Grantee Research Project Results
2023 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: Callan, Richard
Project Period: August 1, 2019 through July 31, 2022 (Extended to July 31, 2024)
Project Period Covered by this Report: August 1, 2022 through July 31,2023
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:
To develop a predictive in vitro assay, we had initially obtained concentration-response curves for four reference chemicals that exhibit either strong embryotoxicity or none in vivo and assessed whether we received comparable responses in vitro. We had next selected chemicals from the ToxCastI library with known effects on the skeleton associated with embryonic exposure and separated those into a Training Set and a Test Set. We have completed the screening of the Training Set chemicals and an additional 17 chemicals from the Test Set. Using a biostatistical prediction model, we determined the predictivity of the human cell-based assay to be 95.5% in the Training Set and 94% in the Test Set. Using linear discriminant analysis, the predictivity of the human cell-based assay was also 94%. However, when using mouse cells, the predictivity dropped to 63.6% in the Training Set and to 39% in the Test Set
(biostatistical model), but increased to 83.3% when we applied linear discriminant functions. Thus, our overall results so far suggest that the human assay seems much better suited to predict risk for human exposure than the mouse assay. Next, we have applied this assay to the prediction of skeletal embryotoxicity associated with exposure to constituents in tobacco smoke and identified catechol, coumarin, acetaldehyde and acrolein as strongly embryotoxic to developing bone cells and quinoline and benzo[a]pyrene as weakly skeletally embryotoxic
Future Activities:
We plan to continue our screen with the last chemicals of the Test Set and then move fully into the screening of TSCA priority chemicals as well as thiourea derivatives. These studies are expected to bring new knowledge about chemical effects on the developing skeleton not yet known. We will also continue our efforts to identify ways to automate calcification analyses and cell number from images including identification of suitable staining methods.
Journal Articles on this Report : 1 Displayed | Download in RIS Format
Other project views: | All 6 publications | 3 publications in selected types | All 3 journal articles |
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Madrid JV, Vera-Colon MK, zur Nieden NI. Perturbations in osteogenic cell fate following exposure to constituents present in tobacco:a combinatorial study. Toxics 2023;11(12):998. |
R839502 (2023) |
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Supplemental Keywords:
exposure, risk assessment, sensitive populations, dose-response, teratogen, develop-mental toxicity, pluripotent stem cells, adverse outcome pathway, imaging, chemical derivativesProgress 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.