/ . Embryol. exp. Morph. Vol. 43, pp. 47-54, 1978
Printed in Great Britain © Company of Biologists Limited 1978
47
Teratological evaluation of 5-fluorouracil and
5-bromo-2-deoxyuridine on hamster fetuses
By R A V I N D R A M. SHAH 1 AND R O B E R T A. M A C K A Y
From the Department of Oral Biology, Faculty of Dentistry,
The University of British Columbia
SUMMARY
A single intraperitoneal injection of 5-bromo-2-deoxyuridine (BUDR) between days 9
and 11 and a single intramuscular injection of 5-fluorouracil (FU) between days 8 and 11
of gestation were teratogenic in hamster. The malformation frequencies were related to the
dose and time of BUDR and F U treatment but the pattern and types of malformations
and their frequencies were different. BUDR showed lower embryotoxic effect than FU.
Both drugs impaired general fetal growth. It is suggested that the differences between F U
and BUDR may be related to the chemical nature and metabolism of the drug and their
eventual effect on the species-specific target tissue.
INTRODUCTION
Cancer chemotherapeutic agents are now recognized for their high degree of
chemical reactivity in biological systems, a property used with good effect in
the treatment of neoplasia (Sieber & Adamson, 1975). The clinical activity of
these drugs, however, also extends to normally proliferating cells. The longterm use of anti-cancer drugs thus makes it imperative to consider their effect
in both the developed and developing tissues.
Two antimetabolite chemicals, a fluorinated pyrimidine 5-fluorouracil (FU),
and a halogenated pyrimidine 5-bromo-2-deoxyuridine (BUDR), were teratologically evaluated in the present study because of their growth-inhibiting
properties. The most important effect of FU is probably interference with
synthesis of DNA by preventing the methylation reaction mediated by thymidylate synthetase which converts deoxyridylic acid to thymidylic acid (Ellison,
1961). The only other reported action of FU is indirect inhibition of RNA
(Bosch, Herbers & Heidelberger, 1958). The effect of BUDR is not precisely
understood (Golbus & Epstein, 1974) although incorporation of BUDR into
DNA leads to defective cytodifferentiation (Wessell, 1964; Tencer & Brächet,
1973; Lee, Deshpande & Kalmus, 1974; Sala & Conte, 1975).
The teratogenic effects of FU have been noted by Karnofsky, Murphy &
1
Author's address: Department of Oral Biology, Faculty of Dentistry, University of
British Columbia, Vancouver, B.C. V6T 1W5, Canada.
4-2
R. M. S H A H A N D R. A. M A C K A Y
48
Lacon (1958) in chick, Dagg (1960) and Forsthoefel (1972) in mouse and
Wilson (1964) in rat. The teratogenic potentiality of BUDR was noted in
hamster by Ruffalo & Ferm (1965) and in mouse by Skalko, Packard, Schwendimann & Raggio (1971). The results obtained following FU and BUDR
treatment of pregnant rodents show distinct inter- and intra-species differences.
The teratogenicity of FU has not yet been evaluated in the hamster, since
Ruffalo & Ferm's (1965) study was done prior to the period of organogenesis
(days 8 to 11 of gestation) (Graves, 1945; Hoffman, 1968). The teratological
effect of 5-BUDR during this period is not yet reported. Numerous reports have
emphasized the significance of teratological drug testing in different species of
animals (W.H.O. Report, 1967; Tuchmann-Duplessis, 1975). The present
study compares the effect of FU and BUDR, administered during the period
of organogenesis, on Golden Syrian hamster fetuses.
MATERIALS AND METHODS
Male and female Golden Syrian hamsters weighing approximately 88 ± 5 g
were caged singly and maintained for at least 1 week in 24 ± 1 °C temperature,
50±5% humidity and alternate cycles of dark (6p.m. to 6 a.m.) and light.
The animals were fed a diet of Purina Chow and allowed water ad libitum.
The estrous cycle was determined by the vaginal smear method (Hoffman,
1968). On the appropriate day of estrus, male and female were mated from 7
to 9 p.m. The midpoint of the mating period was taken as day 0 of pregnancy.
Each pregnant animal was given a single intraperitoneal injection of BUDR
dissolved in saline or a single intramuscular injection of an aqueous solution
of FU, in the doses shown in Tables 1 and 3, respectively. A group of animals
was similarly injected with an appropriate solvent at each time. The treated
hamsters were killed on day 15 of gestation. The uteri were exposed and implantation sites and resorptions recorded. The live fetuses were weighed and
then immersed in Bouin's solution for fixation. The fetuses were subsequently
examined for malformations under a dissecting microscope. The types of malformations observed were similar to those reported following hadacidin treatment (Shah, 1977) and will not be documented again in the present report.
Statistical evaluation of malformation data was carried out by procedures
described earlier (Shah, 1977).
RESULTS
Teratogenicity of 5-bromo-2-deoxyuridine
Fetuses from control animals showed no malformations. A single intraperitoneal injection of BUDR, given to pregnant hamsters between days 9 and 11 of
gestation in doses 50-90 mg, was teratogenic to fetuses (Table 1). The malformation frequency varied with the dose and time of BUDR treatment.
Following treatment on days 9 and 10, the frequency decreased as the dose was
Teratogenicity
ofFU
and BUDR
in Hamster
49
Table 1. Teratogenic effect of a single intraperitoneal injection of
5-bromodeoxyuridine on developing hamster fetuses
Gestational
day of
Injection
8
9
10
11
Dose
(mg)
No. of
animals
No. of
live
fetuses
No. of
Mean
No. of malformed fetal weight (g)|
resorptions* fetuses
±S.D.
0
50
3
2
29
60
3
32
0
0
70
3
1
28
3
80
3
0
20
12
0
90
3
13
14
50
3
15
27
0
12
60
3
30
0
70
3
31
1
29
80
3
30
0
29
90
3
33
0
33
50
3
14
26
3
22
60
3
26
70
3
29
29
80
3
30
30
90
3
28
28
2
50
3
29
2
60
3
3
28
32
0
70
3
9
80
3
0
3
29
0
4
3
90
36
* Frequency of resorption in control an mals: 0-12%.
t Mean weight of control fetuses at term: 2-10 + 0-21 g
1-40 ±0-21
1-48 + 019
1-46 ±0-42
1-81 ±0-25
l-34±0-38
1-64 ±0-27
1-66 ±0-21
1-08 ±0-25
1-41 ±0-24
112±015
1-77 + 0-23
1-32 ±0-27
1-44 ± 0 1 1
1-34 ±0-24
1-27 ±0-27
1-67 ± 0 1 5
1-65 ±0-23
1-54 ±0-29
1-54 ± 0 1 4
1-61 ±0-16
lowered from 90 to 50 mg. The same dosages on day 11 of gestation produced
a very low rate of malformation, unrelated to dose. The drug was not teratogenic
following injection on day 8 of gestation. The overall malformation rate was
highest on day 10 (88 %) followed by day 9 (78 %) and day 11 (13 %).
Treatment with different doses of BUDR on days 9, 10 and 11 of gestation
gave no indication of embryotoxicity (Table 1). The resorption rate of the
embryos, however, was significantly high following 80-90 mg drug on day 8
(P < 0-05), indicating that high doses of BUDR, at the beginning of organogenesis, are toxic to the developing embryo.
With the exception of 80 mg dose, BUDR treatment on day 8 of pregnancy
caused significant reduction in fetal weight (P < 0-05) even though there were
no malformations (Table 1). The weight of malformed fetus was, however,
reduced with 70-90 mg BUDR on day 9 and 60-90 mg on day 10 (P < 0-05).
BUDR treatment on day 11 did not show any significant difference in mean
fetal weight between the control and experimental.
The external malformations induced by BUDR treatment included cleft lip,
cleft palate, micrognathia, short and aplastic tail, limb anomalies such as
50
R. M. SHAH AND R. A. MACKAY
Table 2. Incidence of external malformations following different doses of
5-bromodeoxyuridine injected at various times during gestation
No. of fetuses with developmental defects of
Gestational
day of
injection
8
9
10
11
Dose
(mg)
Total no. of
malformed
fetuses
70
50
60
70
80
90
50
60
70
80
90
50
60
70
80
90
1
15
12
29
29
33
14
22
29
30
28
2
3
9
3
4
A
•\
(
Lower
Lip jaw Gut
1
4
2
28
20
29
0
0
0
0
0
0
0
0
0
0
0
2
0
14
16
31
0
8
9
8
28
0
0
0
0
0
0
0
0
0
1
1
0
0
0
1
0
0
0
0
0
0
Tail Limbs Palate
0
3
0
11
10
32
0
11
9
8
28
0
0
0
0
0
0
0
3
12
9
20
13
20
29
30
28
0
0
9
0
0
1
14
12
29
26
32
12
20
29
29
28
2
3
4
3
4
amelia, phocomelia, micromelia and syndactyly and an occasional herniation
of gut. These malformations are analysed in relation to the dose and time of
drug treatment (Table 2). The malformations coincide with periods of rapid
organogenesis (Graves, 1945; Hoffman, 1968). Malformations induced on day
9, in order of decreasing frequency, were of the palate, lip, lower jaw, tail,
limb and gut. After treatment on day 10, the most common malformations were
of the limb, palate, tail and lower jaw. Following treatment on day 11, defective
development occurred only in palate and limbs.
Teratogenicity of 5-fluorouracil
Teratological effects were observed in fetuses when the mother received a
single intramuscular injection of FU between days 8 and 11 of gestation (Table
3). The malformation rate was related to the dose and time of drug administration. As organogenesis advanced, higher doses of FU were required to produce
malformed embryos. The overall malformation rate was highest on day 9 (78 %).
FU was highly toxic to the embryo between days 8 and 11 of gestation
(Table 3) (P < 0-05). The overall resorption rate was higher following treatment
on days 9, 10 and 11 (40-47%) than on day 8 (20%). There was a positive
correlation between the resorption rate and malformation rate on days 9 and 10.
An effect on fetal weight (Table 3) was observed following FU treatment on
days 8-11 of gestation, but was only statistically significant at high doses.
Teratogenicity
of FU and BUDR
in Hamster
51
Table 3. Teratogenic effect of a single intramuscular injection
of' 5-fiuorouracil on developing hamster fetuses
Gestational
day of
injection
10
II
Dose
(mg)
30
3-5
40
AS
5-0
2-5
3-0
3-5
4-0
4-5
50
50
5-5
60
6-5
70
7-5
80
60
6-5
70
7-5
80
8-5
90
No. of
No. of
No. of
No. of malformed
animals live fetuses resorptions* fetuses
4
3
4
3
1
4
4
4
4
3
3
4
4
3
4
3
3
3
3
4
4
4
4
4
4
39
17
29
11
0
45
43
24
32
0
1
47
39
19
13
8
2
0
28
46
28
31
34
24
34
5
13
14
23
9
2
1
17
12
33
31
1
3
11
33
23
19
36
4
0
7
11
6
18
9
1
0
4
3
0
0
27
22
32
0
1
21
18
19
13
8
2
0
0
6
23
25
34
24
34
Mean fetal
weight (g)f
± S.D.
1-59 ±0-27
1-44±(M1J
1-44 ±0-22 J
1-44 ±0-25 %
0
l-82±0-15
l-35±0-26t
1-23 ±0-28$
1-34±015J
0
0-4
1-68 ±0-22
1-60 ±0-25
1-07 ±0-34$
1-23 ±0-37$
l-25±0-23J
M5±0-07î
0
l-73±0-17
1-74 ±0-19
1-43 ±0-28
1-56 ±0-20
1-59 ±0-26
l-46±0-17J
l-23±0-28J
* Frequency of resorption in control animals = 0-10 %.
f Mean weight of control fetuses at term: 1-91 ± 019 g.
% P < 005 for difference from controls.
Commonest anomalies following FU treatment were those of palate, limb
(amelia, micromelia, phocomelia and syndactyly) and tail. They were related
to the dose and time of drug injection (Table 4). In addition, lower jaw, gut,
eye and brain (exencephaly) were malformed with relatively low frequencies.
DISCUSSION
The teratogenic effect of BUDR observed in the present study differs from
Ruffalo & Ferm's 1965 report. These authors observed 98 % prenatal death rate
with 50-100 mg and 82% malformation rate with 40 mg BUDR on day 8 of
gestation. The observed malformation involved eye, brain and rib. In our study
the resorption rate was 20% and only 1/122 fetuses were malformed at the
comparable dose and time of BUDR treatment (Tables 1, 2). There is, however,
52
R. M. SHAH AND R. A. MACKAY
Table 4. Incidence of external malformations following different
doses of 5-fluorouracil at various times during gestation
No . of fetuses with developmental defect of
A
Gestational
No. of
Dose malformed Brain Eye
Gut
Tail Limbs Palate Lower
day of
injection
fetuses
jaw
(mg)
f
8
9
10
11
3-0
40
4-5
30
3-5
40
50
50
5-5
60
6-5
70
7-5
6-5
70
7-5
8-0
8-5
90
1
4
3
27
22
32
1
21
18
19
13
8
2
0
1
0
0
0
0
0
0
0
0
0
0
0
6
0
23*
0
25*
0
0
34*
0
24*
34*
0
* More than 90 %
0
1
0
0
2
0
2
1
0
1
2
3
0
0
0
0
2
3
2
6
3
2
5
16
0
1
0
1
0
1
8
18
0
0
17
9
0
1
19
19
0
0
13
13
0
0
8
8
0
0
2
2
0
0
0
0
0
0
19
19
0
0
10
10
0
0
34
34
0
0
24
24
0
0
34
34
fetuses; were edematous.
0
2
0
27
22
32
1
0
0
0
0
2
11
0
3
7
15
13
8
2
6
23
25
34
24
34
0
0
3
3
1
0
0
0
4
4
0
0
a basic difference between the two studies. Ruffalo & Ferm injected the drug
intravenously whereas we administered it intraperitoneally. It has been repeatedly
noted that the outcome of a teratogological experiment depends on the route of
drug administration (Fraser, 1960; Wilson, 1973 a).
Treatment of pregnant hamsters with BUDR on days 9,10 or 11 of gestation,
not studied by Ruffalo & Ferm, showed a high malformation and low resorption rate. These observations are similar to those of Skalko et al. (1971) on mice.
The teratogenic effect of FU in hamster was also similar to mice (Dagg, 1960;
Forsthoefel, 1972). A teratogenic dose and time dependency was noted for
both drugs in the present study. By increasing the dose it was possible to extend
the sensitive period following FU (Table 3) and not BUDR (Table 1). With
either BUDR or FU, the types and frequencies of malformations were more
extensive in hamsters than Dagg (1960) and Skalko and associates (1971)
found in mice. These comparisons indicate that hamsters may be more sensitive
to teratogenic assault than mice. In earlier studies from our laboratory, hamster
was found to be teratologically more susceptible than rat to insult with glucocorticoid hormones (Shah & Kilistoff, 1976; Shah & Travill, 1976) and hadacidin (Shah, 1977).
Teratogenicity
of FU and BUDR
in Hamster
53
In the present study, there was a positive correlation between incidence of
fetal death and malformation following FU treatment on day 9 and 10 of
gestation but not after BUDR injection. These latter observations are in
agreement with Skalko and associates (1971) but do not confirm Wilson's (1961)
contention that the rates of intrauterine death and malformations are directly
related. However, Wilson (1973 b) noted that prenatal death and malformation
should be regarded as separate manifestations rather than as different degrees
of reaction to the same type of injury. It seems that the presumptive connection,
if any, between fetal death and malformation may depend on the nature of
the teratogenic agent and other pharmacological parameters such as drug
absorption, metabolism and excretion (Burns, 1971).
The mean fetal weight was reduced by high doses of FU on days 10 and 11
and BUDR on days 8, 9 and 10 of gestation. BUDR treatment on day 8 showed
a significant reduction of fetal weight without malformations. In an earlier
study from our laboratory (Shah, 1977), aberrations were shown to be present
at the cellular and tissue level in fetuses with drug-induced growth retardation,
although no gross malformations were seen.
Experimental induction of concurrent cleft palate and cleft lip and isolated
cleft palate have been reported by many investigators following assault with
different teratogens. In our study, both cleft palate combined with cleft lip
and isolated cleft palate were induced with great reliability in hamster fetuses
following various combinations of dose and time of BUDR treatment (Table 2),
although Skalko et al. (1971) never observed simultaneous occurrence of cleft
palate and cleft lip on BUDR-treated mice fetuses. Prenatal assault with FU
produced only isolated cleft palate in hamster (Table 4), and mouse (Dagg,
1960). These results suggest that the chemical nature of the teratogenic stimulus
and its time-controlled species-specific effect on the target tissue are crucial in
induction of isolated cleft palate and cleft palate combined with cleft lip.
Burston (1959), Poswillo (1974) and Tondury (1976) have emphasized that
isolated cleft palate and cleft palate which occurs with cleft lip are two causally
distinct defects.
In brief, the different teratological effects of the two growth-inhibiting drugs,
BUDR and FU, on hamster fetuses may be related to their chemical nature,
mode of metabolism, and/or eventual effect on the species-specific target tissue.
We wish to express our gratitude to Dr Malcolm Greig of the University of British Columbia's Computing Service for statistical analysis of the data, to Miss V. Beretanos and Miss
S. Lindsey for secretarial assistance and Mrs L. Lee for technical assistance. The research
was supported by a grant from the Medical Research Council of Canada.
REFERENCES
L., HERBERS, E. & HEIDELBERGER, C. (1958). Studies on fluorinated pyrimidines. V.
Effects of nucleic acid metabolism in vitro. Cancer Res. 18, 334-343.
BURNS, J. J. (1971). Pharmacological aspects of teratology. In Congenital Malformations (eds
F. C. Fraser & V. A. McKusick). Amsterdam: Excerpta Medica.
BOSCH,
54
R. M. S H A H A N D R. A. M A C K A Y
BURSTON, W . R. (1959). The development of cleft lip and palate. Ann. R. Coll. Sur. Engl. 25,
225-233.
DAGG, C. P. (1960). Sensitive stages for the production of developmental abnormalities in
mice with 5-fluorouracil. Am. J. Anat. 106, 89-96.
ELLISON, R. R. (1961). Clinical applications of the fluorinated pyrimidines. Med. Clins N.
Am. 45, 677-688.
FORSTHOEFEL, P. F . (1972). The effects on mouse development of interactions of 5-fluorouracil with strong luxoid gene and its plus and minus modifiers. Teratology 6, 5-18.
FRASER, F. C. (1960). Some experimental and clinical studies on the causes of congenital
clefts of the palate and of the lip. Archsfr. Pédiat. 11, 151-166.
GOLBUS, M. S. & EPSTEIN, C. J. (1974). Effects of 5-bromodeoxyuridine on preimplantation
mouse embryo development. Differentiation 2, 143-149.
GRAVES, A. P. (1945). Development of the golden hamster, Cricetus auratus water-house,
during first nine days. Am. J. Anat. 11, 219-239.
HOFFMAN, R. A. (1968). The Golden Hamster. Iowa: Iowa University Press.
KARNOFSKY, D . A., MURPHY, M. L. & LACON, C. R. (1958). Comparative toxicologic and
teratogenic effects of 5-fluro-substituted pyrimidines in the chick embryo and pregnant rat.
Proc. Am. Assoc. Cancer Res. 2, 312.
LEE, H., DESHPANDE, A. K. & KALMUS, G. W. (1974). Studies on effects of 5-bromo-deoxyuridine on the development of explanted early chick embryos. J. Embryol. exp. Morph. 32,
835-848.
POSWILLO, D . (1974). Orofacial malformation. Proc. R. Soc. Med. 67, 343-349.
RUFFALO, P. R. & FERM, V. H. (1965). The embryocidal and teratogenic effects of 5-bromodeoxyuridine in the pregnant hamster. Lab. Invest. 14, 1547-1553.
SALA, M. & CONTE, L. (1975). Effects of 5-bromodeoxyuridine on the development of
Xenopus laevis eggs. I. Early stages of development. Acta Embryol exp. 1, 39-54.
SHAH, R. M. (1977). Effects of prenatal administration of hadacidin, a cancer chemotherapeutic agent, on the developmental of hamster fetuses. J. Embryol. exp. Morph. 39,
203-220.
SHAH, R. M. & KILISTOFF, A. (1976). Cleft palate induction in hamster fetuses by glucocorticoid hormones and their synthetic analogues. / . Embryol. exp. Morph. 36, 101-108.
SHAH, R. M. & TRAVILL, A. (1976). The teratogenic effects of hydrocortisone on palatal
development in hamster. J. Embryol. exp. Morph. 35, 213-224.
SIEBER, S. M. & ADAMSON, R. H. (1975). The clastogenic, mutagenic, teratogenic and carcinogenic effects of various antineoplastic agents. In Pharmac. Basis Cancer Chemotherap.
Baltimore: Williams & Wilkins Co.
SKALKO, R. G., PACKARD, D . S., SCHWENDIMANN, R. N . & RAGGIO, J. F . (1971). The tera-
togenic response of mouse embryos to bromodeoxyuridine. Teratology 4, 87-94.
TENCER, R. & BRÄCHET, J. (1973). Studies on the effects of bromodeoxyuridine (BUDR) on
differentiation. Differentiation 1, 51-64.
TONDURY, G. (1976). Zur genese der lippen-kiefer-haumen-spalten. Z. Kinder-chirug. 19, 5-22.
TUCHMANN-DUPLESSIS, H. (1975). Drug Effects on the Fetus. Acton, Massachusetts: Publishing Sciences Group, Inc.
WESSELL, N. K. (1964). DNA synthesis, mitosis and differentiation in pancreatic acinar cells
in vitro. J. Cell Biol. 20, 415-433.
W.H.O. REPORT (1967). Principles for the testing of drugs for teratogenicity. WldHlth Org.
tech. Rep. Ser. 364, 5-18.
WILSON, J. G. (1961). General principles in experimental teratology. In First International
Conference on Congenital Malformation (ed. M. Fishbein). Philadelphia : J. B. Lippincott Co.
WILSON, J. G. C1964\ Teratogenic interaction of chemical agents in the rat. / . Pharmacol,
exp. Therap. 144, 429-436.
WILSON, J. G. (1973 a). Environment and Birth Defects. New York: Academic Press Inc.
WILSON, J. G. (19736). Principles of Teratology. In Pathobiology of Development or Ontogeny
Revisited (ed. E. V. D . Perrin & M. J. Finegold). Baltimore: Williams & Wilkins Co.
{Received
18 February
1977)