Palate Morphology and Fetal Movements in Mice After DPH BRUCE E. WALKER Anatomy Departmenit, Michigan State Un2iversity, Last Iansing, Michigan 48824

Effects of DPI on fetal development were stuidied using a dose which produced 6Sc6 cleft palate in the CD-1 strain. Genieralized developmental retardationi and diminution of fetal muscular movements occurred at the early fetal stage. Interm ediate stages of palate closure persisted to term, indicating interference with palatine shelf rotation. J Dent Res 58(7):1740-1747, July 1979

Introduction. Diphenylhydantoin (DPH) can be teratogenic both to laboratory animals and to human fetuses.1'2 One of the most common anomalies associated with human use of DPH is cleft lip and/or cleft palate.3?7 High frequencies of cleft palate can be induced with DPH in several strains of mice8'9 and in rats.10 DPH crosses the placenta readily,11 13 and most of it can be recovered, unchanged., in the fetus.14 Thus, cleft palate probably arises from a direct interaction of DPH with fetal cells. This hypothesis is further supported by the failure of several DPH metabolites to produce a significant incidence of orofacial anomalies in mice.15 Among the various known actions of DPH, its effects on folate metabolism and cortisone16'17 have been studied in relation to cleft palate formation. Folinic acid did not provide significant protection against DPH-induced cleft palate in mice.18 When DPH was used in conjunction with cortisone, it did not alter the frequency of cleft palate induced by the latter.9 DPH has numerous effects on the nervous system16'7, and these could logi cally be explored in relation to DPH-induced cleft palate. A link has already been established between cleft formation and the neuromuscular system for certain other teratogens. Both cortisone and triamcinolone reduce the frequency of fetal movements during palate development.19 BarReceived for publication August 1, 1978. Accepted for publication October 18, 1978. This investigation was supported bv National Institutes of Health Research Grant no. DEL02852 frorm the National Institute for Dental Research.

biturates and tranquilizers, because of their effects on the nervous system, were tested for their cleft palate-inducing potential and were found to be teratogenic in mice.20 A proposed role for the fetal neuromuscular system in palate closure is withdrawal of the tongue from between the palatine shelves.21 The shelves must move from a sagittal (vertical) to a transverse (horizontal) plane before fusion can occur,22 and the tongue is an obstacle to this movement.23-25 An alternative hypothesis is that contractile material within the shelves provides the force for the rotation of the palatine shelves.26 In either case, interference with contraction should delay or block the process of shelves becoming horizontal and eventually fusing. Such intermediate stages of palate closure have been demonstrated for a number of teratogens.27'31 If DPH induces cleft palate by decreasing fetal neuromuscular movements, then incomplete palatine shelf rotation should be found in treated fetuses beyond the time of normal palate closure. Secondly, fetal neuromuscular movements should be measurably depressed at the same time. The purpose of this experiment with DPH-treated mice is to test for both of these criteria of neuromuscular involvement.

Materials and methods. Strain CD-1 mice were given free access to water and food.* The mice were kept in darkness from 10 AM to 8 PM for experiments on palate morphology. Ovulation was assumed to occur at 5 PM, so mice were

mated during the afternoon. Mice used for timing fetal movements were kept in darkness from 3 AM to 1 PM. Ovulation was assumed to occur at 10 AM, so matings were conducted in the morning. Females with vaginal plugs were isolated, and

*Wayne Mouse Breeder Blox.

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_+

I1 741 PALA TE MORPHOL OG Y A FTF, R DPI7

Vol. 58 No. 7

pregnancy was determined by palpation 1 1 days later. A total of 138 pregnant mice were injected intraperitoneally with sodium diphenylhydantoin,** 87.5 mg/kg, daily for three successive days starting at 11 days, 22 hours (day 11/22) after the assumed time of conception for experiments on timing fetal movements. Injections were started at day 11/16 for studies on palate morphology. Fetuses for the latter expetiments were collected from 29 mice killed at 2-hour intervals from day 14/14 to day 15/8, from 8 mice killed at day 16/16 to 16/23 and from 18 mice killed at day 19/16. Fetuses were fixed in Bouin's fluid while still within the uterine horns. They were dissected out several days later for weighing, morphological rating,28 and evaluation of palate morphology. Control fetuses were collected from 37 mice killed at 2hr intervals from day 14/6 to day 15/6. Statistical evaluation was performed by converting chronological age to a scale in which day 13/0 equaled zero and each 2hour interval was given a value of one. Weights were coded by class intervals; morphological ratings and palate stages retained their original numerical values. The test of significance for lack of parallelism between two slopes (DPH and control) for various combinations of these measurements was based on the formula

(Goldstein) :32

bl

b

t

1+1

Sy x

SSx

SSx1

The test of significance for inequality of Y on the formula:

intercepts was based

a1,

a2

t~~~ 1

(SSE1 + SSE2 ) \ kl+

n2

4

n1

+_ n2

1

+1

SSxiSSx2/

(SPxy)2

SSE,1

SSyl

S.SX SX1

The 10 DPH-treated and 13 control mice used for timing fetal movements were anesthetized with ether at day 13/22, *

*Parke-Davis.

submitted to spinal cord section between the third and fourth lumbar vertebrae, then allowed to recover from the ether.19 Anesthetization below the mid-thoracic level was confirmed, and the mouse was partially submerged in a special fluid medium24 maintained at 370C. The uterine horns were delivered into the fluid medium through an abdominal incision. The uterine wall was cut open to expose the fetus and its membranes. Movements of each fetus were timed with a metronome for five minutes by observing through the amnion. Then, the amnion was removed and the observations were repeated for another five minutes. Seconds of movement were recorded on a laboratory counter separately for tongue, jaw, neck, limbs, and trunk, then summated. Only fetuses with heartbeat were timed, and each was fixed in Bouin's fluid for subsequent weighing and morphological rating. The chronological age of these fetuses ranged from day 14/0 to day 14/4. Among the 67 DPH-treated mice not used in the above experiments, 40 died from the effects of the drug, 19 aborted, and 8 died during spinal cord operations.

Results. Forty-three DPH-treated fetuses collected at day 19/16 had fused palates (stage 7) and 80 fetuses had open palates (stages 1 through 4), giving a cleft palate frequency

of 65%. Among DPH-treated fetuses collected at day 16/16 to 16/23, 55 had open palates and 25 had fusing-to-fused palates, so the cleft palate frequency in this group was 69%. Fetuses collected at earlier time intervals fell within the range of development involving shelf rotation. Table 1 shows the distribution of palate stages in relation to chronological age for control fetuses. Stages of palate closure prior to fusion occurred only up to day 14/20, and all palates were completely fused at days 15/4 and 15/6. In contrast, DPH-treated fetuses showed a full range of palate stages through'out day 14, and vertical palatine shelves were still plentiful at day 15/8 (Table 2). The conclusion that some DPH embryos start the process of palate closure at the normal time, whereas others lag behind, can be supported

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1 74 2

JDent Res July 1979

WALKER TABLE 1 NUMBER OF CONTROL FETUSES AT EACH PALATE STAGE FOR CHRONOLOGICAL AGES O1 14/6 TO 15/8

Palate

Stag,e 1 2 3 4 5 6 7

Chronological Age (days/hours) 14/6 14/8 14/10 14/12 14/14 14/16 14/18 14/20 14/22 15/0 15/2 15/4 15/6 15/8 30

12

1 3

2 4 1 10 1

2

2 2 5

25 1 2

3 4 20 3

1

9 15

1

5 1 5

4

4

2

4

3

3 6

1

17 1

25 20

10 24

4 31

1 6 7

1 30

21

26

TABLE 2

NUMBFR O1 DPH-TREATED FETUSES AT EACH PALATE STAGE FOR CHRONOLOGICAL AGES OF 14/16 TO 15/8 Chronological Age (days/hours)

Palate

Stage 14/6 14/8 14/10 14/12 14/14 14/16 14/18 14/20 14/22 15/0 15/2 15/4 15/6 15/8 35 1

1 2 3 4 5 6 7

32 1 5 6

2

19 3 2

8 3 1 3

5 6

8

5

4 1

1 1

3

15 4

15 1 3 4 5

TABLE 3 NUMBER OF CONTROL FETUSES AT EACH PALATE STAGE FOR MORPHOLOGICAL RATINGS FROM 1 TO 25 Palate

Morphological Rating 12 13 14 15

Stage

1-6

7

8

9

10

11

1 2 3 4 5 6 7

25

7

23

11 2 2

7 1 8 5 2 8

6

1

8 10 8 54

1 1

1 2

1

12 3

statistically by applying the formulae given previously to the data in Tables 1 and 2. There was a non-significant difference in Y intercept (Student's t, 0.48, p>0.50) and a significant difference in slope (Student's t, 3.46, p

Palate morphology and fetal movements in mice after DPH.

Palate Morphology and Fetal Movements in Mice After DPH BRUCE E. WALKER Anatomy Departmenit, Michigan State Un2iversity, Last Iansing, Michigan 48824...
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