| It has been reported that there are about 1.1 billion smokers around the world. More than 30% men are over 15 years old, and the rate is over 6% in women. Therefore, tobacco use is still a serious threat to the health. China has a huge market for tobaccos consumption, and so far the smoking rate in men and women is 47% and 2%, respectively. However, near 50% people are passive smokers, and tobacco use causes about 6.3 million deaths every year.Cigarette smoke is the most common exogenous hazardous factor for human, and it can cause a comprehensive physical damage. Of all the smoking related deaths, cardiovascular diseases account for 40%, lung cancer accounts for 20%, chronic obstructive pulmonary diseases account for 20%, and others accounts for 20%. Recently, many studies had focused on the smoking related cancer,especially lung cancer, in China,it is well known that tobacco use could cause lung cancer, half of which resulted from cigarette smoke.There are more than 5000 kinds of compounds in the cigarette smoke, and more than 60 of them have been identified as carcinogenic compounds, such as polycyclic aromatic hydrocarbons (such as benzopyrene), nitrosamines and aromatic amines. In fact most of these chemicals are precarcinogens and need to be activated by phase I metabolic enzymes (mainly cytochrome P450 enzyme, CYP) to exert their carcinogenic effect.CYP2A13 is a recently found extrahepatic metabolic enzyme which is mainly expressed in the respiratory system. It is abundantly expressed in the trachea and bronchial epithelial cells, slightly expressed in the trachea and bronchial smooth muscle cells and also expressed in peripheral tissues of the lung. Our previous studies had shown that CYP2A13 could metabolize AFB1, and CYP2A13-mediated metabolic activation of AFB1 played a very important role in the cytotoxicity, DNA adduct formation, DNA damage, apoptosis and cell transformation. In addition, many cohort studies had revealed that low activity CYP2A13 variant (R257C) could reduce the risk of lung cancer (adenocarcinoma). CYP2A13 plays a key role in metabolic activation of cigarette smoke carcinogens. As well known, carcinogens in cigarette smoke, N-nitroso nicotine (NNK) could be metabolic activated by CYP2A13 in lung.There are several substrates of CYP2A13 in cigarette smoke, such as nicotine, benzopyrene, naphthalene,3-methyl-indole (3-MI) and so on. 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 NNK, benzopyrene, naphthalene, and 3-methylindole which the content of these chemicals in cigarette smoke are far lower than nicotine.Thus, nicotine may affect the metabolism of cigarette smoke and the evaluation of toxic effects mediated by CYP2A13. Therefore,it is necessary to remove nicotine to explore the metabolic activation of low content of toxic substances (especially indirect carcinogen) mediated by CYP2A13, which will help provide the experimental clues for further study on the role of CYP2A13 in the metabolism of that cigarette smoke and its related respiratory damage and lung cancer. So far,no relevant research has been reported.In this study, cigarette smoke extract (CSE) was prepared using high efficient absorbing liquid solvent in a self-made smoking device, and nicotine and NNK were detected in high performance liquid chromatography (HPLC). Then, the HPLC was used to remove nicotine from CSE, which was then divided into two groups, nicotine section in CSE (CSE-N) and non-nicotine section in CSE (CSE-O). Their toxicity was compared in human bronchial epithelial cells stably expressing CYP2A13 (B-2A13) and vector (B-V). The effects of nicotine on the metabolicactivation of CSE mediated by CYP2A13 were explored, and it will help elucidate the role of CYP2A13 in respiratory damage induced by CSE.Part I Preparation of CSE and removal of nicotineObjectivesThe solvent with strong absorb ability is chosen to prepare CSE, then nicotine and NNK were detected by ultraperformanceliquid chromatography coupled with a massspectrometer (UHPLC-MS). In reference to the relative retention time of standard nicotine, HPLCwas applied to separate CSE samples, CSE-N and CSE-O were obtained. This can provide experimental material for the subsequent evaluation of the effect of nicotine on the metabolism of CSE mediated by CYP2A13.Methods1. Assembly and stability test of cigarette smoke suction deviceThe cigarette smoke suction device was assembled, the flow rate was set at 75 mL/min to make sure that a cigarette should be smoked in about 5min, the absorbance was measured to test the stability of this device.2. The choose of the absorbentFour representative solvents were selected as the alternative absorbent and three cigarettes smoke were absorbed in the absorant each time with predesigned plan.Then the absorbance was detected by the High Performance Liquid Chromatography (HPLC) and a better solvent was selected depending on the highest absorbance and absorption peak of substances.3. CSE extraction, quality control, the detection of nicotine and NNKThe CSE was prepared as follows:The air flow rate was set at 75 mL/min, two porous glass plate absorption tube, filled with 10 mL of dichloromethane were connected in series to absorb 3 cigarettes smoke each time.The absorbent was concentrated to 3 cigarettes per mL as the reserve of CSE sample.The UHPLC-MS was used to detect nicotine and NNK before and after the concentration, the recoveries of nicotine and NNK were calculated to evaluate the reproducibility of this process4. The separation of nicotine in CSEThe HPLC method was continuously optimized to separate the chromatogram peak in CSE as far as possible. The peak of nicotine in CSE was confirmed by comparing the relative retention times of its standard. Then HPLC was used to remove nicotine from CSE, and CSE was divided into two groups, nicotine section in CSE (CSE-N) and non-nicotine section in CSE (CSE-O).Results1. Determination of the absorption wavelengthThe absorbance was detected by DU800 UV-visible spectrophotometer under scan model. The absorption peak of nicotine was highest when the wavelength was 260 nm, meanwhile the absorbance of CSE was also strong. Thus 260 nm is suitable to evaluate the absorption efficiency for CSE.2. The choose of the absorbentCompared with water, hexane or methanol, the absorption efficiency of dichloromethane for cigarette smoke was stronger at 260 nm wavelength in HPLC. In detail, more peaks and peak area were found in dichloromethane absorption liquid when analyzed by HPLC. Thus, dichloromethane was used as cigarette smoke absorbent.3. CSE preparation, quality control,the detection of nicotine and NNKThe nicotinein CSE was detected by UHPLC-MS, the concentration of nicotine and NNK was 103.655±17.336μg and 12.708±3.823 ng per cigarette, respectively. Their concentrations were 97.76±17.158μg andl2.332±4.823ng after centrating, respectively. These results showed that the stability of the solution was suitable.4. The separation of nicotine in CSEThe relative retention time of nicotine was 20.208 min. CSE was divided into two groups, nicotine section in CSE (CSE-N) and non-nicotine section in CSE (CSE-O). There was no obvious substance peak in 20-23min in CSE-O, but a chromatogram peak (detection wavelength 260 nm) at nicotine retention timein CSE-N was found, and meanwhileits the mass spectra was similar to the molecular weight of nicotine.Part Ⅱ The effects of nicotine on the toxicity of CSE in B-2A13 cellsObjectivesTo explore the effects of nicotine on the metabolism and toxicity of CSE mediated by CYP2A13, the cytotoxicity, apoptosis and DNA damage were compared in B-2A13 and B-Vcells treated with CSE, CSE-N and CSE-O. This will help provide new evidence to explore the role of CYP2A13 in cigarette smoke-induced respiratory system injury, and provide clues in health assessment and tobacco control as well.Methods1. Toxic effects of nicotine and NNK on B-2A13 and B-V CellsB-2A13 and B-V cells were treated with 0,1,10,100,1000μM nicotine or 0, 0.1,1,10,100 μM NNK for 24h. After treatments, cell viability was determined using the CCK-8 assay.2. Toxic effects of CSE, CSE-N and CSE-O on B-2A13 and B-V CellsB-2A13 and B-V cells were treated with CSE (0%,3.33%,5%,10%,20%v/v), CSE-N (0%,3.33%,5%,10%v/v) or CSE-O (0%,0.5%,1%,2%v/v) for 24 h. In addition, B-2A13 cells were treated with CSE-O and in combination with 1μM 8-MOP for 24 h. After the treatments, cell viability was determined using the CCK-8 assay.3. Apoptosis of B-2A13 and B-V Cells induced by CSE, CSE-N and CSE-OB-2A13 and B-V cells were treated with 5% CSE,10% CSE-N and 2%CSE-O for 24h, apoptosis of cells was measured by Hoechst 33258 staining, and the results were then confirmed by flow cytometry.4. Effects of CSE, CSE-N and CSE-O on the expression of apoptosis and DNA damage related proteins in B-2A13 and B-V CellsB-2A13 and B-V cells were treated with 5% CSE,10% CSE-N and 2% CSE-O for 24 h. The total protein of the cells was extracted, and the expression of apoptotic proteins and DNA damage repair proteins were detected by Western blot.Results1. Toxic effects of nicotine and NNK on B-2A13 and B-V CellsNo obvious toxic effects were observed in B-2A13 and B-V cells treated with 1000 μM nicotine for 24 h. The viability of B-2A13 and B-V cells treated with 100 μM NNK for 24 h was 50% and 80%, respectively. It suggested that the metabolic activation of NNK mediated by CYP2A13 played an important role in its cytotoxic effects.2. Toxic effects of CSE, CSE-N and CSE-O on B-2A13 and B-V CellsThe viability of B-2A13 and B-V cells treated with CSE for 24 h decreased dose-dependently. No obvious differences of cytotoxic effects were observed between the two cells at the same concentrations. There was no obvious cytotoxicity in B-2A13 and B-V cells treated with 0-10% CSE-N. The viability of B-V cells was higher than B-2A13 cells treated with 1%-4%CSE-O.8-MOP could inhibit the toxic effect CSE-O in B-2A13 cells. These results showed that there were no differences in the cytotoxicity between B-2A13 and B-V cells treated with CSE or CSE-N. However, the toxic effect of CSE-O on B-2A13 cells was stronger than that in B-V cells, indicating that nicotine in CSE could affect the metabolic activation and toxicity of CSE mediated by CYP2A13.3. Apoptosis of B-2A13 and B-V Cells induced by CSE, CSE-N and CSE-OB-2A13 and B-V cells were treated with 5% CSE,10% CSE-N and 2% CSE-O for 24 hours, and cell apoptosis was measured by Hoechst 33258 staining and flow cytometry. Compared with the control, the apoptosis rate was about 50% in both B-2A13 and B-V cells treated with CSE for 24h. No obvious apoptosis was observed in B-2A13 or B-V cells treated with CSE-N for 24 h. The apoptosis rate was 20% in B-2A13 cells treated with CSE-O for 24 hours, and the apoptosis rate of B-V cells was 8%, near to the control group. These results suggested that nicotine could inhibit cell apoptosis by blockage of the metabolic activation of CSE-O mediated by CYP2A13.4. The effects of CSE, CSE-N and CSE-O on the expression of apoptosis and DNA damage related proteins in B-2A13 and B-V CellsB-2A13 and B-V cells were treated with 5% CSE,10% CSE-N and 2% CSE-O for 24 hours, and CSE treatment resulted in the apoptosis and DNA damage repair in both cells. C-Caspase 3, p-p53, p-Chk1, γ-H2AX and XLF, the protein occurring in apoptosis and DNA damage repair processes, expressed more in B-2A13 cells treated by CSE-O than that of B-V cells, while the apoptosis and DNA damage repair processes induced by CSE-N were not obvious.The results of the experiment suggest that nicotine may affect the cytotoxicity induced by the metabolism of CSE by CYP2A13 through affecting the expression of proteins related to apoptosis and DNA damage repair.Conclusions1. Of all the four solvents (water, several alkyl, methanol and dichloromethane), the absorption capacity of dichloromethane on the cigarette smoke is the best, thus it could be used as the absorption solution of CSE.2. In the present study, nicotine and NNK in CSE samples were detected by UHPLC-MS, their concentration were 103.655±17.336μg and 12.708±3.823 ng per cigarette, respectively.3. Nicotine can be separated from CSE according to its relative retention time, then CSE-O and CSE-N were obtained. In contrast to CSE and CSE-N, treatment with CSE-O was more toxic in B-2A13 than that of B-V, meanwhile the expression of preteins related to apoptosis and DNA damage and repair progression was higher than that of B-V. These effects could be inhibited by 8-MOP, an specific inhibitor of cytochrime P450.4. It suggested that nicotine in the CSE could inhibit the metabolic activation of CSE mediated by CYP2A13. |
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