Analogues of furan – open questions regarding their human metabolism

• Applicant: Professorin Dr. Elke Richling
• Subject Area: Food Chemistry ; Toxicology, Laboratory Medicine
• Term: since 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 490818715

Alkylfurans occur in many different foods and beverages. In 2017 the evaluation of furan and its alkylfuran analogues by the European Food Safety Authority reported uncertainties in the assessment of exposure and incomplete data on toxicologic properties as a main problem in risk evaluation. Furan was found to be a rather potent carcinogen with an unclear mode of action. For example, it is uncertain whether the carcinogenicity results from electrophilic and potentially genotoxic metabolites or other mechanisms, especially cellular and tissue toxicity, occurring directly or as a consequence of depletion of cellular defense constituents (e.g. glutathione). As stated by EFSA, not much is known about the mammalian metabolism of alkylfurans, but metabolic activations as described for furan might occur. Most of the respective metabolites are unidentified, and therefore, the knowledge about their reactivity towards critical cellular targets including proteins, amino acids or DNA bases is rather scarce. This lack of data is especially critical, as alkylfurans are an entire group of different substances of toxicological concern.With our proposal, we will address the following points:(1) We aim to identify phase I metabolites of 2-/3-methylfuran, 5-pentylfuran, 2,5-dimethylfuran, or furfuryl alcohol by incubations of microsomes and isolated enzymes, including a further identification of the products of reaction with cellular nucleophiles. Metabolites will be identified in the microsomal/cellular supernatant using well established techniques, such as HPLC-UV/Vis, UPLC-ESI-MS/MS or UPLC-QToF-MS.(2) The reactivity of alkylfurans and furfuryl alcohol metabolites towards selected amino acids, proteins, glutathione (GSH) will be investigated incubating reactants and using UPLC-ESI-MS/MS or UPLC-QToF-MS for analysis. Further, the conjugates will be quantified via stable isotope dilution analysis (SIDA) using stable isotope labelled standard substances. (3) The occurrence of identified metabolites/conjugates will be verified in metabolically competent cells, such as HepG2 cells. Formed conjugates will be identified and degradation kinetics will be investigated via UPLC-ESI-MS/MS or UPLC-QToF-MS. This allows the investigation of cellular kinetics and concentration dependence of conjugate formation in vitro.(4) The influence of GSH on the cytotoxicity of the investigated alkylfurans and furfuryl alcohol will be determined in primary rat hepatocytes.(5) Incubations of alkylfurans and furfuryl alcohol after metabolic activation with DNA bases and DNA will be performed to assess the reactivity of the test substances towards these cellular nucleophiles. Reactivity of putative reactive metabolites towards DNA components will be investigated using microLC-ESI-MS/MS providing highest sensitivity.Our research approach will finally allow to better evaluate the potential human health risks exerted by consumption of food containing these process-related contaminants.

• DFG Programme: Research Grants