Aflatoxin M1 causes cytotoxicity and intestinal epithelial cell integrity damage in differentiated human Caco-2 cells

Abstract Aflatoxin M1 (AFM1) is a mycotoxin that is commonly found as a milk contaminant, and its presence in milk has been linked to cytotoxicity. The present study aimed to evaluate the acute cytotoxic effects of AFM1 on intestinal CaCo2 cells. Initially, we checked the morphology and viability of CaCo2 cells after treatment with different concentrations of AFM1 (5ng/L, 50ng/L, 250ng/L, 500ng/L, 1000ng/L, and 2000ng/L) for different time intervals (6hrs, 12hrs, and 24hrs). It was found that AFM1 didn’t show any effect on cell morphology and viability. Further, DCFDA assay showed increased ROS production after 6hrs treatments. qPCR analysis showed an increased expression of epithelial specific cytoskeleton markers Cytokeratin, Villin, Vimentin, and JAM1, and a decreased expression of tight junction proteins, Claudin, Occludin, and ZO1. Similarly, we found an increased expression of Cyp1a1 transcript with an increasing AFM1 concentration and incubation time. This gene expression analysis showed AFM1 can causes disruption of gap junctions between intestinal cells, which was further confirmed by a transwell experiment. In conclusion, consumption of AFM1-contaminated milk doesn’t show any effect on cells morphology and viability but decreases the expression of intestinal barrier transcripts that may leads to the disruption of intestinal barrier function and leaky gut.