ECOTOXICOLOGY AND ENVIRONMENTAL SAFETY 23,274-28 1 (1992)

Amelioration by BAL (2,3-Dimercapto-1 -propanol) and DMPS

(Sodium 2,3-Dimercapto-1 -propanesulfonic Acid)
of Arsenite Developmental Toxicity in Mice

Jo& L. DOMINGO, M. ANGELES BOSQUE, JUAN M. LLOBET,

AND JACINTO CORBELLA

Laboratory of Toxicology and Biochemistry, School of Medicine, University of Barcelona,

San Lorenzo 21, E-43201 Reus, Spain

Received July 25, 1991

Inorganic arsenic is embryotoxic and teratogenic in chicks, golden hamsters, mice, and rats.
Certain dithiol chelators have been reported to protect against arsenite-induced lethality and to
decrease arsenic body burden. The present study evaluated the influence of BAL (2,3-dimercapto-
1-propanol) and DMPS (sodium 2,3dimercapto-1-propanestionic acid), a water-soluble analogue
of BAL, on amen&induced embryotoxic and teratogenic effects in the mouse. A series of four
BAL or DMPS injections was administered SCto pregnant mice immediately after a single ip
injection of 12 mg/kg of sodium arsenite given on Day 9 of gestation and at 24,48, and 72 hr
thereafter. Controls received sc corn oil with or without arsenite. Amelioration by BAL and
DMPS of arsenite developmental toxicity was assessedat 1530, and 60 m&kg/day, and 75, 150,
and 300 mg/kg/day, respectively. BAL given following arsenite was not able to ameliorate the
developmentally toxic effectsof arsenite seen in mice, whereas treatment with DMPS at 150 and
300 mg/kg showed sign&ant protective effectsagainst arsenite embryotoxicity and teratogenicity.
DMPS administration at 300 mg/kg also protected the dams against arsenite-induced maternal
toxicity. 0 1992 Academic Press, Inc.

INTRODUCTION
Arsenic is a well-known environmental pollutant. Inorganic arsenic compounds
are generally considered more toxic than organic compounds. Most of environmental
inorganic arsenic is present as arsenate (As+), but arsenite (As3+), generally considered
more toxic than arsenate, is also found in some cases (Vahter, 198 1).
Exposure to arsenic compounds may occur from sources such as herbicides, insec-
ticides, rodenticides, paint pigments, and wood preservatives (Baxley et al., 198 1).
However, environmental arsenic exposure has received attention primarily because
of disease resulting from ingestion of food or water containing arsenic (Franzblau and
Lilis, 1989). Acute ingestion of arsenic may result in immediate gastrointestinal symp
toms, but polyneuropathy represents the subacute secIuela (Goebel et al., 1990). Chronic
effects may involve the cardiovascular, gastrointestinal, hematopoietic, nervous, and
respiratory systems as well as a variety of characteristic skin lesions, namely hyper-
pigmentation, hyperkeratos, and several types of skin cancer (J&up et al., 1989; Shan-
non and Strayer, 1989). Moreover, it is now well established that arsenate and arsenite
can induce teratogenicity and embryolethality in chicks and rodents (Chaineau et al.,
1990; Hood et al., 1977; Morrissey and Mottet, 1983; Peterkova and Puzanova, 1976).

i To whom correspondence should be addressed.

0147-65 13192 $5.00 274

Copyright 0 1992 by Academic Press, Inc.
All rights of reprcduction in any form reserved.
 BAL AND DMPS AMELIORATE ARSENITE TOXICITY IN MICE 275

From the late 1940s the accepted treatment for poisoning with arsenicals is the
administration of 2,3dimercapto- 1-propanol (BAL), a dithiol compound and a strong
chelator of arsenic (Klaassen, 1985). However, recent advances in the treatment of
arsenic toxicity have shown that meso-2,3dimercaptosuccinic acid (DMSA) and so-
dium 2,3dimercaptopropane- 1-sulfonate (DMPS) are orally effective, dithiol chelating
agents useful for treating arsenic poisoning (Aposhian et al., 1984; Aposhian and
Aposhian, 1989). These chelators are less toxic than BAL, and consequently both
DMSA and DMPS can be administered in much higher doses than BAL (Aposhian
and Aposhian, 1989; Kreppel et al., 1990).
Since BAL, DMSA, or DMPS might be administered to an arsenic-intoxicated
pregnant woman, in previous investigations it was determined if BAL and DMSA
could alleviate arsenite-induced teratogenesis in mice. While BAL was not effective
when exposure to arsenite occurred before the chelator was given (Hood and Vedel,
1984), treatment with DMSA significantly reduced the embryolethality and the in-
cidence of gross external and skeletal abnormalities provoked by arsenite (Bosque
et al., 1990a; Domingo et al., 199 1). In the present study, the authors investigated
whether DMPS could ameliorate the developmentally toxic effects of sodium arsenite
seen in mice.

MATERIALS AND METHODS

Sodium arsenite (NaAs02) was purchased from E. Merck (Darmstadt, FRG). BAL
and DMPS were obtained from Sigma Chemical Co. (St. Louis, MO).
Male and female Swiss mice weighing between 27 and 30 g were purchased from
Panlab (Barcelona, Spain). The animals were freely supplied with pelleted dry chow
(Panlab) and were housed in a fully air-conditioned facility with a temperature of 22
+- 2C a relative humidity of 50 + lo%, and a 12-hr light/dark cycle. Female mice
were mated with males (2: 1) overnight and examined the following morning for copu-
latory plugs. The day on which a vaginal plug was found was designated Day 0 of
gestation.
Pregnant mice were divided into eight groups which consisted of one negative control
group, one positive control group, and six experimental groups to which different
concentrations of BAL or DMPS were administered. On Day 9 of gestation, each
pregnant mouse in both the positive and the experimental groups, was given a single
ip injection of aqueous sodium arsenite (12 mg/kg). Day 9 of gestation was expected
to result in a high level of prenatal mortality and abnormal fetuses (Domingo et al.,
1991; Hood et al., 1977; Hood and Vedel, 1984). Subsequently, the animals in the
positive group were given corn oil solutions by sc injection, while those in the exper-
imental groups received BAL or DMPS by SCinjection. A series of four BAL or DMPS
injections were administered at 0,24, 48, and 72 hr after arsenite exposure. BAL was
dissolved in corn oil, and DMPS in 0.9% saline immediately before use. BAL was
given at doses of 15, 30, and 60 mg/kg based on previous studies with BAL and
arsenite or arsenate (Hood and Pike, 1972; Hood and Vedel, 1984). DMPS was injected
at 75, 150, and 300 mg/kg, doses which are not teratogenic by themselves (Bosque et
al., 1990b). Mice in the negative control group were pretreated with 0.9% saline and
then post-treated with corn oil.
Dams were killed by cervical dislocation on gestation Day 18, the fetuses were
delivered by cesarean section, and the status of pregnancy was assessed. The fetuses
276 DOMINGO ET AL.

were first observed for gross external abnormalities and weighed. Approximately two-
thirds of the available fetuses were fixed in 95% ethanol, cleared with potassium hy-
droxide, stained with Alizarin red S, and examined for skeletal malformations and
variations (Dawson, 1926). Fetuses were not examined for visceral abnormalities,
since previous results indicated that their frequency would be low (Baxley et al., 198 1;
Hood and Vedel, 1984).
The nonparametric ANOVA Kruskal-Wallis was used to determine if there was a
significant difference between groups. Where a significant difference was observed,
pairwise comparisons of parameters from experimental groups to control (positive or
negative) groups were made by Students t test or Mann-Whitney U test. Significance
was set at the 0.05 probability level.

RESULTS
The distribution and fate of all mated mice on study are presented in Table 1. The
pregnancy rate was equivalent across all groups. There were no abortions, early deliv-
eries, or deaths in the arsenite-untreated (negative control) group. In contrast, in the
positive control group three dams died, six pregnant animals showed massive he-
morrages, and seven dams were carrying completely resorbed litters detected on ges-
tation Day 18. Treatment with BAL at 60 mg/kg increased the percentage of litters
with fetuses at scheduled termination compared with the positive control group,
whereas no maternal toxicity was seen aher sc administration of DMPS at 300 mg/kg.
Table 2 summarizes the effects of a single ip sodium arsenite injection (12 mg/kg)
to pregnant mice on Day 9 of gestation and the interactions with BAL and DMPS.
Arsenite resulted in a high rate of dead or resorbed fetuses, confirming previous reports
with this protocol (Domingo et al., 1991; Hood, 1972; Hood and Vedel, 1984). Mean
fetal body weights of the positive controls were significantly reduced compared to
those of the negative controls (Table 2). In addition, gross external and skeletal ab-
normalities, predominately exencephaly, exophthalmos, decreased ossification of su-

TABLE 1

EFFECTS OF BAL AND DMPS ON ARSENITE-INDUCED MATERNAL TOXICITY

Groups

NAG W&d 0 12 12 12 12 12 12 12
BAL @x/k) 0 0 15 30 60 0 0 0
DMPS (mg/kg) 0 0 0 0 0 15 150 300

No. in study 24 22 26 23 20 17 19 20
No. deaths 0 3 3 3 0 0 2 0
No. of mice
with massive
hemorrhages 0 6 1 2 4 3 1 0
No. of litters
completely
resorbed 0 7 4 2 4 6 4 0
No. of litters
with fetuses 24 6 12 16 12 8 12 20
 TABLE 2

PREVENTION BY BAL AND DMPS OF SODIUM-ARSENITE-INDUCED EMBRYOTOXIC AND TERATOGENIC EFFECTS IN MICE

Malformed skeletons
Treatment Grossly malformed
Dead or Mean fetal Fetuses
NaAsOr BAL DMPS No. of litters Total resorbed weight No. litters No. fetuses No. litters affected
OWkz) (w/W (w/kg) with fetuses implants* (%I w affected affected affected W)

0 0 0 24 9.8 f 3.0 1.4 f 3.33 1.37 f 0.06* O3 03 lo* 12.j3

12 0 0 6 11.5 f 2.2 76.0 f 41.0' 0.99 f 0.26' 4' 26.4' 6b 84.2'
12 15 - 12 10.9 f 4.1 65.8 + 39.6' 1.28 + 0.16 6' 13.9' lo* 60.0'
12 30 - 16 12.1 + 2.2 43.8 k 32.3h 1.13 + 0.15' 6' 5.72," 12" 33.33,"
12 60 - 12 10.7 2 1.8 46.8 k 37.0* 1.11 f 0.13 6' 12.3' lob 70.9'
I2 - 75 8 10.3 + 3.9 69.8 xk43.3' 1.25 + 0.05 2" 9.4" 8* 93.3'
12 - 150 12 11.3 ic 2.6 47.8 f 40.5' 1.15 + 0.126 6' 4.1* lo* 52.6'*'
I2 - 300 20 10.0? 4.1 34.3 k 12.3',* 1.22 f 0.15 O3 O3 14 35.33.b
-
* The results are expressed as mean values + SD.
.*J Significantly different from arsenite-untreated group (negative control), P < 0.05, P < 0.01, P < 0.001, respectively.
.2.3Significantly different from arsenite + corn oil group (positive control), P < 0.05, P < 0.0 1, P < 0.00 1, respectively.
278 DOMINGO ET AL.

praoccipital, carpus and tarsus, bipartite stemebrae, and fusion of ribs and vertebrae,
were also observed following sodium arsenite administration (Table 3).
Treatment with BAL at 15, 30, and 60 mg/kg did not show statistically significant
protective effects when given alter arsenite exposure. However, the apparent decrease
in the number of dead or resorbed fetuses, as well as in the frequency of gross abnor-
malities and skeletal defects, is suggestive of such an effect (Tables 2 and 3).
On the other hand, the number of dead or resorbed fetuses exposed at 75 or 150
mg/kg of DMPS immediately after arsenite injection and at 24,48, and 72 hr thereafter
appeared to be lower than that observed in litters exposed to arsenite alone, although
the difference was not statistically significant (Table 2). Treatment with DMPS at 300
mg/kg significantly reduced the incidence of prenatal mortality compared to arsenite
alone. Moreover, a significant reduction in the total number of external abnormalities
was seen at 150 mg/kg of DMPS, and no external defects were detected at 300 mg/
kg, while the number of total skeletal abnormalities was significantly decreased at 150
and 300 mg DMPS/kg. Treatment with BAL at 30 mg/kg significantly reduced the
number of grossly malformed fetuses and the number of skeletal anomalies, whereas
there were no statistically significant protective effects of BAL given at 15 or 60 mg/kg.

DISCUSSION
Since the 194Os, BAL has remained the drug of choice for the treatment of arsenic
poisoning even though it has many disadvantages and is far from the ideal drug (Klaas-
sen, 1985; Snider et al., 1990). Treatment SCwith 50 mg BAL/kg 4 hr before, con-
currently with, or 4 hr alter sodium arsenate reduced the frequency or severity of
malformations compared with the effects of arsenate alone (Hood and Pike, 1972).
Nevertheless, BAL given following sodium arsenite on gestation Day 9 afforded no
significant protection against the developmental toxicity of arsenic, although an ap-
parent decrease in gross and skeletal malformations was suggestive of such an effect
(Hood and Vedel, 1984). It was concluded that BAL is unlikely to have a practical
beneficial effect on the at-se&e-exposed conceptus, because it must be administered
prior to arsenite (or perhaps simultaneously with it) to be effective (Hood and Vedel,
1984). In contrast to BAL, treatment sc with DMSA significantly reduced the embry-
otoxicity and teratogenicity of arsenite when given during the first hour after sodium
arsenite injection (Bosque et al., 1990a; Domingo et al., 199 1). The aim of the present
investigation was to evaluate the protective activity of DMPS, a water-soluble chemical
analog of BAL and DMSA, against the developmental toxicity of arsenite and simul-
taneously to compare these effects with those of BAL.
In accordance with earlier studies, treatment with sodium arsenite alone provoked
a significant increase in the number of dead or resorbed fetuses, a decrease in the
mean fetal body weight, and a significant increase in the number of total external and
skeletal abnormalities when arsenite was administered on Day 9 of gestation (Baxley
et al., 1981; Domingo et al., 1991; Hood, 1972; Hood et al., 1977; Hood and Vedel,
1984). According to the results of the present study, sc administration of BAL following
arsenite exposure at doses which are not teratogenic by themselves (Hood and Pike,
1972; Nishimura and Takagaki, 1959) afforded no significant protection against the
arsenic which is in agreement with previous investigations (Hood and Vedel, 1984).
On the other hand, amelioration by DMPS of arsenite developmental toxicity was
not noted at 75 mg/kg, whereas treatment at 150 and 300 mg/kg showed significant
 TABLE 3
EFFECTS OF BAL AND DMPS ON ARSENITE-INDUCED EXTERNAL AND SKELETAL FETAL ABNORMALITIES*

Groups

N~sOZ h&g) 0 12 12 12 12 12 12 12
BAL bw&) 0 0 15 30 60 - - -
DMB Ow/k4 0 0 - - - 75 150 300 5
External abnormalities
Liz
No. of fetuses observed/
z
No. of litters 228124 6816 72112 140/16 114/12 4218 128112 120120
Abdominal 2
hemorrhages O(O) O(O) O(O) x3 W) O(O) x3 O(O) F
Exencephaly w)3 14(2) w-3 w3 w3 wJ)3 O(Q3 W 5
Exophthalmos o(o)* 6(4)b 8(4)b %W 6(4) 4(2)a 4(4) O(O) !z
Missing tail WV WY m w lWb O(O) O(O) O(O) ;;1
Total ow3 18(4) lO(6) S(6)+ 14(6) 4(2)n ws O(O)
Skeletal abnormalities %
No. of fetuses observed/ i+
No. of litters 128124 3816 50112 78116 62112 30/s 16112 90120
Supraoccipital bone, ;;f
decreased ossification 4(4)3 22(6) 10(8), 16( 10),b 14(6)lb 12(6) 14(4)2.4 12(8)3
Carpus, decreased z
ossification W) 18(4) w3 w)3 16(4)b w S(4)* 6(6)3 5
Tarsus, decreased 2
ossification 4(4) 30(6) 18(6), 18(10)2J 38(10) 7x3 12(4)3, 20(8)3*b 2
Bipartite stemebrae W3 24(4) 16(6)b lW3 16(8),b 14(6) 14(8)*+ 14(8)3
Fusion of ribs WJ) 3 20(4) 24(6) 10(8)'* 38(12)' 24(S) 22(6) 20(8)~= g
Fusion of vertebrae ow3 12(6) 22(6) 16(8) 28(10) 12(6) 20(6) 18(lo)c C?
Total 16(lO)r 32(6) 3O(lO)C 26( 12)~~ 44(10) 28(g) 40( 10)qC 32( 14)3.b

* In parentheses, number of affected litters.

OVb*cSignificantlydifferent from arsenite-untreated group (negative control), P < 0.05. P < 0.01, P < 0.001, respectively.
.2.3Significantly different from arsenite + corn oil group (positive control), P < 0.05, P < 0.01. P < 0.001, respectively. 2
m
280 DOMINGO ET AL.

protective effects against arsenite embryotoxicity and teratogenicity. In addition, 300

mg DMPS/kg protected the dams against arsenite maternal toxicity. In a previous
investigation, we demonstrated that DMPS is not a developmental toxicant at levels
up to 300 mg/kg per day (Bosque et al., 1990b). Although the detoxication mechanism
of DMPS (or DMSA) is still obscure, since Hood et al. (1988) concluded that inorganic
arsenite administered to pregnant mice is readily transferred via the placenta and
enters the near-term rodent fetus, it seems quite reasonable that the drug(s) acts as a
chelator of arsenic to obviate the embryotoxic and teratogenic effects of arsenite, and
probably the excretion of arsenic into the urine is then enhanced (Bosque et al., 1990a).

CONCLUSION
Like DMSA (Domingo et al., 1991) and in contrast to BAL (Hood and Vedel,
1984), DMPS offers encouragement with regard to its therapeutic potential for pregnant
women exposed to trivalent arsenic. Both DMSA and DMPS have considerable promise
as clinically useful agents in detoxification As-poisoned individuals. However, since
DMSA is about three times less toxic than DMPS (Aposhian et al., 1984), a comparison
under identical conditions of the amelioration by these drugs of arsenite developmental
toxicity will probably be interesting.

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