Morpho‐functional modifications of human neutrophils induced by aqueous cigarette smoke extract: comparison with chemiluminescence activity

Cigarette smoking plays an important role as a cause of morbidity and mortality in humans, involving respiratory, cardiovascular, digestive and reproductive systems. Tobacco smoke contains a large number of molecules, some of which are proven carcinogens. Although not fully understood, polymorphonuclear leukocytes seem to play a crucial role in the mechanisms by which tobacco smoke compounds are implicated in smoke‐related diseases. In this paper the effects of an aqueous cigarette smoke extract on the expression of adhesion molecules of polymorphonuclear leukocytes together with the changes in the cell morphology have been related to the chemiluminescence activity. The results obtained show that polymorphonuclear leukocytes treated with aqueous cigarette smoke extract are significantly impaired, as suggested by the changes of chemiluminescence activity, of membrane receptors (CD18, CD62), myeloperoxidase expression and of cell morphology. Altogether the present data indicate that treated polymorphonuclear leukocytes are ineffectively activated and therefore unable to phagocytize zymosan particles. Copyright © 2010 John Wiley & Sons, Ltd.     

Farnesol ameliorates massive inflammation, oxidative stress and lung injury induced by intratracheal instillation of cigarette smoke extract in rats: an initial step in lung chemoprevention

Cigarette smoke toxicants are well known for their debilitating effects on lungs. Cigarette smoke toxicities cause various respiratory disorders including pulmonary emphysema, chronic obstructive pulmonary disease (COPD), pulmonary fibrosis and cancer. Farnesol, an isoprenoid, is known to possess anti-inflammatory and chemopreventive properties. In this study we report the protective efficacy of farnesol against massive lung inflammation, oxidative stress and consequent injuries caused by cigarette smoke toxicants. Farnesol was administered by gavage (50 and 100 mg/kg b.wt. in corn oil) one time daily for 7 days. On day 7 lung injuries were induced by intratracheal instillation of aqueous cigarette smoke extract (CSE). LDH, total cell count, total protein, phospholipid content and MDA formation were measured in bronchoalveolar lavage fluid (BALF). In lung tissue H2O2 content, reduced glutathione (GSH), glutathione reductase (GR), glutathione peroxidase (GPx) and catalase activities were evaluated. Prophylactic treatment with farnesol significantly shows lung protection by lowering the levels of LDH, total cell count, total protein and MDA in BALF. Farnesol maintained the phospholipid content of BALF in a positive manner. In lung tissue it positively modulated the CSE altered activities of GR, GPx and catalase. There was a marked increase in GSH content and decrease in H2O2 content of lung tissue by farnesol administration. Histopathological findings correlate with cellular and biochemical parameters of the lungs and potentiate the protective role of farnesol against CSE induced lung inflammation and injuries. These results suggest a potent role of farnesol in protection of lung against cigarette smoke toxicants induced lung injuries.     

Tobacco smoke reduces viability in human lung fibroblasts: protective effect of glutathione S-transferase P1

Cigarette smoking is thought to be a major risk factor in various lung diseases including lung cancer and emphysema. However, the direct effect of cigarette smoke on the viability of lung-derived cells has not been fully elucidated. In this study, we investigated the viability of human lung fibroblast-derived (HFL1) cells to different concentrations of cigarette smoke extract (CSE). CSE induced apoptosis at lower concentrations (10-25%) and necrosis at higher concentrations (50-100%). We also examined the effects of glutathione S-transferase P1 (GSTP1), one of the xenobiotic metabolizing and antioxidant enzymes in the lung, against the cytotoxicity of CSE. Our results indicated that the level of HFL1 cell death was decreased by transfection with a GSTP1 expression vector and was increased by GSTP1 antisense vector transfection. Therefore, transient overexpression and underexpression of GSTP1 appeared to inhibit and enhance the cytotoxic effects of CSE on HFL1 cells, suggesting that GSTP1 may have protective effects against cigarette smoke in the airway cells.     

Oxidative damage in human gingival fibroblasts exposed to cigarette smoke

Cigarette smoke, a complex mixture of over 7000 chemicals, contains many components capable of eliciting oxidative stress, which may induce smoking-related disorders, including oral cavity diseases. In this study, we investigated the effects of whole (mainstream) cigarette smoke on human gingival fibroblasts (HGFs). Cells were exposed to various puffs (0.5-12) of whole cigarette smoke and oxidative stress was assessed by 2',7'-dichlorofluorescein fluorescence. The extent of protein carbonylation was determined by use of 2,4-dinitrophenylhydrazine with both immunocytochemical and Western immunoblotting assays. Cigarette smoke-induced protein carbonylation exhibited a puff-dependent increase. The main carbonylated proteins were identified by means of two-dimensional electrophoresis and MALDI-TOF mass spectrometry (redox proteomics). We demonstrated that exposure of HGFs to cigarette smoke decreased cellular protein thiols and rapidly depleted intracellular glutathione (GSH), with a minimal increase in the intracellular levels of glutathione disulfide and S-glutathionylated proteins, as well as total glutathione levels. Mass spectrometric analyses showed that total GSH consumption is due to the export by the cells of GSH-acrolein and GSH-crotonaldehyde adducts. GSH depletion could be a mechanism for cigarette smoke-induced cytotoxicity and could be correlated with the reduced reparative and regenerative activity of gingival and periodontal tissues previously reported in smokers.     

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.     

Selenium and Vitamin E Modulates Cigarette Smoke Exposure-Induced Oxidative Stress in Blood of Rats

Cigarette smoke contains about 5,000 chemicals that include organic and metallic compounds. The current study was undertaken to investigate the effects of selenium and vitamin E on oxidative stress-induced damage in rats exposed to cigarette smoke. Forty male rats were equally divided into four groups. The first and second groups were used as control and cigarette smoke groups, respectively. Selenium was administered to rats constituting the third group for 27 days. The Se and vitamin E combination was given to animals in fourth group for 27 days. All groups except the control, were exposed to cigarette smoke starting at the third day of the experiment and continuing for 27 days. The blood samples from all groups were taken at the end of 27 days. Plasma lipid peroxidation, triacylglycerol, and total cholesterol levels were higher in the cigarette smoke group than in the control, although erythrocytic superoxide dismutase and glutathione peroxidase activities were lower in the cigarette smoke group than in the control. The plasma lipid peroxidation, triacylglycerol, and total cholesterol levels were lower in cigarette smoke+Se+VE group than in the cigarette smoke group, although erythrocytic superoxide dismutase activity and glutathione peroxidase activity in selenium and vitamin E-administered groups were higher than in the exposed to cigarette smoke group. High-density lipoprotein-cholesterol level was not affect by selenium and vitamin E administrations. In conclusion, selenium and vitamin E seem to have protective effects on the cigarette smoke-induced blood toxicity by supporting the enzymatic antioxidant redox systems.     

Inhibition of IFN-γ-dependent antiviral airway epithelial defense by cigarette smoke

Background

Although individuals exposed to cigarette smoke are more susceptible to respiratory infection, the effects of cigarette smoke on lung defense are incompletely understood. Because airway epithelial cell responses to type II interferon (IFN) are critical in regulation of defense against many respiratory viral infections, we hypothesized that cigarette smoke has inhibitory effects on IFN-γ-dependent antiviral mechanisms in epithelial cells in the airway.

Methods

Primary human tracheobronchial epithelial cells were first treated with cigarette smoke extract (CSE) followed by exposure to both CSE and IFN-γ. Epithelial cell cytotoxicity and IFN-γ-induced signaling, gene expression, and antiviral effects against respiratory syncytial virus (RSV) were tested without and with CSE exposure.

Results

CSE inhibited IFN-γ-dependent gene expression in airway epithelial cells, and these effects were not due to cell loss or cytotoxicity. CSE markedly inhibited IFN-γ-induced Stat1 phosphorylation, indicating that CSE altered type II interferon signal transduction and providing a mechanism for CSE effects. A period of CSE exposure combined with an interval of epithelial cell exposure to both CSE and IFN-γ was required to inhibit IFN-γ-induced cell signaling. CSE also decreased the inhibitory effect of IFN-γ on RSV mRNA and protein expression, confirming effects on viral infection. CSE effects on IFN-γ-induced Stat1 activation, antiviral protein expression, and inhibition of RSV infection were decreased by glutathione augmentation of epithelial cells using N-acetylcysteine or glutathione monoethyl ester, providing one strategy to alter cigarette smoke effects.

Conclusions

The results indicate that CSE inhibits the antiviral effects of IFN-γ, thereby presenting one explanation for increased susceptibility to respiratory viral infection in individuals exposed to cigarette smoke.     

Differential induction of apoptosis by cigarette smoke extract in primary human lung fibroblast strains: implications for emphysema

Cigarette smoke is the principal cause of emphysema. Recent attention has focused on the loss of alveolar fibroblasts in the development of emphysema. Fibroblasts may become damaged by oxidative stress and undergo apoptosis as a result of cigarette smoke exposure. Not all smokers develop lung diseases associated with tobacco smoke, a fact that may reflect individual variation among human fibroblast strains. We hypothesize that fibroblasts from different human beings vary in their ability to undergo apoptosis after cigarette smoke exposure. This could account for emphysematous changes that occur in the lungs of some but not all smokers. Primary human lung fibroblast strains were exposed to cigarette smoke extract (CSE) and assessed for viability, morphological changes, and mitochondrial transmembrane potential as indicators of apoptosis. We also examined the generation of intracellular reactive oxygen species (ROS), 4-hydroxy-2-nonenal, and changes in glutathione (GSH) and glutathione disulfide (GSSG) levels. Each human lung fibroblast strain exhibited a differential sensitivity to CSE as judged by changes in mitochondrial membrane potential, viability, ROS generation, and glutathione production. Interestingly, the thiol antioxidants N-acetyl-L-cysteine and GSH eliminated CSE-induced changes in fibroblast morphology such as membrane blebbing, nuclear condensation, and cell size and prevented alterations in mitochondrial membrane potential and the generation of ROS. These findings support the concept that oxidative stress and apoptosis are responsible for fibroblast death associated with exposure to tobacco smoke. Variations in the sensitivity of fibroblasts to cigarette smoke may account for the fact that only some smokers develop emphysema.     

Analysis of Hydrogen Peroxide in an Aqueous Extract of Cigarette Smoke and Effect of pH on the Yield

An analysis of hydrogen peroxide in an aqueous extract of cigarette smoke, which contains many redox-active compounds, requires a method with high selectivity. An aqueous extract of the particulate phase of cigarette smoke was analyzed by HPLC with an electrochemical detector (ECD). Samples were prepared by collecting the particulate phase of the cigarette smoke on a glass fiber filter and extracting it with a phosphate buffer. The obtained solution was purified by using a Waters Oasis MCX cation-exchange cartridge, and then analyzed by an HPLC-ECD system with a Shodex KS-801 mixed-mode resin column. Pre-injecting hydrogen peroxide at a high concentration into the HPLC instrument stabilized the analytical results. The recovery of hydrogen peroxide by using an extract of the particulate phase of the cigarette smoke was more than 80%. An increase in the amount of hydrogen peroxide was observed during extraction with the phosphate buffer at higher pH values. In contrast, extraction with phosphoric acid did not increase the amount of hydrogen peroxide during extraction.     

Effect of cigarette smoke on lipid peroxidation,antioxidant enzymes and NMDA receptor subunits 2A and 2B concentration in rat hippocampus

The effect of cigarette smoke on lipid peroxidation and antioxidant enzymes such as catalase, superoxide dismutase, glutathione peroxidase and on the concentration of N-methyl-d-aspartate receptor (NMDAR) subunits 2A and 2B in the hippocampus of Sprague-Dawley rats exposed to cigarette smoke for 2h/day for a period of 4 weeks was determined. It was observed that NMDAR 2A and 2B concentrations in the hippocampus were enhanced in the case of animals exposed to cigarette smoke, whereas lipid peroxidation and antioxidant enzyme activities did not show any change as compared to control animals. The results of our study suggest that cigarette smoke induces NMDAR 2A and 2B expression in the hippocampus, and that this is not due to an increased lipid peroxidation, because cigarette smoke has no effect on lipid peroxidation and antioxidant enzyme activities in the hippocampus.     

Exposure to cigarette smoke, a major risk factor for sudden infant death syndrome: effects of cigarette smoke on inflammatory responses to viral infection and bacterial toxins

Exposure to cigarette smoke is a major risk factor for sudden infant death syndrome and also for respiratory infections in children. It has been suggested that toxigenic bacteria colonizing the respiratory tract might play a role in some cases of sudden infant death syndrome and nicotine has been demonstrated to enhance the lethality of bacterial toxins in a model system. Pyrogenic toxins of Staphylococcus aureus have been identified in tissues of infants who died of sudden infant death syndrome. It has been suggested that some of these deaths were due to induction of inflammatory mediators by infectious agents during a period when infants are less able to control these responses. The aim of this study was to assess the effects of a water-soluble cigarette smoke extract on the production of tumor necrosis factor alpha and nitric oxide from human monocytes in response to staphylococcal toxic shock syndrome toxin 1 or infection of the monocytes with respiratory syncytial virus. Cell culture supernatants were examined by a bioassay using mouse fibroblasts (L-929 cell line) for tumor necrosis factor alpha activity and by a spectrophotometric method for nitrite. Compared with monocytes incubated with medium only, monocytes incubated with any of the factors or their combinations tested in the study released higher levels of tumor necrosis factor alpha and lower levels of nitric oxide. Incubation with cigarette smoke extract increased tumor necrosis factor alpha from respiratory syncytial virus-infected cells while it decreased tumor necrosis factor alpha from cells incubated with toxic shock syndrome toxin. Incubation with cigarette smoke extract decreased the nitric oxide production from respiratory syncytial virus-infected cells while it increased the nitric oxide production from cells incubated with toxic shock syndrome toxin. Monocytes from a minority of individuals demonstrated extreme tumor necrosis factor alpha responses and/or very high or very low nitric oxide. The proportion of samples in which extreme responses with a very high tumor necrosis factor alpha and very low nitric oxide were detected was increased in the presence of the three agents to 20% compared with 0% observed with toxic shock syndrome toxin 1 or 4% observed with cigarette smoke extract or respiratory syncytial virus.     

The inactivation of alpha-1-proteinase inhibitor by gas-phase cigarette smoke: protection by antioxidants and reducing species

The abilities of a number of compounds of biological interest to protect alpha-1-proteinase inhibitor (alPI) against the loss of elastase inhibitory capacity (EIC) resulting from exposure to gas-phase cigarette smoke have been tested. We have identified several species that protect AlPI. Amino acids prevent the loss of EIC in a manner that correlates with their pK alpha-values; only the unprotonated amine provides protection. Catalase partially prevents the loss of EIC, suggesting that hydrogen peroxide produced from the reduction of oxygen in cigarette smoke extracts is responsible for at least some of the smoke-induced inactivation. The best protection against smoke-induced loss of EIC was provided by two biologically important antioxidant species: glutathione and ascorbic acid. Both species provide almost complete protection to alPI under the experimental conditions used. The nature of species that protect AlPI against the inactivation caused by exposure to gas-phase smoke provides clues upon which speculations about the mechanism of this inactivation may be based. The identification of protective species could lead to the development of compounds that smokers could take (for example, vitamin C) that would protect their lung tissue against the oxidative damage caused by cigarette smoke.     

Glutathione prevents inhibition of fibroblast-mediated collagen gel contraction by cigarette smoke

Cigarette smoke, the major risk factor for the development of emphysema, contains over 4,700 chemical compounds, including free radicals and other oxidants (10(14)/puff). An imbalance between oxidants and antioxidants has been proposed in the pathogenesis of chronic obstructive pulmonary disease. Inhibition of repair processes has been suggested to be one pathway contributing to the development of emphysema. We hypothesized that cigarette smoke inhibition of repair might result from a shift of the oxidant/antioxidant balance in favor of oxidants. To evaluate this hypothesis, N-acetyl-L-cysteine (NAC), which serves as a substrate for glutathione (GSH) production, and buthionine sulfoximine (BSO), which inhibits GSH production, were incubated in the presence and absence of cigarette smoke extract (CSE) with fibroblasts in three-dimensional collagen gels. Neither agent alone altered gel contraction. CSE inhibition of gel contraction, however, was mitigated by NAC and potentiated by BSO. Parallel effects were observed on cigarette smoke inhibition of fibronectin production and mRNA expression as well as by changes in intracellular GSH content. Pretreatment of fibroblasts with NAC or BSO resulted in similar effects, suggesting that neither agent was acting directly on smoke but, rather, was altering cellular response to smoke. In conclusion, smoke inhibition of fibroblast repair, as reflected by collagen gel contraction and fibronectin production, may be modulated by intracellular GSH levels.     

Effect of vitamin E and selenium on antioxidant enzymes in brain,kidney and liver of cigarette smoke-exposed mice

The present study was conducted to evaluate the protective effects of vitamin E and selenium (Se) application on alteration of antioxidant enzyme activities against cigarette smoking induced oxidative damage in brains, kidneys and liver of mice. Male mice (balb/c) were exposed to cigarette smoke and treated with Se and/or vitamin E. Glutathione transferase (GST), glutathione peroxidase (GPX), glutathione reductase (GRX), superoxide dismutase (SOD) and catalase (CAT) enzyme activities in mice brain, kidney and liver were measured spectrophotometrically. GST, GPX, GRX, SOD and CAT enzyme activities in the brains of smoke-exposed mice were found lower than the enzymes activities of control mice and Se-and vitamin E-treated mice at the end of the three and five months. Opposite to brain, enzyme activities in kidneys and livers of smoke-exposed mice were found higher than the enzymes activities of control mice and Se-and vitamin E-treated mice at the end of the three and five months. Activities of GST, GPX, GRX SOD and CAT in the livers, kidneys and brains of smoke-exposed mice were found statistically different (p < 0.01) compared to control mice and Se-and vitamin E-treated mice. Combined application of vitamin E and Se had an additive protective effect against changing enzymes activities in smoke-exposed mice livers, kidneys and brains at the end of the both application periods. These results suggest that cigarette smoke exposure enhances the oxidative stress, thereby disturbing the tissue antioxidant defense system and combined application of vitamin E and Se protects the brain, kidney and liver from oxidative damage through their antioxidant potential.     

Superoxide formed from cigarette smoke impairs polymorphonuclear leukocyte active oxygen generation activity.

Reactive free radicals contained in cigarette smoke (CS) and compromised phagocytic antimicrobial activities including those of polymorphonuclear leukocytes (PMNs) have been implicated in the pathogenesis of severe CS-related pulmonary disorders. In CS-exposed buffer solutions, O2-. was the predominant generated reactive oxygen species, as demonstrated by lucigenin-amplified chemiluminescence and electron spin resonance (ESR) spin-trapping with 5,5-dimethyl-1-pyrroline N-oxide (DMPO). When PMNs were incubated in this buffer, phorbol 12-myristate 13-acetate (PMA)-stimulated active oxygen production and coupled O2 consumption were strongly impaired without appreciably affecting PMN viability (1-min exposure inhibited active oxygen production by 75%). Superoxide dismutase (SOD) totally protected and an iron chelator, diethylenetriaminepentaacetic acid (DETAPAC), also protected the CS-exposed PMNs, suggesting that generated O2-. was an initiating factor in the impairment and OH. generation was a subsequent injurious factor. Pretreatment of PMNs with antioxidants such as alpha-tocopherol and dihydrolipoic acid (DHLA) was partially protective. The results suggest that (i) O2-. is probably generated in the upper and lower respiratory tract lining fluid when they come in contact with CS; (ii) such generated O2-. can primarily impair PMN capabilities to generate reactive oxygen species; and (iii) since these effects may contribute to the pathogenesis of CS-related lung diseases, prior supplementation with antioxidants such as alpha-tocopherol or DHLA might be successful in preventing these deleterious effects.     

Curcumin ameliorates oxidative stress during nicotine-induced lung toxicity in Wistar rats

Nicotine, a major toxic component of cigarette smoke has been identified as a major risk factor for lung related diseases. In the present study, we evaluated the protective effects of curcumin on lipid peroxidation and antioxidants status in bronchoalveolar lavage fluid (BALF) and bronchoalveolar lavage (BAL) of nicotine treated Wistar rats. Lung toxicity was induced by subcutaneous injection of nicotine at a dose of 2.5 mg/kg body weight (5 days a week, for 22 weeks) and curcumin (80 mg/kg body weight) was given simultaneously by intragastric intubation for 22 weeks. Measurement of biochemical marker enzymes: alkaline phosphatase, lactate dehydrogenase, lipid peroxidation and antioxidants were used to monitor the antiperoxidative effects of curcumin. The increased biochemical marker enzymes as well as lipid peroxides in BALF and BAL of nicotine treated rats was accompanied by a significant decrease in the levels of glutathione, glutathione peroxidase, superoxide dismutase and catalase. Administration of curcumin significantly lowered the biochemical marker enzymes, lipid peroxidation and enhanced the antioxidant status. The results of the present study suggest that curcumin exert its protective effect against nicotine-induced lung toxicity by modulating the biochemical marker enzymes, lipid peroxidation and augmenting antioxidant defense system.     

Cigarette smoke-induced blockade of the mitochondrial respiratory chain switches lung epithelial cell apoptosis into necrosis

Increased lung cell apoptosis and necrosis occur in patients with chronic obstructive pulmonary disease (COPD). Mitochondria are crucially involved in the regulation of these cell death processes. Cigarette smoke is the main risk factor for development of COPD. We hypothesized that cigarette smoke disturbs mitochondrial function, thereby decreasing the capacity of mitochondria for ATP synthesis, leading to cellular necrosis. This hypothesis was tested in both human bronchial epithelial cells and isolated mitochondria. Cigarette smoke extract exposure resulted in a dose-dependent inhibition of complex I and II activities. This inhibition was accompanied by decreases in mitochondrial membrane potential, mitochondrial oxygen consumption, and production of ATP. Cigarette smoke extract abolished the staurosporin-induced caspase-3 and -7 activities and induced a switch from epithelial cell apoptosis into necrosis. Cigarette smoke induced mitochondrial dysfunction, with compounds of cigarette smoke acting as blocking agents of the mitochondrial respiratory chain; loss of ATP generation leading to cellular necrosis instead of apoptosis is a new pathophysiological concept of COPD development.     

Vitamin C prevents cigarette smoke induced oxidative damage of proteins and increased proteolysis

Aqueous extract of cigarette smoke (CS) contains some stable oxidants, which oxidize human plasma proteins, bovine serum albumin, amino acid homopolymers, and also cause extensive oxidative degradation of microsomal proteins. Similar observations are made when the aqueous extract of cigarette smoke is replaced by whole phase CS solution or whole phase cigarette smoke. CS-induced microsomal protein degradation is a two step process: (i) oxidation of proteins by the oxidants present in the CS and (ii) rapid proteolytic degradation of the oxidized proteins by proteases present in the microsomes. Using aqueous extract of CS equivalent to that produced from one-twentieth of a cigarette, the observed initial and postcigarette smoke treated values of different parameters of oxidative damage per milligram of microsomal proteins are respectively: 0.24 and 1.74 nmoles for carbonyl formation, 125.4 and 62.8 fluorescence units for tryptophan loss, 10.2 and 33.4 fluorescence units for bityrosine formation, and 58.3 and 12.2 nmoles for loss of protein thiols. When compared with sodium dodecyl sulphate polyacrylamide gel electrophoresis profiles of untreated microsomal proteins, the extent of microsomal protein degradation after treatment with whole phase CS solution or aqueous extract of CS is above 90%. Ascorbate (100 microM) almost completely prevents cigarette smoke-induced protein oxidation and thereby protects the microsomes from subsequent proteolytic degradation. Glutathione is partially effective, but other antioxidants including superoxide dismutase, catalase, vitamin E, probucol, beta-carotene, mannitol, thiourea, and histidine are ineffective. The gas phase cigarette smoke contains unstable reactive oxygen species such as superoxide (O2*-) and hydrogen peroxide (H2O2) that can cause substantial oxidation of pure protein like albumin but is unable to produce significant oxidative damage of microsomal proteins. Gas phase cigarette smoke-induced albumin oxidation is not only inhibited by ascorbate and glutathione but also by superoxide dismutase, catalase and mannitol. The stable oxidants in the cigarette smoke are not present in the tobacco and are apparently produced by the interaction of O2*-/H2O2/OH* of the gas phase with some components of the tar phase during/following the burning of tobacco.     

Synergistic induction of hydroxyl radical-induced DNA single-strand breaks by chromium(VI) compound and cigarette smoke solution

Chromium(VI) compounds and cigarette smoke are known human carcinogens. We found that K2Cr2O7 and cigarette smoke solution synergistically induced DNA single-strand breaks (0.23+/-0.04 breaks per DNA molecule) in pUC118 plasmid DNA. K2Cr2O7 alone or cigarette smoke solution alone induced much less strand breaks (0.03+/-0.01 or 0.07+/-0.02 breaks per DNA molecule, respectively). The synergistic effect was prevented by catalase and by hydroxyl radical scavengers such as deferoxamine, dimethylsulfoxide, d-mannitol, and Tris, but not by superoxide dismutase. Ascorbic acid enhanced the synergism. Glutathione inhibited strand breakage only at high concentrations. Electron spin resonance (ESR) studies using a hydroxyl radical trap demonstrated that hydroxyl radicals were generated when DNA was incubated with K2Cr2O7 and cigarette smoke solution. Hydroxyl radical adduct decreased dose-dependently when strand breakage was prevented by catalase, deferoxamine, dimethylsulfoxide, d-mannitol or Tris, but not significantly by superoxide dismutase. We also used ESR spectroscopy to study the effects of different concentration of ascorbic acid and glutathione. The results showed that hydroxyl radical, which is proposed as a main carcinogenic mechanism for both chromium(VI) compounds and cigarette smoke solution was mainly responsible for the DNA breaks they induced.