Human metabolism of ethyl- and pentylfuran - open research questions

• Applicant: Professorin Dr. Elke Richling
• Subject Area: Food Chemistry ; Toxicology, Laboratory Medicine
• Term: since 2026
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 576019341

In 2017, the European Food Safety Authority's (EFSA) evaluation of furan and its alkylfuran analogues identified uncertainties in exposure assessment and incomplete data on toxicological properties. In our research project RI 1176/13-1, we have investigated the metabolic activation of 2-methylfuran (2-MF) and 2,5-dimethylfuran. 2-MF is metabolised by CYP P450 2E1 to the highly reactive acetylacrolein, which forms protein and DNA adducts. The metabolism of 2,5-dimethyfuran is still under investigation in our group, as liver enzymes lead to both, ring-opening of the furan ring and side-chain oxidation of the methyl group. Recently, µg/kg levels of 2-ethylfuran (2-EF) and 2-pentylfuran (2-PF) were analyzed in breakfast cereals. As stated by EFSA, not much is known about the metabolism of alkylfurans in mammals. Most of the relevant metabolites have not yet been identified and therefore knowledge of their reactivity with critical cellular targets such as proteins, amino acids or DNA bases is limited. In our proposal, we will focus on the metabolism - especially the metabolic activation - of 2-EF and 2-PF as well as the formation of protein and DNA adducts in vitro using our new high resolution MS (UPLC-tims-ToF-MS) and metabolic competent cells. We will address the following points: (1) We aim to identify phase I metabolites of 2-EF and 2-PF by incubation of microsomes and metabolically competent liver cells (HepG2 2E1). As we now know that ring-opening and side-chain oxidation can occur, we will identify metabolites in the microsomal/cellular supernatants using established techniques such as UHPLC-tims-ToF-MS, UHPLC-ESI-MS/MS, and HPLC-UV/Vis, and if side-chain oxidation occurs via phase I metabolism, our established headspace GC-MS methods will be modified and used instead. (2) The cytochrome P450 isoenzyme(s) responsible for the conversion of 2-EF and 2-pentylfuran to their respective metabolites will be identified using isolated CYP enzyme isoform containing systems like Baculosomes™. (3) The reactivity of the metabolites towards selected amino acids, glutathione (GSH), and proteins will be investigated by incubation of the reactants and analysis by untargeted UHPLC-tims-ToF-MS and UHPLC-ESI-MS/MS. (4) Using SULT-competent liver cells to verify the formation of a potential carbocation and its reactivity towards DNA bases. (5) Incubations of 2-EF and 2-PF (or their respective reactive metabolite(s)) with salmon DNA and SULT-competent cells will be performed followed by untargeted identification of possible DNA adducts. The respective identified DNA bases will be synthesized. The reactivity of putative reactive metabolites (or their pro-forms) towards DNA components and their presence in metabolically competent cells after 2-EF or 2-PF treatment will be investigated using microLC-ESI-MS/MS, which provides the highest sensitivity. Our research approach will allow a better assessment of the potential risks of food containing 2-EF and 2-PF.

• DFG Programme: Research Grants