Abstract
Epidemiological evidence has indicated that arsenic and cigarette smoking exposure act synergistically to increase the incidence of lung cancer. Since oxidative damage of DNA has been linked to cancer, our hypothesis is that aerosolized arsenic and cigarette smoke work synergistically to increase oxidative 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 were for 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 in both the reduced and total glutathione levels compared with arsenic or smoke alone. This correlated with a 5-fold increase in DNA oxidation as shown by 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.
Introduction
Human exposure to arsenic has been correlated to lung cancer, both through inhalation and through ingestion. Arsenic is at best a weak mutagen and is considered a co-carcinogen. It is therefore plausible that co-exposure of arsenic and other lung carcinogens, such as cigarette smoke could act synergistically. Human exposure to cigarette smoke has been shown to induce the initiation and promotion of lung cancer (Lubin et al., 2000). Epidemiological evidence has indicated that cigarette smoke and inhaled arsenic exposure act synergistically to increase the incidence of lung cancer in smelter workers (Xu et al., 1989; LaPaglia et al., 1996; Chen et al., 2004). However, no animal data are present that examine the mechanisms of this effect. Reports investigating the possible synergy between cigarette smoke and arsenic in animal models are scarce (LaPaglia et al., 1996; Chen et al., 2004). Our aim was to investigate whether the toxicity induced by exposure to cigarette smoke and/or inhaled arsenic is related to their ability to increase oxidative stress. Arsenic has been proposed to cause toxicity through increased oxidative stress (Shi et al., 2004). This may occur either directly through cycling between oxidative states or indirectly by decreasing antioxidant defenses (glutathione) or by increasing ROS production through inflammation.
Inflammation is one of the responses of the lung to inhlaed toxic agents and when the affected tissues and adjacent blood vessels respond to the injurious agent then the area becomes heavily populated with inflammatory cells. These include macrophages, neutrophils and eosinophils. These cells respond to toxicants with production of reactive oxygen species (ROS) and cytokines, which participate in airway inflammation. (Vallyathan et al., 1998; Kinnula, 2005). Tumor necrosis factor-α (TNF-α) is an important mediator in many cytokine-dependent inflammatory events. It is known that TNF-α can up regulate adhesion molecules and facilitate the immigration of inflammatory cells into the airway wall thus playing a role in the initiation of airway inflammation. Proinflammatory cytokines like TNF-α increase oxidative stress via the initiation of production of reactive oxygen species (ROS) (Barrett et al., 1999) and reactive nitrogen species (RNS) (Kofler et al., 2005). Production of these cytokines and radicals can contribute to the pathogenesis of cancer (Ekmekcioglu et al., 2005; Yao et al., 2005).
The pathogenesis of cigarette smoke or arsenic-induced lung injury may involve the participation of toxic metabolites of both cigarette smoke and arsenic that elicit an inflammatory response resulting in oxidative stress that may lead to neoplastic transformation of cells (Chungman et al., 2001; Wie et al., 2002). Production of ROS/RNS from sources including cigarette smoke and arsenic can cause severe oxidative stress in cells through the formation of oxidized cellular macromolecules, including lipids, proteins, and DNA (Hartwig et al., 1997; Bolton et al., 2000). Increased oxidative stress resulting in oxidized macromolecules could occur by several mechanisms. First, cigarette smoke contains a broad range of carcinogens derived from tobacco and its pyrolysis products, including free radicals, which induce oxidative stress and subsequent lipid peroxidation (Godschalk et al., 2002). Second, cigarette smoke or arsenic induced inflammatory processes can lead to increased ROS/RNS and these compounds may compromise oxidant defense systems (Tsou et al., 2005).
ROS/RNS may be related to lung carcinogenesis (Chen et al., 2001; Ray et al., 2002). ROS/RNS has been shown to be involved in the initiation step of carcinogenesis, either in the oxidative activation of a procarcinogen, such as benzo(a)pyrene (BAP) found in cigarette smoke, or through direct oxidative DNA damage (Pryor, 1997). The molecular mechanisms involved in cigarette smoke-induced tumors involves the reduction of oxygen to superoxide and hence hydrogen 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 with carcinogenesis (Bolton et al., 2000). The glutathione system buffers the rise in oxidants such as hydrogen peroxide and the hydroxyl radical (Maehira et al., 1999). Glutathione prevents ROS/RNS-mediated damage and loss of lung cell function and lung tissue injury (Lantz et al., 2001; Kaplowitz, 2002). Cigarette smoke and arsenic metabolites may promote depletion of GSH with consequent effects on proteins, lipids, and DNA including DNA oxidation (Comhair et al., 2000).
Synergistic effects between arsenic and other potential carcinogens has been demonstrated in a skin model of carcinogenesis (Rossman et al., 2004). Combined exposure to UV radiation and arsenic in drinking water lead to an increased level of DNA oxidation (Uddin et al., 2005). Our working hypothesis is that, similar to what has been seen in the skin, the effects of cigarette smoke on the lung will act synergistically to produce oxidative damage. To gather evidence for our hypothesis, we used an animal model of inhalation exposure to fresh mainstream cigarette smoke and/or to arsenic trioxide. Our aim was to investigate whether combined exposure to cigarette smoke and arsenic would synergistically increase oxidative stress, either through initiation of inflammation or through interaction with glutathione.
Materials and Methods
Male Syrian golden hamsters were used in these experiments. Four groups of animals were used in the exposure protocol. These included unexposed animals (controls exposed to room air), animals exposed to smoke only, animals exposed to arsenic only and animals exposed to both smoke and arsenic. Animals were exposed 1 hour/day, 5 days/week for either 5 or 28 days in order to examine the progression of changes seen in the lung. Animals were housed and cared for in an AALAC-approved facility. All protocols were approved by the Institutional Animal Care and Use Committee at the University of Arizona.
Exposure Systems
Two exposure systems were used in these experiments, one for smoke exposure and a second for exposure to arsenic. For the cigarette smoke exposure, fresh mainstream smoke was collected from burning reference cigarettes (1R4, University of Kentucky, Smoking and Health Effects Laboratory, Lexington, KY). Smoke was mixed in a chamber with fresh air to provide the appropriate dilution. Relative humidity, temperature and total suspended particulates were measured. Animals were exposed in nose only fashion to 5 mg/m3 total suspended particulates over a 30-minute period. Particulate concentration was determined from changes in plate weights from a 7-stage cascade impactor (IN-TOX, Albuquerque, New Mexico). The particulate concentration was determined immediately after each exposure trial. Cascade impactor plates were weighed on an electronic analytical balance (Mettler Instrument Corporation, Hightstown, New Jersey).
For the arsenic exposure a fluidized bed particle aerosol generator was used to generate aerosols of arsenic trioxide (IN-TOX, Albuquerque, NM). Pressures and flows for the fluidized bed particle aerosol generator were first adjusted to aerosolize titanium dioxide at an average concentration of 200 μg/m3 for an 8-hour exposure (time-weighted average or TWA). After establishing conditions for titanium dioxide, arsenic compounds (As2O3, As2S3, or Ca(AsO4)2) were introduced and tested. Pressures and flows were adjusted and samples were collected and analyzed for arsenic. Concentrations were set for a 30-minute exposure with a final 8-hour TWA concentration of 200 μg/m3. This required that the concentration during the 30-minute exposure be set at 3.2 mg/m3. Once this concentration was achieved in the exposure chamber, we began exposure of hamsters to arsenic.
Combined arsenic and smoke exposures were performed on the same days. After stabilizing the airflow, animals were placed in the arsenic exposure chamber and exposed for 30 minutes to 3.2 mg/m3 as arsenic using arsenic trioxide. Air samples were collected for daily determination of the arsenic concentrations. Following arsenic exposure, animals were rested for 30 minutes prior to nose only exposure to cigarette smoke.
Exposures were carried out 5 days a week for either 5 or 28 days of exposure. One day following the final exposure, animals were then sacrificed for determination of the effects of the arsenic, cigarette smoke, or the potentially synergistic effects of the two exposures.
Lung Lavage and Fixation
After sacrifice, the heart and lungs were removed in bloc immediately after exsanguination by transection of the abdominal aorta. The esophagus and cardiovascular structures were carefully dissected away and the tracheopulmonary bloc weighed. Total lung lavage was performed using a 14-gauge catheter and 3 ml lavage volumes (repeated 3 times) delivered via the trachea (n = 5 animals for each group). Return volumes were quantitated as percent returned and lavage fluid was saved for total cell counts. An aliquot of cell suspension from bronchoalveolar lavage was used to determine total cell count. Cells were spun onto glass slides using a cytospin and stained with DiffQik to evaluate differential cell counts. Lungs from each group were fixed in situ with gluteraldehyde fixative (2% gluteraldehyde, 2.5% formalin, and 0.04% picric acid in 0.1 molar HEPES) at 20 centimeters H2O pressure. The lungs were fixed for 1 hour before being removed in bloc and immersed in fixative for 24 hours @ 4°C. Paraffin embedded sections from the fixed lungs were taken from the left and the right inferior lobes and were processed for light microscopy and immunohistochemistry. Light microscopic sections were stained with hematoxylin and eosin (H&E). Intratracheal fixed tissue sections (H&E) were also examined for signs of inflammation.
Glutathione and Oxidized DNA Determination
Oxidized and reduced glutathione were determined from the whole lung using an HPLC method (Reed et al., 1980). DNA oxidation (8-oxo-2′-deoxyguanosine (8-oxo-dG)) was quantified from genomic DNA extracted from lungs of animals exposed to arsenic and/or cigarette smoke (Shigenaga et al., 1994) with several modifications. The DNA was hydrolyzed by enzymatic digestion to individual nucleosides by nuclease P1 and alkaline phosphatase. The nucleosides were separated by HPLC (C18 column [4.6 × 150 mm] mobile phase 50 mM sodium acetate pH 5.5 with 2% acetonitrile). Unmodified nucleosides were detected by UV absorption at 278 nm and 8-oxo-dG was quantified by electrochemical detection using a potential of +300 mV. These detectors were run in series to allow quantification of both normal and 8-oxo-dG from a single injection. Results are reported as 8-oxo-dG/105.
8-oxo-dG Immunostaining
Tissues were deparaffinized and the slides were placed in citrate buffer in a microwave on high for 2–5 minutes and then an additional 5 minutes on defrost for antigen retrieval. The slides were left to cool at room temperature for 30 minutes and then rinsed with deionized water. After rinsing with deionized water, slides were incubated with RNAse for 1 hour in a humid chamber. DNA was denatured by treatment with 4 N HCl for 7 minutes at room temperature. The pH was adjusted with 50 mM Trizma base for 5 minutes at room temperature. Cells were permeabilized with 3 drops of 0.1% NP-40 in PBS for 10 minutes. The slides were then incubated in blocking serum for 20 minutes at room temperature to block nonspecific binding. Slides were incubated with primary antibody (1:100) (R&D Systems, Minneapolis, MN) at 4°C overnight.
Slides were treated with goat antimouse IgG conjugated to biotin at 37°C for 60 minutes and then incubated with cy-5 conjugated streptavidin at 37°C in a light-tight box in a humid chamber for 60 minutes. Slides were incubated with Yo Yo iodide in a light-tight box for 15 minutes at room temperature to identify nuclei and subsequently mounted with Dako mounting medium and stored in a light-tight box at 4°C overnight. The tissues were imaged with a LEICA TCS-4D confocal microscope with an Argon-Krypton laser that simultaneously scanned the slides with FITC and Cy-5 laser lines. This microscope processed the images with SCAN-WARE software.
Statistical Analysis
Data were analyzed for significant differences by 2 factor analyses of variance (smoke and arsenic) (Winer, 1971). This provides statistical analysis of both main factors as well as interaction between each. Levels of p < 0.05 were considered as significant.
Results
Inflammation Following 5-Day Exposure
Both cigarette smoke particulate exposure and exposure to arsenic can lead to increased inflammation. Therefore, combined exposure to both agents may lead to an enhanced inflammatory response and increased oxidative stress from inflammatory cells. Three different arsenical compounds (calcium arsenate, Ca2As5;arsenic trioxide, As2O3; and arsenic trisulfide, As2S3) were tested at 5-day exposures to see if there were any differences in the early inflammatory response (Figure 1). The level of inflammation was determined by total cell count from bronchoalveolar lavage. There were no significant differences between any of the arsenicals. Neither smoke alone, nor any of the arsenicals by themselves led to significantly increased cell counts above control, untreated levels. Combining arsenic and smoke also did not lead to any synergistic increases in BAL total cell counts. Differential cells counts were also unchanged.
Inflammation Following 28-Day Exposure
In the 5-day exposure animals, arsenic trioxide and cigarette smoke showed an increase over arsenic trioxide alone, but this increase was not significant. Arsenic trioxide is the predominant arsenic form that is emitted during smelting processes. This form of arsenic was therefore used to determine the effects of longer (28 days) exposure. Figure 2 shows total BAL cell counts from 28-day exposures to arsenic trioxide, smoke alone and to both compounds. Only exposure to cigarette smoke alone led to significant increases in total cell counts compared to control animals. This increase was due predominantly to an increase in the numbers of polymorphonuclear leukocytes. Combining arsenic and smoke did not lead to any synergistic increases in BAL total cell counts. While the arsenic and arsenic plus smoke (arsenic/smoke) showed elevated cell counts, these data did not reach significance. Our original expectation was that arsenic would enhance the inflammation caused by cigarette smoke. However, arsenic exposure decreased cigarette smoke-induced increases in the lung lavage cell count. These results were also validated by microscopic inspection of the lungs. No apparent inflammation was evident in the arsenic-and smoke-treated animals compared to controls (Figure 3).
Alterations in Glutathione
Protection from oxidative stress in the cell is provided in part by the glutathione cycling system. We therefore examined the levels and oxidative states of the glutathione system. The levels of reduced, oxidized and total gluathione after single or combined exposures to arsenic and/or cigarette smoke were measured after 28 days of exposure. Individual exposures to either arsenic or smoke alone did not significantly alter reduced, oxidized or total glutathione levels (Figure 4A–C). Arsenic exposure did lead to increased GSSG/GSH ratios (Figure 4D). Two-factor analysis of variance showed that, independent of smoke exposure GSSG/GSH ratios were increased by arsenic, indicating a possible arsenic-induced oxidative stress. While individual exposure did not lead to other significant changes, total glutathione levels were significantly decreased by combined exposure to arsenic trioxide and cigarette smoke (Figure 4C). While GSH levels were drastically reduced by the combined exposure, this was not accompanied by a corresponding increase in GSSG levels. Rather, both GSH and GSSG levels were reduced by the combined exposure.
DNA Oxidation in Whole Lung
Concomitant with the decreases in reduced and total glutathione, combined arsenic and cigarette smoke exposure led to significant increases in DNA oxidation (Figure 5). Levels of DNA oxidation were increased 5 fold in the lungs of animals exposed to both cigarette smoke and arsenic. Exposure to either compound alone did not lead to alterations in DNA oxidation at the levels tested. In order to identify the site(s) of DNA oxidation, lungs from animals exposed to both arsenic and cigarette smoke were processed for immunostaining using an antibody against 8-oxo-dG. Minimal staining is seen in the control animals (Figure 6A). However combined exposure resulted in detection of 8-oxo-dG in airway epithelial cells and in cells adjacent to the airways (Figure 6B).
Discussion
Epidemiological evidence indicates that cigarette smoking and arsenic exposure act synergistically to increase the incidence of lung cancer (Hughes, 2002). In order to examine the mechanism(s) involved, we designed experiments to test the hypothesis that the combined exposure to cigarette smoke and arsenic would result in increased oxidative stress. Our results suggest that arsenic and cigarette smoke act by altering the glutathione system, making the lung more susceptible to DNA oxidative damage.
An emerging hypothesis in the mechanistic studies of metal carcinogenesis is that of oxidative stress caused by exposure. However, arsenic is at best only weakly mutagenic, and it has been suggested that arsenic is a co-carcinogen exerting its carcinogenic effects in combination with other carcinogens such as those found in cigarette smoke (Hughes, 2002). The synergistic effects of arsenic with other carcinogens has previously been examined in a skin model. It has recently been shown that sodium arsenite in drinking water and UV irradiation in mice synergistically increase tumorigenicity in these animals (Rossman et al., 2004). Combined exposure to both UV and arsenite caused increased DNA oxidation in the skin, compared to either compound alone (Uddin et al., 2005). Increased tumor incidence in this model may be due in part to suppression of apoptosis (Wu et al., 2005).
Our experiments were designed to explore the possible genotoxic synergy between cigarette smoke and arsenic in the lung. Formation of 8-oxo-dG, one of the major oxidative DNA adducts, was found to be significantly increased in cigarette smoke and arsenic co-exposed animals. In addition, immunohistochemistry showed an increase in 8-oxo-dG antibody staining in airway epithelium of the co-exposed animals. Experiments were carried out at high, but environmentally relevant levels of exposure for both arsenic and cigarette smoke. Neither agent alone caused any significant changes. However combined exposures acted synergistically to reduce glutathione levels and increased DNA oxidation.
Our original hypothesis was that combined exposure to arsenic and cigarette smoke, by inhalation, would lead to an increased inflammatory response that would correlate with increased oxidative stress and DNA oxidation. Cigarette smoke- and arsenic-induced inflammation in the lung has been well established. Following cigarette smoke exposure, an influx of inflammatory cells (i.e., neutrophils, macrophages and lymphocytes) was demonstrated in mice exposed for 24 weeks, which progressively accumulated both in the airways and lung parenchyma (D’hulst et al., 2005). Chronic human smokers also show an increase in neutrophils in the lung. Gallium arsenide (GaAs), indium arsenide (InAs), and arsenic trioxide (As2O3) particles instilled intratracheally twice a week for a total of 16 installations, showed slight-to-severe inflammatory responses, which was characterized by an accumulation of neutrophils and macrophages (Tanaka et al., 2000). Arsenic, in the form of copper smelter dust, was intratracheally instilled in the mouse lung and biochemical markers, and inflammatory cell number and type demonstrated that copper smelter dust bears distinct inflammatory properties (Broeckaert et al., 1999).
However, for the doses used in this study, that was not the case. While a 28-day exposure to cigarette smoke alone led to increased BAL cell counts, arsenic exposures actually decreased the cigarette smoke-induced increases. Arsenic treatment alone caused a slight, but not significant, increase in BAL cell counts. Differences in our results from other reports in the ability of arsenic particulates to produce inflammation is most likely due to the type of exposure (inhalation versus instillation) and in the doses used. Our doses, while high, were in a range that would be seen in occupational exposures. Our results also indicate that the form of arsenic did not affect our results. Inhalation of arsenic trioxide, arsenic trisulfide, or calcium arsenate either alone or in combination with cigarette smoke did not result in increases in cell counts. This was not entirely unexpected. Alveolar macrophages lavaged from animals that had received intratracheal instillation of soluble and insoluble arsenic compounds did not show any increases in basal or stimulated superoxide production in trivalent species (Lantz et al., 1994, 1995). In addition, in vitro exposure of control macrophages to arsenicals inhibited the ability of the cells to produce superoxide in response to stimulation. Based on these results, inflammation does not appear to play a significant role in the observed synergistic effects between arsenic and cigarette smoke.
The glutathione redox system is the major antioxidant system in the cell and changes in the ratio of intracellular reduced and disulfide forms of glutathione (GSH/GSSG) can affect signaling pathways that participate in various physiological events. 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 regulation of proteins that control the synthesis of the intracellular antioxidant glutathione.
We have shown that combined exposure to arsenic and cigarette smoke leads to depletion of total glutathione stores in the lung. This suggests that, rather than just altering the GSH/GSSG ratio, as would be expected during oxidative stress, the combined exposure alters the synthesis/degradation pathways for glutathione homeostasis. Arsenic- and cigarette smoke-induced perturbations of the glutathione system have been demonstrated using a variety of approaches. Reduced glutathione could be due to conjugation and export. The multidrug resistance protein (MRP1), co-transports xenobiotics with glutathione (GSH). MRP1 also confers resistance to arsenic in association with GSH by transporting it as a tri-GSH conjugate (Leslie et al., 2004). Intracellular reduced glutathione (GSH) was significantly depleted by arsenite exposure in human pulmonary epithelial cells (BEAS-2B) (Castranova and Vallyathan, 2005). In male Wistar rats exposed to 100 ppm arsenic for 10 weeks, there was a significant decrease in GSH in the brain (Flora et al., 2005). Other studies have shown increased glutathione levels in response to arsenic. In porcine endothelial cells (PAECs), trivalent arsenic compounds; arsenic trioxide (As2O3), sodium arsenite (NaAsO2), and sodium arsenate (Na2HAsO4), increased total glutathione (Yeh et al., 2002). In addition, short term, in vitro exposures of human keratinocytes increased gamma-glutamyl-cysteine ligase levels (Schuliga et al., 2002). The effect of arsenic on glutathione appears to be dose, time, and cell-type specific.
As with arsenic, the levels of GSH in cigarette smoke exposed animals can also be significantly lower than that of control animals (Cigremis et al., 2004). The redox state of the GSH/GSSG couple in plasma of smokers and nonsmokers revealed that there was an increase in GSSG in smokers versus nonsmokers, and GSH was lower in smokers than in nonsmokers (Moriarty et al., 2003). The cytotoxicity of gas phase cigarette smoke was found to be dose-dependent, and exposure resulted in the depletion of cellular GSH levels (Piperi et al., 2003). Cigarette smoke exposure for 10 weeks resulted in reduction in GSH levels in the mouse heart (Koul et al., 2003). Exposure of solutions of GSH to gas phase cigarette smoke resulted in its rapid depletion, and about 50% of this depletion could be accounted for by reaction with acrolein and crotonaldehyde, the 2 major alpha, beta-unsaturated aldehydes in cigarette smoke (Reddy et al., 2002).
Regardless of the mechanism, reduction in glutathione levels leads to increased sensitivity to arsenic exposure. In cultured lung epithelial cells, arsenic-induced toxicity increased as arsenic decreased cellular GSH. Buthionine sulfoximine (BSO), a GSH depletor, potentiated the arsenic toxicity in these cells (Lim et al., 2002). Fetal fibroblasts from gamma-glutamyl-cysteine ligase knock-out mice (Gclm (−/−)), which lack the modifier subunit of glutamate-cysteine ligase, the rate-limiting enzyme in glutathione biosynthesis are 8 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 concentrations of arsenic and cigarette smoke, as were used in our experiments, induce a multicomponent response that leads to depletion of glutathione. Arsenic in conjunction with cigarette smoke demonstrates a synergistic effect on this system as both total and reduced glutathione are significantly decreased in arsenic- and cigarette smoke-exposed animals. The exact mechanism of action of combined arsenic and cigarette smoke in the present studies is unclear. Alone, neither toxicant reduced the overall levels of glutathione. Arsenic, independent of cigarette smoke, did lead to an altered GSSG/GSH ratio, indicative of oxidative stress. However, only the combined exposures decreased overall glutathione levels. This could be due to a synergistic inhibition of the glutathione synthesis pathways. While short-term exposures have been reported to increase gamma-glutamyl-cysteine ligase levels (Schuliga et al., 2002), effects of long term in vivo exposures have not been reported. In addition, the combined exposures could result in increased secretion of glutathione complexes, resulting in loss of total glutathione. Xenobiotic burdens deplete GSH as GST conjugates it to the xenobiotic with subsequent export of the conjugated molecule, and GSH, out of the cell. Arsenic alone or cigarette smoke alone did not deplete the tissue of GSH, and it may be that the lung can accommodate either toxicant, but when they insult the lung together the glutathione system is strained and the GSH stores are depleted.
While we have not directly measured the production of ROS, we have evaluated the direct effects of arsenic, cigarette smoke, or the combination on glutathione levels and on glutathione redox status. Reductions in the levels of glutathione as seen with the combined arsenic and smoke exposures will be expected to elevate the levels of hydrogen peroxide in the cells (Rahman et al, 2006). In addition, cigarette smoke contains numerous radicals that can oxidize DNA, including hydrogen peroxide, hydroxyl radicals, and peroxyradicals. Reduction of antioxidant defenses in the lungs, as we have seen, would result a higher rate of DNA lesions from these oxidants. While we have not specifically determined the ROS species involved, we have shown that the ROS are not the result of increased presence of inflammatory cells.
The mutagen and major DNA adduct, 8-oxo-dG, has been demonstrated in both cigarette smoke and arsenic studies. Auto-oxidation of major constituents in cigarette smoke can result in hydroxylation of deoxyguanosine residues in isolated DNA to 8-oxo-dG (Asami et al., 1996). Levels of 8-oxo-dG were significantly higher in the leukocytes of current smokers versus nonsmokers (Asami et al., 1996). Environmental tobacco smoke (ETS)-related oxidative damage in rat lung was demonstrated by a significant enhancement of 8-oxo-dG accompanied by a significant depletion of GSH (Izzotti et al., 1999). Rats exposed to side stream cigarette smoke showed significant increases in the accumulation of 8-oxo-dG (Maehira et al., 1999). Inhalation of cigarette smoke resulted in a significant decrease in GSH along with increased 8-oxo-dG in DNA in the rat lung. When these rats were treated with buthionine sulfoximine to deplete GSH, the oxidative effect of cigarette smoke was greatly potentiated (Park and Gwak, 1998).
In addition to cigarette smoke-induced increases in 8-oxo-dG, arsenic has demonstrated its potential to increase this DNA lesion. These changes have been attributed mainly to the methylated metabolites of arsenic. Pentavalent dimethylated arsenic (DMAV), in conjunction with a tumor initiator, increased 8-oxo
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 in skin, lung, liver, and the urinary bladder, whereas arsenite did not. This may be in part due to the ability of DMAV exposure to decrease cellular GSH levels (Sakurai et al., 2004). The dimethylarsenics thus may play an important role in arsenic carcinogenesis through the induction of oxidative damage (Yamanaka et al., 2001).
While cigarette smoke and arsenic can independently cause DNA oxidation in a variety of tissues, we did not see increased DNA oxidation when each of these compounds was administered independently. Only with combined exposures was there a significant increase in DNA oxidation. Since both agents can affect oxidative stress, it appears that it is the overall level of oxidative stress that determines the effects. The glutathione system works to detoxify arsenic-and cigarette smoke-induced increases in DNA oxidation. If the major sulfhydryl-containing enzymes in the glutathione system are overwhelmed or impaired by arsenic, this could potentially 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 are high compared to other reported levels (Takabayashi et al., 2004). Over the past several years, organizations such as the European Standards Committee on Oxidative DNA Damage (ESCODD) have tried to resolve problems associated with measurements of background DNA damage. (ESCODD 2002a, 2002b, 2003). Samples sent to numerous laboratories using differing techniques for isolation and measurement of 8-oxo-dG resulted in determinations that differed by greater than 2 orders of magnitude. The reasons for these discrepancies are not entirely clear, since high levels were reported from a laboratory that took the most rigorous precautions to prevent oxidation. While the reasons for the variation in the basal levels of 8-oxo-dG are still being debated, what is clear is that, independent of basal levels, HPLC-electochemical detectors are able to detect dose-response relationships for production of 8-oxo-dG (ESCODD, 2003; Collins, 2005). Therefore, while our basal levels are high, we are still able to detect significant increases in the levels of 8-oxo-dG following combined inhalation of arsenic and cigarette smoke.
In conclusion we hypothesize that arsenic and cigarette smoke deplete GSH synergistically and alter the redox status of the cell. Our studies showed a significant decrease in GSH in combined arsenic/cigarette smoke exposure over the cigarette smoke or arsenic exposed groups. Potentially, arsenic may be interfering with the enzymatic activity of the major proteins of the glutathione system and this disruption then allows for an increase in DNA oxidation. This increase in DNA oxidation was demonstrated both chemically (HPLC) and immunohistochemically. It does not appear that arsenic enhances the effects of cigarette smoke exposure by inducing a chronic inflammatory response in the distal lung. Rather it appears that the overall oxidative stress level may be the determining factor leading to DNA oxidation.
Footnotes
Acknowledgments
This research was funded in part by NIH grants P30 ES00694 and R01 ES005561 and by Arizona Disease Control Commission Grant 9703. The authors wish to thank Madel Balagtas for her assistance.