Multi-toxicant exposure to beauvericin, arsenic trioxide, and glyphosate enhances cytotoxicity and intracellular ROS levels in NIH-3T3 fibroblast cells

Introduction Human exposure to environmental contaminants typically occurs through complex chemical mixtures rather than individual compounds; however, the toxicological interactions among co-occurring contaminants remain poorly understood. This study investigated the cytotoxic interactions of the mycotoxin beauvericin (BEA), the pesticide glyphosate (GLY), and arsenic trioxide (ATO) in binary and ternary combinations using NIH‐3T3 fibroblasts. Methods Cell viability was assessed using the MTT assay. Toxicological interactions were quantitatively characterized using the Chou‐Talalay method based on combination index (CI) and dose reduction index (DRI) analyses. Intracellular reactive oxygen species (ROS) generation was measured to evaluate oxidative stress. Results Among the individual toxicants, ATO exhibited the greatest cytotoxicity, followed by BEA and GLY. Combination analysis revealed distinct interaction profiles depending on mixture composition. BEA + ATO and BEA + GLY consistently exhibited synergistic cytotoxicity, whereas ATO + GLY showed predominantly additive effects. Notably, the ternary mixture displayed a dose‐dependent interaction pattern, transitioning from antagonism at a low effect level (IC 10 ) to synergism at a high effect level (IC 50 ). These interaction profiles were accompanied by increased intracellular ROS generation, with synergistic combinations showing higher ROS levels than individual toxicants. Discussion Overall, these findings demonstrate that the toxicity of chemical mixtures cannot be predicted from the toxicity of individual components alone, as toxicological interactions depend on both mixture composition and effect level. The results highlight the importance of incorporating mixture‐specific approaches into cumulative environmental and food safety risk assessment.