Chronic environmental caffeine exposure induces gut transcriptomic reprogramming in zebrafish

Aquatic contaminants are of growing concern due to their widespread presence and potential impacts on aquatic organisms. Caffeine, a commonly detected emerging contaminant, enters water bodies largely through widespread consumption and by insufficient wastewater treatment. Although previous studies have examined caffeine's effects on aquatic species, most have focused on short-term exposures that may not reflect chronic, low-level environmental conditions. Here, we investigate the physiological and transcriptomic impacts of both acute and chronic caffeine exposure in zebrafish (Danio rerio). Acute exposure to high concentrations (100-1000 mg/L) induced pronounced, often non-linear, bradycardia and mortality, revealing a complex dose-time-response relationship. In contrast, chronic exposure to environmentally relevant concentrations (0.1 and 10 µg/L) elicited tissue-specific gene expression changes. RNA-seq analysis of gut tissue revealed a threshold effect, with the higher dose causing extensive dysregulation of genes related to retinol metabolism, lysosomal function, and immune processes. RT-qPCR across tissues indicated systemic disruption of key pathways, including downregulation of the dopamine receptor drd2a in brain and muscle, compensatory downregulation of the adenosine receptor adora1 in the brain, and in the liver, upregulation of keap1 suggesting potential suppression of the antioxidant response pathway. Overall, our results show that caffeine, across a wide concentration range, can induce significant cardiac toxicity and systemic molecular reprogramming, highlighting its capacity to disrupt metabolic, neurological, and oxidative stress pathways in aquatic organisms.