics in an in vitro model of testis development. Toxicol In Vitro 32:123-31 |
| Harris, Sean; Hermsen, Sanne A B; Yu, Xiaozhong et al. (2015) Comparison of toxicogenomic responses to phthalate ester exposure in an organotypic testis co-culture model and responses observed in vivo. Reprod Toxicol 58:149-59 |
| Amory, John K; Hong, SungWoo; Yu, Xiaozhong et al. (2014) Melphalan, alone or conjugated to an FSH-? peptide, kills murine testicular cells in vitro and transiently suppresses murine spermatogenesis in vivo. Theriogenology 82:152-9 |
| Wegner, Susanna; Hong, Sungwoo; Yu, Xiaozhong et al. (2013) Preparation of rodent testis co-cultures. Curr Protoc Toxicol Chapter 16:Unit 16.10 |
