Protective effect of bacoside A on cigarette smoking-induced brain mitochondrial dysfunction in rats.

Chronic exposure to cigarette smoke affects the structure and function of mitochondria, which may account for the pathogenesis of smoking-related diseases. Bacopa monniera Linn., used in traditional Indian medicine for various neurological disorders, was shown to possess mitrochondrial membrane-stabilizing properties in the rat brain during exposure to morphine. We investigated the protective effect of bacoside A, the active principle of Bacopa monniera, against mitochondrial dysfunction in rat brain induced by cigarette smoke. Male Wistar albino rats were exposed to cigarette smoke and administered bacoside A for a period of 12 weeks. The mitochondrial damage in the brain was assessed by examining the levels of lipid peroxides, cholesterol, phospholipid, cholesterol/phospholipid (C/P) ratio, and the activities of isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, malate dehydrogenase, NADH dehydrogenase, and cytochrome C oxidase. The oxidative phosphorylation (rate of succinate oxidation, respiratory control ratio and ADP/O ratio, and the levels of ATP) was evaluated for the assessment of mitochondrial functional capacity. We found significantly elevated levels of lipid peroxides, cholesterol, and C/P ratio, and decreased levels of phospholipids and mitochondrial enzymes in the rats exposed to cigarette smoke. Measurement of oxidative phosphorylation revealed a marked depletion in all the variables studied. Administration of bacoside A prevented the structural and functional impairment of mitochondria upon exposure to cigarette smoke. From the results, we suggest that chronic cigarette smoke exposure induces damage to the mitochondria and that bacoside A protects the brain from this damage by maintaining the structural and functional integrity of the mitochondrial membrane.     

Hes1在烟草诱导人支气管上皮细胞恶性转化过程中的作用

目的:探讨转录因子发状分裂相关增强子1(hairy and enhancer of split,Hes1)在香烟烟气凝集物(cigarette smoke condensate,CSC)诱导永生化人支气管上皮细胞BEP2D恶性转化中的作用。方法:CSC(1L空气中点燃1支香烟)慢性染毒BEP2D细胞至第70代,软琼脂集落形成实验检测CSC诱导的细胞恶性转化表型;采用RT-PCR和Western blot法检测各代细胞的Hes1表达;MTT法、细胞集落形成实验和流式细胞术检测Notch通路阻断剂DAPT或脂质体转染Hes1-siRNA对CSC染毒BEP2D细胞增殖与凋亡的影响。检测吸烟大鼠外周小气道组织中Hes1的表达;采用免疫组化法和RT-PCR法检测非小细胞肺癌组织及正常气道组织中Hes1的表达。结果:第70代BEP2D细胞具备恶性转化表型;Hes1在CSC染毒BEP2D细胞中的表达总体呈逐渐增高的趋势;DAPT和Hes1-siRNA均能通过下调Hes1显著抑制第70代BEP2D细胞的增殖,诱导其凋亡;Hes1在卷烟烟气暴露大鼠气道黏膜1月和6月组的表达较同期对照组显著增高;吸烟显著诱导肺癌组织和正常气道表达Hes1。结论:Hes1可能通过促进凋亡与增殖失衡,参与吸烟诱导的肺癌发生。     

Cardiovascular and Mood Responses to Quantified Doses of Cigarette Smoke in Oral Contraceptive Users and Nonusers

Previous research suggests that the female sex hormones may moderate cardiovascular and mood responses to cigarette smoking and abstinence. To test this possibility, acute effects of cigarette smoking on cardiovascular reactivity and mood were examined in 12 oral contraceptive users and 12 nonusers across two menstrual phases (early and late cycle). After overnight deprivation, each participant attended two sessions in which they first sham-smoked and then smoked two standard cigarettes, via a quantified smoke delivery system. Oral contraceptive users exhibited larger cigarette smoking-induced increases in heart rate compared with nonusers. In addition, cigarette smoking-induced cardiovascular changes varied with both the phase of the menstrual cycle and oral contraceptive use. No menstrual phase-dependent effects were observed for tobacco withdrawal symptoms, premenstrual symptoms, or moods prior to smoking. Cardiovascular hyperreactivity to cigarette smoke in oral contraceptive users may help explain the mechanisms by which smoking and oral contraceptive use contribute to an elevated risk for coronary heart disease.     

Cigarette smoke extract induces DNA damage but not apoptosis in human bronchial epithelial cells

Whether DNA damage caused by cigarette smoke leads to repair or apoptosis has not been fully elucidated. The current study demonstrates that cigarette smoke induces single-strand DNA damage in human bronchial epithelial cells. Cigarette smoke also stimulated caspase 3 precursors as well as intact poly (ADP-ribose) polymerase (PARP) production, but did not activate caspase 3 or cleave PARP, while the alkaloid camptothecin did so. Neither apoptosis nor necrosis was induced by cigarette smoke when the insult was removed within a designated time period. In contrast, DNA damage following cigarette smoke exposure was repaired as evidenced by decreasing terminal dUTP-biotin nick-end labeling positivity. The PARP inhibitor, 3-aminobenzamide blocked this repair. Furthermore, cells subjected to DNA damage were able to survive and proliferate clonogenically when changed to smoke-free conditions. These results suggest that cigarette smoke-induced DNA damage in bronchial epithelial cells is not necessarily lethal, and that PARP functions in the repair process. Our data also suggest that the potency of cigarettes as a carcinogen may result from their ability to induce DNA damage while failing to trigger the apoptotic progression permitting survival of cells harboring potentially oncogenic mutations.     

Smoking induces oxidative stress inside the Graafian follicle

BACKGROUND: A growing body of evidence indicates that pro-oxidant/antioxidant balance inside ovarian follicles plays an important role in folliculogenesis. Over 20% of women of reproductive age in Europe and the USA regularly smoke cigarettes. The impact of tobacco smoking on the intrafollicular markers of oxidative stress has not been fully elucidated. The objective of the present study was to test the hypothesis that cigarette smoking affects the intrafollicular redox milieu. METHODS: In follicular fluid samples originating from 108 IVF patients, lipid peroxidation was assessed by the thiobarbituric reactive substances method and total antioxidative capacity was quantified by the luminol enhanced chemiluminescence method. The level of patients' exposure to the cigarette smoke was evaluated by measuring the follicular fluid cotinine concentration by means of radioimmunoassay. RESULTS: Intrafollicular exposure to cigarette smoke metabolites was associated with a significant increase in follicular lipid peroxidation intensity (P < 0.001), which was accompanied by a significant decrease in the local antioxidative potential (P = 0.004). CONCLUSION: The results indicate that active smoking affects the pro-oxidant/antioxidant balance inside the pre-ovulatory ovarian follicle by inducing intrafollicular oxidative stress. This provides another possible explanation for impaired folliculogenesis in female smokers.     

Cigarette smoke activates caspase-3 to induce apoptosis of human umbilical venous endothelial cells

We explored an hypothesis that cigarette smoking-induced endothelial injury is mediated by accelerated apoptosis by treating human endothelial cells with cigarette smoke extracts (CSE). In cells treated with an increasing doses of CSE (0.005-0.03 cigarette equivalents/mL), we found a dose-dependent increase in the proportion of endothelial cells stained positive for apoptotic changes (8.3 +/- 0.7 to 50.7 +/- 2.2%, P < 0.01), accompanied by changes in caspase-3 activities and p53 protein levels. We suggest that excessive endothelial apoptosis may contribute to cigarette smoke-induced endothelial dysfunction and hence atherogenesis.     

Differential expression of heat shock protein 70 (hsp70) in human monocytes rendered apoptotic by IL-4 or serum deprivation

Apoptosis of monocytes is regulated by the balance between pro- and antiapoptotic triggers and pathways and may strongly influence inflammatory disorders. The major heat shock protein, hsp70, is an effective inhibitor of apoptosis in lymphocytic and monocytic tumor cell lines, but the implications in the regulation of apoptosis of freshly isolated human monocytes have not been elucidated. In this study, we examined whether two different triggers of monocyte apoptosis, serum deprivation and IL-4, respectively, altered hsp70 expression and whether expression levels correlated with monocyte survival. Monocyte apoptosis was determined quantitatively by flow cytometry detecting annexin V binding or nuclear stainability with propidium iodide (PI). Hsp70 expression was analyzed by semiquantitative RT-PCR and immunoblotting. Exposing monocytes to heat shock (47 degrees C, 20 min) induced a rapid and marked upregulation of hsp70 without evoking injury or apoptosis, suggesting that hsp70 conferred protection and survival. In accordance, when monocytes were rendered apoptotic by serum deprivation, a drastic downregulation of hsp70 occurred, which was accompanied by a reduced synthesis of the constitutive family member hsc70. However, induction of monocyte apoptosis by IL-4 increased hsp70 expression in a concentration and time-dependent fashion. A neutralizing antibody against IL-4 abolished hsp70 expression and apoptosis induction after IL-4 treatment and so excluded indirect effects. LPS rescued monocytes from apoptosis but did not alter hsp70 formation significantly. These findings suggest that, in monocytes, distinct apoptotic triggers induce different responses of hsp70 so that this molecule does not exert protection against cell death directly or in general.     

Arsenic and cigarette smoke synergistically increase DNA oxidation in the lung

Epidemiological evidence has indicated that arsenic and cigarette smoking exposure act synergistically to increase the incidence of lung cancer. Since oxidative damage of DNA has been linked to cancer, our hypothesis is that aerosolized arsenic and cigarette smoke work synergistically to increase oxidative stress and increase DNA oxidation in the lung. To test this hypothesis male Syrian golden hamsters were exposed to room air (control), aerosolized arsenic compounds (3.2 mg/m3 for 30 minutes), cigarette smoke (5 mg/m3 for 30 minutes), or both smoke and arsenic. Exposures were for 5 days/week for 5 or 28-days. Animals were sacrificed one day after the last exposure. In the 28-day group, glutathione levels and DNA oxidation (8-oxo-2'-deoxyguanosine (8-oxo-dG)) were determined. Our results show that in the 28-day arsenic/smoke group there was a significant decrease in both the reduced and total glutathione levels compared with arsenic or smoke alone. This correlated with a 5-fold increase in DNA oxidation as shown by HPLC. Immunohistochemical localization of 8-oxo-dG showed increase staining in nuclei of airway epithelium and subadjacent interstitial cells. These results show that dual exposure of arsenic and cigarette smoke at environmentally relevant levels can act synergistically to cause DNA damage.     

香烟烟雾暴露对幼年大鼠海马DNA甲基化的影响

目的:探讨香烟烟雾暴露对新生大鼠海马细胞DNA甲基化的水平的影响。方法:18只的SD幼年大鼠随机分为对照组(control)、香烟烟雾暴露组(cigarette smoke),香烟烟雾暴露组大鼠进行香烟烟雾暴露30 d,利用商品化试剂盒检测各组大鼠海马组织5’甲基胞嘧啶(5-mC)的变化,real time RT-PCR检测各组大鼠海马DNA甲基转移酶1(DNMT1)、DNA甲基转移酶3a(DNMT3a)及DNA甲基转移酶3b(DNMT3b) mRNA表达水平,Western Blot检测各组大鼠海马DNMT1、DNMT3a及DNMT3b蛋白水平表达。结果:与对照组相比,香烟烟雾暴露可导致大鼠海马5-mC水平增加,海马DNMT1 mRNA和蛋白表达增加。结论:幼年大鼠香烟烟雾暴露会上调海马组织DNMT1的表达,进而增加DNA甲基化水平。     

Cigarette smoking alters epithelial apoptosis and immune composition in murine GALT

Smokers have a twofold increased risk to develop Crohn's disease (CD). However, little is known about the mechanisms through which smoking affects CD pathogenesis. Especially Crohn's ileitis is negatively influenced by smoking. Interestingly, the ileum and, more in particular, the Peyer's patches in the terminal ileum are also the sites where the first CD lesions are found. Several chemokines are implicated in the pathogenesis, among which is the CCL20-CCR6 pathway. Here, we studied the gut-associated lymphoid tissue in C57BL/6 wild-type mice and in CCR6-deficient mice after exposure to air or cigarette smoke for 24 weeks. Apoptotic index of the follicle-associated epithelium overlying the Peyer's patches was evaluated. We found that chronic smoke exposure induced apoptosis in the follicle-associated epithelium. Furthermore, immune cell numbers and differentiation along with chemokine expression were determined in Peyer's patches. Important changes in immune cell composition were observed: total dendritic cells, CD4+ T cells (including regulatory T cells) and CD8+ T cells increased significantly after smoke exposure. The CD11b+ dendritic cell subset almost doubled. Interestingly, these changes were accompanied by an upregulated mRNA expression of the chemokines CCL9 and CCL20. However, no differences in the increase of dendritic cells were observed between wild-type and CCR6-deficient mice. Our results show that cigarette smoke exposure increases apoptosis in the follicle-associated epithelium and is associated with immune cell accumulation in Peyer's patches.     

Antinociception induced by chronic exposure of rats to cigarette smoke

To investigate if chronic exposure to cigarette smoke induces analgesia, rats were exposed to concentrated cigarette smoke in an environmental chamber over four successive 5-day blocks (6 h/day), with 2 smoke-free days between blocks. A control group was exposed to room air. Tail flick latencies increased significantly (analgesia) during each smoke exposure block, with a relative decline in analgesia across blocks (tolerance) and a return to control levels during the first three smoke-free interludes while remaining higher after the conclusion of the 4-week exposure period. Mechanical (von Frey) withdrawal thresholds declined over time in smoke-exposed and control groups, with the smoke-exposed group showing significantly lower thresholds. Plasma nicotine reached 95.4 +/- 32 (S.D.) ng/ml at the end of weekly smoke exposure and declined to 44.9 +/- 10.6 ng/ml 24 h after withdrawal. Rats lost weight during smoke exposure and quickly regained weight during smoke-free interludes and at the cessation of smoke exposure. Analgesia may contribute to the initiation of smoking, and rapid reversal of the analgesic effect following acute exposure may contribute to the difficulty in quitting smoking.     

Reciprocal modification of Fas activation and stress protein response decides apoptosis or resistance development of cells

Activation of heat shock factor (HSF)-1 DNA binding and heat shock protein (hsp)-70 expression enable resistance of cells to various forms of stress and maintain cell survival. Fas, a membrane-bound protein, is a central pro-apoptotic factor. Its activation leads to a cascade of events resulting in programmed cell death. Herein, these two mechanisms with contrary functions, promoting either cell survival or death, were addressed for their potential to inhibit each other's activation. Induction of Fas-mediated signalling was followed by a rapid decrease of HSF1 DNA binding and inducible hsp70 expression. Inhibition of HSF1 DNA binding was demonstrated to be based on absent hyperphosphorylation of HSF1 during FAS-signalling. These effects of Fas-activation on the HSF1/hsp70 stress response were blocked by ICE (caspase 1)-inhibitors, suggesting an ICE-mediated process. Furthermore, inhibition of HSF1/hsp70 was accompanied by an increase of apoptosis rates from 20% to 50% in response to heat stress. When analyzing Fas-mediated apoptosis in the presence of HSF1/hsp70 activation, decreased apoptosis rates were detected with induced expression of hsp70 but not with activation of HSF1-DNA binding alone. Thus, we conclude that inhibition of the HSF1/hsp70 stress response during Fas-mediated apoptosis and vice versa may facilitate a cell to pass a previously chosen pathway, stress resistance or apoptosis.     

The Effect of Different Doses of Cigarette Smoke in a Mouse Lung Tumor Model

Few studies have used Balb/c mice as an animal model for lung carcinogenesis. In this study, we investigated the effect of different doses of cigarette smoking in the urethane-induced Balb/c mouse lung cancer model. After injection of 3mg/kg urethane intraperitoneally, the mice were then exposed to tobacco smoke once or twice a day, five times a week, in a closed chamber. The animals were randomly divided into four groups. The control group (G0) received urethane only. The experimental groups (G1, G2 and G3) received urethane and exposure to the smoke of 3 cigarettes for 10 minutes once a day, 3 cigarettes for 10 minutes twice a day, and 6 cigarettes for 10 minutes twice a day, respectively. The mice were sacrificed after 16 weeks of exposure, and the number of nodules and hyperplasia in the lungs was counted. The results showed no statistically significant difference in the mean number of nodules and hyperplasia among the different groups, suggesting that the Balb/c mice are not suitable to study the pathogenesis of tobacco smoking-induced tumor progression in the lungs.     

Chronic smoke exposure induces rheumatoid factor and anti-heat shock protein 70 autoantibodies in susceptible mice and humans with lung disease

The impact of cigarette smoke (CS), a risk factor for rheumatoid arthritis (RA), on sauto-antibody production was studied in humans and mice with and without chronic lung disease (LD). Rheumatoid factor (RF), anti-cyclic citrullinated peptides (CCPs), and anti-HSP70 autoantibodies were measured in several mouse strains and in cohorts of smokers and nonsmokers with and without autoimmune disease. Chronic smoking-induced RFs in AKR/J mice, which are most susceptible to LD. RFs were identified in human smokers, preferentially in those with LD. Anti-HSP70 auto-antibodies were identified in CS-exposed AKR/J mice but not in ambient air exposed AKR/J controls. Whereas inflammation could induce anti-HSP70 IgM, smoke exposure promoted the switch to anti-HSP70 IgG autoantibodies. Elevated anti-CCP autoantibodies were not detected in CS-exposed mice or smokers. AKR/J splenocytes stimulated in vitro by immune complexes (ICs) of HSP70/anti-HSP70 antibodies produced RFs. The CD91 scavenger pathway was required as anti-CD91 blocked the HSP70-IC-induced RF response. Blocking Toll-like receptors did not influence the HSP70-IC-induced RFs. These studies identify both anti-HSP70 and RFs as serological markers of smoke-related LD in humans and mice. Identification of these autoantibodies could suggest a common environmental insult, namely CS, in a number of different disease settings.     

Tobacco smoke: Unraveling a controversial subject

Cigarettes are a modern and industrial form of tobacco use and obviously involve more than just tobacco. A multitude of physical processes and chemical reactions occur inside the burning zone of a cigarette. Cigarette smoke is an aerosol of liquid droplets (the particulate phase) suspended within a mixture of gases and semi-volatile compounds. Two kinds of smoke with different composition and properties are produced during smoking: mainstream smoke inhaled by the smoker and sidestream smoke, which is released into the environment between puffs from the lit end of the cigarette. Several techniques and modifications have altered the design of the cigarette during the last 50 years and changed smoke composition, with the effect of lower tar and nicotine smoke yields. An enormous amount of research has been done since the 1950s on smoke composition. With regard to the numerous toxic or carcinogenic constituents identified in tobacco smoke, there is a strong focus in the industry and with the authorities on the over 40 compounds, called "Hoffmann analytes". The yields of tar and nicotine in mainstream smoke of a cigarette brand as printed on the pack are measured with smoking machines under highly standardized conditions. Yields must comply with regulatory limits set in a number of countries. Smoking by machine is different from the smoking behavior of humans. There is a growing movement to develop more "realistic" methods to estimate smoke yields. But it is unclear whether alternative smoking regimens are more representative of human smoking behavior and provide better predictions of human exposure. Tobacco smoke has strong biological and toxicological effects in vitro and in vivo. There is an obvious need for developing a unified and validated testing approach particularly for the assessment of additives and the evaluation of new potentially reduced exposure products (PREPs). This paper gives a comprehensive overview of cigarette design, the composition and toxicity testing of smoke, and the way machines and people smoke - with links to the more detailed literature.     

Reversible cigarette smoke extract-induced DNA damage in human lung fibroblasts

Cigarette smoke contains thousands of chemicals, many of which may contribute to cytotoxicity and carcinogenesis. Using assays detecting DNA strand breaks (terminal transferase dUTP nick end labeling [TUNEL]) and DNA content (flow cytometry), we evaluated the genotoxic effect of cigarette smoke extract (CSE) on human fetal lung fibroblasts (HFL-1) cultured in three-dimensional collagen gels as well as in monolayer culture. When HFL-1 cells were exposed to CSE, DNA strand breaks were detected in most, as determined by TUNEL. This effect was dependent on CSE concentration, duration of CSE exposure, and the density of HFL-1 cells cast into the collagen gels. Buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis, significantly increased DNA damage induced by 1% CSE, and N-acetylcysteine, a glutathione precursor, blocked 5% CSE from inducing DNA damage. After CSE exposure, most cells were TUNEL-positive, but DNA quantification revealed no hypodiploid cells, indicating that apoptosis was not occurring during the CSE exposure. CSE-induced DNA damage was reversible, and cells proliferated when CSE was removed after 24 h exposure. These results demonstrate that cigarette smoke can induce DNA damage in HFL-1 cells cultured in both three-dimensional collagen gels and monolayer cultures, and that oxidants likely play a role in this damage. Moreover, this DNA damage is reversible, with cells surviving and TUNEL positivity reversing when CSE is removed within 24 h.     

Cigarette smoking behaviors and time since quitting are associated with differential DNA methylation across the human genome

The impact of cigarette smoking can persist for extended periods following smoking cessation and may involve epigenetic reprogramming. Changes in DNA methylation associated with smoking may help to identify molecular pathways that contribute to the latency between exposure and disease onset. Cross-sectional cohort data from subjects in the International COPD Genetics Network (n = 1085) and the Boston Early-Onset COPD study (n = 369) were analyzed as the discovery and replication cohorts, respectively. Genome-wide methylation data on 27 578 CpG sites in 14 475 genes were obtained on DNA from peripheral blood leukocytes using the Illumina HumanMethylation27K Beadchip in both cohorts. We identified 15 sites significantly associated with current smoking, 2 sites associated with cumulative smoke exposure, and, within the subset of former smokers, 3 sites associated with time since quitting cigarettes. Two loci, factor II receptor-like 3 (F2RL3) and G-protein-coupled receptor 15 (GPR15), were significantly associated in all three analyses and were validated by pyrosequencing. These findings (i) identify a novel locus (GPR15) associated with cigarette smoking and (ii) suggest the existence of dynamic, site-specific methylation changes in response to smoking which may contribute to the extended risks associated with cigarette smoking that persist after cessation.     

Involvement of bcl-2 and caspase-3 in apoptosis induced by cigarette smoke extract in the gastric epithelial cell

Previous studies have shown that short-term passive cigarette smoking can increase apoptosis in rat gastric mucosa. However, the mechanism is not yet defined. Chloroform and ethanol extracts were used to investigate whether cigarette smoke could induce apoptosis in a human gastric epithelial cell line (AGS) as well as the roles of bcl-2, caspase-3, and cytochrome c in this process. AGS cell lines were treated with either chloroform extract (CE) or ethanol extract (EE) for 5 hours, and the level of bcl-2, the activity of caspase-3, and the level of cytosolic cytochrome c in these cells were determined. Time course studies on the effects of cigarette smoke extracts (CSEs) on DNA fragmentation and cytochrome c relocalization were also performed. Data showed that only CE induced apoptosis in a dose- and time-dependent manner in AGS cells, along with a decrease of bcl-2 and an increase of caspase-3 activity. Pretreatment with Z-DEVD-FMK (specific inhibitor of caspase-3) dose-dependently blocked the DNA fragmentation induced by the CE. Moreover, CE could time- and dose-dependently increase the level of cytochrome c in the cytoplasm, which might activate caspase-3. In conclusion, CSE triggers apoptosis in AGS cells through the inhibition of bcl-2 and the activation of a mitochondria-related pathway.     

Parental bad habits breed bad behaviors in youth: Exposure to gestational smoke and child impulsivity

In utero exposure to cigarette smoke has been shown to have an adverse effect on healthy brain development in childhood. In the present study, we examine whether fetal exposure to mild and heavy smoking is associated with lower levels of impulsivity and cognitive control at age 10. Using a sample of 2120 children from the Québec Longitudinal Study of Child Development, we examine the association between gestational cigarette smoke exposure and fourth grade teacher reports of impulsivity and classroom engagement which represent behavioral indicators of executive functions. When compared to children of non-smokers, children of mothers who reported smoking heavily during pregnancy (10 or more cigarettes per day) were rated by their fourth grade teachers as displaying higher levels of impulsive behavior, scoring.112 standard deviation units higher than children of non-smokers. Children of mothers who smoked heavily were also less engaged in the classroom, scoring.057 standard deviation units lower than children of women who did not smoke. These analyses were adjusted for many potentially confounding child and family variables. Exposure to perinatal nicotine may compromise subsequent brain development. In particular, fetal nicotine may be associated with impairment in areas recruited for the effortful control of behavior in later childhood, a time when task-orientation and industriousness are imperative for academic success.