/ . Embryol. exp. Morph. Vol. 57,pp. 119-128, 1980
Printed in Great Britain
Morphological study of cleft palate development
in 5-fluorouracil-treated hamster fetuses
By RAVINDRA M. SHAH 1 AND DAVID T. W. WONG
From the Department of Oral Biology, Faculty of Dentistry,
The University of British Columbia
SUMMARY
Morphogenesis of palate was studied in normal and 5-fluorouracil-treated hamster fetuses.
The results showed that normal palatal development was completed between days 12 and
13 of gestation. In 5-fluorouracil-assaulted palate the reorientation of shelves from a vertical
to horizontal plane was delayed. Crown-rump length, gestational age and fetal weight were
reliable predictors of the stages of normal palatal development, whereas the morphological
rating system was not. Following 5-fiuorouracil treatment, however, crown-rump length,
weight and morphological rating were poor indicators of the stage of palatal development.
INTRODUCTION
Since the publication of Baxter & Fraser's study (1950) on cortisone-induced
cleft palate in the mouse fetus, the relative significance of environmental
factors in cleft palate development has been repeatedly recognized. Numerous
chemicals which produce cleft palate in the different laboratory animals have
been identified (Kalter & Warkany, 1959; Cohen, 1964; Dagg, 1966; Shah,
19796). However, detailed morphological analysis of cleft palate development
has been reported only in a few instances, i.e. X-ray, Vitamin A and glucocorticoid treatment, (Walker & Fraser, 1957; Walker & Crain, 1960; Callas &
Walker, 1963; Kochhar & Johnson, 1965; Dostal & Jelinek, 1972; Shah &
Travill, 19766; Shah, 1979a). Such studies contributed significantly toward
understanding the mechanism of drug-induced malformations. For example,
cortisone treatment of pregnant mouse seems to affect the reorientation stage
of palatal development (Walker & Fraser, 1957) whereas hydrocortisone assault
of pregnant hamster appears to interfere with the fusion stage (Shah & Travill,
1976 a, b,).
A growth suppressive agent, 5-fluorouracil (5-FU), when injected into
hamster on day 11 of pregnancy, induces cleft palate in all the fetuses (Shah &
MacKay, 1978). The drug is a pyrimidine analogue and it is suggested to
1
Author's address: Department of Oral Biology, Faculty of Dentistry, The University
of British Columbia, Vancouver, British Columbia, Canada V6T 1W5.
120
R. M. SHAH AND D. T. W. WONG
effect the synthesis of ribonucleic acid (Bosch, Herbers & Heidelberger, 1958),
and deoxyribonucleic acid (Ellison, 1961).
The mechanism by which a malformation is induced may vary, depending
upon, among other factors, the chemical nature of drugs used. It is, therefore,
reasonable to compare the mechanism of cleft palate formation by 5-FU with
other teratogens. With the exception of a histological study in mice (Arvystas
& Cohen, 1971), a sequential morphological observation of 5-FU-induced
cleft palate development is not reported in any species.
The present study reports morphogenesis of 5-FU-induced cleft palate in
hamster fetuses.
MATERIALS AND METHODS
Golden Syrian hamsters (Simonson Laboratory, California) weighing approximately 100 ± 5 g were individually caged and maintained for at least one week
in 50 ± 5 % humidity, 34 ± 1 °C temperature and alternate cycles of light
(6.00 a.m. to 6.00 p.m.) and dark. The food and water was allowed ad libitum.
Following environmental conditioning, males and females were mated from
7.00 to 9.00 p.m. The midpoint of the mating period was taken as day 0 of
pregnancy.
Each pregnant animal was treated with a single intramuscular injection of
either 81 mg/kg 5-FU in saline or 1 ml of saline on day 11 of pregnancy.
Fetuses were obtained at 6 h intervals between days 11:06 (eleventh day and
sixth hour) and 13:00, and thereafter at 24 h intervals until day 15:00 of
gestation. They were immersed in Bouin's solution for 24-36 h for fixation
and then dehydrated in ascending concentration of ethyl alcohol. Upon reaching
70% alcohol, the fetuses were measured for crown-rump length (CRL),
individually weighed, and examined for the development stage of the palate,
limbs, eyes and ears as described by Walker & Crain (1960). Subsequently, they
were processed for histological examination. Serial sections of 7/tm were
obtained and stained with hematoxylin and eosin.
Palatal development was staged according to Shah & Travill (1976/?) with
one exception - stages 5 and 6 were combined and considered as stage 5. Two
criteria of general fetal growth, weight and crown-rump length, were subjected
to linear regression analysis. The staged development of palate was analysed
in relation to CRL, weight, gestational age and according to numerical system
of morphological rating as described by Walker & Crain (1960). For statistical
evaluation of the data on palatal development, standard normal curves
illustrating the probability of each palatal stage in relation to CRL, weight,
gestational age and morphological rating were obtained. To determine the
fitness of the curve, the 50 % point on the curve was evaluated for significance
by x2 statistics. The details of statistical methods are described by Berkson
(1957).
5-Fluorouracil-treated cleft palate
121
RESULTS
The gross and histological appearances of different stages of normal and
abnormal palatal development in hamsters have been described and documented
(Shah & Chaudhry, 1974; Shah & Travill, 19766), and are not repeated in
this report.
General fetal growth
Observations on two criteria of fetal growth, i.e. weight and CRL, are
presented in Table 1. One may deduce from the table that between days 11:06
and 15:00 the CRL and weight of both the control and treated fetuses increased
progressively. Statistical analysis of mean weight data showed that the regression
line is expressed by the equation Y = - 310-89 + 21-05X in control fetuses
(where X is number of hours from the time of treatment and Y is mean fetal
weight in mg), and by Y = -11-592+ 10-32Zin treated ones. When the weight
data are analysed during the period of palatal closure, i.e. between days 11:06
and 13:00 of gestation, the regression line is expressed by the equation Y =
-8-85 + 8-93Xin control fetuses and Y = 43-24 + 3-51X in the treated fetuses.
In both instances there was a significant difference between the two regression
lines (P < 0-001) indicating that 5-FU causes cleft palate and retards weight
gain over the same period.
Similar analysis of CRL between days 11:06 and 15:00 indicated that the
regression line is expressed by Y = 7-20 + 0-19^ in control fetuses (where
Xis the number of hours from the time of treatment and Fis mean crown-rump
length), and by 7 = 7-26 + 0-13X in the 5-FU-treated ones. An analysis of
CRL data during the period of palatal closure showed that the regression line
in control fetuses is expressed by the equation Y = 8-29 + 0-15A" and in the
treated ones by Y = 8-46 + 0-079X In both instances there was no significant
difference between the two regression lines. After day 12, however, the mean
CRL of the 5-FU-treated fetuses was significantly lower than control ones
(P < 0001) (Table 1), thus indicating that 5-FU causes cleft palate and retards
CRL gain during the period of palatal closure.
Gestation age and palatal development
Control data on the morphological stages of fetal palatal development in
relation to chronologic age are summarized in Table 2. Stage 2 (reorientation
of the shelves from a vertical to a horizontal position), stage 3 (horizontal
shelves) and stage 4 (approximation of shelves) of the palatal development
were accomplished in almost all fetuses by day 12:06 of pregnancy. In approximately 95 % of fetuses, stage 5 (closure of the middle and posterior third of
the hard palate) was achieved by day 12:12 and stage 6 (closure of the soft
palate) by day 12:18 of pregnancy. Finally stage 7, indicating closure of the
122
R. M. SHAH AND D. T. W. WONG
Table 1. Average weights and crown-rump lengths of control and 5-fluorouracil-treated
fetuses at different times during gestation
Gestation Number of fetuses
,
period
(days: hours) Control Treated
11:06
11:12
11:18
12:00
12:06
12:12
12:18
13:00
14:00
15:00
60
66
66
64
49
64
55
59
59
62
51
58
58
52
51
55
54
52
41
37
Mean crown-rump length
(mm)±s.D.
A
c
Mean fetal weight (mg) ± S.D.
r
Control
Treated
Control
Treated
9-56±0-55
10-32±0-59
10-72 ±0-55
ll-52±0-57
12-94 ±0-60
131O±O-81
13-83 ±1-09
16-58 ±104
21-12 ±1-70
26-85 ±2-03
9-54 ±0-47
9-49 ±0-59
9-45 ±0-52
9-51 ±0-67
ll-3O±O-53
11-97±M2
11-71 ±1-10
12-71 ±1-51
1805 ±2-74
20-03 + 2-84
8805 ±14-5
115-59± 15-88
134-63 + 22-43
175-69 ±25-22
231-95 + 30-81
272-92 ±49-80
324-12 ±65-20
514-58 ±7810
1023-76± 185-89
2093-99 ±296-11
90-68 ±1M4
91-54 ±12-93
91 07 ±17-66
95 06 ±20-70
160-55 + 22-35
157-97 ±46-52
194-31+ 58-74
236-50± 75-87
728-40 ±258-23
955-83 ±455-74
anterior third of the secondary palate was accomplished in almost all the
fetuses by day 13 of pregnancy.
An analysis of standard normal curve for probability of palatal shelves in
stage 2, along with shelves that had completed stage 2, revealed that 50 % of
fetuses had achieved this stage by day 11:23 of pregnancy (S.D. 2-22; x2 8-09;
P < 0-05). Similarly 50% point for stage 3 on a standard normal curve was
at day 12:00 (S.D. 1-29; # 2 2-56; P < 0-1). For subsequent stages the 50%
points on the curves were: stage 4 - 12:04 (S.D. 3-2; x2 2-39; P < 0-1); stage 5 12:06 (S.D. 4-25; ,Y2 3-68; P < 0-1); stage 6-12:14 (S.D. 1-75; x2 H 4 ; P < 0-1)
and stage 7 - 12:20 (S.D. 2-46; x 2 3-98; P < 0-1).
In 5-FU-treated fetuses (Table 2) stages 2 and 3 were accomplished in most
fetuses after day 13:00 of pregnancy, i.e. after the closure was completed in
the control fetuses. On day 14:00, 78% of the fetuses had horizontal palatal
shelves (stage 3) and 17% approximated (stage 4). Subsequently, however, the
palate did not close and at day 15 all the shelves remained horizontal. The
standard normal curves for stages 2 and 3 showed that 50 % of fetuses were
in this stage or had completed the stage by day 12:21 (S.D. 2-7; x2 4-07; P < 0-1).
Fetal weight and palatal development
Data on the relationship between the fetal weight and the stages of palatal
development are presented in Table 3. One may deduce that stages 2-5 are
accomplished in all control fetuses weighing 250 mg; stage 6 in all fetuses
weighing 400 mg and stage 7 when the weight reaches 450 mg. The 50 % point
on the standard normal curve for stage 2 was at 209 mg (S.D. 50; # 2 0-35;
P < 0-1). The corresponding points for subsequent stages were: stage 3 239 mg (S.D. 50; A'2 2-04; P < 0-1); stage 4 - 2 4 4 mg (S.D. 64; x2 H ; P < 0-1);
stage 5 - 294 mg (S.D. 64; x2 4-32; P < 0-1); stage 6 - 329 mg (S.D. 79; x2 0-81;
P < 0-1) and stage 7 - 409 mg (S.D. 50; x2 0-75; P < 0-1).
123
5-Fluorouracil-treated cleft palate
Table 2. Staged development of control and 5-fluorouracil-treated hamster
fetal palate in relation to gestational age
Number of fetuses at: the stage of palatal development
r
f rpc^Qtinn
vJCMallUll
rent,id
Control
Treated
T^pn^
(days: hours)
11:06
11:12
11:18
12:00
12:06
12:12
12:18
13:00
14:00
15:00
A
1
2
—
—
—
2
—
—
1 —
— —
— —
— —
60
65
66
39
—
—
7
1
2
3
4
— .— — — —
51
57
58
52
44
50
46
27
1
2
—
1
—
—
4
1
3
—
1
—
—
—
—
—
1
4
4
19
32
34
—
—
—
—
2
—
1
6
7
1
3
—
—
4
1
—
6
2
-—
-—
-—
.—
—
•
1
2
2
—
—
—
—
•
5
—
—
16
41
54
3
—
—
—
6
—
—
—
2
8
31
1
—
—
—
—
—
—
—
20
58
59
62
5
6
7
——
——
——
——
——
——
——
——
——
——
—
—
—
—
—
—
—
—
—
—
Table 3. Staged development of control and 5-fluorouracil-treated
hamster fetal palate mi relation to fetal weight
Number of fetuses at: the stage of palatal development
Control
Fetal Weight
(mg)
50-100
101-150
151-200
201-250
251-300
301-350
351-400
401-450
451-500
501-550
551-600
601-650
651-over
Treated
A
f
1
59
125
47
—
—
—
—
—
—
—
—
—
—
2
3
4
5
6
7
— — .— — —
2
— —
1 — —
2
1
6 23 1
1 5
— 40 4
1
1
—
2 34 14 —
—
— 17 17
7
— —
—
6 11
— — —
—
9
—
— — — 16
—
— — — 22
—
— — — 14
— — —
—
5
—
— — — 109
—
•
—
—
.
—
•
—
•
—
•
—
•
—
•
—
•
—
•
A
t
1
166
106
76
29
9
—
—
—
—
—
—
—
—
2
4
—
—
1
— — —
3
6 4
3 11 —
— 12 2
2
1 4
—
5 2
—
3 —
—
1
3
— 4 —
— 2 —
2
—
2
— 43
5
3
5 6
——
——
——
——
——
——
——
——
——
——
——
7
—
—
—
—
—
—
—
—
—
—
—
—
—
Following 5-FU administration, stage 2 was accomplished in all the fetuses
weighing 301 mg, and stage 3 in all the fetuses reaching 351 mg. The 50%
point on the standard curve for stage 2 was at 294 mg (S.D. 64; x2 0-23; P < 0-1)
and for stage 3 - at 329 mg (S.D. 79; f 0-31; P < 0-1).
Morphological rating and palatal development
The relationship between the morphological rating and stages of normal
palatal development is shown in Table 4. Stages 2-4 are accomplished in
80% of fetuses when the morphological rating is between 3 and 4; stage 5 in
124
R. M. SHAH AND D. T. W. WONG
Table 4. Staged development of control and 5-fluorouracil-treated
hamster fetal palate in relation to morphological rating
Number of fetuses at the stage of palatal development
Treated
Control
Morphological
rating
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
r-
A
1
2
3
4
5
5
57
156
10
3
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
2
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
3
1
1
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
1
4
2
2
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
1
14
7
6
7
—
—
—
—
2
34 —
25
3
—
—
—
—
—
—
—
17 2 —
12 18 1
4
—
—
—
—
—
—
—
—
—
—
—
—
—
A
1
45
140
159
26
2
10
1
—
—
2
1
5
6
3 9
—
3
— 2
—
2
— 12
—
7
— 16
— 101
—
5
— 14
—
7
— 12
1
—
2
—
—
—
1
—
—
—
—
—
—
9
2
3
— —
—
1
4
1
— —
1 4
4 7
1 4
—
3
—
6
8
—
—
2
—
1
—
3
—
1
—
1
—
3
—
6
—
2
— —
— —
—
3
— 25
— 13
4
—
—
—
—
1
1
—
—
—
—
—
—
—
2
3
2
2
—
—
—
1
5
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
c
—
—
6
7
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
__
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
Table 5. Staged development of control and 5-fluorouracil-treated
hamster palate in relation to crown-rump length
Crown-rump ,
Length (mm) 1
U-10
10-11
11-12
12-13
13-14
14-15
15-16
16-17
17-18
18-19
19-20
20-over
Number of fetuses at the stage of palatal development
,
*
^
Control
Treated
A
A
*
,
2 3 4 5 6 7
1 2 3 4 5 6
66 —
115 —
48
2
—
—
_
—
_
_
—
—
2
—
—
—
_
—
_
_
—
—
—
—
—
—
—
—
— —
— —
3 9 20 1
2 — 40 8
— — 51 25
— —
3 8
_ _ _ _
— — — —
_ _ _ _
_ _ _ _
— — — —
— — — —
—
—
2
13
14
28
22
6
6
108
205
75
69
36
2
1
—
—
—
—
—
— —
—
2
7 3
1 7
1 9
1 12
—
6
—
3
—
8
—
8
7
—
29
>
7
— —
— —
—
2
5
2
—
2
—
1
1
—
—
—
—
4 —
—
—
5-Fluorouracil-treated cleft palate
125
92% of fetuses when the rating is 5; stage 6 in all fetuses when the rating is
10; and stage 7 in all fetuses when the rating is 12.
A standard normal curve analysis revealed that 50 % point on the curve for
stage 2 corresponded to a morphological rating of 2-79 (s.D. 1-87; /\/22#48;
P < 0-1). For subsequent stages the ratings were: stage 3-2-90 (s.D. 1-0;
X22-13; P < 0-1); stage 4-2-97 (s.D. 1-58; x2197; P < 0-1); stage 5-3-12
(s.D. 2-45; x2 9-33; P < 0-1); stage 6 - 7-69 (s.D. 2-45; x2 2-14; P < 0-1); stage 7
- 11-09 (s.D. 1-58; x2 0-36; P < 0-1).
Following 5-FU treatment (Table 4), stage 2 was accomplished in most
instances at the rating of 6. Subsequently, almost all the palate remained in
stage 3. The 50 % point on the standard curve for stage 2 was at 5-49 (s.D. 1-87;
X22-76;P < 0-1) and for stage 3 at 5-79 (s.D. 2-16; x2 4-59; P < 0-1).
Crown-rump length and palatal development
The observations on staging of palatogenesis in relation to CRL in control
fetuses are arranged in Table 5. It can be deduced that stages 2-5 are achieved
in 96% of the fetuses measuring 13 mm CRL. Stage 6 is accomplished in
88 % of the fetuses measuring 14 mm, and stage 7 in all fetuses by 16 mm CRL.
The 50% point on the standard normal curve was at 12-19 (s.D. 1; x21*86;
P < 0-1) for stages 2-4. The corresponding point for stage 5 was at 12-89 (s.D.
1-29; x2 3-9; P < 0-1), stage 6 at 13-59 (s.D. 1-58; x2 0-52; P < 0-1), and stage 7
at 15-19 (s.D. l;x2O-27;P < 0-1).
In 5-FU-treated fetuses, stage 2 was accomplished in 88 % of the fetuses
measuring 14 mm, and stage 3 in almost all the fetuses measuring 15 mm CRL.
The 50 % point on the curve for stage 2 was at 13-29 (s.D. 1-87; x2 2-34; P < 0-1)
and for stage 3 at 14-28 (s.D. 1-87; x2 2-40; P < 0-1).
DISCUSSION
Observations of normal palatal development made during the present study
resembled those reported earlier (Shah & Chaudhry, 1974; Shah & Travill,
19766; Shah, 1979a). The usefulness of hamsters in the studies of normal and
cleft palate development is once again confirmed.
Two distinct morphological mechanisms for teratogen-induced cleft palate
development have been proposed in the literature. Earlier morphological
studies in rats, mice and hamsters have shown that cortisone, triamcinolone,
radiation, folic acid deficiency, diazo-oxo-norlucine, high doses of vitamin A
and 5-fluoro-2-desoxyuridine induce cleft palate by delaying transposition of
the palatal shelves from a vertical to a horizontal plane (Walker & Fraser,
1957; Walker & Crain, 1960; Asling et al 1960; Callas & Walker, 1963;
Kochhar & Johnson, 1965; Coleman, 1965; Ross & Walker, 1967; Dostal &
Jelinek, 1972; Ferguson, 1977; Shah, 1979a; Diewert, 1979; Diewert & Pratt,
1979). In contrast, recently hydrocortisone treatment in hamsters has been
S>
EM 13 57
126
R. M. SHAH AND D. T. W. WONG
•shown to induce cleft palate by preventing the epithelia of the opposing
horizontal palatal shelves from fusing (Shah & Travill, 1976a, b). The results
of our present study have shown that 5-FU treatment of pregnant hamsters
produces cleft palate in the fetuses by delaying the reorientation of the palatal
.shelves and thus agrees with the earlier morphological observations.
Both the CRL and weight have generally been accepted as appropriate
criteria to assess the general development of the fetus. In case of normal and
abnormal palatal development these criteria have been used not only to determine whether the palatal clefting is an isolated anomaly or is a part of an
overall growth retardation (Fraser & Fainstat, 1951; Chaudhry, Schwartz,
Schwartz & Schmutz, 1967; Shah & Travill, 1976 c; Shah & Kilistoff, 1976;
Ferguson, 1977; Shah, 1979 a), but also to predict the stages of normal palatal
•development and the stage at which the palatal development is inhibited or
prevented by the teratogenic insult (Callas & Walker, 1963; Walker, 1971;
Dostal & Jelinek, 1972; Shah & Travill, 19766; Ferguson, 1978; Shah, 1979a).
In the present study, 5-FU simultaneously caused cleft palate and inhibited
both the fetal weight and the CRL (Table 1), and thus supported the hypothesis
that induced cleft palate may be related to general fetal growth retardation
rather than being an isolated defect.
These observations are in contrast to an earlier study in which triamcinoloneinduced cleft palate was found to be an isolated anomaly (Shah, 1979 a), since
it was not related to general fetal growth retardation.
The seriation according to length and weight has been successfully used in
determining the sequential steps of normal palatal development (Callas &
Walker, 1963; Walker, 1971; Dostal & Jelinek, 1972; Shah & Travill, 19766;
Ferguson, 1978; Shah, 1979a). The results of the present study, showing that
not only the CRL and fetal weight but also the chronological age is a reliable
criterion for predicting the stages of normal palatal development in hamsters,
thus agree with these studies. In contrast, the numerical system of morphological
rating, originally used by Walker & Fraser (1956) to correlate it with the
stages of mouse palatal development, did not show good correlation with
stages of hamster fetal palatal development. This may perhaps be due to great
variability in the development timings of external features themselves as suggested by Dostal & Jelinek (1972), or due to the relatively fast organogenesis,
and consequently, the shorter period of palatal development, in the hamster
than in mice, or both.
Both the fetal weight and CRL in 5-FU-treated fetuses showed good correlation with the palatal staging on the standard normal curve. These developmental criteria, however, are not useful in analysing morphological mechanisms
•of experimentally induced cleft palate. For example, in control fetuses stages 2-5
•of palatal development were achieved in fetuses weighing 250 mg and measuring
13 mm, but in treated ones of similar weight and length, most of the shelves
"were vertical. Similar observations were also made following hydrocortisone
5-Fluorouracil-treated cleft palate
127
and triamcinolone treatments (Shah & Travill, 19766; Shah, 1979a). One
may, therefore, suggest that in teratogen-treated fetuses, both the CRL and
weight are poor indicators of the stage of palatal development in the hamster.
In brief, the foregoing analysis of observations and discussion clearly suggests that 5-fluorouracil induces cleft palate in the hamster fetuses by delaying
reorientation of the palatal shelves from a vertical to a horizontal plane.
Gestational age, crown-rump length and fetal weight are useful criteria for
predicting stages of normal palatal development while the numerical system
of morphological rating is not. None of the general fetal growth indicators
were useful predictors of stages of experimentally induced cleft palate.
The work was supported by a grant from the Medical Research Council of Canada. We
extend our gratitude to Mrs Margaret Law and Miss Ruth Scheuing for their assistance.
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{Received 2 October 1979, revised 10 December 1979)