ABSTRACT
Cigarette smoke is a major risk factor for several diseases, including chronic obstructive pulmonary and cardiovascular diseases. The toxicity of the cigarette smoke can be determined in vitro. The cytotoxicity test of the cigarette smoke is commonly conducted using the cigarette smoke condensate (CSC) and cigarette smoke extract (CSE). The CSC and CSE methods are well known for sampling of the particles and water-soluble compounds in the cigarette smoke, respectively. In this study, the CSC and CSE were analyzed by using a gas chromatography-mass spectrometry (GC-MS) system equipped with a wax column for separation of the volatile organic compounds. The cytotoxic effect of the CSC and CSE were evaluated thoroughly by comparing the analytical results of the CSC and CSE samples. The total concentration of the volatile organic compounds detected in the CSC sample was similar to that in the CSE sample based on the peak area. Except for the dimethyl sulfoxide solvent, nicotine had the highest concentration in the CSC sample, while acetonitrile had the highest concentration in the CSE sample. The compositions were as follows: (1) CSC sample: 55.8% nicotine, 18.0% nicotyrine, 3.20% 1,2,3-propanetriol, triacetate, 1.28% ethyl chloride, 1.22% phenol, etc. and (2) CSE sample: 18.7% acetonitrile, 18.0% acetone, 12.5% 2-hydroxy-2-methyl-propanenitrile, 8.98% nicotine, 5.86% nicotyrine, etc. In this manner, to accurately examine the cytotoxicity of the cigarette smoke using CSC or CSE, the components and their concentrations in the CSC and CSE samples should be considered.
Subject(s)
Acetone , Cardiovascular Diseases , Dimethyl Sulfoxide , Ethyl Chloride , Gas Chromatography-Mass Spectrometry , In Vitro Techniques , Nicotine , Phenol , Risk Factors , Smoke , Tobacco Products , Volatile Organic CompoundsABSTRACT
Cigarette smoke is a major risk factor for several diseases, including chronic obstructive pulmonary and cardiovascular diseases. The toxicity of the cigarette smoke can be determined in vitro. The cytotoxicity test of the cigarette smoke is commonly conducted using the cigarette smoke condensate (CSC) and cigarette smoke extract (CSE). The CSC and CSE methods are well known for sampling of the particles and water-soluble compounds in the cigarette smoke, respectively. In this study, the CSC and CSE were analyzed by using a gas chromatography-mass spectrometry (GC-MS) system equipped with a wax column for separation of the volatile organic compounds. The cytotoxic effect of the CSC and CSE were evaluated thoroughly by comparing the analytical results of the CSC and CSE samples. The total concentration of the volatile organic compounds detected in the CSC sample was similar to that in the CSE sample based on the peak area. Except for the dimethyl sulfoxide solvent, nicotine had the highest concentration in the CSC sample, while acetonitrile had the highest concentration in the CSE sample. The compositions were as follows: (1) CSC sample: 55.8% nicotine, 18.0% nicotyrine, 3.20% 1,2,3-propanetriol, triacetate, 1.28% ethyl chloride, 1.22% phenol, etc. and (2) CSE sample: 18.7% acetonitrile, 18.0% acetone, 12.5% 2-hydroxy-2-methyl-propanenitrile, 8.98% nicotine, 5.86% nicotyrine, etc. In this manner, to accurately examine the cytotoxicity of the cigarette smoke using CSC or CSE, the components and their concentrations in the CSC and CSE samples should be considered.
Subject(s)
Acetone , Cardiovascular Diseases , Dimethyl Sulfoxide , Ethyl Chloride , Gas Chromatography-Mass Spectrometry , In Vitro Techniques , Nicotine , Phenol , Risk Factors , Smoke , Tobacco Products , Volatile Organic CompoundsABSTRACT
AIM: To investigate the role of transcription factor hairy and enhancer of split 1 (Hes1) in the malignant transformation of human bronchial epithelial cell line BEP2D induced by tobacco.METHODS: The BEP2D cells were chronically exposed to cigarette smoke condensate (CSC) at 1 cigarette per L until the 70th generation.The phenotype of malignant transformation of the cells induced by CSC was detected by soft agar clony formation assay.RT-PCR and Western blot were used to determined the expression of Hes1 at mRNA and protein levels in each generation of the cells.The proliferation and apoptosis of the BEP2D cells exposed to CSC were analyzed with the methods of MTT assay, flow cytometry and cell colony formation assay after treatment with Notch pathway bloker DAPT or liposome transfection with Hes1-siRNA.The expression of Hes1 in the peripheral small airway tissues of the smoking rats was evaluated by immunohistochemical staining.The expression of Hes1 in non-small-cell lung cancer and normal airway tissues was also detected by the methods of immunohistochemistry and RT-PCR.RESULTS: The BEP2D cells in the 70th generation had a malignant transformation phenotype.The expression of Hes1 in the BEP2D cells exposed to CSC for different time showed an increa-sing trend.DAPT and liposome transfection with Hes1-siRNA down-regulated the expression of Hes1, inhibited the cell proliferation and induced cell apoptosis.The expression of Hes1 in the airway mucosa of the rats exposed to cigarette smoke for 1 month and 6 months was significantly higher than that in control group.Cigarette smoking induced the expression of Hes1 in lung cancer and normal airway tissues.CONCLUSION: Hes1 may be involved in smoking-induced lung cancer by promoting the imbalance between apoptosis and proliferation.
ABSTRACT
OBJECTIVES: Cigarette smoking is associated with carcinogenesis owing to the mutagenic and genotoxic effects of cigarette smoke. The aim of this study was to evaluate the mutagenic and genotoxic effects of Korean cigarettes using in vitro assays. METHODS: We selected 2 types of cigarettes (TL and TW) as benchmark Korean cigarettes for this study, because they represent the greatest level of nicotine and tar contents among Korean cigarettes. Mutagenic potency was expressed as the number of revertants per μg of cigarette smoke condensate (CSC) total particulate matter whereas genotoxic potency was expressed as a concentration-dependent induction factor. The CSC was prepared by the International Organization for Standardization 3308 smoking method. CHO-K1 cells were used in vitro micronucleus (MNvit) and comet assays. Two strains of Salmonella typhimurium (Salmonella enterica subsp.enterica; TA98 and TA1537) were employed in Ames tests. RESULTS: All CSCs showed mutagenicity in the TA98 and TA1537 strains. In addition, DNA damage and micronuclei formation were observed in the comet and MNvit assays owing to CSC exposure. The CSC from the 3R4F Kentucky reference (3R4F) cigarette produced the most severe mutagenic and genotoxic potencies, followed by the CSC from the TL cigarette, whereas the CSC from the TW cigarette produced the least severe mutagenic and genotoxic potencies. CONCLUSIONS: The results of this study suggest that the mutagenic and genotoxic potencies of the TL and TW cigarettes were weaker than those of the 3R4F cigarette. Further study on standardized concepts of toxic equivalents for cigarettes needs to be conducted for more extensive use of in vitro tests.
Subject(s)
Benchmarking , Carcinogenesis , Comet Assay , DNA Damage , In Vitro Techniques , Kentucky , Methods , Micronucleus Tests , Nicotine , Particulate Matter , Salmonella typhimurium , Smoke , Smoking , Tobacco ProductsABSTRACT
OBJECTIVES: Cigarette smoking is associated with carcinogenesis owing to the mutagenic and genotoxic effects of cigarette smoke. The aim of this study was to evaluate the mutagenic and genotoxic effects of Korean cigarettes using in vitro assays. METHODS: We selected 2 types of cigarettes (TL and TW) as benchmark Korean cigarettes for this study, because they represent the greatest level of nicotine and tar contents among Korean cigarettes. Mutagenic potency was expressed as the number of revertants per μg of cigarette smoke condensate (CSC) total particulate matter whereas genotoxic potency was expressed as a concentration-dependent induction factor. The CSC was prepared by the International Organization for Standardization 3308 smoking method. CHO-K1 cells were used in vitro micronucleus (MNvit) and comet assays. Two strains of Salmonella typhimurium (Salmonella enterica subsp.enterica; TA98 and TA1537) were employed in Ames tests. RESULTS: All CSCs showed mutagenicity in the TA98 and TA1537 strains. In addition, DNA damage and micronuclei formation were observed in the comet and MNvit assays owing to CSC exposure. The CSC from the 3R4F Kentucky reference (3R4F) cigarette produced the most severe mutagenic and genotoxic potencies, followed by the CSC from the TL cigarette, whereas the CSC from the TW cigarette produced the least severe mutagenic and genotoxic potencies. CONCLUSIONS: The results of this study suggest that the mutagenic and genotoxic potencies of the TL and TW cigarettes were weaker than those of the 3R4F cigarette. Further study on standardized concepts of toxic equivalents for cigarettes needs to be conducted for more extensive use of in vitro tests.
Subject(s)
Benchmarking , Carcinogenesis , Comet Assay , DNA Damage , In Vitro Techniques , Kentucky , Methods , Micronucleus Tests , Nicotine , Particulate Matter , Salmonella typhimurium , Smoke , Smoking , Tobacco ProductsABSTRACT
The aim of this study was to investigate the inhibitory effect of Cnidium monnieri fruit (CM) extracts on pulmonary inflammation induced in mice by cigarette smoke condensate (CSC) and lipopolysaccharide (LPS). Pulmonary inflammation was induced by intratracheal instillation of LPS and CSC five times within 12 days. CM extract was administered orally at a dose of 50 or 200 mg·kg(-1). The number of inflammatory cells in the bronchoalveolar lavage fluid was counted using a fluorescence activated cell sorter. Inflammatory mediator levels were determined by enzyme-linked immunosorbent assay. The administration of LPS and CSC exacerbated airway hyper-responsiveness (AHR) and induced an accumulation of inflammatory cells and mediators, and led to histological changes. However, these responses are modulated by treatment with CM, and the treatment with CM extract produces similar or more extensive results than the treatment with cyclosporin A (CSA). CM extract may have an inhibitory effect on pulmonary inflammation related with chronic obstructive pulmonary disease.
Subject(s)
Animals , Female , Anti-Inflammatory Agents , Pharmacology , Therapeutic Uses , Bronchoalveolar Lavage Fluid , Cnidium , Fruit , Lipopolysaccharides , Mice, Inbred BALB C , Phytotherapy , Plant Extracts , Pharmacology , Therapeutic Uses , Pneumonia , Drug Therapy , Pulmonary Disease, Chronic Obstructive , Drug Therapy , Pathology , Smoke , Smoking , Tobacco ProductsABSTRACT
Objective: To establish a chronic malignant transformation model of immortalized human bronchial epithelial cell line BEP-2D by low dose cigarette smoke condensate (simulating smoking environment). Methods: The chronic dose of cigarette smoke condensate was determined by MTT assay and the colony formation test of BEP-2D cells. BEP-2D cells were exposed to cigarette smoke condensate once or for multiple times; unexposed cells were taken as control. The malignant tendency of BEP-2D cells was identified by anti-serum experiment and the malignant features of transformed BEP-2D cells were identified by semisolid agar culture. The differentiation ability of BEP-2D cells in anti-serum experiment and the colony forming rates of BEP-2D cells were compared between different groups. Results: The BEP-2D cells were exposed to 0.5, 1 and 2 μl/ml of cigarette smoke condensate once or for multiple times and were cultured for 25 generations; the differentiation abilities of BEP-2D cells(the 25th generation) was significantly different between the cigarette smoke condensate exposed groups (at 0.5, 1 and 2 μl/ml) and the normal control group (P<0.05). The cell malignant transformation model was successfully established in the cells of the 38th generation; the cells had multi-layer growth and had no contacting inhibition, with chromosome abnormality. The colony forming rates in the semisolid agar culture test was significantly higher in all smoke condensate exposed groups than in control group(P<0.05). The dose-response relationship showed a good linear correlation (r=0.969, y=42x, P<0.05). Conclusion: The malignant transformation of immortalized human bronchial epithelial cells can be successfully induced by cigarette smoke condensate at 0.5-2 μl/ml, which offers an ideal model for simulating smoking environment induced chronic malignant transformation.
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Objective To determine the effect of tea polyphenols on oxidative damage and (apoptosis) in human bronchial epithelial cells induced by low-dose cigarette smoke condensate (CSC).Methods We prepared CSC. 3-(4,5-dimethyl thiazoly) 2,5-diphenyl-tetrazoliun bromide (MTT) assay was used to determine the growth of cultured human bronchial epithelial cells (HBE135-E6E7). Fluorescent-chemiluminescent analyzer was used to measure cell reactive oxygen species (ROS) level. DNA ladder method was used to detect HBE135-E6E7 apoptosis. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect Bcl-2 and Bax mRNA expression.Results Concentration of intracellular ROS in the CSC group and CSC + TP group was significantly higher than that in the control group (P＜0.01); concentration of intracellular ROS in the CSC + TP group was significantly lower than that in the CSC group (P＜0.01). Apparent DNA breakage of the tail belt appeared in the CSC Group,while only a small amount of DNA breakage of the tail belt appeared in the CSC + TP group. Compared with the control group, Bcl-2 mRNA expression was reduced and Bax mRNA expression was increased in the CSC group (all P＜0.01). Compared with the CSC group, Bcl-2 mRNA expression was increased and Bax mRNA expression was reduced in the CSC+TP group (all P＜0.01). Ratio of Bcl-2 mRNA/ Bax mRNA in the CSC group and CSC+TP group was significantly lower than that in the control group (all P＜0.01).Conclusion TP can antagonize CSC-induced airway epithelial cell apoptosis through the effective removal of ROS, promoting Bcl-2 mRNA expression and inhibiting the expression of Bax mRNA.
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Objective:To study p16 methylation of BEP-2D cells during its malignant transformation.Methods:Normal BEP-2D cells and BEP-2D cells treated by cigarette smoke condensate(CSC) for 15 weeks(W-15),25 weeks(W-25),38 weeks(W-38) and 43 weeks(W-43)respectively were chosen to study the p16 methylation status by Nested methylation specific PCR (Nested-MSP).Results:The p16 methylation was found in W-15,W-25,W-38 and W-43 BEP-2D cells,but not in the normal BEP-2D cell.Conclusion:The p16 methylation may be considered as a kind of biology-marker of the early diagnosis of lung cancer because the p16 methylation occurs in the earlier period of lung cancer.
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Objective:To study the change of the survivin mRNA and protein of BEP-2D cells during its malignant transformation.Methods:Normal BEP-2D cell and BEP-2D cells treated by cigarette smoke condensate(CSC)for 15 weeks(P-15),25 weeks(P-25)and 38 weeks(P-38)were respectively chosen to study the survivin gene and protein by RT-PCR(retro-translation PCR,RT-PCR)and immunohistochemical method.Results:The survivin mRNA was found in BEP-2D cells of P-15,P-25 and P-38,respectively with 0.56,0.80,and 0.81,but not in the normal BEP-2D cell.The survivin protein was found in the normal BEP-2D cell,but not in BEP-2D cells of P-15,P-25 and P-38.The levels of survivin protein expressian were different between BEP-2D cells of P-15,P-25 and P-38 and normal BEP-2D cell,with significant difference between P-15 BEP-2D cell and normal BEP-2D cell(P