Aflatoxin M1 absorption and cytotoxicity on human intestinal in vitro model

Abstract Aflatoxin M 1 (AFM 1 ) is the principal hydroxylated Aflatoxin B 1 (AFB 1 ) metabolite and is detected in milk of mammals, after consumption of feed contaminated with AFB 1 . 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 AFM 1 risk (50 ng/kg the EU and 500 ng/kg in the USA). It was demonstrated that if AFB 1 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 AFM 1 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 AFM 1 in absence of metabolic activation. For this reason, while AFM 1 is generally considered a detoxification product of AFB 1 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 AFM 1 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 AFM 1 , 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 AFM 1 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 AFM 1 conjugates have been detected. The present results may be crucial for the evaluation of human risk to AFM 1 exposure, in particular for children's population, due to their large use of milk and derivatives.