| Cytochrome P450 2A13 (CYP2A13) is an extrahepatic metabolic enzyme which predominantly expressed in the respiratory system. There are several substrates of CYP2A13 in cigarette smoke extract (CSE), such as the well-known nicotine Our previous studies show that the non-nicotine section from CSE increases the CYP2A13-medieated metabolic activation in human bronchial epithelial BEAS-2B cells. Therefore, it is necessary to identify the metabolic activation of toxic substances in CSE catalyzed by CYP2A13. Cytochrome P450 2A13 (CYP2A13) is a recently found extrahepatic metabolic enzyme which is mainly expressed in the respiratory system. There are several substrates of CYP2A13 in cigarette smoke. The toxicity of nicotine and its metabolites mediated by CYP2A13 are relatively low, but this might lead to a huge CYP2A13 consumption, and then inhibit the metabolic activation of other chemicals in cigarette smoke. Therefore, it is necessary to remove nicotine to explore the metabolic activation of other toxic substances mediated by CYP2A13.In the present study, the substances in CSE were separated with high performance liquid chromatography (HPLC) and divided into 40 groups (1 group/1 min). Then we exposed human BEAS-2B cells stably expressing CYP2A13 (B-2A13) or the empty vector (B-V) to each group. Consequently, the B-2A13 cells treated with the 5-min group showed significantly remarkable cytotoxic and apoptotic effects. The main component of the 5-min group was identified as 5-hydroxymethylfurfural (5-HMF) by gas chromatograph-tandem mass spectrometry (GC-MS/MS) and nuclear magnetic resonance (NMR) spectroscopy. In vitro metabolism assays confirmed that 5-HMF is a substrate of CYP2A13. Taken together, these results demonstrated that CYP2A13 efficiently metabolically activated 5-HMF in CSE. This paper explored the metabolic activation of larger content of toxic substances mediated by CYP2A13, which provide the experimental clues for further study on the role of CYP2A13 in the metabolism of that cigarette smoke and its related respiratory damage.Part I Screening and identification of 5-HMF, a new metabolic substrtate of CYP2A13 in CSEObjectives:To separate and identify the significant toxic substance of CYP2A13 from CSE.Methods:Cigarette smoke extraction (CSE) was separated by high performance liquid chromatography (HPLC). Subsequently, the cytotoxicity of them were compared in human bronchial epithelial BEAS-2B cells stably expressing CYP2A13 (B-2A13) or the empty vector (B-V), and identified the significant toxic substance by NMR and GC-MS/MS.Results:The B-2A13 cells treated with the 5-min group showed significantly remarkable cytotoxic effects. The main component of the 5-min group was identified as 5-hydroxymethylfurfural (5-HMF) by gas chromatograph-tandem mass spectrometry (GC-MS/MS) and nuclear magnetic resonance (NMR) spectroscopy. In vitro metabolism assays confirmed that 5-HMF is a substrate of CYP2A13.Conlusion:These results demonstrated that CYP2A13 efficiently metabolically activated 5-HMF in CSE. This paper explored the metabolic activation of larger content of toxic substances mediated by CYP2A13, which provide the experimental clues for further study on the role of CYP2A13 in the metabolism of that cigarette smoke and its related respiratory damage.Part Ⅱ Study on the in-vitro metabolic activity of 5-HMF by human CYP2A13Objectives:Construsting in vitro metabolic reaction system to evaluate the metabolic activity of CYP2A13 on 5-HMF, and to define the relationship between them.Methods:1. To obtain functional characterization and highly active CYP2A13 proteins in baculovirus/sf9 system, we constructed and verified recombinant Ac-Bacmid-CYP 2A13-POR; sf9 cells were infected with bvCYP2A13-POR; then we tried co-expression of CYP2A13 and POR by infection of bvCYP2A13-POR in the system.2. Western blot, CO differential spectrometry and Cytochrome C reduction method were carried to detect the protein expression of CYP2A13 and POR. The metabolic activity of CYP2A13 and POR were tested by NNK as metabolic substrate.3. CYP2A13 was used to study the metabolism of 5-HMF and its metabolites were analyzed.Results:1. Immuno blotting showed brighter and specific CYP2A13 and POR protein bands which obtained from microsomal, which indicated the success of co-expression of CYP2A13 and POR protein in Baculovirus/Sf9 system in vitro.2. CO differential spectrometry and Cytochrome C reduction method showed that the activity of CYP2A13 and POR were 0.62 pmol/min/pmol and 38.9nmol/min/mg protein, respectively. NNK, typical substrate of CYP2A13 was used to verify the activity of the co-expression CYP2A13. The result demonstrated that it could significantly metabolic NNK to generate its metabolites NNAL, and it’s metabolic activity as follows, Vmax (pmol/min/pmol):2.5 ± 0.4; (μM):12.0±2.6; Vmax /Km:0.208.3.5-HMF could be completely metabolized by CYP2A13 to 5-HMFA in 120min, the metabolic activity, Vmax (pmol/min/pmol):2.7 ± 0.2;Km (μM):50.9 ± 8.3;Vmax/Km:0.05.Conlusion:Highly active CYP2A13 and POR proteins were successfully expressed in vitro and 5-HMF was a new substrate of CYP2A13.Part Ⅲ Effects of CYP2A13/CYP2A5 on 5-HMF-induced toxicity in respiratory tract in miceObjectives:To preliminarily elucidated the role of CYP2A13 in the metabolism of 5-HMF and the acute injury on respiratory system in mice.Metods:Mouse CYP2A5 is homologous with and human CYP2A13, and they have closely function. The CYP2A5-/- mice were used for the functional study without abtaining CYP2A13 transgenic mice.1. Using pLJM lentivirus to establish BEAS-2B cells which stably expressing CYP2A5 (B-2A5).2. Identified the B-2A5 cell lines, and then the metabolic toxicity of 5-HMF was detected and comparied with B-2A13 cells.3. CYP2A5-/-mice and wild type mice exposed 5-HMF through intranasal instillation, Histopathological examination of the nasals and lungs, the number and types of inflammatory cells and the expression of inflammatory cytokines were comparied between the two groups.Results:1. The green fluorescence of the proposed B-2A5 cells was bright and full, and the transfection efficiency was close to 100% verified by GFP,2. The cytotoxicity results showed that the toxicity of 5-HMF on B-2A5 and B-2A13 cells was almost consistent, and they all could be reversed by 8-MOP, which suggested that CYP2A5 also had similar metabolic activation effect on 5-HMF.3. Animal experiments showed that the structure of the nasal septum mucosa was damaged, and the cilia were broken off, and a large number of eosinophils were infiltrated in 5-HMF treated wild type mice. However, the structure of the CYP2A5-/- mouse is complete, and the epithelial cells are arranged in order. Obvious inflammation in lungs and marked thickening of the alveolar wall cells in wild-type mice were detected, while not in CYP2A5-/-mouse lung; Macrophages, neutrophils, lymphocytes in the bronchoalveolar lavage fluid (BALF) from 5-HMF treated wild-type mice were significantly higher than those of 5-HMF treatment mice CYP2A5-/-, and The concentration of IFN-γ、IL-6、TNF-α in BALF was also significantly increased in wild-type.Conlusion:CYP2A5 could significantly metabolic 5-HMF to produce respiratory toxicity. Due to the homology of CYP2A5 and CYP2A13 and consistent metabolism activation on 5-HMF. Therefore, we inferred that the 5-HMF could be metabolized by CYP2A13, induced injury of respiratory system. |
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