Do Arsenosugars Pose a Risk to Human Health? The Comparative

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Environ. Sci. Technol. 2004, 38, 4140-4148

Do Arsenosugars Pose a Risk to Human Health? The Comparative Toxicities of a Trivalent and Pentavalent Arsenosugar PAUL ANDREWES,† DAVID M. DEMARINI,† KUNIHIRO FUNASAKA,‡ KATHLEEN WALLACE,† VIVIAN W. M. LAI,§ HONGSUI SUN,§ WILLIAM R. CULLEN,§ AND K I R K T . K I T C H I N * ,† Environmental Carcinogenesis Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711, Department of Atmospheric Environment, Osaka City Institute of Public Health and Environmental Sciences, Osaka, Japan, and Department of Chemistry, University of British Columbia, Vancouver, British Columbia, V6T 1Z1 Canada

Seafood frequently contains high concentrations of arsenic (∼10-100 mg/kg dry weight). In marine algae (seaweed), this arsenic occurs predominantly as ribose derivatives known collectively as arsenosugars. Although it is clear that arsenosugars are not acutely toxic, there is a possibility of arsenosugars having slight chronic toxicity. In general, trivalent arsenicals are more toxic than their pentavalent counterparts, so in this work we examine the hypothesis that trivalent arsenosugars might be significantly more toxic than pentavalent arsenosugars in vitro. We compared the in vitro toxicity of (R)-2,3dihydroxypropyl-5-deoxy-5-dimethylarsinoyl-β-D-riboside, a pentavalent arsenosugar, to that of its trivalent counterpart, (R)-2,3-dihydroxypropyl-5-deoxy-5-dimethylarsino-β-Driboside. The trivalent arsenosugar nicked plasmid DNA, whereas the pentavalent arsenosugar did not. The trivalent arsenosugar was more cytotoxic (IC50 ) 200 µM, 48 h exposure) than its pentavalent counterpart (IC50 > 6000 µM, 48 h exposure) in normal human epidermal keratinocytes in vitro as determined via the neutral red uptake assay. However, both the trivalent and the pentavalent arsenosugars were significantly less toxic than MMA(III), DMA(III), and arsenate. Neither the pentavalent arsenosugar nor the trivalent arsenosugar were mutagenic in Salmonella TA104. The trivalent arsenosugar was readily formed by reaction of the pentavalent arsenosugar with thiol compounds, including, cysteine, glutathione, and dithioerythritol. This work suggests that the reduction of pentavalent arsenosugars to trivalent arsenosugars in biology might have environmental consequences, especially because seaweed consumption is a significant environmental source for human exposure to arsenicals. * Corresponding author phone: (919)541-7502; fax: (919)685-3276; e-mail: [email protected]. † U.S. Environmental Protection Agency. ‡ Osaka City Institute of Public Health and Environmental Sciences. § University of British Columbia. 4140

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Introduction Arsenic is a carcinogen in humans, causing skin, lung, and urinary bladder cancers (1, 2). Chronic toxicity is observed from exposure to drinking water that contains ppb levels of inorganic arsenic (1, 2). Higher doses of arsenic are acutely toxic (LD50, mice ∼10 mg sodium arsenite/kg) (3). On the basis of this, the World Health Organization (WHO) has set a tolerable daily intake for arsenic of 0.15 mg/day for a 70 kg person (4). Seawater contains trace quantities of arsenic (∼2 µg/L), which is naturally bioaccumulated by marine organisms by factors of up to 100 000 (5). The majority of arsenic in marine organisms occurs as arsenobetaine (the main species found in fish and shrimp) and arsenosugars (the main species found in marine algae, i.e., seaweed) (6). Arsenosugar is a broad term that collectively refers to a range of carbohydrate compounds containing arsenic. At least 15 different arsenosugars have been identified in the marine environment (6). The most common type of arsenosugar is represented by the dimethylarsinoyl ribosides that contain dimethylated arsenic(V) at C5 of D-ribose derivatives; the most ubiquitous of these compounds is (R)-2,3-dihydroxypropyl-5-deoxy-5dimethylarsinoyl-β-D-riboside (Figure 1). Human exposure to arsenosugars is relatively high in Asia because of the more frequent use of seaweed in cooking (e.g., Nori, one of the products used to wrap Sushi), which typically contains arsenic levels as high as 100 mg/kg dry weight. As a result, dietary intake of arsenic can reach 1 mg/ day (e.g. in Japan) (7), far exceeding the WHO guideline. While it is clear that such use is not associated with any known acute toxic effects, chronic toxicity is a possibility. Arsenobetaine is rapidly excreted in human urine unchanged (8) and is assumed to have negligible toxicity because of its high LD50 in mice (>10 000 mg/kg) (9). In comparison, arsenosugars appear to be less inert than arsenobetaine. However, the toxicity and metabolism of arsenosugars have only been sparsely studied. Erroneously, the published literature often states that arsenosugars are inert and harmless like arsenobetaine (1). Beyond epidemiology, there is no evidence supporting this assertion. In epidemiological studies, no evidence for chronic toxicity associated with seaweed consumption was found. In fact, seaweed consumption has been associated with reduced risk of cancers, including breast (10) and colorectal (11) cancers, probably because of the high fiber and vitamin content of seaweed. One mode by which arsenosugars may exert chronic toxicity is via metabolism to other arsenicals such as dimethylarsinic acid (DMA(V)), a tumor promoter and complete carcinogen in experimental animals (12, 13). Human volunteers fed a synthetic arsenosugar excreted 80% of the dose over 4 days, the major metabolite being DMA(V) (14). In other studies, human volunteers excreted substantially higher amounts of DMA(V) in their urine after consumption of seaweed containing arsenosugars (15-17). Arsenosugars do not appear to decompose when seaweed is cooked (17) nor do arsenosugars appear to be readily decomposed by stomach acid (16, 18), so the occurrence of DMA(V) after arsenosugar ingestion has been attributed to either enzymatic or microbial activity in the human body (16). In a unique series of studies, sheep were examined that had eaten a diet consisting almost entirely of seaweed since birth (19, 20). In this study, the arsenosugar content of the seaweed corresponded to an arsenic intake of approximately 45-90 mg of arsenic per sheep daily. The disposition of 10.1021/es035440f CCC: $27.50

 2004 American Chemical Society Published on Web 06/22/2004

sugars have not yet been detected in vivo, we know of no attempts to measure these compounds. In this work, we examined the cytotoxicity in normal human epidermal keratinocytes of the pentavalent arsenosugar, (R)-2,3-dihydroxypropyl-5-deoxy-5-dimethylarsinoylβ-D-riboside, and its trivalent counterpart, (R)-2,3-dihydroxypropyl-5-deoxy-5-dimethylarsino-β-D-riboside, via the neutral red uptake method. We also examined the ability of these two compounds to nick plasmid DNA in vitro and their mutagenicity in Salmonella TA104. The trivalent arsenosugar either was generated in situ by reduction of the pentavalent arsenosugar with cysteine, glutathione, or dithioerythritol or was synthesized in a separate step.

Materials and Methods

FIGURE 1. General structure of a dimethylarsinoyl riboside (a). To date, at least 11 different types of dimethylarsinoyl ribosides (different R groups) have been identified in the marine environment. In this study, we used only one representative synthetic arsenosugar with the R group shown in (b). Reduction of arsenosugars produces trivalent arsenosugars represented in (c). arsenosugars in these sheep was similar to inorganic arsenic, and arsenic tissue accumulation was evident. Arsenic concentrations in the urine, blood, and wool of seaweed-eating sheep were at least 100× greater than arsenic concentrations in grass-eating sheep. The main arsenosugar metabolite detected in the sheep’s blood and urine was DMA(V) (∼95%). No toxic effects were observed in the sheep resulting from this unique diet, but long-term illness could not be excluded as the sheep were slaughtered (for human consumption) at a young age. The authors concluded that the high absorption of arsenosugars and the similarities that arsenosugars share with inorganic arsenic in terms of metabolism and accumulation indicate that arsenosugars may be more toxic than previously thought. Another possibility is that arsenosugars directly exhibit toxic effects, but most tests of arsenosugar toxicity have yielded essentially negative results (21-24). In one study, an arsenosugar ((R)-2,3-dihydroxypropyl-5-deoxy-5-dimethylarsinoyl-β-D-riboside) induced chromosomal aberrations in human fibroblasts but only at very high concentrations (24). In other studies, the same arsenosugar was weakly cytotoxic in alveolar macrophages (IC50 ) 8000 µM) and BALB/c 3T3 cells (IC50 ) 6000 µM), but conversely at similar concentrations enhanced the viability of peritoneal macrophages (21, 22). Pure arsenosugars are difficult to obtain in sufficient quantities for toxicity testing, and to date only this one pentavalent arsenosugar has been tested. In light of recent findings that methylated trivalent arsenicals are more toxic than methylated pentavalent arsenicals and inorganic arsenicals (25-35), we thought it would be prudent to examine the toxicity of trivalent arsenosugars. As part of the synthesis of trimethylarsonioribosides, Francesconi et al. (36) reported that dimethylarsinoyl ribosides could be reduced using the dithiol compound 2,3dimercaptopropanol. In general, the reduction of arsenic(V) to arsenic(III) compounds by thiols and dithiols is well-known (37, 38) and may occur in vivo with or without enzyme catalysis (31, 39). Thus, there is a possibility that pentavalent arsenosugars ingested in seaweed will be metabolized to trivalent arsenosugars in vivo. Although trivalent arseno-

Caution: Many arsenic compounds are toxic and carcinogenic and should be handled using appropriate safety measures. Chemicals. The pentavalent arsenosugar (Figure 1a), (R)2,3-dihydroxypropyl-5-deoxy-5-dimethylarsinoyl-β-D-riboside, was synthesized by modification of a published method (40). The trivalent arsenosugar (Figure 1c), (R)-2,3-dihydroxypropyl-5-deoxy-5-dimethylarsino-β-D-riboside, was prepared by hydrolysis of (R)-2,3-dihydroxypropyl-5-deoxy-5dimethylarsino-2,3-O-isopropylidene-β-D-riboside, a precursor prepared in the synthesis of the pentavalent arsenosugar, in acidic aqueous solution. The two arsenosugar compounds were characterized via HPLC-ICPMS and 1H NMR spectroscopy (D2O). The NMR spectrum of the pentavalent arsenosugar (see Figure 1 in Supporting Information) was in agreement with previously reported data (40). The major difference of 1H NMR spectra between the trivalent arsenosugar (see Figure 2 in Supporting Information) and the pentavalent arsenosugar is the upfield shifts of the methyl groups and H5. The resonance of the two methyl groups gives rise to a singlet at 0.89 ppm (whereas in the pentavalent arsenosugar, two singlets are seen at 1.74 and 1.76 ppm). The signal for the H5 (CH2) protons is found as a multiplet at 1.72 ppm (two well-separated dd signals at 2.39 and 2.54 ppm for the pentavalent arsenosugar). Three minor impurities were detected in the trivalent arsenosugar via HPLCICPMS; these could not be identified but are most likely arsenosugar sugar compounds with differences in the sugar group (see Results section). The trivalent arsenosugar, although surprisingly stable, slowly oxidizes in solution; therefore, it was stored as a concentrated aqueous stock solution in glass ampules under nitrogen until the day an experiment was performed. Monomethylarsine oxide (41) (MMA(III)), iododimethylarsine (42) (DMA(III)), and trimethylarsine oxide (43) (TMAO) were synthesized using methods described previously. Monosodium methylarsonate (MMA(V), 99.2% purity) was obtained from Chem Service (West Chester, PA), dimethylarsinic acid (DMA(V), 99.5% purity) was obtained from Ansul (Weslaco, TX), and disodium arsenate heptahydrate and sodium arsenite were obtained from Fisher Scientific (Fairlawn, NJ). A series of dilutions of the trivalent arsenosugar stock solution were prepared in PBS. The resulting dosing solutions were immediately used to treat the normal human epidermal keratinocytes (NHEK) and used for the DNA nicking assay. Likewise, solutions of MMA(III) and DMA(III) were prepared fresh each time an experiment was performed. All arsenical dosing solutions were prepared in PBS, and where necessary the pH was adjusted to 8, using sodium hydroxide solution. L-Cysteine (hydrochloride, monohydrate), glutathione (GSH, reduced form, anhydrous), and dithioerythritol (DTE) solutions were prepared fresh each time an experiment was performed by dissolving the appropriate amount of solid in water and, in the case of cysteine and GSH, adjusting the pH to 8 using sodium hydroxide. Supercoiled plasmid DNA (pBR VOL. 38, NO. 15, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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322) was purchased from Roche (Indianapolis, IN). Sodium lauryl sulfate (SLS) and neutral red stock solution (3.3 g/L in D-PBS) were purchased from Sigma (St. Louis, MO). Cell Cultures. Salmonella strain TA104 (hisG428, rfa, ∆uvrB, pKM101) was obtained from Dr. B. N. Ames, University of California, Berkeley, CA. Cryopreserved NHEK from pooled neonatal foreskin tissue was obtained from Cambrex Bioscience (Walkersville, MD). Keratinocytes were grown at 37 °C in a humidified incubator containing 5% CO2. Calcium free keratinocyte basal medium (serum free) was supplemented with bovine pituitary extract (30 µg/mL), human recombinant epidermal growth factor (0.0001 ng/mL), insulin (5 µg/mL), hydrocortisone (0.5 µg/ mL), gentamicin (30 µg/mL), and amphotericin-B (15 ng/ mL), all obtained from Cambrex Bioscience (Walkersville, MD). Arsenic Speciation via HPLC-ICPMS. The HPLC system consisted of a Waters model 510 delivery pump, a Rheodyne model 7010 injector valve with a 20 µL sample loop, and a reverse phase C18 column (GL Sciences Inertsil ODS, 250 mm × 4.6 mm) equipped with a C18 guard column (Supelco, 2 cm). The HPLC system was connected to the ICP nebulizer via a PTFE tube (20 cm × 0.4 mm) and appropriate fittings. A double-focusing magnetic sector field ICPMS (Element2, Thermo Finnigan, Germany) equipped with a conikal nebulizer was used as a detector. The instrument was operated at low-resolution mode (R ) 300). The mass analyzer was set to monitor both the m/z ) 75 signal peak corresponding to As+ and the m/z ) 77 peak corresponding to the interference possibly caused by the chloride in the samples (ArCl+). Because m/z ) 77 also corresponds to 77Se+, 82Se+ was also monitored to correct for the Se portion of the counts collected under m/z ) 77 signal peaks. The signals were acquired continuously over the time of the chromatography. All signals were collected, and the data were manipulated on a separate computer (MS Excel). The eluent contained 10 mM tetraethylammonium hydroxide (TEAH), 4.5 mM malonic acid, 0.1% MeOH, pH ) 6.8 (by HNO3), and the eluent flow-rate was 0.8 mL/min. All samples were filtered (0.45 µm) prior to injection onto the column. Arsenic compounds in the samples were identified by matching the retention times of the peaks in the chromatograms with those of standards. DNA Nicking Assay. In one series of experiments, pBR 322 plasmid DNA (250 ng in 1 µL of TE) was mixed with various concentrations of the trivalent arsenosugar (5 µL of a 10× dosing solution) and TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) to give a total volume of 45 µL. These mixtures were incubated for 2 h at 37 °C. The amounts of damaged (nicked) and intact DNA were determined via agarose gel electrophoresis methods described elsewhere (26, 44). Briefly, the reaction mixture, containing plasmid DNA, was mixed with 5 µL of gel loading solution (0.05% bromophenol blue, 50% v/v sucrose). The mixtures were loaded in 1% agarose gels containing 0.5 µg/mL ethidium bromide (eight lanes per gel). Supercoiled (SC) fast migrating DNA was separated from linear (L) DNA (double-strand break) with moderate electrophorectic mobility and open circular (OC) slow migrating DNA (single-strand break) by electrophoresis for 2-3 h at 50 V (constant voltage). Ethidium bromide-stained DNA bands were visualized under UV light. For comparison, other arsenical species, including the pentavalent arsenosugar, were examined under equivalent conditions. In a second series of experiments, we examined the ability of a mixture of the pentavalent arsenosugar and a thiolbased reducing agent (either DTE, cysteine, or glutathione) to nick pBR 322 plasmid DNA. In this case, DNA nicking is due to the reduction of the inactive pentavalent arsenosugar to the DNA damaging trivalent arsenosugar. Thus, the amount 4142

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of DNA damage observed is related to the efficiency of the reduction and the activity of the product. Mixtures were prepared containing DNA (250 ng in 1 µL of TE), the pentavalent arsenosugar (0.02-4 mM), a reducing agent (either DTE, cysteine, or glutathione, 0.02-5 mM), and TE buffer to give a total volume of 45 µL. The reducing agent was always the last component added to the mixtures. A variety of controls were also run where the arsenical or the reducing agent or both were omitted. The mixtures were incubated for between 2 and 24 h at 37 °C, and the amount of DNA damage was determined by agarose gel electrophoresis as described above. In this paper, DNA damage was considered to have occurred when the intensity of the OC DNA band in the sample lane was clearly greater than that of the OC DNA band in the control lane as determined by visual examination. Salmonella Mutagenicity Assay. The Salmonella mutagenicity plate-incorporation assay (45) was performed using the preincubation method with strain TA104 in the absence of S9 as described previously (35). Assays were performed using a range of doses between 0.7 and 3500 nmol of arsenosugar (dissolved in water) per plate either in the presence or in the absence of DTE (3500 nmol/plate). When the arsenosugar was assayed in the presence of DTE, the arsenosugar was mixed with the DTE and incubated for 2 h at 37 °C before dosing the Salmonella to allow time for the DTE to reduce the pentavalent arsenosugar to the trivalent arsenosugar. The following controls were used: (a) methylglyoxal (50 µg/plate) positive control; (b) water; (c) DTE (35 µmol/plate); (d) DTE oxidized with hydrogen peroxide (3.5 µmol/plate). Human Keratinocyte Cytotoxicity. NHEK cell viability was determined via the neutral red uptake assay. This assay has been previously used to determine the cytotoxicity of inorganic and methylated pentavalent and trivalent arsenicals in NHEKs (46). Cell cultures were established in 25 cm2 flasks ((6.25-22.5) × 104 cells/flask) and fed every 2 days. After reaching 50-80% confluence, cells were subcultured into 96-well plates (Corning COSTAR, Corning, NY) and incubated for 24-48 h before adding the test chemical. All arsenical dosing solutions were prepared in PBS at either 10× or 100× the final concentration. Thus, each well of the 96-well plate contained 250 µL of medium and was treated with either 2.5 or 25 µL of dosing solution. One 96-well plate was used for each test chemical. The plate’s peripheral wells contained only medium, and these wells served as blanks. This left 10 columns containing cells (six wells per column), and eight of these columns were dosed with eight different concentrations of the test chemical; the remaining two columns, placed at either end of the plate, served as negative controls, which contained cells and the dosing vehicle. Cells were treated with the test chemical for either 24, 48, or 72 h. Treatment medium was then removed, neutral red medium (containing KGM and 33 µg of neutral red/mL) was added, and the cells were incubated for an additional 3 h. The neutral red medium was removed, and the cells were rinsed once with PBS. Neutral red dye was released from the cells via a solution of 50% ethanol and 1% acetic acid. Neutral red absorbance was measured using a Wallac microplate reader. SLS was used as a positive control. The results from this assay were always confirmed by microscopic observations. In one series of experiments, we examined the effect of either cysteine or glutathione on the toxicity of either MMA(III), DMA(III), or the trivalent arsenosugar. To do this, 96well plates were prepared with columns of test chemical at each concentration, as described above, and then all wells (including controls) had 2.5 µL of either cysteine or GSH (both at 100 mM) added to give a final concentration of either cysteine or GSH of 1 mM.

FIGURE 3. Agarose gel electrophoresis of pBR 322 plasmid DNA (250 ng) that was incubated with the trivalent arsenosugar (0.1-6 mM) for 2 h at 37 °C in TE buffer (pH 8.0) (SC, supercoiled form of plasmid DNA; OC, open circular form of plasmid DNA (after a singlestrand break)).

FIGURE 2. HPLC-ICPMS (ion pairing) chromatograms of the pentavalent arsenosugar (a) and the trivalent arsenosugar (b).

Results HPLC-ICPMS. The synthetic trivalent arsenosugar was analyzed via HPLC-ICPMS (ion pairing) (Figure 2). In all of our experiments, no interference from ArCl+ occurred, as determined by monitoring signals at m/z ) 77 and 82 (chromatograms not shown). The retention time of the trivalent arsenosugar was exactly the same as that of the pentavalent arsenosugar (Figure 2). However, the trivalent arsenosugar peak was broader than the pentavalent arsenosugar peak and had a large tail. The detection of trivalent arsenosugars in biological samples will require the development of an HPLC method where the trivalent arsenosugar can be resolved from the corresponding pentavalent arsenosugar. Although the NMR data indicated that the trivalent arsenosugar is pure, three unidentified compounds were apparent in the HPLC-ICPMS chromatogram. These did not correspond to any simple methylated (e.g., MMA(III) or MMA(V)) or inorganic arsenicals (e.g., arsenite or arsenate) as determined by comparing retention times of standards. The organoarsenic impurities observed in the HPLC-ICPMS at these levels should be apparent in the NMR. Because they are not apparent in the NMR, they must either be analytical artifacts or be trivalent arsenosugars with differences in the sugar group (e.g., where the dihydroxypropyl group has hydrolyzed off). The pentavalent arsenosugar was incubated with a thiolbased reducing agent, cysteine, using the same conditions as were used in the plasmid DNA nicking assay. When the resulting mixture was analyzed via HPLC-ICPMS, no new arsenic compounds were detected, indicating that only reduction is occurring under these conditions and that no hydrolysis is taking place. However, because the trivalent arsenosugar coelutes with the pentavalent arsenosugar, it was not possible, using this method, to ascertain what percentage of the pentavalent arsenosugar had been reduced. DNA Nicking Assay. When pBR 322 plasmid DNA was incubated with the pentavalent arsenosugar for up to 24 h at 37 °C, no DNA damage was observed for pentavalent arsenosugar concentrations as high as 7 mM (see control

lanes in Figure 4a and b). In contrast, when the trivalent arsenosugar was incubated with pBR 322 plasmid DNA for 2 h at 37 °C, DNA damage was clearly observed for arsenosugar concentrations as low as 0.6 mM (Figure 3). The ability of the trivalent arsenosugar to damage pBR 322 DNA was compared to that of other arsenicals. The trivalent arsenosugar exhibited at least equal potency at nicking pBR 322 plasmid DNA when compared to DMA(III), which is known to be genotoxic to naked DNA (26, 44) and to be a potent clastogen in cells (35). In a second series of experiments, the trivalent arsenosugar was generated in situ by incubating the pentavalent arsenosugar with a thiol-based reducing agent (either DTE, GSH, or cysteine). Neither DTE, GSH, cysteine, nor the pentavalent arsenosugar alone damaged plasmid DNA in vitro. However, when pBR 322 plasmid DNA was incubated with the pentavalent arsenosugar and a reducing agent (either DTE, GSH, or cysteine), DNA damage was clearly evident (Figure 4). The amount of DNA damage produced by the reduced arsenosugar was dependent on the concentration of the arsenosugar, the concentration of the reducing agent, and the incubation time. For any combination of arsenosugar and reducing agent, increasing the incubation time caused an increase in the amount of DNA damage observed. When a 24 h incubation period was used, the lowest concentrations of arsenosugar that caused DNA damage in combination with excess (7 mM) cysteine (Figure 4a) and glutathione (Figure 4b) were 0.07 and 0.4 mM, respectively. Conversely, when the reducing agent was titrated, the lowest concentration of cysteine (Figure 4c) and glutathione (Figure 4d) required, in combination with excess pentavalent arsenosugar (7 mM), to damage DNA (24 h exposure) was 0.2 mM. The arsenosugar does not decompose into other arsenicals when incubated with reducing agents (see above), and neither the pentavalent arsenosugar nor any of the reducing agents (or oxidized reducing agents) are active alone; thus, the activity seen for the combination of the pentavalent arsenosugar and the reducing agent must be due to formation of the DNA damaging trivalent arsenosugar. When the activity of the synthetic trivalent (>90% trivalent by NMR) arsenosugar (Figure 3) is compared to that of the trivalent arsenosugar generated in situ (using either GSH or cysteine, Figure 4), the synthetic arsenosugar is seen to be more active. This suggests that either the in situ generation of the trivalent arsenosugar is not 100% efficient or GSH and cysteine may be acting as ROS scavengers or both. Both glutathione and cysteine are found in millimolar concentrations in vivo. Therefore, the reduction of the pentavalent VOL. 38, NO. 15, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 4. Agarose gel electrophoresis of pBR 322 plasmid DNA after a 24 h incubation at 37 °C with the pentavalent arsenosugar and either 7 mM glutathione (a) or 7 mM cysteine (b) in TE buffer (pH 8.0). In comparison, under the same conditions, the pentavalent arsenosugar concentration was held constant, and either the glutathione (c) or the cysteine (d) concentration was increased (SC, supercoiled form of plasmid DNA; L, linear form of plasmid DNA (after a double-strand break); OC, open circular form of plasmid DNA (after a single-strand break)). arsenosugar, with GSH and cysteine, is efficient enough to be considered biologically relevant. Salmonella Mutagenicity Assay. Neither the pentavalent arsenosugar nor the trivalent arsenosugar (pentavalent arsenosugar + DTE) were mutagenic (Table 1 in Supporting Information) at doses as high as 1 mg/plate (3500 nmol/ plate). In this work, we defined mutagenicity as being at least a 2-fold increase relative to the negative control. Our results for the arsenosugar compounds are consistent with 4144

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all other arsenicals being negative for gene mutation in this assay (35). Strain TA104 was chosen because it is mutated by some ROS generators, including hydrogen peroxide and Paraquot. Possible reasons why trivalent arsenicals do not revert Salmonella TA104 are given in ref 35. There was no evidence for pentavalent arsenosugar cytotoxicity in Salmonella; however, the trivalent arsenosugar (arsenosugar + DTE) displayed cytotoxicity at doses above 700 nmol/plate (Table 1 in Supporting Information). The

TABLE 1. Estimated IC50 Values (µM) for NHEKS That Were Exposed to Various Arsenicals for 24 h at 37 °Ca arsenic(III) compounds arsenite MMA(III) DMA(III) arsenosugar(III)

10 1 1 500

arsenic(V) compounds arsenate MMA(V) DMA(V) TMAO arsenosugar(V)

100 >6000 3000 >6000 >6000

a

The IC50 values were estimated by measuring the neutral red uptake over 3 h.

cytotoxicity of the pentavalent arsenosugar in combination with the DTE was not attributable to DTE because DTE was not cytotoxic at a dose of 35 000 nmol/plate (10 times the dose used here); also oxidized DTE (3500 nmol/plate) was not cytotoxic. Thus, the cytotoxicity of the pentavalent arsenosugar in combination with DTE must be due to the reduction of the pentavalent arsenosugar to the trivalent arsenosugar. Human Keratinocyte Cytotoxicity. We determined the viability of normal human epidermal keratinocytes after exposure of the cells to arsenicals, including the trivalent and pentavalent arsenosugar, for 24, 48, or 72 h via the neutral red uptake assay. Cytotoxicity data for arsenite, arsenate, MMA(III), MMA(V), DMA(III), and DMA(V) in NHEKs have already been reported by others (34, 46), and we obtained similar results, which we list here to make a valid comparison with the two arsenosugar compounds. In this assay, the IC50 of the SLS positive control was approximately 5 µg/mL. The IC50 (24 h exposure) values for the arsenicals we examined are listed in Table 1. In general, trivalent arsenicals were orders of magnitude more toxic than their pentavalent counterparts, and, as seen in Table 1, the trivalent arsenosugar was much more toxic than its pentavalent counterpart. However, the trivalent arsenosugar was not nearly as toxic as the other trivalent arsenic compounds but still was almost as toxic as arsenate and was more toxic than any of the pentavalent organoarsenicals. The trivalent arsenosugar decreased cell viability, as measured by neutral red uptake, in a dose-dependent manner (Figure 5). Increasing exposure time from 24 to 48 h (Figure 5) caused only a minimal decrease in IC50 (2-fold), which was also the case for DMA(III) and MMA(III) but not arsenite (where the IC50 decreased 5-fold). The decrease in neutral red uptake corresponded to clear changes in cell morphology and density. In contrast, the pentavalent arsenosugar did not exhibit toxicity at concentrations up to 6000 µM (Figure 5), and no changes in cell morphology were observed. This is in agreement with other data for pentavalent arsenosugar cytotoxicity, where the lowest IC50 reported was 6000 µM in BALB/c 3T3 cells (22). Given the lower toxicity of the trivalent arsenosugar in comparison to the other trivalent arsenicals examined, the possibility that the observed activity is due to these compounds occurring as impurities in the trivalent arsenosugar needs consideration. A DMA(III) or MMA(III) impurity of 0.2% or an arsenite impurity of 2% in the trivalent arsenosugar could explain our results. The most likely way for simple methyl arsenical impurities to arise is via hydrolysis of the arsenic sugar bond. Thus, we consider DMA(III) to be the most likely toxic impurity to occur in the trivalent arsenosugar. Further decomposition to produce MMA(III) or arsenite would require more severe conditions, so if the possibility of DMA(III) impurities can be effectively argued against then the possibility of MMA(III) and arsenite impurities occurring would be very low. Analysis of the trivalent arsenosugar via 1H NMR spectroscopy showed it to be over 90% pure, and no pentavalent arsenosugar or other organoarsenicals were detected. Analysis

FIGURE 5. Viability of normal human epidermal keratinocytes on exposure to a trivalent and pentavalent arsenosugar. Keratinocytes were incubated with either the trivalent (2, 9) or the pentavalent (*, ×) arsenosugar for either 24 (9, *) or 48 (2, ×) hours at 37 °C. Cell viability was then determined by neutral red uptake, over 3 h (mean ( SD, n ) 6). of the trivalent arsenosugar via HPLC-ICPMS indicated the presence of small amounts of three unidentifiable impurities, which are not arsenite or arsenate or any simple methylated arsenical (e.g., MMA(III/V) or DMA(III/V)). These unidentified compounds are most likely arsenosugar compounds with differences in the sugar group, which would not have substantially different toxicity from the test compound. Although analytical characterization of the trivalent arsenosugar gave no reason to suspect that impurities in the compound could be responsible for its toxicity, a 0.2% impurity would probably not be detected via HPLC-ICPMS or NMR. Nevertheless, we do not believe that arsenite, MMA(III), or DMA(III) impurities could account for the observed results because the behavior of these three compounds in this assay is different from the behavior of the trivalent arsenosugar in the following ways: (a) DMA(III) (10 µM) always volatilized into adjacent control wells of the 96-well plate, causing at least a 50% decrease in viability of the control cells. If a DMA(III) impurity was responsible for the trivalent arsenosugar’s activity, the required impurity level of 0.2% would correspond to a DMA(III) concentration of 12 µM, at the highest concentration of trivalent arsenosugar tested, which would be high enough for volatilization into control wells to occur. In the case of the trivalent arsenosugar, no decrease in viability of controls due to volatilization from adjacent high-dose wells was seen. (b) The IC50 of arsenite decreases greatly (from 10 to 2 µM) when the exposure time is increased from 24 to 72 h, whereas the IC50 for the arsenosugar decreased only a little (from 500 to 400 µM), which suggests that the activity seen in the trivalent arsenosugar cannot be due to arsenite impurities. (c) The toxicities of both MMA(III) and DMA(III) were decreased considerably in the presence of the thiol compounds, glutathione or cysteine (both at 1 mM), whereas under equivalent conditions the toxicity of the trivalent arsenosugar compound was changed only slightly (Figure 6). Both glutathione and cysteine are assumed to decrease the toxicity of MMA(III) and DMA(III) either by inhibiting VOL. 38, NO. 15, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 6. Viability of normal human epidermal keratinocytes on exposure to either MMA(III) (a), DMA(III) (b), or the trivalent arsenosugar (c). Keratinocytes were incubated with the test chemical for 24 h at 37 °C, either alone (9) or with 1 mM glutathione (∆) or 1 mM cysteine (×). Cell viability was then determined by neutral red uptake, over 3 h (mean ( SD, n ) 6). transport of these compounds into the cell (by forming a complex) or by acting as ROS scavengers or both. In the case of the trivalent arsenosugar, the only means for cysteine and glutathione to decrease toxicity is by acting as ROS scavengers because these compounds cannot bind to the trivalent arsenosugar. In conclusion, all of our results are consistent with the toxicity of the trivalent arsenosugar being due to the trivalent arsenosugar itself and not a highly toxic trace arsenical impurity (e.g., DMA(III), MMA(III), or arsenite). Trivalent arsenosugar impurities with slight differences in the sugar group might be present in the test compound. Yet, these impurities are unlikely to be significantly different in toxicity from the test compound and thus are unlikely to confound our results.

Discussion One key chemical characteristic of arsenic is the ease with which pentavalent and trivalent arsenicals can interconvert (5, 37, 47, 48). It has long been known that trivalent arsenicals are in general much more active than their pentavalent 4146

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counterparts (49), for instance, as enzyme inhibitors (33), cytotoxins (32, 34, 41), genotoxins (26, 35, 44, 50, 51), and clastogens (35). This generalization may be applicable to more complex arsenicals such as arsenosugars. In this work, we examined this possibility by performing experiments with a pentavalent arsenosugar and its trivalent counterpart. We determined the activity of these two compounds in a plasmid DNA nicking assay, a mutagenicity assay, and a cytotoxicity assay. The pentavalent arsenosugar was not active in any of these assays at doses as high as 7 mM, consistent with what others have reported (21-24). However, the trivalent arsenosugar was at least 50-fold more active than the pentavalent arsenosugar in the plasmid DNA nicking assay and the cytotoxicity assay (based on IC25). The mechanisms by which arsenic compounds exert toxicity are unclear (3). The most likely mechanisms include induction of oxidative stress (52) and inhibition of key cellular enzymes by the binding of arsenic(III) compounds to enzyme sulfhydryls (3). When DNA damage is considered as a toxic end point, the most probable mechanism for DNA damage to occur is via the generation of ROS (28, 52). This has been clearly demonstrated in model systems with dimethylarsine (53), MMA(III) (44), and DMA(III) (44). Furthermore, in both in vivo and in vitro studies, biomarkers of oxidative damage to DNA are commonly detected after exposure to a number of different arsenicals (28, 52). We previously demonstrated that when TMAO (a good structural model for the pentavalent arsenosugar) is incubated with thiol compounds, it is reduced to DNA damaging trimethylarsine (50). We assume that both trimethylarsine and the trivalent arsenosugar exert their genotoxic effects via the production of ROS. In support of this notion, we found that plasmid DNA damage was inhibited when the nicking assay was performed with the trivalent arsenosugar under anaerobic conditions (unpublished observations). The trivalent arsenosugar was much less toxic (500× less toxic) in the NHEK cytotoxicity assay than MMA(III) or DMA(III). However, the trivalent arsenosugar was much more toxic than the pentavalent arsenosugar (at least 50-fold more toxic, based on IC25). Thus, in this study, the generalization that reduction of pentavalent arsenic to trivalent arsenic increases toxicity holds true, but other structural characteristics are even more important (the presence of the sugar group). When cytotoxicity is considered as an end point, the most likely mechanisms by which arsenic acts are probably induction of oxidative stress (for instance, causing DNA damage, as noted above) and inhibition of key cellular enzymes. If DNA damage serves as an important mechanism for cytotoxicity, then, from the results of the nicking assay, it might be expected that the trivalent arsenosugar would be as cytotoxic as DMA(III), as both compounds have equal potency in the nicking assay. Both MMA(III) and DMA(III), which were shown to be potent at damaging naked DNA in the DNA nicking assay (25), were subsequently shown to be potent clastogens in cells (35). However, in the case of the trivalent arsenosugar, bulk and hydrophilicity may preclude transport into the cell and nucleus, whereas MMA(III) and DMA(III), probably un-ionized, should readily diffuse passively across cell membranes. In future work, it would be useful to determine the relative uptake of the trivalent arsenosugar as compared to other arsenicals. A key difference between the trivalent arsenosugar compound and other trivalent arsenicals (i.e., arsenite MMA(III) and DMA(III)) is that the arsenic in the arsenosugar is surrounded by three alkyl groups and so cannot easily form complexes with enzyme sulfhydryls. Therefore, the toxicity of arsenite, MMA(III), and DMA(III) might be partially attributed to inhibition of key cellular enzymes, but the toxicity of the trivalent arsenosugar compound cannot be attributed to this. Thus, we believe that the trivalent

arsenosugar acts mostly by the induction of oxidative stress. It is also possible that the cytotoxicity of the trivalent arsenosugar might be due to metabolism of the compound to produce more toxic arsenicals, such as DMA(III). More detailed studies with trivalent arsenosugars should give useful clues to the mechanisms of arsenic toxicity. Although this is only the first study of a trivalent arsenosugar, it does raise some concerns with respect to seaweed consumption as a significant environmental source of human exposure to arsenicals. Human exposure to arsenosugars, from seaweed consumption, can be as high as 1 mg/day (7). Furthermore, in the United States, seaweed consumption is rapidly increasing because of increasing consumption of Asian foods and the use of seaweed products as a health supplement. Recently, the concern was voiced that if arsenosugars are metabolized to DMA(V), they may pose a health risk because DMA(V) is a complete carcinogen in rats (54). Indeed, it appears that the human metabolism of arsenosugars to DMA(V) is very efficient (14-17). Our work further suggests that arsenosugars may also be toxic via other mechanisms, by reduction to trivalent arsenosugars that are significantly more toxic than their pentavalent counterparts. However, the question remains, if arsenosugars do have a toxic effect, then why have no effects been observed in people who consume large quantities of seaweed? Although the epidemiology does suggest that arsenosugars are benign, that is not sufficient reason to consider them completely safe as there may be other confounding factors involved. Further research on arsenosugars is required. In summary, we demonstrated that a trivalent arsenosugar is significantly more active in the DNA nicking assay and a cytotoxicity assay than the corresponding pentavalent arsenosugar. Furthermore, the trivalent arsenosugar is as potent as DMA(III) in the DNA nicking assay but is much less cytotoxic than DMA(III). The reduction of pentavalent arsenosugars is expected to occur readily in vivo via reaction with thiol compounds. Therefore, pentavalent arsenosugars might be more of a risk to human health than was previously believed. These preliminary results provide an impetus for further study of arsenosugars and edible seaweed as a significant source of human exposure to arsenicals.

Acknowledgments This work was partially supported (P.A.) by a cooperative agreement between the National Research Council, Washington, DC, and the U.S. Environmental Protection Agency. We thank Don Delker, Mike Hughes, Jeff Ross, and R. Julian Preston for their comments on the manuscript. We are also grateful to Bert Mueller for technical assistance with the ICPMS, and Alan Tennant and Andrew Kligerman for their genotoxicity expertise. This manuscript has been reviewed by the National Health and Environmental Effects Research Laboratory at the U.S. Environmental Protection Agency and approved for publication. The views expressed in this paper are those of the authors and do not necessarily represent the views or policy of the U.S. Environmental Protection Agency.

Supporting Information Available Two figures and one table. This material is available free of charge via the Internet at http://pubs.acs.org.

Nomenclature DMA(V)

dimethylarsinic acid

DMA(III)

dimethylarsinous acid

DTE

dithioerythritol

KGM

keratinocyte growth medium

MMA(V)

monomethylarsonic acid

MMA(III) monomethylarsonous acid NHEK

normal human epidermal keratinocyte

SLS

sodium lauryl sulfate

TE

10 mM Tris-HCl, 1 mM EDTA, pH 8.0

TMAO

trimethylarsine oxide

ROS

reactive oxygen species

SC

supercoiled form of plasmid DNA

OC

open circular form of plasmid DNA (after a single-strand break)

L

linear form of plasmid DNA (after a doublestrand break)

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Received for review December 21, 2003. Revised manuscript received May 15, 2004. Accepted May 18, 2004. ES035440F