Gas Phase Of Cigarette Smoke Research Articles

Glutathione and cysteines suppress cytotoxicity of gas phase of cigarette smoke by direct reacting with unsaturated carbonyl compounds in the gas phase

Unsaturated carbonyl compounds, such as acrolein (ACR) and methyl vinyl ketone (MVK), are environmental pollutants, and are contained in smoke, automobile exhaust, and heated oil. We have previously reported that major cytotoxic factors in the gas phase of cigarette smoke are ACR and MVK. ACR and MVK induce cell damage by reactive oxygen species generation via protein kinase C and NADPH oxidases, and antioxidants, such as glutathione (GSH) and N-acetylcysteine (NAC), can effectively suppress their cytotoxic activities. In this study, we attempted to elucidate the molecular mechanism(s) for suppression of ACR- and MVK-induced cytotoxic activities by these antioxidants. GSH, NAC, L- and D-cysteines completely suppressed cell damage induced by gas phase extract of cigarette smoke. The results of HPLC and mass spectrometry showed that GSH and NAC directly reacted with ACR and MVK. Cysteines and cysteine derivatives suppressed ACR-induced GAPDH carbonylation, a representative protein for carbonylation. The current results suggest that GSH, NAC, and cysteines directly reacted with ACR and MVK, and suppressed these unsaturated carbonyl compounds-induced cell damage by inhibition of protein carbonylation.

Intracellular Ca2+ is an essential factor for cell damage induced by unsaturated carbonyl compounds

The unsaturated carbonyl compounds are known as the environmental pollutants. Acrolein (ACR) and methyl vinyl ketone (MVK) are representative unsaturated carbonyl compounds. ACR is contained in smoke, automobile exhaust, industrial waste, and several foods. MVK is widely used as the industrial chemical. Although ACR and MVK are highly toxic, the molecular mechanism for their cytotoxicity has been unclear. We have previously reported that ACR and MVK are major cytotoxic compounds in the gas phase of cigarette smoke, and protein kinase C (PKC) inhibitor and NADPH oxidases inhibitor partially rescued cells from ACR- or MVK-induced cell death (Noya etal., Toxicology, 314, 1-10, 2013). PKC translocation, which is hallmark for PKC activation, and cell damage were induced by treatment of cultured cells with ACR or MVK. Intracellular Ca2+ chelator completely suppressed ACR- or MVK-induced PKC translocation to the cell membrane and cell damage, while extracellular Ca2+ chelator had no effects on ACR- and MVK-induced cytotoxicity. These results suggest that intracellular Ca2+ is an essential factor for cell damage caused by both PKC-dependent and PKC-independent pathways, and mobilization of Ca2+ from intracellular Ca2+ stores is induced by ACR or MVK.

A Standardized Method for the Preparation of a Gas Phase Extract of Cigarette Smoke.

The gas phase of cigarette smoke is important from the viewpoint of human health, because it can pass through alveolar epithelium and enter the circulation. There is no standard method for the preparation of a gas phase extract of cigarette smoke (CSE), although CSE is widely used for research instead of whole cigarette smoke. We have established a standard method for the preparation of CSE. One cigarette per trial is continuously combusted under a reduced pressure generated by an aspiration pump with a velocity of 1.050 L/min: the main stream of the smoke is passed through a Cambridge filter to remove tar, and subsequently, bubbled through a glass ball filter (pore size, 20-30 µm) into 15 mL of phosphate-buffered saline (PBS). To express the concentration of CSE, a virtual tar concentration is introduced, which is calculated assuming that tar trapped on the Cambridge filter is dissolved in the PBS. CSEs prepared from smaller numbers of cigarettes (original virtual tar concentration≤15 mg/mL) show similar concentration-response curves for cytotoxicity versus virtual tar concentrations. CSEs prepared from various brands of cigarettes and by different smoking regimes (continuous and puff smoking) show similar cytotoxic potency if the virtual tar concentrations are the same. In conclusion, using the standardized method for CSE preparation in combination with the virtual tar concentration, it becomes possible to simply and rapidly prepare standard CSEs with defined concentrations from any brand of cigarettes, which are toxicologically equivalent to CSE prepared by puff smoking.

Online Characterization of Isomeric/Isobaric Components in the Gas Phase of Mainstream Cigarette Smoke by Tunable Synchrotron Radiation Vacuum Ultraviolet Photoionization Time-of-Flight Mass Spectrometry and Photoionization Efficiency Curve Simulation

A newly developed, qualitative and quantitative method based on tunable synchrotron radiation vacuum ultraviolet photoionization time-of-flight mass spectrometry (SR-VUV-PI-TOFMS) and photoionization efficiency (PIE) curve simulation was applied for the online analysis of isomers and isobaric compounds in the gas phase of mainstream cigarette smoke. After blocking the particulate phase components by the Cambridge filter pad, a puff of fresh gas-phase cigarette smoke was immediately introduced into a vacuum ionization chamber through a heated capillary, then was photoionized, and analyzed by a TOF mass spectrometer. The PIE curves for the mass peaks up to m/z = 106 were measured between 8.0 and 10.7 eV. Some components could be directly identified by their discriminated ionization energies (IEs) on the PIE curve. By simulating the PIE curve with the sum of scaled absolute photoionization cross sections (PICSs), complex isomeric/isobaric compounds along with their mole fractions could be obtained when the best-fitting was realized between experimental and simulated PIE curves. A series of reported toxic compounds for quantification, such as 1,3-butadiene (m/z = 54), 1,3-cyclopentadiene (m/z = 66), benzene (m/z = 78), xylene (m/z = 106), 2-propenal (m/z = 56), acetone and propanal (m/z = 58), crotonaldehyde (m/z = 70), furan and isoprene (m/z = 68), were all found to have other isomers and/or isobaric compounds with considerable abundances. Some isomers have never been reported previously in cigarette smoke, like C5H6 isomers 1-penten-3-yne, 3-penten-1-yne, and 1-penten-4-yne at m/z = 66. Isomeric/isobaric compounds characterization for the mass peaks and mole fraction calculations were discussed in detail below 10.7 eV, an energy value covering several conventional used VUV light sources.

Identification of stable cytotoxic factors in the gas phase extract of cigarette smoke and pharmacological characterization of their cytotoxicity

Smoking is a major risk factor for atherosclerotic vascular diseases, but the mechanism for its genesis is unknown. We have recently shown that the gas phase of cigarette smoke (nicotine- and tar-free cigarette smoke extract; CSE) likely to reach the systemic circulation contains stable substances which cause cytotoxicity like plasma membrane damage and cell death in cultured cells, and also that the plasma membrane damage is caused through sequential activation of protein kinase C (PKC) and NADPH oxidase (NOX) and the resulting generation of reactive oxygen species (PKC/NOX-dependent mechanism), whereas cell death is caused through PKC/NOX-dependent and -independent mechanisms. To identify these stable substances, the CSE was prepared by passing the main-stream smoke of 10 cigarettes through a Cambridge glass fiber filter, trapping of the smoke in a vessel cooled at −80°C, and subsequent dissolution in 10ml of water. The CSE was fractionated into nine fractions using reversed-phase HPLC, and each fraction was screened for cytotoxicity in cultured cells, using propidium iodide uptake assay for cell membrane damage and MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] reduction assay for cell viability. The cytotoxicity was positive in two of the nine fractions (Fr2 and Fr5). After extraction of the active fractions into dichloromethane, GC/MS analysis identified 2-cyclopenten-1-one (CPO) in Fr5 but none in Fr2. After derivatization of the active fractions with O-(2,3,4,5,6-pentafluorobenzyl) hydroxylamine hydrochloride, GC/MS analysis identified acrolein, acetone and propionaldehyde in Fr2, and methyl vinyl ketone (MVK) in Fr5. After 4-h incubation, authentic acrolein and MVK induced concentration-dependent cytotoxicity with EC50 values of 75.9±8.2 and 47.0±8.0μM (mean±SEM; n=3), respectively, whereas acetone, propionaldehyde and CPO were without effect. However, after 24-h incubation, CPO induced concentration-dependent cytotoxicity with an EC50 value of 264.0±16.9μM (n=3). The concentrations of acrolein, MVK and CPO in the CSE were 3368±334, 2429±123 and 392.9±31.8μM (n=4), respectively, which were higher than the cytotoxic concentrations. The cytotoxicity of acrolein and MVK consisted of plasma membrane damage and decreased cell viability: the plasma membrane damage was totally prevented by treatment with an inhibitor of PKC or NOX, whereas the decreased cell viability was only partially prevented by these inhibitors. The cytotoxicity of CPO consisted only of decreased cell viability, which was totally resistant to these inhibitors. These results show that acrolein and MVK are responsible for the acute cytotoxicity of the CSE through PKC/NOX-dependent and -independent mechanisms, whereas CPO is responsible for the delayed cytotoxicity of the CSE through a PKC/NOX-independent mechanism.

The influence of tobacco blend composition on carbon monoxide formation in mainstream cigarette smoke.

The aim of this study was to examine the impact of three main tobacco types (flue-cured FC, air-cured AC and sun-cured SC) and two tobacco-based materials (reconstituted tobacco - recon RT and expanded stem) on the formation of carbon monoxide (CO) in the gas phase of mainstream cigarette smoke. The results showed that the type of tobacco examined had a significant impact on the amount of carbon monoxide production in the gas phase of cigarette smoke. AC and SC tobaccos had the most evident impact. The amount of tobacco in mixtures M1, M2 and M3 as well as the addition of expanded stems had an impact on the amount of CO formed in the cigarette smoke. There is weak correlation between CO content in the smoke and the chemical composition of the tobacco. Draw resistance had an impact on CO production. The research results are of great importance, since tobacco selection is the first step in the production of cigarettes with reduced emission of harmful elements contained in the smoke.

Cigarette smoke induces endoplasmic reticulum stress response and proteasomal dysfunction in human alveolar epithelial cells

Cigarette smoking is the major risk factor for chronic obstructive pulmonary disease. Cigarette smoke (CS) causes oxidative stress and severe damage to proteins in the lungs. One of the main systems to protect cells from the accumulation of damaged proteins is the ubiquitin-proteasome pathway. In the present study, we aimed to find out whether exposure of alveolar epithelial cells to CS induces an endoplasmic reticulum (ER) stress response by accumulation of damaged proteins that are inefficiently degraded by the proteasomes. The hypothesis was tested in a human alveolar epithelial cell line (A549) exposed to gas-phase CS. Exposure to gas-phase CS for 5 min caused an increase in the amount of ubiquitin-protein conjugates within 4 h. Cigarette smoke exposure also induced the ER stress response marker eIF2α, followed by a significant reduction of nascent protein synthesis and increase in the level of free intracellular amino acids. Moreover, CS exposure significantly reduced all three proteasomal activities (caspase-, trypsin- and chymotrypsin-like activity) within 4 h, which was still present after 24 h. It can be concluded that gas-phase CS induces ER stress in A549 alveolar epithelial cells, leading to inadequate protein turnover caused by an accumulation of damaged proteins, reduction in nascent protein synthesis and inhibition of the proteasome. We suggest that prolonged ER stress may lead to excessive cell death with disruption of the epithelial barrier, contributing to development of chronic obstructive pulmonary disease.

Cigarette Smoke Affects Keratinocytes SRB1 Expression and Localization via H2O2 Production and HNE Protein Adducts Formation

Scavenger Receptor B1 (SR-B1), also known as HDL receptor, is involved in cellular cholesterol uptake. Stratum corneum (SC), the outermost layer of the skin, is composed of more than 25% cholesterol. Several reports support the view that alteration of SC lipid composition may be the cause of impaired barrier function which gives rise to several skin diseases. For this reason the regulation of the genes involved in cholesterol uptake is of extreme significance for skin health. Being the first shield against external insults, the skin is exposed to several noxious substances and among these is cigarette smoke (CS), which has been recently associated with various skin pathologies. In this study we first have shown the presence of SR-B1 in murine and human skin tissue and then by using immunoblotting, immunoprecipitation, RT-PCR, and confocal microscopy we have demonstrated the translocation and the subsequent lost of SR-B1 in human keratinocytes (cell culture model) after CS exposure is driven by hydrogen peroxide (H2O2) that derives not only from the CS gas phase but mainly from the activation of cellular NADPH oxidase (NOX). This effect was reversed when the cells were pretreated with NOX inhibitors or catalase. Furthermore, CS caused the formation of SR-B1-aldheydes adducts (acrolein and 4-hydroxy-2-nonenal) and the increase of its ubiquitination, which could be one of the causes of SR-B1 loss. In conclusion, exposure to CS, through the production of H2O2, induced post-translational modifications of SR-B1 with the consequence lost of the receptor and this may contribute to the skin physiology alteration as a consequence of the variation of cholesterol uptake.

Oxidation of Cysteine by Gas-Phase Cigarette Smoke and by Combustion Smoke of Poly-Methyl Methacrylate

Oxidation of Cysteine by Gas-Phase Cigarette Smoke and by Combustion Smoke of Poly-Methyl Methacrylate

Mechanism of Oxidative Broncho-Epithelial Cell Damage with Reference to Gas Phase Cigarette Smoke

Mechanism of Oxidative Broncho-Epithelial Cell Damage with Reference to Gas Phase Cigarette Smoke

Smoking Induces Bimodal DNA Damage in Mouse Lung

To clarify the relationship between DNA damage and free radical generation caused by smoking in vivo, DNA damage was investigated in the mouse lung by single-cell gel electrophoresis assay after exposure to cigarette smoke (CS) or gas phase cigarette smoke (GPCS). Although GPCS did not induce DNA lesions, bimodal peaks of DNA damage were detected in mouse lung exposed to CS, one immediately after exposure and another 15 min later. Pretreatment with a specific hydroxyl radical (•OH) scavenger completely prevented both types of DNA damage induced by CS. Electron spin resonance (ESR) study of the kinetics of free radical generation in CS or GPCS revealed that •OH could be detected immediately after the spin trapping of CS without chelators (first •OH generation), whereas •OH was also generated gradually with a time lag when the spin trapping was performed with chelators (second •OH generation). Our ESR study also indicated that the first •OH peak was probably generated from H(2)O(2) via a metal-independent pathway, whereas the second •OH peak might have been generated from H(2)O(2) and other sources via at least two different metal-masked pathways. The bimodal DNA damage induced in lung by smoking appears to be the result of a time lag between the first •OH generation and second •OH generation after exposure to the tar in CS.

The mode of lymphoblastoid cell death in response to gas phase cigarette smoke is dose-dependent

BackgroundCigarette smoke (CS) is the main cause in the development of chronic obstructive pulmonary disease (COPD), the pathogenesis of which is related to an extended inflammatory response. In this study, we investigated the effect of low and high doses of gas phase cigarette smoke (GPS) on cultured lymphocyte progenitor cells, using techniques to assess cell viability and to elucidate whether cells die of apoptosis or necrosis upon exposure to different doses of GPS.MethodsIn our approach we utilised a newly-established system of exposure of cells to GPS that is highly controlled, accurately reproducible and simulates CS dosage and kinetics that take place in the smokers' lung. This system was used to study the mode of cell death upon exposure to GPS in conjunction with a range of techniques widely used for cell death studies such as Annexin V staining, activation of caspase -3, cytoplasmic release of cytochrome C, loss of mitochondrial membrane potential and DNA fragmentation.ResultsLow doses of GPS induced specific apoptotic indexes in CCRF-CEM cells. Specifically, cytochrome C release and cleaved caspase-3 were detected by immunofluorescence, upon treatment with 1-3 puffs GPS. At 4 h post-exposure, caspase-3 activation was observed in western blot analysis, showing a decreasing pattern as GPS doses increased. Concomitant with this behaviour, a dose-dependent change in Δψm depolarization was monitored by flow cytometry 2 h post-exposure, while at 4 h Δψm collapse was observed at the higher doses, indicative of a shift to a necrotic demise. A reduction in DNA fragmentation events produced by 5 puffs GPS as compared to those provoked by 3 puffs GPS, also pointed towards a necrotic response at the higher dose of GPS.ConclusionCollectively, our results support that at low doses gas phase cigarette smoke induces apoptosis in cultured T-lymphocytes, whereas at high doses GPS leads to necrotic death, by-passing the characteristic stage of caspase-3 activation and, thus, the apoptotic route.

Investigation of Tobacco Pyrolysis Gases and Puff-by-puff Resolved Cigarette Smoke by Single Photon Ionisation (SPI) - Time-of-flight Mass Spectrometry (TOFMS)

Abstract The work presented deals with the application of Single Photon Ionisation- Time-of-Flight Mass Spectrometry (SPI-TOFMS) for the investigation of tobacco smoke. SPI-TOFMS is a modern analytical technique, which enables the simultaneous analysis of a large number of organic species in complex gas mixtures in real time. The paper is a summary of a PhD thesis (1) and seven research articles, which were recently published in various scientific journals (2-8). Consequently, more detailed information on particular aspects can be found in there. The experimental part covers two different approaches, and therefore, it is divided into two sub-sections. In the first one, the SPI-TOFMS is coupled to a pyrolysis furnace. The objective is to examine the thermal behaviour of tobacco under various controlled conditions. In so doing, three tobacco types (Virginia, Oriental, and Burley) were pyrolysed in two reaction gas compositions (nitrogen and synthetic air) and seven different furnace temperatures (400 °C, 500 °C, 600 °C, 700 °C, 800 °C, 900 °C, and 1000 °C). Results can help to unravel the complex formation and decomposition reactions taking place when tobacco is heated. In the second part the SPI-TOFMS is connected to a cigarette smoking machine in order to investigate the behaviour of cigarette smoke constituents on a puff-by-puff basis. The work incorporates the comparison of whole smoke and gas phase of cigarette smoke, a puff-resolved quantification of several hazardous smoke constituents, and the overall chemical characterisation of the individual smoking puffs. In addition, a critical consideration of the prevailing smoking procedure is given when applied to single puff analysis. A further study examines the influence of five different cigarette lighting devices (gas lighter, electric lighter, candle, match, and burning zone of another cigarette) on the chemical composition of the first puff.

Carbon-centered radicals in cigarette smoke: acyl and alkylaminocarbonyl radicals.

The widely accepted mechanism of formation for carbon-centered radicals in the gas-phase cigarette smoke involves reactions of NO(2) and alkadienes. However, specific examples of such radicals have never been isolated from fresh cigarette smoke or their structure determined. We have identified two previously unrecognized classes of carbon-centered radicals, alkylaminocarbonyl and acyl radicals, that are unrelated to radicals that form by NO(x) chemistry. The combined abundance of these mainstream smoke radicals is significantly higher than the alkyl radicals previously quantified by electron paramagnetic resonance (EPR) solution spin-trapping methods. The new radicals were trapped directly from smoke with either 3-amino-proxyl (3AP) or 3-cyano-proxyl radical on a solid support and identified by combination of chemical synthesis, deuterium labeling, high-resolution mass spectrometry, nuclear magnetic resonance (NMR) spectroscopy, and ab initio quantum mechanical calculations. 3AP-R adducts were quantified both by high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) and by high-performance liquid chromatography with fluorescence detection (HPLC/FLD). Seven acyl and 11 alkylaminocarbonyl radicals were identified in the whole smoke of cigarettes made from single tobacco varieties and blended tobacco research cigarettes. The overall yield of these radicals was measured to be 168-245 nmol/cigarette from machine-smoked cigarettes under Federal Trade Commission (FTC) conditions. The yield was significantly reduced when the gas-phase smoke was separated from whole smoke by filtration through a 0.1 microm Cambridge filter pad or upon aging whole smoke in an inert tube.

Poly(ethylene glycol)-Based Stable Isotope Labeling Reagents for the Quantitative Analysis of Low Molecular Weight Metabolites by LC−MS

Stable isotope labeling (SIL) in combination with liquid chromatography-mass spectrometry is one of the most widely used quantitative analytical methods due to its sensitivity and ability to deal with extremely complex biological samples. However, SIL methods for metabolite analysis are still often limited in terms of multiplexing, the chromatographic properties of the derivatized analytes, or their ionization efficiency. Here we describe a new family of reagents for the SIL of primary amine-containing compounds based on pentafluorophenyl-activated esters of 13C-containing poly(ethylene glycol) chains (PEG) that addresses these shortcomings. A sequential buildup of the PEG chain allowed the introduction of various numbers of 13C atoms opening extended multiplexing possibilities. The PEG derivatives of rather hydrophilic molecules such as amino acids and glutathione were successfully retained on a standard C18 reversed-phase column, and their identification was facilitated based on m/z values and retention times using extracted ion chromatograms. The mass increase due to PEG derivatization moved low molecular weight metabolite signals out of the often noisy, low m/z region of the mass spectra, which resulted in enhanced overall sensitivity and selectivity. Furthermore, elution at increased retention times resulted in efficient electrospray ionization due to the higher acetonitrile content in the mobile phase. The method was successfully applied to the quantification of intracellular amino acids and glutathione in a cellular model of human lung epithelium exposed to cigarette smoke-induced oxidative stress. It was shown that the concentration of most amino acids increased upon exposure of A549 cells to gas-phase cigarette smoke with respect to air control and cigarette smoke extract and that free thiol-containing species (e.g., glutathione) decreased although disulfide bond formation was not increased. These labeling reagents should also prove useful for the labeling of peptides and other compounds containing primary amine functionalities.

Epidermal growth factor receptor exposed to cigarette smoke is aberrantly activated and undergoes perinuclear trafficking

Exposure to hydrogen peroxide (H2O2), one of the reactive oxidants in the gas phase of cigarette smoke (CS), induces aberrant phosphorylation of the epidermal growth factor receptor (EGFR), resulting in the lack of ubiquitination by c-Cbl, and impaired degradation. EGFR activation without the feedback regulation of normal degradation leads to uncontrolled cell growth and tumor promotion. Using immunoprecipitation, immunoblotting, and confocal microscopy, we now demonstrate that the pattern of EGFR activation by CS is similar to H2O2. We found that exposure of human airway epithelial cells to CS, as with exposure to H2O2, not only results in an increase in EGFR activation over time, but the EGFR activated by H2O2 or CS is neither ubiquitinated nor subsequently degraded due to its inability to bind the E3 ubiquitin ligase, c-Cbl, either directly or indirectly via the Grb2 adapter protein. Moreover, the stabilized H2O2- and CS-activated EGFR remains plasma membrane-bound, while a population of the receptor is trafficked to a perinuclear region. Concomitantly, CS exposure results in the activation of downstream Akt and ERK1/2 survival and proliferation pathways. Therefore, exposure to CS, like exposure to H2O2, results in prolonged signaling by the EGFR and may contribute to uncontrolled lung cell growth.

Hydroxytyrosol protects retinal pigment epithelial cells from acrolein‐induced oxidative stress and mitochondrial dysfunction

Hydroxytyrosol (HTS) is a natural polyphenol abundant in olive oil. Increasing evidence indicates HTS has beneficial effect on human health for preventing various diseases. In the present study, we investigated the protective effects of HTS on acrolein-induced toxicity in human retinal pigment epithelial cell line, ARPE-19, a cellular model of smoking- and age-related macular degeneration. Acrolein, a major component of the gas phase cigarette smoke and also a product of lipid peroxidation in vivo, at 75 μmol/L for 24 h caused significant loss of cell viability, oxidative damage (increase in oxidant generation and oxidative damage to proteins and DNA, decrease in antioxidants and antioxidant enzymes, and also inactivation of the Keap1/Nrf2 pathway), and mitochondrial dysfunction (decrease in membrane potential, activities of mitochondrial complexes, viable mitochondria, oxygen consumption, and factors for mitochondrial biogenesis, and increase in calcium). Pre-treatment with HTS dose dependently and also time dependently protected the ARPE-19 cells from acrolein-induced oxidative damage and mitochondrial dysfunction. A short-term pre-treatment with HTS (48 h) required > 75 μmol/L for showing protection while a long-term pre-treatment (7 days) showed protective effect from 5 μmol/L on. The protective effect of HTS in this model was as potent as that of established mitochondria-targeting antioxidant nutrients. These results suggest that HTS is also a mitochondrial-targeting antioxidant nutrient and that dietary administration of HTS may be an effective measure in reducing and or preventing cigarette smoke-induced or age-related retinal pigment epithelial degeneration, such as age-associated macular degeneration.

Oxidants in the Gas Phase of Cigarette Smoke Pass Through the Lung Alveolar Wall and Raise Systemic Oxidative Stress

Cigarette smoking-induced oxidative stress plays a key role in the pathogenesis of atherosclerosis in smokers. Aqueous cigarette smoke extract (CSE) contains stable oxidants, peroxynitrite-like reactants, which have the ability to oxidize and nitrate low-density lipoprotein (LDL). We examined whether oxidants in CSE can penetrate into the blood through the lung alveolar wall and cause oxidative vascular injury. The oxidants in CSE and sodium peroxynitrite could easily pass through the reconstituted basement membrane. When CSE or sodium peroxynitrite solution was infused into the alveolar air space of an isolated rat lung mounted in tyrosine solution, CSE gradually increased the 3-nitrotyrosine levels in the external tyrosine solution while sodium peroxynitrite caused a rapid increase. CSE did not activate the rat alveolar macrophages. When rats were acutely exposed to the gas phase of cigarette smoke from which tar and nicotine had been removed, both serum levels of 3-nitrotyrosine and 8-hydroxy-2’-deoxyguanine, oxidative stress markers, rapidly increased. Our results demonstrate that relatively stable oxidants in CSE can pass through the pulmonary alveolar wall into the blood and induce systemic oxidative stress, which most likely facilitates oxidative modification of LDL and endothelial dysfunction, explaining early key events in the development of atherosclerosis.

Early specific free radical-related cytotoxicity of gas phase cigarette smoke and its paradoxical temporary inhibition by tar

Electron paramagnetic resonance (EPR) spin trapping studies demonstrated aqueous tar particulate matter (TPM) and gas phase cigarette smoke (GPCS) to behave as different sources of free radicals in cigarette smoke (CS) but their cytotoxic implications have been only assessed in CS due to its relevance to the natural smoking process. Using a sensitive spin trapping detection with 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO), this study compared the respective roles of CS- and GPCS-derived free radicals on smoke-induced cytotoxicity and lipid peroxidation of filtered and unfiltered, machine-smoked experimental and reference cigarettes yielding a wide range of TPM yields. In buffer bubbled with CS the DEPMPO/superoxide spin adduct was the major detected nitroxide. Use of appropriate control experiments with nitric oxide radical (NO*) or carbonyl sulfide, and a computer analysis of spin adduct diastereoisomery showed that the hydroxyl radical (HO*) adduct of DEPMPO seen in GPCS-bubbled was rather related to metal-catalyzed nucleophilic synthesis than to direct HO* trapping. Unexpectedly a protective effect of TPM on murine 3T3 fibroblasts was observed in early (<3h) free radical-, GPCS-induced cell death, and carbon filtering decreased free radical formation, toxicity and lipid peroxidation in three cell lines (including human epithelial lung cells) challenged with GPCS. These results highlight an acute, free radical-dependent, harmful mechanism specific to the GPCS phase, possibly involving NO* chemistry, whose physical or chemical control may be of great interest with the aim of reducing the toxicity of smoke.

Analysis of the Gas Phase of Cigarette Smoke by Gas Chromatography Coupled with UV-Diode Array Detection

A gas chromatography method, coupled with diode array photometric spectral detection in the ultraviolet region (167-330 nm), was developed for the analysis of the gas phase of cigarette smoke. The method enabled us to identify more than 20 volatiles present in the vapor phase of cigarette smoke. In that way, all major volatile organic compounds (including aldehydes, conjugated dienes, ketones, sulfides, furans, and single-ring aromatics), as well as nitric oxide (NO) and hydrogen sulfide (H(2)S), can be analyzed in a straightforward manner through a single chromatographic run of <50-min duration. The method can easily be applied by the introduction of a small volume of the gas-phase stream into the GC injection loop directly through the smoking apparatus exhaust circuit, thus providing an excellent alternative to available methods, which usually require extraction or concentration steps prior to any chromatographic analysis. Furthermore, all problems concerning aging of the gas phase are eliminated. Twelve compounds (including NO) were chosen for quantification through the use of appropriate calibration standards. Comparison of the vapor phase yields of these compounds for the reference cigarette Kentucky 1R4F with already reported data indicates that this method is very reliable as far as accuracy and reproducibility of the results are concerned. Finally, the proposed methodology was used to compare the concentration of these cigarette smoke gas-phase constituents among individual puffs.