Role of oxidative damage in the genotoxicity of arsenic

Free Radical Biology & Medicine, Vol. 37, No. 5, pp. 574 – 581, 2004 Copyright D 2004 Elsevier Inc. Printed in the USA. All rights reserved 0891-5849/$-see front matter

doi:10.1016/j.freeradbiomed.2004.02.003

Serial Review: Redox-active Metal Ions, Reactive Oxygen Species, and Apoptosis Serial Review Editor: Balaraman Kalyanaraman ROLE OF OXIDATIVE DAMAGE IN THE GENOTOXICITY OF ARSENIC TOM K. HEI *,y and METKA FILIPIC z *Center for Radiological Research, College of Physician and Surgeons, and y Department of Environmental Health Sciences, Joseph Mailman School of Public Health, Columbia University, New York, NY 10032, USA; and z National Institute of Biology, Department of Genetic Toxicology and Cancer Biology, Ljubljana, Slovenia (Received 6 November 2003; Revised 15 January 2004; Accepted 2 February 2004) Available online 4 March 2004

Abstract—Arsenic is a well-established human carcinogen and is ubiquitous in the environment. For decades, arsenic has been considered to be a nongenotoxic carcinogen because it is only weakly active or, more often, completely inactive in bacterial and mammalian cell mutation assays. In this review, evidence is presented that when assayed using model systems in which both intragenic and multilocus mutations can readily be detected, arsenic is, indeed, found to be a strong, dose-dependent mutagen which induces mostly multilocus deletions. Furthermore, the roles of reactive oxygen and reactive nitrogen species in mediating the genotoxic response are presented in a systematic and logical fashion in support of a working model. The data suggest that antioxidants may be a useful interventional treatment in reducing the deleterious effects of arsenic. D 2004 Elsevier Inc. All rights reserved. Keywords—Arsenite, Reactive oxygen species, Reactive nitrogen species, Multilocus mutation, Free radicals

INTRODUCTION

Arsenic is a known human carcinogen and induces a variety of human diseases in addition to cancers of the lung, skin, bladder, kidneys, and liver. The mechanism(s) by which arsenic induces cancer, however, remains poorly understood. Although multiple pathways such as inhibition of DNA repair, methylation status, and cocarcinogenesis with other environmental toxicants have been proposed, one common theme that has emerged is the role of reactive radical species in the pathogenesis of arsenic-induced diseases. Free radicals generated as a consequence of arsenic exposure are linked to cell signaling, apoptosis, and mutagenesis. In this review, we discuss the role of reactive oxygen and reactive nitrogen species in the genotoxicity of this carcinogenic metal species.

This article is part of a series of reviews on ‘‘Redox-active Metal Ions, Reactive Oxygen Species, and Apoptosis.’’ The full list of papers may be found on the home page of the journal. Dr. Tom K. Hei received his B.Sc. degree from the University of Wisconsin and his Ph.D. degree in Pathology from Case Western Reserve University. He is currently a Professor of Radiation Oncology and a Professor of Environmental Health Sciences at Columbia University. His research focuses on mechanisms of lung and breast cancers, particularly those pertaining to radiation and environmental origins. Dr. Metka Filipic received her B.Sc. and Ph.D. degrees at the Biotechnical Faculty, University of Ljubljana, Slovenia. Currently she is a senior researcher at the National Institute of Biology, Ljubljana, and an Assistant Professor at the Faculty of Pharmacy, University of Ljubljana. Her current research work focuses on cellular and molecular mechanisms of environmental and foodborne genotoxins, particularly the role of oxidative stress and DNA repair. Address correspondence to: Dr. Tom K. Hei, Center for Radiological Research, Vanderbilt Clinic 11-218, College of Physicians and Surgeons, Columbia University, 630 West 168th Street, New York, NY 10032, USA; Fax: (212) 305-3229; E-mail: [email protected].

ARSENIC AS AN ENVIRONMENTAL CARCINOGEN

Arsenic, as trivalent arsenite (AS3+) or pentavalent arsenate (AS5+), is naturally occurring and ubiquitous in 574

Oxidative damage and arsenic mutagenesis

the environment. Epidemiological data have shown that chronic exposure of humans to inorganic arsenical compounds is associated with liver injury, peripheral neuropathy, keratosis of the skin, and an increased incidence of cancer of the lung, skin, bladder, kidneys, and liver [1,2]. However, the mechanism(s) underlying its carcinogenicity remains unknown. The U.S. 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]. It has been 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 [6,7]. Although the water supplies in the United States are generally low in arsenic, there have been reports of arsenic contamination of ground water in several Southwest states with levels in the hundreds of micrograms per liter and, in few cases, more than 1000 Ag/l [8,9], a level that is 100 times higher than the current U.S. maximum contaminant level of 10 Ag/l (10 ppb). Occupational exposure occurs mainly through inhalation via nonferrous ore smelting, semiconductor and glass manufacturing, or power generation by the burning of arsenic-contaminated coal [8]. There is evidence that underground uranium miners who are also exposed to arsenic have a 10-fold increase in lung cancer risk compared with miners without previous history of arsenic exposure [10]. Although epidemiological data have firmly established inorganic arsenic to be a human carcinogen, animal studies are less well defined. The carcinogenicity of inorganic arsenite in animals, primarily rodents, has been the subject of debate due to the limited data set and the perceived inadequacy in the design of some of these earlier studies [11,12]. However, there is increasing evidence that methylated arsenic, particularly dimethylarsinic acid (DMAV), is carcinogenic to both rats and mice, at extremely high doses in the range 200 – 400 ppm [13,14]. Furthermore, the trivalent methylated metabolite dimethylarsinous acid (DMAIII) may be more toxic than inorganic arsenite and, perhaps, genotoxic as well [15]. These findings are paradoxical as methylation of arsenic has long been considered to be a detoxification route. An inducible arsenic tolerance state in rodent species has been suggested to account for the inability of arsenic to induce tumors in animal models [16]. Inducible tolerance to arsenic 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 [17].

575

ARSENIC AS A GENOTOXIC CARCINOGEN

For decades, arsenic has been considered by many to be a nongenotoxic carcinogen [18 for review]. While arsenic and arsenical compounds are toxic and induce morphological transformants in Syrian hamster embryo and C3H 10T1/2 cells [19,20], they are nonmutagenic both in bacteria or at the hypoxanthine – guanine phosphoribosyl transferase [hprt] or ouabain loci in mammalian cells [19,21]. The failure of arsenic to induce gene mutations in mammalian cells has been taken as evidence that a nongenotoxic pathway such as induction of DNA hypomethylation [22] or inhibition of DNA ligation [23] may be important in the carcinogenesis of arsenic. 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 [24,25]. The negative gene mutation data suggest either that arsenic is a nongenotoxic carcinogen or, given the chromosomal data, that mutants induced at these loci are nonviable. Using the human – hamster hybrid (AL) cell mutagenic assay, which is highly sensitive in detecting both intragenic and multilocus mutations, arsenite has been found to be mutagenic at the CD59 locus and induce mostly multilocus deletions involving millions of base pairs [26,27]. In contrast, among the same population of arsenic-treated cells, there are few, if any, mutations scored at the hprt locus of the hamster X chromosome. The finding is consistent with the observation that very few hprt mutants are recovered from the lymphocytes of copper workers exposed to high levels of arsenic [28]. Because the hprt gene is on the X chromosome, multilocus deletions in the region of the chromosome containing essential genes could be lethal and any mutants induced would not be viable [29]. In contrast, the single copy of chromosome 11 that encodes the CD59 locus at 11p13 is the only human chromosome that the cells carried and, with the exception of a small segment near the Ras gene at 11p15.5, is not required for survival of the hybrid cells. Thus, the entire human chromosome 11 can act as a target for mutagens. In general, deletions in excess of 3 megabases are rarely recovered at the hprt locus and would be completely lethal at the Na/K ATP-ase gene used to measure ouabain-resistant mutants [30]. In this regard, no oua mutants (3 mM ouabain) from arsenite-treated AL cell populations were recovered [26]. On the other hand, a deletion that inactivates the ouabain binding site, which is necessary for the generation of oua mutants, also codeletes the ATP binding sites. Loss of the ATP binding sites renders the mutants ouabain-sensitive, because the enzyme ATPase also cannot bind ATP and is, therefore, inactive [31]. The finding that arsenic induces predominately large mutations is consistent with the ample body

576

T. K. HEI and M. FILIPIC

of data on induction by arsenic of chromosome aberrations and micronuclei. The observation is also consistent with the recent reports that arsenic induces both small and large mutations in the PZ189 shuttle vector system in cultured mammalian cells [32] as well as intrachromosomal recombinations in the hprt gene of CHO cells [33]. Thus, it appears that when studied in mutation assays that detect large chromosomal mutations, arsenic is in fact a potent mutagen/clastogen. DETECTION OF OXYRADICALS IN ARSENIC-TREATED CELLS

The nonfluorescent dye 5V,6V-chloromethyl-2V,7Vdichlorodihydrofluorescein diacetate (CM-H2DCFDA) passively diffuses into cells where the acetates are cleaved by intracellular esterase [34,35]. The resulting diol is more polar and is better retained inside the plasma membrane. The diol can then be oxidized by ROS to the fluorescent form that has absorbency at 504 nm. To localize and semiquantify the induction of ROS by sodium arsenite, human –hamster hybrid (AL) cells are preloaded with a 1 AM dose of CM-H2DCFDA for 40 min at 37jC [27] and exposed to sodium arsenite with or without concurrent treatment with the radical scavenger dimethyl sulfoxide (DMSO). Fluorescence images are then quantified using an image analysis software and confocal microscopy. There is evidence that cells exposed to sodium arsenite exhibit a dosedependent increase in fluorescence level within minutes of treatment when compared with controls, indicative of higher intracellular oxidant levels [27]. In AL cells treated with a 2 Ag/ml dose of arsenite (15 AM), a 3fold increase in the average fluorescence intensity relative to control has been observed. The oxyradical nature behind the increase in fluorescence intensity is further supported by including the radical scavenger DMSO in the reaction mixture, which reduces the signals to near-background levels. Because CM-H2DCFDA is a nonspecific radical detector, to identify the radical species induced by arsenic in mammalian cells, it is necessary to employ other, more definitive assays. The spin-trap probe Tempol-H is a hydroxylamine, which reacts with free radicals to form the more stable nitroxide Tempol, which can be detected and quantified by electron spin resonance (ESR) spectroscopy. Tempol-H readily penetrates plasma membranes and detects free radicals, particularly hydroxyl radicals and superoxide anions, with high sensitivity and specificity [36]. Using ESR in conjunction with superoxide dismutase and catalase to quench superoxide anions and hydrogen peroxide, respectively, has provided clear evidence that arsenite increases the levels of superoxide-driven hydroxyl radicals in AL cells [27]. The next

question then becomes: Are free radicals responsible for arsenic-induced mutagenesis? EVIDENCE THAT FREE RADICALS MEDIATE ARSENIC GENOTOXICITY

Effects of depleted intracellular glutathione level on arsenic mutagenesis Cellular nonprotein sulfhydryls (NPSHs) consist essentially of glutathione (f95%) and other low-molecular-weight aminothiols such as cysteine and cysteamine [37]. As cellular sulfhydryls have significant free radical scavenging abilities, the effect of glutathione depletion on the mutagenicity of arsenic has been examined using buthionine S,R-sulfoximine (BSO), a competitive inhibitor of the enzyme g-glutamyl cysteine synthetase used in the biosynthesis of glutathione. Treatment of cells with BSO (25 AM) for 24 h decreases the NPSH level to less than 2 nmol per 107 AL, a level that is less than 5% of the normal level [38,39]. Pretreatment of cells with BSO enhances both the cytotoxicity and mutagenicity of arsenic to similar extents. In contrast, pretreatment of cells with glutathione and cysteine protects mammalian cells against the toxic effects of arsenite [40]. Furthermore, low concentrations of arsenite have been shown to induce a transient increase in cellular glutathione levels in bovine vascular endothelial cells [41]. The upregulation is thought to be a ‘‘secondary’’ stress response directly regulated by the thiol reactivity of arsenite [42]. These findings are consistent with the observation that arsenite activates the transcription factor nuclear factor nh, which regulates response genes intrinsic to oxidative stress [43]. Effects of antioxidants on arsenic-induced genotoxicity A second, complementary approach that delineates the contribution of ROS in the genotoxicity of arsenite includes the use of the antioxidants, superoxide dismutase, and catalase. The deleterious effect of oxygen toxicity is normally held in check by the delicate balance between the rate of generation of these radicals and the rate of their removal by various antioxidant enzymes. Superoxide dismutase catalyzes the dismutation of superoxide anions, whereas catalase removes hydrogen peroxides and prevents the subsequent formation of hydroxyl radicals [44 for review]. Addition of either superoxide dismutase (400 U/ml) or catalase (5000 U/ ml) can partially suppress both the toxicity and the mutagenic potential of sodium arsenite [45]. In contrast, treatment with heat-inactivated catalase results in essentially no protection. The findings that catalase and SOD can reduce the mutagenic potential of arsenic are consistent with data obtained with other genotoxic endpoints. For example, using CHO cells and an x-ray-

Oxidative damage and arsenic mutagenesis

hypersensitive DNA repair-deficient mutant, XRS-5, Wang and Huang have shown that arsenite induces a dose-dependent increase in micronuclei that is blocked by exogenous catalase [46]. In addition, heme oxygenase, an oxidative stress protein, and peroxidase are induced by sodium arsenite in various human cell lines [47]. Furthermore, antioxidant enzymes such as SOD reduce the incidence of sister chromatid exchanges induced by arsenite in cultured human lymphocytes [48]. As 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 phagocytozed. The ability of SOD and catalase to suppress the mutagenicity of arsenic in mammalian cells is consistent with the ESR findings described above: that arsenic induces hydrogen peroxide as a precursor of hydroxyl radicals in AL cells [27]. Because hydrogen peroxide is freely diffusible between intracellular and extracellular space, addition of extracellular antioxidants is likely to reduce the intracellular oxidative stress induced by arsenite treatment and, subsequently, result in reduced genotoxic damage. The exact pathway, however, remains to be elucidated. Induction of oxidative DNA damage by arsenic in mammalian cells If generation of ROS is one of the major pathways for arsenic-mediated genotoxicity, then it should be expected to induce specific DNA lesions consistent with oxidative damage. One of the most common oxidative DNA lesions is 8-hydroxy-2V-deoxyguanosine (8OHdG). Use of a monoclonal antibody specific for 8OHdG with immunoperoxidase staining has provided evidence that arsenic treatment (4 Ag/ml for 24 h) increases the level of 8-OHdG in AL cells by more than 2-fold compared with that in nontreated controls [45]. Furthermore, addition of SOD and catalase reduces this increase by 75%. These findings provide additional proof that ROS mediate the genotoxic response in mammalian cells on treatment with sodium arsenite. Furthermore, there is evidence that 8-OHdG has also been detected in the skin of patients with arsenic-related Bowen’s disease [49] and in the liver of rats exposed to DMAV [50]. ROLE OF ROS IN MEDIATING THE GENOTOXICITY OF METHYLATED ARSENIC

As mentioned earlier in this article, inorganic arsenic exists in nature in two major forms: the trivalent As3+ and the pentavalent As5+ [8]. Trivalent arsenic is significantly more toxic and carcinogenic than the pentavalent form. In mammals, ingested inorganic arsenic is readily metabolized by methylation into the

577

pentavalent methylated species, namely, the monomethylarsonic acid (MMAV) and dimethylarsinic acid (DMAV) [51,52]. The methylation process has been considered for many years to be a critical detoxifying process [8,53]. These methylated species are much less toxic and genotoxic than the inorganic arsenic species [54,55] and are readily detectable in the urine of exposed human population. However, with improved analytical assays, there is recent evidence that the trivalent methylated species, monomethylarsonous acid (MMAIII) and dimethylarsinous acid (DMAIII), are often detected in human urine samples as well [56]. Furthermore, these trivalent methylated arsenic species are far more cytotoxic and DNA damaging than the inorganic metalloid [55,57]. Using the BX174 DNA nicking assay together with known inhibitors of ROS activities, there is evidence that both MMAIII and DMAIII exert their DNA-damaging effects through an intermediate involving ROS production [57]. Furthermore, studies using ESR and the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) suggest that these radical species to be hydroxyl radicals [57]. WHERE DO THE ROS COME FROM?

Although research over the past decade strongly endorsed the role of radical species in the pathogenicity of arsenic-induced diseases, the mechanism and pathway whereby these ROS are generated remain largely unknown. Figure 1 provides a working model depicting the

Fig. 1. Working model on the induction and pathways of ROS and NOS mediation of the genotoxicity of arsenic in mammalian cells. Trivalent sodium arsenite induces ROS formation within minutes of entering cells [27]. ROS and RNS induce an increase in intracellular oxidative stress that results in the induction of 8-OHdG and mutagenesis. Both mitochondrial membrane damage and the induction of lipid peroxidation contribute to the genotoxicity of arsenic. Stimulation of membrane-bound NADPH oxidase by arsenic enhances ROS production [62,85]. Damage to the mitochondrial membrane may result in the leakage of superoxide anions into the cytosol, and the subsequent production of peroxynitrite anions [82] exasperates the increase in the intracellular oxidative stress level.

578

T. K. HEI and M. FILIPIC

potential genotoxic mechanisms based on data published thus far. Arsenic has been shown to induce lipid peroxidation of membranes and the formation of lipid peroxide both in animal models [58,59] and in human subjects exposed to arsenic through contaminated drinking water [60,61]. Lipid peroxidation increases intracellular oxidative stress and promotes oxidative damage. As describe previously, there is evidence that arsenite increases hydroxyl radical production through a superoxide-driven hydrogen peroxide-mediated process [27]. The finding is consistent with the observation that arsenic activates the GTP-binding protein Rac1 and facilitates assembly of the RacI/NADPH oxidase complex to result in an increase in intracellular superoxide and hydrogen peroxide levels [62]. The presence of hydroxyl radicals then promotes oxidative DNA damage and mutagenesis as described above. If ROS do indeed mediate the genotoxicity of arsenite, why has arsenic not been reported to induce point mutations that are frequently being detected in bacterial assays and among some hprt mutants in mammalian cells exposed to ROS [63]? To begin with, the types of mutants recovered depend on several factors including the mutagen used, the conditions for mutant selection, DNA repair rate and fidelity, and, more importantly, the proficiency of the assay for selecting a full range of mutants including multilocus deletions. Most mutagenic studies using bacterial assays have reported that oxyradicals induce either base substitution or frame-shift mutations [64]. In mammalian cells, it has been reported that f64% of the hprt mutants induced by ROS have no discernable alterations in the gene (i.e., changes <30 bp which can be either a point mutation or no change) and the remaining 36% of the mutants have either partial or total deletions [63]. In contrast, using the AS52 system, which uses a bacterial gpt gene functionally integrated in the CHO genome, Hsie et al. have demonstrated convincingly that assays that allow the recovery of multilocus deletions will score predominately deletion mutations when challenged with ROS [65]. These findings are consistent with the induction of dicentric and chromosomal deletions in hydrogen peroxide-treated human fibroblasts [66]. Similarly, a significantly higher mutant yield has been demonstrated with the L5178 mouse lymphoma cell line, which is hemizygous at the thymidine kinase (TK)+/ locus when exposed to either ROS [67,68], arsenite [54], or agents whose biological effects are known to be mediated by ROS, including ionizing radiation and mitomycin C ([69] for review). The enhanced recovery of large deletions in the L5178 assay system is due to the presence of active copies of the linked essential genes on the homologous chromosome 11 [68]. In this regard, the result is consistent with the observation that 67% of the gpt mutants induced by

arsenic were total deletions [70]. This is in stark contrast with reports that arsenite has been determined to be nonmutagenic with either the bacterial reversion assay or at the hprt locus in CHO cells ([16], for review). Because mitochondria are the metabolic center of a cell, they are intimately involved in the production of ROS, mainly superoxides and hydrogen peroxides. Approximately 2 –4% of the oxygen consumed by mitochondria is converted to superoxides by the electron transport system [71]. Mitochondrial structures, on the other hand, are also very susceptible to oxidative damage through membrane lipid peroxidation, protein oxidation, and mitochondrial DNA damage. Evidence implicating mitochondria as a possible target of arsenic toxicity has been obtained mainly through the induction of apoptosis in various leukemia and cancer cell lines [72,73]. In this regard, it is paradoxical that arsenite, which has long been acknowledged to be a human carcinogen, has been used successfully in the treatment of promyelocytic leukemia recently [74]. There is evidence that arsenic induces apoptosis by its direct effect on mitochondrial transmembrane potential [75], which leads to cytochrome c release and the subsequent activation of the caspase pathway to promote apoptosis [62]. These observations are consistent with the finding that the antioxidant Nacetylcysteine completely suppresses arsenic-induced apoptosis in HeLa cells by preventing mitochondrial membrane depolarization [76]. Mitochondrial membrane damage may result in an increase in intracellular superoxide levels. There is evidence that arsenic upregulates intracellular nitric oxide concentration [77,78]. A possible consequence of mitochondrial damage is, therefore, the secondary production of peroxynitrites, a strong oxidant formed by the diffusion-controlled reaction of superoxides and nitric oxide. There is evidence that in tissues, peroxynitrite not only escapes the scavenging by most low-molecular-weight antioxidants [79], but can also activate stress responsive pathways and kinases of the src family to modulate the cellular signal transduction cascade [80,81]. As mitochondria constitute a primary locus for the intracellular formation and reactions of peroxynitrite, which has a much longer half-life compared with hydroxyl radicals and can readily diffuse across biomembranes [82], it is likely that multiple radical species are involved in the genotoxic response of arsenic. In this regard, the role of peroxynitrite in the genotoxicity of arsenic is supported by the recent data that NG-methyl-L-arginine (L-NMMA), a competitive inhibitor of the enzyme nitric oxide synthase essential for the in vivo biosynthesis of nitric oxide, partially blocked the induction of CD59 mutants in arsenite-treated AL cells (Hei et al., unpublished data). These findings are consistent with the observation that nitric oxide, an upstream molecule in the biosynthesis of

Oxidative damage and arsenic mutagenesis

peroxynitrite, has been implicated in endothelial cell damage associated with arsenic exposure [83]. In addition to mitochondrial contribution, there is evidence based on cell-free assays that ferritin may contribute to oxidative stress induced by arsenic [84]. The ability of both inorganic and methylated arsenic species to mobilize iron from horse spleen ferritin has been evaluated. Both DMAV and DMAIII induce the release of iron followed by the formation of ROS through a Fenton-like reaction. Furthermore, the release of iron is more pronounced under anaerobic conditions. The specific role of ferritin in mediating ROS production by arsenic in mammalian cells has yet to be determined. CONCLUSION

Arsenic is an important environmental carcinogen and its contamination of drinking water is a serious environmental calamity worldwide because of the millions of people at risk, particularly in developing countries such as West Bengal in India and Bangladesh. The extent of human suffering bestowed by arsenic exposure simply cannot be overstated. Although the precise mechanism whereby arsenic induces human cancer is far from clear, it is likely to be multifactorial in nature and depends on the type of tumor. For arsenic-induced skin cancer, the confounding effect of UV light is highly relevant. Similarly, the interactions of radon, tobacco smoke, and arsenic in lung cancer incidence among underground miners have also been examined. Other biological effects of arsenic, including its ability to affect levels of DNA methylation, gene amplification, apoptosis, and DNA repair, no doubt play a vital, although, perhaps, secondary role in its genotoxicity and carcinogenicity. However, evidence in support of the role of oxidative damage in arsenic genotoxicity/carcinogenesis is overwhelming. In light of the observations that modulators of cellular redox status have a profound effect on the mutagenicity of arsenic, it is not surprising that clinical trials are currently underway in Bangladesh to evaluate the protective effects of selenium and vitamin E in preventing arsenic-induced keratosis and other related diseases including cancers.

[3]

[4] [5]

[6] [7]

[8] [9]

[10]

[11] [12] [13]

[14]

[15]

[16] [17] [18]

Acknowledgments — The authors thank Dr. Mohammad Athar, Dr. Charles Waldren, and Dr. Vladimir Ivanov for helpful discussions. Work was supported by National Institutes of Health Grants ES05786 and ES11804, Superfund Grant P42 ES10349, and Environmental Center Grant ES09089.

[19]

[20] REFERENCES

[1] Leonard, A.; Lauwerys, R. R. Carcinogenicity, teratogenicity, and mutagenicity of arsenic. Mutat. Res. 75:49 – 62; 1980. [2] Chapell, W. R.; Beck, B. D.; Brown, K. G.; Chaney, R.; Cothern,

[21]

579

R.; Irgolic, K. L.; North, D. W.; Thornton, I.; Tsongas, T. A. Inorganic arsenic: a need and an opportunity to improve risk assessment. Environ. Health Perspect. 105:1060 – 1067; 1997. U.S. EPA, Soil Screening Guidance Technical Background Document. Office of Solid Waste and Emergency Response. EPA/540/ R-95/128. Washington, DC: U.S. Environmental Protection Agency; 1996. Barrett, J. C.; Lamb, P. W.; Wang, T. C.; Lee, T. C. Mechanisms of arsenic induced cell transformation. Biol. Trace Element Res. 21:421 – 429; 1989. Chowdhury, U. K.; Biswas, B. K.; Chowdhury, T. R.; Samanta, G.; Mandal, B. K.; Basu, G. C.; Chanda, C. R.; Lodh, D.; Saha, K. C.; Mukherjee, S. K.; Roy, S.; Kabir, S.; Quamruzzaman, Q.; Chakraborti, D. Ground water arsenic contamination in Bangladesh and West Bengal, India. Environ. Health Perspect. 108: 393 – 397; 2000. Nickson, R.; McArthur, J.; Burgess, W.; Ahmed, K. M.; Ravenscroft, P.; Rahman, M. Arsenic poisoning of Bangladesh groundwater. Nature 395:338; 1998. Dhar, R. K.; Biswas, B. K.; Samanta, G.; Mandal, B. K.; Chakraborti, D.; Roy, S.; Jafar, A.; Islam, A.; Ara, G.; Kabir, S.; Khan, A. W.; Ahmed, S. A.; Hadi, S. A. Groundwater arsenic calamity in Bangladesh. Cur. Sci. 73:48 – 59; 1997. Chan, P. C.; Huff, J. Arsenic carcinogenesis in animals and in humans: mechanistic, experimental and epidemiological evidence. Environ. Carcinogen. Ecotoxicol. Rev. 15:83 – 122; 1997. Warner, M. L.; Moore, L. E.; Smith, M. T.; Kalman, D. A.; Fanning, E.; Smith, A. H. Increased micronuclei in exofoliated bladder cells of individuals who chronically ingested arseniccontaminated water in Nevada. Cancer Epidemiol. Biomarkers Prevent. 3:583 – 590; 1994. Xuan, X. Z.; Lubin, J. H.; Li, J. Y.; Yang, L. F.; Lou, A. S.; Lan, Y.; Wang, J. Z.; Blot, W. A cohort study in Southern China of tin miners exposed to radon and radon decay products. Health Phys. 64:120 – 131; 1993. Monograph on the Evaluation of the Carcinogenic Risk of Chemicals to Humans, Suppl. 4. Lyon: IARC; 1982:50-51. Huff, J.; Chan, P.; Nyska, A. Is the human carcinogen arsenic carcinogenic to laboratory animals. Toxicol. Sci. 55:17 – 23; 2000. Wei, M.; Wanibuchi, H.; Morimura, K.; Iwai, S.; Yoshida, K.; Endo, G.; Nakae, D.; Fukushima, S. Carcinogenicity of dimethylarsinic acid in male F344 rats and genetic alterations in induced urinary bladder tumors. Carcinogenesis 23:1387 – 1397; 2001. Yamanaka, K.; Takabayashi, F.; Mizoi, M.; An, Y.; Hasegawa, A.; Okada, S. Oral exposure of dimethylarsinic acid, a main metabolite of inorganic arsenic, in mice leads to an increase in 8-oxo-2Vdeoxyguanosine level, specially in the target organs for arsenic carcinogenesis. Biochem. Biophys. Res. Commun. 287:66 – 70; 2001. Petrick, J. S.; Ayala-Fierro, F.; Cullen, W. R.; Carter, D. E.; Aposhian, H. V. Monomethylarsonous acid is more toxic than arsenite in Chang human hepatocytes. Toxicol. Appl. Pharmacol. 163:203 – 207; 2000. Wang, Z.; Rossman, T. G. The carcinogenicity of arsenic. In: Chang, T. ed. Toxicology of Metals. Boca Raton FL: CRC Press; 1996:221-229. Lee, T. C.; Ko, J. L.; Jan, K. Y. Differential cytotoxicity of sodium arsenite in human fibroblasts and CHO cells. Toxicology 56:289 – 299; 1989. Simeonova, P. P.; Luster, M. I. Mechanisms of arsenic carcinogenicity: genetic or epigenetic mechanisms? J. Environ. Pathol. Toxicol. Oncol. 19:281 – 286; 2000. Lee, T. C.; Oshimura, M.; Barrett, J. C. Comparison of arsenic induced cell transformation, cytotoxicity, mutation and cytogenetic effects in Syrian hamster embryo cells in culture. Carcinogenesis 6:1421 – 1426; 1985. Landolph, J. Molecular and cellular mechanisms of transformation of C3H 1OT1/2 and diploid human fibroblasts by unique carcinogenic, non-mutagenic metal compounds. Biol. Trace Element Res. 21:459 – 467; 1989. Rossman, T. G.; Stone, D.; Molina, M.; Troll, W. Absence of

580

[22]

[23] [24] [25]

[26] [27]

[28]

[29] [30]

[31]

[32]

[33]

[34]

[35]

[36] [37] [38]

[39]

[40]

T. K. HEI and M. FILIPIC arsenite mutagenicity in E. coli and Chinese hamster cells. Environ. Mutagen. 2:371 – 379; 1980. Zhao, C. Q.; Young, M. R.; Diwan, B. A.; Coogan, T. P.; Waalkes, M. P. Association of arsenic induced malignant transformation with DNA methylation and aberrant gene expression. Proc. Natl. Acad. Sci. USA 94:10907 – 10912; 1997. Lynn, S.; Lai, H. T.; Gurr, G. R.; Jan, K. Y. Arsenite retards DNA break rejoining by inhibiting DNA ligation. Mutagenesis 12:353 – 358; 1997. Nakamuro, K.; Sayato, Y. Comparative studies of chromosomal aberrations induced by trivalent and pentavalent arsenic. Mutat. Res. 88:73 – 80; 1981. Kochhar, T. S.; Howard, W.; Hoffman, S.; Brammer-Carleton, L. Effect of trivalent and pentavalent arsenic in causing chromosomal alterations in cultured Chinese hamster ovary cells. Toxicol. Lett. 84:37 – 42; 1996. Hei, T. K.; Liu, S. X.; Waldren, C. A. Mutagenicity of arsenic in mammalian cells: role of reactive oxygen species. Proc. Natl. Acad. Sci. USA 95:8103 – 8107; 1998. Liu, S. X.; Athar, M.; Lippai, I.; Waldren, C. A.; Hei, T. K. Induction of oxyradicals by arsenic: implications for mechanisms of genotoxicity. Proc. Natl. Acad. Sci. USA 98:1643 – 1648; 2001. Harrnongton-Brock, K.; Cabrera, M.; Collard, D. D.; Doerr, C. L.; McConnell, R.; Moore, M. M.; Sandoval, H.; Fuscoe, J. C. Effects of arsenic exposure on the frequency of HPRT mutant lymphocytes in a population of copper roasters in Antofagasta, Chile: a pilot study. Mutat. Res. 431:247 – 257; 1999. Thilly, W. G. Dead cells don’t form mutant colonies: a serious source of bias in mutation assays. Environ. Mutagen. 7:255 – 258; 1985. Yamada, Y.; Park, M. S.; Okinaka, R. T.; Chen, D. J. Molecular analysis and comparison of radiation-induced large deletions of the HPRT locus in primary human skin fibroblasts. Radiat. Res. 145:481 – 490; 1996. Kakar, S. S.; Huang, W. H.; Askari, A. Properties of the Na+, K+ and ATP binding sites of the ouabain complexed-ATPase: implications for the mechanism of ouabain action. Prog. Clin. Biol. Res. 268:211 – 218; 1988. Wiencke, J. K.; Yager, J. W.; Varkonyi, A.; Hultner, M.; Lutze, L. H. Study of arsenic mutagenesis using the plasmid shuttle vector pZ189 propagated in DNA repair proficient human cells. Mutat. Res. 386:335 – 344; 1997. Helleday, T.; Nilsson, R.; Jenssen, D. Arsenic(III) and heavy metal ions induce intrachromosomal homologous recombination in the hprt gene of V79 Chinese hamster ovary cells. Environ. Mol. Mutagen. 35:114 – 122; 2000. LeBel, C. P.; Ischiropoulos, H.; Bondy, S. C. Evaluation of the probe 2V,7V-dichlorofluorescein as an indicator of reactive oxygen species formation and oxidative stress. Chem. Res. Toxicol. 5: 227 – 231; 1992. Szejda, P.; Parce, J’W.; Seeds, M. S.; Bass, D. A. Flow cytometric quantification of oxidative product formation by polymorphonuclear leukocytes during phagocytosis. J. Immunol. 133: 3303 – 3307; 1984. Hahn, S. M.; Krishna, M. C.; DeLuca, A. M.; Coffin, D.; Mitchell, J. B. Evaluation of the hydroxylamine Tempol-H as an in vivo radioprotector. Free Radic. Biol. Med. 28:953 – 958; 2000. Meister, A.; Anderson, M. E. Glutathione. Annu. Rev. Biochem. 52:711 – 760; 1983. Xu, A.; Wu, L. J.; Santella, R.; Hei, T. K. Role of reactive oxygen species in the mutagenicity and DNA damage induced by crocidolite fibers in mammalian cells. Cancer Res. 59:5615 – 5624; 1999. Hei, T. K.; Geard, C. R.; Hall, E. J. Effects of cellular non-protein sulfhydryl depletion in radiation induced oncogenic transformation and genotoxicity in mouse 10T1/2 cells. Int. J. Radiat. Oncol. Biol. Phys. 10:1255 – 1259; 1984. Liu, L.; Trimarchi, J. R.; Navarro, P.; Blasco, M. A.; Keefe, D. L. Oxidative stress contributes to arsenic induced telomere attrition, chromosomal instability and apoptosis. J. Biol. Chem. 278: 31998 – 32004; 2003.

[41] Deneke, S. M. Induction of cysteine transport in bovine pulmonary artery endothelial cells by sodium arsenite. Biochim. Biophys. Acta 1109:127 – 131; 1992. [42] Engel, R. R.; Hopenhayn-Rich, C.; Receveur, O.; Smith, A. H. Vascular effects of chronic arsenic exposure. Epidemiol. Res. 16:184 – 208; 1994. [43] Barchowsky, A.; Dudek, E. J.; Treadwell, M. D.; Wetterhahn, K. E. Arsenic induces oxidant stress and NF-kappa B activation in cultured aortic endothelial cells. Free Radic. Biol. Med. 21:783 – 790; 1996. [44] Imlay, J. A.; Linn, S. DNA damage and oxygen radical toxicity. Science 240:1302 – 1309; 1988. [45] Kessel, M.; Liu, S. X.; Xu, A.; Santella, R.; Hei, T. K. Arsenic induces oxidative DNA damage in mammalian cells. Mol. Cell. Biochem. 234:301 – 308; 2002. [46] Wang, T. S.; Huang, H. Active oxygen species are involved in the induction of micronuclei by arsenite in XRS-5 cells. Mutagenesis 9:253 – 257; 1994. [47] Applegate, L. A.; Luscher, P.; Tyrrell, R. M. Induction of heme oxygenase: a general response of oxidant stress in cultured mammalian cells. Cancer Res. 51:974 – 978; 1991. [48] Nordenson, I.; Beckman, L. Is the genotoxic effect of arsenic mediated by oxygen free radicals. Hum. Hered. 41:71 – 73; 1991. [49] Matsui, M.; Nishigori, C.; Toyokuni, S.; Takada, J.; Akaboshi, M.; Ishihawa, M.; Imamura, S.; Miyachi, Y. The role of oxidative DNA damage in human arsenic carcinogenesis: detection of 8OhdG in arsenic related Bowen’s disease. J. Invest. Dermatol. 113:26 – 31; 1999. [50] Wanibuchi, H.; Hori, T.; Meenakshi, V.; Ichihara, T.; Yamamoto, S.; Yano, Y.; Otani, S.; Nakae, D.; Konishi, Y.; Fukushima, S. Promotion of rat hepatocarcino-genesis by dimethylarsinic acid: association with elevated ornithine decarboxylase activity and formation of 8-hydroxydeoxyguanosine in the liver. Jpn. J. Cancer Res. 88:1149 – 1154; 1997. [51] Le, X. C.; Lu, X.; Ma, M.; Cullen, W. R.; Aposhian, H. V.; Zheng, B. Speciation of key arsenic metabolic intermediates in human urine. Anal. Chem. 72:5172 – 5177; 2000. [52] Styblo, M.; Delnomdedieu, M.; Thomas, D. J. Mono- and dimethylation of arsenic in rat liver cytosol in vitro. Chem. Biol. Interact. 99:147 – 164; 1996. [53] Wagner, S. L.; Weswig, P. Arsenic in blood and urine of forest workers as indices of exposure to cacodylic acid. Arch. Environ. Health 28:77 – 79; 1974. [54] Moore, M. M.; Harrington-Brock, K.; Doerr, C. L. Relative genotoxic potency of arsenic and its methylated metabolites. Mutat. Res. 386:279 – 290; 1997. [55] Andrewes, P.; Kitchin, K. T.; Wallace, K. Dimethyarsine and trimethylarsine are potent genotoxins in vitro. Chem. Res. Toxicol. 16:994 – 1003; 2003. [56] Le, X. C.; Ma, M.; Cullen, W. R.; Aposhian, H. V.; Lu, X.; Zheng, B. Determination of monomethylarsonous acid, a key arsenic methylation intermediate in human urine. Environ. Health Perspect. 108:1015 – 1018; 2000. [57] Nesnow, S.; Roop, B. C.; Lambert, G.; Kadiiska, M.; Mason, R.; Cullen, W. R.; Mass, M. J. DNA damage induced by methylated trivalent arsenicals is mediated by reactive oxygen species. Chem. Res. Toxicol. 15:1627 – 1634; 2002. [58] Maiti, S.; Chatterjee, A. K. Differential response of cellular antioxidant mechanism of liver and kidney to arsenic exposure and its relation to dietary protein deficiency. Environ. Toxicol. Pharmacol. 8:227 – 235; 2000. [59] Ramanathan, K.; Shila, S.; Kumaran, S.; Panneerselvam, C. Ascorbic acid and alpha-tocopherol as potent modulators on arsenic induced toxicity in mitochondria. J. Nutr. Biochem. 14:416 – 420; 2003. [60] Pi, J.; Yamauchi, H.; Kumagai, Y.; Sun, G.; Yoshida, T.; Aikawa, H.; Hopenhayn-Rich, C.; Shimojo, N. Evidence for induction of oxidative stress caused by chronic exposure of Chinese residents to arsenic contained in drinking water. Environ. Health Perspect. 110:331 – 336; 2002. [61] Lin, T. H.; Huang, Y. L.; Tseng, W. C. Arsenic and lipid perox-

Oxidative damage and arsenic mutagenesis

[62] [63] [64] [65]

[66]

[67] [68]

[69] [70] [71] [72] [73]

[74]

idation in patients with blackfoot disease. Bull. Environ. Contam. Toxicol. 54:488 – 493; 1995. Smith, K. R.; Klei, L. R.; Barchowsky, A. Arsenite stimulates plasma membrane NADPH oxidase in vascular endothelial cells. Am. J. Physiol. Lung Cell Mol. Physiol. 280:442 – 449; 2001. Oller, A. R.; Thilly, W. G. Mutation spectra in human B-cells: spontaneous, oxygen and hydrogen peroxide induced mutations at the hprt gene. J. Mol. Biol. 228:813 – 826; 1992. Moody, C. S.; Hassan, H. M. Mutagenicity of oxygen free radicals. Proc. Natl. Acad. Sci. USA 79:2855 – 2859; 1982. Hsie, A. W.; Xu, W.; Yu, Y.; Sognier, M. A.; Hrelia, P. Molecular analysis of reactive oxygen species induced mammalian gene mutation. Teratogen. Carcinogen. Mutagen. 10:115 – 124; 1990. Oya, Y.; Yamamoto, K.; Tonomura, A. The biological activity of hydrogen peroxide: induction of chromosome type aberrations susceptible to inhibition by scavengers of hydroxyl radicals in human embryonic fibroblasts. Mutat. Res. 172:245 – 253; 1986. Kruszewski, M.; Szumei, I. DNA damage induced by hydrogen peroxide treatment at 4 degree and 37 degrees C in murine lymphoma L5178Y sublines. Acta Biochem. Pol. 41:120 – 121; 1994. Evans, H. H.; Mencl, J.; Jorng, M. F.; Ricanati, M.; Sanches, C.; Hozier, J. Locus specificity in the mutability of L5178 mouse lymphoma cells: the role of multilocus lesions. Proc. Natl. Acad. Sci. USA 83:4379 – 4383; 1986. Evans, H. H. Failla Memorial Lecture: the prevalence of multilocus lesions in radiation induced mutants. Radiat. Res. 137: 131 – 144; 1994. Meng, Z.; Hsie, A. W. Polymerase chain reaction based deletion analysis of spontaneous and arsenite enhanced gpt mutants in CHO-AS52 cells. Mutat. Res. 356:255 – 259; 1996. Chance, N.; Sies, H.; Boveris, A. Hydroperoxide metabolism in mammalian organs. Physiol. Rev. 59:527 – 605; 1979. Miller, W. H., Jr.; Hyman, M. S.; Lee, J. S.; Singer, J.; Waxman, S. Mechanisms of action of arsenic trioxide. Cancer Res. 62: 3893 – 3903; 2002. Shen, Z. Y.; Shen, W. Y.; Chen, M. H.; Shen, J.; Cai, W. J.; Zeng, Y. The alteration of mitochondria is an early event of arsenic trioxide induced apoptosis in esophageal carcinoma cells. Int. J. Mol. Med. 9:385 – 390; 2000. Shen, Z. X.; Chen, G. Q.; Ni, J. H.; Li, X. S.; Xiong, S. M.; Qiu, Q. Y.; Zhu, J.; Tang, W.; Sun, G. L.; Yang, K. Q.; Chen, Y.; Zhou, L.; Fang, Z. W.; Wang, Y. T.; Ma, J.; Zhang, P.; Zhang, T. D.;

[75]

[76]

[77]

[78]

[79] [80] [81] [82] [83]

[84] [85]

581

Chen, S. J.; Chen, Z.; Wang, Z. Y. Use of arsenic trioxide in the treatment of acute promyelocytic leukemia: II. Clinical efficacy and pharmacokinetics in relapsed patients. Blood 89:3354 – 3360; 1997. Larochette, N.; Decaudin, D.; Jacotot, E.; Brenner, C.; Marzo, I.; Susin, S. A.; Zamzami, N.; Xie, Z.; Reed, J.; Kroemer, G. Arsenic induces apoptosis via a direct effect on the mitochondrial permeability transition pore. Exp. Cell Res. 249:413 – 421; 1999. Woo, S. H.; Park, I. C.; Park, M. J.; Lee, H. C.; Lee, S. J.; Chun, U. L.; Lee, S. H.; Hong, S.; Rhee, C. H. Arsenic trioxide induces apoptosis through a reactive oxygen species dependent pathway and loss of mitochondrial membrane potential in HeLa cells. Int. J. Oncol. 21:57 – 63; 2002. Kao, Y. H.; Yu, C. L.; Chang, L. W.; Yu, H. S. Low concentrations of arsenic induce vascular endothelial growth factor and nitric oxide release and stimulate angiogenesis in vitro. Chem. Res. Toxicol. 16:460 – 468; 2003. Yuan, S. S.; Hou, M. F.; Chang, H. L.; Chan, T. F.; Wu, Y. H.; Wu, Y. C.; Su, J. H. Arsenic induced nitric oxide generation is cell cycle dependent and aberrant in NBS cells. Toxicol. In Vitro 17:139 – 143; 2003. Lymer, S.; Hurst, J. K. Rapid reaction between peroxynitrite ion and carbon dioxide: implications for biological activity. J. Am. Chem. Soc. 117:8867 – 8868; 1995. Klotz, L.; Schroeder, P.; Sies, H. Peroxynitrite signaling: receptor tyrosine kinases and activation of stress responsive pathways. Free Radic. Biol. Med. 33:737 – 743; 2002. Minetti, M.; Mallozzi, C.; Michela Di Stasi, A. M. Peroxynitrite activates kinases of the src family and upregulates tyrosine phosphorylation signaling. Free Radic. Biol. Med. 33:744 – 754; 2002. Radi, R.; Peluffo, G.; Alvarez, M. N.; Naviliat, M.; Cayota, A. Unraveling peroxynitrite formation in biological systems. Free Radic. Biol. Med. 30:463 – 488; 2001. Lee, M. Y.; Jung, B. I.; Seung, M. C.; Bae, O. N.; Lee, J. Y.; Park, J. D.; Yang, J. S.; Lee, H.; Chung, J. H. Arsenic induced dysfunction in relaxation of blood vessels. Environ. Health Perspect. 111:513 – 517; 2003. Ahmad, S.; Kitchin, K. T.; Cullen, W. R. Arsenic species that cause release of iron from ferritin and generation of activated oxygen. Arch. Biochem. Biophys. 382:195 – 202; 2000. Li, J. M.; Shah, A. M. ROS generation by non-phagocytic NADPH oxidase: potential relevance in diabetic nephropathy. J. Am. Soc. Nephrol. 14:S221 – S226; 2003.