Developmental opioid exposure results in lasting transcriptome alterations in zebrafish (Danio rerio) embryos

The opioid epidemic has introduced significant public health challenges with little knowledge regarding the consequences of opioid exposure during embryonic development. While neurobehavioral effects of developmental opioid exposure are well-documented, early effects of exposure remain largely unexplored. We investigated the effects of oxycodone and fentanyl exposure on gene expression in zebrafish (Danio rerio) embryos using whole embryo RNA sequencing. Embryos were exposed to environmentally relevant (Oxycodone HCl 10.6pg/mL and Fentanyl Citrate 0.629pg/mL) and therapeutically relevant (Oxycodone HCl 35.14ng/mL and Fentanyl Citrate 3.14ng/mL) from 2 to 24 hours post-fertilization (hpf), followed by another 24hrs of opioid-free development. RNA sequencing at 48hpf revealed dose and drug specific gene expression changes. Lower doses of oxycodone and fentanyl both induced more differentially expressed genes (DEGs) than higher doses, potentially indicative of opioid receptor desensitization occurring at higher concentrations. In total, 892 DEGs were identified across all conditions indicating continued differential gene expression well after cessation of opioid exposure. Gene ontology analysis revealed changes in gene expression relating to extracellular matrix (ECM) organization, cell adhesion, and visual and nervous system formation. Key pathways include axon guidance, synapse formation, and ECM biosynthesis/remodeling all of which have potential implications on neural connectivity and sensory development. These findings demonstrate that developmental exposure to opioids induced persistent transcriptomic changes which may have lasting implications for structural integrity and function in vertebrate nervous systems, providing insights into the molecular mechanisms of opioid-induced alterations during development. Zebrafish embryos were exposed to one of the following conditions: fentanyl effluent (FE; 0.629 pg/mL), fentanyl therapeutic (FT; 3.14 ng/mL), oxycodone effluent (OE; 10.6 pg/mL), and oxycodone therapeutic (OT; 35.14 ng/mL). Opioid exposure occurred from 0 to 24 hpf in zebrafish embryos, embryos were washed at 24hpf and allowed to develop in non-opioid solution until 48 hpf. At 48hpf, 50 embryos were sample were harvested for total RNA, cDNA library prep, and RNAseq.