Aflatoxin M1 absorption and cytotoxicity on human intestinal in vitro model.

Aflatoxin M1 (AFM1) is the principal hydroxylated Aflatoxin B1 (AFB1) metabolite and is detected in milk of mammals, after consumption of feed contaminated with AFB1. As it is classified as probable human carcinogen (group 2B of the IARC), most countries have regulated its maximum allowed levels in milk in order to reduce AFM1 risk (50 ng/kg the EU and 500 ng/kg in the USA). It was demonstrated that if AFB1 must be converted into its reactive epoxide to exert its effects, and the protein binding may play an important role in its cytotoxicity. Conversely, the AFM1 epoxidation in human liver microsomes is very limited and studies with human cell line (MCL5), expressing or not expressing cytochrome P450 enzymes, demonstrated a direct toxic potential of AFM1 in absence of metabolic activation. For this reason, while AFM1 is generally considered a detoxification product of AFB1 relatively to carcinogenicity and mutagenicity property, this is not always true for cytotoxicity activity. Aim of this work is to evaluate the intestinal absorption of AFM1 using a human in vitro model, the Caco-2 cell line. Either the parental Caco-2 cell line or its derived clone TC7, with higher metabolic competence, have been used. They were treated with different concentrations of AFM1, that mirror the milk contamination level (0.3-32 nM corresponding to 10-10,000 ng/kg), either in undifferentiated or in differentiated phase of growth. After 48 h of treatment in serum free medium, a dose dependent absorption of AFM1 has been detected in both cell lines, especially in differentiated cells, while, no appreciable effects on cell viability were observed, except for a general cellular suffering, revealed by LDH release, particularly evident in the undifferentiated cells. As well, no metabolites or AFM1 conjugates have been detected. The present results may be crucial for the evaluation of human risk to AFM1 exposure, in particular for children's population, due to their large use of milk and derivatives.

Absorption of fumonisin B1 and aminopentol on an in vitro model of intestinal epithelium; the role of P-glycoprotein.

The aim of the present paper is to evaluate the absorption of fumonisin B1 and its principal metabolite, aminopentol on a human intestinal model, Caco-2 cells, cultured on semi-permeable inserts, that reproduces the two different intestinal compartments: luminal (apical) and serosal (basolateral) side. Following separate exposure in apical and in basolateral compartments, aminopentol passage through the cell layer (in particular from basolateral to apical direction) was shown, while it was not observed for the parent compound. The different aminopentol distribution between the two compartments of the culture system, and its variation in presence of verapamil or probenecid (P-gp and MRP inhibitors respectively), strongly suggests the involvement of P-glycoprotein in the influx/efflux mechanisms of aminopentol in the intestinal cells, reducing its oral bioavailability.