High susceptibility of neonatal mice to molecular, biochemical and cytogenetic alterations induced by environmental cigarette smoke and light

Our recent studies have shown that both cigarette smoke and UV-containing light, which are the most widespread and ubiquitous mutagens and carcinogens in the world, cause systemic genotoxic damage in hairless mice. Further studies were designed with the aim of evaluating the induction of genotoxic and carcinogenic effects in Swiss albino mice exposed to smoke and/or light since birth. We observed that a 4-month whole-body exposure of mice to mainstream cigarette smoke, starting at birth, caused an early and potent carcinogenic response in the lung and other organs. Our further experiments showed that exposure of mice to environmental cigarette smoke, during the first 5 weeks of life, resulted in a variety of significant alterations of intermediate biomarkers, including cytogenetic damage in bone marrow and peripheral blood, formation of lipid peroxidation products, increase of bulky DNA adduct levels, induction of oxidative DNA damage, and overexpression of OGG1 gene in lung, stimulation of apoptosis, hyperproliferation and loss of Fhit protein in pulmonary alveolar macrophages and/or bronchial epithelial cells, and early histopathological alterations in the respiratory tract. Moreover, exposure of mice to UV-containing light, mimicking solar irradiation, significantly enhanced oxidative DNA damage and bulky DNA adduct levels in lung, and synergized with smoke in inducing molecular alterations in the respiratory tract. The baseline OGG1 expression in lung was particularly high at birth and decreased in post-weanling mice. Oxidative DNA damage and other investigated end-points exhibited differential patterns in post-weanling mice and adult mice. The findings of these studies provide a mechanistic clue to the general concept that the neonatal period and early stages of life are critical in affecting susceptibility to carcinogens.     

Role of protein kinase N2 (PKN2) in cigarette smoke-mediated oncogenic transformation of oral cells

Smoking is the leading cause of preventable death worldwide. Though cigarette smoke is an established cause of head and neck cancer (including oral cancer), molecular alterations associated with chronic cigarette smoke exposure are poorly studied. To understand the signaling alterations induced by chronic exposure to cigarette smoke, we developed a cell line model by exposing normal oral keratinocytes to cigarette smoke for a period of 12 months. Chronic exposure to cigarette smoke resulted in increased cellular proliferation and invasive ability of oral keratinocytes. Proteomic and phosphoproteomic analyses showed dysregulation of several proteins involved in cellular movement and cytoskeletal reorganization in smoke exposed cells. We observed overexpression and hyperphosphorylation of protein kinase N2 (PKN2) in smoke exposed cells as well as in a panel of head and neck cancer cell lines established from smokers. Silencing of PKN2 resulted in decreased colony formation, invasion and migration in both smoke exposed cells and head and neck cancer cell lines. Our results indicate that PKN2 plays an important role in oncogenic transformation of oral keratinocytes in response to cigarette smoke. The current study provides evidence that PKN2 can act as a potential therapeutic target in head and neck squamous cell carcinoma, especially in patients with a history of smoking.     

Cigarette Smoke Exposure during Pregnancy Alters Fetomaternal Cell Trafficking Leading to Retention of Microchimeric Cells in the Maternal Lung

Cigarette smoke exposure causes chronic oxidative lung damage. During pregnancy, fetal microchimeric cells traffic to the mother. Their numbers are increased at the site of acute injury. We hypothesized that milder chronic diffuse smoke injury would attract fetal cells to maternal lungs. We used a green-fluorescent-protein (GFP) mouse model to study the effects of cigarette smoke exposure on fetomaternal cell trafficking. Wild-type female mice were exposed to cigarette smoke for about 4 weeks and bred with homozygote GFP males. Cigarette smoke exposure continued until lungs were harvested and analyzed. Exposure to cigarette smoke led to macrophage accumulation in the maternal lung and significantly lower fetal weights. Cigarette smoke exposure influenced fetomaternal cell trafficking. It was associated with retention of GFP-positive fetal cells in the maternal lung and a significant reduction of fetal cells in maternal livers at gestational day 18, when fetomaternal cell trafficking peaks in the mouse model. Cells quickly clear postpartum, leaving only a few, difficult to detect, persisting microchimeric cells behind. In our study, we confirmed the postpartum clearance of cells in the maternal lungs, with no significant difference in both groups. We conclude that in the mouse model, cigarette smoke exposure during pregnancy leads to a retention of fetal microchimeric cells in the maternal lung, the site of injury. Further studies will be needed to elucidate the effect of cigarette smoke exposure on the phenotypic characteristics and function of these fetal microchimeric cells, and confirm its course in cigarette smoke exposure in humans.     

Dose-responsiveness and persistence of microRNA expression alterations induced by cigarette smoke in mouse lung

Our previous studies demonstrated that exposure to cigarette smoke (CS), either mainstream or environmental, results in a remarkable downregulation of microRNA expression in the lung of both mice and rats. The goals of the present study were to evaluate the dose responsiveness to CS and the persistence of microRNA alterations after smoking cessation. ICR (CD-1) neonatal mice were exposed whole-body to mainstream CS, at the doses of 119, 292, 438, and 631mg/m(3) of total particulate matter. Exposure started within 12h after birth and continued daily for 4 weeks. The levels of bulky DNA adducts and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) were measured by (32)P postlabeling procedures, and the expression of 697 mouse microRNAs was analyzed by microarray. The highest CS dose was lethal. Exposure to CS caused a dose-dependent increase of DNA alterations. DNA adducts and, even more sharply, 8-oxodGuo were reverted 1 and 4 weeks after smoking cessation. Exposure to CS resulted in an evident dysregulation of microRNA expression profiles, mainly in the sense of downregulation. The two lowest doses were not particularly effective, while the highest nonlethal dose produced extensive microRNA alterations. The expression of most downregulated microRNAs, including among others 7 members of the let-7 family, was restored one week after smoking cessation. However, the recovery was incomplete for a limited array of microRNAs, including mir-34b, mir-345, mir-421, mir-450b, mir-466, and mir-469. Thus, it appears that microRNAs mainly behave as biomarkers of effect and that exposure to high-dose, lasting for an adequate period of time, is needed to trigger the CS-related carcinogenesis process in the experimental animal model used.     

Imbalance in zinc homeostasis enhances lung Tissue Loss following cigarette smoke exposure

Cigarette smoke exposure is a major cause of chronic obstructive pulmonary disease. Cadmium is a leading toxic component of cigarette smoke. Cadmium and zinc are highly related metals. Whereas, zinc is an essential metal required for normal health, cadmium is highly toxic. Zrt- and Irt-like protein 8 (ZIP8) is an avid transporter of both zinc and cadmium into cells and is abundantly expressed in the lung of smokers compared to nonsmokers. Our objective was to determine whether disturbed zinc homeostasis through diet or the zinc transporter ZIP8 increase susceptibility to lung damage following prolonged cigarette smoke exposure.MethodsCigarette smoke exposure was evaluated in the lungs of mice subject to insufficient and sufficient zinc intakes, in transgenic ZIP8 overexpressing mice, and a novel myeloid-specific ZIP8 knockout strain.ResultsModerate depletion of zinc intakes in adult mice resulted in a significant increase in lung cadmium burden and permanent lung tissue loss following prolonged smoke exposure. Overexpression of ZIP8 resulted in increased lung cadmium burden and more extensive lung damage, whereas cigarette smoke exposure in ZIP8 knockout mice resulted in increased lung tissue loss without a change in lung cadmium content, but a decrease in zinc.ConclusionsOverall, findings were consistent with past human studies. Imbalance in Zn homeostasis increases susceptibility to permanent lung injury following prolonged cigarette smoke exposure. Based on animal studies, both increased and decreased ZIP8 expression enhanced irreversible tissue damage in response to prolonged tobacco smoke exposure. We believe these findings represent an important advancement in our understanding of how imbalance in zinc homeostasis and cadmium exposure via tobacco smoke may increase susceptibility to smoking-induced lung disease.     

Cigarette smoke-induced DNA damage in cultured human lung cells: role of hydroxyl radicals and endonuclease activation.

Cigarette smoke can cause DNA single strand breaks in cultured human lung cells (T. Nakayama et al., Nature, 314 (1985) 462-464) but the mechanisms behind this DNA damage have not been clearly elucidated. In the present study we have investigated the possibility that one of the major constituents in cigarette smoke, hydroquinone, may be important for mediating smoke-induced DNA damage in the human epithelial lung cell line, A 549, and the mechanisms behind this damage. Cells were exposed to cigarette smoke, hydrogen peroxide, or hydroquinone, in the absence and presence of different inhibitors, and the resulting DNA damage was assessed either as DNA single strand break formation or formation of the oxidative DNA adduct, 8-hydroxydeoxyguanosine. It was found that (i) exposure to cigarette smoke, hydrogen peroxide or hydroquinone causes a rapid decrease in the intracellular thiol level and a considerable DNA single strand break formation, (ii) the formation of DNA single strand breaks in cells exposed to cigarette smoke is inhibited by catalase, dimethylthiourea, and o-phenantroline, suggesting that hydroxyl radicals generated from iron-catalyzed hydrogen peroxide dissociation are involved in the DNA damage, (iii) hydroquinone causes considerable DNA strand break formation that is blocked by aurintricarboxylic acid, an inhibitor of endonuclease activation, and by BAPTA, an intracellular calcium chelator, (iv) addition of hydroquinone to a smoke condensate greatly enhances its ability to cause DNA single strand breaks, and (v) smoke, but not hydroquinone, causes formation of 8-hydroxydeoxyguanosine, a DNA damage product induced by the action of hydroxyl radicals on the DNA base, deoxyguanosine. These findings suggest that the ability of cigarette smoke to cause DNA single strand breaks in cultured lung cells is due to mechanisms involving hydroxyl radical attack on DNA and endonuclease activation. They also suggest that hydroquinone is an important contributor to the DNA damaging effect of cigarette smoke on human lung cells.     

Alterations in Skeletal Muscle Cell Homeostasis in a Mouse Model of Cigarette Smoke Exposure

Background

Skeletal muscle dysfunction is common in chronic obstructive pulmonary disease (COPD), a disease mainly caused by chronic cigarette use. An important proportion of patients with COPD have decreased muscle mass, suggesting that chronic cigarette smoke exposure may interfere with skeletal muscle cellular equilibrium. Therefore, the main objective of this study was to investigate the kinetic of the effects that cigarette smoke exposure has on skeletal muscle cell signaling involved in protein homeostasis and to assess the reversibility of these effects.

Methods

A mouse model of cigarette smoke exposure was used to assess skeletal muscle changes. BALB/c mice were exposed to cigarette smoke or room air for 8 weeks, 24 weeks or 24 weeks followed by 60 days of cessation. The gastrocnemius and soleus muscles were collected and the activation state of key mediators involved in protein synthesis and degradation was assessed.

Results

Gastrocnemius and soleus were smaller in mice exposed to cigarette smoke for 8 and 24 weeks compared to room air exposed animals. Pro-degradation proteins were induced at the mRNA level after 8 and 24 weeks. Twenty-four weeks of cigarette smoke exposure induced pro-degradation proteins and reduced Akt phosphorylation and glycogen synthase kinase-3β quantity. A 60-day smoking cessation period reversed the cell signaling alterations induced by cigarette smoke exposure.

Conclusions

Repeated cigarette smoke exposure induces reversible muscle signaling alterations that are dependent on the duration of the cigarette smoke exposure. These results highlights a beneficial aspect associated with smoking cessation.     

Cigarette Smoke Causes Caspase-Independent Apoptosis of Bronchial Epithelial Cells from Asthmatic Donors

Background

Epidemiologic studies have demonstrated important links between air pollution and asthma. Amongst these pollutants, environmental cigarette smoke is a risk factor both for asthma pathogenesis and exacerbation. As the barrier to the inhaled environment, the bronchial epithelium is a key structure that is exposed to cigarette smoke.

Objectives

Since primary bronchial epithelial cells (PBECs) from asthmatic donors are more susceptible to oxidant-induced apoptosis, we hypothesized that they would be susceptible to cigarette smoke-induced cell death.

Methods

PBECs from normal and asthmatic donors were exposed to cigarette smoke extract (CSE); cell survival and apoptosis were assessed by fluorescence-activated cell sorting, and protective effects of antioxidants evaluated. The mechanism of cell death was evaluated using caspase inhibitors and immunofluorescent staining for apoptosis-inducing factor (AIF).

Results

Exposure of PBEC cultures to CSE resulted in a dose-dependent increase in cell death. At 20% CSE, PBECs from asthmatic donors exhibited significantly more apoptosis than cells from non-asthmatic controls. Reduced glutathione (GSH), but not ascorbic acid (AA), protected against CSE-induced apoptosis. To investigate mechanisms of CSE-induced apoptosis, caspase-3 or -9 inhibitors were tested, but these failed to prevent apoptosis; in contrast, CSE promoted nuclear translocation of AIF from the mitochondria. GSH reduced the number of nuclear-AIF positive cells whereas AA was ineffective.

Conclusion

Our results show that PBECs from asthmatic donors are more susceptible to CSE-induced apoptosis. This response involves AIF, which has been implicated in DNA damage and ROS-mediated cell-death. Epithelial susceptibility to CSE may contribute to the impact of environmental tobacco smoke in asthma.     

Enhancement of pre-existing DNA adducts in rodents exposed to cigarette smoke

Exposure to tobacco smoke has been implicated in the increased incidence of cancer and cardiovascular diseases. This report describes various experimental studies in animals that were carried out to determine the ability of cigarette smoke to form DNA adducts and to define chromatographic nature of the major adducts. Tissues from rodents exposed to mainstream or sidestream cigarette smoke in nose-only and whole-body exposure systems, respectively, for different durations were analyzed for DNA adducts by 32P-postlabeling assay. The results showed essentially similar qualitative patterns in various respiratory (lung, trachea, larynx) and non-respiratory (heart, bladder) tissues of smoke-exposed rats. However, adduct pattern in the nasal mucosa was different. The mean total DNA adducts in various tissues expressed as per 1010 nucleotides exhibited the following order: heart (700)>lung (420)>trachea (170)>larynx (150)>bladder (50). Some qualitatively identical adducts were routinely detected in tissues from sham-treated rats but at greatly reduced levels (5- to 25-fold). The levels of lung DNA adducts increased with the duration of exposure up to 23 weeks and returned to control levels 19 weeks after the cessation of exposure. Species-related differences in adduct magnitude and patterns were observed among rats, mice and guinea pigs; mouse being the most sensitive to DNA damage and guinea pig the least sensitive. Whole-body exposure of rats to sidestream cigarette smoke also enhanced the pre-existing DNA adducts by several fold in different tissues. Selective chromatography, and extractability in butanol suggested lipophilic nature of smoke-associated DNA adducts, which were, however, recovered significantly better in nuclease P1 than butanol enrichment procedure. The major smoke-associated adducts were chromatographically different from any of the reference adducts of polycyclic aromatic hydrocarbons (PAHs) co-chromatographed with the smoke DNA samples. Because PAH-DNA adducts are recovered with equal efficiency by the two enrichment procedures, the above observations suggested that smoke-associated adducts are not related to typical PAHs, like benzo[a]pyrene. It is concluded that cigarette smoke increased the levels of pre-existing endogenous DNA adducts (the so-called I-compounds) in animal models and that these adducts are unrelated to those formed by typical PAHs.     

NF-kappaB mediates the survival of human bronchial epithelial cells exposed to cigarette smoke extract

Background

We have previously reported that low concentrations of cigarette smoke extract induce DNA damage without leading to apoptosis or necrosis in human bronchial epithelial cells (HBECs), and that IL-6/STAT3 signaling contributes to the cell survival. Since NF-κB is also involved in regulating apoptosis and cell survival, the current study was designed to investigate the role of NF-κB in mediating cell survival in response to cigarette smoke exposure in HBECs.

Methods

Both the pharmacologic inhibitor of NF-κB, curcumin, and RNA interference targeting p65 were used to block NF-κB signaling in HBECs. Apoptosis and cell survival were then assessed by various methods including COMET assay, LIVE/DEAD Cytotoxicity/Viability assay and colony formation assay.

Results

Cigarette smoke extract (CSE) caused DNA damage and cell cycle arrest in S phase without leading to apoptosis in HBECs as evidenced by TUNEL assay, COMET assay and DNA content assay. CSE stimulated NF-κB -DNA binding activity and up-regulated Bcl-XL protein in HBECs. Inhibition of NF-κB by the pharmacologic inhibitor curcumin (20 μM) or suppression of p65 by siRNA resulted in a significant increase in cell death in response to cigarette smoke exposure. Furthermore, cells lacking p65 were incapable of forming cellular colonies when these cells were exposed to CSE, while they behaved normally in the regular culture medium.

Conclusion

The current study demonstrates that CSE activates NF-κB and up-regulates Bcl-XL through NF-kB activation in HBECs, and that CSE induces cell death in cells lacking p65. These results suggest that activation of NF-κB regulates cell survival following DNA damage by cigarette smoke in human bronchial epithelial cells.     

Exogenous heat shock protein-70 inhibits cigarette smoke-induced intimal thickening

Cigarette smoke is associated with increased carotid intimal thickening or stroke. Preliminary work showed that exposure to smoke resulted in a 4.5-fold reduction of heat shock protein-70 (HSP70) expression in spleens of mice using gene microarray analysis. In the current study, we investigated the role of extracellular HSP70 in carotid intimal thickening of mice exposed to cigarette smoke. Intimal thickening was induced by placement of a cuff around the right carotid artery of mice. Cuff injury resulted in increased HSP70 mRNA expression in carotid arteries that persisted for 21 days. Cigarette smoke exposure decreased arterial HSP70 expression and significantly increased intimal thickening compared with mice exposed to air. Treatment of mice exposed to cigarette smoke with intravenous recombinant HSP70 attenuated intimal thickening through reduced phosphorylated extracellular signal-regulated kinase (pERK) expression in the arterial wall. In vitro experiments with rat aortic smooth muscle cells confirmed that recombinant HSP70 decreases pERK and proliferating cell nuclear antigen (PCNA) expression in cells exposed to cigarette smoke extract and H(2)O(2). Our study suggests that decreased expression of arterial HSP70 is an important mechanism by which exposure to cigarette smoke augments intimal thickening. The effects of recombinant HSP70 suggest a role for extracellular HSP70.     

STAT3 activation inhibits human bronchial epithelial cell apoptosis in response to cigarette smoke exposure

We have previously reported that cigarette smoke can induce DNA damage in human lung cells without leading to apoptosis or necrosis. In this study, we report that STAT3 is required for the survival of human bronchial epithelial cells (HBECs) following cigarette smoke-induced DNA damage. Cigarette smoke extract (CSE) exposure increases STAT3 phosphorylation (Tyr 705) and DNA binding activity in HBECs. CSE also stimulates IL-6 release and mRNA expression. Anti-IL-6 neutralizing antibody partially blocks STAT3 activation and renders the cells sensitive to CSE-induced DNA damage. Suppression of STAT3 by siRNA results in severe DNA damage and cell death in response to CSE exposure. These findings suggest that STAT3 mediates HBEC survival in response to CSE-induced DNA damage, at least in part, through the IL-6/STAT3 signaling pathway.     

Passive cigarette smoking increases isoprostane formation

Passive smoking has been demonstrated to exert a variety of deleterious effects eventually resulting in vascular damage. Isoprostanes, a reliable marker of in vivo oxidation injury, have been shown to increase in active cigarette smoking. Data for passive smoking are lacking. We were examining the isoprostane 8-epi-PGF2alpha in 12 smokers and non-smokers exposed daily to passive cigarette smoke for 12 days. Plasma samples stored at liquid nitrogen from people having been examined earlier were used. Prevalues of 8-epi-PGF2alpha are higher in cigarette smokers. Exposure to passive smoking causes a significant increase in 8-epi-PGF2alpha in non-smokers, while in smokers there is only a trendwise increase. After repeated passive smoke exposure, 8-epi-PGF2alpha in non-smokers approaches the respective values of smokers. There is a significant correlation of 8-epi-PGF2alpha to the thromboxane (plasma, serum, conversion from exogenous precursor, 11-dehydro-TXB2) parameters (MDA, HHT- conversion) examined in these patients before. The findings document a significant temporary increase in in vivo oxidation injury due to passive smoke favouring development and/or progression of vascular disease.     

Role of CXCR2 in cigarette smoke-induced lung inflammation

It has been hypothesized that the destruction of lung tissue observed in smokers with chronic obstructive pulmonary disease and emphysema is mediated by neutrophils recruited to the lungs by smoke exposure. This study investigated the role of the chemokine receptor CXCR2 in mediating neutrophilic inflammation in the lungs of mice acutely exposed to cigarette smoke. Exposure to dilute mainstream cigarette smoke for 1 h, twice per day for 3 days, induced acute inflammation in the lungs of C57BL/6 mice, with increased neutrophils and the neutrophil chemotactic CXC chemokines macrophage inflammatory protein (MIP)-2 and KC. Treatment with SCH-N, an orally active small molecule inhibitor of CXCR2, reduced the influx of neutrophils into the bronchoalveolar lavage (BAL) fluid. Histological changes were seen, with drug treatment reducing perivascular inflammation and the number of tissue neutrophils. beta-Glucuronidase activity was reduced in the BAL fluid of mice treated with SCH-N, indicating that the reduction in neutrophils was associated with a reduction in tissue damaging enzymes. Interestingly, whereas MIP-2 and KC were significantly elevated in the BAL fluid of smoke exposed mice, they were further elevated in mice exposed to smoke and treated with drug. The increase in MIP-2 and KC with drug treatment may be due to the decrease in lung neutrophils that either are not present to bind these chemokines or fail to provide a feedback signal to other cells producing these chemokines. Overall, these results demonstrate that inhibiting CXCR2 reduces neutrophilic inflammation and associated lung tissue damage due to acute cigarette smoke exposure.     

Cyto-genotoxic effects of smoke from commercial filter and non-filter cigarettes on human bronchial and pulmonary cells

Cigarette smoke is a complex mixture of chemicals, some of which are known as carcinogens. The cyto-genotoxic effects of cigarette-smoke extract (CSE) from commercial cigarettes without (A and B) and with filter (C and D) were evaluated at different CSE concentrations on A549 and BEAS-2B cells. The particle content of the cigarette smoke and the metal composition of the CSE were also analyzed. The cells were exposed to 1–10% of the CSE from one cigarette per experiment. Cytotoxicity was evaluated by use of the MTT assay after 24 h, and the lactate dehydrogenase (LDH) assay after 30 min and 24 h. The Fpg-modified comet assay was used to evaluate direct-oxidative DNA damage on cells exposed for 30 min. As expected, unfiltered cigarette smoke (particularly from the B cigarette) contained a higher number of particles than filtered smoke. With smoke extract from the B cigarette we found a decrease in cell viability only in BEAS-2B cells. The results of the LDH test showed membrane damage for B-cigarette smoke extract, particularly in BEAS-2B cells. Extracts from unfiltered cigarette smoke induced significant direct DNA damage, to a larger extent in A549 cells. Filtered cigarette-smoke extract induced a significant direct DNA damage at 5–10%. A significant induction of oxidative DNA damage was found at the highest CSE concentration in both cell types (by smoke extracts from B and C cigarettes in A549 cells, and from A and D cigarettes in BEAS-2B cells). Smoke extracts from filter cigarettes induced less direct DNA damage than those from unfiltered cigarettes in A549 cells, probably due to a protective effect of filter. In BEAS-2B cells the smoke extract from the B-cigarette showed the highest genotoxic effect, with a concentration-dependent trend.These findings show a higher cyto-genotoxicity for smoke extracts from the B-cigarette and oxidative effects for those from the A and D cigarettes, particularly in BEAS-2B cells. Moreover, there was a higher responsiveness of A549 cells to genotoxic insult of CSE, and a cigarette-dependent genotoxicity in BEAS-2B cells. Our experimental model demonstrated to be suitable to sensitively detect early genotoxic response of different lung-cell types to non-cytotoxic concentrations of complex inhalable mixtures.     

A relation between TGF-β and mast cell tryptase in experimental emphysema models

Chronic obstructive pulmonary disease (COPD) is a multicomponent disease characterized by emphysema and/or chronic bronchitis. The aim of this study was to investigate the effect of cigarette smoke exposure on mast cells and mast cell function in vitro and in vivo in order to get further insight in the role of mast cells in the pathogenesis of emphysema. Cigarette smoke conditioned medium (CSM) induced the expression of mast cell tryptase (MMCP-6) in primary cultured mast cells. This tryptase expression was caused by the CSM-stimulated production of TGF-β in culture and neutralization of TGF-β suppressed the CSM-induced expression of tryptase in mast cells. An increase in mast cell tryptase expression was also found in an experimental model for emphysema. Exposure of mice to cigarette smoke increased the number of mast cells in the airways and the expression of mast cell tryptase. In accordance with the in vitro findings, TGF-β in bronchoalveolar lavage fluid of smoke-exposed animals was significantly increased. Our study indicates that mast cells may be a source of TGF-β production after cigarette smoke exposure and that in turn TGF-β may change the tryptase expression in mast cells.     

Cell wall reconstruction and DNA damage repair play a key role in the improved salt tolerance effects of He-Ne laser irradiation in tall fescue seedlings

The improved salt tolerance effects of He–Ne laser were further studied through the estimation of ROS levels, cell viability, DNA damage phenomena, physicochemical properties, and monosaccharide compositions of cell wall polysaccharides in tall fescue seedlings. Salt stress produced deleterious effects on seedlings growth and development. ROS levels and genomic DNA damage were markedly increased compared with controls. Physicochemical activities and monosaccharide proportions of cell wall polysaccharide were also pronouncedly altered. He–Ne laser irradiation improved plant growth retardation via increasing cell viability and reverting physicochemical parameters. According to the results of Fourier transform infrared (FTIR) scanning spectra and DNA apopladder analysis, He–Ne laser was showed to efficiently ameliorate cell wall polysaccharide damage and DNA fragmentation phenomena. The treatment with DNA synthesis inhibitor further demonstrated that DNA damage repair was correlated with the improvement effects of the laser. Therefore, our data illustrated that He–Ne laser irradiation resulted in cell wall reconstruction and genomic DNA injury repair in vivo in salt-stressed seedlings, then enhanced salt tolerance probably via interactions between plant cell wall and related resistance gene expression pattern.     

Cigarette smoke induces anaphase bridges and genomic imbalances in normal cells

Exposure to cigarette smoke has long been linked to carcinogenesis, but the emphasis has been placed on mutational changes in the DNA sequence caused by the carcinogens in smoke. Here, we report an additional role for cigarette smoke exposure in contributing to chromosomal aberrations in cells. We have found that cigarette smoke condensate (CSC) induces anaphase bridges in cultured human cells, which in a short time lead to genomic imbalances. The frequency of the induced bridges within the entire population decreases with time, and this decrease is not dependent upon the p53-mediated apoptotic pathway. Additionally, we show that CSC induces DNA double stranded breaks (DSBs) in cultured cells and purified DNA. The reactive oxygen species (ROS) scavenger, 2' deoxyguanosine 5'-monophosphate (dGMP) prevents CSC-induced DSBs, anaphase bridge formation and genomic imbalances. Therefore, we propose that CSC induces bridges and genomic imbalances via DNA DSBs. Furthermore, since the amount of CSC added to the cultures was substantially less than that extracted from a single cigarette, our results show that even low levels of cigarette smoke can cause irreversible changes in the chromosomal constitution of cultured cells.     

Nitric oxide from both exogenous and endogenous sources activates mitochondria-dependent events and induces insults to human chondrocytes

During inflammation, overproduction of nitric oxide (NO) can damage chondrocytes. In this study, we separately evaluated the toxic effects of exogenous and endogenous NO on human chondrocytes and their possible mechanisms. Human chondrocytes were exposed to sodium nitroprusside (SNP), an NO donor, or a combination of lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) as the exogenous and endogenous sources of NO, respectively. Administration of SNP or a combination of LPS and IFN-gamma in human chondrocytes increased cellular NO levels but decreased cell viability. Exposure to exogenous or endogenous NO significantly induced apoptosis of human chondrocytes. When treated with exogenous or endogenous NO, the mitochondrial membrane potential time-dependently decreased. Exposure to exogenous or endogenous NO significantly enhanced cellular reactive oxygen species (ROS) and cytochrome c (Cyt c) levels. Administration of exogenous or endogenous NO increased caspase-3 activity and consequently induced DNA fragmentation. Suppression of caspase-3 activation by Z-DEVD-FMK decreased NO-induced DNA fragmentation and cell apoptosis. Similar to SNP, exposure of human chondrocytes to S-nitrosoglutathione (GSNO), another NO donor, caused significant increases in Cyt c levels, caspase-3 activity, and DNA fragmentation, and induced cell apoptosis. Pretreatment with N-monomethyl arginine (NMMA), an inhibitor of NO synthase, significantly decreased cellular NO levels, and lowered endogenous NO-induced alterations in cellular Cyt c amounts, caspase-3 activity, DNA fragmentation, and cell apoptosis. Results of this study show that NO from exogenous and endogenous sources can induce apoptotic insults to human chondrocytes via a mitochondria-dependent mechanism.     

Aerosolized substance P protects against cigarette-induced lung damage and tumor development.

Both first-hand and second-hand [i.e. side-stream cigarette smoke (SSCS)] exposure to cigarette smoke is known to damage the lungs, alter the immune system, and predispose individuals to the development of emphysema and lung cancer. Previous work from these laboratories has demonstrated that administration of aerosolized substance P (SP) was capable of protecting the pulmonary and immune systems from damage due to environmental toxicants (e.g. hydrocarbon exposures). In the present study, the effects of SP on SSCS exposure were examined. Treatment of mice exposed to SSCS with aerosolized SP prevented pathological cellular and functional changes in the lung as reflected by prevention of damage to airway basement membranes/endothelial cells and preservation of normal airway dynamic compliance. Further, SP treatment reduced and/or prevented the occurrence of micronuclei formation in cells isolated from mice exposed in vivo to SSCS (an indicator of DNA/genetic damage). Finally, in an experimental in vivo lung cancer model, SP therapy significantly reduced the numbers of lung tumors, increased animal survival, and activated pulmonary immune defense mechanisms. Thus, aerosolized SP therapy appears to be capable of inhibiting preventing and/or reversing the cellular and genetic precursors of emphysema and malignancy that often result from exposure to cigarette smoke.