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Effect of Cigarette Smoking on the Oxidant/Antioxidant Balance in Healthy Subjects

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Effect of Cigarette Smoking on the Oxidant/Antioxidant Balance in Healthy Subjects
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  Effect of Cigarette Smoking on the Oxidant/Antioxidant Balance in Healthy Subjects  Jennifier Cha´vez, MD, 1 Clı´maco Cano, PhD, 2 * Aida Souki, MgSc, 2 Valmore Bermu´dez, MD, 2 Mayerlim Medina, MD, 2 Ana Ciszek, MD, 2 Anilsa Amell, MD, 2 Maria E. Vargas, MD, 2 Nadia Reyna, MgSc, 2 Abdon Toledo, MD, 2 Raquel Cano, MD, 2 Gustavo Sua´rez, MD, 1 Freddy Contreras, MD, 3 Zafar H. Israili, PhD, 4 Rafael Herna´ndez-Herna´ndez, MD, 5 and Manuel Valasco, MD 3 Background and Purpose:  Cigarette smoking has been associated with the development of cardiovascular disease and cancer. Even though the molecular mechanism(s) are not clear, thepathology has been related to oxygen free radicals present in cigarette smoke. Thus, the mainobjective of this study was to establish the changes in the oxidation/antioxidation balance induced by cigarette smoking. Methods:  Thirty healthy subjects (15 smokers and 15 nonsmokers) of both sexes were studied. Thesmokers group had smoked a mean of 14 cigarettes per day for an average of 4.5 years. Fastingserum levels of malondialdehyde (MDA), a marker of oxidative stress, nitric oxide (NO), reducedglutathione (GSH), and vitamin C (ascorbic and dehydroascorbic acids) were measured. Results:  Fasting NO concentration was significantly higher in smokers (51.3  6  5.3  m M) than innonsmokers (35.2 6 4.8  m M,  P , 0.05). The smokers had significantly higher serum dehydroascorbicacid levels (2.4  6  0.5 mg/dL,  P  ,  0.03) than the nonsmokers (1.08  6  0.08 mg/dL). No significantdifferences were observed in the levels of ascorbic acid, MDA, and GSH between the smokers andnonsmokers. Conclusions:  Our results suggest that exposure to cigarette smoke increases NO synthesis, such thatNO may act in a compensatory way as an inhibitor of lipid peroxidation. Smoking also activates otherantioxidative mechanisms such as involving vitamin C. These protective mechanisms appear to beenough in preventing accumulation of oxidative products such as MDA and avoiding oxidative damage. Keywords:  cigarette smoking, malondialdehyde, nitric oxide, vitamin C, ascorbic acid, glutathione INTRODUCTION Cigarette smoking increases the risk of cardiovasculardisease, stroke, cancer, and chronic lung disease. 1–5 However, the mechanism(s) by which smoking con-tributes to vascular disease and cancer are not yetcompletely understood, although these pathologieshave been related to oxygen free radicals. 6 Reactiveoxygen species (ROS) and reactive nitrogen species arenow thought to play an important role in the onset of various vascular pathologies. 7 Collectively, ROS refers  From the  1 Physiology Department, School of Medicine, Universityof Zulia, Maracaibo, Venezuela;  2 Center of Endocrine and MetabolicResearch, University of Zulia. School of Medicine, Maracaibo,Venezuela;  3 Unidad de Farmacologı´a Clı´nica, Escuela de MedicinaVargas, La Universidad Central de Venezuela, Caracas, Venezuela; 4 Department of Medicine, Emory University School of Medicine, Atlanta, GA; and  5 Clinical Pharmacology Unit and HypertensionClinic, School of Medicine, Universidad Centroccidental ‘‘Lisandro Alvarado,’’ Barquisimeto, Estado Lara, Venezuela.*Address for correspondence: Facultad de Medicina, Universidaddel Zulia, Centro Endocrino-Metabo´lico (1er piso Frente a laBiblioteca). E-mail: climacoc@hotmail.com American Journal of Therapeutics 14, 189–193 (2007) 1075–2765    2007 Lippincott Williams & Wilkins  to free radicals and oxidants derived from 1-electronreduction of molecular oxygen, including superoxide(O 2 ), hydrogen peroxide (H 2 O 2 ), hydroxyl radical (OH),lipid peroxy radicals (LOO), lipid hydroperoxides(LOOH), and reactive aldehyde products such as4-hydroxynonenal. 7 In an organism, superoxide (O 2 ), the primary oxygenfree radical produced by mitochondria, can be con-verted within the cell to H 2 O 2  by the action of super-oxide dismutases (SOD1, SOD2, and SOD3). In turn,H 2 O 2  can react with reduced transition metals to pro-duce the highly reactive OH, a far more damagingmolecule to the cell. O 2  can also react with nitric oxide(NO)togeneratecytotoxic peroxynitrite anion (ONOO),which can lead to protein damage by way of the for-mation of nitrotyrosine and lipid peroxides. 8 Cigarette smoke, which contains high amounts of free radicals, peroxyradicals, and other oxygen-derivedspecies, 9,10 causes oxidative stress. 11 The toxic productsresulting from both the direct and secondary reactionswith cigarette smoke are thought to activate inflam-matory immune responses, 12 which may play animportant role in smoking-related oxidative tissuedamage. 13 In general, the ROS are detoxified by biological anti-oxidants, including glutathione (GSH),  a -tocopherol(vitamin E), carotenoids, and ascorbic acid (vitamin C).In addition, the antioxidant enzymes, catalase and GSHperoxidase, detoxify H 2 O 2  by converting it to O 2  andH 2 O.However,whenROSlevelsexceedtheantioxidantcapacity of a cell, a deleterious condition known asoxidative stress occurs. 8 Thus, the purpose of this study was to evaluatechanges in the oxidation/antioxidation balance induced by cigarette smoking by measurement of serum levelsof malondialdehyde (MDA), a biomarker of oxidativestress, 14 NO, ascorbic acid, dehydroascorbic acid, andreduced GSH in smokers and nonsmokers. METHODS Subjects Thirty healthy volunteers (13 men and 17 women) wereallocatedto two groups:nonsmokers (n = 15;mean age,26.2 6 2.2yr)andsmokers(n=15;meanage,32.8 6 2.2yr).Smoking status was ascertained by asking the par-ticipants whether they were current smokers at thetime of the interview and whether they had smokedmore than 100 cigarettes in their lifetime. Participantswere classified as current smokers if they answered‘‘yes’’ to both questions. Subjects who were notsmoking at the time of the interview and those whoanswered ‘‘no’’ to smoking more than 100 cigarettes intheir lifetime were classified as nonsmokers. Thesequestions and classifications were identical to a smok-ing status measure used in a national survey. 15 Thesmokers had smoked a mean of 14 cigarettes per dayduring the past 4.5 years (equivalent to moderatesmoking). Distribution of study subjects with regard toage, sex, and weight was similar in both groups.Medical histories were recorded. Exclusion criteriaincluded body mass index less than 18.5 kg/m 2 orgreater than 30 kg/m 2 , use of antioxidants (vitamin C,vitamin E,  a -lipoic acid,  b -carotene, probucol, carve-dilol, and iron chelators), or pro-oxidants (primaquineand iron) within the last 3 months, respiratory infectionwithin 2 weeks before the study, asthma, diabetes,hypertension, cardiovascular disease, stroke, transientischemic attack, or chronic diseases involving the cen-tral nervous system, and pregnancy. Laboratory methods Metabolic parameters were evaluated in venous blooddrawn after a 12 hour overnight fast (Table 1). Serumwas obtained by centrifugation at 1500  g  at roomtemperature for 10 minutes. Fasting glucose, triglycer-ides, total cholesterol, high density lipoprotein (HDL)cholesterol, MDA, NO, ascorbic acid, and dehydroas-corbic acid and reduced GSH were measured in theserum. Glucose, total cholesterol, and triglyceride lev-els were determined using an enzymatic colorimetricassay. Low density lipoprotein cholesterol and verylow density lipoprotein cholesterol concentrations werecalculated using the Friedewald formula. 16 HDL cho-lesterol was quantified using a commercial kit (HumanGesellschaft fu¨r Biochemica und Diagnostica mbH). Table 1.  Baseline characteristics of smokersand nonsmokers.Nonsmokers SmokersAge, yr 26.3  6 2.2 32.8 6  2.4No. (men/women) 15 (9/6) 15 (8/7)Weight, kg 62.8  6 2.2 65.4 6  2.8Height, cm 167.6  6 2.9 169.3 6  3.4Fasting glucose, mg/dL 81.1  6 2.0 87.6 6  3.0Total cholesterol, mg/dL 172.2  6 5.4 174.9 6  6.0Triglycerides, mg/dL 87.9  6 7.6 106.7 6  12.0HDL-c, mg/dL 44.2  6 1.8 42.6 6  2.5LDL-c, mg/dL 103.0  6 7.1 110.2 6  7.3VLDL-c, mg/dL 19.4  6 2.0 24.0 6  3.3 Values are mean 6  standard error.HDL-c, high density lipoprotein-cholesterol; LDL-c, low densitylipoprotein-cholesterol; VLDL-c, very low density lipoprotein-cholesterol.  American Journal of Therapeutics (2007)  14 (2) 190  Cha´vez et al   Levels of MDA, the marker of oxidative stress, 14 were measured by way of the formation of thiobarbi-turic acid-reactive substances. 17 NO levels were mea-sured using a colorimetric assay for determination of total nitrites. 18 Levels of ascorbic acid and dehydroas-corbic acid were measured using the method of Schwarz and Williams, 19 and reduced GSH was deter-mined with a commercial colorimetric kit (GSH-400Assay, Oxis International, Inc., Foster City, CA). Statistical analysis All results are expressed as mean  6  standard error.Data were analyzed using Students’  t  test and analysisof variance. Statistical analysis was performed usingSPSS software for Windows version 12.0 (Chicago, IL).Results were considered significant when the corre-sponding  P  value was less than 0.05. RESULTS The demographic and laboratory data of the smokersand nonsmokers are presented in Table 1. There wereno significant differences among the two groups withrespect to mean age, weight, systolic and diastolic blood pressure, fasting plasma glucose, and lipidprofile.The MDA levels were higher in smokers (1.75  6 0.33  m M) than in nonsmokers (1.03  6  0.16  m M), butthe difference was not significant (Fig. 1). The NOconcentration was significantly higher ( P  ,  0.05) insmokers (51.3  6  5.3  m M) than in nonsmokers (35.2  6 4.8  m M) (Fig. 2). There was no significant differencein the levels of GSH between smokers (309.1  6 6.2  m g/mL) and nonsmokers (293.0  6  9.0  m g/mL)(Fig. 3). The smokers had significantly higher levels of serumdehydroascorbicacid(2.4 6 0.5mg/dL, P , 0.03)compared with the nonsmokers (1.08  6  0.08 mg/dL), but serum ascorbic levels were similar (Fig. 4) in thetwo groups (smokers 0.9 6 0.2 mg/dL vs. nonsmokers0.8 6  0.1 mg/dL). DISCUSSION Our results suggest that to avoid the toxic effects of cigarette smoking, the organism activates some anti-oxidative defense mechanisms, as demonstrated by an FIGURE1.  Basalserummalondialdehydeconcentrationinsmokers and non-smokers. NS, no significant difference. FIGURE 2.  Basal serum nitric oxide concentration insmokers and non-smokers. * P  , 0.05. FIGURE3.  Basal GlutathioneLevels insmokersandnon-smokers. NS, no significant difference.  American Journal of Therapeutics (2007)  14 (2) Cigarette Smoking and Oxidative Stress   191  increase in NO levels and conversion of some ascorbicacid to its oxidized form (dehydroascorbic acid). Thesemechanisms prevent the accumulation in the body of free radicals present in cigarette smoke and the lipidperoxidation products, such as MDA, formed by theinteraction of free radicals with lipids. 14 There is evidence that the polyphenols present in thecigarette smoke particulate phase are the main sourceof O 2  and H 2 O 2 . 20,21 On the other hand, the vaporphase contains a factor, identified as carbonyl sulfide,which produces OH from H 2 O 2 . 21 Nevertheless, cig-arette smoke increases NO synthesis, as also demon-strated by cell culture studies that report the presenceof ONOO, the product of the reaction of NO andO 2 . 22,23 Cigarette smoke probably up-regulates theinducible, soluble isoform of NO-synthase (mainlyNO-synthase-2) expressed in a wide variety of celltypes, including bronchoalveolar macrophages, mono-cytes, platelets, respiratory tract epithelial cells, amongothers; this induction is most probably in response tothe free radicals, including O 2 . 24 NO isanunstable freeradical that plays a critical rolein the regulation of lipid oxidation induced by ROS(O 2 , H 2 O 2 , OH, and OOH). NO exhibits a dual naturein that at low concentration, it promotes lipidperoxidation, whereas at high concentration, it inhibitslipid peroxidation induced by O 2  and ONOO. 25 Thestimulation of lipid peroxidation by NO occurs whenits production rate is lower or equivalent to theproduction rate of O 2 . 26–28 However, when the rateof formation of NO exceeds that of O 2 , lipid perox-idation is inhibited, and instead the reactive speciesreact with thiobarbituric acid to form MDA. 25 There are multiple mechanisms by which theorganism is protected against different types of freeradicals acting at different sites. For example, a pro-tective mechanism involves vitamin C, a water solublevitamin and the first line of antioxidant defense inplasma, which is oxidized in the first 60 minutes of exposure to free radicals from exogenous sources, suchas cigarette smoke, thus protecting lipids from perox-idation. 29 Vitamin C (ascorbic acid) is oxidized firstto ascorbyl radical and then to dehydroascorbate andin the process scavenges free radicals and preventsradical-induced damage of lipoproteins and othermacromolecules. Ascorbic acid may act like NO in blocking free radical chain propagation reactionthrough a radical-radical reaction with the cytotoxicROS species. Because ascorbate is used up in protectingthe organism from oxidative damage, it is considereda sacrificial and preventive antioxidant. 29,30 A second line of defense against oxygen-derivedtoxic substances involves GSH, which, in the presenceof a selenium-dependent peroxidase, reduces H 2 O 2 . Inthe process, GSH is oxidized to oxidized GSH (GSSG),which is then rapidly reduced back to GSH by anicotinamide adenine dinucleotide phosphate-oxidase-dependent reductase, creating a closed loop. 31 In thepresent study, there was no significant change in GSHlevels in this group of moderate smokers, possibly becasueofitsrapidregenerationbythereductase.Itispos-sible that heavy smokers may have lower levels of GSH.The findings of this study suggest that exposureto cigarette smoke in humans leads to stimulation of NO synthesis by O 2  present in the smoke, with theorganism reacting to counteract the oxidative stressand inhibit lipid peroxidation. It also activates otheranti-oxidative mechanisms, such as by way of rapidconversion of ascorbic acid to its oxidized form(dehydroascorbic acid), which provides protectionagainst oxidative damage and accumulation of oxida-tion products, such as MDA. These results are inagreement with previous studies that show that thelevel of MDA is not modified by smoking 32 and thatthe antioxidant capacity is increased in smokers. 33,34 The protective role of NO and ascorbic acid (vitaminC) as antioxidants appears to be sufficient, with noneed for activating other defense mechanisms, asdemonstrated by the fact that there was no change inGSH levels in moderate smokers. Therefore, it can beconcluded that in moderate smokers, NO and vitaminC appear to effectively prevent oxidative damagecaused by the free radicals in cigarette smoke, avoidingan imbalance between oxidation and antioxidation thatwould have led to the development of atherosclerosisand cancer. The point where this compensatory effect isovercome, and toxic effects are initiated that lead to thedevelopment of cancer or atherosclerosis, the precursorof cardiovascular disease, is not known. It may be that FIGURE 4.  Basal serum ascorbic and dehydroascorbiclevels in smokers and non-smokers. White columnsrepresentnon-smokers;blackcolumnsrepresentsmokers.NS, no significant difference. * P  , 0.03.  American Journal of Therapeutics (2007)  14 (2) 192  Cha´vez et al   heavy smoking or genetic factors increase the risk of pathologic consequences. There is a need for furtherstudies to understand the pathophysiologic mecha-nisms by which cigarette smoke is involved in thegenesis of these diseases. REFERENCES 1. Pryor WA. Cigarette smoke radicals and the role of freeradicals in chemical carcinogenicity [Review].  Environ Health Perspect . 1997;105(Suppl 4):875–882.2. Alberg AJ, Samet JM. Epidemiology of lung cancer. Chest 2003;123(Suppl 1):21S–49S.3. AhsanH,Thomas DC.Lung canceretiology:independentand joint effects of genetics, tobacco, and arsenic.  JAMA .2004;292:3026–3029.4. Leone A. 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