/. Embryol. exp. Morplu, Vol. 14, Part 1, pp. 63-73, August 1965
Printed in Great Britain
The susceptibility of the offspring of alloxandiabetic mice to a teratogen
by KIICHI TAKANO, TAKASHI TANIMURA
HIDEO NISHIMURA
and
1
From the Department of Anatomy, Faculty of Medicine, Kyoto University
M A T E R N A L diabetes, or pre-diabetes, has been considered as a possible factor
in the aetiology of human congenital malformations by several investigators
(De Costa, 1955; Hoet et al, 1960; Hiekkala & Koskenoja, 1961; Nicholson &
De Maria, 1963; Pederson et al, 1964; Lenz & Maier, 1964). However, a few
authors have reported no increase in the incidence of anomalies among infants
of diabetic women (Given et al, 1950; Hall & Tillman, 1951; Rubin & Murphy,
1958). Alloxan-induced maternal diabetes in animals has also been studied,
and an increase of stillbirth and foetal macrosomia has usually been recognized
(Davis et al, 1947; Lindan & Morgans, 1950; Bartelheimer & Kloos, 1952;
Angervall, 1959; Solomon, 1959; Kim et al, 1960; Love etal, 1960; Koskenoja
1961). The incidence of malformations, however, was not increased in most
cases, the exception being those reported by Ross & Spector (1952), Fujimoto
et al (1958) and Watanabe & Ingalls (1963).
Recently, Runner & Dagg (1960) and Wilson (1964a, b) suggested that an
interaction of multiple environmental factors might be important and that a
potentiation or reinforcing effect might occur between two or more agents in
regard to teratogenesis. In this respect there have been several animal experiments in which the influence of a maternal factor on the action of a certain
teratogen was investigated. A hypocaloric diet was shown to have a synergistic
effect on the teratogenicity of cortisone (Kalter, 1960); simultaneous administration of certain vitamins or hormones influenced the activity of chemical
teratogens such as cortisone (Stream & Peer, 1956; Nishihara et al, 1960),
vitamin A (Millen & Woolam, 1958), urethan (Nishimura, 1961) or triethylene
thiophosphoramide (Tanimura, 1962). Most of those factors were short acting,
and do not correspond to such chronic modification of metabolic states as
occurs in humans.
The present study is an attempt to investigate the susceptibility of the foetuses
1
Authors' address: Department of Anatomy, Faculty of Medicine, Kyoto University,
Kyoto, Japan.
64
K. TAKANO, T. TANIMURA and H. NISHIMURA
in alloxan-diabetic mice to a known teratogen, as well as the effect of maternal
alloxan treatment, itself, on the development of the offspring.
MATERIALS AND METHODS
Japanese colony bred ddS stock mice maintained at Animal Center, Kyoto
University were used. Nulliparous females at 12 to 17 weeks of age were
placed in the evening with mature males from the same colony. Copulation
was checked by the presence of a vaginal plug next morning. The day when
the vaginal plug was found was designated as 0 (zero) day of gestation. On the
8th day of gestation, pregnancy of the animals was ascertained by inspection
of the vaginal smear. Animals were kept on the diet of pellets N.M.F. (Crude
protein, 26-5 per cent; crude fat, 6*1 per cent.; crude fibre, 4-1 per cent.
N.F.E., 49-8 per cent.; Total ash, 6-5 per cent.) supplied by Oriental Co.,
Tokyo, and fresh tap water ad libitum. The pregnant animals were treated with
either or both of the two chemical compounds being tested.
A preliminary experiment showed that intraperitoneal injection of 4 per cent,
solution of alloxan at 200 mg. per kg. of body weight induced an increased
blood sugar level and glucosuria within 2 days in most animals. In the main
experiment this dose was injected once, immediately after the preparation of
the solution, intraperitoneally to about half the available pregnant mice either
on the 8th or 9th day of gestation.
As the teratogen, triethylene thiophosphoramide (thio-TEPA) was used: its
potent teratogenicity to mouse foetuses had been ascertained in our laboratory (Tanimura & Nishimura, 1962). The agent was administered by a single
intraperitoneal injection at 5-0 or 2-5 mg. per kg. of body weight in 0 • 1 or
0-05 per cent, solution in physiological saline to about two-thirds of the
alloxan-treated and a similar number of non-treated pregnant mice, on the
10th or 11th day.
A further twenty-five animals were used as controls without any treatment.
The pregnant mice were sacrificed on the 18th day of gestation and the
foetuses were examined for intrauterine death and for external deformities.
In a separate investigation of placental transmission of thio-TEPA, an
additional experiment using 32P-labelled thio-TEPA was carried out. Alloxan
(200 mg./kg., i.p.) was injected into twenty pregnant mice on the 9th day.
Twenty non-treated pregnant mice were used as controls. Freshly prepared
aqueous solutions of the labelled compound were injected intraperitoneally to
both groups of pregnant mice on the 1 lth day at 5 • 0 mg. per kg. of body weight.
Five animals of the treated and five of the control group were sacrificed at
times, 1£, 3, 6 and 24 hr. after the injection, and two live foetuses and their
placentae were removed from each litter at random. After finding the fresh
weight of each foetus and placenta, they were homogenized and diluted to
0-5 ml. with distilled water. One-fifth, i.e. 0-1 ml., of the sample was put on a
Susceptibility to a teratogen
65
sample dish and desiccated. The radioactivity of each sample was counted by a
gas flow counter (Shimadzu Seisakusho Ltd., Kyoto). The specific activity of
the labelled thio-TEPA was about 6 • 95 me. per g. at the time of its injection
and 5 • 20 me. per g. at the time of counting.
RESULTS
All the mice survived until the 18th day of gestation, when they were sacrificed.
All uteruses were found to have live foetuses or nodes of placenta. The results
are summarized in Tables 1, 2 and 3.
Effect of alloxan
The mortality rates of the foetuses were 12-9 per cent, in group le and
9-7 per cent, in group 2e respectively (Tables 1, 2). The former rate is significantly higher than that in the normal control N(p <0-01), but the latter is not
significantly higher.
The incidence of malformation in surviving foetuses was 7 • 1 per cent, (ten
cases) in group le, and of these three showed polydactylia and seven had clubfeet. In group 2e, 12-2 per cent, (seventeen cases) were malformed of which one
showed oligodactylia, thirteen clubfeet, and three cleft palates. Both of these
incidences are significantly higher than those in the control without treatment
(p<0-01).
The average body weight of living foetuses in the group le was slightly lower
than that in the control N(p<0-05). In group 2e, however, the foetal weight
was almost the same as that in the control N.
Effect of thio-TEPA upon normal animals
Although not statistically significant, the foetal mortality rate in groups lc
and 2c tended to be higher than that in the control N. In groups Id and 2d
such tendencies were not shown.
The incidences of malformed foetuses among survivors were significantly
higher in every experimental group than in the normal control. In regard to
the types of malformation, polydactylia of the hindfeet was the most common
in the group lc and Id. In the experimental groups 2c and 2d such digital
malformations were not shown, but clubfoot was a common abnormality.
The average body weight of live foetuses was significantly lower in the
experimental groups than in the controls, especially when treated on the 9th day.
Effect of thio-TEPA upon animals pre-treated with alloxan
The foetal mortality rate was remarkably high in the injected groups with
pre-treatment: the rates were from 23 to 38 per cent. In all these experimental
Malformed^
(%)
52-1
47-3
11-9
13-5
7-1
1-0
38-0
25-4
12-9
4-8
9-3
5-5
(%)
t Percentage of total living foetuses.
TABLE 2
158
201
204
164
162
210
19
23
25
19
20
25
* Percentage of total implants.
Total
implants
No. of
mice
i
Dead*
Weight (alive)
Average ± Sx (range)
(g.)
0-957±0014 (0-74-1-25)
1-020±0-016 (0-61-1 -62)
1-136±0-008 (0-74-1-33)
1-161+0-011 (0-82-1-64)
1-119 + 0-021 (0-61-1-74)
1-185 + 0-008 (0-84-1-60)
2a
2b
2c
2d
2e
N
Group
—
—
+
Pre-treatment
with alloxan
+
21
21
20
20
20
25
No. of
mice
173
177
178
163
154
210
Total
implants
* Percentage of total implants.
50
2-5
50
2-5
—
Dose of
thio-TEPA
(mg./kg.)
living foetuses.
10
12-2
9-6
34-1
17-6
13-3
(%)
Malformed\
4-3
9-7
4-8
t Percentage of total
7-3
25-4
23 0
(%)
Dead*
0-938±0021
(0-42-1-33)
0
1 123 ±0-009 (0-63-1-43)
1 043 ±0-010 (0-70-1-35)
1 105±0-012 (0-82-1-47)
1 164±0011 (0-80-1-55)
1 185±0O08 (0-84-1-60)
(if.)
Weight (alive)
average ± Sz (range)
Effect of intraperitoneal injection of thio-TEPA at 11 days of gestation on the foetuses of mice with or without
pre-treatment at 9 days of 200 mg./kg. ofalloxan
Foetuses
Group
\a
\b
\c
\d
\e
N
Pre-treatment
with alloxan
+
+
Dose of
thio-TEPA
(mg./kg.)
5-0
2-5
50
2-5
Effect of intraperitoneal injection of thio-TEPA at 10 days of gestation on the foetuses of mice with or without
pre-treatment at 8 days of 200 mg.jkg. alloxan
Foetuses
TABLE 1
X
vx
X
a-
ON
ON
8
8
—
—
8
9
9
—
—
9
5-0
2-5
5-0
2-5
—
(day 11)
(day 11)
(day 11)
(day 11)
—
Dose of
thio-TEPA
(mg./kg.)
5-0 (day 10)
2-5 (day 10)
5-0 (day 10)
2-5 (day 10)
98
150
185
155
141
129
136
165
156
139
200
No. of
live
foetuses
40:50
4:11
0:0
0:0
0:1
0:0
2-4
0
0-7
0
0:0
0:0
0:0
24-0
10-3
70
8-4
2-1
0:0
0:0
0:0
2:4
9:3
2:4
0:0
0:0
0:0
Foetuses*
Type and number
with
Bd
Od
malformed
digits (%) fh
fh
45-9
8:0
1:0
1:0
36-7
21:1
9:0
4:0
1:0
0:0
0:0
0:0
0:2
0:0
0:0
0:0
0:0
sd
fh
A
33
55
15
15
3
0
0
0
0
0
0
±Mdh
Pdh
10
19
1
1
0
0
0
0
0
0
0
alone
Mdh
of digital malformations^
9-6
9-3
10
11-5
4-9
5-1
50
6-2
5-1
14-3
11-3
(%)
(%)
20
2-7
0
0
0
130
2-2
0
0
2-2
0
Foetuses* Foetuses*
with
with cleft
clubfoot
palate
* Percentage of total living foetuses.
t Bd, Brachydactylia; f, forefeet; h, hindfeet; Mdh, Macrodactylia at hindfeet; Od, oligodactylia; Pdh, polydactylia at hindfeet; Sd, Syndactilia;
± , with or without.
la
lb
lc
Id
le
2a
2b
2c
2d
2e
N
Group
Day of pretreatment
with
alloxan
Incidence of malformations induced by thio-TEPA injection with or without alloxan pre-treatment
TABLE 3
I
ON
I
1
•8
K. TAKANO, T. TANIMURA and H. NISHIMURA
68
groups (la, \b, 2a, 2b) the rates exceeded the sum of the rates in the corresponding groups to which either alloxan or thio-TEPA alone were administered.
It is interesting that the tendency to potentiation was also clearly shown in
the incidence of malformations. As shown in Tables 1 and 2, the percentage of
75
70
65
.Foetus
60
j
•Placenta I
AUoxan
pre-treated
55
50
s
45
&
40
—x
Foetus
.- x
Placenta
Control
1 35
1
30
25
20
15
10
2
4
6
8
10
Hours after
12
32
14
16
.18
20
22
24
P-thio-TEPA injection
TEXT-FIG. 1. Comparison of placental transmission of 32P-labelled thio-TEPA between the
alloxan-diabetic animal and the non-treated control. The injection of alloxan C200 mg./kg.,
i.p.) was done on the 9th day of gestation and that of 32 P-thio-TEPA (5-0 mg./kg., i.p.)
on the 11th day.
malformed foetuses among the survivors in the groups to which both agents
were applied significantly exceeded the sum of the percentages shown in the
groups to which one agent alone was administered, except in the group 2b. In
this group, however, a conspicuous potentiation was revealed in regard to the
incidence of digital malformations as shown in Table 3. Potentiation was not
Susceptibility to a teratogen
69
consistently seen in respect of the incidences of clubfoot or of cleft palate. As
far as the malformed digits are concerned, a high susceptibility to teratogenesis
is presumed in the foetuses of all groups pre-treated with alloxan.
The growth of the surviving foetuses was severely suppressed in groups \a
and 2a, where 5 • 0 mg. per kg. body weight of thio-TEPA was applied after the
alloxan pre-treatment.
Placental transmission of labelled thio-TEPA
Ten foetuses and placentae at a time were examined for their radioactivity in
each group. Text-fig. 1 represents the mean value of counts per min. per mg.
of wet weight.
Noticeable radioactivity was already shown in both groups \\ hr. after the
administration of the labelled agent and the amount in the embryos was significantly higher than that in the placentae. However, no significant difference of
the radioactivity either in foetuses or in placentae was shown between the pretreated and the control group.
DISCUSSION
The following adverse effects on foetuses of alloxan diabetes in pregnant
mothers have earlier been reported: increase of intrauterine or neonatal death,
abnormal growth of surviving foetuses, and the induction of congenital
malformations.
High foetal mortalities were reported in mice (Koskenoja, 1961; Watanabe &
Ingalls, 1963) and in rats (Davis et al, 1947; Bartelheimer & Kloos, 1952;
Angervall, 1959) in which alloxan diabetes was induced during pregnancy. The
lethal effect to offspring was also recognized when the alloxan treatment had
occurred before pregnancy in rabbits (Fujimoto et ah, 1958) and in rats (Lindan
& Morgans, 1950; Kim et al, 1960; Lawrence & Contopoulos, 1960; Love
et ah, 1960). Lazarow et al (1960) reported that the foetal mortality increases
even in sub-diabetic rats to as much as three times the normal control level.
Love et al. (1960) in rat and Koskenoja (1961) in mice, however, did not find
such tendencies in pre-diabetic or mildly diabetic animals.
Overweight has been reported in the live born of mother animals with patent
diabetes (Angervall, 1959) or with mild diabetes or sub-diabetes (Solomon,
1959; Lazarow et al, 1960; Koskenoja, 1961). Kim et al (1960) and Koskenoja
(1961) recognized that the variation in body weights of the offspring of diabetic
animals was wider than that in the controls, although a similar average body
weight was shown. In other words, the frequency of neonatal overweight
might be higher in the offspring of diabetics.
As regards congenital malformations induced by maternal alloxan diabetes,
Bartelheimer & Kloos (1952) found only two anomalies (short tail and cataract)
70
K. TAKANO, T. TANIMURA and H. NISHIMURA
among fifty-nine living newborn rats whose mothers had been given alloxan
at various stages of pregnancy. Fujimoto et al. (1958) found ten cases of
malformed foetuses, mostly affected in the central nervous system, among
ninety-three live-born from alloxan-diabetic rabbits. In mice, Ross & Spector
(1952) reported that the incidence of malformed foetuses was 29 per cent, from
mothers which had received alloxan on three successive days during pregnancy,
and most of the malformed foetuses were likely to have died by the time of
observation. However, Koskenoja (1961) found only a few cases with zonular
cataract from alloxan-diabetic mice. Recently, Watanabe & Ingalls (1963)
treated female Phipps mice with a single injection of alloxan between 8 • 5 and
13-5 days after copulation and found a fairly high incidence of malformed
foetuses: 21-6 per cent, and 12-9 per cent, of live foetuses were deformed after
treatment at 8 • 5 and 9 • 5 days after copulation respectively. The 8 • 5 days after
copulation in their report corresponds to the 8th day in our usage.
In comparison with the reports mentioned above, the foetal mortality in our
results with alloxan-treated mice was lower, and the mean weight of living
foetuses was only slightly affected. The frequency of malformed foetuses was,
however, significantly increased. The low incidences of congenital malformations in most of the former reports might be due to the fact that the offspring
were only examined after delivery.
The raised susceptibility of alloxan-pre-treated mice to teratogenesis by thioTEPA might make one suspect that the metabolism of thio-TEPA in them and/or
the permeability of their placentae to the agent were modified by the induced
diabetes of their mothers and that the amount of the agent transferred to the
foetuses in diabetic mothers, was larger than in the control animal. In this respect,
Dixon et al. (1961) have found that diabetes induced by alloxan decreases the
activity of certain hepatic drug-metabolizing enzymes in rats. However, the
concentration of thio-TEPA in foetuses and in placentae from alloxan-pretreated mothers was almost the same as that in the control so far as our present
research using 32P-labelled thio-TEPA was concerned. Therefore, it might be
assumed that the foetuses in both groups were equally exposed to thio-TEPA.
Considering also the fact that the types of deformity induced by both treatments
were similar to those caused by the single administration of thio-TEPA, it seems
likely that the susceptibility of the foetuses to the agent was intensified in the
alloxan-diabetic mice.
Furthermore, it might be suggested that high incidence of foetal death and of
malformations in diabetic mothers ascertained by several investigators is due
to the combined effects of intensified susceptibility and of unknown exogenous
factors.
SUMMARY
1. Pregnant ddS stock mice were treated in three ways: the first group of
animals was given a single intraperitoneal injection of 200 mg. of alloxan per
kg. of body weight on the 8th or 9th day of gestation; the second group was
Susceptibility to a teratogen
71
given 2-5 mg. or 5 -0 mg. of thio-TEPA per kg. of body weight on the 10th or
11th day of gestation; the third group received both treatments with alloxan
and with thio-TEPA at the same times as the first and the second group. The
fourth, untreated, group was used as the control. The foetuses from 233
pregnant mice of all groups were examined near term.
2. After alloxan treatment alone a significantly raised incidence of gross
deformities, including clubfoot, cleft palate, polydactylia and oligodactylia, was
observed, while the foetal mortality rate and the mean weight of live foetuses
were only slightly affected in comparison with the normal control.
3. After successive administration of the two agents, the incidence of digital
malformations and of foetal death significantly exceeded the sum of the effects of
their separate actions, and the mean weight of surviving foetuses was notably
diminished.
4. In a supplementary experiment to examine placental transmission of
32
P-labelled thio-TEPA, detectable radioactivity was found in foetuses and
placentae \\ and 3 hr. after the administration of the labelled agent both in the
alloxan-pre-treated mothers and in the control animals. However, no significant
difference in the level of radioactivity either in foetuses or in placentae was
shown between the pre-treated and the control group.
RESUME
Sensibilite a un agent teratogene de la descendance de souris rendues diabetiques
par injection d'alloxane
1. Des Souris gravides de souche ddS ont ete soumises a trois traitements
differents. Les animaux d'un premier groupe ont recu en une seule injection
intraperitoneale 200 mg. d'alloxane par kg. de poids au 8eme ou 9eme jour de
la gestation. Dans le second groupe, les Souris ont recu 2,5 ou 5 mg. de thioTEPA par kg. au lOeme ou 11 erne jour de la gestation. Les animaux du
troisieme groupe ont subi les deux injections, alloxane et thio-TEPA, en meme
temps que les premier et deuxieme groupes. Un quatrieme groupe temoin est
forme d'animaux non traites.
Les foetus de 233 Souris gestantes des differents groupes ont ete examines
peu avant la naissance.
2. Apres le seul traitement a Palloxane, on a constate une augmentation
significative de graves malformations, telles que pieds-bots, fentes palatines,
polydactylie et oligodactylie. En revanche, le taux de mortalite foetale et le
poids moyen des foetus vivants sont peu affectes par rapport aux temoins.
3. La frequence des malformations des doigts et de la mortalite foetale apres
injection successive des deux agents est significativement superieure a la somme
des effets obtenus avec l'un et l'autre des agents. Le poids moyen des foetus
vivants est sensiblement diminue.
4. Une autre serie experimentale a ete faite pour examiner la transmission
72
K. TAKANO, T. TANIMURA and H. NISHIMURA
placentaire du thio-TEPA marque au 32 P. On a decele de la radioactivite dans
les foetus et les placentas \\ et 3 hr. apres administration de l'agent marque,
aussi bien chez les meres traitees prealablement a l'alloxane que chez les temoins.
Toutefois, on n'a observe aucune difference significative dans le taux de radioactivite presente par les foetus ou les placentas dans les groupes temoin et
traites.
ACKNOWLEDGEMENT
We are indebted to the Sumitomo Chemical Company Ltd., Osaka, for the sample of
thio-TEPA and to Takarazuka Radiation Laboratory of Sumitomo Atomic Energy Industries
Ltd., Takarazuka, for supplying 32P-labelled thio-TEPA. This investigation was supported
by a grant, HD00074-01 from National Institutes of Health, Public Health Service, U.S.A.
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