Aflatoxin M1 decreases the expression of genes encoding tight junction proteins and influences the intestinal epithelial integrity

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 Caco-2 cells. Initially, we checked the morphology and viability of Caco-2 cells after treatment with different concentrations of AFM1 (5 ng/L, 50 ng/L, 250 ng/L, 500 ng/L, 1000 ng/L, and 2000 ng/L) for different time intervals (6 h, 12 h, and 24 h). It was found that AFM1 did not show any effect on cell morphology, but 10% decrease in viability above 1000 ng/L after 12 h. Furthermore, DCFDA assay showed increased ROS production after 6 h treatments. qPCR analysis showed an increased expression of epithelial-specific cytoskeleton marker genes, Cytokeratin, Villin, Vimentin, and JAM-1, and a decreased expression of tight junction protein genes, Claudin-1, Occludin, and ZO-1. Similarly, we found an increased expression of Cyp1a1 transcript with an increasing AFM1 concentration and incubation time. This gene expression analysis showed AFM1 can cause disruption of tight junctions between intestinal cells, which was further confirmed by a transwell experiment. In conclusion, consumption of AFM1-contaminated milk does not show any effect on cells morphology and viability but decreases the expression of intestinal barrier transcripts that may lead to the disruption of intestinal barrier function and leaky gut.

Keywords: AFM1-cytotoxicity; Caco-2 cells; Cyp1a1; Epithelial transcript; Milk contaminants.