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Developmental anomalies in mice induced by 2 3-dimercaptopropanol (BAL).

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Developmental Anomalies in Mice Induced by
2,3-Dimercaptopropanol (BAL)'
H I D E 0 NISHIMURA AND SUMIKO TAKAGAKI
Department of A n a t o m y , Kyoto University, Kyoto, J a p a n
Formerly the causes of congenital malformations in mammals were generally believed to be genetic ones and no importance was attached to exogenous factors.
However. in recent years reports on malformations produced by environmental
means have appeared in rapid succession.
Teratogenic effects by chemical means has
been ascribed to nutritional deficiencies,
the injection of hormone and azo dyes and
treatment with several chemicals which
are regarded as mitotic poisons, such as
nitrogen mustard and ethylurethan (Grunwald, '47; Fraser et al., '51a; Hickey, '52;
Wilson, '54; Nishimura, '56; Kalter et al.,
'59).
An experimental application of 2,3dimercaptopropanol (BAL) on the bone
marrow of the mouse indicated that i t
possesses qualities of a mitotic poison of
the colchicine type (Dustin, '47). Since its
effect on the development of mammals has
not been investigated, the experiments to
be reported here were carried out to observe any teratogenic effects that this
agent might have on mice.
MATERIALS AND METHODS
The mice used in these experiments
were Japanese hybrid mice that have been
maintained in our laboratory for two or
three generations. Females two to 5
months old were fed on a diet consisting
of crushed rice 75 parts, crushed wheat 25
parts, yeast one part, salt one part, calcium carbonate one part, supplemented
with dried fish and vegetables at random.
A total of 65 female mice were chosen for
the experiment two to 15 days following
copulation, as determined by the presence
of a vaginal plug.
Most of the pregnant females were injected one to 4 times subcutaneously at
the nape with a BAL solution prepared by
The Daiichi Seiyaku Co., Ltd., Osaka (containing 10% 2,3-dimercaptopropanol dissolved in a refined peanut oil, with 10%
benzyl benzoate added). The quantity in
each injection was 0.001 ml per gm of
body weight. The mice thus injected were
sacrificed mostly at or near term or at
some time during the third trimester of
pregnancy. Some mice were allowed to go
to delivery Fetuses or pups obtained were
fixed in 10% formalin. As controls 277
near-term fetuses and 127 fetuses on the
12th or 13th day of gestation were taken
from 63 normal mice of the same colony.
Also 58 newborn mice delivered spontaneously were added to the control group.
Furthermore, 8 mice were injected with
0.025 to 0.05 ml of the solvent of the BAL
solution on day 8%2 to 12th day of gestation, and 42 fetuses were taken near term
for examination. Some of the mice with
macroscopically recognized malformations
and their placentae were examined histologically after paraffin sectioning and
staining with hematoxylin and eosin or by
the azan method.
RESULTS
ControZ data. No malformations of the
type induced by BAL injection were found
in 404 fetuses removed near term or in
the third trimester or in 58 newborn pups
from normal mother mice. Also no malformations were detected in 42 fetuses
from the mothers treated with the solvent
of the BAL solution.
E f f e c t of BAL. Observations were made
on fetuses at or near term or on newborn
pups from 52 pregnant mice which were
1 This investigation w a s supported by a Grantin-Aid for Fundamental Scientific Research f r o m
t h e Ministry of Education in Japan.
26 1
262
HIDE0 NISHIMURA AND SUMIKO TAKAGAKI
injected with BAL solution during the
second to 15th day of gestation. Treatment once or twice on any day between
the second and 8th day of gestation in
18 mothers caused neither abnormalities
nor suppression of growth in the offspring.
It was suspected that such treatment prevented implantation or resulted in resorption of the embryos in some cases. In order to make sure of this, we examined on
the 12th or 13th day of gestation the fetuses from 13 mother mice which were injected similarly at the 7th and 8th day.
Only a slightly increased intrauterine death
rate as compared to that of controls was observed.
The results of treatment at the later
stages observed at term or newly born stage
are summarized in table 1.
It is apparent that this drug often has a
little or no lethal effect. Reference to the
data on body weights shows that BAL has
a slight suppressing effect on fetal growth.
Developmental malformations frequently
occurred in the fetuses after treatment of
mothers on the 9th to the 13th day. More
than half of the mother mice treated during this period produced malformed fetuses. All the malformations involved the
skeletal system and the extremities were
the most frequent sites of maldevelopment.
An especially high incidence of digital
deviation was observed, followed by an
appreciable number of brachydactyly, and
a small number of aplasia or hypoplasia
of the ungual process, syndactyly, shortness of foot or fore limb and malformation
of the joints of the extremities. There was
TABLE 1
Effect i n the offspring of pregnant mice injected with BAL
(0.001 ml/gm of body weight in 10% peanut oil solution)
Fetuses or pups
Day of
injection
&
:;
Total
Dead
no.
%
Congenital
anomalies
Number and
types of
malformation'
average
body weight
of fetuses ( F )
%
9
8
60
Experimental group
8
18
3D(f), 13D(t)
9-10
5
28
4
43
2T, lCr, l C u , lBa,
W f ) , 1S(t),4D(f),
17D(t)
F 0.60-0.75
11
11-12
2
5
10
34
30
6
30
65
1 D ( f ) , 2D(t)
lPs, lPe, 7Bd(f),
6Bd(t), 1 S ( f ) , 5S(t),
12D(f), 16D(t)
F 0.90-1 .OO
F 0.75-0.88
F 0.87-1.05
P 0.80-1.35
P 1.10-1.40
12
2
13
8
69
5Mm, lPv, l B d ( f ) ,
5Bd(t), 6D(f)
P 1.15-1.20
12-13
8
40
38
48
3Ps, lMm, 1Pd(t),
97Bd(f), 98Bd( t ),
F 0.73-0.97
P 0.77-1.30
13-14
14-15
2
2
8
13
0
0
0
0
52
335
Uninjected controls
4.8
0
P 1.35-1.45
P 1.20-1.30
F 1.01-1.22
P 1.14-1.41
1 Malformations: Ba, shortness of fore-arm; Bd(f), brachydactyly (finger); Bd(t), (toe);
Cr, protrusion in cranial region (cranial fault and cerebral hernia); Cu, elbow-joint i n
extension; D ( f ) , digital deviation (finger); D ( t ) , (toe); Mm, shortness of fore-foot ( h a n d ) ;
P d ( f ) , polydactyly (finger); P d ( t ) , (toe); Pe, pes equinus; Ps, cleft palate; Pv, clubfoot;
S ( f ) , syndactyly (finger); S ( t ) , (toe); T, various external structures (eyelid, auricle,
umbilicus and digits) retarded.
263
MALFORMATIONS BY 2,3-DIMERCAPTOPROPANOL
little difference betweeen the incidence of
anomalies in front and hind limbs with
the possible exception of a higher incidence
of the digital deviations of the hind foot.
A few cases of cleft palate and one of
cerebral hernia were found.
Most cases of the malposition of digits
were found to be the result of deviations
of the end-phalanx towards a lateral or
medial direction (fig. 2). In rare cases a
digit was bent to the palm or to the sole.
In the fore-foot deviations occurred most
frequently in the second digit, less so in
the third digit; and with low frequency in
the fourth and fifth digits. In the hindfoot, abnormality occurred most frequently
in the third digit, then in the second and
the fourth digits; with least frequency in
the fifth digit. Occasionally two or three
adjacent digits deviated together in a converging or diverging manner. Syndactyly
occurred most often between the second
and third or the third and fourth digit
(fig. 3). Sometimes i t occurred among
three digits. Brachydactyly was recognized mostly in the second, third or fourth
digit, accompanied by deviation in the
majority of cases. In a few cases an ungual process was not recognizable (fig. 5)
and in others all digits were short and divergent, almost resembling a starfish in
shape (fig. 6).
From a histological standpoint, the
shortened digits showed shortness of all
phalanges. The digit apparently without
an ungual process exhibited an underdeveloped unguis which is buried under
the epidermis at the tip of digit. Cases of
the starfish-shaped short digits were char-
acterized by shallow epithelial ingrowth
between adjacent digits and sometimes,
were accompanied by a retarded ossification of the metatarsus. In those cases it
appeared that the development of the foot
plate was arrested at some embryonal
stage. One case of polydactyly was found
to be due to an extra small end-phalanx,
which probably originated from a fission
of the end-phalanx. The cleft palates were
mostly complete and bilateral. They are
characterized by having the nasal septum
entirely free from the roof of the palate
and the oral surface covered with a stratified squamous epithelium. One case
showed a partial cleft localized on the
posterior part of the palate.
Critical period for malformations in the
digits, particularly the more frequently encountered ones are estimated.
It is seen from table 2 that the period
for inducing various digital anomalies falls
between the 9th to 12th day of gestation
and there is no clear difference during that
period between the digits of the fore-foot
and those of hind-foot.
The placentae of the malformed fetuses
were examined grossly in order to determine whether the above abnormalities
might be caused by a placental disturbance. Except for the placentae of the
prenatally dead fetuses, no remarkable abnormalities were recognized in size, shape
or color. Placentae of several fetuses with
severe malformations were fixed in formalin and examined histologically. In no case
was degeneration or other abnormality
found.
TABLE 2
Relation between the time o f BAL injection and occurrence of digital
malformations i n fetuses
Day of
injection
(days after
copulation)
Total no.
of fetuses
9
9-10
11
11-12
12
12-13
13-14
48
34
10
39
13
35
15
Fetuses with
malformed digits
of fore-foot
Fetuses with
malformed digits
of hind-foot
%
%
6
15
10
28
54
19
24
20
44
15
46
49
0
0
264
H I D E 0 N I S H I M U R A A N D SUMIKO TAKAGAKI
DISCUSSION
As stated previously BAL in adequate
doses and at the correct time h a s a teratogenic effect and also a growth retarding
action upon the mouse fetuses. It may be
presumed that BAL directly affected the
fetuses but only for a short time after
injection; it is of such a size as to cross
the placenta and chemically it is of labile
character. Regarding the mechanism of
this effect, it is assumed that this chemical
acts as a mitotic poison, like colchicine,
with a toxic effect on certain enzyme
(Peters et al., '45; Dustin, '47).
As for the types of malformations, BAL
is characterized by a predilection for inducing anomalies of the extremities. Although various skeletal anomalies have
been induced by such exogenous factors
as nutritional deficiencies, anoxia, certain
hormones and chemicals in recent years,
the malformations reported in none of
these experiments was entirely similar to
the results of our experiment, either as to
the types of defects or their frequencies.
In this respect, BAL may be considered
to have a specific teratogenic effect.
As was shown i n tables 1 and 2, the
period for inducing various skeletal anomalies ranges from the 9th to 12th day
of gestation. We shall now refer to the
critical periods for similar anomalies in
mice caused by other chemical agents.
Fraser and Fainstat ('51b) found in their
experiments on cortisone administration
to mice that a critical period for inducing
cleft palate ranged from the 9th to 17th
day. However, Nishimura and Kuginuki
('58) recognized that a critical period for
inducing various skeletal malformations
by ethylurethan injection ranged from 9th
to 12th day, which is very similar to our
case. Thalhammer and Heller-Szollosy
('55) injected nitrogen mustard into inbred mice and found that various osseous
malformations were induced by a n injection given on the 11th to 13th day of
gestation. Takagaki ('57) ascertained i n
a similar experiment on mice of the SMA
inbred strain in Japan that the critical
period of teratogenic effect on the skeletal
system for nitrogen mustard falls i n the
interval between 6% and 13% days of
gestation. Recently, Nishimura and co-
workers determined that TEM (Nishimura
et al., '57), 8-azaguanine (Nishimura and
Nimura, 'SS), and nicotine (Nishimura
and Nakai, '58) have teratogenic effect
mainly upon the osseous system in mice.
The critical period for the effect of TEM
in mice of Japanese dd strain was recognized to be the interval between 9 % and
14?h days of gestation, that of 8-azaguanine between the 8th and 15th day and
that of nicotine between the 6th and 14th
day. Those results mean that a rough
similarity as well as some difference can
be noted in respect to the period of sensitivity to various chemical agents. To
explain this fact the differences in the
duration of action of those chemicals and
i n the strains of mice may be considered.
I n regard to the developmental stage of
the extremities during the sensitive period,
we found that on the 9th day the primordium of the extremities begins to appear and on the 12th day the fore-foot
was at the state where the anterior footplate has just formed, with the rnesenchymal tissue differentiating into a compact cellular precartilage. As for the palate, Kanamori ('58) reported that the palatine process began to appear on the 11th
or 12th day in mice of our colony. In view
of the above facts, it seems permissible to
hypothesize that BAL acts upon the organs
during the sensitive period when the primordia are developing actively and in so
doing impedes or suppresses their organogenesis.
The fact should be mentioned that there
may be differences in the teratogenic effects between litters or between littermates.
This is clearly shown in table 1. Moreover,
anomalies in a n individual do not always
coincide on the two sides of the body. As
reasons for these, the following points may
be mentioned: (1) There exist genetic
differences between different litters or even
among littermates; ( 2 ) Having a different
condition of its placenta and circulatory
organ, each littermate is not always equally
influenced by the same agents; ( 3 ) It is
probable that the individuals in each litter
show detectable variations i n their developmental state.
Next we wish to consider the action of
this drug when applied before the sensitive
MALFORMATIONS BY 2,3-DIMERCAPTOPROPANOL
period. It is probable that BAL sometimes
caused a lethal effect or disturbed implantation at this time. According to Russell ('501, when x-ray was applied to fetuses of l/2 to 4142 days (probably 5?h
days) of fetal age, many embryos died although those which survived grew normally. Evans et al. ('53) in their experiments
involving folic acid deficiency in pregnant
rats found that treatment during two to
three days preceding implantation caused
no developmental disturbance in the embryos. These observations are roughly in
accord with our results. It seems that
generally in experiments with rodents
when the agent is applied to fetuses before
implantation, it may cause a lethal effect
but induce no malformation.
SUMMARY
1. BAL (2,3-dimercaptopropanol) in
10% oil solution was injected into pregnant mice of a Japanese colony bred hybrid strain. It was found that this agent
has a teratogenic effect, a slight growth
retarding effect and a moderate effect on
the mortality of embryos in early life.
2. When mice were injected with 0.001
ml of BAL solution per gm of body weight
on the 9th to 12th day of gestation teratogenic effects on the fetuses were manifest.
3. When BAL was applied to mice earlier than the 9th day of gestation, it was
likely that the drug disturbed the embryo's
implantation, or gave a lethal effect in
some cases.
4. The malforamtions occurred mostly
in the skeletal system of the extremities.
Digits with abnormal direction and situation were most frequently found, and other
malformations such as shortness of the
limb or foot, brachydactyly, syndactyly,
adactyly and polydactyly were occasionally
found. A few cases of cleft palate and one
case of cerebral hernia were also recognized.
5. In macroscopic and routine histological observations no placental abnormality was recognized.
6. It is probable that the teratogenic
effects of this drug resulted from its action
as a mitotic poison to the cells of the
embryo, in consequence to which disturb-
265
ance of development of the primordia of
the affected organs occurred.
ACKNOWLEDGMENTS
The authors are indebted to The Daiichi
Seiyaku Co., Ltd., Osaka for the generous
supply of the BAL and wish to thank Dr.
J. G. Wilson, Professor of Anatomy of
Florida for his helpful criticism in this
work and his advice in the preparation
of the manuscript.
LITERATURE CITED
Dustin. P. 1947 Some new aspects of mitotic
poisoning. Nature, Lond., 159: 794-797.
Evans, H. M., H. V. Wright, C. W. Asling and
M. NI. Nelson 1953 Congenital skeletal anomalies resulting from transitory maternal folic
acid deficiency. Anat. Rec., 115: 303 (Abst.).
Fraser, F. C., and T. D. Fainstat 1951a Causes
of congenital defects (A review). A. M. A. J.
Dis. Child., 82: 593-603.
1951b Production of congenital defects
in the offspring of pregnant mice treated with
cortisone. Pediatrics, 8: 527-533.
Griinwald, P. 1947 Mechanism of abnormal
development. A. M. A. Arch. Path., 44: 398436.
Hickey, M. F. 1952 Genes and mermaids:
Changing theories of causation of congenital
abnormalities. Med. J. Australia, 1I 649-667.
Kalter, H., and J. Warkany 1959 Experimental
production of congenital malformations in
mammals by metabolic procedure. Physiol.
Rev., 39: 69-115.
Kanamori, H. 1958 Embryological studies on
various malformations in the offspring of pregnant mice treated with ethylurethan. 1. The
developmental process of the cleft palate.
Kaibo. Z. (Acta Anat. Nipponica), 33: 55-63
(Japanese ) .
Nishimura, H. 1956 How fetal environmental
factors control the innate constitution? Saishin-Igaku, l l : 2783-2806 (Japanese).
Nishimura, H., and M. Sakaue 1957 Teratogenic effect of some antitumor chemicals
(TEM, 8-Azaguanine etc.) upon fetal mice.
Kaibo 2. (Acta, Anat. Nipponica), 32: 63-64
(Japanese) (Abst.).
Nishimura, H., and K. Nakai 1958 Develop
mental anomalies i n offspring of pregnant mice
treated with nicotine. Science, 127: 877-878.
Nishimura, H., and M. Kuginuki 1958 Congemtal malformations induced by ethylurethan
i n mouse embryos. Okajimas Fol. Anat.
Japonica, 31: 1-10.
Nishimura, H., and H. Nimura 1958 Congenital malformations in mouse embryos induced by 8-azaguanine. J. Embryol. Exp.
Morph., 6 : 593-596.
Peters, R. A., L. A. Stocken and R. H. S. Thompson 1945 British anti-lewisite (BAL). Nature, Lond., 156: 616-619.
266
HIDE0 NISHIMURA AND SUMIKO TAKAGAKI
Russell, L. B. 1950 X-ray induced developmental abnormalities in the mouse and their
use in the analysis of embryological patterns.
J. EXP. ZOO^., 114: 545-596.
Takagaki, S. 1957 Congenital malformations in
mice induced by nitrogen mustard. Kaibo 2 .
(Acta Anat. Nipponica), 32: 248-256 (Japanese).
Thalhammer, O . , and E. Heller-Szollosy 1955
Exogene Bildungsfehler (“Missbildungen”)
durch Lostinjektion bei der graviden Maus.
Zschr. Kinderh., 76: 351-365.
Wilson, J. G. 1954 Influence on the offspring
of altered physiologic states during pregnancy
i n the rat. Ann. N. York Acad. Sci., 57:
517-525.
PLATE 1
E X P L A N A T I O N O F FIGURES
Various malformations i n the fetuses at term after BAL injection.
1 Partial cleft at the posterior part of the palate (arrow).
x
8.
x
2
Digit no. 2 (arrow) of left fore-foot showing abnormal direction.
3
Syndactyly between digits no. 3 and no. 4 (arrow) of left hind-foot.
4
Right clubfoot. X 6.
5
Digits no. 3 and no. 4 (arrows) of left hind-foot, lacking ungual processes. X 13.
6
Brachydactyly in all digits of left hind-foot (starfish shape). X 13.
16.
x 16.
MALFORMATIONS BY 2,3-DIMERCAPTOPROPANOL
Hideo Nishimura and Sumiko Takagaki
PLATE 1
267
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