Refining the AOP for retinoid-induced teratogenicity: Insights into RAR/RXR overactivation and RXR cross-talk with retinoic acid and thyroid hormone signaling

Retinoic acid (RA), a vitamin A metabolite, plays a crucial and evolutionarily conserved role in vertebrate development. Chemical disruption of retinoid signaling can severely affect organisms; yet, despite its teratogenicity and pathway interactions, this disruption remains understudied. Increasing research aims to address these gaps by developing comprehensive frameworks like Adverse Outcome Pathways (AOPs). This study refines a previously proposed AOP network on retinoid-induced teratogenicity by: (1) empirically confirming overactivation of the Retinoic Acid Receptor (RAR)/Retinoid X Receptor (RXR) heterodimer as the molecular initiating event (MIE), through morphological rescue of 5 dpf zebrafish co-exposed to all-trans Retinoic Acid (ATRA), a RAR ligand, and either BMS493 (RAR inverse agonist) or UVI3003 (RXR antagonist); (2) Identifying the window of sensitivity for MIE through time-stage co-exposure (4-24, 4-48, 4-72, and 4-120 hpf); (3) integrating key molecular and cellular events from existing knowledge. The study also brings new knowledge on how RXR signaling disruption contributes to RA and Thyroid Hormone (TH) signaling disruption using in vitro reporter assays and in vivo morphological endpoints. Results show that BMS493 and, unexpectedly, diclazuril (pesticide identified as a thyroid hormone receptor antagonist in vitro) inhibit Retinoic Acid Response Element (RARE) activity in vitro. UVI3003-ATRA co-exposure induced additive effect on RARE activity. UVI3003-TH co-exposure inhibited Thyroid Hormone Response Element activity. In zebrafish, co-exposure of ATRA with BMS493 or diclazuril rescued ATRA-induced malformations, i.e., craniofacial and tail malformations, and microphthalmia - confirming RAR/RXR overactivation as MIE. It also rescued posterior swim bladder inflation and retinal layer defects -revealing novel role for RAR/RXR in these phenotypes. Additionally, UVI3003-ATRA co-exposure increased zebrafish embryos mortality, and UVI3003 alone increased fluorescence expressed in thyrocytes of thyroglobulin-mCherry zebrafish. Altogether, these findings reveal RXR's involvement in endocrine crosstalk and highlight the critical role of retinoid signaling in developmental toxicity and the need for its inclusion in hazard assessment.