Chemoprevention of genotoxic damage in lung cells of rats exposed to cigarette smoke]

Male Sprague-Dawley rats were exposed to cigarette smoke (CS) according to a complex protocol which lasted 40 days. Some of the groups were pre-treated with N-acetyl-L-cysteine (NAC) by gavage (1 g/kg b.w.) 5 h before each exposure. Bronchoalveolar lavage was performed in each animal at the end of each exposure period in order to recover pulmonary alveolar macrophages (PAM). Cells were identified and counted under the microscope, and the number of micronucleated (MN) and binucleated (BN) PAM was registered. The results showed an increase in the number of MN PAM, which was already evident after 8 days of CS exposure; this increase remained constant after 28 and 40 days of exposure. A significant decrease in the number of MN PAM was observed in the animals pre-treated with NAC. BN PAM were significantly increased after 28 and 40 days of exposure; again, a slight yet not significant decrease was detected in NAC-pre-treated animals. On the whole, this study demonstrates that CS is clearly clastogenic to alveolar macrophages and that NAC can efficiently prevent this cytogenetic damage.     

Regional heterogeneity in murine lung fibroblasts from normal mice or mice exposed once to cigarette smoke

Chronic obstructive lung disease (COPD) is characterized by matrix deposition in the small airways but matrix loss from the parenchyma, phenomena which must depend on the ability of local fibroblasts to produce matrix after smoke exposure. To investigate this idea, we exposed C57Bl/6 mice once to cigarette smoke or to air (control) and prepared primary cultures of lung fibroblasts by microdissecting large airways (trachea, LAF), medium size airways (major bronchi, MAF) and parenchyma (PF). Control PF showed the lowest rate of wound closure and wound closure was depressed in all lines by a single in vivo smoke exposure. Gene expression of matrix proteins differed considerably among the sites; decorin, which may sequester TGFβ, was markedly higher in PF. PF showed higher intrinsic ratios of pSmad2/Smad2. Smoke caused much greater increases in secreted and matrix deposited collagens 1 and 3 in PF than in LAF or MAF. Expression of Thy-1, a gene that suppresses myofibroblast differentiation, was increased by smoke in PF. We conclude that there is considerable regional heterogeneity in murine lung fibroblasts in terms of matrix production, either basally or after in vivo smoke exposure; that PF have lower ability to repair wounds and higher intrinsic TGFβ signaling; and that a single exposure to smoke produces lasting changes in the pattern of matrix production and wound repair, changes that may be mediated in part by smoke-induced release of TGFβ. However, PF still retain the ability to repair by producing new matrix after a single in vivo smoke exposure.     

5-nitro-gamma-tocopherol increases in human plasma exposed to cigarette smoke in vitro and in vivo

We hypothesized that the high concentrations of reactive nitrogen species in cigarette smoke and the known stimulatory effects of cigarette smoke on the inflammatory immune systems would lead to the formation of 5-nitro-γ-tocopherol (NGT). In order to assess γ-tocopherol nitration, human plasma was exposed in vitro to gas phase cigarette smoke (GPCS) or air for up to 6 h. A liquid chromatography-mass spectrometry (LC-MS) method was developed to quantitate NGT. Detector response was linear from 0.1 to 3 pmol NGT, with a detection limit of 20 fmol. After a 1 h lag time, 6 h plasma exposure to GPCS depleted 75% of -T, 60% of γ-T and increased NGT from 3 to 134 nmol/l. The increase in NGT accounted for 20% of the γ-T decrease. NGT also correlated (R² = 0.9043) with nitrate concentrations in GPCS-exposed plasma. The physiologic relevance of NGT was evaluated in a group of healthy humans. Smokers (n = 15) had plasma NGT concentrations double those of nonsmokers (n = 19), regardless of corrections using lipids or γ-T; plasma -T and γ-T concentrations were similar between the groups. Our results show that LC-MS can be successfully used for NGT quantitation in biologic samples. Importantly, NGT in smokers' plasma suggests that cigarette smoking causes increased nitrosative stress.     

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.     

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.     

Evaluation of level of DNA damage in blood leukocytes of non-diabetic and diabetic rat exposed to cigarette smoke

The objective of the present study was to use the comet assay to evaluate the steady-state level of DNA damage in peripheral blood leukocytes from diabetic and non-diabetic female Wistar rats exposed to air or to cigarette smoke. A total of 20 rats were distributed into four experimental groups (n=5 rats/group): non-diabetic (control) and diabetic exposed to filtered air; non-diabetic and diabetic exposed to cigarette smoke. A pancreatic beta (beta)-cytotoxic agent, streptozotocin (40 mg/kg b.w.) was used to induce experimental diabetes in rats. Rats placed into whole-body exposure chambers were exposed for 30 min to filtered air (control) or to tobacco smoke generated from 10 cigarettes, twice a day, for 2 months. At the end of the 2-month exposure period, each rat was anesthetized and humanely killed to obtain blood samples for genotoxicity analysis using the alkaline comet assay. Blood leukocytes sampled from diabetic rats presented higher DNA damage values (tail moment=0.57+/-0.05; tail length=19.92+/-0.41, p<0.05) compared to control rats (tail moment=0.34+/-0.02; tail length=17.42+/-0.33). Non-diabetic (tail moment=0.43+/-0.04, p>0.05) and diabetic rats (tail moment=0.41+/-0.03, p>0.05) exposed to cigarette smoke presented non-significant increases in DNA damage levels compared to control group. In conclusion, our data show that the exposure of diabetic rats to cigarette smoke produced no additional genotoxicity in peripheral blood cells of female Wistar rats.     

Neutrophil capture by selectins on endothelial cells exposed to cigarette smoke

We used a novel perfusion system to expose cultured human umbilical vein endothelial cells (HUVEC) to water-soluble components of cigarette smoke and study subsequent adhesion of flowing neutrophils. Neutrophils did not bind to HUVEC immediately after it had been exposed to cigarette smoke, but many adhered 90-150 min after exposure. The effect was reduced if the exposed medium was made serum-free, but this reduction was partially reversed if low density lipoprotein was added. Treatment of smoke-exposed HUVEC with antibodies against E-selectin or P-selectin reduced adhesion by approximately 50% or 75%, respectively; a combination of both antibodies essentially abolished adhesion. Enzyme-linked immunosorbent assay confirmed that exposure to smoke caused HUVEC to upregulate surface expression of E- and P-selectin. Thus, water-soluble constituent(s) of cigarette smoke cause efficient selectin-mediated capture of flowing neutrophils. This pro-inflammatory response may contribute to pathology associated with smoking, especially in tissues remote from the lung.     

Susceptibility to pulmonary hypertension in inbred strains of mice exposed to cigarette smoke.

Cor pulmonale is a significant cause of morbidity and mortality in patients with emphysema, but it is not known whether alveolar destruction is directly involved in the disease pathogenesis. The purpose of this study was to examine the relationship between susceptibility to smoking-induced cor pulmonale and alveolar destruction in eight inbred strains of mice: 129XI/SvJ, A/J, A/HeJ, BALB/cJ, C3H/HeJ, C57BL/6J, DBA/2J, and SWR/J. The mice were exposed to filtered air or mainstream cigarette smoke at a concentration of 250 mg/m(3) for 5.5 h/day, 5 days/wk for 5 mo, housed for 4 more months, and killed. The ratio of the weight of the right ventricle/left ventricle plus septum [RV/(LV + S)] was used to assess right ventricular hypertrophy. Alveolar mean linear intercept was used to quantify severity of alveolar destruction. Morphometric determination of blood vessel muscularization was done on sections from four mouse strains. Smoke exposure resulted in significant increases in RV/(LV + S) in the A/J and A/HeJ strains compared with air-exposed controls. The magnitude of the smoking-induced increase in RV/(LV + S) decreased as a function of the genetic distance of the other strains from the A/J and A/HeJ strains. Pulmonary vascular muscularization was significantly increased in smoke-exposed A/J and BALB/cJ mice but not in C3H/HeJ and C57BL/6 mice. Also, mouse strain susceptibility to smoking-induced pulmonary vascular muscularization did not correlate with changes in mean linear intercept. The data from this study suggest that alveolar destruction by itself is not sufficient to cause smoking-induced cor pulmonale in inbred mice.     

Increased sister-chromatid exchange in bone-marrow cells of mice exposed to whole cigarette smoke

Male Wistar rats were fed diets of varying selenium content in order to obtain selenium-deficient and selenium-supplemented rats. After 5-6 weeks on the respective diet, the rats were used to investigate how selenium influences the effect of dimethylnitrosamine (DMN) on some liver enzymes and related reactions. The selenium-dependent glutathione peroxidase activity in postmicrosomal supernatant from liver was about 1% in selenium-deficient rats as compared to selenium-supplemented rats or rats fed a standard diet. The highest DMN-demethylase activity was observed in postmitochondrial supernatant from selenium-deficient rat liver, and the lowest in selenium-supplemented rats. No dietary effect was observed on hepatic microsomal cytochrome P450 levels. C-Oxygenation of N,N-dimethylaniline (DMA) was not affected by the selenium level. On the other hand, selenium deficiency seemed to reduce N-oxygenation of DMA. The mutagenicity of DMN in Chinese hamster V79 cells after metabolic activation by the isolated perfused rat liver, was approximately doubled when selenium-deficient livers were used as compared to selenium-supplemented livers and livers from rats fed a standard diet. A negative correlation between DMA-N-oxygenation and mutagenicity from DMN was observed, whereas no correlation between DMA-C-oxygenation and mutagenicity from DMN was found.     

Differential thiol status in blood of different mouse strains exposed to cigarette smoke

C57Bl/6J, DBA/2 and ICR mouse strains are known to possess different susceptibilities to developing emphysema after exposure to cigarette smoke with DBA/2 and C57Bl/6J strains being significantly more susceptible to pulmonary damage than the ICR strain. This study was aimed at analysing the occurrence of systemic oxidative stress in the blood of these different mouse strains after exposure to cigarette smoke. This study did not observe a significant decrease in glutathione in erythrocytes or in plasma cysteine, cysteinylglycine, homocysteine and glutathione in C57Bl/6J or DBA/2 mice, whereas a significant increase in the corresponding oxidized forms was observed in plasma. However, the ICR strain showed a significant increase in glutathione in erythrocytes and a significant decrease in most of the oxidized forms of cysteine, cysteinylglycine, homocysteine and glutathione in plasma after the same exposition. These experiments demonstrate that exposure to cigarette smoking induces systemic oxidative stress only in some mouse strains which are susceptible to developing emphysema.     

Antioxidant action of propolis on mouse lungs exposed to short-term cigarette smoke

Propolis is a natural product with antioxidant properties. In this study, we tested the efficacy of propolis against acute lung inflammation (ALI) caused by cigarette smoke (CS). C57BL6 male mice were exposed to CS and treated with propolis (200 mg/kg orally, CS+P) or only with propolis (P). A Control group treated with propolis was sham-smoked (Control+P). We collected the lungs for histological and biochemical analyses. We observed an increase in alveolar macrophages and neutrophils in the CS group compared with the Control+P. These counts reduced in the CS+P group compared to the CS group. The treatment with propolis normalized all biochemical parameters in the CS+P group compared with the CS group, including nitrite, myeloperoxidase level, antioxidant enzyme activities (superoxide dismutase, catalase and glutathione peroxidase), reduced glutathione/oxidized glutathione ratio and malondialdehyde. Additionally, TNF-α expression reduced in the CS+P group when compared with the CS group. These data imply a potential antioxidant and anti-inflammatory role for propolis with regard to ALI caused by CS in mice.     

Increased transglutaminase activity was associated with IL-6 release in cultured human gingival fibroblasts exposed to dental cast alloys

Summary. Molecular mechanisms underlying gingival and periodontal inflammation caused by dental alloys are still poorly understood. Recently, it has been demonstrated that tissue transglutaminase can be involved in inflammatory cell response. The aim of this study was to evaluate effects of exposure to orthodontic materials on transglutaminase in cultured human gingival fibroblasts. The incubation with Ni–Ti heat-activated (T3) or Ni–Ti super-elastic (T4), and with Ni–Cr–Co (T2) alloys produced respectively 2.5-fold and 8-fold increases in IL-6 release compared with control cultures. Transglutaminase activity was significantly increased in cells exposed to T3 and T4 alloys (about 170% of control; p < 0.05), where it was mainly localized close to inner part of cell membrane. The exposure to T3 and T4 specimens significantly up-regulated also tTG expression compared with control cultures. These data first show an association between IL-6 release and tissue transglutaminase increases, suggesting that TGase-mediated reactions may play a major role in periodontal inflammation.     

Oxidative radioiodination damage to human lactoferrin.

Oxidative iodination of human lactoferrin (Lf) as commonly performed by using the chloramine-T, the Iodogen or the lactoperoxidase method produces an unreliable tracer protein because of excessive and heterogeneous polymer formation. Before iodination a minor tetramer fraction may be demonstrable in iron-saturated Lf only. Iodination-induced polymerization of iron-poor as well as iron-saturated Lf occurs independently of the presence or absence of 10 mM-EDTA and the 125I-/Lf molar ratio used for iodination. 125I-Lf polymers are mainly covalently linked, as suggested by the lack of substantial dissociation in SDS/polyacrylamide-gel electrophoresis. Damage to the 125I-Lf monomer may be another consequence of oxidative iodination. This is demonstrated in SDS/polyacrylamide-gel electrophoresis where 50% of the radioactivity of apparently normal monomer (Mr 75,000) is displaced to a lower-Mr region (30,000-67,000) after reduction with dithiothreitol. Non-oxidative iodination by the Bolton-Hunter technique produces an antigenetically stable tracer that is not being subjected to polymerization and monomer degradation as judged by high-performance gel chromatography and SDS/polyacrylamide-gel electrophoresis with and without dithiothreitol treatment. It is concluded that oxidation in itself leads to covalent non-disulphide cross-linking between human Lf molecules and, possibly, to intramolecular peptide-bond breaking becoming unmasked under reducing conditions. In biological experiments with human 125I-Lf this problem should be carefully considered.     

Oxidative damage induced genotoxic effects in human fibroblasts from Xeroderma Pigmentosum group A patients

Xeroderma Pigmentosum A protein plays a pivotal role in the nucleotide excision repair pathway. Through site-directed binding of rigidly kinked double-stranded DNA, it verifies damaged DNA for subsequent excision and incision. Although Xeroderma Pigmentosum A-deficient cells have shown to be defective in oxidative base-lesion repair, the effects of oxidative assault on such cells have not been fully explored. Therefore, we sought to determine the involvement of Xeroderma Pigmentosum A in oxidative DNA damage-repair by treating primary fibroblasts from a patient suffering from Xeroderma Pigmentosum A with sodium arsenite and hydrogen peroxide. Our results show dose-dependent increase in genotoxicity with little change in cytotoxicity with both arsenite and H2O2 in Xeroderma Pigmentosum A-deficient cells compared to control cells. Xeroderma Pigmentosum A-deficient cells displayed increased susceptibility and reduced repair capacity when subjected to DNA damage induced by oxidative stress. Superarray results of apoptotic genes revealed differential expression of approximately 10 genes between Xeroderma Pigmentosum A-deficient and normal cells following arsenite treatment. Interestingly, we noted that arsenite did not inflict as much damage in the cells compared to H2O2. Lack of functional Xeroderma Pigmentosum A seems to increase the susceptibility of oxidative stress-induced genotoxicity while retaining cell viability posing as a potential cancer risk factor of Xeroderma Pigmentosum A patients.     

Levels of DNA damage in blood leukocyte samples from non-diabetic and diabetic female rats and their fetuses exposed to air or cigarette smoke

The objective of the present study was to evaluate DNA damage level in blood leukocytes from diabetic and non-diabetic female Wistar rats exposed to air or to cigarette smoke, and to correlate the findings with levels of DNA damage detected in blood leukocyte samples from their fetuses. A total of 20 rats were distributed into four experimental groups: non-diabetic (control; G1) and diabetic exposed to filtered air (G2); non-diabetic (G3) and diabetic (G4) exposed to cigarette smoke. Rats placed into whole-body exposure chambers were exposed for 30min to filtered air (control) or to tobacco smoke generated from 10 cigarettes, twice a day, for 2 months. Diabetes was induced by a pancreatic beta-cytotoxic agent, streptozotocin (40mg/kgb.w.). At day 21 of pregnancy, each rat was anesthetized and humanely killed to obtain maternal and fetal blood samples for genotoxicity analysis using the alkaline comet assay. G2, G3 and G4 dams presented higher DNA damage values in tail moment and tail length as compared to G1 group. There was a significant positive correlation between DNA damage levels in blood leukocyte samples from G2 and G3 groups (tail moment); G3 and G4 groups (tail length) and G3 group (tail intensity) and their fetuses. Thus, this study showed the association of severe diabetes and tobacco cigarette smoke exposure did not exacerbate levels of maternal and fetal DNA damages related with only diabetes or cigarette smoke exposure. Based on the results obtained and taking into account other published data, maternal diabetes requires rigid clinical control and public health and education campaigns should be increased to encourage individuals, especially pregnant women, to stop smoking.     

Dose-responsive increase in sister-chromatid exchanges in bone-marrow cells of mice exposed nose-only to whole cigarette smoke

The effect of whole cigarette smoke exposure on bone-marrow sister-chromatid exchanges (SCEs) was studied in B6C3F1 mice. Animals were exposed nose-only to 10% (v/v) cigarette smoke 5 days/week for 2 weeks. Four dose levels of cigarette smoke (1, 4, 9 and 18 exposures/day) were studied using 2 cigarette types, Kentucky reference 3A1 (3A1) and American Blend (AB). A single exposure represented approximately 1 cigarette. A dose-dependent increase in SCEs was observed for both the 3A1 and AB cigarettes at dose levels which had no effect on bone-marrow cell-replication kinetics. These findings represent the first demonstration of a dose-responsive increase in cigarette smoke-induced SCEs in a rodent model system.     

Early detection of susceptibility to acute lung inflammation by molecular imaging in mice exposed to cigarette smoke

Matrix metalloproteinases (MMPs) are extracellular proteolytic enzymes involved in acute lung inflammation in response to cigarette smoke exposure (CSE). We present the in vivo detection of MMP activity using a specific MMP-activatable, near-infrared, polymer-based proteolytic probe in strains of mice with different susceptibility to developing smoking-induced emphysema (susceptible mice, C57BL/6j, and resistant mice, 129S2/SvHsd) to characterize the distinctive profile of CSE-induced acute inflammation. In vivo imaging of pulmonary inflammation expressing MMPs revealed a significantly different median ratio twofold higher in smoker than in nonsmoker susceptible mice (C57BL/6j) and no significant differences between the smoker and the nonsmoker group in resistant mice (129S2/SvHsd). Ex vivo imaging of the lungs of each group of mice confirmed the same in vivo experiment results obtained for both strains of mice. In the biochemical study of lung tissue, the proteolytic signal colocalized with the endogenously expressed MMP protein levels, with MMP-9 levels that are 2.2 times higher than in the nonsmoke-exposed group in C57BL/6j mice and no significant differences in the 129S2/SvHsd mice. The MMP-activatable probe provides a useful reagent for the in vivo and ex vivo detection of MMP-selective proteolytic activity. We are able to distinguish between susceptible and resistant strains of mice in terms of the profile of MMP activity in the early stages of pulmonary disease.     

Metals in cigarette smoke

Metals are vital for a huge number of physiological processes in the human body, but can also destroy health when the concentration is not within the physiologically favourable range. Cigarette smoking interferes with the carefully controlled metal homeostasis of the human body. This review focuses on the consequences of metal delivery to the human body by cigarette smoking and discusses the body's responses. The metal content of tobacco plants, smoke, the circulation, and various organs is discussed. Finally, we link individual cigarette smoke contained metals to the genesis of human diseases.