Aflatoxin M 1 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.