Gas Phase Of Cigarette Smoke Research Articles

Gas-phase cigarette smoke inhibits plasma lecithin-cholesterol acyltransferase activity by modification of the enzyme's free thiols

Cigarette smoking is associated with an increased risk of premature atherosclerosis. The underlying mechanisms responsible for this association are unknown. Recent work from this laboratory has shown that ex vivo exposure of plasma to gas-phase cigarette smoke (CS) produces a rapid inhibition of lecithin-cholesterol acyltransferase (LCAT) activity and crosslinking of HDL-apolipoproteins. The goal of the present study was to investigate the mechanism(s) by which CS inhibited LCAT and modified HDL. When dialyzed human plasma (12 ml) was exposed to the gas-phase of an equivalent of l 8 of a cigarette (one ‘puff’) at 15 min intervals for 3 h, LCAT activity was reduced by 76 ± 1% compared to controls; supplementation of plasma with glutathione produced a dose-dependent protection of LCAT activity where at the highest concentration (l mM) 78% protection was observed. A similar protection was obtained with N-acetyl cysteine (l mM). In addition to LCAT inhibition, HDL-apolipoproteins were crosslinked after 3 h exposure of plasma to CS; crosslinking was reduced by the addition of either glutathione or N-acetyl cysteine to plasma. The amino compounds N-acetyl lysine, N-acetyl arginine, and aminoguanidine failed to protect LCAT and HDL indicating a specificity with regard to the ability of free thiols to buffer the deleterious components of CS which inhibited LCAT and crosslinked HDL-apolipoproteins. Since LCAT contains two free cysteine residues (Cys-31 and -184) near the active site of the enzyme, we tested whether pretreatment of plasma with the reversible sulfhydryl modifying compound, 5,5′-dithiobis-2-nitrobenzoic acid (DTNB), could protect LCAT from CS-induced inhibition. Plasma pretreated with DTNB prior to CS exposure retained an additional 50% of LCAT activity compared to CS treatment in the absence of DTNB. These results indicate that covalent modification of free cysteine residues of LCAT was, in part, responsible for the inhibition of plasma LCAT activity by CS.

Copper and gas-phase cigarette smoke inhibit plasma lecithin:cholesterol acyltransferase activity by different mechanisms.

Cigarette smokers have reduced levels of plasma high density lipoprotein (HDL) compared to nonsmokers and are at risk of premature cardiovascular disease. Previous work from this laboratory has shown that exposure of human plasma to gas-phase cigarette smoke (CS) inhibited the activity of lecithin:cholesterol acyltransferase (LCAT), the enzyme that catalyzes the formation of cholesteryl ester in HDL and thereby promotes HDL maturation. As CS contains free radicals that could potentially oxidize plasma lipoproteins, we examined the involvement of lipid peroxidation in LCAT inhibition. Results obtained with CS were compared with those obtained by initiating lipid peroxidation with copper ions. Exposure of dialyzed human plasma to an equivalent of one-eighth of a cigarette at 15-min intervals resulted in a progressive loss of LCAT activity (50 and 90% reductions by 1 and 6 h, respectively). A similar pattern of LCAT inhibition was produced with copper (0.5 mM) where 50 and 97% reductions were observed at 1 and 6 h, respectively. To determine whether LCAT inhibition was related to lipid peroxidation, lipoprotein fractions corresponding to VLDL-IDL, LDL, and HDL were isolated from plasma exposed to CS or copper and analyzed for changes in TBARS, the polyunsaturated fatty acid arachidonate relative to palmitate (20:4/16:0 ratio), and vitamin E concentrations. Exposure of plasma for 6 h to CS had no effect on the levels of TBARS and 20:4/16:0 ratio; however, 6 h copper treatment (0.5 mM) caused a 3.0-, 4.0-, and 1.4-fold increase in TBARS and a 17, 25, and 13% reduction in the 20:4/16:0 ratio in VLDL-IDL, LDL, and HDL fractions, respectively. In addition, a complete depletion of lipoprotein vitamin E was observed with CS, whereas copper decreased vitamin E levels by approximately 50% in each fraction. Supplementation of plasma with either vitamin C (85 microM) or butylated hydroxytoluene (BHT, 0.45 mM) was unable to protect LCAT from CS. In contrast, BHT completely protected LCAT activity from inhibition by copper. We conclude that unlike copper, CS-induced inhibition of plasma LCAT activity was unrelated to free radical-induced lipid peroxidation. The inhibition of LCAT activity by cigarette smoke may contribute to the development of atherosclerosis by impairing HDL metabolism and the reverse cholesterol transport process.

Molecular mechanisms of damage by excess nitrogen oxides: Nitration of tyrosine by gas-phase cigarette smoke

Nitric oxide (nitrogen monoxide, •NO) plays important physiological roles, but an excess can be toxic. •NO is present in cigarette smoke (CS) at up to 500 ppm, and probably represents one of the greatest erogenous sources of •NO to which humans are exposed. We show here that gas-phase CS is capable of converting tyrosine to 3-nitrotyrosine (3-NO 2-Tyr) and dityrosine, to an extent dependent on time of exposure and pH. Glutathione, ascorbic acid and uric acid decreased the CS-induced formation of 3-NO 2-Tyr and dityrosine. We suggest that nitrogen oxides in CS can modify proteins in the respiratory tract and may contribute to CS toxicity.

Cigarette smoke chemistry: conversion of nitric oxide to nitrogen dioxide and reactions of nitrogen oxides with other smoke components as studied by Fourier transform infrared spectroscopy

Nitric oxide is both a critical biological toxin and an important messenger molecule, signalling events as diverse as nerve transmission and smooth muscle relaxation. Fresh cigarette smoke contains from 300 to 500 ppm nitric oxide. While there have been reports of the rate of oxidation of nitric oxide to nitrogen dioxide in cigarette smoke, none have utilized a real-time method and provided detailed kinetic data and modelling. In this paper we present a Fourier transform infrared spectroscopy (FT-IR) method for the simultaneous determination of nitric oxide and nitrogen dioxide in gas phase cigarette smoke and in a number of gaseous mixtures that model smoke. The method uses multivariate least-squares regression analysis, which allows simultaneous quantitation of several components even in the presence of overlapping peaks, and fast data acquisition for kinetic analysis. Model systems containing mixtures of nitric oxide and isoprene, methanol, and/or acrolein and acetaldehyde in air were studied. The concentrations of nitric oxide, isoprene, etc., in the model systems were chosen to duplicate those in authentic cigarette smoke. In our best model (a mixture of nitric oxide, methanol, and isoprene in air) the disappearance of nitric oxide and the appearance of nitrogen dioxide follow time courses that closely duplicate those for cigarette smoke. Furthermore, the production of nitrogen dioxide follows a time course that agrees with our previously published rate of the development of organic free radicals in cigarette smoke. The present work, therefore, substantiates the steady-state mechanism we previously proposed for the production of free radicals in gas phase cigarette smoke.

Modification of LCAT activity and HDL structure. New links between cigarette smoke and coronary heart disease risk.

The mechanism(s) through which smoking influences the progression of atherosclerosis is poorly understood. Recent evidence suggests that oxidants present in the gas phase of cigarette smoke are involved. We exposed human plasma to the filtered gas phase of cigarette smoke to assess its effects on plasma components involved in the antiatherogenic reverse cholesterol transport pathway. In our model, freshly isolated plasma (24 mL) was exposed to filtered air or gas-phase cigarette smoke for up to 6 hours at 37 degrees C. Lecithin-cholesterol acyltransferase (LCAT) activity was dramatically inhibited by cigarette smoke. A single 15-minute exposure to the smoke from an eighth of a cigarette was sufficient to reduce LCAT activity by 7%; additional exposures resulted in further decreases in activity. At 6 hours, only 22% of control LCAT activity remained in plasma exposed to smoke. Compared with control, gas-phase cigarette smoke-exposed plasma possessed high-density lipoprotein (HDL) with increased (16%) negative charge and with cross-linked apolipoproteins AI and AII. These data demonstrate that gas-phase cigarette smoke can inhibit a key enzyme (LCAT) and modify an integral lipid transport particle (HDL) that are essential components for the normal function of the reverse cholesterol transport pathway. Gas-phase cigarette smoke-induced modification of the reverse cholesterol transport pathway may provide a new mechanistic link between cigarette smoke and coronary heart disease risk.

Lipoic and dihydrolipoic acids as antioxidants. A critical evaluation.

A detailed evaluation of the antioxidant and pro-oxidant properties of lipoic acid (LA) and dihydrolipoic acid (DHLA) was performed. Both compounds are powerful scavengers of hypochlorous acid, able to protect alpha 1-antiproteinase against inactivation by HOCl. LA was a powerful scavenger of hydroxyl radicals (OH.) and could inhibit both iron-dependent OH. generation and peroxidation of ox-brain phospholipid liposomes in the presence of FeCl3-ascorbate, presumably by binding iron ions and rendering them redox-inactive. By contrast, DHLA accelerated iron-dependent OH. generation and lipid peroxidation, probably by reducing Fe3+ to Fe2+. LA inhibited this pro-oxidant action of DHLA. However, DHLA did not accelerate DNA degradation by a ferric bleomycin complex and slightly inhibited peroxidation of arachidonic acid by the myoglobin-H2O2 system. Under certain circumstances, DHLA accelerated the loss of activity of alpha-antiproteinase exposed to ionizing radiation under a N2O/O2 atmosphere and also the loss of creatine kinase activity in human plasma exposed to gas-phase cigarette smoke. Neither LA nor DHLA reacted with superoxide radical (O.2-) or H2O2 at significant rates, but both were good scavengers of trichloromethylperoxyl radical (CCl3O2.). We conclude that LA and DHLA have powerful antioxidant properties. However, DHLA can also exert pro-oxidant properties, both by its iron ion-reducing ability and probably by its ability to generate reactive sulphur-containing radicals that can damage certain proteins, such as alpha 1-antiproteinase and creatine kinase.

Modification of plasma proteins by cigarette smoke as measured by protein carbonyl formation.

Exposure of human plasma to gas-phase (but not to whole) cigarette smoke (CS) produces oxidative damage to lipids [Frei, Forte, Ames & Cross (1991) Biochem. J. 277, 133-138], which is prevented by ascorbic acid. The ability of CS to induce protein damage was measured by the carbonyl assay and by loss of enzyme activity and protein -SH groups. Both whole and gas-phase CS caused formation of carbonyls in human plasma, which was partially inhibited by GSH but not by ascorbic acid or metal-ion-chelating agents. Isolated albumin exposed to CS showed much faster carbonyl formation (per unit protein) than did whole plasma; damage to isolated albumin was partially prevented by chelating agents. Isolated creatine kinase (CK) lost activity upon exposure to CS much faster than did CK in plasma. Direct addition to plasma of mixtures of some or all of the aldehydes reported to be present in CS caused protein carbonyl formation and inactivation of CK, but neither occurred to the extent produced by CS exposure.

Antioxidant vitamins and prevention of lung disease.

Although the evidence for oxidative stress for air pollution in the human lung is fragmentary, the hypothesis that oxidative stress is an important, if not the sole, mechanism of toxicity of oxidizing air pollutants and tobacco smoke is compelling and growing. First, biochemical mechanisms have been worked out for oxidation of lung lipids by the gas phase of cigarette smoke, NO2 and O3. The oxidation of lung lipids can be prevented by both vitamins C and E. Vitamin C is more effective in preventing oxidation by NO2, and vitamin E is more effective against O3. Second, multiple species of experimental animals develop lung disease similar to human bronchitis and emphysema from exposure to NO2 and O3, respectively. The development of these diseases occurs over a near lifetime exposure when the levels of NO2 or O3 are at near ambient air pollution values. Third, isolated human cells are protected against oxidative damage from NO2 and O3 by both vitamins C and E. Fourth, the vitamin C level in the lung either declines on exposure to NO2 for short-term exposures or increases on chronic cigarette smoke exposure. The effects of cigarette smoking on serum vitamin C is apparently complex and may be related to the daily intake of vitamin C as well as smoking. Serum vitamin C levels may be poor indicators of lung demands when daily vitamin C intakes are above 100 mg/day. Fifth, vitamin C supplementation protects against the effects of ambient levels of air pollution in adults as measured by histamine challenge. An augmented response to histamine challenge may represent increased lung permeability brought about by air pollution. In experimental animal and human experiments, the amount of vitamin C or E that afforded protection was in excess of the current recommended dietary allowance. Although animal studies do not provide evidence for complete protection against NO2 or O3, they do illustrate that current recommended daily allowances are inadequate for maximum protection against air pollution levels to which over 100 million Americans are exposed. The problem of air pollution and its effects on humans is truly of global concern. Air pollution is not restricted to North America or Japan where it was first recognized, but is a major public health problem in Europe as well. When data are available, air pollution probably will be shown to be a major public health problem in all urban areas of the world.(ABSTRACT TRUNCATED AT 400 WORDS)

Studies on toxicological mechanisms of gas-phase cigarette smoke and protection effects of GTP

Gas-phase cigarette smoke (GPCS) was able to induce lipid peroxidation in lecithin liposomes, rat liver microsomes, and rat lung cells (RLC), and change the membrane fluidity of RLCs. Lipid free radicals were trapped in a GPCS-treated microsomal suspension by using 4-POBN as the spin trap. In addition, it was found that GPCS-peroxidized liposomes in appropriate degree of lipid peroxidation had the ability to increase the generation of superoxide anions in rat peritoneal neutrophils (RPN). Effects of green tea polyphenols (GTP) on the GPCS-induced damages were investigated The results showed that GTP was capable of inhibiting the GPCS-induced damages.

Release of iron from ferritin by aqueous extracts of cigarette smoke.

This study demonstrates the ability of aqueous extracts of cigarette smoke to reduce iron and cause its release from ferritin. Superoxide dismutase (SOD) increases the rates of iron release with the less filtered smoke extracts, but has no effect on the rate of iron release caused by aqueous extracts of well-filtered gas-phase cigarette smoke. Faster rates of iron release are observed under anaerobic conditions, and the reducing power of the cigarette smoke extracts is prolonged when incubated in argon. Hydroquinone and catechol, two of the major polyhydroxybenzenes in cigarette smoke, increase in concentration in the smoke extracts as these are subjected to less filtration, and higher concentrations of polyhydroxybenzenes correlate with higher rates of iron release from ferritin. Concomitant with iron release, depletions of amino acids in ferritin are observed. Depletion of histidine is partially prevented by bathophenanthrolinedisulfonate and mannitol, while lysine and arginine depletions remain unaffected. These observations suggest that cigarette smoke components react directly with these amino acid residues in ferritin. Cigarette smoke induced release of iron could alter iron metabolism in the lungs of chronic smokers and contribute to the increase in the total oxidative burden on the lungs of smokers.

Experimental studies on some aspects of toxicological effects of gas phase cigarette smoke (GPCS)

We have studied the toxicological effects of gas phase cigarette smoke (GPCS) on: 1) lecithin-formed liposome lipid peroxidation; 2) the liposomal membrane fluidity; 3) conformation changes of rat lung cell membrane proteins; and 4) respiratory burst of human polymorphonuclear leukocytes. The results of these investigations suggested that by stimulating lipid peroxidation, GPCS can cause changes of membrane fluidity and alterations of membrane protein conformations as well as damages to membrane-bound enzymes. We conclude that oxidative damage to cells and tissues is an important toxicological property of GPCS.

Inhibitory effect of gas phase cigarette smoke on breathing: role of hydroxyl radical

Inhaling cigarette smokes evokes immediate bradypnea in rats, resulting from stimulation of vagal bronchopulmonary C-fiber afferents by smoke constituent(s) other than nicotine. To determine the contribution of the gas phase of smoke to this irritant effect, the acute respiratory responses to both cigarette smoke and gas phase smoke were studied and compared in anesthetized Sprague-Dawley rats; smoke (6 ml, 50%) was generated by a machine from low-nicotine research cigarettes and the gas phase was obtained by passing the smoke through a glass-fiber Cambridge filter. Inhalation of gas phase smoke alone evoked a transient inhibitory effect on breathing, prolonging expiratory time (Te) to peak of 159 ± 6% of the base line; this response was very similar to that triggered by inhaling the unified smoke (Te = 177 ± 12%). The bradypnea started within 1–4 breaths after the onset of smoke inhalation, lasted for 3–5 breaths and was completely abolished by vagotomy. This inhibitory effect of gas phase smoke on breathing was also largely prevented after a pretreatment with either intravenous infusion or aerosol inhalation of hydroxyl radical scavenger, dimethylthiourea. These results suggest that the gas phase is primarily responsible for eliciting the reflexogenic bradypneic response to cigarette smoke in anesthetized rats and that hydroxyl radicals released endogenously in the lungs may be involved.

The Effects of Drugs, Other Foreign Compounds, and Cigarette Smoke on the Synthesis of Protein by Lung Slices

The incorporation of 14C-leucine into rabbit lung slices was monitored in the absence and presence of selected drugs and chemicals relevant to the perturbation of lung function and the development of lung disease. Known inhibitors of protein synthesis (cycloheximide and ricin) inhibited the incorporation of 14C-leucine. Marked inhibition was also recorded with the lung toxins paraquat and 4-ipomeanol. By contrast, orciprenaline, salbutamol, and terbutaline were without effect although some response was recorded with isoprenaline. The filtered gas phase of cigarette smoke and acrolein, one of its components, were inhibitory but protection was afforded by N-acetylcysteine. It is suggested that the inhibitory effects of cigarette smoke may be due to its acrolein content. It is further suggested that the use of lung slices and measurements of 14C-leucine incorporation provide valuable means for monitoring potential pulmonary toxins.

The effects of drugs, other foreign compounds, and cigarette smoke on the synthesis of protein by lung slices.

The incorporation of 14C-leucine into rabbit lung slices was monitored in the absence and presence of selected drugs and chemicals relevant to the perturbation of lung function and the development of lung disease. Known inhibitors of protein synthesis (cycloheximide and ricin) inhibited the incorporation of 14C-leucine. Marked inhibition was also recorded with the lung toxins paraquat and 4-ipomeanol. By contrast, orciprenaline, salbutamol, and terbutaline were without effect although some response was recorded with isoprenaline. The filtered gas phase of cigarette smoke and acrolein, one of its components, were inhibitory but protection was afforded by N-acetylcysteine. It is suggested that the inhibitory effects of cigarette smoke may be due to its acrolein content. It is further suggested that the use of lung slices and measurements of 14C-leucine incorporation provide valuable means for monitoring potential pulmonary toxins.

Analysis of 1,3-butadiene and other selected gas-phase components in cigarette mainstream and sidestream smoke by gas chromatography-mass selective detection

An analytical procedure was developed for the analysis of 1,3-butadiene, acrolein, isoprene, benzene and toluene in the gas phase of cigarette smoke and environmental tobacco smoke (ETS) utilizing cryogenic gas chromatography-mass selective detection (GC-MSD). The MSD was operated in the selective ion monitoring (SIM) mode. The compounds of interest eluted in less than 15 min. The gas phase of freshly generated mainstream smoke was introduced into the GC-MSD via a 10-port gas sampling valve on a puff-by-puff basis. This method minimizes the ageing of tobacco smoke. The levels of 1,3-butadiene in the mainstream smoke ranged from 16 to 75 micrograms/cigarette. The gas phase of sidestream smoke was trapped in methanol using three midget impingers at -78 degrees C. The amount of 1,3-butadiene in the sidestream smoke ranged from 205-361 micrograms/cigarette. The concentration of 1,3-butadiene in ETS in a smoke-filled bar amounted to 2.7-4.5 micrograms/m3.

Gas phase cigarette smoke (CS) induces lipid peroxidation in human plasma

Gas phase cigarette smoke (CS) induces lipid peroxidation in human plasma

Cigarette smoke degrades hyaluronic acid

Cigarette smoke has been implicated in the pathogenesis of a variety of diseases that affect connective tissues. The effect of the gas phase of cigarette smoke on hyaluronic acid was investigated in vitro. The smoke markedly reduced the chain length of purified hyaluronic acid as assessed by both viscometry and gel chromatography. The hyaluronate exposed to smoke, nevertheless, could aggregate proteoglycans, although the viscosity of the aggregates was lower than in those formed with the unexposed material. The degradation of the hyaluronate was a rapid phenomenon and was inhibited by dimethylsulfoxide, a known scavenger of hydroxyl radicals. We conclude that free radicals in the gas phase of cigarette smoke degrade hyaluronic acid in vitro.

Quantitative Fourier Transform Infrared Analysis of Gas Phase Cigarette Smoke and Other Gas Mixtures

Abstract A new method for the analysis of selected components in complex gas mixtures has been developed utilizing a relatively inexpensive Fourier transform infrared spectrometer and a continuous flow gas cell. the method was used to monitor nitric oxide and nitrogen dioxide concentrations in cigarette smoke with time. Using multivariate least-square regression analysis, it is possible to simultaneously quantitate both NO and NO2, even in the presence of overlapping peaks. Using this method, the oxidation of nitric oxide in the presence of isoprene in cigarette smoke and in a model system was followed with time. the method also can be applied to other compounds in smoke or to any other gaseous mixture.

Characterization of two distinct transglutaminases of murine bone marrow-derived macrophages: effects of exposure of viable cells to cigarette smoke on enzyme activity.

The present study examines the effects of water soluble extracts of gas-phase cigarette smoke on intracellular transglutaminase activities of intact, murine, bone marrow-derived macrophages maintained in culture. Western blotting of cell lysates utilizing noncross-reactive antisera indicate that mouse bone marrow-derived macrophages contain both tissue-type transglutaminase and factor XIII-associated transglutaminase. This finding is also supported by data indicating that the intracellular transglutaminase activity of these cells contains thrombin-dependent and -independent components. Macrophages incubated with cigarette smoke solutions for 15 minutes at 37 degrees C display a dose-dependent decrease (maximum inhibition = 55%, p less than .001) in tissue-type (thrombin-independent) transglutaminase activity, as compared to control cells incubated with phosphate-buffered saline. Factor XIII (zymogen) is not inactivated following incubation of macrophages with smoke extracts. Smoke exposure under the conditions employed has no effect on either cell viability or adherence. Incubation with 2 microM retinoic acid for 24 hours leads to a modest (2-fold) induction of tissue transglutaminase, but does not induce factor XIII; in contrast, incubation with 10% homologous serum for 24 hours results in a decrease in factor XIII, but does not affect tissue transglutaminase. These data indicate that: bone marrow-derived macrophages contain factor XIII as well as tissue-type transglutaminase; and gas-phase cigarette smoke can inactivate tissue transglutaminase within viable murine bone marrow-derived macrophages, but cannot inactivate zymogenic factor XIII.

Inactivation of alveolar macrophage transglutaminase by oxidants in cigarette smoke.

The present study demonstrates the existence of tissue transglutaminase in rabbit alveolar macrophages and measures the effects of cigarette smoke extracts on the activity of enzyme partially purified from these cells. The effects of smoke on transglutaminase purified from guinea pig liver are also measured. A water soluble component of gas-phase cigarette smoke inhibits both enzymes in a dose-dependent manner, with a maximum inhibition of 65% occurring at concentrations of smoke as low as 1% in the case of the rabbit enzyme. The chemical oxidant N-chlorosuccinimide mimics the dose-dependent inhibitory effects of cigarette smoke on both enzyme systems. The thiol reducing agent, dithiothreitol, can prevent enzyme inactivation mediated by both smoke and N-chlorosuccinimide. Dithiothreitol is also capable of reversing inactivation mediated by cigarette smoke, but does not reverse inactivation caused by chemical oxidation with N-chlorosuccinimide. These data suggest that cigarette smoke inhibits transglutaminase activity via an oxidative mechanism that may selectively attack the active-site cysteine residue.