Environ. Sci. Technol. 2005, 39, 2169-2176
Thermodynamic Analysis of Arsenic Methylation P A U L M . D O M B R O W S K I , †,‡ W E I L O N G , † K E V I N J . F A R L E Y , †,§ J O H N D . M A H O N Y , † JOSEPH F. CAPITANI,| AND D O M I N I C M . D I T O R O * ,§,⊥ Department of Environmental Engineering and Department of Chemistry, Manhattan College, Riverdale, New York 10471, HydroQual, Inc., Mahwah, New Jersey 07450, Department of Civil and Environmental Engineering, University of Delaware, Newark, Delaware 19716, and Metcalf and Eddy, Inc., Wakefield, Massachusetts 01880
The Challenger mechanism for the methylation of arsenic is a repeating sequence of a two-electron reduction of pentavalent arsenic As(V) species to trivalent arsenic As(III) species followed by a methylation-oxidation reaction forming the successive methyl As(V) species. This unusual oxidation-reduction sequence prompted an examination of the thermodynamics of these reactions. Quantum chemical methods are employed to estimate the thermodynamic parameters for the methyl arsenic species. The sequence is thermodynamically favored at neutral pH for redox potentials with pe < 0 and methyl cation activities pCH3+ < -3 to -7 depending on the precise situation analyzed. The observed distribution of methyl arsenic species in human urine, which is remarkably constant across many studied populations, can be reproduced using an equilibrium model if the formation of TMA species is prevented. The estimated thermodynamic parameters are sufficiently accurate to evaluate questions of thermodynamic plausibility but not the precise details of speciation.
Introduction In the commonly accepted biomethylation pathwaysthe Challenger mechanismsarsenic undergoes an alternating sequence of reduction and oxidative methylation reactions (1-4) schematically represented in Figure 1 (4). Considerable attention has been given to these reactions that transform inorganic to organic arsenic species. Once believed to be a detoxification mechanism for arsenic, the methylated species may actually be more toxic and reactive along the methylation pathway (5, 6). The initial reaction is the reduction of inorganic arsenate, AsO(OH)3 denoted by iAs(V), to arsenite, As(OH)3 denoted by iAs(III), via the loss of the oxygen double bonded to the arsenic, AsdO. This reduction is followed by the methylationoxidation of iAs(III) to MMA(V), monomethylarsonic acid CH3AsO(OH)2, The AsdO bond is reformed, and OH is replaced with CH3. The sequence repeats with the reduction of MMA(V) to MMA(III), monomethylarsonous acid CH3* Corresponding author telephone: (302)831-4092; fax: (302)8313640; e-mail:
[email protected]. † Department of Environmental Engineering, Manhattan College. ‡ Metcalf and Eddy, Inc. § HydroQual, Inc. | Department of Chemistry, Manhattan College. ⊥ University of Delaware. 10.1021/es0489691 CCC: $30.25 Published on Web 02/19/2005
2005 American Chemical Society
FIGURE 1. Schematic of the Challenger pathway for biomethylation, with the alternating sequence of reduction and oxidative methylation of arsenic (4). As(OH)2, followed by the methylation-oxidation of MMA(III) to form DMA(V), dimethylarsinic acid (CH3)2AsO(OH)1. The sequence continues to form DMA(III), dimethylarsinous acid, (CH3)2As(OH)1, then TMA(V), trimethylarsine oxide (CH3)3AsO, and finally TMA(III), trimethylarsine (CH3)3As. Challenger proposed this sequence of reactions from an analysis of methylation mechanisms for arsenic, selenium, and tellurium by various molds and bacteria (1). Improved analytical techniques that distinguish and quantify the methyl arsenite and methyl arsenate species have strengthened Challenger’s conclusions (7-10). The reducing agent is a thiol, and glutathione is involved in the reduction step (3). S-Adenosylmethionine (SAM) is the methyl group donor, as it is for many biological reactions (4, 11). In humans iAs(V) and MAs(V) are reduced enzymatically by specific reductases (12). A specific methyl transferase was believed to be required to catalyze each step (3). However, more recent evidence points to one enzyme that can catalyze both mono- and dimethylation reactions (11) and possibly the complete series of reactions converting iAs(III) to methylated species (6). This unusual oxidation-reduction sequence that alternates between oxidized As(V) and reduced As(III) species as methylation proceeds (iAs(III) f MMA(V) f MMA(III) f DMA(V) etc.) prompted an investigation of the thermodynamics of the reactions and the speciation that would be expected at equilibrium. To carry out the analysis, the thermodynamic properties of the methyl arsenic species that have not been experimentally determined need to be estimated. The prediction of thermodynamic properties has been a long-standing goal of computational quantum chemistry (13). Improvements in computing power and computational methods have progressed to the point that reasonably accurate estimates of the Gibbs free energy of formations accurate to approximately 2-5 kcal/mol for neutral molecules in the aqueous phasescan be made (13). Occurrence and Distribution of Methyl Arsenic. Methyl arsenic compounds are commonly observed when arsenic is present. A summary is presented in Figure 2A and Table S1. Earlier studies did not distinguish between the oxidation statesAs(III) or As(V)sof inorganic or methyl arsenic species. In those data sets where oxidation state was differentiated, the arsenic species below are denoted with (III) or (V). In both fresh and marine waters containing arsenic, MMA(V) and DMA(V) are the principal methyl arsenic species observed (14-17). MMA(III) and DMA(III) have been measured in lake water but only in minor amounts (16). MMAs, DMAs, and TMAs have been measured in the freshwater green algae Chlorella vulgaris when cultured in arsenite containing VOL. 39, NO. 7, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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tamination (25). Consequently, there is an abundance of analytical data for methyl arsenic speciation in human urine (Tables S2-S3). Arsenic speciation is remarkably uniform throughout the reported samples of populations exposed to inorganic arsenic (Figure 2B,C), and it is independent of the concentration being ingested (Tables S2-S3). In human urine, generally 10-20% of total arsenic is iAs, 10-20% is MMA, and 60-80% is DMA (21, 25, 28, 32). This distribution is observed in virtually all of the studies examined including a population in China whose only exposure to arsenic is airborne As2O3 (33). One notable exception is native populations in the Andes Mountains that have been exposed to arsenic in their drinking water for thousands of years who excrete very low fractions of MMA, ∼2%, and increased DMA (34). An average distribution computed from several recent urinary methyl arsenic studies that did differentiate between As(III) and As(V) species was used to compare to equilibrium species distributions: 7% iAs(V), 12% iAs(III), 12% MMA(V), 3% MMA(III), 55% DMA(V), and 11% DMA(III) (7-10, 24, 30) (Figure 2C). Humans also consume arsenic in marine seafood and seaweed, mostly as arsenobetaine ((CH3)3AsCH2COO). It is believed to leave the body metabolically unchanged (25, 35). However, elevated DMA concentrations are measured in urine after seafood consumption (27, 36). Volunteers from the studies listed in Table S3 were asked to abstain from seafood consumption for at least 2 days prior to giving urine samples to eliminate the effects of dietary intake on analysis. Therefore, arsenobetaine is not included in the equilibrium calculations presented below.
Methods FIGURE 2. (A) Methyl arsenic speciation in various natural media: hamster liver tissue (23), human hepatocytes (9), human blood plasma (10), Lake Biwa, Japan (16), Tosa Bay, Japan (17) (Table S1). (B) Methyl arsenic distribution in human urine (Table S2). Distribution of iAs, total MMA, and total DMA. (C) Methyl arsenic distribution in human urine (Table S3). media. However, the TMA species were not observed until after 36 h of exposure to arsenic (18). The direct methylation of arsenic by algae is observed in batch experiments under axenic conditions (19). Certain bacteria and fungi are known to produce dimethylarsine gas, AsH(CH3)2, and TMA(III) gas in the presence of inorganic arsenic (1, 2, 4, 20). In mammals, arsenic methylation occurs primarily in the liver, but the reactions may occur in other tissues (21). In both hamster liver tissues (22, 23) and human hepatocytes (9), dimethylated arsenic species, DMA(V) and DMA(III), are the most prevalent methyl arsenic species. In human red blood cells, ∼80% of the total arsenic detected was DMA(V) (24). In human blood plasma (24) and urine (7-10, 24-30), the primary arsenic species is detected is DMA along with a smaller fraction of MMA. More recent analyses have shown that the largest part of the DMA and MMA in urine is DMA(V) and MMA(V), respectively (7-10, 24, 30), which have both been shown to be less acutely toxic than inorganic arsenite (7, 8, 31). Mice and dogs excrete very little MMA in their urine; the arsenic is almost entirely as DMA (21). For the many biological media that have been sampled, DMA(V) is the most commonly measured methyl arsenic species (Figure 2A). Arsenic in Human Urine. Contaminated drinking water is the usual exposure route for humans, and 60-80% of ingested inorganic arsenic is excreted in urine (25, 32). Urine has been shown to be the most reliable biological indicator of arsenic exposure and metabolic conversion, as arsenic remains in the bloodstream for only a few hours after ingestion, and hair and nails are subject to surface con2170
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Quantum Chemical and Thermodynamic Computations. Thermodynamic data are available for iAs(III) and iAs(V) (37) but not for the methyl arsenic species. The necessary values were computed using quantum chemical methods. For molecules in the aqueous phase, a two-step procedure is used. First the molecule is analyzed in the gas phase, and then the solvation energy is computed. For the gas phase computations, the Gaussian 2 (G2) method (38) was chosen within the Gaussian98 (39) suite of programs. G2 is a compound method that calculates the thermodynamic properties of molecules at their equilibrium geometries by combining the results of several different computational methods to approximate a single high-level calculation that would otherwise be computationally impractical. The errors in G2 thermodynamics estimates has been extensively investigated (38) and found to be within 2-5 kcal/mol for small molecules containing atoms in the first two complete rows of the periodic table (H-Ar) and for molecules containing third row (Ga-Kr) atoms (40). The Gibbs free energy of formation in the gas phase ∆Gfqc(g) is calculated using the G2 method for all the arsenic species as well for the arsenic, carbon, hydrogen, and oxygen atoms (Table 1A). The standard state employed in quantum chemical computations is the nuclear skeleton with all the electrons at infinite distance. This is denoted by the superscript “qc”. A more useful quantity is the free energy of atomization ∆Gfa(g) for which the standard states are the gaseous neutral atoms. These atomization energies ∆Gfa(g) (Table 1B) can be used for the thermodynamic analysis. However, it is useful to convert to ∆Gf°(g) using the standard states employed in the National Bureau of Standards (NBS) tabulation (11) so that comparisons with experimental values can be made. The conversion can be made since the atomization energy of a molecule will be the same regardless of the standard states used since only energy differences are involved (Supporting Information, eqs S1-S4).
TABLE 1. Thermodynamic Dataa A ∆Gfqc(g) (hartree)
B ∆Gfa(g) (kcal/mol)
C ∆Gf°(g) (kcal/mol)
D ∆Gsolv° (kcal/mol)
E ∆Gf°(aq) (kcal/mol)
F ∆Gf°(aq) (kcal/mol)
G ∆Gf°(aq) (kcal/mol)
H ∆Gf°(aq) (kcal/mol)
AsO(OH)3 As(OH)3 AsO(CH3)(OH)2 As(CH3)(OH)2 AsO(CH3)2(OH) As(CH3)2(OH) AsO(CH3)3 As(CH3)3 H2O CH4
-2536.738666 -2461.611654 -2500.811413 -2425.670235 -2464.875436 -2389.730181 -2428.936283 -2353.799971 -76.349648 -40.428194
-514.86 -596.45 -753.06 -662.58 -904.20 -811.15 -1053.34 -965.91 -207.93 -368.18
Aqueous Phase Species -166.80 -16.24 -140.59 -7.87 -121.21 -10.90 -86.11 -5.30 -70.14 -7.55 -32.48 -1.53 -17.08 -5.89 14.97 3.60 -55.39 -6.05 -13.44 1.81
-183.04 -148.46 -132.11 -91.41 -77.69 -34.01 -22.97 18.57 -61.44 -11.63
-183.08 -152.95 na na na na na na -56.675 -8.21
-183.04 -148.46 -133.49 -97.24 -80.42 -41.19 -27.04 10.04 -61.44 -11.62
-183.04 -148.46 -132.15 -95.90 -77.73 -38.50 -23.01 14.08 -56.675 -8.21
As(g) C(g) H(g) O(g)
-2234.268273 -37.798846 -0.510654 -74.996982
Gas-Phase Species 62.4 160.434 48.577 55.385
a A: G2 free energy (1 hartree ) 627.51 kcal/mol). B: G2 atomization energy eq S2. C: G2 atomization energy using NBS standard state eq S4. D: COSMO solvation energy. Computed using Density Functional Theory (DFT) with the B3LYP functional and the 6-311++G(3df,2pd) basis set that is large enough to reach the basis set limit. E: G2-COSMO estimates. F: From NBS tables (11). G: G2-COSMO estimates using reaction 2. H: G2-COSMO estimates using reaction 2 with ∆Gf°(aq) for H2O and CH4 from NBS tables (11).
TABLE 2. Thermodynamic Parameters and Stoichiometric Coefficients νij i /j
species
1 2 3 4 5 6 7 8 9 10 11
AsO(OH)3 As(OH)3 MMA(V) MMA(V)MMA(V)2MMA(III) DMA(V) DMA(V)DMA(III) TMA(V) TMA(III) a
∆Gf°(aq)a AsO43- CH3+ e- H+ H2O 5 log10 K (kcal/mol) 1 2 3 4 -183.04 -148.56 -132.11 -126.52 -114.65 -91.41 -77.69 -69.23 -34.01 -22.97 17.57
1 1 1 1 1 1 1 1 1 1 1
0 0 1 1 1 1 2 2 2 3 3
0 2 2 2 2 4 4 4 6 6 8
3 5 4 3 2 6 5 4 7 6 8
0 -1 -1 -1 -1 -2 -2 -2 -3 -3 -4
21.34 40.81 25.53 21.44 12.73 37.25 27.12 20.99 36.73 28.64 40.48
Table 1E.
The aqueous phase free energy of formation ∆Gf°(aq) is obtained from the gas phase ∆Gf°(g) by adding the solvation energy ∆Gsolv°, the energy liberated by transferring a gasphase molecule to the aqueous phase:
∆Gf°(aq) ) ∆Gf°(g) + ∆Gsolv°
(1)
The solvation energies are computed using COSMO (41), a polarizable continuum model that is a component of the Gaussian98 suite of programs (Table 1D). For neutral species the estimation errors are again approximately 2-5 kcal/mol (41). Therefore, the overall error in the estimation procedure is on the order of 3-5 kcal/mol. It is difficult to be more precise about the absolute level of accuracy since arsenic is in the fourth row of the periodic table, which has not been as thoroughly explored as the rows 1-3. Both MMA(V) and DMA(V) exist primarily as singly deprotonated anions at neutral pH. Aqueous formation energies for these anions are computed using published pKa values (16) for MMA(V) and DMA(V), see eqs S5-S7 in the Supporting Information. The accuracy of the free energy estimates can sometimes be improved using experimentally determined ∆Gf°(aq) as part of the estimation procedure by using a hypothetical reaction for which all but one of the ∆Gf°(aq) are known (42). For example, each methyl arsenic species can be formed
FIGURE 3. Gibbs free energy of reaction for methylations (striped bars) and reductions (solid bars). (A) Estimated using G2-COSMO atomization energy energies (Table 1E). (B) Estimated from using experimental values for water, methane and inorganic arsenic species (Table 1H). from inorganic arsenic, methane, and water:
AsO(OH)3 + CH4 f CH3AsO(OH)2 + H2O
(2)
For DMA(V) and TMA(V), the methane and water stoichiometric coefficients are 2 and 3, respectively. The trivalent methyl arsenic species are formed using As(OH)3. The VOL. 39, NO. 7, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 4. Equilibrium methyl arsenic species distribution for As(V) (A, C) and As(III) (B, D) vs redox potential (pe) at neutral pH for various methyl cation activities. (A, B) pCH3+ ) -2, (C, D) pCH3+ ) -7. Parameters in Table 2. reaction free energy change ∆Grxn can be computed as the difference in free energies of the products and reactants using the G2-COSMO computed ∆Gf°(aq) values (Table 1E). This reaction energy is likely to be more accurate than the individual G2-COSMO energies since reaction 2 involves breaking and forming only two bonds. Therefore, only two bond energies need to be estimated, whereas all the bond energies need to be estimated to compute the atomization energies. Using the known experimental values of ∆Gf°(aq) for H2O, CH4, AsO(OH)3, and As(OH)3 (11), reaction 2 and similar reactions are used to calculate ∆Gf°(aq) for the methyl arsenic species (Table 1G). All the computed ∆Gf°(aq) values (Table 1G) are equal or lower in free energy than the direct G2-COSMO estimates (Table 1E). One additional correction was made in order to improve the accuracy of the estimates. There is approximately a 4 kcal/mol difference between the G2-COSMO computed and experimental values for water and methane (Table 1E-F). If the experimental values are used in reaction 2, then only the differences in computed and experimental values for the inorganic arsenic species, AsO(OH)3 and As(OH)3, are included (Table 1H). The sensitivity of the results to these various estimates is discussed below. Chemical Equilibrium Computations. The chemical equilibrium computations are performed using the equilibrium equations in tableau form (43). The components are AsO4-3, CH3+, e-, H+, and H2O, and all necessary parameters for the species included in the equilibrium calculations are shown in Table 2. The concentration [Si] of each species Si can be computed from the activities {Cj} and stoichiometric coefficients νij of the components Cj and the equilibrium 2172
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constant Ki:
log[Si] ) log[Ki] + νi1 log{AsO43-} + νi2 log{H+} +
νi3 log{e-} + νi4 log{CH3+} (3)
Activities are used for the components and concentrations for the species for notational convenience only. The activity coefficients are all assumed to be unity. The equilibrium constant Ki is obtained from the reaction free energy: 4
∆Grxn°[Si] ) ∆Gf°[Si] -
∑ν ∆G °[C ] ij
f
j
(4)
j)1
via
∆Grxn°[Si] ) - RT ln(Ki) ) - 1.364 log(Ki)
(5)
for T ) 25 °C where νij are the stoichiometric coefficients and ∆Gf°[Cj] is the component free energy of formation for component j and species i (Table 1E). Free electrons do not normally exist in aqueous solutions, but pe ) -log{e-} is used to quantify the activity of the electron in a chemical equilibrium system (44). Similarly, the speciation of the methyl cation CH3+ is not explicitly considered since that would require knowing the thermodynamics and concentrations of the methyl cation donor. Rather pCH3+ ) -log{CH3+} is used to quantify the methyl cation activity. As a consequence, the formation energies of e- and CH3+ are set to zero since the component activities are specified directly. The formation energies for the arsenic
FIGURE 5. Equilibrium methyl arsenic species distribution vs methyl cation activity (pCH3+) at neutral pH using parameters in Table 2. As(V) species (black) and As(III) species (gray). (A) pe ) -1.0. (B) pe ) -1.0, inhibition of TMA species formation. (C) pe ) -2.0. (D) pe ) -2.0, inhibition of TMA species formation. species are obtained from the quantum chemical computations described above (Table 1E). The equilibrium calculations are performed at a fixed pe, pH, and pCH3+. The only remaining unknown in eq 3 is the activity of the arsenate ion, AsO43-. It is determined from the mass balance equation for total arsenic: N
[AsT] )
∑
Ki{AsO43-}{CH3+}νi2{e-}νi3{H+}νi4
(6)
i)1
or
can be written as
(CH3)nAsO(OH)3-n + 2H+ + 2e- f (CH3)nAs(OH)3-n + H2O (8) where n ) 0, 1, 2, and 3 corresponds to the reduction of arsenate, MMA(V), DMA(V), and TMA(V). The change in free energy for the reduction reaction ∆Grxn°(Rn) is
∆Grxn°(Rn) ) ∆Gf°((CH3)nAs(OH)3-n) + ∆Gf°(H2O) N
{AsO43-} ) [AsT]/
∑
Ki{CH3+}νi2{e-}νi3{H+}νi4
(7)
∆Gf°((CH3)nAsO(OH)3-n) - 2∆Gf°(H+) - 2∆Gf°(e-) (9)
i)1
where N ) 11, the number of species, and {CH3+} ) 10-pCH3+, {e-} ) 10-pe, and {H+} ) 10-pH. Total arsenic concentration is set arbitrarily to [AsT] ) 1 M for all computations since the fractional distribution of As species is independent of the total As concentration used in the computation.
Results and Discussion Energetics of Methylation. The Challenger mechanism proposes alternating methylation-oxidation and reduction reactions (Figure 1). The thermodynamic plausibility can be analyzed using the estimated Gibbs free energies ∆Gf°(aq) for the arsenic species (Table 1E). The reduction reactions
where (Rn) denotes the reduction reaction for iAs(V) (n ) 0), MMA(V) (n ) 1), DMA(V) (n ) 2), and TMA(V) (n ) 3). The proton and electron free energies are related to pH and pe via
∆Gf(H+) ) ∆Gf°(H+) + RT ln{H+} ) - 1.364pH ∆Gf(e-) ) ∆Gf°(e-) + RT ln{e-} ) - 1.364pe (10) where both ∆Gf°(H+) ) 0 and ∆Gf°(e-) ) 0 (Table 2). Therefore, the energy change for the reduction of the As(III) VOL. 39, NO. 7, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 6. Computed and observed (Table S3) methyl arsenic equilibrium speciation. (A) G2-COSMO thermodynamic parameters (Table 1E) for all methyl arsenic species. pe ) -1.84 and pCH3+ ) -2.73. Error bars indicate the change in percent of total arsenic for that species when changing ∆Gf°(aq) by (1 kcal/mol. (B) Inhibition of TMA species, pe ) -2.3 and pCH3+ ) -3.1. species ∆Grxn°(Rn) is
∆Grxn(Rn) ) ∆Gf°((CH3)nAs(OH)3-n) ∆Gf°((CH3)nAsO(OH)3-n) + ∆Gf°(H2O) + 2.73(pH + pe) (11) which is a function of pH and pe. An analogous derivation for the methylation-oxidation step proceeds from the reaction
(CH3)nAs(OH)3-n + CH3+ f (CH3)n+1AsO(OH)2-n + H+ (12) where n ) 0, 1, 2 correspond to iAs(III), MMA(III), and DMA(III). The result is that the free energy change associated with the methylation-oxidation reaction ∆Grxn(Mn) is
∆Grxn(Mn) ) ∆Gf°((CH3)n+1AsO(OH)2-n) -
∆Gf°((CH3)nAs(OH)3-n) - 1.364(pH - pCH3+) (13)
where (Mn) denotes the methylation-oxidation reaction for iAs(III) (n ) 0), MMA(III) (n ) 1), DMA(III) (n ) 2), and TMA(III) (n ) 3). Figure 3A illustrates the reaction energies at neutral pH over a range of redox potential and methyl cation activities. The solid bars are the reduction reactions, and the striped bars are the methylation-oxidation reactions. All of the reduction and methylation reactions have positive ∆Grxn in oxic water (pe ) 13) with pCH3+ ) 0. The methyl arsenic reduction reactions ∆Grxn°(Rn) are particularly unfavorable, and the second methylation reaction is the most endothermic. Under more reducing conditions (pe ) 0), the reduction reactions become thermodynamically favorable with ∆Grxn°(Rn) ≈ -10 kcal/mol. In order for all of the methylation reactions to proceed spontaneously, the methyl cation activity must be pCH3+ ) -6. A similar analysis using the alternate free energies computations (Table 1H) is presented in Figure 3B. Since the Gibbs free energies are lower for the trivalent methyl 2174
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arsenic species, the reduction reaction energies are more negative at both pe ) 13 and pe ) 0. For the methylation reactions, a higher methyl cation activity is required, and all of the methylation reactions are not spontaneous unless the pCH3+ ) -7. Equilibrium Distribution of Methyl Arsenic Species. The second objective of this thermodynamic analysis was to investigate if the Challenger sequence corresponds to a continuous reduction in redox potential. Cellular pH is buffered at pH ) 7.2-7.4 (45), and even when habitat pH varies, the intracellular pH remains circumneutral (46). Therefore the pH of cell cytoplasm was assumed to be neutral (pH ) 7) for these calculations. Figure 4 presents the equilibrium methyl arsenic speciation computed using the parameters in Table 2 and eqs 3 and 7, as a function of redox potential at neutral pH and two methyl cation activities. The pentavalent (Figure 4A,C) and the trivalent (Figure B,D) arsenic species are presented separately. For pCH3+ ) -2 (Figures 4A and 4B) as pe is lowered, reduction and methylation proceed from iAs(V) to both MMA(V) and iAs(III) and then to TMA(III). There are smaller peaks of DMA(V) and the other species. For pCH3+ ) -7 (Figure 4C,D), iAs(III) is completely suppressed, and the sequence is iAs(V), MMA(V), TMA(V), and TMA(III). Neither of these titrations reproduce the observed coexistence of trivalent and pentavalent methyl arsenic species nor the dominance of DMA(V) (Figure 2C). An alternative to changing the redox potential is varying the methyl cation activity. Figure 5A presents the resulting distributions at pH ) 7 and pe ) -1.0, the redox potential where a transition between MMA(V) and TMA(V) is observed in Figure 4. Methylation proceeds with increasing methyl cation activity (a more negative pCH3+), and the predicted sequence is iAs(III), MMA(V), and TMA(V). At its peak, the DMA(V) concentration is an order of magnitude lower than MMA(V) and TMA(V). If the formation of the TMA species is prevented (Figure 5B), simulating a kinetic inhibition, then the predicted sequence of formation is iAs(III), MMA(V), and DMA(V) with smaller concentrations of MMA(III) and DMA(III) forming simultaneously with MMA(V) and DMA(V), respectively. It is interesting to note that the distribution of the isomethyl arsenic species (e.g., MMA(V) and MMA(III)) are parallel since they have the same CH3+ stoichiometry (Table 2). The separation is determined by the pe. As pe is lowered from pe ) -1 to pe ) -2 (Figure 5C,D), the concentration lines are closer together. Therefore, both pe and pCH3+ are important in controlling the speciation. Modeling Urinary Arsenic Distribution. The speciation of arsenic in human urine is remarkably uniform with a predominance of DMA(V) and lesser concentrations of the other species (Figure 2C). This suggests that a specific combination of pe and pCH3+ exists where the observed urinary arsenic speciation would be predicted at equilibrium. A nonlinear fitting routine (Solver in Excel) was used to find the pe and pCH3+ that minimized the difference between the observed average distribution of species in urine (Table S3) and the calculated equilibrium distribution. Computations were performed with [AsT] ) 1 M for all computations. This is for convenience only since the species distribution would be identical if the total As concentration were set to that measured in urine, ∼100 µg/L (Table S3). Figure 6A shows the resulting solution at pe ) -1.84 and pCH3+ ) -2.73. The result is not very satisfactory since most of the arsenic is MMA(V) and TMA(III). Since the accuracy of the quantum chemical estimates is not greater than a few kcal/mol (see Table 1E-H for the various estimates), a sensitivity analysis was performed. The ∆Gf°(aq) for each species was adjusted by (1 kcal/mol, and the resulting difference in percent of total arsenic of that
species is shown by the error bars in Figure 6A. MMA(V) and TMA(III) are especially sensitive to changes in ∆Gf°(aq), increasing by nearly 40% when the ∆Gf°(aq) is lowered by 1 kcal/mol. Inhibition of TMAs. In human populations exposed to inorganic arsenic in drinking water, TMA species are not observed in their urine. One data set suggests that trace amounts of TMA(V) (∼5% of total arsenic) may be detected in urine when given an oral dose of DMA(V). However, greater than 80% of the total arsenic was excreted unchanged as DMA by humans, mice, and hamsters (47). It may be that the addition of a third methyl group is inhibited within human cells. Removing TMA(V) and TMA(III) from the equilibrium computation simulates such a situation. The results are shown in Figure 6B for pe ) -2.3 and pCH3+ ) -3.1. The speciation is now quite close to the observations, where DMA(V) is the dominant species followed by MMA(V). Smaller amounts of DMA(III), MMA(III), and iAs(III) are correctly predicted. Only the observed presence of iAs(V) cannot be rationalized. It should be pointed out that this result may well be fortuitous since the accuracy of the estimated thermodynamic parameters is limited. Nevertheless, it is clear that the Challenger mechanism is thermodynamically plausible and that a metastable equilibrium can be reproduce the speciation observed in human urine.
(12) Radabaugh, T. R.; Aposhian, H. V. Enzymatic reduction of arsenic compounds in mammalian systems: reduction of arsenate to arsenite by human liver arsenate reductase. Chem. Res. Toxicol. 2000, 12 (1), 26-30.
Acknowledgments
(21) Vahter, M. Mechanisms of arsenic biotransformation. Toxicology 2002, 181-182, 211-217.
This work was funded by the National Institute of Environmental Health Superfund Basic Research Program Grant 42ES10344.
Supporting Information Available The formulas for converting the thermodynamic parameters to the NBS standard state (eqs S1-S4), the free energy of the ions (eqs S5-S7), and the data and references used for Figure 2 (Tables S1-S3). This material is available free of charge via the Internet at http://pubs.acs.org.
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Received for review July 6, 2004. Revised manuscript received November 16, 2004. Accepted January 4, 2005. ES0489691
