Arsenite and its Mono- and Dimethylated Trivalent Metabolites

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Arsenite and its Mono- and Dimethylated Trivalent Metabolites Enhance the Formation of Benzo[a]pyrene Diol Epoxide-DNA Adducts in Xeroderma Pigmentosum Complementation Group A Cells Shengwen Shen,† Jane Lee,‡ William R. Cullen,§ X. Chris Le,*,† and Michael Weinfeld*,‡ Department of Laboratory Medicine and Pathology, 10-102 Clinical Sciences Building, UniVersity of Alberta, Edmonton, Alberta, Canada, T6G 2G3, Department of Oncology, UniVersity of Alberta, Cross Cancer Institute, 11560 UniVersity AVenue, Edmonton, Alberta, Canada, T6G 1Z2, and Department of Chemistry, UniVersity of British Columbia, 2036 Main Mall, VancouVer, British Columbia, Canada, V6T 1Z1 ReceiVed September 5, 2008

Recently, inorganic arsenite (iAsIII) and its mono- and dimethylated metabolites have been examined for their interference with the formation and repair of benzo[a]pyrene diol epoxide (BPDE)-induced DNA adducts in human cells (Schwerdtle, T., Walter, I., and Hartwig, A. (2003) DNA Repair 2, 1449-1463). iAsIII and monomethylarsonous acid (MMAIII) were found to be able to enhance the formation of BPDE-DNA adducts, whereas dimethylarsinous acid (DMAIII) had no enhancing effect at all. The anomaly manifested by DMAIII prompted us to further investigate the effects of the three trivalent arsenic species on the formation of BPDE-DNA adducts. Use of a nucleotide excision repair (NER)-deficient Xeroderma pigmentosum complementation group A cell line (GM04312C) allowed us to dissect DNA damage induction from DNA repair and to examine the effects of arsenic on the formation of BPDE-DNA adducts only. At concentrations comparable to those used in the study by Schwerdtle et al., we found that each of the three trivalent arsenic species was able to enhance the formation of BPDE-DNA adducts with the potency in a descending order of MMAIII > DMAIII > iAsIII, which correlates well with their cytotoxicities. Similar to iAsIII, DMAIII modulation of reduced glutathione (GSH) or total glutathione S-transferase (GST) activity could not account for its enhancing effect on DNA adduct formation. Additionally, the enhancing effects elicited by the trivalent arsenic species were demonstrated to be highly time-dependent. Thus, although our study made use of short-term assays with relatively high doses, our data may have meaningful implications for carcinogenesis induced by chronic exposure to arsenic at low doses encountered environmentally. Introduction Epidemiological studies have revealed that chronic exposure to relatively high concentrations of inorganic arsenic leads to increased risk of skin, lung, bladder, and other internal cancers (1-3). However, the mechanism of arsenic carcinogenicity still remains to be fully elucidated, due largely to the general lack of animal models for studying arsenic carcinogenesis. The metabolism of arsenic plays a pivotal role in its toxicological effects, including its carcinogenicity. Biomethylation is the major metabolic pathway for inorganic arsenic (iAs)1 in humans and in most animal species (4, 5). A classical reduction and oxidative methylation pathway has been proposed * To whom correspondence should be addressed. E-mail: michaelw@ cancerboard.ab.ca (M.W.), [email protected] (X.C.L.). † Department of Laboratory Medicine and Pathology, University of Alberta. ‡ Department of Oncology, University of Alberta. § University of British Columbia. 1 Abbreviations: iAsIII, arsenite; iAsV, arsenate; ATP, adenosine-5′triphosphate; BaP, benzo[a]pyrene; BPDE, benzo[a]pyrene diol epoxide; CDNB, 1-chloro-2,4-dinitrobenzene; CE-LIF, capillary electrophoresis laserinduced fluorescence; DMAIII, dimethylarsinous acid; DMAV, dimethylarsinic acid; DMSO, dimethyl sulfoxide; GSH, reduced glutathione; GST, glutathione S-transferase; MMAIII, monomethylarsonous acid; MMAV, monomethylarsonic acid; MRP2, multidrug resistance protein 2; NER, nucleotide excision repair; PBS, phosphate-buffered saline; ROS, reactive oxygen species; THF, tetrahydrofuran; XP, Xeroderma pigmentosum.

by Challenger (6) and extensively studied thereafter (7-10). It involves two types of reactions: (i) the reduction of pentavalent arsenicals to trivalent species and (ii) the stepwise methylation of trivalent arsenicals to pentavalent methylated species. The scheme is as follows: iAsV f iAsIII f monomethylarsonic acid (MMAV) f monomethylarsonous acid (MMAIII) f dimethylarsinic acid (DMAV) f dimethylarsinous acid (DMAIII) f trimethylarsine oxide (TMAOV). Because MMAV, DMAV, and TMAOV, the pentavalent methylated metabolites, are less acutely toxic than iAs and more readily excreted in the urine, the biomethylation of iAs has been historically considered as a mechanism of detoxification (4, 11). However, this view did not take into account the formation of the intermediate metabolites, MMAIII and DMAIII, which have been confirmed to be present in vitro (12, 13), in animal tissues (14-16), and in the urine of humans exposed to iAs in drinking water (17-21). Notably, both MMAIII and DMAIII have been shown to be more active than iAs for enzyme inhibition (22-24), cytotoxicity, and genotoxicity (25-31). Although much of the evidence obtained so far has suggested that arsenic (mainly iAsIII, iAsV, MMAV, and DMAV) is only a cancer promoter, or a cocarcinogen (32-43), there are a few successful animal models showing that iAsIII can act as a transplacental carcinogen (44, 45), and DMAV as a complete carcinogen (46). iAsV, MMAV, and TMAOV were also shown to be carcinogenic (47-49). The fact that MMAIII and DMAIII are highly reactive

10.1021/tx800335p CCC: $40.75  2009 American Chemical Society Published on Web 01/15/2009

As(III) Compounds Enhance DNA Adduct Formation

and exert much higher toxicity than their pentavalent counterparts has precipitated the realization of their potential contributions to the tumorigenic action induced by MMAV or DMAV. Cohen et al. (50) even proposed that it was DMAIII formed in vivo from DMAV, instead of DMAV itself, that played a role in DMAV-induced bladder carcinogenesis observed in their rat model. Besides MMAIII and DMAIII, dimethylarsine and trimethylarsine have also been suggested as likely candidates for the causal carcinogenic species of arsenic because they showed more potent genotoxicity than did MMAIII and DMAIII and are possibly generated in humans exposed to arsenic as well as in animals (41, 51). Examination of the carcinogenicity of trivalent methylated species has attracted considerable attention. Currently, animal studies on the carcinogenicity of trivalent methylated arsenic species are emerging. MMAIII was shown to be carcinogenic in K6/ODC and C57BL/6J transgenic mice (52, 53), whereas DMAIII was shown to be cocarcinogenic in mice (54). It is generally believed that arsenic species, including MMAIII and DMAIII, are not mutagens and they do not react with DNA directly (29, 42). However, it has been well documented that iAs enhanced persistence of DNA damage induced by UV light, benzo[a]pyrene (BaP), X-rays, alkylating agents, and DNA cross-linking compounds in cultured mammalian cells and potentiated mutagenicity of the damaging agents (42, 55). Inhibition of DNA repair and induction of oxidative stress have been implicated in the comutagenicity of arsenic (39-42, 56). Binding of trivalent arsenic to critical thiol groups on DNA repair-related enzymes may be responsible for the DNA repair inhibition by arsenic (41, 57-62). On the other hand, DNA adduct formation (covalent DNA modifications by exogenous or endogenous reactive chemical agents) is the preceding step to DNA mutation and has been shown to be necessary for tumorigenesis (63, 64). The effect of arsenic on the formation of DNA adducts induced by BaP or its reactive metabolite benzo[a]pyrene diol epoxide (BPDE) has also been investigated in a few studies. However, the results generated in those studies were contradictory. Maier et al. (65) showed that exposure of mouse hepatoma Hepa-1 cells to low concentrations of iAsIII increased BaP-induced BPDE-DNA adduct levels by as much as 18-fold although it did not alter the cellular adduct removal kinetics. An in vivo study from the same research group reported that iAsIII cotreatment increased the average BPDE-DNA adduct levels in both mouse lungs and skin, with the increase (∼2-fold) in the lungs being statistically significant (P ) 0.038) (66). In another in vivo study using mice, no additional stable BPDE-DNA adducts were observed in the group exposed to iAsIII plus BaP than in the group exposed to BaP alone (67). However, in a study with Sprague-Dawley rats, iAsIII was shown to decrease BaP-induced BPDE-DNA adduct formation (68). Recently, five arsenic speciessiAsIII, MMAIII, DMAIII, MMAV, and DMAVshave been examined by Schwerdtle et al. regarding their effects on the formation and repair of BPDE-DNA adducts in A549 human lung cancer cells (69). They observed that iAsIII and MMAIII increased the BPDE-DNA adduct formation starting at 25 and 2.5 µM and yielding 40% and 60% more adducts at 75 and 7.5 µM, respectively. Surprisingly, DMAIII had no enhancing effect on the formation of BPD-DNA adducts in the concentration range under investigation (0-7.5 µM). This result appears counterintuitive because, as intermediates in the metabolic methylation process of inorganic arsenic in human cells (17-21), both DMAIII and MMAIII are more potent than inorganic arsenic for enzyme inhibition (22-24),

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cytotoxicity, and genotoxicity (25-31). It is also noteworthy that DMAIII was demonstrated to be more toxic than MMAIII for A549 cells in their study (69). Therefore, we felt it necessary to reexamine the effects of the three trivalent arsenic compounds on the formation of BPDE-DNA adducts in order to reach a more comprehensive understanding of iAsIII and its two trivalent methylated metabolites in this regard. In the present study, a nucleotide excision repair (NER)deficient SV40-transformed Xeroderma pigmentosum complementation group A (XPA) human skin fibroblast (GM04312C) cell line was chosen so that changes in adduct levels could unambiguously be attributed to the effects of arsenic on adduct formation. NER is responsible for removing bulky lesions such as BPDE-DNA adducts from the genome. Abrogation of the p53 function by SV40 transformation could further diminish DNA repair (70). Although the methylating capacity of this cell line has not been determined, it is expected to be insignificant according to reports on the methylation of fibroblast cell lines in general (42, 71). The results of this study indicate that MMAIII and DMAIII are more potent than iAsIII in enhancing the formation of BPDE-DNA adducts and the enhancing effect was shown to be time-dependent.

Materials and Methods Caution: Inorganic arsenic and B[a]P are classified as human carcinogens. The following chemicals should be considered as hazardous and handled with care: iAsIII, methylarsine oxide, dimethyliodoarsine, and BPDE. Chemicals. Racemic anti-BPDE was supplied by the NCI Chemical Carcinogen Repository (Midwest Research Institute, Kansas City, MO). To avoid hydrolysis of the epoxide, a stock solution of BPDE was always prepared fresh by dissolving BPDE in anhydrous tetrahydrofuran (THF) (>99.9% purity, SigmaAldrich, St. Louis, MO) immediately before use. iAsIII (99.4% purity) was obtained as an arsenic atomic absorption standard solution from Aldrich (Milwaukee, WI) and used as a stock solution with a concentration of 13.3 mM. Methylarsine oxide (CH3AsIIIO) and dimethyliodoarsine ((CH3)2AsIIII) were used as the precursors to MMAIII and DMAIII (30, 31), respectively. The stock solutions of MMAIII and DMAIII were prepared by dissolving the precursors in deionized water to a final concentration of 10 mM. When preparing the stock solution of DMAIII, three volumes of dimethyl sulfoxide (DMSO) were used to dissolve (CH3)2AsIIII before adding deionized water. To minimize oxidation of the trivalent methylated arsenic species, their stock solutions were prepared shortly before use. Cells and Cell Cultures. The GM04312C (SV-40 transformed XPA human skin fibroblast) cell line was obtained from the NIGMS Human Genetics Cell Repository (Camden, NJ) and demonstrated to be lacking in global genomic repair of BPDE-DNA adducts in our previous study (72). Cells were cultivated in Dulbecco’s modified Eagle’s medium/nutrient mixture F-12 (DMEM/F12) (1:1 ratio) (Gibco BRL, Rockville, MD) supplemented with 10% fetal bovine serum, penicillin (50 U/mL), streptomycin (50 mg/mL), L-glutamine (2 mM), nonessential amino acids (0.1 mM), and sodium pyruvate (1 mM). The cells were seeded in 100 mm dishes at a density of 1 × 106 cells per dish and maintained at 37 °C in humidified air containing 5% CO2. The cells were grown to about 80-90% confluence for treatments unless otherwise stated. Treatment of Cells. GM04312C cells were pretreated with various arsenic species at the indicated concentrations for the respective experiments in complete growth medium for 24 h. The cells were washed twice with phosphate-buffered saline (PBS) before the addition of BPDE in serum-free medium at a final concentration of 0.5 µM. After a 30 min incubation, BPDE was removed and the cells were washed with PBS three times, and their DNA was extracted for measurement of the BPDE-DNA adducts.

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The concentrations of organic solvent did not exceed 0.001%, to avoid the direct influence of DMSO or THF on the cells. Cell Counting and Colony-Forming Assay. We determined the cytotoxic responses of GM04312C cells to trivalent arsenic species by cell counting and colony-forming assay. The 80-90% confluent GM04312C cells were treated for 24 h with the test arsenic compounds. The cells were trypsinized and counted using a Z2 Coulter particle count and size analyzer (Beckman Coulter, Coulter Electronics Inc., Hialeah, FL). For the colony-forming assay, exponentially growing GM04312C cells were trypsinized and resuspended in DMEM/F12 medium. The cells were seeded into 60 mm dishes at densities from 300 to 105 cells per dish and allowed to attach overnight. The cells were treated with arsenic species at various concentrations for 24 h. After treatment, the cells were washed three times with ice-cold PBS and restored in fresh growth medium for colony formation. After 2-3 weeks, the colonies were stained with 0.25% methylene blue and counted, and the cloning capability was calculated based on the plating efficiency of untreated control cells. DNA Isolation. Cells were lysed in DNAzol reagent (Invitrogen Life Technologies), and the genomic DNA was precipitated with ice-cold 99.9% ethanol and washed twice with cold 70% ethanol. The DNA pellet was air-dried and resuspended in deionized water, and the solution was placed in an incubator at 37 °C overnight to facilitate the redissolution of DNA. DNA concentrations were measured at 260 nm using a SmartSpec 3000 spectrometer (BioRad Laboratories, Cambridge, MA). Detection of BPDE-DNA Adducts. A capillary electrophoresis laser-induced fluorescence (CE-LIF) based immunoassay, as described previously (73, 74), was used in this study to detect BPDE-DNA adducts. Typically, 1 µg of DNA was heat-denatured at 100 °C for 5 min followed by cooling on ice for 3 min. Denatured DNA was incubated with a mouse anti-BPDE antibody (clone 8E11, isotype IgG1, Trevigen Inc., Gaithersburg, MD) and a goat antimouse antibody provided in a Zenon Alexa Fluor 546 mouse IgG1 labeling kit (Molecular Probes, Eugene, OR). An incubation buffer (10 mM Tris and 80 mM glycine adjusted with acetic acid to pH 7.8) was used to bring the total sample volume to 20 µL. After overnight incubation on ice in the dark, samples were electrokinetically injected into the capillary using an injection voltage of 10 kV for 10 s. The separation was carried out at room temperature with a separation voltage of 20 kV. The running buffer was a Tris-glycine mixture containing 30 mM Tris and 170 mM glycine, at pH 8.3. Between runs, the capillary was rinsed electrophoretically for 5 min with 0.02 M NaOH and 5 min with the running buffer. Measurement of the Cellular GSH Level. Cellular GSH content was determined by using a Bioxytech GSH-400 colorimetric assay kit (Oxis International, Portland, OR). For higher sensitivity, a fluorometric method reported by Hissin and Hilf (75) can be employed. Cells (106-107) were trypsinized, centrifuged, and washed with PBS. Cells were then resuspended in 100 µL of icecold metaphosphoric acid. After four cycles of freeze-thaw, the solution was centrifuged at 10 000g at 4 °C for 10 min. The clear supernatant was collected at 4 °C for the subsequent assay. Reagent R1 and NaOH from the assay kit were added to the supernatant. After incubation at 25 °C for 10 min in the dark, the absorbance of the solution was measured at 400 nm. GSH concentrations in the solution were calculated from the absorbance and a prestored calibration curve of a GSH standard. Cellular GSH content is expressed as nanomoles of GSH per million cells. Measurement of Total GST Activity. Cellular GST activity was measured by a GST colorimetric activity assay kit (Biovision, Cedarlane Laboratory Ltd., ON, Canada) using 1-chloro-2,4dinitrobenzene (CDNB) as substrate. Briefly, the cells were trypsinized, centrifuged, and homogenized in 100 µL of GST sample buffer. After centrifugation at 10 000g at 4 °C for 10 min, the supernatant was collected for the subsequent assay. GSH and CDNB in GST assay buffer were added to the supernatant and mixed. The absorbance was read at 340 nm using a SPECTRA MAX 190 microplate spectrophotometer controlled by SOFTmax PRO 4.0

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Figure 1. Effect of iAsIII, MMAIII, and DMAIII on the cell viability of GM04312C cells examined by a colony-forming assay (A) and direct cell number counting (B). Error bars indicate the standard error of four determinations from two experiments.

(Molecular Devices Corporation, Sunnyvale, CA). Absorbance readings were repeatedly taken for a minimum of five time intervals, to obtain enzyme kinetic information. GST activity was expressed as nanomoles of CDNB reduced per minute per million cells.

Results Cytotoxicity of Trivalent Arsenic Compounds to GM04312C Cells. It has previously been reported that MMAIII and DMAIII are more toxic than iAsIII to human cells. We therefore examined the relative toxicities of these compounds in GM04312C (XPA) cells. The cytotoxicity was determined both by cell number counting immediately after exposure to arsenic and by the colony-forming assay, which requires cell growth for 2-3 weeks after exposure. The colony-forming assay is known to be a very sensitive method of toxicity evaluation, but it may underestimate early cell survival (76). As shown in Figure 1, the trivalent methylated metabolites MMAIII and DMAIII exerted higher cytotoxicity on GM04312C cells compared to iAsIII. Assessed by the colony-forming assay (Figure 1A), treatment with 1 µM



Figure 2. Electropherograms showing the analyses of DNA extracted from GM04312C cells treated with various concentrations of BPDE for 30 min. The cells were lysed, and DNA was extracted for analysis of BPDE-DNA adducts using the CE-LIF immunoassay. Peak 2 corresponds to the antibody complex with the BPDE-damaged DNA, and peak 1 corresponds to the free unbound antibodies.

MMAIII or 5 µM DMAIII for 24 h reduced survival to 2-4% of the control. After treatment with 10 µM iAsIII, approximately 28% of cells were able to form colonies. At higher concentrations of MMAIII or DMAIII or at 100 µM iAsIII, no colonies were observed. Cell counts of cells handled in the same manner and at the same confluence as the cells in the adduct formation experiments were obtained and are shown in Figure 1B. Treatment with 100 µM iAsIII, 10 µM MMAIII, or 25 µM DMAIII for 24 h spared 40%, 10%, or 15% of the control cells from cell killing, respectively (Figure 1B). At 25 µM iAs(III) approximately 55% of the control cells survived (Figure 1B). The difference between colony formation and survival based on cell counting is clear. Similar results were obtained with A549 and HeLa S3 cells where colony formation was dramatically reduced after the cells were exposed to comparable concentrations of trivalent arsenic compounds for 18 h (28, 69). Dose-Response for BPDE-DNA Adduct Formation in GM04312C Cells. The GM04312C (XPA) cells were incubated for 2 h with BPDE at concentrations ranging from 0 to 10 µM, and the extracted DNA was subjected to the CE-LIF immunoassay. The resulting electropherograms are shown in Figure 2. Electrophoretic separation resulted in two resolved peaks. The later-eluting peak corresponds to the antibody complex with the BPDE-damaged genomic DNA, and its peak area thus represents the level of the BPDE-DNA adducts. The peak area of that peak increased with increasing concentrations of BPDE, demonstrating an expected dose-response relationship between the BPDE concentration and the BPDE-DNA adduct level. Nonspecific binding between antibody and DNA was reflected in the control sample (0 µM). For examining the influence of iAsIII and its trivalent methylated metabolites, we chose a dose of 0.5 µM BPDE. Effect of Trivalent Arsenic Compounds on the Formation of BPDE-DNA Adducts. Each of the three trivalent arsenic compounds was shown to be capable of enhancing the formation of BPDE-DNA adducts (Figures 3-5). iAsIII increased adduct formation by 39% at 10 µM and by 87% at 100 µM (Figure 3), whereas MMAIII increased adduct formation by 100% at 5 µM and by 209% at 10 µM (Figure 4). DMAIII was found to increase adduct formation by 21% at 5 µM and by 90% at 25 µM (Figure 5). Effect of Trivalent Arsenic Compounds on the GSH level and GST Activity. As shown in Table 1, after a 24 h treatment, iAsIII almost doubled the cellular GSH level (from

Figure 3. Effect of iAsIII on the formation of BPDE-DNA adducts. GM04312C cells were pretreated with iAsIII at the indicated concentrations for 24 h and then incubated with 0.5 µM BPDE for 30 min. The cells were lysed, and DNA was extracted for analysis of BPDE-DNA adducts using the CE-LIF immunoassay. The electropherograms are shown in panel A. Peak 2 corresponds to the antibody complex with the BPDE-damaged DNA, and peak 1 corresponds to the excess antibodies. The peak area percentage of peak 2 was used to calculate the level of BPDE-DNA adducts. The relative adduct levels are represented in panel B. Adduct levels obtained from BPDE incubation without prior As exposure were used as controls. The data in the bar graphs indicated with /// are statistically significantly different from the controls with p < 0.01 using the one-way Student’s t test. Error bars indicate the standard deviation from three experiments.

approximately 6 to 11 nmol/106 cells) at 10 µM. An increase in iAsIII concentration to 50 µM did not increase the GSH level further. Similarly, 5 µM DMAIII increased the GSH level by more than 2-fold and 10 µM DMAIII did not elicit a further rise in the GSH level. On the other hand, the same treatment with iAsIII or DMAIII did not lead to any appreciable change in GST activity. Enhancement of BPDE-DNA Adduct Formation Is Time-Dependent. To determine if the enhancement of BPDEDNA adduct formation is time-dependent, we pretreated cells with iAsIII or MMAIII for different time periods: 30 min, 6 h, or 24 h. We observed that both iAsIII and MMAIII increased

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Figure 5. Effect of DMAIII on the formation of BPDE-DNA adducts. The electropherograms are shown in panel A. The peak area percentage of peak 2 represents the level of BPDE-DNA adducts. The relative adduct levels are shown in panel B. Adduct levels obtained from BPDE incubation alone were used as controls. The data in the bar graphs marked with // and /// are statistically significantly different from the controls with p < 0.05 and p < 0.01, respectively, using the oneway Student’s t test. Error bars indicate the standard deviation from three experiments.

III

Figure 4. Effect of MMA on the formation of BPDE-DNA adducts. The electropherograms are shown in panel A. The peak area percentage of peak 2 represents the level of BPDE-DNA adducts. The relative adduct levels are shown in panel B. Adduct levels obtained from BPDE incubation alone were used as controls. The data in the bar graphs indicated with /// are statistically significantly different from the controls with p < 0.01 using the one-way Student’s t test. Error bars indicate the standard deviation from three experiments.

adduct formation in a time-dependent manner, as shown in Figure 6. When the pretreatment time increased, the enhancement by 100 µM iAsIII or 5 µM MMAIII also increased. After a 6 or 24 h treatment with 5 µM MMAIII, the formation of BPDE-DNA adducts was enhanced by 35% and 100%, respectively. Only after 24 h of treatment did 10 µM iAsIII enhance the adduct formation significantly (by 41%) while 100 µM iAsIII enhanced the adduct formation significantly after any time period under investigation.

Discussion Schwerdtle et al. (69) initiated a systematic study of five arsenic species, iAsIII (0-75 µM), MMAIII (0-7.5 µM), DMAIII (0-7.5 µM), MMAV (0-500 µM), and DMAV (0-500 µM),

Table 1. Cellular GSH Level and GST Activity after Arsenic Treatment for 24 ha arsenic compound

GSH level (nmol/106 cells)

GST activity (nmol/min/106 cells)

control iAsIII 10 µM 50 µM DMAIII 5 µM 10 µM

5.9 ( 1.2 11.1 ( 1.4* 11.0 ( 1.4* 16.2 ( 1.3* 13.6 ( 1.4*

4.2 ( 0.9 5.3 ( 0.7 4.9 ( 1.0 5.0 ( 0.1 4.9 ( 0.3

a Each value represents the mean ( SD of three experiments; / denotes statistically significant differences from control (p < 0.05), using one-way Student’s t test.

on their effects on the formation and repair of BPDE-DNA adducts. They found that all of the five arsenic species inhibited the repair of BPDE-DNA adducts at noncytotoxic concentrations. The inhibitory potencies of these arsenic species correlated more or less with their cytotoxicities, in a descending order of MMAIII > DMAIII > iAsIII > DMAV ≈ MMAV (69). This was confirmed in our laboratory with comparable results (data not shown). However, they found that iAsIII and MMAIII were able to enhance the formation of BPDE-DNA adducts, whereas, quite surprisingly, DMAIII had no enhancing effect at concentrations up to 7.5 µM. These observations would imply that DMAIII behaved differently from iAsIII and MMAIII in modulating DNA


Figure 6. Effect of iAsIII (A) and MMAIII (B) on the enhancement of BPDE-DNA adduct formation is time-dependent. Data points marked with an asterisk are statistically different from the control values (p < 0.05 using the one-way Student’s t test). Error bars indicate the standard deviation from three experiments.

damage induced by other agents. The anomaly manifested by DMAIII prompted the present study to reexamine the effects of these three trivalent arsenic compounds on the formation of BPDE-DNA adducts. Through the use of XPA cells, which lack the capacity to remove bulky DNA adducts, we found that each of the three trivalent arsenic compounds was capable of enhancing the formation of BPDE-DNA adducts (Figures 3-5). Their potencies of enhancing the formation of BPDE-DNA adducts in descending order are MMAIII > DMAIII > iAsIII. Our findings suggested that iAsIII, MMAIII, and DMAIII enhanced the formation of BPDE-DNA adducts, possibly via a shared mechanism. To date no data are available on the relative cocarcinogenicity of these arsenic compounds, although iAsIII was shown to enhance the tumorigenicity of solar UV irradiation in hairless mice (32, 33) and DMAIII was shown to be cocarcinogenic in hairless mice (54). In the study of Schwerdtle et al. (69), iAsIII started to enhance adduct formation at 25 µM and achieved 40% more adducts at 75 µM, whereas MMAIII was observed to enhance BPDE adduct formation at a dose as low as 2.5 µM and yield 60% more adducts at 5 µM. The difference in the magnitude of enhancement between their study and ours might be due to different treatment protocols or different cell lines used. In their study, exponentially growing A549 cells were preincubated with arsenic for 16 h and then coincubated with 50 nM BPDE for 2 h. It is noteworthy that DMAIII is easily oxidized (27, 77); therefore, DMAIII was freshly prepared and used in our study. Although impaired expression of CYP1A1 (an enzyme required for BaP metabolism) and suppressed antioxidant enzymes have been associated with the XPA


phenotype (78-82), we thought that these changes in enzyme expression would have little or no bearing on our observations because we used BPDE, the ultimate metabolite of BaP, to induce BPDE-DNA adducts, and suppression of antioxidant enzymes and resultant metabolic activity may only affect the induction and repair of oxidative DNA damage rather than bulky adducts (83-87). Studies by States and co-workers (88, 89) also showed that BPDE-induced cytotoxicity in XPA cells was due to formation of BPDE-DNA adducts and BPDE was only slightly more cytotoxic to XPA cells than to normal human skin fibroblasts. Therefore, possible interaction between arsenic and CYP1A1 or antioxidant enzymes is unlikely to be the mechanism responsible for enhancing the BPDE-DNA adduct formation in XPA cells observed in our present study. GST-catalyzed GSH conjugation of BPDE is believed to be the most important enzymatic pathway to inactivate BPDE (90-92). Therefore, it is conceivable that trivalent arsenic may modulate BPDE-DNA adduct levels via its effects on GSH levels and GST activity. GSH and GSH-related enzymes, including GST, are known to maintain an intracellular reducing environment and to protect against excessive generation of reactive oxygen species (ROS). They have been found to play important roles in arsenic detoxification by ameliorating arsenicinduced oxidative stress. More directly, GSH may conjugate with iAsIII, MMAIII, and DMAIII to form AsIII(SG)3, MMAIIIII I(SG)2, and DMA SG, respectively (93-97). These GSH conjugates have recently been suggested to constitute a novel pathway of iAs metabolism (98). Additionally, potential binding of the trivalent arsenic compounds to critical cysteine residues on GSH-related enzymes may also affect the activity of the enzymes and GSH turnover (99-101). The cellular GSH level may be significantly raised or lowered depending on the arsenic speciation, the dose, the time after exposure, and the cell type (99, 102-107). Similarly, increases, decreases or no change in GST activity after arsenic treatment have been reported (99, 108, 109). Consistent with a study by Maier et al. (65) and our previous observation (72), our results showed that a 24 h treatment with iAsIII or DMAIII did not deplete cellular reduced GSH; rather, both arsenic species increased the GSH level by 2-3-fold (p < 0.05) (Table 1). However, the arsenic-elicited increase in the GSH level did not neutralize BPDE and reduce BPDE-DNA adduct formation. Thus, effects on the GSH level could not account for the enhancement of BPDE-DNA adduct formation starting at 10 µM iAsIII or 5 µM DMAIII for these two arsenic species (Figures 3 and 5). The mechanism for the GSH level increase remains to be determined; however, it is likely that GSH synthesis was up-regulated in response to oxidative stress induced by iAsIII and DMAIII during the 24 h exposure or GSH was initially depleted and then bounced back at the time of GSH measurement (107). Treatment with the same two arsenic species did not change GST activity from its basal level, implying that inhibition of GST enzymes was also irrelevant to the enhancement of DNA adduct formation. Couchane and Snow (100) had previously shown that direct interaction of As species with GSH-related enzymes, including GST, did not significantly effect enzyme activity. Table 2 summarizes the effective concentrations at which significant changes in BPDE-DNA adduct formation, GSH levels, GST activity, and cell viability were observed. We previously reported that iAsIII increased cellular uptake of BPDE, which likely led to the enhanced formation of BPDE-DNA adducts (72). The reactive trivalent arsenic species may bind to thiol groups in membrane proteins, thus preventing adsorption of BPDE to the cell membrane and facilitating the

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Table 2. Effective Concentrations (Micromolar) of Arsenic Species at which Significant Changes in Each Biochemical Parameter Were Observeda iAsIII GSH GST cell number colony-forming ability BPDE-DNA adducts

0 n.s. 4.5 1 10

MMAIII n.d. n.d 0.75 0.1 5

DMAIII 5 n.s. 0.75 0.2 5

a n.d., not determined; n.s., not statistically significant. For cell number and colony-forming ability, the effective concentrations were estimated by interpolating from the respective curves in Figure 1, parts A and B, based on the viability approximating 90% of the control. It should not necessarily be concluded that the above effects could not occur at lower concentrations.

uptake of BPDE. Alternatively, the trivalent arsenic species may also have a negative impact on the adenosine-5′-triphosphate (ATP)-dependent efflux of BPDE-glutathione (GSH) conjugates by inhibiting the activity of transporter enzymes or by depleting ATP (110-112). There is a positive correlation between intracellular accumulation of BPDE-GSH conjugates and increased formation of BPDE-DNA adducts in cells lacking multidrug resistance protein 2 (MRP2) (113). The results of our study showed that the three trivalent arsenic species increased the formation of BPDE-DNA adducts in a dose-dependent manner (Figures 3-5). Additionally, we demonstrated that iAsIII and MMAIII elevated the adduct formation in a time-dependent manner (Figure 6). It should be pointed out that the lower arsenic concentrations used in this study (e.g., e10 µM) are relevant to some real-world exposure scenarios and in biological systems (2, 100, 114). Because trivalent arsenic compounds increased carcinogen-DNA adduct formation in our study, this observation may help explain why long-term lowconcentration human exposures to arsenic can lead to cancer. Acknowledgment. This work was supported by the National Cancer Institute of Canada with funds from the Terry Fox Foundation and the Canadian Institutes of Health Research (to X.C.L. and M.W.), Natural Sciences and Engineering Research Council of Canada, Canadian Water Network, Alberta Health and Wellness, and Canada Research Chairs Program (to X.C.L.).

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TX800335P

Arsenite and its Mono- and Dimethylated Trivalent Metabolites

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