Mechanisms of action and adaptive responses to diclofenac and meloxicam during the early life stages of Oryzias melastigma

Non-steroidal anti-inflammatory drugs (NSAIDs), though designed to target specific molecular pathways, pose significant environmental risks to non-target organisms, particularly marine fish. This study investigated the toxicity mechanisms and adaptive responses to diclofenac (DCF) and meloxicam (MEX) during the early life stages (ELS) of Oryzias melastigma at environmentally relevant concentrations over a 31-day period. Mechanistic investigations of sub-lethal effects were conducted using Enzyme-Linked Immunosorbent Assay (ELISA), RNA sequencing (RNA-Seq) and quantitative PCR (qPCR). The results revealed that cyclooxygenase (COX) inhibition disrupted the renin-angiotensin system, leading to an accumulation of angiotensin II and cardiovascular developmental defects. Additionally, downregulation of the pla2 gene reduced substrates essential for COX enzyme activity, exacerbating the effects. Although NSAIDs are known to affect the digestive system, no significant effects on developmental factors were observed. RNA-Seq and qPCR analyses revealed an adaptive upregulation of key genes, including ace2 and cyp7a1, involved in cardiovascular and metabolic regulation. Furthermore, 16S rRNA sequencing identified shifts in the microbial community, particularly in g_Rubritalea and g_Sphingomonas genera. Both the upregulated genes and the altered microbial taxa likely played a role in mitigating toxic effects and promoting homeostasis. Moreover, molecular docking suggested that MEX exhibited stronger sub-lethal effects than DCF, likely due to its higher binding affinity to COX. These findings provide valuable insights into NSAID toxicity mechanisms in marine fish, highlighting the importance of adaptive responses in countering environmental stress and underscoring the long-term ecological risks of chronic NSAID exposure.