Chem. Res. Toxicol. 1993,6, 102-106
102
Reactions of Arsenic(II1) and Arsenic(V) Species with Glutathione Nelson Scott,+Kristina M. Hatlelid,J Neil E. MacKenzie,! and Dean E. Carter*J Department of Chemistry, California Polytechnic State University, Pomona, California 91 768, and Department of Pharmacology & Toxicology and Department of Pharmaceutical Sciences, College of Pharmacy, University of Arizona, Tucson, Arizona 85721 Received October 23, 1992
Arsenic is metabolized by living systems using opidation-reduction and methylation reactions, and reduced glutathione (GSH) has been shown to be important in that metabolism. In this study, the solution reactions between GSH and arsenate, arsenite, and their methylated metabolites, monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA), were characterized using 'Hand 13CNMR under a nitrogen atmosphere. Binding to GSH through the thiol group was primarily followed by shifts in the carbon atom bonded to the sulfhydryl group of the cysteinyl residue, i.e., the CHZcarbon atom and the protons bonded to it. The methylated metabolites also showed shifts in the methyl groups attached to the arsenic atom after reaction with GSH. Sodium arsenite, As(III), bound to GSH to form an As(SG)3 complex in solution as indicated by NMR spectra. The identity of the complex was confirmed by FAB-MS after isolation of the compound. Mixtures of sodium arsenate, As(V), and GSH showed that arsenate oxidized GSH in DzO solutions a t pH 7 to form oxidized glutathione (GSSG). When the molar ratio of As:GSH exceeded 1:2,evidence for the formation of As(SG)3 was observed. MMA and DMA are both As(V) species, and mixtures with GSH showed oxidation to GSSG initially followed by formation of CH3*As(SG)zand (CH~)Z*A~.SG, respectively. The effects of GSH on arsenic metabolism may result from direct reactions between the two compounds. Introduction Arsenic is an important environmental toxicant which has both natural and industrial sources. Arsenic predominantly exists in two oxidation states, As(V) and As(III), and each species is hypothesized to act through a different mechanism ( 11. The most important mechanism for As(II1)toxicity is postulated to be through binding to sulfhydryl-containingenzymeswhile As(V)has been shown to substitute for phosphate in enzyme-catalyzedreactions where it can disrupt normal function. Comparing the toxicity of these two species is complicated by the fact that As(II1) and As(V) can be converted to the other species in the body (2),and the mechanism for these oxidationreduction reactions is unclear. In addition, inorganic arsenic can be methylated to form two As(V) species, monomethylarsonic acid and dimethylarsinic acid, and this metabolism appears to be a detoxification process since the products are less acutely toxic than the inorganic species (I). Glutathione, a sulfhydryl-containingtripeptide, is found in high (millimolar) concentrations in cells and has been identified as an important compound in the cellular defense against toxicants (3). Glutathione was identified by Voegtlin and co-workers in 1924 (4)as a compound which could interfere with the actions of As(III), and this reaction has been used to study the active sites of enzymes which contain sulfhydryl groups (5).The study described here examined the interactions between glutathione and the As(III)/As(V) species using nuclear magnetic resonance spectroscopy to investigate the im-
portance of oxidation-reduction and binding reactions and to characterize the products of those reactions. Materials and Methods Chemicals. Reduced glutathione, GSH,' was obtained from Aldrich Chemical Co. (Milwaukee, WI). Oxidized glutathione were purchased from (GS-SG) and dimethylarsinic acid (DMA) Sigma Chemical Co.(P.O. Box 14508,St. Louis, MO). Sodium arsenite (NaAsOz), ACS grade, waa purchased from Fisher Scientific Co. (Fairlawn, NJ) and sodium arsenate (Nar HAs01.7H20) from J. T. Baker Chemical Co. (Phillipsburg, NJ). Monomethylarsonic acid, disodium salt (MMA), waa purchased fromPfaltz and Bauer, Inc. (Waterbury, CT). Allother reagente and chemicale used were AR grade and were used without further purification. Caution: The following chemicals are hazardous
and should be handled carefully: sodium arsenite and sodium arsenate are human lung and skin carcinogens that should be handled using appropriate worker protection for carcinogens. Synthesis of Methylarsine Oxide. Methylarsine oxide waa synthesized according to a method reported in the literature (6). This involved reducing MMA, diesolved in the minimum quantity of water, with SO2 gaa. The solution waa refluxed for about 4 h in order to complete the reaction. The water was evaporated to dryness under reduced pressure at ambient temperature. The white residue left waa extracted with 4 X 25-mL portions of hot benzene, and the mixture was filtered. The filtrate waa evaporated to dryness under reduced pressure without heating to obtain a white solid. This solid waa characterized to be CHaA s 4 (mp 95 OC; FAB-MS, mlz values of 318 and 424 for the trimer and tetramer of C H b 4 ) . Syntheses of Complexes of GSH with Arsenic Species. The complexes of GSH with arsenite, methylamine oxide, and dimethylarsinic acid were Synthesized and isolated. The general
+ California Polytechnic State University.
Department of Pharmacology & Toxicology, College of Pharmacy, University of Arizona. 8 Department of Pharmaceutical Sciences, College of Pharmacy, University of Arizona.
Abbreviations: glutathione, GSH; oxidized glutathione, GS-50; monomethylareonic acid, MMA;dimethylareinic acid, DMA, fast atom bombardment mase spectrometry, FAB-MS.
CD1993 American Chemical Society
Chem. Res. Toxicol., Vol. 6, No. 1, 1983 103
Arsenic Reactiom with Glutathione procedure used was that reported by Cullen and co-workers (7) which involved reacting the arsenic compound with GSH in the stoichiometric ratio expected for each reaction. The solutions were mixed for 5-6 h at room temperature under an atmosphere of nitrogen. The experimental conditions used for each reaction were as follows: (i) Sodium arsenite 10.085 g (6.5 X lo-' mol)] and GSH [0.6 g (1.96X 109 mol)] were dissolved in 1 mL of distilled degassed (dd) water. Methanol was added to precipitate the product of the reaction and the mixture was filtered. Any residual water was removed under reduced pressure at ambient temperature. mol)] and GSH [1.6g (5.2 X (ii) DMA [0.224g (1.4X mol)] were dissolved in 18 mL of dd water. At the end of the reaction the water was evaporated to dryness under reduced pressure at ambient temperature. The resulting powder was extracted with 4 X 30mL of methanol, and the combined extracts were evaporated to dryness under reduced pressure without heating. The resulting powder was recrystallized from methanol,' water (l:l), fiitered, and dried. (iii) Methylarsine oxide [O.lOg (9.4 X lO-'mol)] and GSH [0.6 mol)] were dissolved in 2 mL of dd water. The g (1.96 X product was precipitated with ethanol, filtered, and dried. NMR Experiments. NMR spectra were obtained in 0.5 M potassium phosphate buffer solutions, pH 7.0-7.1, in DzO. The concentration of the arsenic solutionsused for the lH NMR specta was 2.5 mM as arsenic, and for the 13C spectra it was 0.3 M as arsenic. All experiments were carried out under a nitrogen atmosphere and at room temperature. 13Cspectra were obtained under broad-band decoupling conditions on a Bruker AM 250 spectrometer (Bruker Instrument Co., San Jose, CA) operating at 62.5 MHz, and 'H spectra were obtained on a Bruker AM 500 spectrometer operating at 500 MHz. Dioxane, in a coaxial capillary, was used as a chemical shift (6) reference for both 13C (66.50ppm) and lH (3.740ppm) with respect to TMS at 0.0 ppm. Mass Spectrometry. FAB mass spectra of the arsenic glutathione complexes were obtained with a Finnigan MAT 90 mass spectrometer (Finnigan Instrument Co., San Jose, CA) operated at an accelerating voltage of 6 kV and resolution of 1OOO. The mass spectrometer was equipped with a cesium ion gun (AMD Bremen, West Germany) operated at 11 kV. The sample was introduced in a glycerol matrix. The masses were scanned from 400 to 1050 amu. The mass spectrum of CH3A s 4 was obtained in the E1 ionization mode at an accelerating voltage of 6 kV and resolution of 1OOO. E1 conditions included an electron energy of 70 eV, an emission current of 1 mA, and a source temperature of 200 OC.
Results NMR Spectra of Reduced and Oxidized Glutathione. Glutathione can exist in the reduced form, GSH, and/or the oxidized form, GS-SG. In the pH range 3.39.0, the structures of the two forms can be represented by structures a and b, respectively, of Figure 1 (8). Since oxidation of GSH involves formation of the dimer, GSSG, via the cysteinyl sulfhydryl groups, the greatest differences in the NMR spectra between GSH and GS-SG were seen at themethylenegroup bonded to the sulfhydryl group of the cysteinyl residue, i.e., the &CH2 carbon atom and the protons attached to it (Table I). The adjacent carbon atom, the a-C atom, and ita methine proton were also shifted but to a reduced extent. The two cysteinyl P-CH2 protons of GSH appeared as closely spaced multiplets at 6 2.67-2.74 ppm. One of the two &CH2 protons of GS-SG appeared as a multiplet in an identical position, 6 2.74-2.77 ppm, while the other gave a multiplet at 3.073.09 ppm. NMR Spectra and Reactions of Sodium Arsenite and Glutathione Mixtures. The 'H NMR spectra of and GSH were different from mixtures of arsenite (AsO~~-)
+w
H O
0
H--C--CH-CH-C--NH-C--C--NH-CH-C~o I II I l l
I
2
I
2
o+ C \ o -
2
\o-
CHZ
I I S I
S
+NH, H-
CHZ
cI -CH- c H - P;c -NH- cI -c -NH-CH-c+O 2 Lo2 z I I II C H
o+ \ o -
O
b
Figure 1. Structure of (a) reduced glutathione (GSH)and (b) oxidized glutathione (GS-SG) in aqcleous solutions at pH 7.0. Table I. NMR Data of GSH,GS-SG, and A e ( I I I ) 4 S H
Mixtures
6, ppm
GSH
CYSB-CH2
Cys a-CH
GS-SG 'H NMRa 2.74 (5.19) 2.77 (9.51) 2.71 (5.37) 2.74 (9.60) 2.70 (7.20) 3.09 (4.55) 2.67 (7.27) 3.07 (4.54) 4.32(5.25) 4.53 4.31 (5.47)
13CNMR 56.83 53.82 26.75 39.91 CY6 B cysC=O 172.96 173.10 Gly a 44.01 44.71 GlyC=O 176.44 177.54 Glu a 55.21 55.27 27.42 27.49 Glu B 32.54 32.61 Glu y Glu a C=O 175.03 175.31 Glu y C - 0 175.97 176.22 Parenthetical numbers are JHH values in Hz. cys n
As(III)-SG 2.98 (8.11) 2.96 (8.08) 3.12 (4.81) 3.09 (4.70) 4.44 (4.89) 4.43 (5.00) 65.75 34.46 172.28 44.75 177.20 55.21 27.50 32.71 175.16 175.78
those of either GS-SG or GSH. In these mixtures, one of the cysteinyl &CH2 protons appeared as a multiplet at 6 2.96-2.98 ppm, and the other gave a multiplet at 6 3.093.12 ppm. This change in the peak positions of the cysteinyl &CH2 protons indicates that GSH binds via ita sulfhydryl group to arsenic. This was futher evidenced by the difference in the coupling constants, JHH,of the &CH2 protons of GSH, GS-SG, and the As03*-GSH mixture (Table I) and the differences in 13Cchemical shifta of the cysteinyl 8-C atom of GSH, GS-SG, and the AsO~~--GSH mixture (Table I). When the molar ratio, As0s3-:GSH, was varied from 1.00.5to 1.03.5,the presence of unchanged GSH was first evidenced at a ratio of 1.03.5 (small peaks at 6 2.68 and 4.32 ppm). This indicates that the stoichiometry of the complex of As0s3- with GSH is 1:3. Hence the complex can be postulated to be As(SG13 and the reaction can be represented as
3H' + As0;- + 3 GSH + As(SG), + 3H,O (1) The identity of the complex formed between AsOsSand GSH was established unambiguously by synthesizing and isolating the reaction product. FAB-MS of the white
104 Chem. Res. Toxicol., Vol. 6, No. 1, 1993
I
I
4.5
4.0
I 3.5
I 3.0
1 2.5
Scott et al.
I 2.0
PPm Figure 2. lH NMR spectrum of a mixture of AsOd3- and GSH
(molar ratio 1:3) in DzO solution at pH 7.1. Peaks characteristic of GSSG (A) and of the As(II1)-SG complex (B)are labeled. Their assignments are found in Table I.
powder obtained as the reaction product gave an intense peak for the (M + H)+ ion at an mlz value of 994. This corresponds to the molecular weight of [As(SG)3 + H+l. The peaks that were considered to be characteristic in the 1H and 13CNMR spectra of the reaction solutions discussed above were confirmed to be those of the complex, As(SG)3, by the lH NMR and 13C spectra of the isolated product (lH: 6 2.96-2.98 ppm; 13C: 6 34.46 ppm). NMRSpectraof Sodium Arsenate andGlutathione Mixtures. Examination of mixtures of arsenate ( A ~ 0 4 ~ - ) and GSH showed that arsenate oxidized GSH in DzO solutions maintained at a pH value of 7.0. Air oxidation of GSH in these solutions was prevented by maintaining an atmosphere of nitrogen over the mixtures throughout the experiment. The 'H NMR spectrum showed peaks associated with both GS-SG and the As(II1)-SG complex (Figure 2). When the molar ratio, arsenate:GSH, was increased from 1:l to 1:2 and then to 1:3, the signals at 6 2.98 and 4.44 ppm increased in intensity. These signals were due to the cysteinyl b-CH2 protons and the cysteinyl a-CH proton, respectively, of the As(II1)-SG complex. At the molar ratio for arsenate:GSH of 1:1, the 'H NMR signalscharacteristic of the As(II1)-SG complexwere much lower in intensity than those found for GS-SG (6 2.742.77 and 3.07-3.09 ppm). Likewise, the 13C spectrum of an equimolar mixture of GSH and h o d 3 - in D20 solution did not show any evidence for the formation of the As(111)-SG complex. This suggests that the main reaction between excess arsenate and GSH is the oxidation of GSH to GS-SG with the concomitant reduction of arsenate to arsenite. Hence the reaction may be represented as 3H+ + As0;-
+ 5GSH
As(SG), + GS-SG + 4Hz0
=i
(2) NMR Spectra of DMA and Glutathione Mixtures. lH NMR spectroscopy of mixtures of dimethylarsinic acid (DMA) and GSH in D2O solution at a pH value of 7.0 showed both oxidation and complexation of GSH. Oxidation of GSH to GS-SG by DMA was shown by the appearance of signals a t 6 3.03-3.07 and 2.70-2.77 ppm for &CH2 and 4.49-4.51 ppm for the a-CH in the 'H NMR
spectrum. The formation of GS-SG in mixtures of GSH and DMA was also indicated by 13CNMR spectra (peaks at 6 39 and 53.8 ppm). Further, there was evidence that the nature of DMA had changed during this reaction since the position of the CH3 proton singlet of DMA changed from 6 1.50 ppm in solutions containing DMA only to 6 1.10 ppm in solutions containing mixtures of DMA and GSH. The 13C chemical shift value of the CH3 group of DMA changed from 6 18.54ppm in solutions of unchanged DMA to 6 14.80 ppm in mixtures of DMA and GSH. Complexation of GSH was also indicated because one of the two P-CH2 cysteinyl protons of GSH gave a signal at 6 2.89 ppm. The peaks that appeared at 6 32.67 and 56.42 ppm were unique, were not found in the 13C spectra of DMA, GSH, or GS-SG,and may be attributed to the DMASG complex. 'H NMR spectra of a series of solutions containing different molar ratios of DMA:GSH (in the range 1.0:0.5 to 1.03.5) were obtained. At molar ratios, DMA:GSH, lower than 1:3, the CH3 proton signal of DMA appeared as a singlet at 6 1.50 ppm due to uncomplexed DMA and as a singlet at 6 1.10 ppm due to a DMA species complexed to GSH. When the molar ratio, DMA:GSH, was increased to 1.0:3.0, the signal a t 6 1.50 ppm disappeared, indicating that all of the DMA species had undergone complexation. Hence the stoichiometry between DMA and GSH in the reaction is 1:3and the reaction may be represented as follows: GS-SG + (CH,&As*SG + 2HZO (3) The compound (CH&+bSG was synthesized and isolated from the reaction mixture. Its identity was confirmed by FAB-MS [a peak at mlz 412 for the (M + H)+ion and a peak at mlz 434 for the (M + Na)+ ion]. This compound gave peaks at 6 2.89 ppm in the 'H NMR spectrum and a t 6 32.67 and 56.42 ppm in the 13C spectrum, confirming that these were characteristic of the compound (CH3)rAwSG. NMR Spectra of MMA and GlutathioneMixtures. The reaction between monomethylarsonic acid (MMA) and GSH was similar to that between DMA and GSH because it resulted in the formation of GS-SG and a different MMA species followed by the binding of this MMA species to excess GSH. The CH3 protons of MMA gave a sharp singlet at b 1.56 ppm in the absence of GSH. The position of this peak changed to 6 1.45 ppm in the presence of GSH. Similarly, the frequency of the 13Csignal of the CH3 group of MMA changed from 6 17.92 ppm in the absence of GSH to 6 16.25 ppm in the presence of GSH. The @-CHzand a-CH protons of the cysteinyl residue gave broad peaks at 6 2.90 and 4.38 ppm, respectively (Figure 3). Since these peaks are not found in the 'H NMR spectrum of either GSH or GS-SG, it is likely they are due to the complex CHvAs(SG)z. Evidence for the oxidation of GSH came from both 'H NMR and 13C NMR spectra (1H NMR multiplets at 6 2.75, 3.09, and4.50 ppm, 13C: peaks at 39.83 and 53.78 ppm). Hence the reaction between GSH and MMA may be represented as follows: (CHJZ*As.O.OH + 3GSH
+
GS-SG + CH,*As(SG), + 3HzO (4) The compound CH3*As(SG)zwas isolated as the product of the reaction between CH3*As=Oand GSH. Again ita identity was established by spectrometry [mlz 703 for the CH3*As-O.(OH), 4GSH
Chem. Res. Toxicol., Vol. 6, No. 1, 1993 101
Arsenic Reactions with Glutathione
C
4.5
4.0
3.5
3.0
2.5
2.0
1.5
PPm
Figure 3. lH NMR spectrum of a mixture of MMA and GSH (molar ratio 1:2) in DzO solution at pH 7.1. Peaks characteristic of GSSG (A) and of the MMA-SG complex (B)are labeled, and the assignments are found in Table I. The peaks from the methyl group of the free MMA (C)and the bound MMA (D)are shown.
+
(M H)+ ion]. Characteristic peaks at 6 1.45 ppm in the lH NMR spectrum and at 6 16.25 ppm in the 13C NMR spectrum were observed.
Discussion The present investigation describes the redox reactions which occur between As(V) species and glutathione and the binding reactions which occur between As(II1) species and glutathione. These reactions have been described previously with MMA and DMA; Cullen and co-workers (7)isolated the glutathione complexes with MMA and DMA. Others have shown the reactions between phenyldichloroarsine and diphenylchloroarsine with glutathione using NMR spectroscopy (9-11). In addition, Jacobson and Murphy (12) found a loss of sulfhydryl groups for 2-mercaptoethanol, L-cysteine, dithiothreitol, and glutathione when DMA was added to an aqueous solution of these compounds buffered at pH 5.5. Thus, alkyl arsenicals, such as DMA and MMA, in the +V oxidation state can be reduced by thiols and can subsequently react in the +I11 state. Sulfhydryl binding to arsenic(1II)species has been shown to be important from in vivo experimental results. Stevenson et al. (13) have shown the inhibition of the enzyme complex, pyruvate dehydrogenase, presumably through binding to the lipoic acid which is essential for enzyme activity. Early work with organoarsenicals developed for the treatment of infectious diseases showed that glutathione could antagonize the effects of the arsenical. The toxicity of 3-amino-4-hydroxy-1-phenylarsenious oxide (arsenoxide) toward T r y p a n o s o m a equiperdum in blood was reduced by the addition of glutathione both when added in vitro and in vivo. This effect was not unique to glutathione; compounds like cysteine,thioglycolate,thiolactate, and thiosalicylatewere also found to inhibit the toxicity (4). Complexes between thiols and arsenite [As(III)I or arsenate [As(V)l seem to have been more difficult to make than their alkylarsenic analogues. Our study focused on these reactions because the mechanisms for the arsenic
redox reactions in vivo have not been characterized and the species involved in cellular arsenic transport are unknown. Glutathione was chosen for study instead of other sulfhydryl compounds because of its potential involvement in the metabolism of arsenic. Glutathione is a likely candidate as a reducing agent because it is present in all cells at high concentrations (generally millimolar) and it exists primarily in the reduced form. Recent in vitro work on arsenic metabolism has shown that glutathione was necessary for full activity of the arsenic methylation reactions. The effects of GSH were regulated through several mechanisms: facilitation of As(II1) diffusion through the cells, stimulation of the first methylation reaction, and increase of DMA excretion by the cells (14, 15). When glutathione levels were reduced in vivo, urinary MMA and DMA were decreased and hepatic arsenic levels were increased (16). These authors postulated that an unidentified glutathione conjugate with As(111)was excreted in the bile because the absence of GSH resulted in increased liver levels. They also postulated that the methylation of arsenic occurred by As(II1) being methylated to the monomethyl As(II1) species followed by methylation to the dimethyl As(II1) species. These Aa(II1)species were then oxidized to MMA and DMA by an unknown mechanism before being excreted in the urine. Our results demonstrate the existence of the As(III)-(GS)s complex and the ability of GSH to reduce arsenate, arsenic in the +V state. Previous work on methylation mechanisms has suggested that the methylation occurred through the transfer of a carbenium ion (17). This reaction would result in a oxidation of the As(II1) species to the methylated As(V) species. This would require that arsenic must be reduced to an As(II1) species between each methylation step, and several investigators have suggested this as a mechanism (18-21; also, references 13-16 in ref 14). Our results indicate that glutathione can serve to reduce the h ( V ) species and suggest that, since the glutathione complexes form after reduction, they may be the substrates for the methylating enzymes. The significance of the arsenic(III)-glutathione complexes in transport and metabolism and whether these complexes exist in vivo are unknown and will require further studies. In addition, the role of oxidation of the arsenic must be important in vivo since arsenic +V species (arsenate, MMA, and DMA) are the predominant arsenic species found in the urine ( 2 ) and no methylated metabolites in the +I11 oxidation state have been found. It is unclear how or where the oxidation reactions occur or how they may relate to the toxicity. Our solution studies were done under a nitrogen atmosphere, a nonphysiological state. This was done to protect the GSH from air oxidation. The in vitro liver cytosol incubation experiments performed by Buchet and Lauwerys (14) also used a nitrogen atmosphereto maintain GSH in a reduced state. This is necessary in order to model the cell, which has a number of enzyme systems to maintain reduced GSH in millimolar concentrations. In summary, the reduction of As(V) species to As(II1) and their subsequent complexation with GSH have been described. The reduction step is necessary for the two mechanisms proposed for arsenic methylation in the literature. The methylation mechanisms differ in which As species forms after the methylation reaction [As(III) or As(V)I and whether the reduction by GSH is necessary
106 Chem. Res. Toxicol., Vol. 6, No. 1, 1993
between methylation steps. Also, neither mechanism has considered whether glutathione complexes may be important in the methylation reaction. It is also unclear how the reduced species are oxidized before their appearance in the urine as MMA and DMA or how the redox reactions of arsenic may be important in our understanding of the toxic mechanisms of arsenic. Acknowledgment. The support provided by BristolMeyers Company Research Corporation Fund Grant C-2679 (N.S.) and Superfund Basic Research Grant ES4940 (D.E.C.) was greatly appreciated. Additional thanks go to Mike Kopplin, Dr. Paul R. Gooley,Dr. Quintus Fernando, Dr. Kenneth Nebensy, and David Collins for their contributions.
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Thieme Publishers, Stuttgart. (9) Dill, K., Adams, E.R., O’Connor, R. J., Chong, S., and McGown, E. L. (1987) One-Dimensionaland Two-DimensionalNuclear Magnetic
Resonance Studies of the Reaction of Phenyldichloroarsine with Glutathione. Arch. Biochem. Biophys. 257, 293-301. (10) Dill, K., O’Connor,R. J., and McGown, E. L. (1987) Spin-echoNMR Investigation of the Interaction of Phenyldichloroarsine with Glutathione in Intact Erythrocytes. Inorg. Chim.Acta 138,9597. (11) Adams, E. R., Kolis, J. W., and Dill, K. (1988) 1% NMR Spectral Analysis of Mono and Diphenylarsine Adducts of Glutathione in DMSO. Inorg. Chim.Acta 152, 1-2. (12) Jacobson, K. B., and Murphy, J. B. (1972) Reaction of cacodylicacid with organic thiols. FEBS Lett. 22, 80-83. (13) Stevenson, K. J., Hale, G., and Perham, R. N. (1978) Inhibition of Pyruvate Dehydrogenase Multienzyme Complex from Escherichia Coli with Mono- and Bifunctional Arsenoxides. Biochemistry 17, 2189-2192. (14) Buchet, J. P., and Lauwerys, R. (1988) Role of thiols in the in vitro
methylation of inorganic arsenic by rat liver cytosol. Biochem. Pharmacol. 37, 3149-3153. (15) Georis, B., Cardenas, A., Buchet, J. P., and Lauwerys, R. (1990) Inorganic arsenic methylation by rat tissue slices. Toxicology 63, 73-84. (16) Buchet, J. P.,andLauwerys,R.R. (1987)Studyoffactorainfluencing (17)
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