Grantee Research Project Results

2022 Progress Report: Skeletal Teratogenicity of Industrial and Environmental Chemicals Predicted with Human Pluripotent Stem Cells in Vitro

EPA Grant Number: R839502
Title: 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: Packard, Benjamin H
Project Period: August 1, 2019 through July 31, 2021 (Extended to July 31, 2025)
Project Period Covered by this Report: August 1, 2021 through July 31,2022
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 whether the chemicals affect 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 17 chemicals with human and mouse cells, completing our Training Set chemicals with the exception of one chemical. In addition, we have obtained concentration-response curves for four reference chemicals that exhibit either strong embryotoxicity or none. All chemicals tested so far have been evaluated with an existing biostatistical prediction model, which found to only partially predict embryotoxicity classes according to the chemical’s known in vivo effects when screened using mouse cells. However, when human cells were used, only one chemical was incorrectly predicted. In addition to the Training Set chemicals, we also screened 4 chemicals from the Test Set in all human endpoints (hESC viability, hESC differentiation, human fibroblast viability) and another 8 in both hESC endpoints
(fibroblast endpoint to be completed in the coming months). When the half-maximal inhibitory concentrations for cell viability and differentiation were charted using a canonical plot applying the boundaries found with the Training Set, all human Test Set chemicals classified correctly according to their in vivo embryotoxic potential. The results to date put as well on the trajectory towards achieving our objectives as proposed. Towards the identification of adverse outcome pathways, we have harvested cells for RNA isolation from 17 chemicals and a solvent control, in 5 replicates each.

Future Activities:

Finalizing the chemicals screen for the Test Set in cell types of both mouse and human will help establish the predictivity of our in vitro assay for human risk associated with exposure and the development of a new biostatistical prediction model, specifically geared toward risk for skeletal malformations. Once we regain access to equipment on our campus, currently non-usable due to COVID-19 prevention measures, we will evaluate alternative, less expensive endpoints for cytotoxicity (viability) and differentiation inhibition. The coming months will see RNA library preparations as well as sequencing runs. We also intend to generate an hESC reporter cell line that will fluoresce in different colors depending on the embryonic origin of the osteoblasts that differentiate in culture (neural crest-purple, paraxial mesoderm orange and lateral plate mesoderm green) and repeat exposures coupled with quantitative fluorescent imaging.

Journal Articles on this Report : 1 Displayed | Download in RIS Format

Publications Views
Other project views:	All 8 publications	3 publications in selected types	All 3 journal articles

Publications
Type	Citation	Project	Document Sources
Journal Article	Karmach O, Madrid J, Dasgupta S, Volz D, zur Nieden N. Embryonic Exposure to Cigarette Smoke Extract Impedes Skeletal Development and Evokes Craniofacial Defects in Zebrafish. MOLECULAR SCIENCES 2022;23(17):9904	R839502 (2022)	Full-text from PubMed Associated PubMed link Full-text: MDPI - Full Text HTML Exit

Supplemental 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 Abstract

The 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.