Computational model for fetal skeletal defects potentially linked to disruption of retinoic acid signaling

Abstract

All-trans retinoic acid (ATRA) gradients determine skeletal patterning morphogenesis and can be disrupted by diverse genetic or environmental factors during pregnancy, leading to fetal skeleton defects. Adverse Outcome Pathway (AOP) frameworks for ATRA metabolism, signaling, and homeostasis allow for the development of new approach methods (NAMs) for predictive toxicology with less reliance on animal testing. Here, a data-driven model was constructed to identify chemicals associated with both ATRA pathway bioactivity and prenatal skeletal defects. The phenotype data was culled from ToxRefDB prenatal developmental toxicity studies and produced a list of 363 ToxRefDB chemicals with altered skeletal observations. Defects were classified regionally as cranial, post-cranial axial, appendicular, and other (unspecified) features based on ToxRefDB descriptors. To build a multivariate statistical model, high-throughput screening bioactivity data from >8,070 chemicals in ToxCast/Tox21 across 10 in vitro assays relevant to the retinoid signaling system were evaluated and compared to literature-based candidate reference chemicals in the dataset. There were 48 chemicals identified for effects on both in vivo skeletal defects and in vitro ATRA pathway targets for computational modeling. The list included 28 chemicals with prior evidence of skeletal defects linked to retinoid toxicity and 20 chemicals without prior evidence. The combination of thoracic cage defects and DR5 (direct repeats of 5 nucleotides for RAR/RXR transactivation) disruption was the most frequently occurring phenotypic and target disturbance, respectively. This data model provides valuable AOP elucidation and validates current mechanistic understanding. These findings also shed light on potential avenues for new mechanistic discoveries related to ATRA pathway disruption and associated skeletal dysmorphogenesis due to environmental exposures.

Development and validation of CYP26A1 inhibition assay for high‐throughput screening

“…These chemicals were selected based on their demonstrated activity against diverse protein targets that are linked to the retinoid system, which contributes to developmental processes and toxicities. [28,29] This test set included twelve known CYP26 A retinoid with higher affinity toward RAR-β and -γ. [42] Aldrin (309-00-2) 13.0 ± 1.8 (−95.35 ± 4.06) An organochlorine pesticide with agonistic activity at RARβ and RARγ.…”

“…These chemicals were selected based on their demonstrated activity against diverse protein targets that are linked to the retinoid system, which contributes to developmental processes and toxicities. [ 28,29 ] This test set included twelve known CYP26 inhibitors, various other CYP inhibitors, eight retinoids, two rexinoids (RXR‐selective retinoids), and three RAMBAs. The assay performed well with S/B ratio exceeding 25‐fold, CV of 8.9%, and a Z’ of 0.7.…”

“…These compounds were previously predicted to be potential developmental toxicants associated with atRA signaling pathway disruptions. [28,29] The assay was performed in three independent runs (n = 3). The highest concentration of each compound tested in the CYP26A1 assay was 115 µM, except for propargite (1.7 mM), and SSR126768 (109 µM).…”

Read‐Across Application for Food or Feed Ingredients

“…CYP26 is not measured in ToxCast, but two surrogate enzymes CYP1A1 and CYP2C8. Relevant assays tested for retinoic acid pathways from ToxCast were suggested by Pierro et al, 2022 regarding All-trans retinoic acid (ATRA) gradients. Results indicated inactivity for most assays related to ATRA.…”

“…Table 17: Assays related to ATRA for most similar compounds identified by biological similarity (Pierro et al, 2022) Propiconazole Tetraconazole…”