Purpose: This investigation was conducted to determine the impact of continuous exposure to low doses of cereulide on the behavior of intestinal cells. Caco-2 cells were used as model of the intestinal mucosa.
Methods: First, the limit of CER toxicity in undifferentiated Caco-2 cells was evaluated after a three-day exposure to low concentrations. Next, cells were exposed to varying concentrations around the predicted limit of CER toxicity for 18 days, to investigate the effect of longer exposure. To explore the mechanisms involved in cytotoxic response and mitochondrial function, the Seahorse Bioscience XFe24 analyzer (Massachusetts, USA) was used, in combination with well-established assays for mitochondrial activity (MTT), to observe changes in protein content (SRB). The effects of cereulide on the mitochondrial oxygen consumption rate (OCR) in Caco-2 were assessed using the Seahorse Bioscience XF Cell Mito Stress Test kit. In this assay, modulators of cellular respiration (oligomycin, FCCP, and mix of rotenone and antimycin A) were serially injected, providing insight into different aspects of mitochondrial function.
Results: Both MTT and SRB assays showed toxicity on undifferentiated cells at 0.125 ng CER/mL, after three days exposure. The three-day low CER concentration treatment on mitochondrial respiration in intact cells showed perturbations in mitochondrial respiration at a concentration of 0.125 ng/ml.
Significance: These in vitro data suggest that repeated exposure to CER might injure intestinal cells, even at relatively low doses. Cereulide appears to be more toxic than other cyclodepsipeptide toxins with ionophoretic properties, like valinomycin and beauvericin.