Grantee Research Project Results
2020 Progress Report: Skeletal Teratogenicity of Industrial and Environmental Chemicals Predicted with Human Pluripotent Stem Cells in Vitro
EPA Grant Number: R839502Title: Skeletal Teratogenicity of Industrial and Environmental Chemicals Predicted with Human Pluripotent Stem Cells in Vitro
Investigators: zur Nieden, Nicole I , Volz, David C.
Institution: University of California - Riverside
EPA Project Officer: Aja, Hayley
Project Period: August 1, 2019 through July 31, 2022 (Extended to July 31, 2024)
Project Period Covered by this Report: August 1, 2019 through July 31,2020
Project Amount: $849,811
RFA: Advancing Actionable Alternatives to Vertebrate Animal Testing for Chemical Safety Assessment (2018) RFA Text | Recipients Lists
Research Category: Chemical Safety for Sustainability
Objective:
We hypothesize that cultures of human embryonic stem cells (hESCs) can be utilized to predict the skeletal embryotoxicity of industrial and environmental chemicals in vitro. The objectives of this study are to differentiate hESCs into bone-forming osteoblasts with concomitant chemical exposure (the test bank includes selected chemicals from the ToxCast I library with known effects on the skeleton). Detrimental effects of the chemical are evaluated based on a reduction in matrix mineralization (as a surrogate of skeletal maturation). To identify suitable assays for matrix mineralization that adequately predict human risk, we compare inexpensive absorbance-based assays and image analysis tools recently developed by us. Hierarchies of chemical potency and efficacy will be established by setting the half-maximal inhibitory doses of differentiation inhibition in relation to the half-maximal cytotoxic dose using a biostatistical model, canonical linear discriminants, and integration of our new data into EPA’s Toxicology Priority Index to create a skeletal embryotoxicity score. A case study will determine whether the scoring system is sufficiently sensitive to distinguish between closely related chemical derivatives and will consequently establish whether the assay can be used to identify safer chemical alternatives. Lastly, testing the chemicals with differentiation protocols that generate only neural crest or mesodermal osteoblasts coupled with next-generation sequencing will identify potential modes-of-action (disruption of organogenesis) and mechanisms of toxicity (molecular-level initiating event).
Progress Summary:
In order to develop a predictive in vitro assay, we have selected chemicals from the ToxCastI library with known effects on the skeleton associated with embryonic exposure. From this list, we have so far tested Benz[a]anthracene (B(a)A), Coumarin, Triadimenol, Triadimefon, Cyclopamine, Quinoline, 17α-Estradiol, Chlorpyrifos, and DEET. Concentration response curves have been obtained for absorbance-based assay endpoints. The first six of these chemicals were then evaluated with an existing biostatistical prediction model found to only partially predict embryotoxicity classes according to the chemical’s known in vivo effects. This was an expected result and lends evidence to the hypothesis that a different prediction model needs to be created, as is the objective of this proposal. In preparation for this, we have additionally screened three of four reference chemicals including two that serve as positive and one that serves as a negative control. These three chemicals classified correctly when using the existing prediction model. The results to date put as well on the trajectory towards achieving our objectives as proposed.
Future Activities:
Screening the same chemicals with mouse cells and comparing results to those obtained with human cells will help establish the predictivity of our in vitro assay for human risk associated with exposure. Finalizing chemical screening with the human cells will allow the development of a new biostatistical prediction model, specifically geared toward risk for skeletal malformations. Similarly, once we regain access to equipment on our campus, currently non-usable due to COVID-19 prevention measures, alternative, less expensive endpoints will be evaluated for predictivity, which has the potential to bring down the costs for the in vitro assay and effectively allow widespread application.
Journal Articles:
No journal articles submitted with this report: View all 6 publications for this projectSupplemental Keywords:
exposure, risk assessment, sensitive populations, dose-response, teratogen, develop-mental toxicity, pluripotent stem cells, adverse outcome pathway, imaging, chemical derivatives.Progress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.