Inactivation of glutathione transferase zeta by dichloroacetic acid and other fluorine-lacking α-haloalkanoic acids

Article abstract of DOI:10.1021/tx990085l

Dichloroacetic acid (DCA) is a contaminant of chlorinated drinking water supplies, is carcinogenic in rats and mice, and is a therapeutic agent used for the treatment of congenital lactic acidosis. The biotransformation of DCA to glyoxylic acid is catalyzed by glutathione transferase zeta (GSTZ). Treatment of rats and human subjects with DCA increases its plasma elimination half-life and reduces the extent of DCA biotransformation in rat hepatic cytosol. In the investigation presented here, the kinetics of the DCA-induced inactivation of GSTZ, the turnover of GSTZ, and the susceptibility of GSTZ to inactivation by a panel of α-haloacids were studied. DCA rapidly inactivated GSTZ in both rat hepatic cytosol and intact Fischer 344 rats. The time course of inactivation in vivo was mirrored by a concomitant loss of immunoreactive GSTZ protein. The turnover of GSTZ in rat liver was 0.21 day^-1, which corresponded to a half-life of 3.3 days. The degree of GSTZ inactivation after daily administration of DCA could be predicted from the amount of inactivation after a single treatment. Other fluorine-lacking dihaloacetic acids also inactivated GSTZ, whereas α- monohaloacids and fluorine-containing dihaloacetic acids failed to inactivate GSTZ. These data show that the observed DCA-induced decrease in the level of DCA metabolism is caused by the inactivation of GSTZ.

Full text of DOI:10.1021/tx990085l

1144
Chem. Res. Toxicol. 1999, 12, 1144-1149
In a ctiva tion of Glu ta th ion e Tr a n sfer a se Zeta by
Dich lor oa cetic Acid a n d Oth er F lu or in e-La ck in g
r-Ha loa lk a n oic Acid s
Wayne B. Anderson,† Philip G. Board,‡ Bryan Gargano,† and M. W. Anders*,†
Department of Pharmacology and Physiology, University of Rochester Medical Center,
601 Elmwood Avenue, Box 711, Rochester, New York 14642, and J ohn Curtin School of Medical
Research, Australian National University, GPO Box 34, Canberra ACT 2601, Australia
Received May 17, 1999
Dichloroacetic acid (DCA) is a contaminant of chlorinated drinking water supplies, is
carcinogenic in rats and mice, and is a therapeutic agent used for the treatment of congenital
lactic acidosis. The biotransformation of DCA to glyoxylic acid is catalyzed by glutathione
transferase zeta (GSTZ). Treatment of rats and human subjects with DCA increases its plasma
elimination half-life and reduces the extent of DCA biotransformation in rat hepatic cytosol.
In the investigation presented here, the kinetics of the DCA-induced inactivation of GSTZ,
the turnover of GSTZ, and the susceptibility of GSTZ to inactivation by a panel of R-haloacids
were studied. DCA rapidly inactivated GSTZ in both rat hepatic cytosol and intact Fischer
344 rats. The time course of inactivation in vivo was mirrored by a concomitant loss of
immunoreactive GSTZ protein. The turnover of GSTZ in rat liver was 0.21 day-1, which
corresponded to a half-life of 3.3 days. The degree of GSTZ inactivation after daily administra-
tion of DCA could be predicted from the amount of inactivation after a single treatment. Other
fluorine-lacking dihaloacetic acids also inactivated GSTZ, whereas R-monohaloacids and
fluorine-containing dihaloacetic acids failed to inactivate GSTZ. These data show that the
observed DCA-induced decrease in the level of DCA metabolism is caused by the inactivation
of GSTZ.
acetylglycine, and carbon dioxide (11-13). The biotrans-
formation of DCA to glyoxylic acid is catalyzed by
glutathione-dependent cytosolic enzymes (14, 15). Recent
studies show that GSTZ catalyzes the biotransformation
of DCA to glyoxylic acid (16) and that glyoxylic acid is
the sole product of the GSTZ-catalyzed biotransformation
of DCA (17). The formation of glycolic acid and oxalic acid
can be explained by the lactate dehydrogenase-catalyzed
reduction and oxidation, respectively, of glyoxylic acid.
R-Ketoglutarate:glyoxylate carboligase catalyzes the con-
version of glyoxylic acid to carbon dioxide, and the
transamination of glyoxylic acid affords glycine, which
is metabolized to hippuric acid. GSTZ is identical with
maleylacetoacetate isomerase, which catalyzes the isomer-
ization of maleylacetoacetate to fumarylacetoacetate, the
penultimate step in the tyrosine degradation pathway
(18).
In tr od u ction
Trihalomethanes and haloacetic acids, including dichlo-
roacetic acid (DCA)1 and bromochloroacetic acid, are the
most abundant disinfection byproducts found in chlori-
nated drinking water supplies in the United States (1,
2). DCA is also formed as a metabolite of tetrachloro-
ethene, trichloroethene, trichloroacetic acid, and chloral
hydrate (3-6).
DCA is hepatocarcinogenic in male B6C3F1 mice (7)
and in male Fischer 344 rats (8), which are estimated to
be about 10 times more sensitive than male B6C3F1 mice
to the induction of hepatocellular neoplasia by DCA (8).
The mechanism by which DCA induces liver tumor
formation has not been determined.
DCA, which activates pyruvate dehydrogenase second-
ary to its inhibition of pyruvate dehydrogenase kinase,
is used clinically in the management of congenital lactic
acidosis (9). DCA has been proposed for use as a neuro-
protective agent (10) and in the management of other
disease-associated lactic acidoses.
Pharmacokinetic studies show that repeated treatment
with DCA increases its plasma elimination half-life in
both rats and humans (13, 19-22). Furthermore, the
extent of glutathione-dependent biotransformation of
DCA to glyoxylic acid is reduced in hepatic cytosol from
rats given DCA (15).
DCA is metabolized to chloroacetic acid, glyoxylic acid,
glycolic acid, oxalic acid, glycine, hippuric acid, phenyl-
The objective of the experiments described here was
to explore further the mechanisms of the apparent DCA-
induced reduction in the level of DCA metabolism. We
report herein the kinetics of the DCA-induced inactiva-
tion of GSTZ, the turnover of GSTZ, and the susceptibil-
ity of GSTZ to inactivation by a panel of R-haloacids.
These data provide the first direct evidence that GSTZ
is irreversibly inactivated by DCA.
* To whom correspondence should be addressed: Department of
Pharmacology and Physiology, University of Rochester Medical Center,
601 Elmwood Ave., Box 711, Rochester, NY 14642. Telephone: (716)
275-1681.
Fax:
(716)
0244-9283.
E-mail:
anders@
pharmacol.rochester.edu.
† University of Rochester Medical Center.
‡ Australian National University.
1 Abbreviations: DCA, dichloroacetic acid; GSTZ, glutathione trans-
ferase zeta.
10.1021/tx990085l CCC: $18.00 © 1999 American Chemical Society
Published on Web 10/29/1999

Inactivation of Glutathione Transferase Zeta
Chem. Res. Toxicol., Vol. 12, No. 12, 1999 1145
Ma ter ia ls a n d Meth od s
Ch em ica ls a n d Rea gen ts. Dichloroacetic acid, difluoroacetic
acid, 2-chloropropionic acid, 2,2-dichloropropionic acid, and
bromofluoroacetic acid ethyl ester were obtained from Aldrich
Chemical Co. (Milwaukee, WI). Dibromoacetic acid was pur-
chased from J anssen Chimica (Beerse, Belgium). Chlorofluoro-
acetic acid ethyl ester was obtained from Lancaster Synthesis,
Inc. (Windham, NH). Bromochloroacetic acid was kindly pro-
vided by A. B. DeAngelo (U.S. Environmental Protection Agency,
Research Triangle Park, NC). Phenylhydrazine, potassium
ferricyanide, phenylmethanesulfonyl fluoride, and mono- and
dibasic potassium phosphates were purchased from Sigma
Chemical Co. (St. Louis, MO). Dithiothreitol was purchased from
Eastman Kodak (Rochester, NY). Rabbit anti-human GSTZ1-1
polyclonal antibody was prepared as described previously (23).
Reagents for SDS-PAGE, Western analysis, and protein quan-
tification were obtained from Bio-Rad Laboratories (Hercules,
CA). Other reagents were purchased from commercial suppliers.
Syn th eses. The method of Tong et al. (17) was used with
slight modification to prepare bromofluoro- and chlorofluoro-
acetic acids; 2 g of the ethyl esters was heated with stirring at
50 °C in 40 mL of 1 N H2SO4 until the immiscible ester
disappeared (3-4 h). The reaction mixture was then extracted
four times with ethyl acetate, and the combined extracts were
concentrated under vacuum. The crude product was purified by
Kugelrohr distillation (24). 1H NMR spectroscopy indicated that
the acids were >99% pure.
An im a l Tr ea tm en ts a n d P r ep a r a tion of Liver Cytosol.
Male Fischer 344 rats (200-225 g, Charles River Laboratories,
Wilmington, MA) were kept on a 12 h light/dark cycle and were
provided with food and water ad libitum. Rats were given
R-haloalkanoic acids as neutralized solutions in 0.9% NaCl by
ip injection between 9 a.m. and noon. Control animals were
given 0.9% NaCl. At the indicated times, the rats were anes-
thetized with diethyl ether and killed by decapitation. The livers
were homogenized in 4 volumes (w/v) of 20 mM potassium
phosphate buffer (pH 7.4) containing 1.15% KCl, 2 mM EDTA,
2 mM DTT, and 100 µM phenylmethanesulfonyl fluoride. The
cytosolic fraction was prepared by differential ultracentrifuga-
tion, as previously described (25), and dialyzed overnight with
two buffer changes against 30 volumes of homogenization buffer
(without 1.15% KCl). Dialyzed cytosol was stored at -80 °C until
it was used. No reduction in GSTZ activity was observed after
storage for several months under these conditions. Protein
concentrations were determined by the method of Bradford (26)
with bovine serum albumin as the standard.
F igu r e 1. DCA-induced inactivation of GSTZ in vitro. Reaction
mixtures contained rat liver cytosol (0.5 mg of protein/mL), 1
mM glutathione, and 100 mM phosphate buffer (pH 7.4) and
were incubated for 3 min at 37 °C, and reactions were then
initiated by addition of 0.5 mM DCA. At the indicated times,
the reactions were quenched by addition of trifluoroacetic acid,
and glyoxylic acid formation (b) was quantified as described in
Materials and Methods. Where indicated (dashed lines), the
reaction mixtures were incubated and analyzed for glyoxylic acid
as described above except that after incubation for 30 min a
second addition of either rat liver cytosol (O) or DCA (4) was
made, and the reaction mixtures were incubated for 0-10 min.
The reactions were quenched, and glyoxylic acid formation was
quantified, as described above. Data are shown as means ( SD
(n ) 3). The asterisk indicates a significant difference (p < 0.05)
from the complete system (b) or after the second addition of
DCA (4).
phosphatase conjugate. The alkaline phosphatase substrates
5-bromo-4-chloro-3-indoyl phosphate and nitroblue tetrazolium
were employed for detection. Developed blots were scanned as
gray-scale images, and the density in each band was determined
with the GelPlot2 macro of ScionImage â3b software
(www.scioncorp.com). The linearity of the response was con-
firmed by analyzing 3.2-16 µg samples of protein from control
rat liver cytosol. Only samples from the same blot were
compared when determining relative immunoreactivities.
Estim a tion of P r otein Tu r n over . The turnover rate of
GSTZ in rat liver was estimated from the time course of
inactivation (see Figure 2A) by the method of Price et al. (29).
[Discussions of this and other methods for determining the rate
of protein turnover are available (30, 31).] Briefly, after admin-
istration of an irreversible inhibitor, the rate at which an
enzyme recovers activity is a function of two competing pro-
cesses: synthesis and degradation. This relationship is described
in eq 1, where e is defined as enzyme activity:
Activity Assa y. GSTZ activity was determined by measuring
the extent of product formation with a discontinuous assay, as
previously described (16). Reaction mixtures contained rat liver
cytosol (0.5 mg of protein/mL) and glutathione (1 mM) in a final
volume of 1 mL of 0.1 M phosphate buffer (pH 7.4). Reaction
mixtures were incubated for 3 min at 37 °C; the reaction was
initiated by addition of 0.5 mM DCA. At the indicated times, or
routinely after 20 min, reactions were quenched by addition of
50 µL of trifluoroacetic acid. The precipitated proteins were
removed by centrifugation, and the supernatant was analyzed
for glyoxylic acid by the method of Vogels and Van Der Drift
(27).
de
dt
) ksynthesis - kdegradation × e
(1)
The rates of synthesis and degradation are zero- and first-order
processes, respectively. At steady state,
de
dt
) 0 and ksynthesis ) kdegradation × ess
(2)
Wester n An a lysis a n d Qu a n tifica tion of Im m u n or ea c-
tive GSTZ. For quantification of immunoreactive GSTZ, rat
liver cytosol (16 µg of protein) from all rats at all time points
was resolved by electrophoresis on 12% gels according to the
method of Laemmli (28) and then electrophoretically transferred
to 0.45 µm nitrocellulose membranes. For the data shown in
Figure 2B, 15% gels were used, and 20 µg of liver cytosolic
protein from rats at each time point with median enzyme
activity was analyzed. Western analysis was performed accord-
ing to instructions provided with the Bio-Rad goat anti-rabbit
IgG-alkaline phosphatase Immun-Blot assay kit. Membranes
were incubated for 1 h with a 1:1000 dilution of rabbit anti-
human GSTZ1-1, which cross-reacts with rat GSTZ (16), and
for 1 h with a 1:3000 dilution of goat anti-rabbit IgG-alkaline
Substitution of eq 2 into eq 1 and rearrangement yield the first-
order relationship
de
dt
) kdegradation(ess - e)
(3)
Integration of eq 3 gives
ln(ess - e) ) -kdegradation × t + c
(4)
The degradation rate of the enzyme is obtained from the slope
of a plot of the natural logarithm of the difference between the
enzyme activity at steady state and the enzyme activity at
several times during the course of recovery versus time. The

1146 Chem. Res. Toxicol., Vol. 12, No. 12, 1999
Anderson et al.
F igu r e 3. DCA-induced inactivation of GSTZ after five daily
treatments. Male Fischer 344 rats (n ) 3/treatment group) were
given five daily doses of 0.3 mmol of DCA/kg or saline ip.
Animals were killed 24 h after the last dose of DCA. GSTZ
activity was measured in liver cytosol, as described in Materials
and Methods. Data are expressed as the percent of control
(means ( SD, n ) 3). Activity remaining 24 h after giving a
single dose of 0.3 mmol of DCA/kg (Figure 2) was used to predict
that GSTZ activity would be reduced to ∼2% [(%A24h)5] after
five daily doses.
nism, a second addition of either DCA or cytosol was
made after the reaction reached a plateau. After addition
of cytosol, the reaction proceeded at the initial rate,
whereas the addition of DCA did not result in further
glyoxylic acid formation, indicating the enzyme was
inactivated (Figure 1). To determine whether GSTZ was
stable under the incubation conditions, rat liver cytosol
was incubated for 30 min before the addition of DCA. No
reduction in the initial rate of reaction or change in the
overall time course of formation of glyoxylic acid from
DCA was observed (data not shown).
In a ctiva tion of GSTZ in Vivo by DCA. The dose
dependency and kinetics of the DCA-induced inactivation
of GSTZ in rats were studied. Treatment of rats with
0.15, 0.30, and 0.60 mmol of DCA/kg reduced GSTZ
activity 24 h after treatment to 79 ( 3%, 55 ( 4%, and
24 ( 3%, respectively, compared with saline-treated rats.
The kinetics of GSTZ inactivation was investigated
after giving rats 0.30 mmol of DCA/kg. Administration
of DCA resulted in a rapid loss of both enzymatic activity
and immunoreactive GSTZ protein (panels A and B of
Figure 2). The nadir of both activity and the amount of
immunoreactive GSTZ protein occurred 12 h after treat-
ment, and GSTZ activity and the amount of immunore-
active GSTZ protein did not return to initial values until
10-12 days after treatment (Figure 2A). Furthermore,
administration of five daily doses of 0.30 mmol of DCA/
kg to Fischer 344 rats reduced GSTZ activity to about
5% of control values (Figure 3) and immunoreactive
GSTZ protein concentrations to less than 10% of control
values (data not shown). The observed degree of inactiva-
tion was similar to that predicted by extrapolating the
activity remaining 24 h after a single administration to
5 days [(activity24h)5] (Figure 3).
F igu r e 2. DCA-induced inactivation of GSTZ in vivo. (A) Male
Fischer 344 rats (n ) 3/time point) were given 0.3 mmol of DCA/
kg ip and were killed at the indicated times. GSTZ activity (b)
and the amount of immunoreactive GSTZ protein (O) were
measured in liver cytosol as described in Materials and Methods
and are expressed as the percent of the 0 h value. Data are
shown as means ( SD (n ) 3). (B) Representative immunoblots
of rat liver cytosol (20 µg of protein/lane) collected at the
indicated times after DCA administration.
half-life for protein turnover is related to the degradation rate
constant by the expression
t1/2 ) ln 2/kdegradation ) 0.693/kdegradation
(5)
For this study, steady-state activity was taken as the activity
determined 12 days after giving DCA.
Sta tistica l An a lysis. Statistical analyses were carried out
with Microsoft Excel 97. Data are expressed as means ( SD.
Some data were analyzed with a two-tailed Student’s t test. A
p value of e0.05 was chosen for acceptance or rejection of the
null hypothesis.
Resu lts
In a ctiva tion of GSTZ in Vitr o by DCA. To inves-
tigate whether the observed decrease in the extent of
biotransformation of DCA seen in hepatic cytosol from
rats treated with DCA was due to the inactivation of
GSTZ, the kinetics of the biotransformation of DCA to
glyoxylic acid by rat liver cytosol was studied. The extent
of biotransformation of DCA to glyoxylic acid was not
linear with time and showed no further increase after
incubation for 20-30 min (Figure 1), after which time
an estimated 5% of the DCA had been converted to
glyoxylic acid. The nonlinearity of the biotransformation
of DCA could be due to the establishment of an equilib-
rium between substrate and product, competitive inhibi-
tion, or inactivation of GSTZ. To determine the mecha-
Tu r n over of GSTZ. The data presented above indi-
cated that GSTZ was irreversibly inactivated by DCA and
targeted for proteolysis in vivo. It was, therefore, impor-
tant to determine the time course of recovery of activity.
The method of Price et al. (29) was used to determine
the rate of turnover of GSTZ in vivo. GSTZ was degraded
in rat liver at the rate of -0.21 day-1, which corresponded
to a t1/2 of 3.3 days (Figure 4).
In a ctiva tion of GSTZ by DCA An a logu es. GSTZ
catalyzes the biotransformation of a range of R-haloal-

Inactivation of Glutathione Transferase Zeta
Chem. Res. Toxicol., Vol. 12, No. 12, 1999 1147
restart the reaction indicated that the enzyme was
irreversibly inactivated. If the reaction had stalled
because of the establishment of an equilibrium between
substrate and product or competitive inhibition, a second
addition of DCA would have been expected to restart the
reaction. Also, dialysis of DCA-inactivated recombinant
GSTZ1-1 did not result in significant recovery of activity,2
which also indicates that DCA irreversibly inactivates
GSTZ. Gonzalez-Leon et al. (32) recently reported that
the Vmax for the biotransformation of dibromoacetic acid,
bromochloroacetic acid, and DCA was decreased after
their administration in the drinking water, but did not
alter the Km of the enzyme for their biotransformation,
which is consistent with the inactivation of GSTZ.
F igu r e 4. Determination of the rate of GSTZ turnover in rat
liver. The activity data from the time course of inactivation
experiment (Figure 2) were used to determine the degradation
rate constant, as described in Materials and Methods. The
degradation rate constant for GSTZ was -0.21 day-1 (r2 ) 0.96),
which corresponded to a half-life of 3.3 days.
DCA-induced inactivation of GSTZ in vivo was rapid
and dose-dependent. The maximal reduction of GSTZ
activity occurred 12 h after the administration of a single
dose of 0.3 mmol of DCA/kg. This dose is similar to the
therapeutic doses used in the management of lactic
acidosis (33), but is much larger than the estimated
human intake from drinking water containing DCA,
which amounts to about 4 µg kg-1 day-1 (33). The
elimination t1/2 of DCA in Fischer 344 rats is 2.4 h (22);
hence, after 5 t1/2 (or 12 h), more than 95% of the DCA
would be eliminated and unavailable to inactivate GSTZ.
Quantitative Western analysis revealed that the decrease
in GSTZ activity paralleled the decrease in the amount
of immunoreactive GSTZ protein during the time course
of enzyme inactivation, indicating that DCA-inactivated
GSTZ was rapidly degraded. The rapidity of the loss of
GSTZ activity and protein in vivo complements the in
vitro observation that DCA inactivates GSTZ directly.
Hence, other mechanisms of downregulation, e.g., de-
creased levels of transcription or translation or decreased
mRNA stability, are not involved in the observed reduc-
tion in the level of DCA metabolism. Furthermore, the
finding that the GSTZ activity remaining 5 days after
the daily administration of DCA could be predicted from
the activity remaining 24 h after a single treatment with
DCA indicates that GSTZ activity is not induced by
repeated DCA treatment.
F igu r e 5. Inactivation of GSTZ in vivo by R-haloalkanoic acids.
Male Fischer 344 rats (n ) 3/treatment group) were given 0.3
mmol of R-haloalkanoic acids/kg ip and were killed 12 h after
treatment. GSTZ activity was measured in liver cytosol, as
described in Materials and Methods. Data are expressed as
means ( SD (n ) 3). The asterisk indicates a significant
difference (p <0.05) from rats given saline. BFA, bromofluoro-
acetic acid; CPA, (R,S)-2-chloropropionic acid; CFA, chlorofluo-
roacetic acid; DFA, difluoroacetic acid; DCPA, 2,2-dichloropro-
pionic acid; DCA, dichloroacetic acid; BCA, bromochloroacetic
acid; DBA, dibromoacetic acid.
kanoic acids (17). Hence, the inactivation of GSTZ by
R-haloalkanoic acids was studied in vivo. Treatment of
rats with 2,2-dichloropropionic acid, DCA, bromochloro-
acetic acid, and dibromoacetic acid reduced GSTZ activity
12 h after treatment to 78 ( 0%, 55 ( 2%, 19 ( 4%, and
17 ( 0% (n ) 3 rats/treatment group), respectively,
compared with saline-treated animals. Administration of
bromofluoroacetic acid, (R,S)-2-chloropropionic acid, chlo-
rofluoroacetic acid, and difluoroacetic acid had no effect
on GSTZ activity (Figure 5).
Inspection of the immunoblots (Figure 2B) showed the
appearance of an immunoreactive protein with a molec-
ular mass slightly higher than that of GSTZ in samples
taken 1.5, 3, and 6 h after DCA administration, which
may indicate that GSTZ is inactivated through a covalent
modification involving DCA or glutathione, or both. The
higher-molecular mass, immunoreactive GSTZ was not
present in na¨ıve rats or at any time during the course of
GSTZ recovery, which further implicates this as an
inactivated form of the enzyme.
The rate of turnover of hepatic GSTZ was 0.21 day-1
in Fischer 344 rats. The determination of the turnover
rate is important for understanding DCA metabolism in
vivo, because it allows prediction of the time required for
restoration of GSTZ activity after any level of GSTZ
inactivation. Curry et al. (20) showed that increased
plasma DCA elimination half-lives can persist for as long
as 3 months after DCA exposure in humans, indicating
a different turnover rate in humans and rats. Also,
Gonzalez-Leon et al. (32) reported that DCA metabolic
activity recovers much faster in mice than in rats after
continuous DCA treatment for 2 weeks.
Discu ssion
The study presented here provides the first direct
evidence that the DCA-induced reduction in its extent
of metabolism is caused by the irreversible inactivation
of GSTZ.
In vitro biotransformation experiments showed that
the rate of biotransformation of DCA to glyoxylic acid by
rat liver cytosol was linear with time for about 10 min
and then reached a plateau. The reason for the decrease
in the rate of GSTZ-catalyzed biotransformation of DCA
was investigated by making a second addition of either
enzyme or DCA to the stalled reaction. The observation
that a second addition of enzyme, but not of DCA, could
2
H.-F. Tzeng and M. W. Anders, unpublished observations.

1148 Chem. Res. Toxicol., Vol. 12, No. 12, 1999
Anderson et al.
Sch em e 1
Tong et al. (17) recently reported that GSTZ catalyzes
the biotransformation of a range of dihaloacetic acids and
R-halopropionic acids. In the studies presented here,
fluorine-lacking dihaloacetic acids (DCA, bromochloro-
acetic acid, and dibromoacetic acid) and 2,2-dichloropro-
pionic acid inactivated GSTZ, but no inactivation was
observed after giving fluorine-containing dihaloacetic
acids (bromofluoroacetic acid, chlorofluoroacetic acid, and
difluoroacetic acid) or (R,S)-2-chloropropionic acid. The
inactivation of GSTZ by fluorine-lacking dihaloacetic
acids (Scheme 1, 1, R ) H, X1 ) X2 ) Br or Cl) may be
explained by the formation of an S-(R-halocarboxymeth-
yl)glutathione intermediate (Scheme 1, 2, R ) H, X2 )
Br or Cl), which may react with a nucleophilic site on
the enzyme to give a covalently modified, inactivated
enzyme (Scheme 1, 3, R ) H). The hydrolysis of the S-(R-
halocarboxymethyl)glutathione intermediate would af-
ford the hemithioacetal S-(R-hydroxycarboxymethyl)-
glutathione (Scheme 1, 4, R ) H), which may eliminate
glutathione to give glyoxylic acid (Scheme 1, 5, R ) H).
Hence, hydrolysis of S-(R-halocarboxymethyl)glutathione
(Scheme 1, 2) would compete with inactivation of the
enzyme. Similarly, GSTZ-catalyzed reaction of 2,2-dichlo-
ropropionic acid (Scheme 1, 1, R ) CH3, X1 ) X2 ) Cl)
with glutathione may afford an S-(R-methyl-R-halocar-
boxymethyl)glutathione intermediate (Scheme 1, 2, R )
CH3, X2 ) Cl), which may react with a nucleophilic site
on the enzyme (Scheme 1, 3, R ) CH3) or may undergo
hydrolysis to pyruvic acid (Scheme 1, 5, R ) CH3). The
reason for the failure of monofluorine-containing diha-
loacetic acids, which are substrates for GSTZ, to inacti-
vate the enzyme is not fully understood. It is possible to
speculate that the S-(R-fluorocarboxymethyl)glutathione
intermediate (Scheme 1, 2, R ) H, X2 ) F) cannot form
a resonance-stabilized zwitterionic intermediate because
fluoride is a strong base and, therefore, a poor nucleofuge
(34). Hence, this fluorine-containing intermediate may
be more stable than the bromine- or chlorine-containing
analogues and may undergo hydrolysis to give product
rather than producing detectable inactivation. The failure
of (R,S)-2-chloropropionic acid (Scheme 1, 6) to inactivate
GSTZ is consistent with the formation of the stable
glutathione conjugate S-(R-methylcarboxymethyl)glu-
tathione (Scheme 1, 7) (17). Difluoroacetic acid is not a
substrate for GSTZ (17). Hence, neither (R,S)-2-chloro-
propionic acid nor difluoroacetic acid forms a reactive
intermediate that inactivates GSTZ.
The development of isozyme-selective inhibitors of
glutathione transferases that are effective in vivo has
received much recent attention (for a review, see ref 35).
Presently, few isozyme-selective, irreversible inhibitors
of glutathione transferases have been identified; haloenol
lactone (36), N-ethylmaleimide (37), and derivatives of
ethacrynic acid (38) are isozyme-selective, irreversible
inhibitors of glutathione transferase π. In addition, the
in vivo effectiveness of glutathione transferase inhibitors
has only been demonstrated for ethanol (39) and (R)-5-
(ethyloxycarbonyl)-2-γ-(S)-(glutamylamino)-N-2-heptyl-
pentamide (40). The findings presented here demonstrate
that DCA is an effective in vivo inactivator of GSTZ. Also,
DCA is expected to exhibit isozyme selectivity since,
unlike the other irreversible inhibitors that are active
site-directed, DCA is apparently a mechanism-based or
kcat inhibitor of GSTZ.3 In addition, the biotransformation
of DCA to glyoxylic acid does not consume or oxidize
glutathione (17), indicating that DCA, unlike ethacrynic
acid and other inhibitors that are substrates for glu-
tathione transferases, is unlikely to deplete cellular
glutathione concentrations and perturb cellular redox
status. Further investigations into the isozyme selectivity
and mechanism of inactivation of GSTZ by R-haloal-
kanoic acids are warranted.
Ack n ow led gm en t. This research was supported by
National Institute of Environmental Health Sciences
Grant ES03127. B.G. was supported by a University of
Rochester Strong Children’s Research Center Summer
Fellowship. We thank Dr. Zeen Tong (Wyeth-Ayerst,
Princeton, NJ ) and Dr. Robert Freeman (University of
Rochester, Rochester, NY) for helpful advice and as-
sistance and Ms. Sandra E. Morgan for her assistance
in the preparation of the manuscript.
Refer en ces
(1) Krasner, S. W., McGuire, M. J ., J acangelo, J . G., Patania, N. L.,
Reagan, K. M., and Aieta, E. M. (1989) The occurrence of
disinfection by-products in US drinking water. J . Am. Water
Works Assoc. 81, 41-53.
(2) Weisel, C. P., Kim, H., Haltmeier, P., and Klotz, J . B. (1998)
Exposure estimates to disinfection by-products of chlorinated
drinking water. Environ. Health Perspect. 107, 103-110.
3 H.-F. Tzeng, P. G. Board, and M. W. Anders, unpublished observa-
tions.

Inactivation of Glutathione Transferase Zeta
Chem. Res. Toxicol., Vol. 12, No. 12, 1999 1149
(3) Dekant, W., Metzler, M., and Henschler, D. (1984) Novel me-
tabolites of trichloroethylene through dechlorination reactions in
rats, mice and humans. Biochem. Pharmacol. 33, 2021-2027.
(4) Larson, J . L., and Bull, R. J . (1992) Species differences in the
metabolism of trichloroethylene to the carcinogenic metabolites
trichloroacetate and dichloroacetate. Toxicol. Appl. Pharmacol.
115, 278-285.
(5) Henderson, G. N., Yan, Z., J ames, M. O., Davydova, N., and
Stacpoole, P. W. (1997) Kinetics and metabolism of chloral hydrate
in children: identification of dichloroacetate as a metabolite.
Biochem. Biophys. Res. Commun. 235, 695-698.
(6) Vo¨lkel, W., Friedewald, M., Lederer, E., Pa¨hler, A., Parker, J .,
and Dekant, W. (1998) Biotransformation of perchloroethene:
dose-dependent excretion of trichloroacetic acid, dichloroacetic
acid, and N-acetyl-S-(trichlorovinyl)-L-cysteine in rats and hu-
mans after inhalation. Toxicol. Appl. Pharmacol. 153, 20-27.
(7) DeAngelo, A. B., Daniel, F. B., Stober, J . A., and Olson, G. R.
(1991) The carcinogenicity of dichloroacetic acid in the male
B6C3F1 mouse. Fundam. Appl. Toxicol. 16, 337-347.
(8) DeAngelo, A. B., Daniel, F. B., Most, B. M., and Olson, G. R. (1996)
The carcinogenicity of dichloroacetic acid in the male Fischer 344
rat. Toxicology 114, 207-221.
(9) Stacpoole, P. W. (1989) The pharmacology of dichloroacetate.
Metabolism 38, 1124-1144.
(10) Peeling, J ., Sutherland, G., Brown, R. A., and Curry, S. (1996)
Protective effect of dichloroacetate in a rat model of forebrain
ischemia. Neurosci. Lett. 208, 21-24.
(11) Larson, J . L., and Bull, R. J . (1992) Metabolism and lipoperoxi-
dative activity of trichloroacetate and dichloroacetate in rats and
mice. Toxicol. Appl. Pharmacol. 115, 268-277.
(12) Lin, E. L. C., Mattox, J . K., and Daniel, F. B. (1993) Tissue
distribution, excretion, and urinary metabolites of dichloroacetic
acid in the male Fischer 344 rat. J . Toxicol. Environ. Health 38,
19-32.
(13) J ames, M. O., Yan, Z., Cornett, R., J ayanti, V. M. K. M.,
Henderson, G. N., Davydova, N., Katovich, M. J ., Pollock, B., and
Stacpoole, P. W. (1998) Pharmacokinetics and metabolism of [14C]-
dichloroacetate in male Sprague-Dawley rats. Identification of
glycine conjugates, including hippurate, as urinary metabolites
of dichloroacetate. Drug Metab. Dispos. 26, 1134-1143.
(14) Lipscomb, J . C., Mahle, D. A., Brashear, W. T., and Barton, H.
A. (1995) Dichloroacetic acid: metabolism in cytosol. Drug Metab.
Dispos. 23, 1202-1205.
(15) J ames, M. O., Cornett, R., Yan, Z., Henderson, G. N., and
Stacpoole, P. W. (1997) Glutathione-dependent conversion to
glyoxylate, a major metabolite of dichloroacetate biotransforma-
tion in hepatic cytosol from humans and rats, is reduced in
dichloroacetate-treated rats. Drug Metab. Dispos. 25, 1223-1227.
(16) Tong, Z., Board, P. G., and Anders, M. W. (1998) Glutathione
transferase Zeta catalyzes the oxygenation of the carcinogen
dichloroacetic acid to glyoxylic acid. Biochem. J . 331, 371-374.
(17) Tong, Z., Board, P. G., and Anders, M. W. (1998) Glutathione
transferase Zeta-catalyzed biotransformation of dichloroacetic
acid and other R-haloacids. Chem. Res. Toxicol. 11, 1332-1338.
(18) Ferna´ndez-Can˜o´n, J . M., and Pen˜alva, M. A. (1998) Characteriza-
tion of a fungal maleylacetoacetate isomerase gene and identifica-
tion of its human homologue. J . Biol. Chem. 273, 329-337.
(19) Curry, S. H., Chu, P.-I., Baumgartner, T. G., and Stackpoole, P.
W. (1985) Plasma concentrations and metabolic effects of intra-
venous sodium dichloroacetate. Clin. Pharmacol. Ther. 37, 89-
93.
(22) Gonzalez-Leon, A., Schultz, I. R., Xu, G., and Bull, R. J . (1997)
Pharmacokinetics and metabolism of dichloroacetate in the F344
rat after prior administration in drinking water. Toxicol. Appl.
Pharmacol. 146, 189-195.
(23) Board, P. G., Baker, R. T., Chelvanayagam, G., and J ermiin, L.
S. (1997) Zeta, a novel class of glutathione transferases in a range
of species from plants to humans. Biochem. J . 328, 929-935.
(24) Williamson, K. L. (1987) Microscale Organic Experiments, D. C.
Heath, Lexington, MA.
(25) Dohn, D. R., and Anders, M. W. (1982) Assay of cysteine conjugate
â-lyase activity with S-(2-benzothiazolyl)cysteine as the substrate.
Anal. Biochem. 120, 379-386.
(26) Bradford, M. M. (1976) A rapid and sensitive method for the
quantitation of microgram quantities of protein utilizing the
principle of protein-dye binding. Anal. Biochem. 72, 248-254.
(27) Vogels, G. D., and Van Der Drift, C. (1970) Differential analyses
of glyoxylate derivatives. Anal. Biochem. 33, 143-157.
(28) Laemmli, U. K. (1970) Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature 227, 680-685.
(29) Price, V. E., Sterling, W. R., Tarantola, V. A., Hartley, R. W., J r.,
and Rechcigl, M., J r. (1962) The kinetics of catalase synthesis
and destruction in vivo. J . Biol. Chem. 237, 3468-3475.
(30) Goldberg, A. L., and Dice, J . F. (1974) Intracellular protein
degradation in mammalian and bacterial cells. Annu. Rev. Bio-
chem. 43, 835-869.
(31) Smith, K., and Rennie, M. J . (1996) The measurement of tissue
protein turnover. Baillieres Clin. Endocrinol. Metab. 10, 469-
495.
(32) Gonzalez-Leon, A., Merdink, J . L., Bull, R. J ., and Schultz, I. R.
(1999) Inhibition of metabolism by chlorinated and brominated
di-haloacetates and differential recovery in B6C3F1 mice and
F344 rats. Toxicol. Sci. 48 (1-S), 206.
(33) Stacpoole, P. W., Henderson, G. N., Yan, Y., Cornett, R., and
J ames, M. O. (1998) Pharmacokinetics, metabolism, and toxicol-
ogy of dichloroacetate. Drug Metab. Rev. 30, 499-539.
(34) Wempe, M. F., Anderson, W. B., Tzeng, H.-F., Board, P. G., and
Anders, M. W. (1999) Glutathione transferase zeta-catalyzed
biotransformation of deuterated dihaloacetic acids. Biochem.
Biophys. Res. Commun. 261, 779-783.
(35) Mulder, G. J ., and Ouwerkerk-Mahadevan, S. (1997) Modulation
of glutathione conjugation in vivo: how to decrease glutathione
conjugation in vivo or in intact cellular systems in vitro. Chem.
Biol. Interact. 105, 17-34.
(36) Zheng, J ., Mitchell, A. E., J ones, A. D., and Hammock, B. D. (1996)
Haloenol lactone is a new isozyme-selective and active site-
directed inactivator of glutathione S-transferase. J . Biol. Chem.
271, 20421-20425.
(37) Tamai, K., Satoh, K., Tsuchida, S., Hatayama, I., Maki, T., and
Sato, K. (1990) Specific inactivation of glutathione S-transferases
in class pi by SH-modifiers. Biochem. Biophys. Res. Commun. 167,
331-338.
(38) Ploemen, J . H. T. M., Bogaards, J . J . P., Veldink, G. A., van
Ommen, B., J ansen, D. H. M., and van Bladeren, P. J . (1993)
Isoenzyme selective irreversible inhibition of rat and human
glutathione S-transferases by ethacrynic acid and two brominated
derivatives. Biochem. Pharmacol. 45, 633-639.
(39) Aragno, M., Tamagno, E., Danni, O., Chiarpotto, E., Biasi, F.,
Scavazza, A., Albano, E., Poli, G., and Dianzani, M. U. (1996) In
vivo potentiation of 1,2-dibromoethane hepatotoxicity by ethanol
through inactivation of glutathione S-transferase. Chem.-Biol.
Interact. 99, 277-288.
(40) Ouwerkerk-Mahadevan, S., van Boom, J . H., and Mulder, G. J .
(1996) Isoenzyme-selective inhibition of glutathione conjugation
in vivo: selective inhibition of the conjugation of S-2-bromoiso-
valerylurea in the rat. J . Pharmacol. Exp. Ther. 276, 923-928.
(20) Curry, S. H., Lorenz, A., Chu, P.-I., Limacher, M., and Stacpoole,
P. W. (1991) Disposition and pharmacodynamics of dichloroace-
tate (DCA) and oxalate following oral DCA doses. Biopharm. Drug
Dispos. 12, 375-390.
(21) Henderson, G. N., Curry, S. H., Derendorf, H., Wright, E. C., and
Stacpoole, P. W. (1997) Pharmacokinetics of dichloroacetate in
adult patients with lactic acidosis. J . Clin. Pharmacol. 37, 416-
425.
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