Molecular and Cellular Biochemistry 234/235: 301–308, 2002. © 2002 Kluwer Academic Publishers. Printed in the Netherlands.
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Arsenic induces oxidative DNA damage in mammalian cells Maris Kessel,1 Su Xian Liu,1 An Xu,1 Regina Santella2 and Tom K. Hei1,2 1
Center for Radiological Research, College of Physicians and Surgeons; 2Department of Environmental Health Sciences, Joseph Mailman School of Public Health, Columbia University, NY, USA
Abstract Although arsenic is a well-established human carcinogen, the underlying carcinogenic mechanism(s) is not known. Using the human-hamster hybrid (AL) cell mutagenic assay that is sensitive in detecting mutagens that induce predominately multilocus deletions, we showed previously that arsenite is indeed a potent gene and chromosomal mutagen and that oxyradicals may be involved in the mutagenic process. In the present study, the effects of free radical scavenging enzymes on the cytotoxic and mutagenic potential of arsenic were examined using the AL cells. Concurrent treatment of cells with either superoxide dismutase or catalase reduced both the cytotoxicity and mutagenicity of arsenite by an average of 2–3 fold, respectively. Using immunoperoxidase staining with a monoclonal antibody specific for 8-hydroxy-2′-deoxyguanosine (8-OHdG), we demonstrated that arsenic induced oxidative DNA damage in AL cells. This induction was significantly reduced in the presence of the antioxidant enzymes. Furthermore, reducing the intracellular levels of non-protein sulfhydryls (mainly glutathione) using buthionine S-R-Sulfoximine increased the total mutant yield by more than 3-fold as well as the proportion of mutants with multilocus deletions. Taken together, our data provide clear evidence that reactive oxygen species play an important causal role in the genotoxicity of arsenic in mammalian cells. (Mol Cell Biochem 234/235: 301–308, 2002) Key words: arsenic, mutagenicity, oxidative stress, antioxidant enzymes, 8-OHdG
Introduction Arsenic, as trivalent arsenite (AS3+) or pentavalent arsenate (AS5+), is naturally occurring and ubiquitously present in the environment. Epidemiological data have shown that chronic exposure of humans to inorganic arsenical compounds is associated with liver injury, peripheral neuropathy, and an increased incidence of cancer of the lung, skin, bladder, and liver [1, 2]. However, the mechanism(s) underlying its carcinogenicity remains unknown. The United States Environmental Protection Agency has placed arsenic at the top of its Superfund contamination list [3]. Biologically, the trivalent sodium arsenite is significantly more active than the pentavalent sodium arsenate [4]. Arsenic contamination of drinking water is a serious environmental problem worldwide because of the large number
of contaminated sites that have been identified and the large number of people at risk [5]. The risk of developing arsenicinduced human diseases from environmental exposure is particularly high in many developing countries. For example, it is estimated that as many as 50 million people are at risk in Bangladesh alone, where both acute and chronic arsenic poisoning as well as increased cancer incidence have been reported [6]. Although the water supplies in the United States are generally low in arsenic, there have been reports of arsenic contamination of ground water in the Southwest with levels in the hundreds, and in few cases, more than 1,000 µg/l [7, 8], a level that is 20 times higher than the current U.S. maximum contaminant level of 50 µg/l. Occupational exposure occurs mainly through inhalation via nonferrous ore smelting, semiconductor and glass manufacturing, or power generation by the burning of arsenic-contaminated coal [7,
Address for offprints: T.K. Hei, Center for Radiological Research, Columbia University, VC 11-205, 630 West 168th Street, NY 10032, USA (E-mail:
[email protected])
302 9]. There is evidence that underground uranium miners who are also exposed to arsenic have a 10-fold increase in lung cancer risk compared to miners without previous history of arsenic exposure [10]. Arsenic is unusual because it is one of the few demonstrated human carcinogens for which carcinogenicity in laboratory animals has not been firmly established [11]. An inducible arsenic tolerance state in rodent species has been suggested to account for this discrepancy [12]. Inducible tolerance to arsenic-induced toxicity has not been observed in human cells. There is also evidence that human cells are, in fact, more sensitive to arsenite than cells of rodent origin [13]. In the absence of suitable animal models to study the mechanisms of arsenic-induced carcinogenicity, in vitro studies have been conducted to provide information on the cellular mechanisms involved. While arsenic and arsenical compounds are toxic and induce morphological transformants in Syrian hamster embryo and C3H 10T1/2 cells [14, 15], it is inactive as a gene mutagen at either the hypoxanthine guanine phosphoribosyl transferase [HPRT] or ouabain loci [14, 16]. Arsenic compounds, however, are potent clastogens in many cell types and induce sister chromatid exchanges and chromosomal aberrations in both human and rodent cells in culture [4, 17]. One possible scenario is that arsenic induces mostly multilocus deletions that are incompatible with cell survival when selected at gene loci located in closed proximity to other essential genes. Using the AL mutagenic assay system which is sensitive in detecting multilocus deletions [18, 19], we have shown that this is, in fact, the case with the trivalent sodium arsenite [20, 21]. At equivalent dose of arsenite, the mutant yield at the CD59 locus was ~ 35 fold higher than the corresponding HPRT locus. In addition, the majority of the CD59– mutants induced were multilocus deletions. In the present study, we examined the effects of the antioxidant enzymes catalase and superoxide dismutase on mutagenicity of arsenite at the CD59 locus of the human hamster hybrid (AL) cells. We further characterized the induction of the oxidative DNA damage product, 8-hydroxydeoxyguanosine (8-OHdG) as well as the modulating effects of sulfhydryl depletion on the induction and types of mutations induced in arsenite treated cells.
Materials and methods Cell culture The human-hamster hybrid (AL) cell, formed by fusion of human fibroblasts and the gly–A mutant of the Chinese hamster ovary cells (CHO) [22], was used in these studies. In addition to a standard set of CHO-K1 chromosomes, these hybrid cells contain a single copy of human chromosome 11. Chromosome 11 encodes several cell-surface antigenic mark-
ers that render the cells sensitive to killing by specific monoclonal antibodies in the presence of rabbit serum complement (HPR, Denver, PA, USA). Antibody E7.1 specific to the CD59 (formerly known as S1) antigen was produced from hybridoma culture and used as described [19, 20]. Cells were cultured in Ham’s F-12 medium supplemented with 8% heated inactivated fetal bovine serum (Hyclone Laboratory, Logan, UT, USA), 2 × 10–4 M glycine, and 25 µg/ml gentamycin at 37°C in a humidified 5% CO2/95% air incubator and passaged as described [18–20]. Determination of arsenic cytotoxicity Stock solution of sodium arsenite (Sigma Chemical, St. Louis, MO, USA) at 1 mg/ml was prepared in doubled-distilled water and sterilized by passing through a 0.22 µ syringe filter. Working concentrations were prepared by diluting the stock with complete F12 medium. To determine the dose response cytotoxicity of AL cells to arsenite, exponentially growing cells were trypsinized and replated into 25-cm2 tissue-culture flasks at a density of 1 × 105 cells per flask, and treated 48 h after plating with arsenite for either 1 or 5 days. After treatment, cultures were washed twice with balanced salt solution, trypsinized, and replated into 100-mm diameter petri dishes for colony formation. Cultures were incubated for 7– 12 days, at which time they were fixed with formaldehyde and stained with Giemsa. The number of colonies was counted to determine the surviving fraction as described [20, 21]. Treatment with antioxidants Stock copper/zinc superoxide dismutase (SOD) from bovine liver (Diagnostic Data, Inc., Mountainview, CA, USA) was weighted out, dissolved in phosphate buffered saline, filtered through a 0.22 µ syringe filter and stored at –20°C until use. Working solutions of 400 U/ml were prepared fresh each time from stock solution by dilution with complete medium. Since catalase (Sigma Chemicals, St. Louis, MO, USA) is relatively unstable in aqueous solution, it was prepared fresh each time just before use. Stock catalase solution was membrane filtered and diluted with complete F12 medium to a working concentration of 5000 U/ml. To ascertain the role of oxyradicals in arsenite mutagenesis, exponentially growing AL cells were exposed to arsenite for 24 h with or without concurrent treatment with doses of either SOD or catalase. SOD and catalase, at the doses used, were non-toxic and nonmutagenic and had been shown to be an effective free radical scavenger in a variety of in vitro and in vivo studies [23, 24]. After treatment, cultures were trypsinized, counted with a Coulter electronic counter (Coulter Corporation, Miami, FL, USA), and replated for both survival and mutagenesis as described.
303 Non-protein sulfhydryl depletion by buthionine S-R sulfoximine (BSO) Exponentially growing AL cells (5 × 105) in 25 cm2 tissue culture flasks were treated with BSO at 10 µM (Chemalog, S. Plainfield, NJ, USA) for 24 h prior to arsenite treatment. The doses of BSO used were shown previously to be non-toxic and non-mutagenic and to reduce the NPSH level to less than 5% of the control level [21, 25]. After treatment with arsenite in the presence of BSO for another 24 h, cultures were trypsinized and replated to determine survival and mutagenesis as described above.
Mutations assay After treatment, cultures were replated in T-75 flasks and cultured for 5–7 days. This expression period is needed to permit surviving cells to recover from the temporary growth lag from arsenite treatment and to multiply such that the progeny of the mutated cells no longer express lethal amounts of the CD59 surface antigen. To determine mutant fractions, 5 × 104 cells were plated into each of six 60 mm dishes in a total of 2 ml of growth medium as described [18–21]. The cultures were incubated for 2 h to allow for cell attachment, after which 0.2% CD59 antiserum and 1.5% freshly thawed complement (v/v), were added to each dish. The cultures were further incubated for 7–8 days, at which time they were fixed, stained, and the number of CD59– mutants scored. Controls included identical sets of dishes containing antiserum alone, complement alone, or neither agent. The cultures derived from each group were tested for mutant yield for two consecutive weeks to ensure full expression of the mutations. Mutant fractions were calculated as the number of surviving colonies divided by the total number of cells plated after correction for any non-specific killing due to complement alone.
94°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 1 min. After the last cycle, samples were incubated at 72°C for an additional 20 min, electrophoresed on 3% agarose gels, and stained with ethidium bromide.
Immunoperoxidase staining for 8-OHdG Exponentially growing AL cells were inoculated into Lab-Tek glass chambered slides (Nunc Inc., Naperville, IL, USA) at 5 × 104 and cultured for 24 h. The cultures were exposed to graded doses of arsenite for 24 h with or without 0.5% DMSO. The dose of DMSO was nontoxic, non-mutagenic, and had been shown to be an effective free radical scavenger [20, 26]. After treatment, cultures were rinsed twice with PBS and fixed with 5% acid-alcohol at –20°C. Immunoperoxidase staining for 8-OHdG was performed as described previously [25, 27]. Briefly, fixed cultures were treated with RNase (100 µg/ml) for 1 h at 37°C and proteinase K (10 µg/ml) for 10 min at room temperature. DNA was denatured with 4 N HCl for 10 min at room temperature and 10% normal horse serum was used to block nonspecific antibody binding sites. The cultures were then incubated with the primary antibody 1F7 (1:50 dilution) at 4°C overnight followed by goat antimouse IgG conjugated to biotin at 37°C for 30 min. Endogenous peroxidase was blocked by treating the cultures with 3% H2O2 in methanol for 30 min at room temperature. ABC reagent, avidin conjugated to horseradish peroxidase was added and the slides were incubated for 30 min at 37°C, followed by extensive washing. Cells were treated with diaminobenzidine to localize peroxidase, cleaned with xylene and mounted with cover glass using Permount. A Cell Analysis System CAS 200 microscope and a Cell Measurement Software package (Becton Dickinson, San Jose, CA, USA) were used to quantify the relative staining intensity from 50 randomly selected cells per group. Data presented are the average absorbance multiplied by 1000.
Analyses of mutant spectrum by multiplex PCR Statistics CD59– mutants were isolated by cloning and expanded in culture as described [18–21]. Five marker genes located on either the short arm (WT, PTH, CAT, RAS) or the long arm (APO-A1) of human chromosome 11 were chosen for multiplex PCR analysis because of their mapping positions relative to the CD59 gene and the availability of PCR primers for the coding regions of these genes. PCR amplifications were performed for 30 cycles using a DNA thermal cycle model 480 (Perkin-Elmer/Cetus) in 20 µl reaction mixtures containing 0.2 µg of the EcoRI-digested DNA sample in 1 × Stoffel fragment buffer, all four dNTPs (each at 0.2 mM), 3 mM MgCl2, 0.2 mM each primer, and 2 units of stoffel fragment enzyme. Each PCR cycle consisted of denaturation at
Statistical analysis of data was carried out using Student’s ttest. Differences between means are regarded as significant if p < 0.05.
Results Effects of SOD and catalase on cell killing and mutagenesis by arsenite Figure 1 shows the surviving fraction and induced CD59 mutations in AL cells treated with either a 1.5 or 2.0 µg/ml
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Fig. 1. Effects of exogenous SOD (400 U/ml) on induced mutant fractions in AL cells treated concurrently with graded doses of sodium arsenite for 24 h. Induced mutant fractions are the total mutant yield minus background, which amounts to 46 ± 10 mutants per 10 5 survivors among the AL cells used in these studies. The survival fraction of the various treatment groups is shown above each bar. Data were pooled from 3–5 experiments. Error bars represent ± S.D.
dose of sodium arsenite for 24 h, with or without concurrent exposure to SOD (400 U/ml). The normal plating efficiency of AL cells used in these studies ranged from 81–89%. Over the range of arsenic concentration examined, the dose response survival of AL cells was consistent with our previously published data [22, 23]. Addition of SOD to the culture medium had essentially no effect of the clonogenic survival of control cells. In contrast, SOD treatment significantly reduced the clonogenic toxicity of arsenic at both the 1.5 µg/ml dose (0.31 ± 0.06 vs. 0.44 ± 0.08 with SOD, p < 0.05) and 2 µg/ml dose level (0.18 ± 0.07 vs. 0.62 ± 0.13 with SOD, p < 0.025). Likewise, addition of SOD to the culture medium reduced the mutagenicity of arsenic in AL cells. The average number of spontaneous CD59– mutants per 105 survivors in AL cells used for these experiments averaged 46 ± 10. Treatment of cells with a 1.5 and 2.0 µg/ml dose of arsenite resulted in induced mutant fractions (total mutant fraction minus background) of 95 ± 24 and 125 ± 35, respectively. While SOD treatment by itself induced no CD59– mutations, its presence in the culture medium during arsenic treatment reduced the mutant fractions by 3.2 and 2 fold to 30 ± 8 and 64 ± 16, respectively for the 1.5 and 2.0 µg/ml dose treatment (Fig. 1). The possible contribution of hydrogen peroxide in the cytotoxic and genotoxic effects of arsenite was ascertained using catalase in the culture medium as shown in Fig. 2. Similar to the findings with SOD, catalase, at a concentration of 5000 U/ml, significantly reduced the clonogenic toxicity of
Fig. 2. Effects of exogenous catalase (5,000 U/ml) on induced mutant fractions in AL cells treated concurrently with graded doses of sodium arsenite for 24 h. The survival fraction of the various treatment groups is shown above each bar. Data were pooled from 3 experiments. Error bars represent ± S.D.
arsenic at both the 1.5 µg/ml dose (0.39 ± 0.06 vs. 0.84 ± 0.12 with catalase, p < 0.025) and 2 µg/ml dose level (0.28 ± 0.07 vs. 0.64 ± 0.09 with catalase, p < 0.025). Likewise, catalase treatment reduced the mutagenicity of arsenic in AL cells, being more effective at lower dose of the naturally occurring metalloid. While catalase treatment by itself induced no CD59– mutations, its presence in the culture medium during arsenic treatment reduced the mutant fractions by 2.1 and 1.9 fold to 80 ± 25 and 120 ± 20, respectively for the 1.5 and 2.0 µg/ml dose treatment (Fig. 2). On the other hand, treatment of catalase with heat obliterated its suppressive effect on arsenite-induced mutagenic yield (data not shown). It should be noted that the batch of AL cells used in the catalase series of experiments had a higher mutant background as well as induction level. However, the overall trend remains similar to that of the SOD studies.
Induction of oxidative DNA damage by arsenic in AL cells If generation of reactive oxygen species is one of the major pathways for arsenic-mediated genotoxicity, then it should be expected to induce specific DNA lesions consistent with oxidative damages. One of the most common oxidative DNA lesions is 8-hydroxy-2′-deoxyguanosine (8-OHdG). Using a monoclonal antibody specific for 8-OHdG coupled with immunoperoxidase staining, we determined the formation of the oxidative DNA damage product in AL cells treated with a 4 µg/ml dose of sodium arsenite for 24 h as shown in Fig. 3.
305 8-OHdG was localized mainly in the nucleus of both control and arsenite treated cells. Although a faint, background staining was evident in the control cultures, treatment of AL cells with arsenic resulted in a dose dependent increase in 8-OHdG levels. Quantification of staining intensity from 50–80 randomly selected cells treated with a 4 µg/ml dose of arsenite indicated a 2.1 fold increase in staining intensity above background (Fig. 4).
Effects of SOD and catalase on the formation of 8-OHdG by arsenic Figure 4 shows the suppressive effect of SOD (400 U/ml) and catalase (5000 U/ml) on the formation of 8-OHdG induced by a 4 µg/ml dose of sodium arsenite in AL cells. The relative staining intensity decreased from an arbitrary unit of 333 to 207 and 212 in the presence of SOD and catalase, respectively. SOD and catalase by themselves, however, had little or no effect on the formation of 8-OHdG among control AL cells yielding a staining intensity in arbitrary units of 156 and 162, respectively.
Fig. 4. Effects of SOD and catalase on the induction of 8-OHdG in AL cells treated concurrently with a 4 µg/ ml dose of sodium arsenite for 24 h. Data were pooled from 3 experiments. Bars represent ± S.D.
Effects of BSO treatment on toxicity and mutagenicity of arsenic Figure 5 shows the CD59– mutant fraction induced by a 0.5 µg/ml dose of sodium arsenite with or without pretreatment with 10 µM BSO. The average number of spontaneous CD59– mutants per 105 survivors in AL cells used for these experiments was 43 ± 18. The induced mutant fraction in arsenitetreated AL cells was ~ 1.7 fold higher than background. BSO treatment by itself induced a low and non-significant increase in the background mutant yield. In contrast, BSO pretreatment enhanced the mutagenic potential of arsenite such
Fig. 3. Representative immunoperoxidase staining for 8-OHdG in AL cells. (A) control cells; (B) treated with a 4 µg/ ml dose of sodium arsenite for 24 h (×200).
Fig. 5. Induction of CD59– mutants in AL cells treated with a 0.5 µg/ml dose of sodium arsenite for 24 h with or without pretreatment with BSO (10 µM). Induced mutant fractions are the total mutant yield minus background. Data were pooled from 3 independent experiments. P.E. – plating efficiency, S.F. – surviving fraction. Bars represent ± S.D.
306 that there was a 3-fold increase in CD59– mutant yield in cells treated with both BSO and arsenite as compared to those treated with arsenite alone (p < 0.025). Furthermore, pretreatment of AL cells with BSO also enhanced their sensitivity to the cytotoxicity of arsenite (Fig. 5). These results indicate that intracellular antioxidant status has a profound effect on the mutagenic response of AL cells to arsenic treatment and suggest that ROS play a significant role in mediating the mutagenic process.
Effect of sulfhydryl depletions on mutant spectrum by arsenite To determine the types of mutation associated with the CD59– phenotype in arsenite treated cells with or without BSO, we isolated individual independent clones and applied multiplex PCR to determine the presence or absence of five chromosome 11 markers located on either side of the CD59 gene. The primers and PCR conditions were selected to amplify only the human genes and not their CHO cognates [19, 20, 28]. Previous studies have shown that a small segment of the human chromosome 11 near the RAS gene is required for survival of CD59– mutants, the obligate presence of this region identified here by the present of RAS probe in all the mutants provides a convenient internal PCR control [28]. As
Fig. 6. Mutation spectra CD59– mutants of spontaneous origin or from cells exposed to a 0.5 µg/ml dose of sodium arsenite with or without pretreatment with BSO (10 µM). Each line depicts a single mutant. Blank space depicts missing markers on chromosome 11 as determined by multiplex PCR.
shown in Fig. 6, the majority of spontaneous CD59– mutants (19 of 25 or 76%) as well as those treated only with BSO (22 of 26 or 85%) retained all five of the chromosome 11 markers, whereas only 11 of 23 or 48% of those treated with a 0.5 µg/ml dose of sodium arsenite were of this type. Pretreatment of cells with BSO at a dose of 10 µM, which reduced the NPSH level to less than 10% of control, significantly increased the proportion of mutants with large multilocus deletions such that only 4 of 22 mutants or 18% retained all five of the markers examined. In fact, 9 of 22 mutants or 41% in this group lost the four markers that spanned both arms of the human chromosome 11. Southern analysis using the centromeric probe p82H indicated that these mutants lost the centromere of the human chromosome 11 as well (data not shown).
Discussion Arsenic is a well-established human carcinogen based on epidemiological studies. However, the mechanism(s) underlying its carcinogenicity remains unclear. Besides being a human carcinogen, arsenic is also a risk factor for atherosclerosis, diabetes, and peripheral neuropathy [7]. The lack of suitable animal models as well as a poor understanding of its carcinogenic/genotoxic mechanism hamper accurate risk assessment of the health effects of arsenite on both humans and animals, and necessitates reliance on in vitro studies to illuminate the cellular and molecular pathways involved. Using the human hamster hybrid (AL) cells assay that is efficient in the recovery multilocus deletions, we showed previously that arsenic is indeed a potent gene and chromosomal mutagen [20, 21]. Herein we present evidence that the mutagenicity is largely mediated by oxyradicals. Reactive oxygen species such as superoxide anions, hydroxyl radicals and hydrogen peroxides are the intermediates formed during oxidative metabolisms. The observations that antioxidants such as dimethyl sulfoxide, catalase, and Nacetyl cysteine reduce the in vitro biological activities of arsenic [19, 29, 30] suggest that active oxygen species may contribute to the carcinogenic/mutagenic process of the metalloid. The deleterious effect of oxygen toxicity is normally held in check by the delicate balance between the rate of generation of these radicals and their removal by various antioxidant enzymes. Superoxide dismutase catalyzes the dismutation of superoxide anions while catalase removes hydrogen peroxides and prevents the subsequent formation of hydroxyl radicals (see [31] for review). These hydroxyl radicals are far more damaging to cells than other radical species and have been associated with arsenic induced mutagenicity [20]. Our findings that catalase and SOD can reduce the mutagenic potential of arsenic in mammalian cells are consistent with data obtained with other genotoxic endpoints.
307 Using CHO cells and a X-ray hypersensitive, DNA repair deficient mutant, XRS-5, Wang and Huang showed that arsenite induced a dose dependent increase in micronuclei which was blocked by exogenous catalase [32]. In addition, heme oxygenase, an oxidative stress protein, and peroxidase are induced by sodium arsenite in various human cell lines [33]. Furthermore, antioxidant enzymes such as superoxide dismutase reduced the incidence of sister chromatid exchanges (SCE) induced by arsenite in cultured human lymphocytes [34]. These data are consistent with our recent findings using ESR spin trapping assay that arsenite increases the levels of superoxide-driven hydroxyl radicals in mammalian cells [20]. However, the origin of these radical species remains unknown. DNA damage induced by reactive oxygen species is important in mutagenesis and carcinogenesis [31]. 8-OHdG is one of the most abundant oxidized DNA bases and has been shown to be a mutagenic DNA lesion [35]. While there are a variety of methods for quantifying 8-OHdG in mammalian DNA including HPLC-EC, GC/MS, and 32P post-labeling, we choose the immunoperoxidase assay which offers several advantages. This assay, based on a monoclonal antibody specific for 8-OHdG (1F7), allows the detection of 8-OHdG in single cells and in a highly reproducible manner [27]. The specificity of 1F7 for 8-OHdG has previously been demonstrated using competitive ELISA assay [36]. Furthermore, using DNA extracted from mammalian cells exposed to inducers of oxidative damages (e.g. hydrogen peroxide), the yield of 8-OHdG formations determined with the immunoperoxidase assay correlated well with that obtained using the more conventional HPLC-EC method [36]. Since the immunoperoxidase method does not require isolation and processing of DNA, the potential for artificial generation of oxidative damage is eliminated. Treatment of AL cells with sodium arsenite induced a dose dependent increase in the formation of 8-OHdG. Such increases can be significantly reduced in the presence of the antioxidant enzymes. These data are consistent with the mutation data shown in Figs 1 and 2. Since SOD and catalase are relatively large molecules with molecular weights of 30 and 250 Kda, respectively, they are highly unlikely to pass across the cell membrane without being phagocytosized. Our data with the antioxidants, therefore, are consistent with the previous finding with electron spin resonance (ESR) and the spin trap Tempol-H that arsenic induces hydrogen peroxide as a precursor of hydroxyl radicals in AL cells [21]. Since hydrogen peroxide is freely diffusible between intracellular and extracellular space, addition of extracellular antioxidants are likely to reduce the intracellular oxidative stress induced by arsenite treatment. However, the exact pathway remains to be elucidated. Cellular non-protein sulfhydryls consists essentially of glutathione (~ 95%) and other low molecular weight aminothiols
such as cysteine and cysteamine [37]. These sulfhydryls scavenge free radicals and contributes to the maintenance of cellular integrity. Although a decrease in cellular glutathione may not in itself result in cell death, sulfhydryl depletion has been shown to enhance the cytotoxicity of a variety of agents, including ionizing radiation, heavy metals, oxidative stress, and certain chemotherapeutic drugs [38]. There is also evidence that such cellular thiols as glutathione and cysteine protect mammalian cells against the toxic effects of arsenite [30, 39]. Furthermore, low concentrations of arsenite have been shown to induce a transient increase in cellular glutathione levels in bovine vascular endothelial cells [40]. The up-regulation is thought to be a ‘secondary’ stress response directly regulated by the thiol reactivity of arsenite [41]. Our present findings indicate that an increase in cellular oxidative stress status not only enhances the cytotoxicity and mutagenicity of arsenite but alters the spectrum of mutants generated as well. The relative proportion of mutants that lost all 4 additional genes increased from 4–41% in arsenitetreated cells with diminished glutathione content compared with the corresponding controls. As a naturally occurring metalloid, arsenic is a serious environmental concern world-wide, because of the large number of known contamination sites and millions of people at risk from drinking arsenic-contaminated water. A better understanding of the mutagenic/carcinogenic mechanisms of arsenic should provide a basis for better interventional approach in both treatment and prevention.
Acknowledgement This work was supported by National Institute of Health Grants ES 08821, Superfund Grant P42 ES10349 and Environmental Health Center grant P30 ES09089. Maris Kessel was a senior at the Bronx High School in Science in New York City while conducting the study. Part of this study was awarded a semifinal entry title to the Intel Science competition in December 2000.
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