Aflatoxin M sub(1) absorption and cytotoxicity on human intestinal in vitro model

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