Abstract
Cytochrome P450 2A13 (CYP2A13), an extrahepatic enzyme mainly expressed in the human respiratory system, has been reported to mediate the metabolism and toxicity of cigarette smoke. We previously found that nicotine inhibited 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) metabolism by CYP2A13, but its influence on other components of cigarette smoke remains unclear. The nicotine component of cigarette smoke extract (CSE) was separated, purified, and identified using high-performance liquid chromatography (HPLC) and ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS), splitting CSE into a nicotine section (CSE-N) and nicotine-free section (CSE-O). Cell viability and apoptosis by Cell Counting Kit-8 (CCK-8) and flow cytometry assays were conducted on immortalized human bronchial epithelial (BEAS-2B) cells stably expressing CYP2A13 (B-2A13) or vector (B-V), respectively. Interestingly, CSE and CSE-O were toxic to BEAS-2B cells whereas CSE-N showed less cytotoxicity. CSE-O was more toxic to B-2A13 cells than to B-V cells (IC50 of 2.49% vs. 7.06%), which was flatted by 8-methoxypsoralen (8-MOP), a CYP inhibitor. CSE-O rather than CSE or CSE-N increased apoptosis of B-2A13 cells rather than B-V cells. Accordingly, compared to CSE-N and CSE, CSE-O significantly changed the expression of three pairs of pro- and anti-apoptotic proteins, Bcl-2 Associated X Protein/B cell lymphoma-2 (Bax/Bcl-2), Cleaved Poly (Adenosine Diphosphate-Ribose) Polymerase/Poly (Adenosine Diphosphate-Ribose) Polymerase (C-PARP/PARP), and C-caspase-3/caspase-3, in B-2A13 cells. In addition, recombination of CSE-N and CSE-O (CSE-O/N) showed similar cytotoxicity and apoptosis to the original CSE. These results demonstrate that the nicotine component decreases the metabolic activation of CYP2A13 to CSE and aids in understanding the critical role of CYP2A13 in human respiratory diseases caused by cigarette smoking.
Highlights
Tobacco use is a serious threat to health, and there are approximately 1.1 billion smokers worldwide [1]
These results demonstrate that the nicotine component decreases the metabolic activation of Cytochrome P450 2A13 (CYP2A13) to cigarette smoke extract (CSE) and aids in understanding the critical role of CYP2A13 in human respiratory diseases caused by cigarette smoking
Following the analysis of the total CSE, CSE was separated into two groups: the nicotine section (CSE-N; 8.8–10.8 min) and nicotine-free section (CSE-O; 0–8.8 and 10.8–25 min)
Summary
Tobacco use is a serious threat to health, and there are approximately 1.1 billion smokers worldwide [1]. PAHs in cigarette smoke, such as pyrene, 1-hydroxypyrene, 1-nitropyrene and 1-acetylpyrene, can be metabolically activated by CYP2A13 and are thought to be associated with lung cancer [18]. Heterocyclic amines constitute another type of substrate of CYP2A13. At a high alkaline pH, nicotine is in a non-ionized state and can be readily absorbed across the epithelium of the lung, oral mucosa, nose, and skin [20] It undergoes metabolism by CYP2A13 and CYP2A6 in the liver and lung to generate the metabolite cotinine [21]. The study aims to understand the role of CYP2A13 in the metabolic activation of toxic components of cigarettes and related respiratory diseases, including lung cancer
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