Glucose Co-Treatment Modulates Caffeine-Induced Transcriptomic Alterations During Embryogenesis in Zebrafish (Danio rerio)

Caffeine exposure during pregnancy has been associated with developmental abnormalities; however, the combined effects of caffeine and glucose on embryogenesis remain unclear. In this study, zebrafish embryos were exposed to caffeine or caffeine plus glucose co-treatment from 3 h post-fertilization to 96 h post-fertilization to evaluate developmental, physiological, and transcriptomic changes. Morphological analyses revealed developmental abnormalities in treatment groups, including pericardial edema, abnormal body curvature, delayed tail extension, and enlarged yolk sac morphology. Significant alterations in hatch rate and heart rate were also observed following treatment exposure. RNA sequencing analysis demonstrated that caffeine induced broad transcriptomic disruption involving neuronal signaling, embryonic development, metabolism, mitochondrial activity, immune regulation, and protein turnover pathways. Compared with caffeine treatment alone, caffeine plus glucose co-treatment was associated with differences in differential gene expression and developmental and metabolic signaling pathways. Alternative splicing analysis further identified widespread exon-skipping events and altered splicing patterns in genes associated with developmental and neuronal regulation. Collectively, these findings indicate that glucose co-treatment was associated with altered developmental and transcriptomic responses relative to caffeine exposure alone during zebrafish embryogenesis. Because a glucose-only treatment was not included in the present study, these findings should not be interpreted as demonstrating a definitive caffeine–glucose interaction.