Comparative inhibition of yeast glutathione reductase by arsenicals and arsenothiols

Article abstract of DOI:10.1021/tx960139g

Tri(γ-glutamylcysteinylglycinyl)trithioarsenite (As(III)(GS)₃) is formed in cells and is a more potent mixed-type inhibitor of the reduction of glutathione disulfide (GSSG) by yeast glutathione (GSH) reductase than either arsenite (As(III)) or GSH. The present work examines the effects of valence and complexation of arsenicals with GSH or L-cysteine (Cys) upon potency as competitive inhibitors of the reduction of GSH disulfide (GSSG) by yeast GSH reductase. Trivalent arsenicals were more potent inhibitors than their pentavalent analogs, and methylated trivalent arsenicals were more potent inhibitors than was inorganic trivalent As. Complexation of either inorganic trivalent As or methylarsonous diiodide (CH₃As(III)I₂) with Cys or GSH produced inhibitors of GSH reductase that were severalfold more potent than the parent arsenicals. In contrast, dimethylarsinous iodide ((CH₃)₂As(III)I) was a more potent inhibitor than its complexes with either GSH or Cys. Complexes of CH₃As(III) with GSH (CH₃-As(III)(GS)₂) or with Cys (CH₃As(III)(Cys)₂) were the most potent inhibitors, with K(i)'s of 0.009 and 0.018 mM, respectively. Inhibition of GSH reductase by arsenicals or arsenothiols was prevented by addition of meso-2,3-dimercaptosuccinic acid (DMSA) to a mixture of enzyme, GSSG, and inhibitor before addition of NADPH. DMSA added to the reaction mixture after NADPH reversed inhibition by (CH₃)₂As(III)I but had little effect on inhibition by CH₃As(III)I₂, CH₃As(III)(GS)₂, CH₃As(III)(Cys)₂, or As(III)(GS)₃. Partial redox inactivation of the enzyme with NADPH increased the inhibitory potency of CH₃As(III)I₂ and (CH₃)₂As(III)I and changed the mode of inhibition for CH₃As(III)I₂ from competitive to noncompetitive. The greater potency of methylated trivalent arsenicals and arsenothiols than of inorganic trivalent As suggests that biomethylation of As could yield species that inhibit reduction of GSSG and alter the redox status of cells.

Full text of DOI:10.1021/tx960139g

Chem. Res. Toxicol. 1997, 10, 27-33
27
Com p a r a tive In h ibition of Yea st Glu ta th ion e Red u cta se
by Ar sen ica ls a n d Ar sen oth iols
Miroslav Styblo,† Spiros V. Serves,‡ William R. Cullen,‡ and David J . Thomas*,§
Curriculum in Toxicology, University of North Carolina at Chapel Hill,
Chapel Hill, North Carolina 27599-7270, Department of Chemistry, University of British Columbia,
Vancouver, British Columbia, Canada V6T 1Y6, and Pharmacokinetics Branch, Experimental
Toxicology Division, National Health and Environmental Effects Research Laboratory, U.S.
Environmental Protection Agency, Research Triangle Park, North Carolina 27711
Received August 6, 1996X
Tri(γ-glutamylcysteinylglycinyl)trithioarsenite (AsIII(GS)3) is formed in cells and is a more
potent mixed-type inhibitor of the reduction of glutathione disulfide (GSSG) by yeast glutathione
(GSH) reductase than either arsenite (AsIII) or GSH. The present work examines the effects
of valence and complexation of arsenicals with GSH or L-cysteine (Cys) upon potency as
competitive inhibitors of the reduction of GSH disulfide (GSSG) by yeast GSH reductase.
Trivalent arsenicals were more potent inhibitors than their pentavalent analogs, and
methylated trivalent arsenicals were more potent inhibitors than was inorganic trivalent As.
Complexation of either inorganic trivalent As or methylarsonous diiodide (CH3AsIIII2) with
Cys or GSH produced inhibitors of GSH reductase that were severalfold more potent than the
parent arsenicals. In contrast, dimethylarsinous iodide ((CH3 )2AsIIII) was a more potent
inhibitor than its complexes with either GSH or Cys. Complexes of CH3AsIII with GSH (CH3-
AsIII(GS)2) or with Cys (CH3AsIII(Cys)2) were the most potent inhibitors, with Ki’s of 0.009 and
0.018 mM, respectively. Inhibition of GSH reductase by arsenicals or arsenothiols was
prevented by addition of meso-2,3-dimercaptosuccinic acid (DMSA) to a mixture of enzyme,
GSSG, and inhibitor before addition of NADPH. DMSA added to the reaction mixture after
NADPH reversed inhibition by (CH3 )2AsIIII but had little effect on inhibition by CH3AsIIII2,
CH3AsIII(GS)2, CH3AsIII(Cys)2, or AsIII(GS)3. Partial redox inactivation of the enzyme with
NADPH increased the inhibitory potency of CH3AsIIII2 and (CH3)2AsIIII and changed the mode
of inhibition for CH3AsIIII2 from competitive to noncompetitive. The greater potency of
methylated trivalent arsenicals and arsenothiols than of inorganic trivalent As suggests that
biomethylation of As could yield species that inhibit reduction of GSSG and alter the redox
status of cells.
and (CH3)2As from pentavalency to trivalency and forms
stable complexes with (CH3)2AsIII (1, 5) and phenyldi-
chlorarsine (C6H5AsIIICl2) (6, 7). A complex of C6H5AsIII
and GSH is the predominant arsenical present in human
erythrocytes following in vitro exposure to C6H5AsIII Cl2
(8). Given the high intracellular concentration of GSH
and the high affinity of AsIII for thiols, it is likely that
arsenothiols are commonly present in cells. Because
trivalent arsenicals are more acutely toxic than pentava-
lent arsenicals (9), GSH-dependent reduction to triva-
lency may increase the potential toxicity of ingested As.
Several fates have been identified for arsenothiol com-
plexes formed by the GSH-dependent reduction of ar-
senicals. For example, AsIII(GS)3 donates AsIII to dithiol-
containing molecules (10) and is a substrate for the
enzyme from rat liver cytosol that catalyzes the produc-
tion of CH3As (11).
In tr od u ction
The tripeptide GSH (γ-glutamylcysteinylglycine) plays
critical roles in the reduction of pentavalent arsenicals
to trivalency and in the complexation of arsenicals to
form arsenothiols. The conversion of pentavalent inor-
ganic As (AsV) to trivalency by the oxidation of GSH and
the complexation of trivalent inorganic As (AsIII) by GSH
is described by the reaction scheme,
AsV + 5 GSH f AsIII(GS)3 + GSSG
(1)
This reaction occurs in aqueous solution and in intact
rabbit erythrocytes (1-4). GSH reduces AsV in CH3As1
* Address correspondence to David J . Thomas, Pharmacokinetics
Branch, MD-74, ETD, NHEERL, U.S. EPA, Research Triangle Park,
NC 27711. Email: Thomas@herl45.herl.epa.gov.
† University of North Carolina.
The redox cycling of GSH is central to the cellular
response to oxidative stress. In the reaction scheme,
‡ University of British Columbia.
§ U.S. Environmental Protection Agency.
X Abstract published in Advance ACS Abstracts, December 1, 1996.
2GSH + H2O2 f 2H2O + GSSG
(2)
1
Abbreviations: tri(γ-glutamylcysteinylglycinyl)trithioarsenite,
AsIII(GS)3; methylarsonous diiodide, CH3AsIIII2; dimethylarsinous io-
dide, (CH3 )2AsIIII; di(γ-glutamylcysteinylglycinyl)methyldithioarsonite,
CH3AsIII(GS)2; dicysteinylmethyldithioarsenite, CH3AsIII(Cys)2; meso-
2,3-dimercaptosuccinic acid, DMSA; phenyldichlorarsine, C6H5AsIIICl2;
arsenic acid disodium salt, Na2HAsVO4; sodium m-arsenite, NaAsIIIO2;
methylarsonic acid, disodium salt, CH3AsVO(ONa)2; dimethylarsinic
acid, (CH3)2AsVO(OH); tricysteinyltrithioarsenite, AsIII(Cys)3; cys-
teinyldimethylthioarsinite, (CH3)2AsIII(Cys); (γ-glutamylcysteinylgly-
cinyl)dimethylthioarsinite, (CH3)2AsIII(GS).
GSSG + NADPH + H+ f 2GSH + NADP+ (3)
the cycling between GSH and GSSG is linked to the
scavenging of hydroperoxides generated by the dispro-
portionation of O2• to O2 and H2O2 by superoxide dismu-
tase (12). Reaction 2 is catalyzed by GSH peroxidase (EC
S0893-228x(96)00139-7 CCC: $14.00 © 1997 American Chemical Society

28 Chem. Res. Toxicol., Vol. 10, No. 1, 1997
Styblo et al.
Syn th esis of Dicystein ylm eth yld ith ioa r sen ite (CH3AsIII
-
1.11.1.9), and reaction 3 is catalyzed by GSH reductase
(EC 1.6.4.2). The balance between the oxidation of GSH
to GSSG and the rapid reduction of GSSG by GSH
reductase contributes to the maintenance of a cellular
GSH:GSSG ratio of about 300:1 (13, 14). Increasing the
intracellular concentration of GSSG by oxidative stress
is positively correlated with an increase in protein-GSH
mixed disulfides (15). Formation of mixed disulfides of
protein thiols and GSH may affect protein structure and
serve a regulatory function (16, 17). For example,
S-glutathiolation of Cys residues by this process has been
shown to modulate the phosphatase activity of carbonic
(Cys)2), Di(γ-glu t a m ylcyst ein ylglycin yl)m et h yld it h io-
a r son ite (CH3AsIII(GS)2), Cystein yld im eth ylth ioa r sin ite
((CH3)2AsIII(Cys)), a n d (γ-Glu ta m ylcystein ylglycin yl)d i-
m eth ylth ioar sin ite ((CH3)2AsIII(GS)). These compounds were
prepared as previously described (5). The recrystallization of
(CH3)2AsIII(GS) was modified to increase purity and ease of
preparation. Briefly, the compound was dissolved in methanol/
water (1:1 v/v), and acetone was added until the solution was
turbid. The solution was stored at 4 °C overnight, and the white
precipitate was filtered and washed twice with methanol/water
(1:1 v/v). The washed filtrate was dried in vacuo for 1 day.
1
Identity and purity were confirmed by mp, H-NMR, and TLC.
anhydrase III (18). Previous work has shown that AsIII
-
P r ep a r a tion of Stock Solu tion s. Stock solutions of ar-
senicals, arsenothiols, GSH, and Cys were prepared in distilled
deionized water at 130-520 mM immediately before use.
Dropwise addition of concentrated HCl was needed to increase
solubility of some compounds. A stock solution of 260 mM CH3-
AsIIII2 was prepared in 70% ethanol, or 260 mM (CH3)2AsIIII was
prepared in 50% ethanol. DMSA was dissolved by dropwise
addition of 5 N NaOH, and the resulting neutral solution was
diluted in distilled deionized water to a final concentration of
260 mM.
GSH Red u cta se Assa y. Standard assay conditions for GSH
reductase activity were those described by Styblo and Thomas
(19). Here, 0.3 µg of yeast GSH reductase was added to 0.15 M
phosphate buffer (pH 7) containing 6 mM EDTA and 0.1-1.0
mM GSSG. Inhibitor (arsenical, arsenothiol, Cys, or GSH) and/
or DMSA were added as required for the assay. After a 2 min
preincubation at 37 °C, the reaction was initiated by addition
of NADPH to a final concentration of 0.23 mM. The final
volume of the assay mixture was 2.6 mL. The 340 nm absor-
bance of the reaction mixture was monitored at 20-s intervals
for 3 min in a cuvette with a 1 cm light path. To determine the
rate of nonenzymatic oxidation of NADPH, a blank reaction
containing all components of the reaction mixture except enzyme
was monitored at 20-s intervals for 3 min. The net rate of
enzymatic oxidation of NADPH (i.e., the difference between the
rates of the total and nonenzymatic reactions) was used to
calculate enzyme activity (nmol of NADPH consumed/min).
Duplicate assays were performed for all experimental conditions,
and mean data were used in all calculations.
(GS)3 is a mixed-type inhibitor of the reduction of GSSG
by GSH reductase (19). Using purified yeast GSH
reductase, the work reported here examines the depen-
dence of potency of organoarsenicals on valence and the
effect of complexes of organoarsenicals with GSH or Cys
upon NADPH-dependent reduction of GSSG. These
studies demonstrate that trivalent organoarsenicals are
more potent inhibitors than their pentavalent analogs
and that arsenothiols are a novel class of potent inhibi-
tors of GSH reductase. Either trivalent organoarsenicals
generated during biomethylation of As or arsenothiols
formed in the cell by reaction with GSH or Cys could alter
the cellular GSH:GSSG ratio by the inhibition of GSH
reductase.
Exp er im en ta l P r oced u r es
Ca u tion : Inorganic arsenic is classified as a human carcino-
gen (20), and appropriate precautions should be taken in its
handling.
Ch em ica ls. GSH reductase (type IV prepared from baker’s
yeast), arsenic acid disodium salt (Na2HAsVO4), sodium m-
arsenite (NaAsIIIO2), Cys, GSH, GSSG, NADPH, and DMSA
were obtained from Sigma (St Louis, MO). Methylarsonic acid,
disodium salt (CH3AsVO(ONa)2), was obtained from Chem
Service (West Chester, PA) and dimethylarsinic acid ((CH3)2-
AsVO(OH)) from Strem (Newburyport, MA).
Ch em ica l Syn th esis. Syn th esis of Meth yla r son ou s Di-
iod id e (CH3AsIIII2). This compound was prepared by the
method of Goddard (21). To a solution of CH3AsO(OH)2 (4.4 g,
24 mmol) in 17 mL of water, KI (8.76 g, 26 mmol) and then 3
mL of concentrated HCl were added. SO2 was bubbled through
the mixture for 4 h. The yellow solid that appeared in the
mixture was collected by filtration, washed with cold water, and
dried in vacuo for 1 h. Fractional distillation of the solid under
reduced pressure gave CH3AsIIII2 (4.2 g, 56% yield) as a yellow
crystalline solid (bp 128 °C/16 mmHg). Identity was confirmed
by 1H-NMR (CDCl3): δ 3.1 (s). To prevent oxidation, the
compound was stored in sealed ampules.
Syn th esis of Dim eth yla r sin ou s Iod id e ((CH3 )2AsIIII).
This compound was prepared by the method of Goddard (21).
To a solution of (CH3)2AsO(OH) (4.14 g, 30 mmol) in 30 mL of
water, KI (5.48 g, 33 mmol) and then 1.7 mL of concentrated
H2SO4 were added. SO2 was bubbled through the mixture for
3 h. During this period, a yellow oil separated. The oil was
collected and dried over Na2SO4. Fractional distillation of the
oil under reduced pressure gave (CH3 )2AsIIII (5.3 g, 76% yield)
as a yellow-orange oil (bp 155 °C). Identity was confirmed by
1H-NMR (CDCl3): δ 2.01 (s). To prevent oxidation, the com-
pound was stored in sealed ampules.
Resu lts
Ch a r a cter iza tion of In h ibitor s of th e Activity of
GSH Red u cta se. The effects of arsenicals, arsenothiols,
Cys, and GSH on the activity of yeast GSH reductase
were measured for substrate concentrations from 0.1 to
1.0 mM GSSG. All trivalent arsenicals and arsenothiols
tested were inhibitors of the enzymatic reduction of
GSSG. A double-reciprocal plot of 1/v versus 1/[GSSG]
for each compound was used to determine the mode of
inhibition. A Ki constant for each compound was calcu-
lated from a replot of slopes of the reciprocal plots versus
the corresponding concentrations of the inhibitor. As an
illustration, Figure 1 shows the double-reciprocal plot and
the replot of slopes for CH3AsIII(GS)2, the most potent
inhibitor tested in the present study. The estimated Ki’s
and the mode of inhibition for each compound are
summarized in Table 1.
All trivalent arsenicals and arsenothiols, except AsIII
-
(GS)3, were competitive inhibitors of GSH reductase with
Ki’s that ranged from 0.009 to 5.7 mM (Table 1). In
contrast, AsIII(GS)3 was a mixed-type inhibitor (Ki ) 0.325
mM). CH3AsIII(Cys)2 and CH3AsIII(GS)2 were the most
potent inhibitors, with Ki’s of 0.018 and 0.009 mM,
respectively. Both CH3AsIIII2 and (CH3)2AsIIII were sub-
stantially more potent inhibitors than NaAsIIIO2 and AsIII-
(GS)3. Cys was found to be a relatively potent competi-
tive inhibitor (Ki ) 0.165 mM). In contrast, GSH was a
Syn th esis of Tr icystein yltr ith ioa r sen ite (AsIII(Cys)3)
a n d Tr i(γ-glu ta m ylcystein ylglycin yl)tr ith ioa r sen ite (AsIII
-
(GS)3). AsIII(Cys)3 was prepared by the procedure of Serves and
associates (22). Identity and purity were confirmed by mp, 1H-
NMR, and TLC. AsIII(GS)3 was prepared as described by Styblo
and Thomas (19). The stability of this complex in the standard
reaction mixture has been demonstrated (19).

Inhibition of Glutathione Reductase by Arsenicals
Chem. Res. Toxicol., Vol. 10, No. 1, 1997 29
F igu r e 2. Relative potency for pentavalent and trivalent
arsenicals, arsenothiols, and thiols as inhibitors of GSSG
reduction by GSH reductase in the presence of 0.1 mM GSSG.
O, CH3AsIII(GS)2; b, CH3AsIII(Cys)2; 2, CH3AsIIII2; 0, (CH3)2-
AsIIII; ), AsIII(Cys)3; +, (CH3)2AsIII(Cys); (, Cys; 4, (CH3)2AsIII
GS; 9, AsIII(GS)3; 1, NaAsIIIO2; 3, GSH; +, Na2HAsVO4.
-
(CH3)2AsVO(OH) did not inhibit GSH reductase activity
(data not shown). To determine whether I- contributed
to the inhibition of GSH reductase by CH3AsIIII2 or (CH3)2-
AsIIII, NaI was tested as an inhibitor of enzyme activity.
Up to 10 mM NaI did not inhibit the enzymatic reduction
of 0.1 mM GSSG (data not shown).
Effect of GSH on th e In h ibition of GSH Red u c-
ta se by CH3AsIIII2, (CH3)2AsIIII, a n d Na AsIIIO2. To
examine further the interaction between GSH and orga-
noarsenicals and its effect on the activity of GSH reduc-
tase, GSH was added to reaction mixtures (buffer,
enzyme, 0.1 mM GSSG, arsenical). To promote the
formation of complexes, GSH was added at a molar ratio
of 2:1 for CH3AsIIII2 and of 1:1 for (CH3)2AsIIII. After a
2-min preincubation at 37 °C, the reaction was started
by addition of NADPH. The concentration dependency
of inhibition by simultaneously-added trivalent orga-
noarsenical and GSH was compared with that for the
parent trivalent organoarsenical or authentic organoarse-
nothiols (Figure 3). The inhibitory effect of CH3AsIIII2
was increased by the addition of a 2-fold molar excess of
GSH (Figure 3a), yielding a pattern of the inhibition that
was identical to that for CH3AsIII(GS)2. The comparable
potency of authentic complex and the mixture of compo-
nents suggested that the complex was formed quickly in
the reaction mixture. In contrast, addition of GSH to a
reaction mixture containing (CH3)2AsIIII resulted in a
decreased inhibition as compared with authentic (CH3)2-
F igu r e 1. Inhibition of GSH reductase by CH3AsIII(GS)2. (a)
Double-reciprocal plots of 1/v vs 1/[GSSG] for 0 mM (O), 0.05
mM (0), 0.1 mM (b), and 0.2 mM (9) CH3AsIII(GS)2.. Mean and
range of duplicate assays shown. (b) Replot of slopes of the
double-reciprocal plots vs corresponding concentrations of CH3-
AsIII(GS)2 for the determination of Ki.
Ta ble 1. Ch a r a cter istics of th e In h ibition of GSH
Red u cta se by Ar sen ica ls a n d Ar sen oth iol Com p lexes
compound
Ki (mM)a
mode of inhibition
NaAsIIIO2
5.71
competitive
competitive
mixed-type
competitive
competitive
competitive
competitive
competitive
competitive
competitive
uncompetitive
AsIII(Cys)3
0.076
0.325
0.074
0.018
0.009
0.056
0.834
0.732
0.165
3.46
AsIII(GS)3
CH3AsIIII2
CH3AsIII(Cys)2
CH3AsIII(GS)2
AsIIIGS (Figure 3b). The reduced effect of (CH3)2AsIII
I
(CH3)2AsIII
I
in the presence of GSH suggests that this tripeptide can
protect the enzyme against the inhibitory effect of this
organoarsenical. In the absence of arsenicals, addition
of up to 2 mM GSH did not inhibit the reduction of GSSG
by GSH reductase (data not shown).
(CH3)2AsIII(Cys)
(CH3)2AsIII(GS)
Cys
GSH
a
Ki value was determined from the replots of the slope of each
Assay conditions were further modified, and the ad-
dition of GSH to the reaction mixture (buffer, enzyme,
0.1 mM GSSG, inhibitor) was delayed until 1 min after
the addition of NADPH. Figure 4 shows the time-
dependent changes in 340 nm absorbance of the reaction
mixtures before and after addition of GSH. Addition of
9 mM GSH to an assay mixture containing 3 mM
NaAsIIIO2 slowed the rate of NADPH oxidation (Figure
4a), suggesting that AsIII(GS)3, a more potent inhibitor
than NaAsIIIO2, was formed in the reaction mixture.
Addition of 0.2 mM GSH to an assay mixture containing
reciprocal plot versus the corresponding inhibitor concentration
(see Figure 1).
weak uncompetitive inhibitor of GSSG reduction (Ki )
3.456 mM).
Because of its relatively low potency, the IC50 value
and not the Ki was estimated for the pentavalent arseni-
cal, Na2HAsVO4. Figure 2 compares the IC50 value for
Na2HAsVO4 (∼30 mM) with those determined for triva-
lent arsenicals and arsenothiols in the presence of 0.1
mM GSSG. Addition of 20 mM CH3AsVO(ONa)2 or

30 Chem. Res. Toxicol., Vol. 10, No. 1, 1997
Styblo et al.
F igu r e 3. Concentration-dependent inhibition of GSH reduc-
tase by concurrently-added trivalent organoarsenicals and GSH
compared with the effects of authentic organoarsenothiols,
parent organoarsenicals, or GSH. Arsenicals and/or GSH were
added to the reaction mixture during the 2-min preincubation
period, and reactions were started by addition of 0.23 mM
NADPH. Concentration of GSSG in the assay mixtures was 0.1
mM. (a) Effect of concurrent addition of CH3AsIIII2 and GSH
(1:2 molar ratio) (0); CH3AsIII(GS)2 (b); CH3AsIIII2 (O); or GSH
(9). (b) Effect of concurrent addition of (CH3)2AsIIII and GSH
(1:1 molar ratio) (0); (CH3)2AsIII(GS) (b); (CH3)2AsIIII (O); or GSH
(9).
0.1 mM CH3AsIIII2 did not affect the rate of NADPH
oxidation (Figure 4b), suggesting that CH3AsIII(GS)2, a
potent inhibitor of GSH reductase, was not formed under
these conditions. Addition of 0.3 mM GSH to an assay
mixture containing 0.3 mM (CH3)2AsIIII increased the
rate of NADPH oxidation to a rate comparable to that
found in uninhibited reaction mixtures (Figure 4c),
suggesting that (CH3)2AsIIIGS was formed under these
conditions.
F igu r e 4. Effect of delayed addition of GSH on the rate of
NADPH oxidation in reaction mixtures that contained 3 mM
NaAsIIIO2 (a), 0.1 mM CH3AsIIII2 (b), or 0.3 mM (CH3)2AsIIII (c).
Following preincubation of enzyme and 0.1 mM GSSG with or
without arsenical for 2 min, reactions were started by addition
of 0.23 mM NADPH. One minute after addition of NADPH, 9
mM GSH was added to (a), 0.2 mM GSH to (b), and 0.3 mM
GSH to (c). Rates of NADPH oxidation (A340) were measured
before and after addition of GSH. O, assay mixture without
arsenical or GSH added; 0, assay mixture with only GSH added;
b, assay mixture with only arsenical added; 9, assay mixture
with arsenical and GSH added.
Effect of DMSA on th e In h ibition of GSH Red u c-
ta se. The effects of DMSA on the inhibition of GSH
reductase by arsenicals and arsenothiols were examined.
In the first experiment, DMSA was added to the reaction
mixture (buffer, enzyme, 0.1 mM GSSG, arsenical, or
arsenothiol) during the preincubation period. Increasing
concentrations of DMSA added before NADPH antago-
nized the inhibitory effects of 0.5 mM CH3AsIIII2, 0.5 mM
(CH3)2AsIIII, 0.2 mM CH3AsIII(Cys)2, and 0.1 mM CH3-
AsIII(GS)2 (Figure 5). The percentage of the recovered
enzymatic activity was proportional to the DMSA con-
centration in the assay mixture. The recovery of activity
was nearly complete at a molar ratio of DMSA:arsenical
of ∼0.5 for (CH3)2AsIIII and of ∼1 to 1.2 for CH3AsIIII2 or
(Cys)2 could not be fully reversed by addition of up to
0.32 mM DMSA (molar ratio ) 1.6). Notably, in the
absence of arsenicals or arsenothiols, addition of greater
than 1 mM DMSA to the reaction mixture was required
to inhibit GSH reductase (data not shown).
In other experiments, DMSA was added to the reaction
mixture (buffer, enzyme, 0.1 mM GSSG, arsenical, or
arsenothiol) 1 min after the addition of NADPH. Figure
6 shows time-dependent changes in A340 for reaction
mixtures before and after the addition of DMSA. Addi-
tion of 0.5 or 1.0 mM DMSA to reaction mixtures that
contained 0.5 mM CH3AsIIII2 increased the rate of NAD-
CH3AsIII(GS)2. The inhibitory effect of 0.2 mM CH3AsIII
-

Inhibition of Glutathione Reductase by Arsenicals
Chem. Res. Toxicol., Vol. 10, No. 1, 1997 31
F igu r e 5. Concentration-dependent effects of DMSA on inhibi-
tion of GSH reductase by trivalent organoarsenicals (a) or
organoarsenothiols (b) in the presence of 0.1 mM GSSG.
Arsenicals and/or DMSA were added to the reaction mixtures
during the 2-min preincubation period, and reactions were
started by addition of 0.23 mM NADPH. ), DMSA alone; O, 0.5
mM (CH3)2AsIIII; 0, 0.5 mM CH3AsIIII2; 9, 0.1 mM CH3AsIII(GS)2;
b, 0.2 mM CH3AsIII(Cys)2.
F igu r e 6. Effect of delayed addition of DMSA on the rate of
NADPH oxidation in reaction mixtures that contained 0.5 mM
CH3AsIIII2 (a) or 0.5 mM (CH3)2AsIIII (b). Following preincuba-
tion of enzyme and 0.1 mM GSSG with or without arsenical for
2 min, reactions were started by addition of 0.23 mM NADPH.
One minute after addition of NADPH, DMSA was added to the
reaction mixture. Rates of NADPH oxidation were determined
before and after addition of DMSA. O, assay mixture without
arsenical or DMSA added; 0, assay mixture with only DMSA
added; b, assay mixture with only arsenical added; 9, assay
mixture with arsenical and 0.5 mM DMSA added; (, assay
mixture with arsenical and 1.0 mM DMSA added.
PH consumption by about 35% (Figure 6a). Similarly,
only partial reversibility of inhibition was observed when
DMSA was added at a molar ratio (DMSA:arsenical) of
4:1 to 10:1 to reaction mixtures containing 1 mM
AsIIII2 or (CH3)2AsIIII in an assay system preincubated
with 10 µM NADPH. Pretreatment with NADPH de-
creased the Ki’s for both organoarsenicals. The estimated
Ki’s for CH3AsIIII2 and (CH3)2AsIIII following preincuba-
tion with NADPH were 0.034 mM and 0.014 mM,
respectively. For (CH3)2AsIIII, the mode of inhibition was
competitive for both the native enzyme and the NADPH-
pretreated enzyme (Figure 7b). For CH3AsIIII2, NADPH
pretreatment changed the mode of inhibition to noncom-
petitive (Figure 7a).
AsIII(GS)3, 0.1 mM CH3AsIII(GS)2, or 0.2 mM CH3AsIII
-
(Cys)2 (data not shown). In contrast, addition of 0.5 mM
DMSA to a reaction mixture that contained 0.5 mM
(CH3)2 AsIIII completely reversed inhibition (Figure 6b).
Effect of P r ein cu ba tion w ith NADP H on In h ibi-
tion by GSH Red u cta se. The preincubation of purified
GSH reductase with low concentrations of NADPH
produces a time-, temperature-, and concentration-de-
pendent reduction in the rate of GSSG reduction (23).
The effects of preincubation with NADPH on the inhibi-
tion of enzymatic activity by organoarsenicals were
examined. Here, the procedure was modified to include
10 µM NADPH in the reaction mixture (buffer, enzyme,
0.1 mM GSSG, and arsenical) during the 2-min prein-
cubation period. Assays were started by the addition of
NADPH to a final concentration of 0.23 mM, and time-
dependent changes in absorbance at 340 nm were moni-
tored. Over the substrate concentration range of 0.1-1
mM GSSG, pretreatment with NADPH reduced the rate
of GSSG reduction to about 50% of the rate found in
control assays. Figure 7 shows double-reciprocal plots
of 1/v versus 1/[GSSG] for different concentrations of CH3-
Discu ssion
The work reported here demonstrates that valence and
complexation with thiols affect the potency of arsenicals
as inhibitors of the NADPH-dependent reduction of
GSSG by GSH reductase. These findings suggest that
the biomethylation of As, a process catalyzed by cytosolic
AdoMet-requiring enzyme(s) (11, 24), yields organoarseni-
cals that are more potent inhibitors of GSH reductase
than the parent inorganic As. The commonly-held belief
that biomethylation is a mechanism for the detoxication

32 Chem. Res. Toxicol., Vol. 10, No. 1, 1997
Styblo et al.
pared to those observed after addition of GSH to the
reaction mixture. Previous work has shown that AsIII is
readily donated from AsIII(GS)3 to DMSA and that DMSA
and its analogs are effective antidotes for acute As
intoxication (27, 28). Addition of DMSA to the reaction
mixture before the addition of NADPH antagonized the
inhibitory effect of CH3AsIIII2, CH3AsIII(GS)2, CH3AsIII
-
(Cys)2, or (CH3)2AsIIII. Activity was almost fully restored
for assays containing CH3AsIIII2, CH3AsIII(GS)2, or (CH3)2-
AsIIII. Previous work has shown that addition of DMSA
to the reaction mixture before addition of NADPH
antagonized the inhibitory effect of AsIII(GS)3 (19). The
failure of DMSA to restore fully the activity of reaction
mixtures that contained CH3AsIII(Cys)2 could be due to
the liberation of Cys during the decomposition of the
complex. Because Cys is a fairly potent competitive
inhibitor of GSH reductase, its release could affect the
restoration of enzymatic activity. Alternatively, a mixed
GSH-DMSA disulfide could be formed and could be an
inhibitor of GSH reductase. For reaction mixtures that
contained As(GS)3, CH3AsIIII2, CH3AsIII(GS)2, or CH3AsIII
-
(Cys)2, the delayed addition of a molar excess of DMSA
after addition of NADPH only partly restored activity.
The inhibitory effect of (CH3)2AsIIII was fully reversed by
addition of an equimolar amount of DMSA. The failure
of DMSA to reverse inhibition of GSSG reduction by some
trivalent arsenicals and their thiol complexes suggests
that in the presence of NADPH these species are bound
to sites in the enzyme that are of higher affinity than
those provided by DMSA. These high-affinity binding
sites could be provided by the 5 Cys residues in each
subunit of dimeric GSH reductase of Saccharomyces
cerevisiae (29).
F igu r e 7. Effect of preincubation with NADPH on inhibition
of GSH reductase by organoarsenicals. Double-reciprocal plots
of 1/v vs 1/[GSSG] for 0 mM (O), 0.025 mM (0), and 0.05 mM
(b) CH3AsIIII2 (a) and for 0 mM (O), 0.05 mM (0), and 0.1 mM
(b) (CH3)2AsIIII (b) in a reaction mixture preincubated with 10
µM NADPH. Following preincubation of enzyme, arsenical, 10
µM NADPH, and 0.1 mM GSSG for 2 min, the reaction was
started by addition of 0.23 mM NADPH.
Finally, the nature of inhibition by arsenicals was
examined using GSH reductase that was exposed to a
low concentration of NADPH before assay of its capacity
to reduce GSSG. GSH reductase is inactivated by
NADPH by an intramolecular modification of the dimeric
protein (30). Inactivation is thought to involve the
formation of an “erroneous disulfide” between a critical
Cys residue of the redox active site of the enzyme and a
neighboring Cys. This effect is reversed by exposure to
mono- and dithiols. Coexposure to NADPH and AsIII
results in greater inhibition of GSH reductase than is
produced by NADPH alone. The effect of both inhibitors
could be reversed with mono- and dithiols (30). The
experiments reported have examined the influence of
NADPH inactivation on the characteristics of inhibition
by CH3AsIIII2 and (CH3)2AsIIII. For both organoarseni-
cals, the NADPH-inactivated enzyme yielded lower Ki’s,
and, for CH3AsIIII2 but not (CH3)2AsIIII, changed the mode
of inhibition from competitive to noncompetitive. These
findings suggest that NADPH inactivation alters the
structure of the enzyme in a manner that affects the
nature of its interactions with organoarsenicals. Because
NADPH may modify a site adjacent to the redox active
center of the enzyme, these findings suggest that the
organoarsenicals may be interacting with a site in close
proximity to the active center of the enzyme.
of As (25) may be altered by results of the current study
that demonstrate the products of the methylation of As
to be potent and specific inhibitors of GSH reductase.
As has been reported for GSH reductases from other
species (14, 26), GSH was found to be an uncompetitive
inhibitor of yeast GSH reductase. The complexation of
arsenicals by GSH or Cys had striking effects on their
potency as inhibitors of GSH reductase. GSH- or Cys-
containing complexes of AsIII and CH3AsIII were more
potent inhibitors than the parent arsenicals. In contrast,
either thiol-containing complex with (CH3)2AsIII was less
potent than (CH3)2AsIIII. Addition of GSH to the reaction
mixture before addition of NADPH to initiate GSSG
reduction resulted in patterns and magnitudes of inhibi-
tion consistent with those observed for authentic com-
plexes of GSH with AsIII, CH3AsIII(GS)2, or (CH3)2AsIII
.
This finding suggested that arsenothiol complexes could
be formed in the reaction mixture. However, if the
addition of GSH was delayed until after preincubation
with NADPH, then the pattern and magnitude of inhibi-
tion for CH3AsIIII2 differ from those observed for authentic
CH3AsIII(GS)2. This finding suggested that the presence
of NADPH prevented the interaction of the arsenothiol
complex formed in the reaction mixture with some critical
site in the enzyme.
Ack n ow led gm en t. M.S. is a postdoctoral fellow
supported by Training Grant T901915 of the U.S. Envi-
ronmental Protection Agency/University of North Caro-
lina Toxicology Research Program with the Curriculum
in Toxicology, University of North Carolina at Chapel
Hill. We thank Ms. Karen Herbin-Davis for excellent
technical assistance. This article has been reviewed in
The effects of addition of DMSA on the potency of
arsenicals as inhibitors of GSH reductase can be com-

Inhibition of Glutathione Reductase by Arsenicals
Chem. Res. Toxicol., Vol. 10, No. 1, 1997 33
mixed disulfides and its relationship to glutathione disulfide.
Biochem. Pharmacol. 32, 2529-2534.
accordance with the policy of the National Health and
Environmental Effects Research Laboratory, U.S. Envi-
ronmental Protection Agency, and approved for publica-
tion. Approval does not signify that the contents neces-
sarily reflect the views and policies of the Agency, nor
does mention of trade names or commercial products
constitute endorsement or recommendation for use.
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