In vitro formation kinetics of two 2,5-dimethylfuran phase-I metabolites: cis-3-hexene-2,5-dione and 5-methylfurfuryl alcohol.

2,5-Dimethylfuran (DMF) is a heat-induced contaminant found in various thermally processed foods. Due to its structural similarity to furan, a well-known hepatotoxin and potential human carcinogen, the presence of DMF in food poses a potential risk to consumers. DMF undergoes cytochrome P450 (CYP)-mediated biotransformation leading to the formation of two primary phase-I metabolites: the reactive cis-3-hexene-2,5-dione (HDO), which can react with cellular nucleophiles, and the primary alcohol 5-methylfurfuryl alcohol (MFA), formed after hydroxylation of the alkyl moiety. To deduce the in vitro formation kinetics of these two phase-I metabolites, we utilised a newly developed HPLC-ESI-MS/MS method to quantify HDO after scavenging with glutathione and monitored the formation of MFA by GC-MS in parallel. Metabolic activation and the formation of HDO was the predominant biotransformation pathway in human liver microsomes, whereas the parallel formation of MFA was significantly less efficient. In line with data on the metabolic activation of furan, CYP2E1 was the most active human CYP-isoform in the potential metabolic activation of DMF. However, CYP3A4 and CYP2D6 also contributed to the HDO formation in vitro. Hydroxylation of DMF and formation of MFA were exclusively catalysed by CYP2E1. Despite the formation of MFA as an alternative metabolic pathway, our kinetic data indicate that DMF is primarily metabolised by CYP2E1 to the reactive cis-enedial intermediate HDO, particularly at physiologically relevant concentrations. Therefore, exposure to DMF may contribute to the overall risk associated with dietary exposure to furans.