不同焦油释放量卷烟全烟气气-液界面暴露致细胞毒性、炎症因子释放和细胞凋亡的研究

目的:探讨两种不同焦油释放量卷烟对人支气管上皮细胞的细胞毒性、炎症因子释放水平和细胞凋亡的影响。方法:选取国际标准化组织(ISO)规定抽吸参数下焦油释放量分别为每支9.4 mg的卷烟样品1和每支14.0 mg的卷烟样品2作为研究对象。实验分为洁净空气对照组和烟气染毒组。利用VITROCELL系统产生的洁净空气或稀释的主流烟气以气-液界面方式暴露染毒Beas-2b细胞,经计算洁净空气对照组剂量为0支卷烟产生的主流烟气,烟气染毒组剂量分别为0.12%支、0.27%支、0.57%支、1%支卷烟产生的主流烟气;利用中性红细胞毒性试验检测细胞存活率;ELISA法检测细胞培养液中的IL-6、IL-8释放水平;Annexin V-FITC/PI流式细胞分析法检测细胞凋亡水平。结果:样品1和样品2产生的主流烟气均可引起Beas-2b细胞存活率降低,炎症因子IL-6、IL-8的释放水平和细胞凋亡率显著增加。在0.12%支的烟气剂量下,样品1与样品2烟气引起的细胞毒性、炎症因子释放水平和细胞凋亡率的差异无统计学意义;在0.57%和1%支的烟气剂量下,样品2的细胞毒性高于样品1。结论:两种不同焦油释放量卷烟样品均可引起细胞毒性、促炎症因子释放和细胞凋亡。在0.57%支~1%支烟气剂量范围内,具有高焦油释放量的卷烟烟气引起的细胞毒性强于低焦油释放量的卷烟烟气。     

Telomerase activity,telomere length and hTERT DNA methylation in peripheral blood mononuclear cells from monozygotic twins with discordant smoking habits

Increased telomerase expression has been implicated in the pathogenesis of lung cancer and, since the primary cause of lung cancer is smoking, an association between telomerase reactivation and tobacco smoke has been proposed. In this work an investigation has been performed to assess the relationship between tobacco smoke exposure and telomerase activity (TA) in peripheral blood mononuclear cells of healthy smokers. The methylation status of the catalytic subunit of telomerase hTERT was concurrently investigated to assess the possible association between epigenetic modifications of hTERT and TA. Besides, the association between smoke and telomere length (TL) has been evaluated. Healthy monozygotic twins with discordant smoking habits were selected as study population to minimize inter‐individual differences because of demographic characteristics and genetic heterogeneity. Statistically significant higher values of TA and TL were observed in smokers compared to nonsmoker co‐twins. The multivariate analysis of data showed, besides smoking habits (P = 0.02), an influence of gender (P = 0.006) and BMI (P = 0.001) on TA and a borderline effect of gender (P = 0.05) on TL. DNA methylation analysis, focused on 100 CpG sites mapping in hTERT, highlighted nine CpG sites differentially methylated in smokers. When co‐twins were contrasted, selecting as variables the intra‐twin difference in TA and hTERT DNA methylation, a statistically significant inverse correlation (P = 0.003) was observed between TA and DNA methylation at the cg05521538 site. In conclusion, these results indicate an association of tobacco smoke with TA and TL and suggest a possible association between smoke‐induced epigenetic effects and TA in healthy smokers. Environ. Mol. Mutagen. 58:551–559, 2017. © 2017 Wiley Periodicals, Inc.     

Changes in sputum cytology,airway inflammation and oxidative stress due to chronic inhalation of biomass smoke during cooking in premenopausal rural Indian women

To perform sputum analysis for verification of pulmonary changes in premenopausal rural Indian women chronically exposed to biomass smoke during cooking.Three consecutive morning sputum samples were collected from 196 women (median age 34 years) cooking with biomass and 149 age-matched control women cooking with cleaner fuel liquefied petroleum gas. Smears made on slides were stained with Papanicolaou and Perl's Prussian blue. Airway oxidative stress was estimated as reactive oxygen species (ROS) generation (by flow cytometry) and superoxide dismutase (SOD) level (by spectrophotometry) in sputum cells. Airway inflammation was measured as sputum levels of interleukin (IL)-6, -8 and tumor necrosis factor- alpha (TNF-α). Particulate matter of diameter less than 10 (PM₁₀) was measured using laser photometer while benzene exposure was monitored by measuring trans, trans-muconic acid (t,t-MA) in urine by HPLC-UV. Compared with control, sputum of biomass users contained more neutrophils, lymphocytes, eosinophils, alveolar macrophages, and showed presence of ciliocytophthoria, Charcot-Leyden crystals, Curschmann's spiral. ROS generation was increased by 2-fold while SOD was depleted by 31% in biomass users. They also had higher sputum levels of IL-6, -8 and TNF-α. Levels of PM₁₀ and t,t-MA were 2.9- and 5.8-times higher in biomass-using women. PM₁₀ and t,t-MA levels were positively associated with cellular changes in the sputum, markers of airway inflammation, and oxidative stress. Cooking with biomass alters sputum cytology, and increases airway inflammation and oxidative stress that might result in further amplification of the tissue damaging cascade in women chronically exposed to biomass smoke.     

SIRT4 inhibits cigarette smoke extracts-induced mononuclear cell adhesion to human pulmonary microvascular endothelial cells via regulating NF-κB activity

Cigarette smoking is an important risk factor for chronic obstructive pulmonary disease (COPD), yet its pathogenic mechanisms are not yet fully understood. Endothelial dysfunction is known to be involved in the pathogenesis of COPD. A detailed understanding of the mechanism involved in its progression would have a substantial impact on the optimization and development of treatment strategies. Here, we report that the expression of SIRT4, a mitochondrial sirtuin, is markedly down-regulated in cigarette smoke extract (CSE)-treated human pulmonary microvascular endothelial cells (HPMECs). Overexpression of SIRT4 significantly inhibits CSE-induced mononuclear cell adhesion to HPMECs. Consistently, we found that overexpression of SIRT4 attenuates the induction of vascular cell adhesion molecule 1 (VCAM-1) and E-selectin. Importantly, SIRT4 was found to negatively regulate CSE-induced NF-κB activation via inhibiting the degradation of IκBα. Moreover, we also found that proinflammatory cytokines interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and IL-6, the downstream target genes of NF-κB, are also inhibited by overexpression of SIRT4. These results suggest that SIRT4 protects HPMECs exposed to CSE stress via a mechanism that may involve the NF-κB pathway. Strategies based on the enhancement of SIRT4 may prove to be beneficial in the treatment of cigarette smoking caused COPD.