. Embryol. exp. Morph. Vol. 35, 1, pp. 213-224, 1976
213
Printed in Great Britain
The teratogenie effects of hydrocortisone on
palatal development in the hamster
By RAVINDRA M. SHAH 1 AND ANTHONY A. TRAVILL 2
From the Department of Oral Biology and Pathology,
University of British Columbia, and the Department of Anatomy,
Queerts University, Kingston
SUMMARY
The effect of different doses of hydrocortisone, administered at various times during
gestation, on the fetal hamster and its palatal development was studied. Both the frequency of
cleft palate and that of resorption depended upon the dose of hydrocortisone and the time of
its administration. Two peaks in the incidence of cleft palate were observed, which appeared
to be due to inhibition of different events.
Incidence of morphologically different types of cleft palate was related to the doses and
gestational time of hydrocortisone treatment. A close association was observed between the
frequency of cleft palate and fetal growth retardation and between the latter and the dose
and time of drug administration. It was suggested that maternal physiology and the placental
barrier may play a crucial role in cleft palate induction.
INTRODUCTION
Palatal clefts are among the most common major malformations in Man
(Stevenson, 1969). A genetic component can only be shown in 12-20% of
cases (Poswillo & Roy, 1965) and it is therefore possible that environmental
factors are at least in part responsible for a large proportion of palatal clefts.
The role of non-genetic factors in the causation of palatal clefts has been
elucidated to some degree by experimental studies in animals (Hale, 1933, 1935,
1937; Warkany & Nelson, 1940, 1941; Warkany & Schraffenberger, 1944,
1946). However, it was not until Baxter & Fraser (1950) observed that cortisone
injection to pregnant mice produced only cleft palate in fetuses that a reliable
experimental system became available for the study of mechanisms that underlie
the formation of clefts. Since then much experimental work has been done in
different inbred strains of mice (Fraser & Fainstat, 1951; Fraser, Fainstat &
Kalter, 1953; Kalter, 1954; Ingalls & Curley, 1957; Chaudhry, Schwartz &
Schmutz, 1966; Chaudhry, Schwartz, Schwartz & Schmutz, 1967; Dostal,
1971; Dostal & Jelinek, 1970, 1971a, b), demonstrating that intraspecies
1
Author's address: Faculty of Dentistry, University of British Columbia, Vancouver,
British Columbia, Canada.
2
Author's address: Department of Anatomy, Queen's University, Kingston, Ontario,
Canada.
214
R. M. SHAH AND A. A. TRAVILL
differences play an important role in determining susceptibility to teratogens
(Saxen & Rapola, 1969). Glucocorticoids have also been shown to induce cleft
palate in the fetuses of rabbit (Fainstat, 1954; Walker, 1967) and rat (Dostal &
Jelinek, 1971c).
Recently Shah & Chaudhry (1973) observed that hydrocortisone is a potent
teratogen when administered to pregnant hamsters and produces cleft palate
in the fetuses. We therefore studied the effect of different doses of hydrocortisone, administered at various times during gestation, on fetal palatal development in hamsters.
MATERIALS AND METHODS
Golden Syrian hamsters, 295 female and 25 male, weighing approximately
90 ± 3 g, were maintained under controlled environmental conditions as
described previously (Shah & Travill, 1975). On the appropriate day of the
estrous cycle, female hamsters were mated with a male from 7 to 9 p.m. The
midpoint of the mating period (8 p.m.) was designated as day 0.
Each pregnant animal was given a single intramuscular injection of an
aqueous suspension of either 15, 20, 25, 30, 40 or 50 mg hydrocortisone (Sigma
Chemicals, St Louis, Missouri, Batch No. 71C-0020). The injections were made
at 4 h intervals between 10 d and 12 h (10:12) and 11 d and 12 h (11:12) of
gestation. A group of animals were similarly injected with water at each time.
Both the experimental and controls were killed on day 15:00 of gestation. The
viable fetuses were weighed and then immersed in Bouin's fluid for fixation.
The fetuses were examined for the status of cleft palate development. For
statistical treatment of data on palatal development, a computer program of
nonlinear regression analysis was performed on 1BM-360 (Dixon, 1970). The
relationship between doses and times to the incidence of total cleft palate was
derived by using the following formula:
^ -
//I
\
eX
P
L 21
Z
"X5
where P = probability of cleft palate; D = dose in mg; TT = 3-14159; ja, a
determined for each time of administration; fi = a + bt + ct*; t = gestation
time (h)-264; a= +18-66 (s.D. 0-542); b = 0-4302 (s.D. 0-0278); c =
+ 0-040503 (s.D. 0-0051); cr = 5-66 (s.D. 0-55).
The constants a, b, c and a were estimated from data using a weighted
least-squares procedure.
Similarly, the relationship between doses and times to the incidence of
resorption was derived by using the following formula:
p = 01 + ( # 2 e x p - ^ ) A
where P = probability of resorption; dx = -0-003722 (s.D. 0-000456); 02 =
0-08903 (s.D. 0-0141); 03 = 0-77253 (s.D. 0-256); D = & (actual dose in mg);
t = jo (gestation time in hours-264).
Cleft palate development
215
RESULTS
Single intramuscular injections of hydrocortisone to pregnant hamsters, in
doses ranging from 25 to 50 mg between days 10:12 and 11:12 of gestation,
produced cleft palate in the fetuses. Cleft palates were also produced in the
fetuses following administration of 15 and 20 mg between days 10:16 and 11:12
of gestation. No other external malformations were found.
•A 3 2 - 1
10:12 :14
:18
:20
:22 11:00 :02
:04
Gestation period (days: hours)
:06
:08
1
Fig. 1. The probability of cleft palate in hamster fetuses in relation to the dose and
the gestational time of hydrocortisone administration.
The data presented in Table 1 indicate that as the dose of hydrocortisone
increases from 15 to 50 mg between days 10:12 and 11:12 of gestation, the
frequency of cleft palate increases. The frequency of cleft palate also varies
according to gestation period. For example, hydrocortisone administration in
doses of 15 to 30 mg, between days 10:12 and 10:20 of gestation, produces
lower incidence of cleft palate than when administered between days 11:00 and
11:12. Also two peaks in the incidence of cleft palate are seen following 15, 20
and 25 mg hydrocortisone injection. The first peak is observed at day 11:00
and the second at day 11:08 of gestation. Similar peaks are not observed
following administration of 30-50 mg hydrocortisone.
Statistical relationships of doses and times to the probability of cleft palate
are summarized in Fig. 1. One may deduce that in order to produce maximum
incidence of cleft palate, a minimum dose of 34 mg hydrocortisone is required
216
R. M. SHAH AND A. A. TRAVILL
Table 1. Incidence of induced fetal cleft palate and resorption sites following
administration of different doses of hydrocortisone to pregnant hamsters at various
times during gestation
Gestation
period
(day:h)
10:12
10:16
10:20
11:00
11:04
11:08
11:12
No. of
live
fetuses
Resorption*
(%)
No. of
cleft
palate (%)
8
8
6
6
37
28
52
54
43
7
33
40
58
49
54
56
94
90
82
71
61
20
94
127
107
133
69
70
84
76
85
82
76
75
53
86
84
68
55
51
3
10
2
37
40
79
3
13
9
9
11
19
I
7
12
28
28
59
2
5
4
9
11
10
0
6
9
6
3
11
5
4
7
15
18
20
0
0
37
56
93
100
3
13
48
73
96
97
5
35
59
85
99
100
49
79
95
96
100
100
24
66
74
97
100
100
40
88
89
96
100
99
3
5
5
6
30
40
51
61
7
22
7
10
4
14
58
69
90
100
100
Dose
(mg)
No. of
animals
15
20
25
30
40
50
15
20
25
30
40
50
15
20
25
30
40
50
15
20
25
30
40
50
15
20
25
30
40
50
15
20
25
30
40
50
3
3
5
6
6
3
3
4
6
5
5
6
8
8
8
8
7
4
8
11
9
12
7
7
7
7
8
8
7
7
15
20
25
30
40
50
5
8
6
68
7
7
78
* Frequency of resorption in control animals: 6-10 %.
Cleft palate development
217
between days 10:22 and 11:12 of gestation. Before day 10:22, much higher
doses are required to produce maximum frequency of cleft palate. Wilson (1957)
has suggested that specific and reproducible effects are best seen when minimally effective doses are used. Higher doses not only increase the incidence but
also the type and severity of defects produced. Therefore, the appropriate dose
to induce cleft palate is one which causes highest frequency of defects in the
fetuses with little or no fetotoxic effect.
| f e Resorption | ^ ^ ^ E E ^
ar^sriHi
I sorption
probability
WMNMNMOM
001-010
10:12
:14
:18
:20
:22 11:00 :02 :04
Gestation period (days: hours)
:06
:08
Fig. 2. The probability of resorption of hamster fetuses in relation to the dose and
the gestational time of hydrocortisone administration.
The frequency of resorption (Table 1) was high when doses of 30, 40 and
50mg were administered at days 10:12 and 10:20 of gestation. However,
similar doses between days 11:00 and 11:12 did not cause undue fetal resorption. An exception was observed with doses of 40 and 50 mg at day 11:08 and
with 20 mg at day 11:12 of gestation when the incidence of fetal resorption
was high. The statistical relationship between doses and times to the frequency
of resorption are summarized in Fig. 2. When 34 mg hydrocortisone is administered after day 11:03 of gestation (S.E. ± 2-5) the fetotoxic effect is minimal.
Before day 10:22 the probability of resorption is dose-dependent, but thereafter
the probability decreases.
Cleft palate was of two types: complete, and incomplete or partial. Complete
cleft palate involved the length of the secondary palate (Fig. 3). The palatal
shelves of complete cleft palate were horizontal in position although the gap
between them varied.
218
R. M. SHAH AND A. A. TRAVILL
Cleft palate development
219
The incomplete or partial clefts varied in morphology: some involved only
the anterior part of the secondary palate, the rest being fused (Fig. 4); others
involved a combination of cleft of anterior and posterior parts of the secondary
palate with a variable length of fusion in the middle (Fig. 5). Occasionally a
partial cleft involved only the soft palate (Fig. 6).
As shown in Table 2, the incidence of partial cleft palate increased as the
doses of hydrocortisone were reduced. Further analysis of morphologically
different partial cleft palate indicates that the frequency of combined cleft of
anterior and posterior third of secondary palate increases as the hydrocortisone doses are increased. This is in contrast to the frequency in cleft of the
anterior third of the secondary palate which is inversely related to the dose of
hydrocortisone.
Average fetal weight following hydrocortisone administration was less than
controls (P < 0-01, Table 3). Fetal weight was less with 25-50 mg hydrocortisone than with 15 mg on any day (P < 0-05), and less with 15, 20, 25 and
30 mg on day 11:00 than at any other time between days 10:12 and 11:12 of
gestation.
DISCUSSION
The present study confirmed the earlier observation of Fraser & Fainstat
(1951), Ingalls & Curley (1957), Chaudhry et al. (1966, 1967) and Dostal &
Jelinek (1971 a) that both the time of glucocorticoid administration and the dose
are important factors in the production of cleft palate. On each gestational day,
the frequency of cleft palate decreased as the dose of hydrocortisone was
reduced from 50 to 25 mg. However, further reduction in dose to 20 and 15 mg
resulted in narrowing of the sensitive period. Using several teratogens in
different strains of mice, Gebhardt & Schade (1969) and Dostal & Jelinek
(1971a) had made similar observations, suggesting that not only the frequency
but also the sensitive period to induce cleft palate in experimental animals is
dose-dependent.
FIGURES
3-6
Ventral view of hydrocortisone-induced cleft palate in hamster fetuses at term. Both
the lower jaw and the tongue have been removed.
Fig. 3. Complete cleft of the secondary palate following 30 mg hydrocortisone on
day 11:00 of gestation. The cleft does not involve the primary palate (P). x 10.
Fig. 4. Partial cleft of the secondary palate following 15 mg hydrocortisone on day
11:00 of gestation. The cleft is present only in the anterior third of the palate (arrow).
xlO.
Fig. 5. Partial cleft of the secondary palate following 25 mg hydrocortisone on day
11:00 of gestation. The cleft (arrows) is present in both the anterior and the posterior
thirds of the palate with fusion in the middle, x 10.
Fig. 6. Partial cleft of the secondary palate following 30 mg hydrocortisone on day
10:20 of gestation. The cleft is present only in the soft palate (arrow), x 10.
220
R. M. SHAH AND A. A. TRAVILL
Table 2. Incidence of induced fetal complete and partial cleft palate following
administration of different doses of hydrocortisone to pregnant hamsters at various
times during gestation
Gestation
period
(day:h)
10:12
10:16
10:20
11:00
11:04
11:08
11:12
Dose
(mg)
No. of
fetuses with
cleft palate
Complete
cleft of
palate
25
30
49
50
15
20
25
30
40
50
15
20
25
30
40
50
15
20
25
30
40
50
15
20
25
30
40
50
15
20
25
30
40
50
15
30
40
7
1
5
28
36
52
54
5
31
48
60
60
20
46
100
101
128
69
70
16
51
63
80
76
75
21
76
75
65
55
50
0
3
9
7
0
0
2
7
24
25
0
0
13
18
19
20
18
35
54
122
69
70
0
9
18
75
76
75
1
1
3
17
32
37
15
20
25
30
40
50
4
23
35
55
68
78
0
1
0
4
9
25
Partial cleft palate
A
^
Total
A*
APf
15
27
31
0
1
5
26
29
28
29
5
31
35
42
41
0
15
22
12
0
1
5
21
16
16
14
5
31
17
38
12
0
26
60
37
3
0
0
0
2
16
0
0
0
5
13
12
15
0
0
17
2
28
0
2
5
17
3
0
0
0
2
15
4
0
0
0
1
12
18
13
13
0
0
0
0
0
0
20
75
72
48
23
13
16
40
30
1
0
0
20
74
60
30
10
0
4
22
35
51
59
53
4
21
21
34
29
13
0
1
14
17
30
40
0
0
0
0
0
0
28
65
47
6
0
0
16
42
45
5
0
0
* Cleft of the anterior part of the secondary palate.
f Cleft of the anterior and posterior part of the secondary palate.
f Cleft of the posterior part of the secondary palate.
n0
3
3
0
0
0
0
0
0
0
0
0
1
2
1
0
0
0
0
0
0
0
0
0
0
0
0
0
Cleft palate development
221
Following administration of 15, 20 and 25 mg hydrocortisone, cleft palate
incidence showed two peaks. Although the exact significance of the biphasic
nature of cleft palate frequency is not yet clear, it may be that there are at least
two critical events in palatal development that are inhibited by hydrocortisone.
A similar biphasic nature of cleft palate had been observed by Russell & Russell
(1954) following X-ray exposure of mouse fetuses and by Kocchar (1973) and
Shenefelt (1972) following retinoic acid treatment of mouse and hamster
Table 3. Average fetal weight {mg) following administration of various
doses of hydrocortisone at different gestation period*
Gestation
period
(days:h)
10:12
10:16
10:20
11:00
11:04
11:08
11:12
Dose (mg)
15
20
25
—
1519
1369
1471
1427
1133
1537
1367
.1217
1140
1468
1113
1737
1340
1132
1661
1312
1120
1467
1669
1290
* Average weight of control fetuses at
30
40
1184
0891
1094
0918
1130
0991
1090
1090
1132
1130
1105
0991
1200
1200
term = 2250 mg.
50
0810
0842
0969
.1063
1044
0922
0991
fetuses. On the other hand, using different glucocorticoids, neither Gebhardt &
Schade (1969) nor Dostal & Jelinek (1971a) observed two peaks of cleft palate
frequency in mouse fetuses.
Partial or incomplete cleft involving only the anterior third of the secondary
palate has been observed earlier in rat fetuses by Giroud & Martinet (1956) and
Buresh & Urban (1964) and in mouse fetuses by Dostal & Jelinek (1971a)
following treatment with different teratogens. Dostal and Jelinek suggested that
such partial cleft palates were not related to the dose of teratogen. In the
present study, however, we observed that with decreasing doses of hydrocortisone the relative incidence of partial cleft palate increased. Furthermore,
several morphological variations of the partial cleft palate were observed
(Figs. 4-6), related both to dose and to gestational time. It may be that some
level of maternal serum hydrocortisone needs to be maintained for several
hours, during a specific interval, in order to produce a particular type of cleft
palate.
From their study, Shah & Chaudhry (1974) have shown that normal closure
of the hamster palate starts in the middle and posterior thirds of the secondary
palate, extends to the soft palate and finally to the anterior third of the secondary
palate. It seems that small teratogenic doses of hydrocortisone selectively
inhibit palatogenesis in hamsters, thereby causing various types of clefts. The
fact that different fetuses within the same litter display individual differences in
222
R. M. SHAH AND A. A. TRAYILL
their developmental stages (Shah & Travill, 1975) may explain differences in
types of cleft with the same teratogenic dose given at the same gestation period.
Also the middle and posterior thirds of the hard palate, the soft palate and the
anterior third of the hard palate (where cartilaginous nasal septum is involved
during normal closure) have different types of adult tissue, and may therefore
differ in their growth rates, differentiation and sensitivity to environmental
agents.
Fraser & Fainstat (1951) and Chaudhry et al. (1967) observed a close correlation between fetal birth weight and cleft palate in cortisone-treated pregnant
mice. A similar observation was made in hamster fetuses in the present study.
Furthermore, fetal growth retardation was associated with the dose, the time
of administration and the type of cleft palate, suggesting perhaps that the
duration of the growth-inhibitory action of hydrocortisone (which in turn
depends on the latter's metabolism in the pregnant mother and on the placental
barrier) is a significant factor in cleft palate induction. We therefore suggest
that the growth-inhibitory effect of larger doses of hydrocortisone is faster and
of longer duration than that of smaller doses.
Cleft palates in hamsters resemble morphologically those in humans to a
certain extent. A complete cleft extending through the length of the secondary
palate was produced with great reliability following hydrocortisone administration. Although partial cleft of the soft palate was observed only occasionally,
other types of partial palatal clefts were produced in high frequencies at certain
dosages and gestation periods. Submucous palatal clefts were not observed in
hamster fetuses at any time, though such clefts have been recognized in humans.
Since experiments in mice, rabbits, rats and hamsters have consistently shown
that glucocorticoid administration during pregnancy was associated with
production of cleft palate, it is possible that glucocorticoid may be teratogenic
to human fetuses. Several cases of malformations, including cleft palate, in
human fetuses following glucocorticoid therapy have been recorded in the
literature (Harris & Ross, 1956; Doig & Coltman, 1956; Bongiovanni &
McFadden, 1960; Popert, 1962; Warrel & Taylor, 1968). However, Woollam
& Millen (1960) and Tuchmann-Duplessis (1970) have expressed caution
against extrapolation of results from animal experiments to man.
The authors wish to express their gratitude to Mr L. Broekhoven for statistical analysis of
the data, to Miss V. Beretanos and Miss J. Cann for their secretarial assistance, to Mr R.
Paton, Mr H. Verstappen and Mrs Judy Nairn for their skilful photographic and technical
assistance and to the Medical Research Council of Canada for the support of this research.
Cleft palate development
223
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(Received 5 September 1975, revised 14 October 1975)