Reversal of arsenic-induced hepatic apoptosis with combined administration of DMSA and its analogues in guinea pigs: Role of glutathione and linked enzymes

Article abstract of DOI:10.1021/tx700315a

Arsenicosis, due to contaminated drinking water in the Indo-Bangladesh region, is a serious health hazard in terms of morbidity and mortality. Reactive oxygen species (ROS) generated due to arsenic toxicity have been attributed as one of the initial signals that impart cellular toxicity, which is controlled by the internal antioxidant glutathione (GSH). In the present study, we investigated (i) the role of GSH and its linked enzymes, glutathione peroxidase and glutathione reductase, in reversing chronic arsenic toxicity using a thiol chelating agent, meso-2,3-dimercaptosuccinic acid (DMSA), or one of its analogues individually or in combination; (ii) if alterations in the carbon side chain of DMSA increased efficacy; and (iii) whether the combination therapy enhance arsenic removal from hepatic tissue and prevent hepatic apoptosis. Results indicated that chronic arsenic exposure led to a ROS-mediated, mitochondrial-driven, caspase-dependent apoptosis in hepatic cells with a significant increase in glutathione disulfide (GSSG) levels and decreased glutathione reductase levels. Monotherapy with DMSA and its analogues did show minimal recovery postchelation. However, the combination of DMSA with long carbon chain analogues like monoisoamyl DMSA (MiADMSA) or monocyclohexyl DMSA (MchDMSA) showed a better efficacy in terms of reducing the arsenic burden as well as reversing altered biochemical variables indicative of oxidative stress and apoptosis. We also observed that GSH and its linked enzymes, especially glutathione reductase, play a vital role in scavenging ROS, maintaining GSH pools, and providing clinical recoveries. On the basis of the above observations, we recommend that combinational therapy of DMSA and its long carbon chain analogues MiADMSA or MchDMSA would be more effective in arsenic toxicity.

Full text of DOI:10.1021/tx700315a

4
00
Chem. Res. Toxicol. 2008, 21, 400–407
Reversal of Arsenic-Induced Hepatic Apoptosis with Combined
Administration of DMSA and Its Analogues in Guinea Pigs: Role of
Glutathione and Linked Enzymes
Deepshikha Mishra, Ashish Mehta, and Swaran J. S. Flora*
DiVision of Pharmacology and Toxicology, Defence Research and DeVelopment Establishment, Jhansi Road,
Gwalior-474 002, India
ReceiVed September 5, 2007
Arsenicosis, due to contaminated drinking water in the Indo-Bangladesh region, is a serious health
hazard in terms of morbidity and mortality. Reactive oxygen species (ROS) generated due to arsenic
toxicity have been attributed as one of the initial signals that impart cellular toxicity, which is controlled
by the internal antioxidant glutathione (GSH). In the present study, we investigated (i) the role of GSH
and its linked enzymes, glutathione peroxidase and glutathione reductase, in reversing chronic arsenic
toxicity using a thiol chelating agent, meso-2,3-dimercaptosuccinic acid (DMSA), or one of its analogues
individually or in combination; (ii) if alterations in the carbon side chain of DMSA increased efficacy;
and (iii) whether the combination therapy enhance arsenic removal from hepatic tissue and prevent hepatic
apoptosis. Results indicated that chronic arsenic exposure led to a ROS-mediated, mitochondrial-driven,
caspase-dependent apoptosis in hepatic cells with a significant increase in glutathione disulfide (GSSG)
levels and decreased glutathione reductase levels. Monotherapy with DMSA and its analogues did show
minimal recovery postchelation. However, the combination of DMSA with long carbon chain analogues
like monoisoamyl DMSA (MiADMSA) or monocyclohexyl DMSA (MchDMSA) showed a better efficacy
in terms of reducing the arsenic burden as well as reversing altered biochemical variables indicative of
oxidative stress and apoptosis. We also observed that GSH and its linked enzymes, especially glutathione
reductase, play a vital role in scavenging ROS, maintaining GSH pools, and providing clinical recoveries.
On the basis of the above observations, we recommend that combinational therapy of DMSA and its
long carbon chain analogues MiADMSA or MchDMSA would be more effective in arsenic toxicity.
Introduction
Arsenic is a metalloid that is ubiquitously present in water,
soil, and air from natural and anthropogenic sources. Arsenic,
which is found in several different chemical and oxidation states
metals, from the cells via GSH-mediated pathways (11, 12).
This GSH pool is mainly controlled by the conversion of GSSG
to GSH by an ubiquitous flavoenzyme, glutathione reductase
(
GR). Glutathione is extensively involved in the metabolism of
inorganic arsenic, specifically in the reduction of pentavalent
arsenicals to their trivalent form, and experiments have shown
that the addition of GSH to rat liver extracts promoted arsenite
methylation (13, 14). Both arsenite and its methylated metabo-
lites have been known to be potent inhibitors of GR in vitro
(
-3, 0, +3, and +5), causes acute and chronic adverse health
effects (1). Over 200 million people worldwide are at risk, out
of which 112 million are residing in West Bengal, India (2),
and Bangladesh, areas where groundwater arsenic concentrations
exceed the World Health Organization maximum permissible
level of 50 µg/L (3). Arsenic is known to interfere with a number
of bodily functions of the central nervous system, hematopoietic
system, liver, and kidneys (4–6) and has also been associated
with cancer (7).
(
15–17); thus, exposure to arsenicals would compromise the
antioxidant mechanisms by consuming GSH and inhibiting the
enzyme responsible for its recycling and may eventually lead
to cell death. Few studies have attempted to unravel the
mechanism of arsenic toxicity in vitro studies and have
demonstrated that arsenic exposure causes apoptosis via caspase
activations (18, 19). Recently, Santra et al. (20) also demon-
strated mitochondrial-dependent apoptosis in mouse liver after
arsenic treatment.
1
The tripeptide GSH (L-γ-glutamyl-cysteinyl-glycine) is the
most important intracellular nonprotein thiol in mammalian cells
(
8). GSH possesses reducing and nucleophilic properties and
can exist in either reduced (GSH) or oxidized (GSSG) form
9). The maintenance of an adequate cellular GSH pool is not
(
only critical for normal cellular redox status (10) but also for
the removal of numerous potentially toxic compounds, including
Chelation therapy with 2,3-dimercaprol (BAL) has been the
basis for the medical treatment of arsenic poisoning, but the
serious side effects of BAL have opened new avenues in this
area. meso-2,3-dimercaptosuccinic acid (DMSA) has been tried
successfully in animals (21, 22) as well as in a few cases of
human arsenic poisoning (23). In an experimental study, we
provided in vivo evidence of oxidative stress in a number of
major organs of arsenic-exposed rats and showed that these
effects can be mitigated by pharmacological intervention that
encompasses combined treatment with N-acetylcysteine and
DMSA (24). However, a double blind, randomized, controlled
*
To whom correspondence should be addressed. Tel: +91 751 2344301.
Fax: +91 751 2341148. E-mail: sjsflora@hotmail.com or sjsflora@
drde.drdo.in.
1
Abbreviations: DMSA, meso-2,3-dimercaptosuccinic acid; MiADMSA,
monoisoamyl DMSA; MchDMSA, monocyclohexyl DMSA; MmDMSA,
monomethyl DMSA; ROS, reactive oxygen species; MMP, mitochondria
membrane potential; SOD, superoxide dismutase; GSH, glutathione; GSSG,
glutathione disulfide; GPx, glutathione peroxidase; GR, glutathione reduc-
tase; TBARS, thiobarbituric acid reactive substances; ALT, alanine ami-
notransaminase; AST, aspartate aminotransaminase; CAT, catalase.
1
0.1021/tx700315a CCC: $40.75

2008 American Chemical Society
Published on Web 12/29/2007

Therapy for Arsenic-Induced Hepatic Apoptosis
Chem. Res. Toxicol., Vol. 21, No. 2, 2008 401
(
Germany). All other analytical laboratory chemicals and reagents
were purchased from Merck (Germany), Sigma, or BDH Chemicals.
Ultrapure water prepared by Millipore (New Delhi, India) was used
throughout the experiment to avoid metal contamination and for
the preparation of reagents/buffers used for various biochemical
assays in our study.
MiADMSA, MchDMSA, and MmDMSA were synthesized in
our synthetic chemistry division, by the controlled esterification of
DMSA, with their corresponding alcohol in acidic medium (27).
The product was purified (purity 99.9%) and characterized using
spectral and analytical methods before experimentation. The
chemicals were stored in desiccators at 4 °C to avoid oxidation
and thermal decomposition. DMSA and MiADMSA were dissolved
in 5% sodium bicarbonate solutions, respectively. All of the antidote
solutions were prepared immediately before use. The dosing volume
amounted to 4 mL/kg body weight.
Animal and Treatments. All of the experiments were performed
on male guinea pigs weighing 400 ( 50 g. Animals were obtained
from the animal house facility of the Defense Research and
Development Establishment (DRDE) (Gwalior). The animal ethical
committee of DRDE (Gwalior, India) approved the protocols for
the experiments. Prior to dosing, they were acclimatized for 7 days
to light from 0600 to 1800 h alternating with 12 h of darkness.
The animals were housed in stainless steel cages in an air-
conditioned room with the temperature maintained at 25 ( 2 °C.
The guinea pigs were allowed standard animal’s chow diet (Lipton,
India; metal content of diet, in ppm dry weight: Zn, 45; Cu, 10;
Mn, 55; Fe, 70; and Co, 5) throughout the experiment. Forty-five
animals were randomized into two groups and were treated as
indicated for a period of 4 months: group I, no treatment (drinking
water) (n ) 5); and group II, 25 ppm sodium arsenite (in drinking
water) (n ) 40).
After 4 months, arsenic-exposed animals were randomly divided
into eight groups and given the following treatments consecutively
for 5 days: group IIA, saline (n ) 5); group IIB, DMSA (0.3 mM/kg,
orally, once daily; n ) 5); group IIC, MiADMSA (0.3 mM/kg, orally,
once daily; n ) 5); group IID, MchDMSA (0.3 mM/kg, orally, once
daily; n ) 5); group IIE, MmDMSA (0.3 mM/kg, orally, once daily;
n ) 5); group IIF, DMSA (0.15 mM/kg, orally, once daily) +
MiADMSA (0.15 mM/kg, ip, once daily; n ) 5); group IIG, DMSA
Figure 1. Structure of chelating agents (A) DMSA, (B) MiADMSA,
(
C) MChDMSA, and (D) MmDMSA.
trial study conducted on patients from arsenic-affected West
Bengal (India) regions with oral administration of DMSA
suggested that it was not effective in producing any clinical or
biochemical benefits or any histopathological improvements of
skin lesions (25). To achieve optimal chelation as compared to
DMSA, a large number of esters of DMSA have been
synthesized. These esters are mainly the mono- and dimethyl
esters of DMSA that have been studied experimentally and have
shown better potential in mobilizing arsenic from tissues in mice
(0.15 mM/kg, orally, once daily) + MchDMSA (0.15 mM/kg, ip, once
daily; n ) 5); and group IIH, DMSA (0.15 mM/kg, orally, once daily)
+
MmDMSA (0.15 mM/kg, ip, once daily; n ) 5).
Arsenic exposure was stopped during chelation therapy. The
selection of the doses of each of the chelators used in the study
was based on our previous studies. These doses were at physi-
ologically attainable concentrations. At the end of treatment, all
animals were sacrificed under light ether anesthesia and blood was
collected through intracardiac puncture for serum separation. Liver
tissue samples were removed and washed with normal saline, and
all the extraneous materials were removed before weighing. Tissues
were kept at ice-cooled conditions at all times.
Reactive Oxygen Species (ROS). The amount of ROS in the
liver was measured using 2′,7′-dichlrofluorescin diacetate (DCF-
DA), which gets converted into highly fluorescent DCF by cellular
peroxides (including hydrogen peroxide). The assay was performed
as described by Socci et al. (28). Briefly, 1% liver homogenate
was prepared in ice-cold 40 mM Tris-HCl buffer (pH 7.4), and
this was further diluted to 0.25% with the same buffer (40 mM
Tris-HCl, pH 7.4) and placed on ice. The samples were divided
into two equal fractions (2 mL each). In one fraction, 40 µL of
(
22, 26).
The present study was planned (i) to address the role of
glutathione and linked enzymes in inducing oxidative stress and
its recovery by chelation therapy; (ii) to determine if alternations
in the carbon side chain of DMSA could help in achieving better
clinical recoveries; (iii) to determine if combined chelation of
succimer (DMSA) (Figure 1A), which is a hydrophilic chelator,
and its analogue [monoisoamyl DMSA (MiADMSA), mono-
cyclohexyl DMSA (MchDMSA), and monomethyl DMSA
(
MmDMSA)] (Figure 1B,C,D), which are lipophilic in nature
and presently have a minimal clinical role, could be better
options for the treatment of chronic arsenic poisoning; (iv) to
determine whether combination therapy mobilizes arsenic from
the target organ; and (v) to determine whether biochemical variables
indicative of oxidative stress and cell death are recovered.
1
.25 mM DCF-DA prepared in methanol was added for ROS
estimation, whereas only 40 µL of methanol was added to the other
fraction that served as a control for tissue autofluorescence. All
samples were incubated for 15 min in a 37 °C water bath. The
fluorescence was determined at 488 nm excitation and 525 nm
emission using a fluorescence reader (Perkin-Elmer, LS-55, United
Kingdom). Liver ROS readings were expressed as arbitrary
fluorescence intensity units (FIU at 530 nm).
Experimental Procedures
Chemicals and Reagents. DMSA was procured from Sigma (St.
Louis, MO), while sodium arsenite was obtained from Merck
Mitochondrial Membrane Potential (MMP) (∆Ψ ). The
m
MMP was measured using a JC-1 probe as described previously

4
02 Chem. Res. Toxicol., Vol. 21, No. 2, 2008
Mishra et al.
with minor modifications (29). Briefly, liver homogenate was
centrifuged at 800g for 5 min, and the supernatant was collected,
which was again recentrifuged at 1000g for 10 min. An equal
number of cells from each group was taken after counting and
incubated for 10 min with 10 µM JC-1 at 37 °C. The cells were
washed and resuspended in phosphate-buffered saline, and the
fluorescence was measured. Subsequently, the changes in fluores-
cence were monitored at two different wavelengths with excitation
at 485 nm and with emission at 530 nm and the other excitation at
before the enzymatic determination as the starting reagent. The assay
was run at 340 nm for 4 min with absorbance readings taken every
30 s.
Hepatic Reduced and Oxidized Glutathione Level. Liver GSH
and GSSG estimation were performed as described in our previous
paper (32). Briefly, 0.25 g of tissue sample was homogenized on
ice with 3.75 mL of 0.1 M phosphate-0.005 M EDTA buffer (pH
8.0) and 1 mL of 25% HPO , which was used as a protein
3
precipitant. The homogenate (4.7 mL) was centrifuged at 100000g
for 30 min at 4 °C. For the GSH assay, 0.5 mL of supernatant and
4.5 mL of phosphate buffer (pH 8.0) were mixed. The final assay
mixture (2.0 mL) contained 100 µL of supernatant, 1.8 mL of
phosphate-EDTA buffer, and 100 µL of O-phthaldehyde (OPT;
1000 µL/mL in absolute methanol, prepared fresh). After they were
mixed, the fluorescence was determined at 420 nm with an
excitation wavelength of 350 nm using a spectrofluorometer (Perkin-
Elmer, LS-55, United Kingdom).
5
35 nm and emission at 590 nm. The ratio of the reading at 590
nm to the reading at 530 nm (590:530 ratio) was considered as the
relative ∆Ψm value.
RNA Extraction and RT-PCR. Liver tissue RNA was extracted
from different treated groups using the TRIzol method. One
microgram of RNA was converted to cDNA using superscript
reverse transcriptase. PCR was performed with the initial denatur-
ation cycle at 94 °C for 5 min, followed by 35 cycles of 94 °C for
3
0 s, 60 °C for 30 s, 72 °C for 60 s, and followed by final extension
For the GSSG assay, 0.5 mL of supernatant was incubated at
room temperature with 200 µL of 0.04 mol/L N-ethylmaleimide
solution for 30 min. To this mixture, 4.3 mL of 0.1 mol/L NaOH
was added. A 100 µL sample of this mixture was taken for the
measurement of GSSG using the procedure described above for
the GSH assay, except that 0.1 mol/L NaOH was used as the
diluents instead of phosphate buffer (32).
at 72 °C for 5 min. The PCR-amplified products were then subjected
to electrophoresis on 1.5% agarose gels. The primers used for the
study were bcl (sense, CTT TGT GGA ACT GTA CGG CCC
CAG CAT GCG; antisense, ACA GCC TGC AGC TTT GTT TCA
TGG TAC ATC; 231 bps; accession #M16506), bax (sense, GGG
AAT TCT GGA GCT GCA GAG GAT GAT T; antisense, GCG
GAT CCA AGT TGC CAT CAG CAA ACA T; 96 bps; accession
2
Catalase (CAT) Activity. The CAT activity was assayed
following the procedure of Pande and Flora (33) at room temper-
ature. A 100 µL amount of tissue extract was placed on an ice
bath for 30 min, and then, for another 30 min at room temperature,
10 µL of Triton-X100 was added to each tube. In a cuvette
containing 200 µL of phosphate buffer and 50 µL of tissue extract
was added 250 µL of 0.066 M H O (in phosphate buffer); a
#
L22472), and ꢀ-actin (sense, TAT GGA GAA GAT TTG GCA
CC; antisense, GTC CAG ACG CAG GAT GGC AT; 300 bps;
accession #J00691). The band intensities were further analyzed
using Image J software, and values were then normalized using
ꢀ
-actin as an internal control to plot the graphs.
Caspase 3. The caspase 3 assay was performed using the Caspase
Colorimetric Assay Kit per the manufacturer’s instructions
2
2
3
decrease in the optical density was measured at 240 nm for 60 s.
The molar extinction coefficient of 43.6 M cm- was used to
determine the CAT activity. One unit of activity was equal to the
mol of H O degraded/min/mg protein.
1
(
Sigma, United States). Briefly, the single cell suspension was
prepared as described above, and an equal number of cells was
taken after counting. The cells were centrifuged, and the pellet
was suspended in 1× lysis buffer and incubated on ice for 15 min,
followed by centrifugation at 20000g for 15 min at 4 °C. The
supernatant was collected. Samples were mixed with assay buffer
with or without caspase 3 inhibitor (Ac-DEVD-CHO) for control.
Finally, caspase 3 substrate (Ac-DEVD-pNA) was added to all of
the samples and incubated at 37 °C for 60 min. The absorbance
was recorded at 405 nm. The data are a representation of three
experiments.
2
2
Thiobarbituric Acid Reactive Substances (TBARS) and
SOD Activity. Hepatic TBARS and SOD were estimated by the
methods described in detail in our earlier publication (31).
Elemental Analysis. The arsenic concentrations in liver and urine
were measured after wet acid digestion using a microwave digestion
system (Anton Paar Multiwave 3000, Austria, Europe). Samples
were brought to a constant volume, and determination of tissue
arsenic was performed using an autosampler (AS-72) and graphite
furnace (MHS) fitted with an atomic absorption spectrophotometer
Alanine Aminotransferase (ALT) and Aspartate Aminotrans-
ferase (AST). Serum ALT and AST were measured following the
method of Flora et al. (30). Briefly, the assay system contained 1
mL of buffer/substrate solution and 0.2 mL of serum and was
incubated for exactly 60 min for ALT and 30 min for AST at 37
(
AAS, Perkin-Elmer model AAnalyst 100).
Statistical Analysis. Data were expressed as means ( SEM. Data
comparisons were carried out using one-way analysis of variance
followed by Tukey’s post-test to compare means between the
different treatment groups. The difference (with or without chela-
tion) with a p value <0.05 was considered significant.
°
C in a water bath. One milliliter of chromogen solution was added,
mixed, and allowed to stand for 20 min at room temperature, and
0 mL of a 0.4 N NaOH was added subsequently. The extinction
1
Results
was read at 505 nm against a blank. The controls were run in
parallel, and the substrate was added after deproteinization.
Glutathione Peroxidase (GPx) and GR. The GPx activity was
measured as described in detail in our earlier publication (31). The
reaction mixture contained 0.3 mL of phosphate buffer (0.1 M, pH
7
0
for measuring the superoxide dismutase (SOD) activity]. The
complete mixture was incubated at 37 °C for 15 min, and the
reaction was terminated by adding 0.5 mL of 5% TCA. Tubes were
centrifuged at 1500g for 5 min, and the supernatant was collected.
Two hundred microliters of phosphate buffer (0.1 M, pH 7.4) and
There was no gross change in the general appearance of the
guinea pigs during arsenic exposure. However, there were signs
of hair loss after 3 months of arsenic exposure. There was no
significant change in the liver weights in various treated groups
when compared to the controls (data not shown). Exposure of
arsenic caused a significant increase (2.5 times) in hepatic ROS,
which was measured by a flurometric dye DCFDA (Figure 2A,
bar 2). Monotherapy with DMSA was effective in reversing
the increased hepatic ROS levels by about 20%, whereas in the
case of MiADMSA and MchDMSA, there was about 40%
reduction in the elevated ROS levels. MmDMSA was found to
be ineffective in reversing increased hepatic ROS (Figure 2A).
Combinational therapy was much more effective than mono-
therapy, but the best results of combination were observed when
DMSA was administered with MiADMSA (Figure 2A, Bar 7).
Because there was a significant ROS increase, we wondered
if this would impair the mitochondrial functioning. The MMP
.4), 0.2 mL of GSH (2 mM), 0.1 mL of sodium azide (10 mM),
.1 mL of H O (1 mM), and 0.3 mL of supernatant [as prepared
2
2
0
.7 mL of DTNB (0.4 mg/mL) were added to 0.1 mL of reaction
supernatant. After they were mixed, the absorbance was recorded
at 420 nm.
To measure the GR activity (31), homogenates were thawed at
room temperature and centrifuged at 700g for 10 min. Twelve
microliters of supernatant was added to quartz cuvettes containing
a fresh solution of 0.44 mM GSSG and 0.30 M EDTA in 0.1 M
phosphate buffer (pH 7.0), and 0.036 M NADPH was added just

Therapy for Arsenic-Induced Hepatic Apoptosis
Chem. Res. Toxicol., Vol. 21, No. 2, 2008 403
Figure 3. Reversal of arsenic-induced changes in gene expression of
bcl and bax. (A) Change in gene expression profile of bcl and bax
2
2
after chelation therapy in arsenic-intoxicated guinea pig liver. ꢀ-Actin
was used as an internal control. (B) Ratio of bax/bcl change in relative
2
gene expression after normalizing with ꢀ-actin. Groups: 1, control (no
treatment); 2, arsenic; 3, DMSA; 4, MiADMSA; 5, MchDMSA; 6,
MmDMSA; 7, DMSA + MiADMSA; 8, DMSA + MchDMSA; and
9
¶
, DMSA + MmDMSA. Values are means ( SE; n ) 5. *, †, ‡, and
indicate differences between values with matching symbol notations
within each column and are not statistically significant at the 5% level
of probability.
Figure 2. Arsenic-induced changes in ROS (A) and mitochondrial
membrane potential (B) and its reversal with administration of DMSA
and its analogues alone or in combination. Groups: 1, control (no
treatment); 2, arsenic; 3, DMSA; 4, MiADMSA; 5, MchDMSA; 6,
MmDMSA; 7, DMSA + MiADMSA; 8, DMSA + MchDMSA; and
9
¶
, DMSA + MmDMSA. Values are means ( SE; n ) 5. *, †, ‡, and
indicate differences between values with matching symbol notations
within each column and are not statistically significant at the 5% level
of probability.
(
∆Ψ ) of hepatic cells was estimated using a potentiometric,
m
fluorescent dye JC-1, and a significant loss of membrane
potential following arsenic treatment was observed (Figure 2B).
DMSA and MmDMSA alone did not have any major influence
on ∆Ψ . Treatment with MiADMSA and MchDMSA showed
m
Figure 4. Alteration in caspase 3 activity after chelation therapy in
hepatic tissue of arsenic-treated guinea pigs. Groups: 1, control (no
treatment); 2, arsenic; 3, DMSA; 4, MiADMSA; 5, MchDMSA; 6,
MmDMSA; 7, DMSA + MiADMSA; 8, DMSA + MchDMSA; and
marked recovery in ∆Ψ (Figure 2B). Combinational therapy
m
of DMSA with MiADMSA or MchDMSA showed significant
recoveries in ∆Ψ , whereas DMSA + MmDMSA did not show
m
9
¶
, DMSA + MmDMSA. Values are means ( SE; n ) 5. *, †, ‡, and
any significant change in ∆Ψm.
indicate differences between values with matching symbol notations
within each column and are not statistically significant at the 5% level
of probability.
A significant rise in ROS and a loss of ∆Ψm indicated cell
death. We estimated the gene expression of bcl and bax, which
2
are known to play a vital role in controlling cell death (Figure
3
). Semiquantitative estimation by RT-PCR showed that there
that there was a significant increase in the caspase 3 activity
following arsenic treatment (Figure 4). Monotherapy with
DMSA and MmDMSA showed only about 10 and 7% recover-
ies, respectively, whereas MiADMSA and MchDMSA showed
a significant decrease of about 41 and 30%, respectively, in
elevated caspase 3 levels. Combinational administration of
DMSA with its analogues further increased the fall in caspase
3 activity after arsenic exposure. Moreover, the best recoveries
were seen with a combination of DMSA + MiADMSA (45%
recovery) followed by DMSA + MchDMSA (41%) and DMSA
+ MmDMSA (23%).
Clinical indications of hepatic damage were observed with a
significant increase in serum AST and ALT levels following
arsenic exposure. These levels were reduced following treatment
with succimer (DMSA) and its analogues alone or in combina-
tion. The combination of DMSA + MiADMSA showed
maximum promise followed by DMSA + MchDMSA in
recovering the AST and ALT levels (Table 1). Because arsenic
was shown to induce oxidative stress by increasing ROS levels,
was a significant decrease in bcl expression after chronic arsenic
exposure. Bcl expression increased after treatment with all three
DMSA analogues, with a maximum expression recovery with
MiADMSA and MchDMSA alone. Combinational therapy too
showed a marked increase in bcl gene expression. On the other
hand, bax expression showed a marked elevation after arsenic
exposure, which was reduced after treatment of MiADMSA,
MchDMSA only. No significant change was observed with
DMSA and MmDMSA. A combinational therapy with DMSA
and its analogues showed a decrease in the elevated bax
expression; however, maximal recovery was seen with the
DMSA + MiADMSA group (Figure 3). Treatment of DMSA
or its analogues alone to animals did not show any significant
2
2
2
change in bcl or bax gene expression (data not shown).
2
Because arsenic treatment affected the mitochondria (loss of
Ψ ) and bax/bcl ratio significantly, there was an indication
m 2
∆
of mitochondrial-dependent cell death or apoptosis. To confirm
this, we estimated the caspase 3 activity in cells and observed

4
04 Chem. Res. Toxicol., Vol. 21, No. 2, 2008
Mishra et al.
a
Table 1. Reversal of Arsenic Induced Hepatic Oxidative Stress and Serum AST and ALT in Guinea Pigs by Thiol Chelators
TBARS (nmol SOD (U min- CAT (µmol-1 min-1 serum AST (nmol-1 min-1 serum ALT (nmol-1 min-1
1
GSH/GSSG ratio mg protein-
1
)
mg protein
-1
)
mg protein )
-1
mg protein-1)
mg protein-1)
normal
arsenic
DMSA
MiADMSA
MchDMSA
MmDMSA
DMSA +
MiADMSA
DMSA +
MchDMSA
DMSA +
MmDMSA
9.1 ( 0.3*
2.4 ( 0.69*
2.35 ( 0.16*
2.93 ( 0.07*
2.92 ( 0.30*
2.82 ( 0.13*
2.76 ( 0.03*
3.05 ( 0.07*
2.76 ( 0.11*
2.77 ( 0.03*
12.5 ( 1.67*
22.3 ( 1.67
†
†
†
‡
‡
†
†
†
†
1.3 ( 0.9
9.6 ( 0.29
7.5 ( 0.59
4.3 ( 0.54
4.1 ( 0.76
7.3 ( 0.60
5.45 ( 0.24
45.7 ( 2.12
73.2 ( 0.45
b
‡
‡
‡
‡
3.6 ( 0.7
3.05 ( 0.05
33.4 ( 1.56
62.1 ( 1.22
b
‡
‡
‡
‡
5.7 ( 0.9
2.97 ( 0.01
23.1 ( 1.35
51.2 ( 1.48
b
‡
‡
‡
4.2 ( 0.2
2.80 ( 0.01*
24.8 ( 1.76
43.3 ( 1.39
b
†
‡
‡
†
1.2 ( 0.7
3.88 ( 0.07
31.3 ( 1.35
62.4 ( 1.93
‡
‡
9.4 ( 0.2*
3.1 ( 0.40*
2.00 ( 0.10*
2.17 ( 0.21*
2.59 ( 0.20*
19.6 ( 1.19
33.2 ( 1.22
b
‡
‡
‡
‡
5.4 ( 0.5
3.7 ( 0.28
2.78 ( 0.25*
2.83 ( 0.14*
24.3 ( 1.20
39.4 ( 1.40
b
†
‡
‡
‡
1.8 ( 0.6
5.4 ( 0.26
26.3 ( 1.16
46.3 ( 2.21
b
a
Values are means ( SE; n ) 5. For entries with symbols *, †, and ‡, differences between values with matching symbol notations within each row
b
are not statistically significant at the 5% level of probability. All of the treated groups were pre-exposed to arsenic and then treated with different
chelating agents.
it was important to evaluate if these elevated levels translated
in altering the biochemical variables. Hepatic oxidative stress
was indicated by a substantial decrease in the GSH/GSSG ratio
and increased TBARS and SOD levels (Table 1). However, no
significant change was observed in CAT activity. All of the
variables showed consistent reversal of altered variables with
chelation therapy (monotherapy or combinational), but no
significant recovery was observed with MmDMSA alone or in
combination (Table 1).
Because the GSH/GSSG ratios showed maximum alterations,
we evaluated the role of GSH and its linked enzymes (GPx
and GR) (Figure 5A). A marked increase in the GPx level was
observed following arsenic exposure, which decreased following
chelation therapy (monotherapy or combinational therapy). GR
levels decreased significantly on arsenic exposure, and chelation
therapy with MiADMSA alone and in combination with DMSA
showed a significant increase in the GR levels (Figure 5A).
Arsenic-induced oxidative stress variable GSSG and GR had a
good correlation with tissue arsenic levels (Figure 5B).
To confirm the correlation between GSSG, GR, and arsenic
levels, statistical analysis using multiple scatter regression plots
was performed. It was noted that with an increase in arsenic
levels, there was a decrease in GR levels, suggesting an inverse
relationship (Figure 6A), whereas there was a direct relationship
between GSSG levels and arsenic levels (Figure 6B). These
relations were also observed with chelation therapy, which
indicated that with the removal of arsenic with chelation, GR
and GSSG levels turned toward normalcy. A strong direct
relationship between an increase of GSSG levels and a decrease
of GR was also observed, pre- and postarsenic exposure (Figure
Figure 5. Influence of mono or combinational therapy on glutathione
peroxidase and glutathione reductase in liver after arsenic intoxication
in guinea pigs. (A) Glutathione peroxidase and glutathione reductase
levels after chelation therapy. Values are means ( SE; n ) 5. *, ‡,
and ¶ indicate differences between values with matching symbol
notations within each column and are not statistically significant at the
6
C).
5
% level of probability. (B) Correlation between arsenic levels and
One of the other major objectives of the study was to
GSSG and GR in arsenic-treated groups. Units: GR, nmol/min/mg
protein; GPx, nmol/min/mg protein; and GSSG, µg/g tissue.
determine whether chelation therapy could mobilize arsenic from
the target organs. It was observed that DMSA and DMSA +
MmDMSA were ineffective in reducing the arsenic burden from
hepatic tissues (Figure 7A). MiADMSA, MchDMSA, MmDM-
SA alone, MiADMSA, and MchDMSA in combination with
DMSA markedly reduced the arsenic burden from the hepatic
tissues. Apart from this, there was a significant increase in
urinary arsenic levels (control, not detected vs arsenic, 1.25 (
Discussion
Arsenic is a serious health hazard in the Indo-Bangladesh
region with no clinical remedies to treat chronic arsenicosis.
Arsenic causes damage to biological system because of its ability
to generate oxidative stress in the cells (24). Arsenic is known
to generate ROS and free radicals like hydrogen peroxide
(34–37), hydroxyl radical (34), nitric oxide (38), superoxide
anion (35, 39), dimethyl arsenic peroxy radical (40, 41), and
dimethylarsinic radical (41, 42) in living systems. In mammals,
liver is the foremost organ that helps in detoxifying arsenic by
its methylation and detoxification (43). During this process, there
is a significant generation of ROS (20), which in turn leads to
0
.2 µg/mL) following chelation therapy (DMSA, 1.35 ( 0.4;
MiADMSA, 4.57 ( 0.5; MchDMSA, 2.89 ( 0.7; MmDMSA,
.56 ( 0.8; DMSA + MiADMSA, 5.28 ( 0.3; DMSA +
2
MchDMSA, 4.23 ( 0.4; and DMSA + MmDMSA, 3.95 ( 0.5
µg/mL). Significant excretion of arsenic in the urine was
observed with the DMSA + MiADMSA group, which suggested
the additive action of the two chelators.

Therapy for Arsenic-Induced Hepatic Apoptosis
Chem. Res. Toxicol., Vol. 21, No. 2, 2008 405
The following study was hence planned to evaluate the role of
glutathione and its linked enzymes (GPx and GR) in regulating
cellular homeostasis and the effect of chelation on cellular
recoveries.
Our results show that chronic arsenic exposure causes a
significant increase in ROS levels, which were measured by
DCFDA, a fluorescent dye, suggesting that free radicals were
involved in oxidative stress. This increased ROS was in turn
responsible for the lipid bilayer damage and caused a significant
fall in ∆Ψ , which suggested mitochondria to be one of the
m
prime targets for ROS. It is known that early loss of ∆Ψm is
indicative of considerable cellular changes that take place due
to the inhibition of the respiratory chain (29). Several studies
have also shown that molecules stimulating the formation of
ROS cause apoptosis (47, 48). Recently, Santa et al. (20) showed
that arsenic affects the mitochondria permeability transitions
in a dose-dependent fashion in mice. They further suggested
that this damage to mitochondria was directly proportional to
the increase in caspase activity inducing cell death. We too
observed that due to a change in ∆Ψ , there was an increase
m
in the bax/bcl ratio, which in turn also increased the caspase 3
2
activity, suggesting that oxidative stress induced mitochondrial-
driven apoptosis of hepatic cells in guinea pig.
Our results showed that there was a significant drop in the
GSH/GSSG ratio after arsenic exposure, suggesting an imbal-
ance in the pro- and antioxidant levels, and there was an
alteration in the enzyme levels that are responsible for cycling
of the two molecules (GSH and GSSG) within the cell,
suggesting that these enzymes may play the role of a rate-
limiting step in the maintenance of the GSH pool. Interestingly,
there was an increase in GPx levels following arsenic exposure,
indicating that GPx efficiently converted GSH to GSSG;
however, a significant drop in the GR levels suggested that
GSSG was not getting converted to GSH, thus inhibiting the
recycling of the two molecules and increasing the GSSG levels.
The literature shows that the primary biochemical mechanism
of arsenic toxicity is binding of the metal to cellular sulfhydryl
groups, resulting in inhibition of a number of cellular enzyme
systems (49, 50). Our data of inhibition of GR further gets
credibility from the fact that trivalent arsenicals are potent
inhibitors of GR (16), which cause inhibition by the interaction
of arsenic with critical thiol groups in these enzymes (1). Thus,
exposure to arsenicals would compromise the antioxidant
mechanisms by consuming GSH and inhibiting the enzyme
responsible for its recycling. Reports have also suggested that
inhibition of GSH and dependent enzymes has been rendered
as a very important factor of arsenic-induced toxicity (11).
Figure 6. Correlation between oxidized glutathione, glutathione per-
oxidase, and arsenic levels in hepatic cells after chelation therapy. (A)
Correlation between GR levels and arsenic levels postchelation therapy.
(
B) Graph shows direct relationship between GSSG levels and arsenic
concentration following treatment with thiol chelators alone or in
combination. (C) Correlation between GSSG and GR after chelation
therapy in liver of arsenic-intoxicated guinea pigs. Values on each dot
are means ( SE; n ) 5.
Because arsenic has a higher affinity to dithiols than mono-
thiols, which is shown by the highly favored transfer of arsenite
from a (GSH) -arsenic complex to the dithiol DMSA (51), it
3
becomes a better choice for arsenicosis. However, previous
findings suggest that DMSA is distributed in an extracellular
manner, and DMSA is incapable of crossing the cell membrane
Figure 7. Liver arsenic concentrations in arsenic-exposed guinea pigs
after chelation. Groups: 1, control (no treatment); 2, arsenic; 3, DMSA;
4
, MiADMSA; 5, MchDMSA; 6, MmDMSA; 7, DMSA + MiADMSA;
(
52). It is also believed that DMSA is incapable of entering
8
, DMSA + MchDMSA; and 9, DMSA + MmDMSA. Values are
cells due to highly charged carboxyl groups (53). However,
because of the limited access of DMSA, the recoveries after
chelation are not impressive in chronic models as they are for
acute models. Our data also shows that in the majority of the
variables studied, DMSA did not show any statistically signifi-
cant recovery, suggesting that in chronic models DMSA did
not provide efficient removal of arsenic and hence did not show
any clinical recoveries. On the other hand, esters of DMSA,
because of their long carbon chains and lipophilic nature, could
get access inside the cell and remove arsenic more efficiently.
means ( SE; n ) 5. *, †, ‡, and ¶ indicate differences between values
with matching symbol notations within each column and are not
statistically significant at the 5% level of probability.
oxidative stress in tissues. Although the downstream pathway
for arsenic-induced cell death is not well-defined, evidence
shows oxidative stress following chronic exposure to arsenic
(
44–46). Our group has previously reported that arsenic binds
with high affinity to GSH, restricts its antioxidant activity, and
inhibits various enzymes, which require GSH as a cofactor (44).

4
06 Chem. Res. Toxicol., Vol. 21, No. 2, 2008
Mishra et al.
Apart from this, because they still retained the functional aspect
of the parent compound (dithiol), they could help in GSH
biosynthesis (54). Ercal and co-workers (54) for the first time
suggested that DMSA, due to the presence of vicinal thiol
groups, could have some antioxidant properties and may also
help in GSH biosynthesis. Hence, a number of DMSA esters
were synthesized, and these esters have been shown to be
superior to DMSA in chelating heavy metals (22, 27, 30, 32, 44).
We too found MiADMSA, the isoamyl derivative, and Mch-
DMSA, the cyclo-hexyl derivative of DMSA, to be more
effective in bringing clinical recoveries than MmDMSA, the
methyl derivative. This variation could be attributed to the length
of the carbon chain in helping the molecule to enter the cell.
Reports from various groups have suggested that DMSA
analogues that have long carbon chains have a ready access
across the cell membranes through endogenous ligands (55) and
make the chelating agents more efficient (26) in removing
arsenicals. However, there was no evidence in terms of clinical
recoveries in these studies. Our results have shown marked
clinical recoveries following treatment with MiADMSA (an
isoamyl derivative) and MchDMSA (a cyclohexyl derivative)
in most of the variables studied. However, a lack of comparable
clinical recoveries with MmDMSA (methyl derivative) could
be attributed to the fact that these molecules had short carbon
chains, which did not facilitate their cellular uptake. Another
major problem associated with chelation therapy is to prevent
redistribution of heavy metals (21, 56). To circumvent this,
combinational therapy of DMSA with its analogue was used.
This provided the ability to remove intracellular (analogue of
DMSA) as well as extracellular (DMSA) arsenic from tissue
sites for effective and speedy clinical recoveries as well as to
prevent redistribution of arsenic. Our data also show that the
combination of MiADMSA or MchDMSA with DMSA was
more effective than MmDMSA + DMSA, due to the presence
of a longer carbon chain and better cellular entry. We also
observed that there was an additive effect in clinical recoveries
in various variables (like AST, ALT, and caspase etc.), even
when the chelators were administered at half the dose (0.15
mM/kg) than monotherapy (0.3 mM/kg). This further suggested
that better recoveries could be obtained at 50% doses of
chelators in combinational therapy, which would further reduce
the chances of side effects of the chelators per se.
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