Radical Model of Arsenic(III) Toxicity: Theoretical and EPR Spin

Apr 22, 2014 - Arsenic is one of the most environmentally significant pollutants and a great global health concern. Although a growing body of evidenc...
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Radical Model of Arsenic(III) Toxicity: Theoretical and EPR Spin Trapping Studies Pedro L. Zamora,† Antal Rockenbauer,‡ and Frederick A. Villamena*,† †

Department of Pharmacology, The Davis Heart and Lung Research Institute, College of Medicine, The Ohio State University, Columbus, Ohio 43210, United States ‡ Department of Physics, Budapest University of Technology and Economics and MTA-BME Condensed Matter Research Group, Budafoki ut 8, 1111 Budapest, Hungary S Supporting Information *

ABSTRACT: Arsenic is one of the most environmentally significant pollutants and a great global health concern. Although a growing body of evidence suggests that reactive oxygen species (ROS) mediate the mechanism of arsenic toxicity, the exact mechanism remains elusive. In this study, we examine the capacity of trivalent arsenic species arsenous acid (iAsIII), monomethylarsonous acid (MMAIII), and dimethylarsinous acid (DMAIII) to generate ROS through a theoretical analysis of their structures, redox properties, and their reactivities to various ROS using a density functional theory (DFT) approach at the B3LYP/6-31+G**//B3LYP/6-31G* level of theory and by employing electron paramagnetic resonance (EPR) spin trapping studies using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as a spin trap. Results show that the oxidized forms (AsIV) are structurally more stable compared to the reduced forms (AsII) that impart elongated As−O bonds leading to the formation of AsIII and hydroxide anion. Enthalpies of one-electron reduction and oxidation indicate that increasing the degree of methylation makes it harder for AsIII to be reduced but easier to be oxidized. The order of increasing favorability for arsenical activation by ROS is O2 < O2•− < HO•, and the oxidation of DMAIII to DMAV is highly exoergic in multiple redox pathways with concomitant generation of radicals. This is followed by MMAIII and by iAsIII being the least favorable. Spin trapping studies showed a higher propensity for methylated arsenicals to generate radicals than iAsIII upon treatment with H2O2. However, in the presence of FeII,III, all showed radical generation where MMAIII gave predominantly Ccentered adducts, while acidified iAs III and DMAIII gave primarily HO-adducts, and their formation was affected in the presence of SOD suggesting a AsIII−OO/OOH radical intermediate. Therefore, our results suggest a basis for the increased redox activity of methylated arsenicals that can be applied to the observed trends in arsenic methylation and toxicity in biological systems.

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of functional enzymes.6 Once inhaled or ingested, inorganic arsenic is transformed to organic arsenicals via a series of enzyme-catalyzed successive reduction and methylation steps (Scheme 1).7 Initially believed to be a detoxification

INTRODUCTION As a naturally occurring element abundant in the earth’s crust, arsenic and its compounds are environmentally pervasive health hazards. Long known for its acute toxicity, arsenic is also understood to act as a potent carcinogen, even at parts per billion concentrations over long periods of exposure. Studies of long-term exposure in humans associate arsenic with increased risk of developing cancer of the bladder, lung, and liver to name a few.1 Besides a possible role in causing cancer, arsenic exposure has also been linked to cardiovascular diseases2 and neurotoxic effects3 where undernourished populations are believed to be at a higher risk for developing these conditions. Hence, the monitoring of levels of arsenic in food, water, and soil is of tremendous importance, particularly in developing countries.4,5 The urgency of this global health initiative has renewed interest in arsenic chemistry, particularly for the purposes of elucidating the mechanism of its carcinogenic properties. Inorganic arsenic in the environment typically exists as the pentavalent AsV (arsenic acid, H3AsO4) or the trivalent AsIII (arsenous acid, H3AsO3). Of the two forms, AsIII (or as it is more commonly named by its conjugate base, arsenites) is more toxic due to its ability to bind to the critical thiol groups © 2014 American Chemical Society

Scheme 1. Biomethylation of Arsenic in Humansa65

a

The reduction steps are GSH-mediated, while the oxidative methylation steps are catalyzed by S-adenosyl methionine (SAM) as the methyl donor to produce de-methylated S-adenosyl-L-homocysteine (SAH). Received: November 12, 2013 Published: April 22, 2014 765

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demonstrating the appropriateness of this level for the study of the H2AsO radical compared to that of other more powerful levels of theory.31,32 The 6-31G* basis set was chosen for its computational speed and its good agreement with experimentally determined bond lengths and angles for the third row atoms.33 These basis set calculations used the standard six Cartesian d functions. Vibrational frequency analyses (B3LYP/6-31G*) for each of the stationary points for all arsenic species and for DMPO and its spin adducts yielded no imaginary vibrational frequencies. A scaling factor of 0.9806 was used for the zero-point vibrational energy (ZPE) corrections with B3LYP/ 6-31G*.34 Samples Preparation. Sodium (meta)arsenite (NaAsO2), As2O3, and cacodylic acid (Me2AsO2H) were all commercially obtained. MeAsI2 was obtained from University of Alberta courtesy of Professor X. Chris Le. Stock solution of NaAsO2 was made in Dulbecco’s phosphate-buffered saline (pH 7.4) as well as with As2O3 with strong agitation. An acidified solution of As2O3 was also prepared using 1 equiv of As2O3 per 6 equiv of HCl. MMAIII was prepared according to the methods of Cullen et al.35 and Scott et al.36 with minor modification. Two grams of As2O3 was dissolved in 6 mL of 5 M aqueous solution of NaOH, and 13 mL of MeI was then added. The bilayered mixture was vigorously refluxed for 24 h. After reflux, 40 mL of ethanol was added, and the precipitated white disodium methylarsonate (MMAV) was collected by filtration. Two grams of MMAV was dissolved in 2 mL of water, and the solution was bubbled with SO2 gas (generated from dropwise addition of 5 M HCl to solid sodium bisulfite) for 5 min inside the hood with NaOH solution as the SO2 trap. The solution was then refluxed for an additional 4 h, and water was evaporated under reduced pressure. To the white residue was added 5 × 10 mL of hot benzene, the mixture was sonicated, and the benzene extracts were collected. Benzene was evaporated under reduced pressure and gave a pungent white solid (ESI-MS positive ion mode, m/z: [(CH3AsO)4 + 4H]4+ calcd for 427.8; found, 427.9). DMAIII was prepared according to the method described by Burrows et al.37 starting with 2 g of cacodylic acid and gave ∼1 mL of dimethyliodoarsine as a dense yellow oil. The oil was separated and dried over sodium sulfate. Small portions of solid of NaHCO3 were added to the oil until the effervescence ceased. Benzene was then added (5 × 2 mL) to the oil, the mixture was sonicated, and benzene extracts were collected and evaporated under reduced pressure yielding a pungent white solid (ESI-MS positive ion mode, m/z: (CH3)2AsO calcd for 120.96; found, 121.02). The MMAIII and DMAIII solids were immediately used for spin trapping by dissolving an appropriate amount in phosphate buffer. EPR Spin Trapping Studies. EPR measurements were carried out on an X-band spectrometer with a high sensitivity resonator at room temperature. General instrument settings are as follows: microwave power, 10 mW; modulation amplitude, 0.5 G; receiver gain 3.17−3.56 × 105; time constant, 82 ms; and time sweep, 42 s. The hyperfine splitting constants of the spin adducts were determined by simulating the spectra using an automatic fitting program.38 DMPO, FeSO4, and 80% H2O2 were all commercially obtained. All solutions for spin trapping were prepared using Dulbecco’s phosphate buffered saline solution at pH 7.4. The total volume of each solution used for the EPR measurement was 70 μL, and it was loaded into a 50 μL micropipet. In a typical experiment, a sample contains 100 mM DMPO, 100 mM AsIII species, 0.9% H2O2, and/or 1 mM FeSO4 in aerobic PBS. From here onward, FeSO4 will simply be referred to as FeII, although a mixture of FeII,III is present in solution. For all the experiments, H2O2 solution was added last to initiate radical production. All spectra presented here are of the same x- and y-scales for ease of comparison.

mechanism, it has been realized that the organic trivalent arsenic species monomethylarsonous acid (MMAIII) and dimethylarsinous acid (DMAIII) are the most toxic of all arsenic metabolites.8 Variations at certain points in the human arsenic methyltransferase (AS3MT) gene may cause differences in methylating ability between individuals,9 but data suggests a standard profile of urinary arsenic in humans as 10−30% inorganic, 10−20% MMA(III+V), and 60−80% DMA(III+V).8,10 The acute toxicity of inorganic AsV has been understood in its capacity to replace phosphate in biomolecules and to uncouple ATP via so-called arsenolysis. Compounding the lack of information is the apparent paradoxical dual action of arsenic as both carcinogen and chemotherapeutic agent. Despite the plethora of evidence linking arsenic exposure to the development of various cancers, inorganic arsenite remains one of the most effective treatments for certain cancers, including acute promyelocytic leukemia (APL).11,12 A growing body of evidence suggests that both the apoptosisinducing antitumor effects and the genotoxic effects of arsenite and other arsenic metabolites are mediated via ROS and oxidative stress pathways.13−15 Previous experiments have demonstrated that methylated trivalent arsenic species, MMAIII and DMAIII, induce the production of ROS in association with DNA damage.16 In the aforementioned study, the spin trapping agent DMPO and other free radical scavengers were shown to inhibit the DNA damaging activities of MMAIII and DMAIII suggesting a direct role of ROS in arsenic-induced genotoxicity. More recent evidence also suggests that biomethylation of arsenic and the resulting methylated arsenic species may be responsible for the observed carcinogenicity.16,17 However, it is currently not known which specific form (or forms) of arsenic is responsible for the observed antitumor and genotoxic effects due to the various transformations arsenic undergoes in vivo. Furthermore, there is a current lack of literature concerning the structural differences between the organic arsenicals and inorganic arsenites and the effects of structure on the capacity to generate ROS. Gaining greater understanding of the electronic properties, reactivity, and differences therein of the inorganic and organic arsenic radical species may help to elucidate arsenic’s toxic mechanisms. In this study, we used electron paramagnetic resonance (EPR) spectroscopy and the spin trap 5,5-dimethyl-1-pyrrolineN-oxide (DMPO) to investigate radical generation from iAsIII, MMAIII, and DMAIII in aqueous solution. We also performed theoretical calculations on the structures and thermodynamics of the arsenical species involved in several proposed mechanisms of oxidation and radical generation.

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EXPERIMENTAL PROCEDURES

Caution: All arsenic-containing chemicals are hazardous and should be handled with extreme care. Arsenites, arsenates, and methylated arsenicals are carcinogens which should be handled with appropriate laboratory protection. General Computational Methods. All calculations were performed at the Ohio Supercomputer Center using Gaussian 09.18 Density functional theory19,20 was applied in this study to determine the optimized geometry, vibrational frequencies, and single-point energy of all stationary points.21−24 Single-point energies were obtained at the B3LYP/6-31+G** level based on the optimized B3LYP/6-31G* geometries, and the B3LYP/6-31+G**//B3LYP/631G* level was used for natural population analyses (NPA).25 The effect of solvation on the gaseous phase calculations was also investigated using the polarizable continuum model (PCM).26−30 The B3LYP level was employed based on previous studies

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RESULTS Structural and Electronic Properties of Arsenic Species and Arsenic Radicals. Theoretical analysis of the electronic structures of the diagmagnetic AsIII species as well as the radical-based AsII and AsIV species was carried out at the PCM(water)/B3LYP/6-31G**//B3LYP/6-31G* level of theory. In this study, the hydrolyzed forms of As species 766

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Figure 1. Bond distances (Å), NPA spin (e), and charge (e) (in parentheses) densities of various arsenic species at the PCM(water)/B3LYP/631+G**//B3LYP/6-31G* level. Note the trend of increasing spin density on the hydroxyl O-atom and AsIV of the reduced and oxidized AsIII, respectively, with increasing methyl substitution.

were employed since these are the common forms that would exist in aqueous solution.39,40 As shown in Figure 1, all structures with the exception of AsII species have trigonal pyramidal geometry and examination of the calculated AsIII structures gave As−C bond distances of 1.94−1.96 Å and As− O bond distances of 1.79−1.83 Å, which are consistent with the X-ray crystallographic bond distances obtained from the cyclo(CH3AsO)4 of 1.906−1.933 Å for As−C and 1.78−1.82 Å for As−O.41 Reduction of AsIII to the paramagnetic AsII species shows significant elongation of As−O bonds leading to an As− O bond breaking process suggesting that the AsII species can lead to the formation of hydroxide and AsIII radicals as evidenced by large spin density distribution on As compared to that on O. The order of increasing spin density on As is DMAII (80%) < MMAII (85%) < iAsII (87%); while it is the reverse for the O atom: iAsII (1%) < MMAII (7%) < DMAII (19%), indicating that DMAII may have a higher propensity to form a hydroxyl radical than a hydroxide ion. Oxidation of AsIII species to the paramagnetic AsIV leads to shorter As−O bonds of 1.75 Å. The spin densities on AsIV atoms are lower compared to that of the AsII analogues, and the trend is reversed, that is, iAsII (46%) < MMAII (54%) < DMAII (64%); while for the O atoms, the spin densities are higher compared to that of the AsII analogues (i.e., 14%). Therefore, increasing methylation character was proportional to the increase in spin density on the As atom for AsIV species, while the reverse is true for AsII due to the gross partitioning of the As−O bond. Also worth noting is that there is a significant increase in the positive charge density on the AsIV atom in general and that the order of decreasing positivity follows that of the increasing degree of methylation, that is, iAsIV (2.13e) < MMAIV (1.90e) < DMAIV (1.66e). Table 1 shows the electron

Table 1. Electron Affinities and Ionization Potentials for Various Trivalent Arsenic Species at the PCM(Water)/ B3LYP/6-31+G**//B3LYP/6-31G* Level of Theory at 298 K

electron affinities ΔHred,aq in kcal/mol (eV) 43.90 (1.91) 42.58 (1.85) 34.27 (1.49)

substitutents iAs (R1 = R2 = OH) MMA (R1 = Me; R2 = OH) DMA (R1 = R2 = Me)

ionization potentials ΔHox,aq in kcal/mol (eV) 167.16 (7.25) 151.19 (6.56) 138.93 (6.03)

affinities and ionization potentials of the AsIII species as derived from the enthalpies of reduction and oxidation, respectively, at the PCM(water)/B3LYP/6-31G**//B3LYP/6-31G* level of theory. Results show that by increasing the degree of methylation, we can decrease the electron affinity and ionization potential of AsIII. This suggests that methylation makes it harder for AsIII to be reduced but easier for it to oxidize. Reactivity of the AsIII Species to Reactive Oxygen Species. We then examined at the same level of theory the mechanisms of AsIII oxidation by common oxidants including O2, O2•−, and H2O2/HO•, which have been previously established as causal agents in the oxidation of AsIII to AsV, with AsIV as the proposed intermediate.42 As shown in Tables 767

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2−4, the calculated free energies reveal an important trend: increasing methylation of the starting trivalent arsenic species is

Table 4. Free Energies (in kcal/mol) of Oxygen-Initiated Radical Oxidation of Arsenic at the PCM(Water)/B3LYP/631G**//B3LYP/6-31G* Level of Theory

Table 2. Free Energies (in kcal/mol) of Hydroxyl RadicalInitiated Radical Oxidation of Arsenics at the PCM(Water)/ B3LYP/6-31G**//B3LYP/6-31G* Level of Theory

proportional to the increasing exoergicity of oxidation reactions with ROS. Activation of arsenic by HO• is the most favorable with exoergic ΔG298 K,aq of addition ranging from −14.8 to −23.2 kcal/mol with DMAIII being the most reactive (Table 2). An exoergic electron-transfer reaction of arsenic with O2•− was only observed for DMAIII (ΔG298 K,aq = −3.1 kcal/mol) followed by an endoergic reaction with MMAIII (ΔG298 K,aq = 8.4 kcal/mol) and a highly endoergic one for iAsIII (ΔG298 K,aq = 25.4 kcal/mol) (Table 3). Oxygen activation of arsenics are unfavorable with highly endoergic ΔG298 K,aq values of 52.9− 81.4 kcal/mol (Table 4). The reactivity of oxygen with AsIV species is most favored for DMAIV (−18.9) followed by MMAIV (−16.7) and iAsIV (−10.7) with the generation of O2•−/HOO•. Thus, the order of increasing favorability for arsenic activation

by ROS is O2 < O2•− < HO•, and the total free energy for the oxidation of trivalent to pentavalent arsenic with concomitant generation of ROS byproducts is more energetically favorable with increasing methyl substitution. EPR Spin Trapping. iAsIII. No EPR signals were observed using DMPO alone, DMPO with H2O2, or DMPO with FeII (Figure S1, Supporting Information). Two different sources of iAsIII were used in this study, the sodium meta-arsenite (NaAsO2) and arsenic trioxide (As2O3). For As2O3, both acidified and neutral solutions were used. Using DMPO as spin trap and NaAsO2, no detectable spin adducts were observed by EPR from spin trapping in the presence of H2O2 alone, FeII alone, or H2O2 with FeII (data not shown). These results are perplexing given that spin trapping in a solution with DMPO, FeII, and H2O2 yields a strong hydroxyl radical signal (Figure S1, Supporting Information). These same results were obtained with As2O3 in neutral solution (data not shown). However, acidification of As2O3 with HCl could give a different behavior since this can drive the reaction toward the formation of more As(OH)3 as the decomposition product of AsCl3. Figure 2 shows that unlike with the unacidified solutions of As2O3 or NaAsO2, acidified As2O3 in the presence of FeII with H2O2 gave a strong OH-adduct signal with concomitant formation of about 15% of an HOO-like adduct which was not quenched in the presence of SOD but showed C-centered adduct formation in the presence of DMSO with a reduced amount of the HOOlike adduct (7%). Spin trapping using acidified As2O3 in the presence of H2O2 only showed a small but significant formation of the HO-adduct, but no adduct formation was observed with FeII alone in the presence of acidified As2O3. MMAIII. The use of MeAsI2 (or with Me2AsI) for spin trapping was not ideal due to oxidation of iodide to iodine by H2O2 that can complicate EPR spectral analysis. Spin trapping with DMPO in solutions of MMAIII or MMAIII with FeII yielded no detectable spin adducts (Figure S2, Supporting Information). Spin trapping in a solution of MMAIII with H2O2

Table 3. Free Energies (in kcal/mol) of Superoxide-Initiated Radical Oxidation of Arsenics at the PCM(Water)/B3LYP/ 6-31G**//B3LYP/6-31G* Level of Theory

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Figure 3. (a) EPR spectra from spin trapping using DMPO in solutions containing MMAIII and H2O2. (b) Computer composite simulation of the experimental spectrum showing (c) a C-centered adduct as the major species (76%) (g = 2.0054, aN = 16.4 G, and aβ‑H = 23.4 G) and (d) a minor species (24%) producing a triplet signal (g = 2.0055, aN = 15.5 G, and 86%). (e) The same as in panel (a) but in the presence of SOD. (f) The same as in panel (a) but in the presence of DMSO.

Figure 2. EPR spectra from spin trapping using DMPO in solutions containing acidified iAsIII, FeIII, and/or H2O2 in the presence and absence of SOD or DMSO. (a) Computer composite simulation of the experimental spectrum shows 86% HO-adduct and 14% HOO-adduct (g = 2.0056, aN = 14.8 G, aβ‑H = 10.9 G, and aγ‑H = 2.0 G). (b) Pure HO-adduct. (c) 95% HO-adduct. (d) No signal. (e) 86% HO-adduct and 14% HOO-adduct. (f) 68% HO-adduct, 22% alkyl-adduct, 7% HOO-adduct, and 3% triplet.

species similar to those in the same solution without DMSO and with a similar signal intensity. Figures S4 and S5 (Supporting Information) show the simulated spectrum of the MMAIII/FeII/H2O2 system in the presence of SOD and DMSO, respectively, both showing the unidentified y-signal and the triplet species as minor species. DMAIII. Spin trapping with DMPO in solutions of DMAIII or DMAIII with FeII yielded no detectable spin adducts (Figure S6, Supporting Information). Shown in Figure 5 is the spin trapping results from a solution of DMAIII with H2O2 which yielded a major species of the HO-adduct (80%) and a minor species of the C-centered adduct (20%) and no detectable spin adducts in the presence of either SOD or DMSO. Spin trapping in a solution containing DMAIII, FeII, and H2O2 also yielded the HO-adduct (68%) primarily with a minor species of the Ccentered adduct (32%) (Figure 6). In the presence of SOD, it yielded similar species but with enhanced signal intensity. However, in the presence of DMSO it yielded a strong signal due primarily to the C-centered adduct (89%), with a minor

yielded predominantly C-centered adducts (76%), with minor species (24%) producing a triplet signal (Figure 3). Spin trapping of a solution of MMAIII, H2O2, and superoxide dismutase (SOD) yielded no detectable spin adducts, while spin trapping of a solution containing MMAIII, H2O2, and DMSO yielded species similar to those in the solution with MMAIII and H2O2 but with a markedly diminished signal intensity and an additional minor (∼16%) species that is still yet to be identified and was assigned as the x-signal (see Figure S3, Supporting Information). Spin trapping of a solution containing MMAIII, FeII, and H2O2 yielded an alkyl radical as a major species (68%), with minor species (18%) giving a triplet signal with g = 2.0055, aN = 15.5 G, and a still yet to be identified signal which is referred to as the y-signal with g = 2.0056, aN = 16.5 G, and aβ‑H = 15.6 G (Figure 4). Spin trapping using the same solution in the presence of SOD yielded similar species but with markedly increased signal intensity. In the presence of DMSO, it yielded 769

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Figure 5. (a) EPR spectra from spin trapping using DMPO in solutions containing DMAIII and H2O2. (b) Computer composite simulation of the experimental spectrum showing (c) a HO-adduct as the major species (80.32%) (g = 2.0055, aN = 15, and aβ‑H = 15 G) and (d) a C-centered adduct as the minor species (19.7%) (g = 2.0054, aN = 16.4 G, and aβ‑H = 23.4 G). (e) The same as in panel (a) but in the presence of SOD. (f) The same as in panel (a) but in the presence of DMSO. Figure 4. (a) EPR spectra from spin trapping using DMPO in solutions containing MMAIII, FeII, and H2O2. (b) Computer composite simulation of the experimental spectrum showing (c) a C-centered adduct as the major species (67.5%) (g = 2.0054, aN = 16.4 G, and aβ‑H = 23.4 G. (d) A minor species (17.8%) producing a triplet signal (g = 2.0055 and aN = 15.5 G) and (e) another minor species (14.8%) producing a “y-signal” (g = 2.0056, aN = 16.5 G, and aβ‑H = 15.6 G). (f) The same as in panel (a) but in the presence of SOD. (g) The same as in panel (a) but in the presence of DMSO.

reactivity particularly with nucleophiles such as thiols36,44 upon oxidation of AsIII by ROS. Calculated ionization potential (IP) and electron affinity (EA) for arsenics as shown in Table 1 indicate that methylation allows facile oxidation but a more difficult reduction. Computational studies on the substituent effect on the phosphorus (an atom with a common electronic resemblance to As) redox properties show lower adiabatic IP (166.2 kcal/mol) and EA (30.5 kcal/mol) for POMe2 than PO(OH)2 with an IP of 184.6 kcal/mol and an EA of 55.1 kcal/mol.45 Moreover, increasing the methylation of phosphorus has the same effect on the adiabatic IP and EA values, that is, for IP (in kcal/mol), PH2 (218.0) > PHCH3 (197.1) > P(CH3)2 (181.2); and for EA (in kcal/mol), PH2 (16.1) > PHCH3 (9.5) > P(CH3)2 (4.9).45 This indicates that the influence of methyl substitution onto the As atom allows for the stabilization of the global positive character of the oxidized arsenic. This lower IP and EA for methylated arsenicals could favor the formation of a more reactive AsIV species which can have implication for their toxicity. For example, the lethal dose low (LDL0, in mmol kg−1) reported for 4-XC6H4As(O)(OH)2 was 0.46 for X = Me and 2.06 X = HO.46 Trivalent arsenics undergo single electron transfers to ROS such as HO• to form the AsIV species and have been demonstrated to be highly kinetically favorable with rates in the order of (1−9) × 109 M−1 s−1.47,48 This further supports the exoergic (ΔG298 K,aq = −14.8 kcal/mol) HO• oxidation of AsIII species as shown in Table 2. The favorability of oxidation of AsIII by O2•− (ΔG298 K,aq = −3 to 25 kcal/mol) is intermediate between that of HO• and O2, which is not surprising considering that O2•− is a weak oxidant and that

species of the HO-adduct (11%) (Figure 6). Figures S7 and S8 (Supporting Information) show the simulated spectrum of the DMAIII/FeII/H2O2 system in the presence of SOD and DMSO, respectively.

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DISCUSSION Optimization at the B3LYP/6-31G* level gave structures whose geometries and bond lengths are consistent with the reported X-ray structure for cyclo-(CH3AsO)441 indicating the reasonable accuracy of this level of theory in predicting the arsenic structures. One-electron reduction and oxidation of the AsIII compounds yielded the radicals, AsII and AsIV, species where the former exhibited significant elongation of the As−O bonds that can lead to the formation of the hydroxide ion and arsenic(III) radical suggesting the instability of these structures. He et al. identified the presence of the H2AsIIIO radical in the gas phase by laser fluorescence and wavelength resolved emission spectroscopy.43 However, the optimized geometries of AsIV species yielded intact structures with spin densities mainly localized around the As atom (∼50%) and the O atoms (∼15%), and with a high positive charge on the As atom. This high positive charge can have significant implication in their 770

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free energy of oxidation of AsIII by O2 is highly endoergic, oxidation of AsIV species by O2 to form HO2•/O2•− has been shown to occur at a higher rate in acidic pH according to eqs 3 and 4:48 As IV ‐O2 + H+ → As V + HO•2 /O•− 2

k ≈ 1010 M−1s−1 (3)

IV

−

V

As ‐O2 + HO → As +

O•− 2

4

−1 −1

k ≈ 10 M s

(4)

•

Therefore, in oxidative conditions, HO could originate mostly from the H2O2 reaction with low valent transition metals via Fenton chemistry and is the relevant oxidant for the activation of AsIII. This is more so with the methylated AsIII analogues, and the activation of O2 by AsIV favorably forms HO2•/O2•−. Although the addition of •AsIV(OH)3 was found to be exoergic with ΔG298 K,aq = −20.1 kcal/mol, none of the AsIII species yielded an As-centered adduct which is expected to give the additional splitting pattern due to the 75As nucleus with I = 3/2. There are major differences observed, however, in the spin trapping behavior between iAsIII, MMAIII, and DMAIII. Addition of H2O2 to As2O3 in neutral solution or NaAsO2 did not give an EPR signal but exhibited a very weak HO-adduct signal using the acidified solution of As2O3 (Figure 2b), while MMAIII gave primarily a robust C-centered adduct (Figure 3) and DMAIII primarily an HO-adduct (Figure 5). This difference in behavior in the radical generating capability of methylated-As compared to iAsIII in the presence of H2O2 alone is consistent with the trend in calculated IPs for various AsIII where MMAIII and DMAIII are shown to be easier to oxidize than iAsIII. The nature of radicals produced from MMAIII and DMAIII with H2O2 are quite different, although both are quenched in the presence of SOD and in the presence of DMSO (a significant decrease in intensity was observed in the case of MMA III). The predominance of the C-centered adduct in the MMAIII/ H2O2 system could only be attributed to the generation of a methyl radical presumably from the MMAIII itself via displacement by the HO2• formed through the complexation of H2O2 to the As atom (see Scheme 2). The proposed

Figure 6. (a) EPR spectra from spin trapping using DMPO in solutions containing DMAIII, FeII, and H2O2. (b) Computer composite simulation of the experimental spectrum showing (c) HO-adduct as the major species (68.1%) (g = 2.0055, aN = 15.1, and aβ‑H = 14.6 G) and (d) a C-centered adduct as the minor species (31.9%) (g = 2.0054, aN = 16.4 G, and aβ‑H = 23.4 G). (e) The same as in panel (a) but in the presence of SOD. (f) The same as in panel (a) but in the presence of DMSO.

Scheme 2. Proposed Mechanisms for the Formation of Hydroxyl and Methyl Radicals from One-Electron Oxidation of DMAIII and MMAIII by H2O2, Respectively, Showing the As-Peroxyl Complex Intermediate

whether O2•− could actual oxidize AsIII is still ambiguous.49 We also explored the favorability of the O2•− addition reaction to AsIII and showed it to be less exoergic with ΔG298 K,aq = −2.5 kcal/mol and that of the addition of HO2• with ΔG298 K,aq = −2.5 kcal/mol according to eqs 1and 2, respectively. The

formation of the HO2−As complex as an intermediate for radical formation is attractive due to the inhibition of radical formation in the presence of SOD. Organoarsenic peroxy radicals have been generated and detected by EPR from the reaction of organoAsIV and O2,50 and dimethylarsenic peroxy radicals (CH3)2AsOO• have been implicated as reactive intermediates in oxidative damage to DNA.51 Although the

addition of O2•− or HO2• to AsIII only resulted in long As−O bond distances of 2.37 and 2.72 Å, respectively, indicating that the interactions are more electrostatic in nature. Although the 771

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proposed mechanism of methyl radical formation from MMAIII is hypothetical at this point, this warrants further investigation. Spin trapping using the AsIII/H2O2/FeII system gave EPR signals for iAsIII, MMAIII, and DMAIII that are predominantly HO-adducts (Figure 2), C-centered adducts (Figure 4), and HO-adducts (Figure 6), respectively. Using neutral solutions of As2O3 and NaAsO2, we observed that no EPR signal was detected in the presence of H2O2 with FeII perhaps due to the high reactivity of iAsIII to HO• hence competing with DMPO for HO•. Dilution of iAsIII by 1/10th of the initially used concentration still gave a weak HO-adduct signal. In the case of acidified solution of iAsIII, an intense HO-adduct signal was observed, and this intensity was significantly reduced in the presence of SOD (Figure 2e) due perhaps to the formation of the HO2−As intermediate and upon O−O cleavage could lead to the formation of HO•, which was confirmed by the formation of C-centered adducts in the presence of DMSO (Figure 2f). The weak signal intensity for the C-centered adduct formed with MMAIII (Figure 4a) was enhanced in the presence of SOD (Figure 4f) and with only a slight increase in intensity with DMSO (Figure 4g). A similar effect with SOD was observed for DMAIII (Figure 6e) with enhancement of HO-adduct formation, while a C-centered adduct in the presence of DMSO was formed indicating that the observed signal originates from HO•. The rationale for the enhanced signal formation in the presence of SOD in the AsIII/H2O2/FeII system is complicated by the fact that FeII,III has been shown to also catalyze the oxidation of AsIII by oxygen or H2O2;52 therefore, the formation of the HO2−As intermediate is compromised with high valent As, leading to less radical formation. This suggests a crucial role of the peroxyl/ hydroperoxyl intermediate in radical generation with H2O2. In spite of the complexity and diversity of the spin trapping behavior observed in this work, these results may corroborate previous findings that methylated arsenic species MMAIII and DMAIII are more potent toxicants53−55 than iAsIII due to their ability to generate radicals. Recent experiments have also shown that hepatocytes of catalase-knockout mice exposed to H2O2 or MMAIII exhibit significantly higher occurrences of DNA strand breaks than in wild type mice with the intact gene for catalase (catalase catalyzes the conversion of H2O2 to oxygen and water, thus limiting the production of damaging hydroxyl radical).56 In the same study, EPR spin trapping with DMPO of the cell contents showed the presence of HO• in hepatocytes of mice exposed to H2O2 or MMAIII, the signal for which was greatly reduced in wild type mice versus catalase-knockout mice. This suggests that the genotoxic effects of organic arsenical exposure are mediated by ROS, in particular HO•. Moreover, the use of common exogenous antioxidants such as resveratrol can indirectly attenuate As2O3-induced hepatoxicity by increasing antioxidant enzyme activities and GSH/GSSG ratio57 or through direct chelation in the case of α-lipoic acid to reduce As2O3-induced cardiotoxicity.58 However, studies in cell-free growth media show vitamin C to be prooxidative by increasing the rate of AsIII oxidation to AsV,59 while in rats vitamin E showed a protective effect by reducing DNA damage and the TNF-α level, and inhibiting caspase cascade activation.60 In consideration of recent studies that link arsenic exposure to oxidative stress and ROS generation, our findings further show that organic arsenicals generate various ROS when exposed to common oxidants in solution. Both H2O2 and O2 have previously been demonstrated to oxidize AsIII to AsV and DMAIII to DMAV,42,61 and our theoretical calculations

suggesting a basis for the increased redox activity of methylated arsenicals can be applied to the observed trends in arsenic methylation and toxicity. That methylation appears to localize electron spin density on arsenic centered radicals may explain the increasing exoergicity of radical oxidation proportional to increasing methyl substitution of arsenic. In turn, the increasing exoergicity of methylated arsenical oxidation in water and thus increasing ROS production may help to explain the trends in arsenic toxicity. Another study compared gene expression and cell viability among iAsIII, MMAIII, and DMAIII in human keratinocytes.62 After analyzing trends in cell proliferation, apoptosis, and the expression of genes promoting cellular oxidative defense, apoptosis factors, and growth factors, the study’s authors concluded that MMAIII was the most cytotoxic species (LC50 = 2.8 μM), followed by DMAIII (LC50 = 5.6 μM), and then iAsIII (LC50 = 17 μM). In human bladder cancer cells, treatment with DMAIII or DMMTAV (dimethylmonothioarsinic acid) drastically reduced GSH and p53 levels compared to those of the control cells. While iAs also decreased p53, it was not as drastic as DMAIII, and treatment actually increased GSH levels compared to those of the control.14 One should not compare these results with the previously described study in human keratinocytes as the cancer cells have already demonstrated a compromised oncosuppresive system, so further toxic insult is not likely to produce the response seen in normal cells exposed to high levels of arsenic. The authors of this study suggest that the increased toxicity of the organic arsenicals was occurring via a cycle of GSH depletion from the reduction of DMAV to DMAIII and subsequent regeneration of the trivalent form. Our theoretical results corroborate this finding, where we found the oxidation of DMAIII to DMAV to be highly exoergic in multiple redox pathways, and our results also suggest that observations63,64 of ROS formation in cells exposed to iAsIII may be due to an indirect metabolic effect of iAsIII rather than direct radical formation.

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ASSOCIATED CONTENT

S Supporting Information *

Thermodynamics parameters and the complete reference for ref 18; and additional spin trapping EPR spectra and ESI-MS for MMAIII and DMAIII. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: 614-292-8215. E-mail: [email protected]. Funding

This work was supported in part by an allocation of computing time from the Ohio Supercomputer Center. We thank the OSU Department of Pharmacology Research Incentive for financial support. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

We thank Professor William R. Cullen of University of British Columbia and to Professor X. Chris Le of University of Alberta for providing MeAsI2 and helpful advice for the synthesis of MMAIII. 772

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ABBREVIATIONS ROS, reactive oxygen species; iAsIII, arsenous acid; MMAIII, monomethylarsonous acid; DMAIII, dimethylarsinous acid; DFT, density functional theory; EPR, electron paramagnetic resonance; IP, ionization potential; EA, electron affinity

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