JOURNAL OF APPLIED TOXICOLOGY, VOL.

17(1), 7174 (1996)

Racemic-2,3-dimercaptosuccinic Acid for

Inorganic Mercury Mobilization in Rats
K. Kostial,1, N. Restek-Samarzija,1 M. Blanusa,1 M. Piasek,1 Lj. Prester,1 M. M. Jones2
and P. K. Singh2,3
1
Department of Mineral Metabolism, Institute for Medical Research and Occupational Health, 10001 Zagreb, Ksaverska
cesta 2, Republic of Croatia
2
Department of Chemistry, Vanderbilt University, Nashville, TN 37235, USA
3
Ivy Laboratory, Toxicology and Food Residue Laboratory, Ellington Agricultural Center, Tennessee Department of
Agriculture, Nashville, TN 37220, USA

Key words: inorganic mercury; rats; meso-2,3-dimercaptosuccinic acid; rac-2,3-dimercaptosuccinic acid; oral chelation
treatment; 203Hg retention; whole body, kidney, liver.

The efficiency of racemic-2,3-dimercaptosuccinic acid (rac-DMSA) compared with meso-2,3-dimercapto-

succinic acid (meso-DMSA) in mobilizing inorganic mercury was evaluated in female rats. Chelators
were administered orally at a dose of 0.5, 1.0 or 2.0 mmol kg21 on four consecutive days, 5 days after
a single intraperitoneal 203Hg injection (with 0.5 mg HgCl2 kg21). Both chelators reduced 203Hg retention
in whole body and kidney and at higher doses also in the liver. Racemic-DMSA was more efficient at
lower dose levels and equal to meso-DMSA at the highest dose level. Kidney retention decreased after
rac-DMSA to 27, 10 and 10% of controls and after meso-DMSA to 68, 39 and 10% of control values
at the 0.5, 1.0 and 2.0 mmol kg21 dose level, respectively. Since meso-DMSA is already approved for
human use, its stereoisomeric form, rac-DMSA, deserves further attention for treatment of mercury
poisoning. 1996 by John Wiley & Sons, Ltd.

The present experiments were performed further to

INTRODUCTION
Removal of mercury from the mammalian body is
most readily affected by chelating agents containing
thiol groups. Of these, meso-2,3-dimercaptosuccinic
acid (meso-DMSA) and dimercaptopropan-1-sulfonate
(DMPS) are found to be the most effective in animal
models and also in humans.1 Dimercaptopropan-1-sul-
fonate is reported to be somewhat more effective in
removing mercury from the kidneys in animal models.
Nevertheless, an equivalent mobilization of mercury
can be achieved by a longer treatment period with the
more readily available DMSA.2 The antidotal efficiency
of DMSA against Hg poisoning was first disclosed in
mice and guinea pigs in China by Ding et al. in
1965.3 This antidotal effect was confirmed by Western
scientists.4,5 Clinical reports show that DMSA would
appear to be the most useful compound for current
clinical treatment of mercury poisoning.6
More than 25 years ago Russian scientists reported
that racemic-2,3-dimercaptosuccinic acid (rac-DMSA),
which was obtained as a by-product in the process of
synthesizing meso-DMSA, has certain advantages over
the meso form in increasing inorganic mercury elimin-
ation and in reducing lethality and pathohistological
changes in organs caused by toxic doses of inorganic
mercury.7,8 These studies were quite preliminary and
did not furnish sufficient biological data. However,
these reports, although abbreviated, indicate that rac-
DMSA might be superior to meso-DMSA as an anti-
dote in mercury poisoning.
Author to whom correspondence should be addressed.
evaluate and extend these early findings. Previous
experiments were primarily performed using early sub-
cutaneous administration of chelation therapy, while
our experiments were designed as a later oral treatment
for mobilizing mercury.
Since meso-DMSA is a chelating agent already
approved for human application for mercury poisoning,
its stereoisomeric form, rac-DMSA, certainly deserves
further investigation.
METHODS
Experimental design
Two experiments were performed (Experiments 1 and
2) on female rats. In Experiment 1, 8-week-old female
Wistar-derived albino rats from the Institute for Medi-
cal Research and Occupational Health in Zagreb breed-
ing farm were used (average weight 190 g). In
Experiment 2, 7-week-old female Wistar albino rats
donated from the animal farm of Pliva Pharmaceutical
Co, Zagreb, Croatia, were used (average weight 180 g).
The design in both experiments was the same; only
the dose level of the treatment with chelating agent
was different. In Experiment 1 the effect of lower dose
was tested and in Experiment 2 the effect of two higher
dose levels was tested.
During the experiments rats were kept in polycarbon-
ate cages (510 in each). Standard rat diet and tapwater
were provided ad libitum. Both experiments lasted for
9 days. On the first day all rats received a single
administration of inorganic 203Hg (HgNO3). Five days

CCC 0260437X/96/01007104 Received 17 July 1996

1996 by John Wiley & Sons, Ltd. Accepted 14 August 1996
72 K. KOSTIAL ET AL.
later they were divided into three groups according to
treatment (control, meso-DMSA or rac-DMSA
therapy). Chelators were administered orally for 4 days
(on days 5, 6, 7 and 8 of the experiments). One day
after the last treatment (day 9 of the experiments) rats
were sacrificed under an ether anesthesia by exsanguin-
ation from abdominal aorta, and organs were removed
for radioactivity measurements.
203
Mercury loading and Hg radioactivity
measurements
203
Radioactive Hg was supplied as nitrate from New
England Nuclear (Massachusetts, USA) with a specific
activity of 12 Ci g21 (370740 GBq g21 ). Mercuric
cloride (HgCl 2) was provided by Kemika (Zagreb,
Croatia).
Rats were injected intraperitoneally with 0.5 mg
HgCl2 kg21 in distilled water containing 2.5 mCi
(92.5 KBq) of 203Hg in a volume of 1.0 ml. Five days
later, i.e. before the beginning of chelation therapy and
24 h after each of the 4-day treatments, whole-body
(WB) retention of 203Hg was determined. At the end
of the experiment 203Hg retention was determined in
kidneys (K), liver (L) and brain (B) in Experiments 1
and 2 and additionally in the femur (F) and pelt (P)
in Experiment 2.
The radioactivity measurements in the whole body
and pelt were performed using a double-crystal scintil-
lation counter (Tobor, Nuclear Chicago). In other
tissues, 203Hg was determined in an automatic gamma
scintillation counter (Nuclear Chicago).
All results were corrected for radioactive decay and
the geometry of the samples.
Chelation therapy
Meso-2,3-dimercaptosuccinic acid (meso-DMSA) was
purchased from Aldrich Chemical Co., Millwaukee,
WI, USA). Racemic-2,3 dimercaptosuccinic acid (rac-
DMSA) was synthesized by using a modified pro-
cedure,9 which provided an improved yield of the rac-
DMSA.10,11 Both chelating agents were stored under
nitrogen and solutions for administration by gastric
intubation (gavage) were freshly prepared in a volume
of 1.0 ml of 5% NaHCO3. In Experiment 1 chelators
were administered at the 0.5 mmol kg21 dose level,
and in Experiment 2 at the 1.0 and 2.0 mmol kg21
dose levels.
Statistical analysis
Results are expressed as a percentage of the 203Hg
dose (% dose) and are presented as the arithmetic
mean and one standard error of the mean. Results are
also presented as a percentage of control values (% C).
The statistical significance of differences between
groups (P , 0.05) was evaluated by Duncans multiple
range test using the CSS Biostatistica program (release
3.1, Statsoft 1991 package).
RESULTS
Results of Experiments 1 and 2 are presented separately
as a percentage of the 203Hg dose and together as a
percentage of control values (Tables 1 and 2, and
Fig. 1). This was necessary since control values due to
differences in the animal populations used (age,
sources, timing of the two experiments) could not be
pooled. The increase of body weights between control
and treated rats was the same in both experiments.
203
Effects on Hg whole-body retention
In Experiment 1 at the 0.5 mmol kg21 dose level, rac-
DMSA was superior to meso-DMSA at all intervals
(i.e. 24 h after the first, second, third and fourth dose),
as shown by different superscripts (Table 1, upper part).
In Experiment 2 at the 1.0 mmol kg21 dose level,
rac-DMSA was again superior to meso-DMSA at all
intervals. At the higher dose level of 2.0 mmol kg21,
rac-DMSA was superior to meso-DMSA after the first
and second treatment (WB 1 and WB 2) but after the
third and fourth treatments no difference was observed
(Table 1, lower part).
In Fig. 1, results of the two experiments are
presented as a percentage of control values to illustrate
the decrease in whole-body retention in relation to
dose and length of the treatment. Both the dose and
length of the treatment influenced the results. Gener-
ally, retention values after rac-DMSA were lower than
after meso-DMSA treatment and lowest retention
values for both chelators were observed at the end of
the treatment. Greatest differences between the
efficiency of the two treatments were observed at the
1.0 mmol kg21 dose level.
203
Effects on Hg tissue retention
Table 2 presents 203Hg retention in the kidneys, liver
and brain from Experiment 1 (upper part) and retention
in the kidneys, liver, brain, femur and pelt from
Experiment 2 (lower part). In both experiments treat-
ment with chelating agents caused a decrease in 203Hg
in most tissues, as indicated by the percentage
reduction of control values. However, a statistically
significant difference after treatment was observed only
in the kidney and at higher dose levels in the liver.
In Experiment 1 at the 0.5 mmol kg21 dose levels,
both meso- and rac-DMSA caused decreased kidney
retention compared with controls. However, rac-DMSA
was more efficient in this respect than meso-DMSA.
In Experiment 2, rac-DMSA was superior to meso-
DMSA in decreasing liver and kidney retention at the
1.0 mmol kg21 dose level and similar to meso-DMSA
at the 2.0 mmol kg21 dose level as shown by the
superscripts in Table 2.
Kidney retention after the meso-DMSA treatment
was reduced to 68, 39 and 10% of control values and
after rac-DMSA to 27, 10 and 10% at the 0.5, 1.0 or
2.0 mmol kg21 dose level, respectively. This indicates
that differences between these two treatments observed
at the lower dose level (highest at the 1.0 mmol kg21
dose level) disappeared at the highest dose level
(2.0 mmol kg21).
 RAC-DMSA FOR INORGANIC MERCURY MOBILIZATION IN RATS 73

203
Table 1. Effect of oral administration of meso- or racemic-DMSA on Hg whole-body retention in ratsa

WB 1 WB 2 WB 3 WB 4

% Dose %C % Dose %C % Dose %C % Dose %C

Experiment 1
Control (8) 66.9 2.6b 65.1 1.3b 69.7 1.5b 60.2 2.2b
4 3 0.5 mmol kg21:
Meso-DMSA (8) 63.9 2.0b 95 61.5 2.2b 94 54.6 1.7c 78 52.9 2.2c 88
Racemic-DMSA (8) 54.1 1.9c 81 48.0 1.5c 74 45.6 1.5d 65 40.8 1.5d 68

Experiment 2
Control (10) 56.7 1.7b 54.6 1.6b 53.1 1.9b 52.1 1.7b
4 3 1.0 mmol kg21:
Meso-DMSA (9) 47.9 2.5c 85 41.0 2.5c 75 36.1 1.8c 68 32.0 1.9c 62
Racemic-DMSA (10) 34.4 1.5d 60 22.7 0.8e 42 18.1 0.7d 34 15.1 0.6d 29
4 3 2.0 mmol kg21:
Meso-DMSA (5) 41.9 1.2d 74 33.5 1.7d 61 22.4 0.7d 42 17.9 0.5d 34
Racemic-DMSA (10) 30.1 1.0e 53 20.7 1.0e 38 17.5 0.9d 33 15.8 1.2d 30
a
Female rats (numbers in parentheses) received 203 Hg with 0.5 mg HgCl2 kg21 body wt. by single i.p. injection. A 4-day oral
treatment started 5 days later. Values obtained 24 h after each treatment at different dose levels (4 3 0.5, 1.0 or 2.0 mmol kg21).
Results expressed as % of 203 Hg dose (arithmetic mean SEM) and as % of controls (% C); WB 1WB 4 refer to the percentage
of remaining whole-body 203Hg after each treatment.
Significant differences (at P , 0.05 by Duncans multiple range test) are indicated by different superscript letters.

203
Table 2. Effect of oral administration of meso- or racemic-DMSA on Hg tissue retention in ratsa

Control (8) Meso-DMSA (8) Racemic-DMSA (8)

% Dose % Dose %C % Dose %C

Dose 0.5 mmol kg21Experiment 1

K 26.4 2.17b 18.0 1.19c 68 7.0 0.92d 27
L 2.2 0.33b 1.9 0.29b 85 1.8 0.25b 82
B 0.014 0.001b 0.012 0.001b 86 0.012 0.001b 86
Dose 1.0 mmol kg21Experiment 2
K 33.6 2.20b 13.2 1.25c 39 3.26 0.19d 10
L 0.78 0.07b 0.73 0.05b 94 0.49 0.03c 63
B 0.025 0.003b 0.026 0.001b 104 0.024 0.002b 96
F 0.034 0.002b 0.037 0.002b 109 0.028 0.002b 82
P 1.60 0.05b 1.61 0.05b 101 1.53 0.05b 89
Dose 2.0 mmol kg1Experiment 2
K 33.6 2.20b 3.28 0.48d 10 3.19 0.29d 10
L 0.78 0.07b 0.49 0.04c 63 0.47 0.04c 60
B 0.025 0.003b 0.022 0.001b 88 0.018 0.001b 72
F 0.034 0.002b 0.03 0.002b 88 0.031 0.003b 91
P 1.60 0.05b 1.39 0.01b 81 1.45 0.08b 84
a
Female rats (numbers in parentheses) received 203 Hg with 0.5 mg HgCl2 kg21 body wt. by single i.p. injection. A 4-day oral
treatment started 5 days later (at doses 4 3 0.5, 1.0 or 2.0 mmol kg21). Rats were sacrificed 24 h after last treatment, i.e. 10 days
after 203Hg administration. Results presented as % of 203Hg dose (arithmetic mean SEM) and as % of control values (% C) for
kidney (K), liver (L), brain (B) (Exps 1 and 2) and for femur (F) and pelt (P) retention (Exp. 2). Significant differences (at P , 0.05
by Duncans multiple range test) are indicated by different superscript letters.

meso-DMSA also reduces mercury retention in the

DISCUSSION
Both rac- and meso-DMSA decreased the body burden
of mercury after late oral administration, as indicated
by decreased whole-body and kidney retention at all
dose levels and also liver retention at higher dose
levels. It is known from previous publications that
brain.35 However, generally, meso-DMSA was not
found notably effective in reducing accumulation of
mercury in the brain of rodents.2 It was explained that
meso-DMSA is quite hydrophilic, and would not be
expected to enter the lipid part of the brain in which
Hg appears to accumulate. For rac-DMSA quantitative
data on kidney and liver retention, although referred
to as reduced after treatment, are missing from the
74 K. KOSTIAL ET AL.
Figure 1. Effect of meso- or rac-DMSA on 203Hg whole-body
retention (% control). Values obtained 24 h after each of the
4-day oral treatments (doses: 4 3 0.5, 1.0 or 2.0 mmol kg21).
Treatments started 5 days after 203Hg i.p. injection.
papers of Okonishnikova et al.7,8 However, the superi-
ority of rac- to meso-DMSA in enhancing mercury
elimination is in agreement with their results. They
also found that the ability of the racemate to prevent
death and development of other morphological changes
was more pronounced than for the meso-form. All
effects of racemate were more apparent after just one
administration. In our experiments rac-DMSA was
superior to meso-DMSA at lower dose levels (0.5 and
1.0 mmol kg21) but equal to meso-DMSA at the highest
dose level (2.0 mmol kg21). An increase in the dose
of meso-DMSA enhanced its efficiency in decreasing
the retention of mercury, while a similar increase in
the dose of rac-DMSA did not improve its effect. The
doseresponse curve for mercury mobilization by rac-
DMSA rises more rapidly as the dose increases than
does the curve for meso-DMSA. The retention of 203Hg
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In a recent study in rats, a comparison of the
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The partition coefficient of rac-DMSA is found to be
2.8 in the n-octanol/water system. 9
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toxicity testing of the two isoforms of DMSA. Prelimi-
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might explain the results obtained at the highest rac-
DMSA dose used in this experiment. In all our experi-
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response. However, due to the higher toxicity of rac-
DMSA, further work should include additional dose-
related experiments. Such studies are justified since
rac-DMSA definitely seems to be superior to meso-
DMSA in decreasing metal retention, especially at the
beginning of the treatment.
Acknowledgements
The present study was supported by the National Institutes of Health
via Grant ES-02638 (M.M.J., P.K.S.) and the Ministry of Science,
Republic of Croatia (K.K., N.R.-S., M.B., M.P., Lj.P.). The skilful
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