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DOI: 10.1080/01926230600824926 2006 34: 396Toxicol Pathol

Allison Marie Hays, Dinesh Srinivasan, Mark L. Witten, Dean E. Carter and R. Clark LantzArsenic and Cigarette Smoke Synergistically Increase DNA Oxidation in the Lung

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Toxicologic Pathology, 34:396404, 2006Copyright C by the Society of Toxicologic PathologyISSN: 0192-6233 print / 1533-1601 onlineDOI: 10.1080/01926230600824926

Arsenic and Cigarette Smoke Synergistically Increase DNA Oxidationin the Lung

ALLISON MARIE HAYS,1 DINESH SRINIVASAN,2 MARK L. WITTEN,3,4 DEAN E. CARTER,2,4 AND R. CLARK LANTZ1,4

1Department of Cell Biology and Anatomy, University of Arizona, Tucson, Arizona 857242Department of Pharmacology and Toxicology, University of Arizona, Tucson, Arizona 85724

3Department of Pediatrics, University of Arizona, Tucson, Arizona 857244Southwest Environmental Health Science Center, University of Arizona, Tucson, Arizona 85724

ABSTRACT

Epidemiological evidence has indicated that arsenic and cigarette smoking exposure act synergistically to increase the incidence of lung cancer. Sinceoxidative damage of DNA has been linked to cancer, our hypothesis is that aerosolized arsenic and cigarette smoke work synergistically to increaseoxidative stress and increase DNA oxidation in the lung. To test this hypothesis male Syrian golden hamsters were exposed to room air (control),aerosolized arsenic compounds (3.2 mg/m3 for 30 minutes), cigarette smoke (5 mg/m3 for 30 minutes), or both smoke and arsenic. Exposures werefor 5 days/week for 5 or 28-days. Animals were sacrificed one day after the last exposure. In the 28-day group, glutathione levels and DNA oxidation(8-oxo-2-deoxyguanosine (8-oxo-dG)) were determined. Our results show that in the 28-day arsenic/smoke group there was a significant decrease inboth the reduced and total glutathione levels compared with arsenic or smoke alone. This correlated with a 5-fold increase in DNA oxidation as shownby HPLC. Immunohistochemical localization of 8-oxo-dG showed increase staining in nuclei of airway epithelium and subadjacent interstitial cells.These results show that dual exposure of arsenic and cigarette smoke at environmentally relevant levels can act synergistically to cause DNA damage.

Keywords. Arsenic; Cigarette Smoke; Inhalation; DNA Oxidation.

INTRODUCTIONHuman exposure to arsenic has been correlated to lung can-

cer, both through inhalation and through ingestion. Arsenicis at best a weak mutagen and is considered a co-carcinogen.It is therefore plausible that co-exposure of arsenic and otherlung carcinogens, such as cigarette smoke could act synergis-tically. Human exposure to cigarette smoke has been shownto induce the initiation and promotion of lung cancer (Lubinet al., 2000). Epidemiological evidence has indicated thatcigarette smoke and inhaled arsenic exposure act synergis-tically to increase the incidence of lung cancer in smelterworkers (Xu et al., 1989; LaPaglia et al., 1996; Chen et al.,2004). However, no animal data are present that examine themechanisms of this effect. Reports investigating the possi-ble synergy between cigarette smoke and arsenic in animalmodels are scarce (LaPaglia et al., 1996; Chen et al., 2004).Our aim was to investigate whether the toxicity induced byexposure to cigarette smoke and/or inhaled arsenic is relatedto their ability to increase oxidative stress. Arsenic has beenproposed to cause toxicity through increased oxidative stress(Shi et al., 2004). This may occur either directly through

Address correspondence to: R. Clark Lantz, College of Medicine, De-partment of Cell Biology and Anatomy, 1501 North Campbell Avenue, P.O.Box 245044, Tucson, AZ 85724-5044; e-mail: [email protected]

Abbreviations: DNA: Deoxyribose nucleic acid; 8-oxo-dG: 8-oxo-2-deoxyguanosine; HPLC: High Pressure Liquid Chromatography; GSH:Glutathione; ROS: Reactive Oxygen Species; TNF-alpha: Tumor Necro-sis Factor-alpha; RNS: Reactive Nitrogen Species; BAP: benzo(a)pyrene;DMAV: Dimethylarsenic Acid; As2O3: Arsenic Trioxide; HCl: Hydrochlo-ric Acid; Ca2As5: Calcium Arsenate; As2S3: Arsenic Trisulfide; GSSG:Reduced Glutathione; GaAs: Gallium Arsenide; InAs: Indium Arsenide;BAL: Bronchoalveolar Lavage; MRP1: Multidrug Resistance Protein;BSO: Buthionine Sulfoximine.

cycling between oxidative states or indirectly by decreasingantioxidant defenses (glutathione) or by increasing ROS pro-duction through inflammation.

Inflammation is one of the responses of the lung to inhlaedtoxic agents and when the affected tissues and adjacent bloodvessels respond to the injurious agent then the area becomesheavily populated with inflammatory cells. These includemacrophages, neutrophils and eosinophils. These cells re-spond to toxicants with production of reactive oxygen species(ROS) and cytokines, which participate in airway inflam-mation. (Vallyathan et al., 1998; Kinnula, 2005). Tumornecrosis factor- (TNF-) is an important mediator in manycytokine-dependent inflammatory events. It is known thatTNF- can up regulate adhesion molecules and facilitatethe immigration of inflammatory cells into the airway wallthus playing a role in the initiation of airway inflammation.Proinflammatory cytokines like TNF- increase oxidativestress via the initiation of production of reactive oxygenspecies (ROS) (Barrett et al., 1999) and reactive nitrogenspecies (RNS) (Kofler et al., 2005). Production of thesecytokines and radicals can contribute to the pathogenesis ofcancer (Ekmekcioglu et al., 2005; Yao et al., 2005).

The pathogenesis of cigarette smoke or arsenic-inducedlung injury may involve the participation of toxic metabo-lites of both cigarette smoke and arsenic that elicit an in-flammatory response resulting in oxidative stress that maylead to neoplastic transformation of cells (Chung-man et al.,2001; Wie et al., 2002). Production of ROS/RNS from sourcesincluding cigarette smoke and arsenic can cause severe ox-idative stress in cells through the formation of oxidized cel-lular macromolecules, including lipids, proteins, and DNA(Hartwig et al., 1997; Bolton et al., 2000). Increased ox-idative stress resulting in oxidized macromolecules could

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occur by several mechanisms. First, cigarette smoke containsa broad range of carcinogens derived from tobacco and itspyrolysis products, including free radicals, which induce ox-idative stress and subsequent lipid peroxidation (Godschalket al.,2002). Second, cigarette smoke or arsenic induced in-flammatory processes can lead to increased ROS/RNS andthese compounds may compromise oxidant defense systems(Tsou et al., 2005).

ROS/RNS may be related to lung carcinogenesis (Chenet al., 2001; Ray et al., 2002). ROS/RNS has been shownto be involved in the initiation step of carcinogenesis, ei-ther in the oxidative activation of a procarcinogen, such asbenzo(a)pyrene (BAP) found in cigarette smoke, or throughdirect oxidative DNA damage (Pryor, 1997). The molecu-lar mechanisms involved in cigarette smoke-induced tumorsinvolves the reduction of oxygen to superoxide and hencehydrogen peroxide and the hydroxyl radical (Bolton et al.,2000). Formation of oxidatively damaged bases such as 8-oxo-dG, via the hydroxyl radical, has been associated withcarcinogenesis (Bolton et al., 2000). The glutathione systembuffers the rise in oxidants such as hydrogen peroxide and thehydroxyl radical (Maehira et al., 1999). Glutathione preventsROS/RNS-mediated damage and loss of lung cell functionand lung tissue injury (Lantz et al., 2001; Kaplowitz, 2002).Cigarette smoke and arsenic metabolites may promote deple-tion of GSH with consequent effects on proteins, lipids, andDNA including DNA oxidation (Comhair et al., 2000).

Synergistic effects between arsenic and other potential car-cinogens has been demonstrated in a skin model of carcino-genesis (Rossman et al., 2004). Combined exposure to UVradiation and arsenic in drinking water lead to an increasedlevel of DNA oxidation (Uddin et al., 2005). Our working hy-pothesis is that, similar to what has been seen in the skin, theeffects of cigarette smoke on the lung will act synergisticallyto produce oxidative damage. To gather evidence for our hy-pothesis, we used an animal model of inhalation exposure tofresh mainstream cigarette smoke and/or to arsenic trioxide.Our aim was to investigate whether combined exposure tocigarette smoke and arsenic would synergistically increaseoxidative stress, either through initiation of inflammation orthrough interaction with glutathione.

MATERIALS AND METHODSMale Syrian golden hamsters were used in these exper-

iments. Four groups of animals were used in the exposureprotocol. These included unexposed animals (controls ex-posed to room air), animals exposed to smoke only, animalsexposed to arsenic only and animals exposed to both smokeand arsenic. Animals were exposed 1 hour/day, 5 days/weekfor either 5 or 28 days in order to examine the progression ofchanges seen in the lung. Animals were housed and cared forin an AALAC-approved facility. All protocols were approvedby the Institutional Animal Care and Use Committee at theUniversity of Arizona.

Exposure Systems. Two exposure systems were used inthese experiments, one for smoke exposure and a secondfor exposure to arsenic. For the cigarette smoke exposure,fresh mainstream smoke was collected from burning ref-erence cigarettes (1R4, University of Kentucky, Smokingand Health Effects Laboratory, Lexington, KY). Smoke was

mixed in a chamber with fresh air to provide the appropriatedilution. Relative humidity, temperature and total suspendedparticulates were measured. Animals were exposed in noseonly fashion to 5 mg/m3 total suspended particulates over a30-minute period. Particulate concentration was determinedfrom changes in plate weights from a 7-stage cascade im-pactor (IN-TOX, Albuquerque, New Mexico). The particu-late concentration was determined immediately after eachexposure trial. Cascade impactor plates were weighed on anelectronic analytical balance (Mettler Instrument Corpora-tion, Hightstown, New Jersey).

For the arsenic exposure a fluidized bed particle aerosolgenerator was used to generate aerosols of arsenic trioxide(IN-TOX, Albuquerque, NM). Pressures and flows for thefluidized bed particle aerosol generator were first adjustedto aerosolize titanium dioxide at an average concentration of200 g/m3 for an 8-hour exposure (time-weighted averageor TWA). After establishing conditions for titanium dioxide,arsenic compounds (As2O3,As2S3, or Ca(AsO4)2) were in-troduced and tested. Pressures and flows were adjusted andsamples were collected and analyzed for arsenic. Concentra-tions were set for a 30-minute exposure with a final 8-hourTWA concentration of 200 g/m3. This required that the con-centration during the 30-minute exposure be set at 3.2 mg/m3.Once this concentration was achieved in the exposure cham-ber, we began exposure of hamsters to arsenic.

Combined arsenic and smoke exposures were performedon the same days. After stabilizing the airflow, animals wereplaced in the arsenic exposure chamber and exposed for30 minutes to 3.2 mg/m3 as arsenic using arsenic trioxide.Air samples were collected for daily determination of thearsenic concentrations. Following arsenic exposure, animalswere rested for 30 minutes prior to nose only exposure tocigarette smoke.

Exposures were carried out 5 days a week for either 5 or28 days of exposure. One day following the final exposure,animals were then sacrificed for determination of the effectsof the arsenic, cigarette smoke, or the potentially synergisticeffects of the two exposures.

Lung Lavage and Fixation. After sacrifice, the heart andlungs were removed in bloc immediately after exsanguina-tion by transection of the abdominal aorta. The esophagusand cardiovascular structures were carefully dissected awayand the tracheopulmonary bloc weighed. Total lung lavagewas performed using a 14-gauge catheter and 3 ml lavagevolumes (repeated 3 times) delivered via the trachea (n = 5animals for each group). Return volumes were quantitatedas percent returned and lavage fluid was saved for total cellcounts. An aliquot of cell suspension from bronchoalveolarlavage was used to determine total cell count. Cells were spunonto glass slides using a cytospin and stained with DiffQik toevaluate differential cell counts. Lungs from each group werefixed in situ with gluteraldehyde fixative (2% gluteraldehyde,2.5% formalin, and 0.04% picric acid in 0.1 molar HEPES) at20 centimeters H2O pressure. The lungs were fixed for 1 hourbefore being removed in bloc and immersed in fixative for24 hours @ 4C. Paraffin embedded sections from the fixedlungs were taken from the left and the right inferior lobesand were processed for light microscopy and immunohis-tochemistry. Light microscopic sections were stained with


398 HAYS ET AL. TOXICOLOGIC PATHOLOGY

hematoxylin and eosin (H&E). Intratracheal fixed tissue sec-tions (H&E) were also examined for signs of inflammation.

Glutathione and Oxidized DNA Determination. Oxi-dized and reduced glutathione were determined from thewhole lung using an HPLC method (Reed et al., 1980). DNAoxidation (8-oxo-2-deoxyguanosine (8-oxo-dG)) was quan-tified from genomic DNA extracted from lungs of animalsexposed to arsenic and/or cigarette smoke (Shigenaga et al.,1994) with several modifications. The DNA was hydrolyzedby enzymatic digestion to individual nucleosides by nucleaseP1 and alkaline phosphatase. The nucleosides were separatedby HPLC (C18 column [4.6 150 mm] mobile phase 50 mMsodium acetate pH 5.5 with 2% acetonitrile). Unmodifiednucleosides were detected by UV absorption at 278 nm and8-oxo-dG was quantified by electrochemical detection usinga potential of +300 mV. These detectors were run in seriesto allow quantification of both normal and 8-oxo-dG from asingle injection. Results are reported as 8-oxo-dG/105.

8-oxo-dG Immunostaining. Tissues were deparaffinizedand the slides were placed in citrate buffer in a microwaveon high for 25 minutes and then an additional 5 minuteson defrost for antigen retrieval. The slides were left to coolat room temperature for 30 minutes and then rinsed withdeionized water. After rinsing with deionized water, slideswere incubated with RNAse for 1 hour in a humid chamber.DNA was denatured by treatment with 4 N HCl for 7 min-utes at room temperature. The pH was adjusted with 50 mMTrizma base for 5 minutes at room temperature. Cells werepermeabilized with 3 drops of 0.1% NP-40 in PBS for 10 min-utes. The slides were then incubated in blocking serum for20 minutes at room temperature to block nonspecific binding.Slides were incubated with primary antibody (1:100) (R&DSystems, Minneapolis, MN) at 4C overnight.

Slides were treated with goat antimouse IgG conjugated tobiotin at 37C for 60 minutes and then incubated with cy-5conjugated streptavidin at 37C in a light-tight box in a hu-mid chamber for 60 minutes. Slides were incubated with YoYo iodide in a light-tight box for 15 minutes at room tem-perature to identify nuclei and subsequently mounted withDako mounting medium and stored in a light-tight box at4C overnight. The tissues were imaged with a LEICA TCS-4D confocal microscope with an Argon-Krypton laser thatsimultaneously scanned the slides with FITC and Cy-5 laserlines. This microscope processed the images with SCAN-WARE software.

Statistical Analysis. Data were analyzed for significantdifferences by 2 factor analyses of variance (smoke and ar-senic) (Winer, 1971). This provides statistical analysis of bothmain factors as well as interaction between each. Levels ofp < 0.05 were considered as significant.

RESULTSInflammation Following 5-Day Exposure

Both cigarette smoke particulate exposure and exposureto arsenic can lead to increased inflammation. Therefore,combined exposure to both agents may lead to an enhancedinflammatory response and increased oxidative stress frominflammatory cells. Three different arsenical compounds(calcium arsenate, Ca2As5;arsenic trioxide, As2O3; and ar-

FIGURE 1.Total Cell Count using different arsenic speciesMale Syriangolden hamsters were exposed to aerosolized calcium arsenate, arsenic trisulfideand arsenic trioxide with or without cigarette smoke for 5 days and on the 6th daybronchoalveolar lavage was performed. There were no significant differencesbetween any of the groups for lavaged cell counts. Also, there were no significantdifferences between any of the three arsenicals. (n = 5 for each group)(Ca2As5 =calcium arsenate, As2S3 = arsenic trisulfide, As2O3 = arsenic trioxide).

senic trisulfide, As2S3) were tested at 5-day exposures to seeif there were any differences in the early inflammatory re-sponse (Figure 1). The level of inflammation was determinedby total cell count from bronchoalveolar lavage. There wereno significant differences between any of the arsenicals. Nei-ther smoke alone, nor any of the arsenicals by themselves ledto significantly increased cell counts above control, untreatedlevels. Combining arsenic and smoke also did not lead to anysynergistic increases in BAL total cell counts. Differentialcells counts were also unchanged.

Inflammation Following 28-Day ExposureIn the 5-day exposure animals, arsenic trioxide and

cigarette smoke showed an increase over arsenic trioxidealone, but this increase was not significant. Arsenic triox-ide is the predominant arsenic form that is emitted dur-ing smelting processes. This form of arsenic was thereforeused to determine the effects of longer (28 days) exposure.Figure 2 shows total BAL cell counts from 28-day exposures

FIGURE 2.Total Cell Count in arsenic trioxide exposed animalsMaleSyrian golden hamsters were exposed to aerosolized arsenic trioxide with orwithout cigarette smoke for 28 days and on the 29th day bronchoalveolar lavagewas performed. Only exposure to cigarette smoke alone led to significant in-creases in total cell counts compared to control animals. While the arsenic andarsenic plus smoke (ars/smoke) showed elevated cell counts, these data didnot reach significance. Arsenic exposure decreased cigarette smoke-induced in-creases in the lung lavage cell count. (A = significantly different from control,p < 0.05). (n = 5 for each group).



FIGURE 3.Light microscopic examination of lung tissue. Hematoxylin andeosin stain. Representative photomicrographs of lung tissue from male Syriangolden hamsters 24 hours after a 28-day exposure to room air or to aerosolizedarsenic trioxide with cigarette smoke. (A) room air control. (B) aerosolizedarsenic trioxide (3.2 mg/m3, for 30 minutes) plus cigarette smoke (5mg/m3 for30 minutes).

to arsenic trioxide, smoke alone and to both compounds.Only exposure to cigarette smoke alone led to significantincreases in total cell counts compared to control animals.This increase was due predominantly to an increase in thenumbers of polymorphonuclear leukocytes. Combining ar-senic and smoke did not lead to any synergistic increasesin BAL total cell counts. While the arsenic and arsenic plussmoke (arsenic/smoke) showed elevated cell counts, thesedata did not reach significance. Our original expectationwas that arsenic would enhance the inflammation causedby cigarette smoke. However, arsenic exposure decreasedcigarette smoke-induced increases in the lung lavage cell

FIGURE 4.Alteration in the glutathione system. Male Syrian golden hamsters were exposed to aerosolized arsenic trioxide (3.2 mg/ m3 for 30 minutes) with orwithout cigarette smoke (5mg/m3 for 30 minutes) for 28 days. Sacrifice was one day after the final exposure. Lungs were processed for HPLC to determine levels ofreduced glutathione (GSH) (A), oxidized glutathione (GSSG) (B), total glutathione (C), and GSSG/GSH ratio (D). While neither arsenic nor cigarette smoke aloneled to significant alterations in GSH levels, combined exposure drastically decreased GSH (A) and total glutathione (C). (measurements are in nmoles/mg tissue).

count. These results were also validated by microscopic in-spection of the lungs. No apparent inflammation was evidentin the arsenic-and smoke-treated animals compared to con-trols (Figure 3).

Alterations in GlutathioneProtection from oxidative stress in the cell is provided in

part by the glutathione cycling system. We therefore ex-amined the levels and oxidative states of the glutathionesystem. The levels of reduced, oxidized and total glu-athione after single or combined exposures to arsenic and/orcigarette smoke were measured after 28 days of expo-sure. Individual exposures to either arsenic or smoke alonedid not significantly alter reduced, oxidized or total glu-tathione levels (Figure 4AC). Arsenic exposure did leadto increased GSSG/GSH ratios (Figure 4D). Two-factoranalysis of variance showed that, independent of smokeexposure GSSG/GSH ratios were increased by arsenic, in-dicating a possible arsenic-induced oxidative stress. Whileindividual exposure did not lead to other significant changes,total glutathione levels were significantly decreased by com-bined exposure to arsenic trioxide and cigarette smoke(Figure 4C). While GSH levels were drastically reducedby the combined exposure, this was not accompanied bya corresponding increase in GSSG levels. Rather, bothGSH and GSSG levels were reduced by the combinedexposure.



FIGURE 5.HPLC determination of DNA oxidation. Male Syrian goldenhamsters were exposed to aerosolized arsenic trioxide (3.2 mg/m3 for 30 min-utes) with or without cigarette smoke (5mg/m3 for 30 minutes) for 28 days.Sacrifice was one day after the final exposure. Lungs were processed for HPLCto investigate the levels of 8-oxo-dG as a marker of DNA oxidation. Concomitantwith the decreases in total glutathione, combined arsenic and cigarette smokeexposure led to significant increases in DNA oxidation. (measurements are8-oxo-dG/105 dG). (A=significantly different from control, p < 0.05). (n =5 for each group).

DNA Oxidation in Whole LungConcomitant with the decreases in reduced and total glu-

tathione, combined arsenic and cigarette smoke exposure ledto significant increases in DNA oxidation (Figure 5). Levelsof DNA oxidation were increased 5 fold in the lungs of ani-mals exposed to both cigarette smoke and arsenic. Exposureto either compound alone did not lead to alterations in DNAoxidation at the levels tested. In order to identify the site(s) ofDNA oxidation, lungs from animals exposed to both arsenicand cigarette smoke were processed for immunostaining us-ing an antibody against 8-oxo-dG. Minimal staining is seenin the control animals (Figure 6A). However combined ex-

FIGURE 6.Immunohistochemical determination of DNA oxidation. Lungs from control (A) and arsenic plus cigarette smoke (B) exposed male Syrian goldenhamsters were processed for immunohistochemistry to investigate the location of 8-oxo-dG as a marker of DNA oxidation. Combined exposure to arsenic andcigarette smoke lead to significant increases in 8-oxo-dG in airway epithelial cells (arrows) and sub adjacent cells.

posure resulted in detection of 8-oxo-dG in airway epithelialcells and in cells adjacent to the airways (Figure 6B).

DISCUSSIONEpidemiological evidence indicates that cigarette smoking

and arsenic exposure act synergistically to increase the inci-dence of lung cancer (Hughes, 2002). In order to examinethe mechanism(s) involved, we designed experiments to testthe hypothesis that the combined exposure to cigarette smokeand arsenic would result in increased oxidative stress. Our re-sults suggest that arsenic and cigarette smoke act by alteringthe glutathione system, making the lung more susceptible toDNA oxidative damage.

An emerging hypothesis in the mechanistic studies of metalcarcinogenesis is that of oxidative stress caused by exposure.However, arsenic is at best only weakly mutagenic, and ithas been suggested that arsenic is a co-carcinogen exertingits carcinogenic effects in combination with other carcino-gens such as those found in cigarette smoke (Hughes, 2002).The synergistic effects of arsenic with other carcinogens haspreviously been examined in a skin model. It has recentlybeen shown that sodium arsenite in drinking water and UVirradiation in mice synergistically increase tumorigenicity inthese animals (Rossman et al.,2004). Combined exposure toboth UV and arsenite caused increased DNA oxidation inthe skin, compared to either compound alone (Uddin et al.,2005). Increased tumor incidence in this model may be duein part to suppression of apoptosis (Wu et al., 2005).

Our experiments were designed to explore the possiblegenotoxic synergy between cigarette smoke and arsenic inthe lung. Formation of 8-oxo-dG, one of the major ox-idative DNA adducts, was found to be significantly in-creased in cigarette smoke and arsenic co-exposed animals.



In addition, immunohistochemistry showed an increase in8-oxo-dG antibody staining in airway epithelium of the co-exposed animals. Experiments were carried out at high, butenvironmentally relevant levels of exposure for both arsenicand cigarette smoke. Neither agent alone caused any sig-nificant changes. However combined exposures acted syn-ergistically to reduce glutathione levels and increased DNAoxidation.

Our original hypothesis was that combined exposure toarsenic and cigarette smoke, by inhalation, would leadto an increased inflammatory response that would corre-late with increased oxidative stress and DNA oxidation.Cigarette smoke- and arsenic-induced inflammation in thelung has been well established. Following cigarette smokeexposure, an influx of inflammatory cells (i.e., neutrophils,macrophages and lymphocytes) was demonstrated in miceexposed for 24 weeks, which progressively accumulatedboth in the airways and lung parenchyma (Dhulst et al.,2005). Chronic human smokers also show an increase inneutrophils in the lung. Gallium arsenide (GaAs), indiumarsenide (InAs), and arsenic trioxide (As2O3) particles in-stilled intratracheally twice a week for a total of 16 in-stallations, showed slight-to-severe inflammatory responses,which was characterized by an accumulation of neutrophilsand macrophages (Tanaka et al., 2000). Arsenic, in the formof copper smelter dust, was intratracheally instilled in themouse lung and biochemical markers, and inflammatorycell number and type demonstrated that copper smelter dustbears distinct inflammatory properties (Broeckaert et al.,1999).

However, for the doses used in this study, that was notthe case. While a 28-day exposure to cigarette smoke aloneled to increased BAL cell counts, arsenic exposures actuallydecreased the cigarette smoke-induced increases. Arsenictreatment alone caused a slight, but not significant, increasein BAL cell counts. Differences in our results from otherreports in the ability of arsenic particulates to produce in-flammation is most likely due to the type of exposure (in-halation versus instillation) and in the doses used. Our doses,while high, were in a range that would be seen in occupa-tional exposures. Our results also indicate that the form ofarsenic did not affect our results. Inhalation of arsenic triox-ide, arsenic trisulfide, or calcium arsenate either alone or incombination with cigarette smoke did not result in increasesin cell counts. This was not entirely unexpected. Alveolarmacrophages lavaged from animals that had received intra-tracheal instillation of soluble and insoluble arsenic com-pounds did not show any increases in basal or stimulatedsuperoxide production in trivalent species (Lantz et al., 1994,1995). In addition, in vitro exposure of control macrophagesto arsenicals inhibited the ability of the cells to produce su-peroxide in response to stimulation. Based on these results,inflammation does not appear to play a significant role in theobserved synergistic effects between arsenic and cigarettesmoke.

The glutathione redox system is the major antioxidant sys-tem in the cell and changes in the ratio of intracellular reducedand disulfide forms of glutathione (GSH/GSSG) can affectsignaling pathways that participate in various physiologicalevents. Adaptation to oxidative (ROS) and nitrosative (RNS)stress is observed in a wide variety of cells including lung

epithelial cells exposed to air-borne pollutants and toxicants.This acquired characteristic has been related to the regula-tion of proteins that control the synthesis of the intracellularantioxidant glutathione.

We have shown that combined exposure to arsenic andcigarette smoke leads to depletion of total glutathionestores in the lung. This suggests that, rather than justaltering the GSH/GSSG ratio, as would be expected dur-ing oxidative stress, the combined exposure alters the syn-thesis/degradation pathways for glutathione homeostasis.Arsenic- and cigarette smoke-induced perturbations of theglutathione system have been demonstrated using a va-riety of approaches. Reduced glutathione could be dueto conjugation and export. The multidrug resistance pro-tein (MRP1), co-transports xenobiotics with glutathione(GSH). MRP1 also confers resistance to arsenic in asso-ciation with GSH by transporting it as a tri-GSH conju-gate (Leslie et al., 2004). Intracellular reduced glutathione(GSH) was significantly depleted by arsenite exposure inhuman pulmonary epithelial cells (BEAS-2B) (Castranovaand Vallyathan, 2005). In male Wistar rats exposed to 100ppm arsenic for 10 weeks, there was a significant decreasein GSH in the brain (Flora et al., 2005). Other studies haveshown increased glutathione levels in response to arsenic.In porcine endothelial cells (PAECs), trivalent arsenic com-pounds; arsenic trioxide (As2O3), sodium arsenite (NaAsO2),and sodium arsenate (Na2HAsO4), increased total glutathione(Yeh et al., 2002). In addition, short term, in vitro expo-sures of human keratinocytes increased gamma-glutamyl-cysteine ligase levels (Schuliga et al., 2002). The effect ofarsenic on glutathione appears to be dose, time, and cell-typespecific.

As with arsenic, the levels of GSH in cigarette smoke ex-posed animals can also be significantly lower than that ofcontrol animals (Cigremis et al., 2004). The redox state of theGSH/GSSG couple in plasma of smokers and nonsmokers re-vealed that there was an increase in GSSG in smokers versusnonsmokers, and GSH was lower in smokers than in non-smokers (Moriarty et al., 2003). The cytotoxicity of gas phasecigarette smoke was found to be dose-dependent, and expo-sure resulted in the depletion of cellular GSH levels (Piperiet al., 2003). Cigarette smoke exposure for 10 weeks resultedin reduction in GSH levels in the mouse heart (Koul et al.,2003). Exposure of solutions of GSH to gas phase cigarettesmoke resulted in its rapid depletion, and about 50% of thisdepletion could be accounted for by reaction with acroleinand crotonaldehyde, the 2 major alpha, beta-unsaturated alde-hydes in cigarette smoke (Reddy et al., 2002).

Regardless of the mechanism, reduction in glutathionelevels leads to increased sensitivity to arsenic exposure.In cultured lung epithelial cells, arsenic-induced toxicityincreased as arsenic decreased cellular GSH. Buthionine sul-foximine (BSO), a GSH depletor, potentiated the arsenic tox-icity in these cells (Lim et al., 2002). Fetal fibroblasts fromgamma-glutamyl-cysteine ligase knock-out mice (Gclm (-/-)), which lack the modifier subunit of glutamate-cysteine lig-ase, the rate-limiting enzyme in glutathione biosynthesis are8 times more sensitive to arsenite-induced apoptotic death.Because of a dramatic decrease in glutathione levels, Gclm(-/-) fibroblasts have a high pro-oxidant status (Kann et al.,2005).



It is possible that environmentally relevant concentra-tions of arsenic and cigarette smoke, as were used in ourexperiments, induce a multicomponent response that leadsto depletion of glutathione. Arsenic in conjunction withcigarette smoke demonstrates a synergistic effect on thissystem as both total and reduced glutathione are signifi-cantly decreased in arsenic- and cigarette smoke-exposedanimals. The exact mechanism of action of combined ar-senic and cigarette smoke in the present studies is un-clear. Alone, neither toxicant reduced the overall levels ofglutathione. Arsenic, independent of cigarette smoke, didlead to an altered GSSG/GSH ratio, indicative of oxida-tive stress. However, only the combined exposures decreasedoverall glutathione levels. This could be due to a synergis-tic inhibition of the glutathione synthesis pathways. Whileshort-term exposures have been reported to increase gamma-glutamyl-cysteine ligase levels (Schuliga et al., 2002), effectsof long term in vivo exposures have not been reported. Inaddition, the combined exposures could result in increasedsecretion of glutathione complexes, resulting in loss of to-tal glutathione. Xenobiotic burdens deplete GSH as GSTconjugates it to the xenobiotic with subsequent export ofthe conjugated molecule, and GSH, out of the cell. Ar-senic alone or cigarette smoke alone did not deplete thetissue of GSH, and it may be that the lung can accommo-date either toxicant, but when they insult the lung togetherthe glutathione system is strained and the GSH stores aredepleted.

While we have not directly measured the production ofROS, we have evaluated the direct effects of arsenic, cigarettesmoke, or the combination on glutathione levels and on glu-tathione redox status. Reductions in the levels of glutathioneas seen with the combined arsenic and smoke exposureswill be expected to elevate the levels of hydrogen perox-ide in the cells (Rahman et al, 2006). In addition, cigarettesmoke contains numerous radicals that can oxidize DNA,including hydrogen peroxide, hydroxyl radicals, and perox-yradicals. Reduction of antioxidant defenses in the lungs, aswe have seen, would result a higher rate of DNA lesionsfrom these oxidants. While we have not specifically deter-mined the ROS species involved, we have shown that theROS are not the result of increased presence of inflammatorycells.

The mutagen and major DNA adduct, 8-oxo-dG, has beendemonstrated in both cigarette smoke and arsenic studies.Auto-oxidation of major constituents in cigarette smoke canresult in hydroxylation of deoxyguanosine residues in iso-lated DNA to 8-oxo-dG (Asami et al., 1996). Levels of8-oxo-dG were significantly higher in the leukocytes of cur-rent smokers versus nonsmokers (Asami et al., 1996). En-vironmental tobacco smoke (ETS)-related oxidative damagein rat lung was demonstrated by a significant enhancementof 8-oxo-dG accompanied by a significant depletion of GSH(Izzotti et al., 1999). Rats exposed to side stream cigarettesmoke showed significant increases in the accumulation of8-oxo-dG (Maehira et al., 1999). Inhalation of cigarettesmoke resulted in a significant decrease in GSH along withincreased 8-oxo-dG in DNA in the rat lung. When these ratswere treated with buthionine sulfoximine to deplete GSH,the oxidative effect of cigarette smoke was greatly potenti-ated (Park and Gwak, 1998).

In addition to cigarette smoke-induced increases in 8-oxo-dG, arsenic has demonstrated its potential to increase thisDNA lesion. These changes have been attributed mainly tothe methylated metabolites of arsenic. Pentavalent dimethy-lated arsenic (DMAV), in conjunction with a tumor initiator,increased 8-oxo-dG in mice. When the mice were topicallytreated with trivalent dimethylated arsenic (DMAIII), a furtherreductive metabolite of DMAV, an elevation of 8-oxo-dG inthe epidermis were observed (Mizoi et al., 2005). The appear-ance of 8-oxo-dG was examined immunohistochemically inarsenic-related and arsenic-unrelated human skin cancer andthe rate of 8-oxo-dG-positive tumors was significantly higherin arsenic-related human skin cancer (100%) versus arsenic-unrelated human skin cancer (15%) (An et al., 2004). In pa-tients chronically poisoned by the consumption of well waterwith elevated levels of arsenate (AsV), elevated 8-oxo-dGconcentrations in urine were also observed (Yamauchi et al.,2004).

Combined UV and arsenic also led to increased skin 8-oxo-dG (Uddin et al., 2005). The oral administration of DMA,in mice, significantly enhanced the amounts of 8-oxo-dG inskin, lung, liver, and the urinary bladder, whereas arsenite didnot. This may be in part due to the ability of DMAV exposureto decrease cellular GSH levels (Sakurai et al., 2004). Thedimethylarsenics thus may play an important role in arseniccarcinogenesis through the induction of oxidative damage(Yamanaka et al., 2001).

While cigarette smoke and arsenic can independentlycause DNA oxidation in a variety of tissues, we did not seeincreased DNA oxidation when each of these compoundswas administered independently. Only with combined ex-posures was there a significant increase in DNA oxidation.Since both agents can affect oxidative stress, it appears thatit is the overall level of oxidative stress that determines theeffects. The glutathione system works to detoxify arsenic-and cigarette smoke-induced increases in DNA oxidation. Ifthe major sulfhydryl-containing enzymes in the glutathionesystem are overwhelmed or impaired by arsenic, this couldpotentially lead to a significant increase, in cigarette smoke-induced DNA oxidation in the form of 8-oxo-dG.

Our baseline 8-oxo-dG levels for control hamster lung arehigh compared to other reported levels (Takabayashi et al.,2004). Over the past several years, organizations such as theEuropean Standards Committee on Oxidative DNA Dam-age (ESCODD) have tried to resolve problems associatedwith measurements of background DNA damage. (ESCODD2002a, 2002b, 2003). Samples sent to numerous laboratoriesusing differing techniques for isolation and measurement of8-oxo-dG resulted in determinations that differed by greaterthan 2 orders of magnitude. The reasons for these discrep-ancies are not entirely clear, since high levels were reportedfrom a laboratory that took the most rigorous precautions toprevent oxidation. While the reasons for the variation in thebasal levels of 8-oxo-dG are still being debated, what is clearis that, independent of basal levels, HPLC-electochemicaldetectors are able to detect dose-response relationships forproduction of 8-oxo-dG (ESCODD, 2003; Collins, 2005).Therefore, while our basal levels are high, we are stillable to detect significant increases in the levels of 8-oxo-dG following combined inhalation of arsenic and cigarettesmoke.



In conclusion we hypothesize that arsenic and cigarettesmoke deplete GSH synergistically and alter the redox sta-tus of the cell. Our studies showed a significant decrease inGSH in combined arsenic/cigarette smoke exposure over thecigarette smoke or arsenic exposed groups. Potentially, ar-senic may be interfering with the enzymatic activity of themajor proteins of the glutathione system and this disruptionthen allows for an increase in DNA oxidation . This increasein DNA oxidation was demonstrated both chemically (HPLC)and immunohistochemically. It does not appear that arsenicenhances the effects of cigarette smoke exposure by induc-ing a chronic inflammatory response in the distal lung. Ratherit appears that the overall oxidative stress level may be thedetermining factor leading to DNA oxidation.

ACKNOWLEDGMENTSThis research was funded in part by NIH grants P30

ES00694 and R01 ES005561 and by Arizona Disease ControlCommission Grant 9703. The authors wish to thank MadelBalagtas for her assistance.

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arsenic and cigarette smoke synergistically increase dna oxidation in the lung. - 2006 - hays et al

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