Congenital Malformations in Mouse Embryos
Induced by 8-Azaguanine
by HIDEO NISHIMURA and HIROAKI NIMURA 1
From the Department of Anatomy, Kyoto University
INTRODUCTION
IN recent years congenital malformations have been induced in higher mammals by such exogenous factors as nutritional deficiencies, anoxia, certain
hormones, and chemicals (Fraser & Fainstat, 1951; Hickey, 1952; Wilson, 1954;
Nishimura, 1956). This study was undertaken to ascertain whether 8-azaguanine,
an antagonist to guanine, has a teratogenic effect on mouse embryos.
METHOD
Female mice of the Japanese dd strain were mated at 3 to 5 months of age.
On days 7 to 15 of pregnancy they were given a single intraperitoneal injection
of 8-azaguanine solution (16 mg. /c.c.) and then were sacrificed shortly before
term to determine state of pregnancy and condition of the foetuses. Routine
histological examinations were made of malformed organs and also of the
placentae of affected embryos. As controls foetuses from 30 untreated mothers
from the same colony were examined.
RESULT
Table 1 shows the results of injection with 8-azaguanine at various times in
gestation.
Of 245 control foetuses from untreated mice, only 8 were dead and one
malformed (with double ungual process of the first digit of the hind foot). It is
clear, therefore, that 8-azaguanine often has a lethal effect. Many embryos seem
to have died and become macerated immediately after injection as indicated
by the frequency of complete resorptions found at term. Where pregnancy was
not interrupted by complete resorption no remarkable alteration in litter size
was found.
It is also clear that 8-azaguanine has a considerable teratogenic effect. The
malformations occurred mostly in the skeletal system, and especially in the
extremities, although there were some cases of cleft palate. Histologically it
1
Authors' address: Department of Anatomy, Faculty of Medicine, Kyoto University, Sakyoku,
Kyoto. Japan.
[J. Embryol. exp. Morph. Vol. 6, Part 4, pp. 593-596, December 1958]
594
H. NISHIMURA AND H. NIMURA
was observed that the deviated digits, the commonest type of anomaly, were
associated with malformed joints and sometimes also with bent proximal bones.
In cases of pes varus or pes valgus, luxation of the talocrural joint, hydrops of
the tarsal joint with atrophy of the tarsal bone cells or irregularity in form of
metatarsal bones was observed. All cleft palates were bilateral; some were
TABLE 1
Effects of a single injection of 8-azaguanine on pregnant mice
No. of
pregnancies
Foetuses at term
Total No. undergoing
complete Total No.
No. with
Days of Dosage of mice
resorption No.
injection (mg./g.) (litters)
dead malformation
1
7
008
8
8
008
4
5
2
0-24
7
8
8
9
0-32
0-4
0-24
9
4
3
10
0
0
0
4
12
22
0
0
4
2
2
—
—
—
1
0
1
1
032
2
2
6
—
—
0
—
—
1
—
—
9
0-4
1
0
8
0
5
10
11
2
5
1
2
1
0
11
0-4
4
3
2
0
0
7
12
12
12
024
0-32
1
4
4
13
13
0-32
2
4
5
2
2
20
7
7
8
—
—
11
0-32
0-24
0-32
0-4
1
14
14
15
0-32
0-4
0-32
1
1
3
1
0
0
0
0-24
0-4
0
7
13
—
7
8
19
1
—
1
0
—
0
0
0
5
2
1
—
3
—
4
4
—
2
2
4
Types and
No. of anomalies*
—
1 Ps, 1 Cd, 2 Pd
1 Ps, 4 Pd, 1 Sd(t),
1 Dd(t)
—
1 Md(t)
—
—
1 Bd(f), 1 Sd(f),
2 Dd(f), 5 DdCt)
—
2 Sd(t)
1 Sd(t)
—
3 Dd(t), 1 Dd(f)
—
2 Ps, 4 Dd(t)
6 Dd(t)
—
2 Dd(t)
3 Dd(t)
4 Dd(t)
* Bd(f): brachydactyly (finger). Bd(t): brachydactyly (toe). Cd: malformation of elbow joint.
Dd(f): deviation of finger. Dd(t): deviation of toe. Md(t): macrodactyly (toe). Pd: malformation of
ankle joint. Ps: cleft palate. Sd(f): syndactyly (finger). Sd(t): syndactyly (toe).
incomplete posteriorly, others complete. It should be noted that the critical
period for digital anomalies, if one exists, covered a lengthy period, from the 8th
to at least the 15th day. No apparent alteration of body-weight was recognized in
the living embryos subjected to 8-azaguanine. Placentae of malformed foetuses
showed no macroscopic or histological abnormalities.
DISCUSSION
Concerning the mechanism of the teratogenic effect of 8-azaguanine little
can be said, except that there was no evidence that the placenta was implicated.
E F F E C T OF 8 - A Z A G U A N I N E ON M O U S E E M B R Y O S
595
The fact that it induced malformations mostly in the skeletal system recalls
the results obtained with such agents as nitrogen mustard (Haskin, 1948; Danforth & Center, 1954; Thalhammer & Heller-Szollosy, 1955; Takagaki, 1957),
and ethylurethan (Sinclair, 1950; Nishimura & Kuginuki, 1958). However, 8azaguanine is peculiar in that it caused numerous slight malformations such
as deviation of interphalangeal joints.
SUMMARY
1. The effect of 8-azaguanine on the development of offspring of mice was
investigated by injecting mothers once intraperitoneally with 0-08 to 0 4 mg. /g.
of body-weight on a day between the 7th and 15th of gestation.
2. The principal teratogenic effect was on the skeletal system. Among the
malformations found were cleft palates and various abnormalities of the extremities; abnormally directed digits due to deviation of interphalangeal joints
were most frequently encountered.
3. When administered between the 7th and 14th days of gestation, this compound is frequently lethal to embryos, causing death immediately or a few
days after injection.
ACKNOWLEDGEMENTS
We are grateful to Professor James G. Wilson of the Department of Anatomy
of the University of Florida, U.S.A., for his assistance in the preparation of
this manuscript.
8-azaguanine was generously supplied by the Tanabe & Co., Osaka.
This investigation was supported in part by a grant in Aid for Fundamental
Scientific Research from the Ministry of Education of Japan.
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