Oxidative Stress in Zebrafish Gut Induced by Individual and Combined Exposure to Amoxicillin, Arsenic and Fluoride: Engagement of Nrf2‐Keap1‐ARE Pathway

ABSTRACT This study investigates the effects of oxidative stress on zebrafish gut as a consequence of exposure to amoxicillin (AMX, 100 mg/L) alone or in combination with arsenic (As₂O₃, 50 μg/L, equivalent to 37.87 μg/L of As), and fluoride (NaF, 15 mg/L, equivalent to 6.8 mg/L of F) for 15 days. While the toxic ramifications of antibiotics and heavy metals have been extensively studied individually, their co‐toxicity on aquatic piscine models and the associated cellular stress responses remain poorly understood. The research focuses on understanding the mechanisms of stress and simultaneous toxicity in this specific scenario. The study revealed elevated levels of cellular ROS, MDA, and GSH, along with increased CAT enzyme activity in all treated groups, indicating oxidative stress. Histological damages, including increased numbers of goblet cells and necrotic spots, further confirmed oxidative injury. Gene expression analysis showed enhancement of stress‐responsive genes such as nrf2 , GPx‐1 , hsp70 , keap1 , nqo1 , Cyp 1a , ucp2 , Cu/Zn‐SOD , and Mn‐SOD , aligning with the observed biochemical changes. The translocation and heightened expression of Nrf2 in the nucleus were notable across all treatments. Furthermore, the combined effects of AMX, As, and F were more pronounced compared to other combinations, while exposure to AMX alone had the least impact. These findings underscore the role of the Nrf2‐Keap1‐ARE pathway in mediating the observed responses and highlight the synergistic toxicity of AMX, As, and F in the environment, posing a significant threat to both ecosystems and human health.