The Uptake of 5-Bromodeoxyuridine by the Chicken Embryo and Its Effects Upon Growth

John Bannigan 1, Jan Langman 2, and Alex van Breda 1

1 Department of Anatomy, University College, Earlsfort Terrace, Dublin 2, Ireland z Department of Anatomy, University of Virginia, Charlottesville, VA, USA

Summary. When embryonic cells in vitro are exposed to bromodeoxyuridine (BUdR) the duration of exposure can be m a d e t o last for several cell generation times. Such exposure is known to prevent embryonic cells undergoing terminal differentiation while leaving cell division and basic cell function unaffected. When BUdR is injected into pregnant mammals, it remains available for incorporation in the D N A for only a fraction of one S phase and causes foetal anomalies that are apparently the result of cell death and a transient slowing of the cell generation time but not o f failure of cell lines to differentiate.

The objectives o f our experiments were to ascertain the availability time of BUdR in the chicken embryo in ovo, to assess its teratogenicity and to examine its effects on the growth o f the embryo.

When 3H – BUdR (0.02 rag) was injected into the albumen space on day 3 of incubation, subsequent scintillation spectrometry and autoradiography showed that the drug was incorporated into the D N A of the embryo for more than 8 h or more than one cell generation time at this stage of development. On the other hand, a trace amount o f tritiated thymidine (3H-TdR) was available for only one hour, the difference being probably due to an expansion of the nucleotide precursor pool in the case o f BUdR.

The injection of 0.02mg BUdR on day 3 caused growth retardation as manifested by differences in weight and in D N A content between BUdR and saline treated embryos. The difference in D N A content was evident 24 h after treatment and was probably due in part o f the cell necrosis in the developing CNS that began 10h after injection. Differences in weight did not become apparent until 4 days after treatment and were thus thought to be due to factors other than cell necrosis.

On day 11 of incubation, the mortality o f BUdR treated embryos became significantly greater than that o f controls and many survivors after this time had ventral body wall defects. When treatment was delayed until days 4 or 6, the

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426 J. Bannigan et al

subsequent development of BUdR and saline treated embryos was in-distinguishable, The sensitivity of day 3 was thought to be due to the fact that embryo D N A content quadrupled between days 3 and 4 whereas it only doubled per 24 h period thereafter.

Key words: BUdR – Chicken embryo - Growth - D N A

Introduction

The thymidine analogue 5-bromodeoxyuridine (BUdR) is known to be a potent suppressor of differentiation when applied to embryonic cells in culture (Holtzer et al. 1972; Wilt and Anderson 1972). At the same time it does not appear to affect cell viability, When BUdR is injected into pregnant animals, it causes a variety of fetal malformations (Skalko etal . 1971). The teratogenic effects of BUdR in vivo do not seem to be exerted through an inhibition of cellular differentiation but rather by causing cell degeneration and prolonging the cell generation time (Bannigan and La~gma~ 1979).

When Packard et el. (1973) injected a teratogenic dose o f ZH-BUdR into mice on day 10 of gestation they found that the availability time of the analogue was about 2 – 4 h and the hal~life of the free nucleoside in the embryo was one hour, Since the D N A synthetic (S) phase at this stage of development in the mouse is 5,5 h (Atlas and Bond 1965), ~t is d e a r that no cell will undergo substitution of all its thymine sites with bromouracil (BU). Indeed Packard etal . (1973) calculated that only 2 of thymine sites were substituted in their experiments. This is in contrast to the 80 substitution of thymine sites reported by Stellwagen and Tomkins (1971) and Fabian and Wilt (1973) for cells in vitro where the exposure lasts for periods longer than one S-phase. Agnish and Kochar (1976) cultured mouse embryos in vitro in the presence of BUdR for 24 h and then continued culture of the limb buds alone for a further 9 days. This procedure resulted in a complete suppression ofchondrogenesis in the limbs. However, as the limbs were only inspected grossly for the presence or absence of cartilage these authors could not comment on the cellular mechanisms responsible for the inhibition of chondrogenesis.

It is possible that the chicken embryo in ovo might provide a system for the study of the effects o f BUdR more similar to the cultured embryonic cell system as a dose of BUdR injected into the albumen space might remain available for incorporation for longer periods than in pregnant mammals. However, very little is known about the teralogenic effects of BUdR on the chicken embryo and nothing is known of the fate and dislribution o f BUdR after injection into the environment of the chicken embryo. Therefore, the purpose of this study was to see how tong BUdR remained avaitaNe for incorporation into the D N A of the chicken embryo after injection into the aibumeri space arid to examine what effects this incorporation had on the subsequent growth and gross development of the embryo.

Materials and Methods

Approximately1500 eggs of the white leghornvarietywereused in this study. The eggs weremaintained 80 at a time in a forced draft incubator at 38~C and relative humidity 60-80 %.

BUdR and Chicken Embryo
427

The uptake of BUdR was studied by injecting 15 p.Ci 3H-BUdR (specific activity 27.7Ci/mM, Amersham) mixed with 0.02 mg unlabelled BUdR (Sigma) in normal saline into the albumen of the egg 60 h after incubation had commenced. Prior to injection 10 embryos from each batch of 80 were surface stained by the method of Hilbelink and Kaplan (1978), in order to determine their stage of development. It was consistently found that after 60h of incubation the embryos had attained to stages 14-16 of Hamilton (~952). At hourly intervals ~fter injection up to 24 h embryos were removed from their shells, washed in ice-cold saline, dried on filter paper, weighed and then homogenized separately in :10~oPCA at 4 ~C. The total embryo DNA was then extracted by the Ogur-Rosen melhod as described by Munro and Fleck (1966). At the same time intervals, samples of the albumen and yolk were recovered. Aliquots (0.2 ml) of albumen, yolk and DNA extract were added to 14 ml Aquasol (New England Nuclear) and their radioactivities determined with a Beckman liquid scintillation counter. The concentrations of DNA in the embryo extracts were also measured by the Giles and Myers (1965) modification of the Burton diphenylamin reaction using calf-thymus DNA as a standard. Five eggs were processed in this way every hour after injection.

To determine which cells of the embryo were incorporating BUdR and to compare their number and distribution with that following injection of tritiated thymidine, an autoradiographic method was used. Thus, eggs were injected with a mixture of labelled and unlabelled BUdR as above or with 15 gCi tritiated thymidine (3H-TdR, specific activity 20 Ci/mM, Amersham) dissolved in 0.2 ml normal saline. Every hour for 6 h after injection 5 embryos from each group were recovered and fixed overnight in 2 paraformaldehyde, 2 ~ glutaraldehyde in 0.1 M sodium cacodylate at pH 7.3 (Karnofsky, 1965). After post-fixation in 1 ~o osmium tetroxide and dehydration in a graded ethanol series the embryos were embedded in araldite and sectioned at 1 gm on a Sorvall Porter-Blum MT2B ultramicrotome. The unstained sections mounted on glass slides were dipped in Ilford K 5 emulsion diluted 1 : I with water and exposed at 4~ for 7-10 days. After development they were stained with 2% azure 11.

To see what effects BUdR had on embryonic growth, eggs were injected on days 3, 4, and 6 with 0.02 mg BUdR in 0.2 ml normal saline. This dose was chosen since a pilot study showed that 0.02 mg was Iethal to 100 ~ of embryos within 24 h when injected on day 2 and 0.04 mg was similarly lethal when injected on day 4. Controls received 0.2 ml normal saline. FolIowing injection embryos were recovered at various intervals up to day 17. On removal from their shells they were weighed and inspected for gross anomalies. In addition, on days 3 through 7 their total DNA content was measured as described above.

To see if BUdR could cause cellular changes, some embryos were treated with aH-BUdR plus 0.02 mg unlabelled BUdR and autoradiographs were prepared 10, 24, and 48 h afterwards. Controls for this experiment were injected with 3H-TdR plus 0.0 16 mg unlabelled TdR. The latter dose is the molar equivalent of the BUdR dose.

Results

i) Distribution of BUdR after Injection

B e t w e e n 15 r a i n a n d 1 h a f t e r i n j e c t i o n t h e r a d i o a c t i v i t y o f t h e a l b u m e n h a l v e d in

v a l u e , t h e r e a f t e r it d e c l i n e d m o r e g r a d u a l l y r e a c h i n g n e g l i g i b l e levels b y 18 h

(Fig . 1). A s m a l l a m o u n t o f r a d i o a c t i v i t y a p p e a r e d in t h e y o l k b u t a t o n l y a f r a c t i o n

o f t h e a l b u m e n levels . T h e r a d i o a c t i v i t y p e r m g D N A i n c r e a s e d a t t h e s a m e r a t e as

t h e c o n c e n t r a t i o n o f B U d R in t h e a l b u m e n d e c l i n e d (Fig . 2). T h e c o n c e n t r a t i o n o f

B U d R in t h e D N A p e a k e d a t 8 h a f t e r i n j e c t i o n a n d d e c l i n e d t o r e a c h a s t e a d y l e v e l

at 18 h. S i n c e at this s t a g e o f d e v e l o p m e n t t h e S p h a s e lasts 5 h, t h e G 2 2.5 h a n d

m i t o s i s 0.5 h w i t h a n e g l i g i b l e G 1 ( L a n g m a n a n d H a d e n 1970), it is c l e a r t h a t t h e

B U d R r e m a i n e d a v a i l a b l e f o r i n c o r p o r a t i o n f o r a t l e a s t t h e d u r a t i o n o f o n e S p h a s e .

W h e n t h e p e r c e n t a g e o f l a b e l l e d cells (the l a b e l l i n g i n d e x ) in t h e n e u r o e p i t h e l i u m o f

t h e s p i n a l c o r d w a s c a l c u l a t e d a t h o u r l y i n t e r v a l s a f t e r t h e i n j e c t i o n o f 3 H – B U d R it

was f o u n d to i n c r e a s e b y 1 0 – 1 4 ~ p e r h o u r f o r t h e first 4 h ( F i g . 3). T h u s all cells t h a t

w e r e e n t e r i n g t h e S p h a s e f o r 4 h a f t e r B U d R w a s i n j e c t e d i n c o r p o r a t e d t h e a n a l o g .

T h i s c o n f i r m e d t h e r e s u l t s o b t a i n e d b y d i r e c t m e a s u r e m e n t o f r a d i o a c t i v i t y . B y

428 J. Bannigan et al.

200

s 150

~o 100

x

~ 50

2 Z, 6 8 10 12 14 16 18 20 22 2/.

Hours after injection of 3H-BUdR into albumen

]Fig, 1. Rate of decline of radioactivity in albumen following injection of 3H-BUdR on day 3. The amount of BUdR in the albumen reaches a steady level at 5 h. Each point represents measurements from
5 eggs

< 300
Z

O
o~ 250
E

% 200

x

13-

0

150

lO0

50

2 z, 6 8 10 12 14 16 18 20 22 2/, Hours after injection into albumen

Fig. 2. Rate of increase of radioactivity per mg DNA extract followinginjection of 3H-BUdR on day 3. Incorporation of BU in the DNA increases constantly for 8 h as more cellsenter the S phase. The decline after 8 h is probably due to the decrease in the quantity of BUdR available for incorporation in combination with an increase in the total amount of DNA due to cell division. Each point represents measurements from 5 eggs

calculating the labelling index at h o u r l y intervals after 3 H - T d R (Fig. 3) it was seen that the percentage of labelled cells did n o t change in the 4 h after injection . Therefore a trace a m o u n t of T d R remains available for a r o u n d 1 h.

The decline in B U d R c o n t e n t of the D N A which begins after 8 h was p r o b a b l y due to the r e d u c t i o n in the c o n c e n t r a t i o n o f the a n a l o g u e in the a l b u m e n in c o m b i n a t i o n with a n increase in the total a m o u n t of D N A due to cell proliferation .

BUdR and Chicken Embryo 429

Table 1. Weights* of embryos (g • S.E.M.). BUdR or saline was injected on day 3. Results analysed by Student's t-test

Day of incubation
3 4 5 6 7 10 11 13
Saline 0.0260.084 0.224 0.539 1.415 3.638 4.699 9.11
_+0.006 _+0.003 _+0.008 _+0.012 _+ 0.014 _+0.068 _+0.077 _+0.305
BUdR - 0.0810 0.207 0.507 1.355 3.119 3.66 7.386
_+_0.003 • _0.014 + 0.022 ___0.073 _+0.105 •
~Difference and - -3.8 -7.3 - 5 - 4.3 - 14 - 22 -18
significance level N.S. N.S. N.S. P<0.05 P<0.001 P<0.001 P<0.001

S.E.M. = standard error of Mean; N.S.= Not significant

* Each figure is the mean of the weights of at least 40 embryos

(ii) Gross Teratological Observations and Effects on Weight Gain

When eggs which had been injected on day 3 of incubation were opened on succeeding days, no gross malformations were seen until day 11, when 47.6 ~ (1 l 9 out o f 250) of the B U d R treated and 14 ~ (28 out of 200) o f the saline treated were dead ( p > 0.001, X2-test). N o n e o f the saline treated had gross malformations but 30 ~o of the B U d R injected had various degrees of ventral body wall defects consisting of gastroschisis with protruding stomach and liver or combined gastroschisis and thoracoschisis with ectopia cordis. By day thirteen, 64 ~ (64 out of 100) of the B U d R treated and 12 ~ (12 out o f 100) o f the saline treated were dead. N o n e of the survivors were grossly malformed, but ventral body wall defects were evident in m a n y o f those that had died as a result of BUdR . When eggs were opened on day seventeen, 70 ~ (70 out o f 100) of the B U d R treated and 15 ~o (15 out of 100) o f saline treated were dead. Since none o f the survivors had ventral body wall defects it was concluded that all of the latter died around day 12.

The wet-weights of embryos treated with BUdR or saline are presented in Table 1. Weight differences between the two groups were not found to be significant until 4 days after injection. Thereafter the growth lag between B U d R and saline treated increased reaching a peak of 22 ~ 8 days after treatment (day 11 o f incubation) and decreasing after this so that the difference was only 10 ~ on day 17. Thus only those embryos with a less severe growth retardation survived until day 17.

The weights and survival rates of those receiving B U d R on days 4 and 6 did not differ significantly from those that received saline on the same days.

(iii) Effects o f BUdR on Embryo D N A Content

On day 3 o f incubation the mean D N A content of the embryos was 26.6 gg (_+ 6.6). Table 2 represents values of D N A content on days 3 through 7 following treatment with B U d R or saline on day 3. The a m o u n t of D N A per embryo more than quadrupled between day three and day four whereas it merely doubled per 24 h

60 9- – - – - – “3H -TdR

=” 3 H – B U d R

50

~o

30

20 i i i I I
I 2 3 /-,
Time in hours

Fig. 3. Rate of increase of labelling index in neuroepithelium of spinal cord following injection of 0.02 mg 3H-BUdR or a trace amount of 3H-TdR into albumen. Note the constant increase in percentage of labelled cells after 3H-BUdR. The percentage of labelled cells remains the same for about 4 h after 3H-TdR

Fig. 4. Neuroepithelium of spinal cord 24 h after injection of 3H-BUdR. Note labelled necrotic cells (arrows). Cell necrosis was not seen after the injection of an equimolar amount of 3H-TdR. L, the lumen; M, matrix layer; N, the neuroblast layer

BUdR and Chicken Embryo
431

period thereafter. Treatment with BUdR on day 3 resulted in a 26 % reduction in embryo DNA content 24 h later compared to controls. On succeeding days the DNA content of the BUdR treated embryos was about 10 % less than that of controls. Injection of BUdR on days 4 or 6 did not cause significant differences in DNA content on succeeding days.

When semithin araldite sections were examined, cell necrosis was observed to begin in the neuroepithelium of the spinal cord i 0 h after BUdR treatment, and was still evident at 24h after injection (Fig. 4). All degenerating cells were labelled indicating that they had incorporated 3H-BUdR. Cell necrosis was not seen in tissues other than the neuroepithelium. No cellular changes were seen in the tissues of embryos treated with 0.016 mg TdR. In addition embryos that were sectioned 48 h after injection showed dilated vascular channels and extravascated blood in the mesenchyme of the lateral body folds.

Discussion

These experiments have shown that when BUdR is injected into the albumen space of the egg, the analog remains available for incorporation into the DNA of the embryo for about 8 h or for a complete cell generation time at this stage of development. The results of experiments with scintillation spectrometry were supported by the autoradiographic experiments. Since G2+ . M lasts 3 h, a short pulse with a labelled nucleoside should not result in a significant increase in the labelling index between one and four hours after the pulse is applied. In our experiments the labelling index increased by around 13 % per hour between 1 h and 4 h after injection of 3H-BUdR. Therefore it was concluded that for a number of hours after giving BUdR all cells entering the S phase had incorporated the analog. The autoradiographic experiments with 3H-TdR showed that in trace amounts the whole dose of the latter is rapidly incorporated into replicating DNA, its availability time being less than one hour. This difference in the kinetics of incorporation of trace and larger doses of nucleosides is similar to the finding of Packard et al. (1973) that the peak of incorporation of 3H-BUdR into mouse embryo DNA occurred later when a teratogenic dose of BUdR was injected along with the labelled analog than when a trace dose was injected alone. Clearly there is a maximum rate at which nucleosides can be incorporated into DNA and the application of a large amount saturates the chemical pathways.

The effects of BUdR on embryo survival are in general agreement with the results of Zamenhof et al. (1971) who found that a dose of 0.02rag on day 3 of incubation allowed only 50 % of the embryos to hatch. In the present study larger doses were lethal within 24 h when injected on day 3 and the same dose was similarly lethal when given on day 2. These findings may be explained by the discovery of Steck et al. (1969) that an excess of any nucleoside can inhibit the entry of heterologous nucleosides into the nucleoside precursor pool and to the related finding of Xeros (1962) that high concentrations of TdR can inhibit DNA synthesis in mammalian cells in culture. Since an equimolar amount of TdR injected on day 3 did not cause cellular degeneration in our experiments, it may be concluded that the dose of BUdR used did not disturb embryo growth by inhibiting the entry of heterologous nucleosides into the precursor pool. Similarly Zamenhof et al. (1971)

432 J. Bannigan et al.

Table2. Embryo DNA content* (Bg• S.E.M.) BUdR or saline was injected on day 3. Results were analysed by Student’s t-test

Day of incubation
3 4 5 6 7
Saline 26.6 122 315.9 705.6 1121
• 1.5 • 6.35 _+ 9.4 • 25.24 _+ 23.29
BUdR - 90.0 284.9 598.4 999.4
+_ 5.18 • 9.62 • 35.8 • 22.6
~Difference and - - 26 - 9.8 - 15 - 10
significance level P<0.001 P<0.025 P<0.01 P<0.001

S.E.M. = Standard error of the mean

* Each figure is the mean ofmeasureme~ztof at least 40 embryosexceptday 3 where 20 measurementsare represented

found that 0.02 mg T d R injected on day 3 had no effects on embryo survival or on brain D N A content at the end of the incubation period.

The dose of B U d R used in this study was only effective in causing growth retardation as measured by slower weight gain and reduced D N A content when applied on day 3 but not on subsequent days. The sensitivity of day 3 can probably be related to the very rapid increase in embryo D N A which takes place between days 3 and 4 (Table 2). In addition Zagris (1979) has found that exposure of the unincubated chick blastoderm in culture to BUdR had no effect on haemoglobin or melanin synthesis when the drug was applied after the second day in culture. Although an exact temporal comparison cannot be made between the embryo in ovo and the cultured blastoderm, the experiments of Zagris have shown that the cells of the chick embryo undergo a change in sensitivity to BUdR early in development.

The differences in D N A content per embryo became significant 24h after treatment on day 3 and were maintained on succeeding days. This difference was probably the result of the cell necrosis which became evident 10 h after injection and was still present after 24h. Since it was previously found in the mouse embryo that the cell generation time was prolonged in those cells which incorporated BU (Bannigan and L a n g m a n 1979), it is possible that a similar process may be partially responsible for the reduction in D N A content reported here. The fact that the difference in D N A content was greatest 24 h after treatment and became less on subsequent days (Table 2) m a y be an indication that a reparative process operates.

It is unlikely that the reduction in embryo wet weight resulting from B U d R treatment is due purely to cell necrosis, as it only became significant 4 days after treatment. Furthermore, cell death was only present in the central nervous system and could not therefore account for the 22 ~ weight difference noted on day 11. It is possible that BUdR treatment causes disturbances in the cardiovascular or endocrine systems which in turn could result in growth retardation. The growth retardation might also have been due to interference with transcription of R N A since Palayoor (1976) found that when mouse embryos were treated with BUdR on

BUdR and Chicken Embryo
433

d a y 6 o f gestation, there was a n 80 ~ r e d u c t i o n in the u p t a k e o f 3 H - u r i d i n e o n d a y

11. S i m i l a r l y Lee et al. (1974) n o t e d t h a t g r o w t h o f e x p l a n t e d c h i c k e m b r y o s in B U d R r e d u c e d t h e u p t a k e o f 3 H - u r i d i n e b y n e u r o e p i t h e l i a l cells.

T h e o c c u r r e n c e o f v e n t r a l b o d y wall defects f o u n d in the p r e s e n t s t u d y c a n n o t yet be s a t i s f a c t o r i l y e x p l a i n e d . T h e s e defects were p r o b a b l y r e l a t e d t o the d i l a t e d
v a s c u l a r c h a n n e l s n o t e d in the m e s e n c h y m e o f the l a t e r a l b o d y folds 48 h a f t e r B U d R t r e a t m e n t . G r a b o w s k i (1966) f o u n d , a m o n g o t h e r a b n o r m a l i t i e s , a b d o m i n a l h e r n i a s a s s o c i a t e d with h a e m a t o m a t a a n d d e r m a l blisters in chicken e m b r y o s injected with c a l c i u m salts a n d similar defects in chicken e m b r y o s e x p o s e d to h y p o x i a ( G r a b o w s k i 1964). G r a b o w s k i p o s t u l a t e d t h a t in b o t h cases the defects were r e l a t e d to d i s t u r b a n c e s in fluid h o m e o s t a s i s . It is p o s s i b l e t h a t in o u r m a t e r i a l , B U d R c a u s e d m e t a b o l i c d i s t u r b a n c e s t h a t r e s u l t e d in v a s c u l a r d i l a t a t i o n s . These in t u r n m a y have i n t e r f e r e d with c l o s u r e o f the b o d y wall.

I n c o n c l u s i o n , it a p p e a r s t h a t in ovo injection o f B U d R in the chicken e m b r y o p r o v i d e s c o n d i t i o n s m o r e similar to t h o s e c o n t a i n i n g in tissue c u l t u r e t h a n the in u t e r o t r e a t m e n t o f m a m m a l i a n e m b r y o s . I n spite o f this, certain cells still d i e d as a result o f B U d R i n c o r p o r a t i o n . It r e m a i n s to be seen f r o m f u r t h e r e x p e r i m e n t s i f o t h e r cells were p r e v e n t e d f r o m differentiating .

Acknowledgements. Supported in part by grant no. NS 06188-14 to J. Langman, and by a Medical Research Council of Ireland grant to J. Bannigan.
We also wish to thank Professor J.W. Harman, Department of Pathology, University College, Dublin, for permitting the use of his laboratory for the DNA estimations.

Our thanks are also due to Ms Maeve Lynch for her excellent technical assistance, to Mr. Sean Black for the photography, to Mrs. Marjorie Markey for typing the manuscript and to Dr. Paul Peters for his critical reading of it.

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Accepted June 2, 1981