Bentonite possible toxic and protective effects against aflatoxin B1: an in vitro study in Caco-2 cell line

Feed and food contamination by aflatoxins (AFs) is a serious economic and health concern for both animals and humans [1, 2]. Great efforts are made to minimize risks and losses due to the presence of AFs in feed and food commodities. Bentonite (BEN), is a commonly used sequestering agent adsorbing AFs when added to livestock diets [3]. However, little is known about its possible side effects on animal health and derived food-products. To answer this question, an in vitro study evaluating the effects of BEN, alone or in combination with aflatoxin B1 (AFB1), was carried out on the Caco-2 cell line, since the gastrointestinal tract represents the first and foremost interface between AFB1, BEN and the host. To increase the expression of one pivotal enzyme involved in AFB1 bioactivation, i.e. the cytochrome P450 3A4 (CYP3A4), cells were pre-treated with 12-o-tetradecanoylforbol-13-acetate and sodium butyrate [4]. BEN cytotoxicity was assessed by the WST-1 assay, while the effects on cell integrity and monolayer permeability estimated measuring the Transepithelial Electrical Resistance (TEER) and the Lucifer Yellow uptake (LY), respectively. Finally, the uptake of AFB1 in the basolateral compartment and the BEN sequestering capability toward AFB1 and its metabolites, i.e. aflatoxin M1 (AFM1) and aflatoxicol (AFL), were assessed by using set up and validated LC-MS/MS analytical methods. After 48 hrs of incubation, BEN showed an 50% inhibitory concentration of cell viability (IC50) of 0.08 mg/mL. When tested in combination with a fixed concentration of AFB1 (0.025 mg/mL), BEN showed the maximum protective effect against AFB1 at 0.1 mg/mL (p≤0.01); by contrast, BEN concentrations over 0.6 mg/mL surprisingly appeared to overcome AFB1 toxicity (p≤0.05). Additionally, BEN alone showed no effects on the cell integrity and permeability (TEER), while AFB1 alone (0.025 mg/mL) negatively affected the integrity of cell monolayer, as expected. Worth noting, the addition of BEN (0.1 mg/mL) restored the control condition. No statistically significant results were obtained with the LY assay. The adsorbent capacity of BEN against AFB1, AFM1, and AFL was higher for AFL (50%) and roughly 40% and 35% for AFB1 and AFM1, respectively. Finally, the transport rate from the apical to the basolateral compartment was not affected by the addition of BEN. Present results confirm the protective effect of BEN against AFB1, in the intestinal tract, thanks to its adsorbent capacity. This point was supported also by the preliminary results of transcriptome analysis (RNA-sequencing) in which BEN alone didn’t show any effect on gene expression, while proved a consistent involvement in the co - treatment with AFB1. Moreover the aluminosilicate appeared not to interfere with the monolayer physical properties; however, at higher concentrations it showed a certain cytotoxicity, which we hypothesize it is due to potential adsorbent capacity towards medium components like vitamins and minerals.