Analogues of furan – open questions regarding their human metabolism

烷基呋喃存在于许多不同的食品和饮料中。2017年，欧洲食品安全局对呋喃及其烷基呋喃类似物的评估报告称，风险评估中的主要问题是暴露评估的不确定性和毒理学特性数据的不完整。呋喃被发现是一种相当强的致癌物质，其作用方式尚不清楚。例如，尚不确定致癌性是由亲电性和潜在的遗传毒性代谢物还是其他机制引起的，特别是直接发生的细胞和组织毒性，还是由于细胞防御成分（如谷胱甘肽）的消耗而引起的。正如欧洲食品安全局所述，哺乳动物对烷基呋喃的代谢知之甚少，但呋喃的代谢激活可能会发生。大多数相应的代谢物都是未知的，因此，关于它们对关键细胞靶点（包括蛋白质、氨基酸或DNA碱基）的反应性的知识相当缺乏。数据的缺乏尤其重要，因为烷基呋喃是一组不同的毒理学关注物质。根据我们的建议，我们将解决以下问题：(1)我们的目标是通过培养微粒体和分离酶来鉴定2-/3-甲基呋喃、5-戊基呋喃、2,5-二甲基呋喃或糠醇的I期代谢物，包括进一步鉴定与细胞亲核试剂反应的产物。代谢物将在微粒体/细胞上清液中使用成熟的技术进行鉴定，如HPLC-UV/Vis， UPLC-ESI-MS/MS或UPLC-QToF-MS。(2)烷基呋喃和糠醇代谢物对选定的氨基酸、蛋白质和谷胱甘肽（GSH）的反应性将通过培养反应物和UPLC-ESI-MS/MS或UPLC-QToF-MS进行分析。此外，使用稳定同位素标记的标准物质，通过稳定同位素稀释分析（SIDA）对共轭物进行定量。(3)鉴定的代谢物/偶联物的出现将在代谢能细胞（如HepG2细胞）中进行验证。形成的共轭物将被识别，降解动力学将通过UPLC-ESI-MS/MS或UPLC-QToF-MS进行研究。这使得研究细胞动力学和体外共轭物形成的浓度依赖性成为可能。(4)将在原代大鼠肝细胞中测定谷胱甘肽对所研究的烷基呋喃和糠醇的细胞毒性的影响。(5)用DNA碱基和DNA进行代谢活化后的烷基呋喃和糠醇孵育，以评估测试物质对这些细胞亲核试剂的反应性。假定的反应性代谢物对DNA组分的反应性将使用灵敏度最高的microLC-ESI-MS/MS进行研究。我们的研究方法最终将使我们能够更好地评估食用含有这些加工过程相关污染物的食品所带来的潜在人类健康风险。

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.