Auramine O, a Synthetic Food Dye, Induces Hepatic Injury by Disrupting <i>NOS2</i> Signaling, Inflammatory Cytokines, and Metabolic Pathways in an <i>In Vivo</i> Zebrafish Model

Synthetic dye, auramine O (AO) commonly utilized in food, textile, and leather manufacturing industries, has raised health concerns; however, its chronic hepatotoxic effects remain underexplored. The current study was designed to assess the chronic hepatotoxic effects of AO using zebrafish as an in vivo model. Zebrafish embryos were exposed to AO (1-100 mg/L) for developmental toxicity assessment, whereas adults and larvae were subjected to environmentally relevant concentrations, 5 and 10 mg/L of AO for 28 days, alongside bisphenol A (BPA, 225 μg/L) serving as a positive control. Oxidative stress and apoptosis were evaluated in 5-day-old larvae using DCFH-DA and acridine orange staining, whereas adult liver samples underwent biochemical, histological, and gene expression analysis. Also, AO accumulation was quantified in the adult liver tissue using high-performance liquid chromatography (HPLC) analysis. AO exposure led to dose-dependent increases in reactive oxygen species (ROS) (24.31% ± 0.17%), apoptosis (17.19% ± 0.35%), lipid peroxidation (27.37 ± 1.90 nmol/mg), LDH (1.71 ± 0.012 U/mL), ALT (0.68 ± 0.001 U/mL), and AST (0.82 ± 0.008 U/mL), with concurrent reductions in superoxide dismutase and catalase activity activities. Metabolic disruption was evident from elevated glucose (131.3 ± 0.88 mg/dL), cholesterol (3.26 ± 0.16 mmol/L), and triglycerides (2.31 ± 0.03 mmol/L) at 10 mg/L. Gene expression analysis showed upregulation of COX-2, iNOS, TNF-α, FAS, and BAX and downregulation of PIK3CD and BCL-2, indicating inflammation and impaired survival signaling. Histopathology showed considerable changes in liver morphology. Also, oil red O and toluidine blue staining indicated that the lipid was significantly increased (37.35% ± 2.20%) and mast cell degranulation occurred (83.41% ± 1.94%) in the group treated with the highest levels of AO. AO liver concentrations were measured by HPLC and demonstrated a dose-dependent association. These data indicate that AO dye alters the antioxidant defense system by increasing oxidative stress, hepatic inflammation, and altering metabolic function; however, regulation and further toxicological testing are necessary based on these results.