Elke Richling

• Furan occurs as heat-induced contaminant in a variety of thermally treated foods and was categorized as hepatotoxic and possibly carcinogenic to humans (IARC 2B). The formation of furan in food is frequently accompanied by co-occurrence of various furan analogues that include, for example, 2-methylfuran, 3-methylfuran, 2,5-dimethylfuran, 2-ethylfuran or 2-pentylfuran. In contrast to furan, far less is known regarding the metabolism or potential toxicity of these alkylfurans. However, due to their structural similarity and frequent co- occurrence, alkylfurans may contribute significantly to the overall dietary furan exposure and to an increased risk for consumers. Analogue to furan, cytochrome-P450 (CYP)-catalyzed metabolic activation to highly reactive cis-enedial intermediates is expected to play a central role for the potential toxicity of alkylfurans. In the context of this project, we investigated the metabolic activation of 2-methylfuran in metabolically competent cells. It was demonstrated that 2-methylfuran is metabolically activated in vitro to the respective reactive intermediate 3acetylacrolein by quantification of corresponding Nα-acetyl-L-lysine adducts in cell supernatants. In addition, CYP2E1 was identified as the main contributor to the metabolic activation of 2-methylfuran. Although, 3-acetylacrolein has shown to be capable of reacting with DNA bases in silico, corresponding DNA adducts could not be detected in in vitro experiments, indicating that formed 3-acetylacrolein reacts primarily with cytosolic proteins. Within the scope of these in vitro experiments, no direct genotoxicity of 2-methylfuran could be confirmed. In the case of 2,5-dimethylfuran, experiments with human liver microsomes confirmed a previous hypothesized side-chain hydroxylation of 2,5-dimethylfuran to 5-methyl- 2-furfuryl alcohol for the first time. However, a comparable biotransformation of 2-methylfuran and 3-methylfuran to the corresponding primary alcohols could not be observed. Subsequently, CYP2E1 was identified as the CYP-isoform that catalyzes the hydroxylation of 2,5-dimethylfuran. Besides the CYP-mediated hydroxylation it could be shown that 2,5-dimethylfuran can be metabolized to the reactive cis-enedial intermediate cis-3-hexene-2,5-dione by CYP2E1 but also CYP3A4 and CYP2D6, respectively. By parallel quantification of 5-methyl-2-furfuryl alcohol and cis-3-hexene-2,5-dione after scavenging with glutathione it was possible to deduce the respective formation kinetics in human liver microsomes and CYP-isoenzyme systems. The kinetic data indicated that the metabolic activation of 2,5-dimethylfuran to the reactive metabolite cis-3-hexene-2,5-dione represents the main route of biotransformation, whereas the parallel side-chain hydroxylation is less pronounced.