Analogues of furan – open questions regarding their human metabolism

呋喃类似物 – 关于其人体新陈代谢的开放性问题

• 批准号: 490818715
• 负责人: Professorin Dr. Elke Richling
• 金额: --
• 依托单位: Fachrichtung Lebensmittelchemie / Toxikologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/490818715?language=en
• 关键词: Analogues; furan; open; questions; regarding

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.

烷基呋喃存在于许多不同的食物和饮料中。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，暴露评估存在不确定性，毒物学性质数据不完整是风险评估的主要问题。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，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，特别是细胞和组织毒性，直接发生还是由于细胞防御成分(如谷胱甘肽)的耗尽。正如EFSA所说，对哺乳动物的烷基呋喃代谢知之甚少，但可能会发生呋喃的代谢激活。大多数各自的代谢物都是未知的，因此，关于它们对包括蛋白质、氨基酸或DNA碱基在内的关键细胞靶点的反应性的知识相当匮乏。在我们的建议中，我们将解决以下几点：(1)我们的目标是通过微生物体和分离的酶的孵育来鉴定2-/3-甲基呋喃、5-戊基呋喃、2，5-二甲基呋喃或呋喃醇的I相代谢物，包括进一步鉴定与细胞亲核剂反应的产物。将使用成熟的技术，如高效液相-UV/VIS、UPLC-ESI-MS/MS或UPLC-QToF-MS，在微体/细胞上清液中鉴定代谢物。(2)将通过孵育反应物和使用UPLC-ESI-MS/MS或UPLC-QToF-MS来研究烷基呋喃和糠醇代谢物与选定的氨基酸、蛋白质、谷胱甘肽(GSH)的反应性。此外，将通过使用稳定同位素标记的标准物质的稳定同位素稀释分析(SIDA)来对偶联物进行定量。(3)已鉴定的代谢物/结合物的出现将在具有代谢能力的细胞中得到验证，如HepG2细胞。将通过UPLC-ESI-MS/MS或UPLC-QToF-MS鉴定形成的偶联物并研究降解动力学。(4)将在原代培养的大鼠肝细胞中测定GSH对所研究的烷基呋喃和呋喃醇细胞毒性的影响。(5)将代谢活化后的烷基呋喃和呋喃醇与DNA碱基和DNA孵育，以评估受试物质对这些细胞亲核性的反应性。我们将使用具有最高灵敏度的MicroLC-ESI-MS/MS来研究假定的反应性代谢物对DNA组分的反应性。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过程相关污染物的食品所带来的潜在人类健康风险。