Key Lab of Modern Toxicology of Ministry of Education, School of Public Health, Nanjing Medical University, 101 Longmian Avenue, Nanjing 211166, PR China; School of Nursing, Nanjing Medical University, 101 Longmian Avenue, Nanjing 211166, PR China.
Key Lab of Modern Toxicology of Ministry of Education, School of Public Health, Nanjing Medical University, 101 Longmian Avenue, Nanjing 211166, PR China; State Key Lab of Reproductive Medicine, Institute of Toxicology, Nanjing Medical University, 101 Longmian Avenue, Nanjing 211166, PR China.
Toxicol Appl Pharmacol. 2018 Nov 15;359:108-117. doi: 10.1016/j.taap.2018.09.031. Epub 2018 Sep 22.
Cytochrome P450 2A13 (CYP2A13) is an extrahepatic enzyme mainly expressed in the human respiratory system and is reported to mediate tobacco-specific N-nitrosamines (TSNA) metabolism in cigarette smoke. This study aimed to identify other new substrates of CYP2A13 in cigarette smoke and their corresponding respiratory toxicity. Following separation by HPLC, GC-MS/MS, NMR and cytotoxicity assays in BEAS-2B cells stably expressing CYP2A13 (B-2A13), 5-Hydroxymethylfurfural (5-HMF) was screened and identified in the 4-5 min section of cigarette smoke extract (CSE). In vitro metabolism results showed that CYP2A13 mediated the fast clearance of 5-HMF and formed the metabolite 5-HMF acid (5-HMFA). CSE 5-HMF (CSE-5-HMF) showed cytotoxicity similar to that of standard 5-HMF in B-2A13 and B-2A5 cells, which was inhibited by 8-methoxypsoralen (8-MOP), a CYP enzyme inhibitor. Mouse CYP2A5, a homologous CYP enzyme to CYP2A13, shares many substrates with CYP2A13 in cigarette smoke. Thus, CYP2A5 mice were generated to explore the role of CYP2A5 in 5-HMF bioactivation. Compared with CYP2A5 mice, WT mice showed serious histological lung and nasal olfactory mucosa damage, as well as increased inflammatory cells and elevated TNF-α and IL-6 levels in bronchoalveolar lavage fluid. Besides, nasal microsomes undertook fast 5-HMFA formation in WT mice than that in CYP2A5 mice, which could be inhibited by 8-MOP. This study is the first to identify 5-HMF as a new toxic substrate of human CYP2A13 in cigarette smoke, it may play a potential role in cigarette smoke-induced respiratory injuries.
细胞色素 P450 2A13(CYP2A13)是一种主要在人体呼吸系统中表达的肝外酶,据报道可介导香烟烟雾中的烟草特异性 N-亚硝胺(TSNA)代谢。本研究旨在鉴定香烟烟雾中 CYP2A13 的其他新底物及其相应的呼吸毒性。通过高效液相色谱法(HPLC)、气相色谱-质谱联用(GC-MS/MS)、核磁共振(NMR)和稳定表达 CYP2A13(B-2A13)的 BEAS-2B 细胞的细胞毒性试验分离后,在香烟烟雾提取物(CSE)的 4-5 分钟部分筛选并鉴定出 5-羟甲基糠醛(5-HMF)。体外代谢结果表明,CYP2A13 介导 5-HMF 的快速清除,并形成代谢产物 5-HMF 酸(5-HMFA)。CSE 5-HMF(CSE-5-HMF)在 B-2A13 和 B-2A5 细胞中表现出与标准 5-HMF 相似的细胞毒性,该毒性可被 CYP 酶抑制剂 8-甲氧基补骨脂素(8-MOP)抑制。与 CYP2A13 同源的小鼠 CYP2A5 与 CYP2A13 一样,在香烟烟雾中有许多共同的底物。因此,生成了 CYP2A5 小鼠以探索 CYP2A5 在 5-HMF 生物活化中的作用。与 CYP2A5 小鼠相比,WT 小鼠表现出严重的肺组织学和鼻嗅黏膜损伤,支气管肺泡灌洗液中炎症细胞增多,TNF-α 和 IL-6 水平升高。此外,WT 小鼠的鼻微粒体比 CYP2A5 小鼠更快地进行 5-HMFA 形成,这可被 8-MOP 抑制。本研究首次鉴定出 5-HMF 是香烟烟雾中人类 CYP2A13 的一种新的毒性底物,它可能在香烟烟雾引起的呼吸损伤中发挥潜在作用。