Incorporation of 54ododeoxyuridine into the DNA of Mouse Embryos: Its Relation to Embryotoxicity RICHARD G . SKALKO, DAVID S. PACKARD, JR.,' D O N N A A . C A N I A N O AND ROBERT D . S A X 2 Embryology Laboratory, Birth Defects Institute, N e w York State Health D e p a r t m e n t , and D e p a r t m e n t of A n a t o m y , Albany Medical College, Albany, N e w York 12208

ABSTRACT Pregnant female ICR mice were administered, ip, either a trace (200 pCi/kg) or teratogenic (200 p C i 300 mglkg) dose of [6-3H]5-iododeoxyuridine (IdU) on day 10 of gestation. Maternal liver, spleen, intestine, and kidneys, and placentas and embryos were removed at various time intervals after iqjection, weighed, and homogenized in cold 0.5 M perchloric acid. The half-lives of IdUderived nucleotides i n the acid-soluble fraction ranged from 31-46 min (trace) to 57-131 min (teratogenic) for the tissues analyzed. [3H]IdU was incorporated into the DNA of all mitotically active tissues after both dosages. The presence of the label in iodouracil was demonstrated by thin-layer chromatography of DNA bases extracted from maternal spleen and embryo. Growth of embryos following injection on day 10 resulted in decreased 3H-specific activity in the DNA fraction and concomitant retention of total activity. It is suggested that the previously demonstrated embryotoxicity of IdU is related to its retention at its presumed intracellular site of action.

+

The embryotoxicity of chemicals results from interactions between a number of interdependent factors. Some of these factors are (1) the species (Skalko and Gold, '74), (2) the stage of intrauterine development at which the compound is administered (Wilson, '61; Skalko et al., '71), (3) the time interval during which the compound or its "active" metabolites are available for placental transport (Gibson and Becker, '71; Kimmel et al., '71), (4) the influence of maternal catabolism on tissue distribution of the compound (Schumacher et al., '69; Gibson and Becker, '71), and (5) the sequential cellular phenomena leading to embryotoxicity (Seegmiller et al., '72). To develop working model systems for analyzing the relation of these factors to the teratogenic effects of a chemical agent it is necessary to relate its presumed mechanism of action to detailed information on maternal physiology, placental function, and embryological development in susceptible animals (Karnofsky, '65). We are attempting to develop a model that relates these interdependent factors utilizing the halogenated deoxyuridines as teratogens and mice as the species. The pyrimidine analogues 5-chlorodeoxyuridine, 5-bromodeoxyuridine, and 5-iodoTERATOLOGY, 12: 157-164

deoxyuridine are teratogen in rats, mice, and hamsters (Chaube and Murphy, '64; Ruffolo and Ferm, '65; Skalko et al., '71; Skalko and Packard, '73). In concentrations of 1.5-15 X 10-6 M they inhibit the development of chick myoblasts in vitro (Coleman et al., '69). The similarity of the van der Waal's radii of the respective halogens (Cl, Br, I) to that of the methyl group of thymidine makes the halogenated deoxynucleosides structural analogues of thymidine (Prusoff et al., '65) and, as such, they are incorporated into the DNA of mitotically active cells (Cheong et al., '60; Djordjevic and Szybalski, '60; Visser et al., '60). Previous studies from this laboratory have shown that, in pregnant mice, 5-bromodeoxyuridine (BrdU) satisfies all of the criteria associated with its being a thymidine analogue in vivo; it is incorporated into the DNA of mitotically active maternal tissues, placentas, and embryos on day 10 (Witschi stage 18) of gestation in both trace and teratogenic doses (Packard et al., '73) and has the same cellular localization as Received Mar. 21, '75. Accepted May 16, '75. Present address: Departmentof Anatomy, State University of New York, Upstate Medical Center, Syracuse, New York 13210. Brown-Hazen Research Fellow, New York State Health Department.

157

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R. G. SKALKO, D. S. PACKARD, JR., D. A. CANIANO AND R. D. SAX

thymidine (dT) when analyzed by autoradiography (Skalko and Packard, '74). At equivalent ip dosages (300 and 500 mg/kg) 5-iododeoxyuridine (IdU) is more embryotoxic than BrdU (Skalko and Packard, '73). In view of the structural similarity between these analogues the present study with IdU was undertaken. It was felt that a rigorous andysis of placental transport, maternal pharmacokinetics, and incorporation of IdU into the DNA of maternal organs, placentas, and embryos would provide a meaningful insight into the documented differential embryotoxicity of IdU and BrdU. MATERIALS AND METHODS

Nulliparous female ICR mice (25-35 g) were used.3 They were housed in a facility maintained at 21-26°C and having a controlled lighting regimen of 20 h of light and 4 h of darkness, the dark period occurring between 2200 and 0200. All animals were fed Purina Mouse Breeder Chow ad libitum. Females were caged with fertile males of the same stock and examined daily for copulation plugs. The day a vaginal plug was found was designated time 0 of pregnancy in accord with criteria previously described (Skalko et al., '71). All experiments were begun at 1300 on day 10 (Witschi stage 18) of gestation and a minimum of 3 pregnant females were used for each experimentaI point. Nucleotide pool kinetics and incorporation into D N A Two experiments were performed. To study the distribution of a trace dose of IdU pregnant females were administered an ip injection of 200 &i/kg [6-3H]IdU (20.6 Ci/ mM). To study a teratogenic dose other females were given an ip injection of 200 pCi/ kg [6-3H]IdU (13.9 Ci/mM) plus 300 mg/ kg unlabeled IdU as a suspension in 0.5% carboxymethylcellulose (CMC; Nutritional Biochemicals, Cleveland, Ohio). The final specific activity of this solution was 0.24 Ci/M.

The animals were killed by cervical dislocation at various times after injection and the uterus was removed and placed in icecold 0.9% NaCI. The maternal liver, spleen, a segment of small intestine distal to the pylorus ("gut"), and both kidneys were rapidly removed and placed in separate centrifuge tubes containing icecold 0.5 M perchloric acid (PCA). The embryos and pla-

centas were dissected free from the uterus and from each other, counted, and also placed in separate tubes containing cold 0.5 M PCA. Tissue wet weights were determined, the tissues homogenized separately in the 0.5 M PCA, and the acid-soluble RNA and DNA fractions isolated from each tissue by a modified Schneider procedure (Schneider, '45; Packard et al., '73). The portion of the tritium activity in the acid-soluble fraction associated with IdU nucleotides was estimated by taking advantage of the fact that nucleotides are selectively and quantitatively adsorbed on activated charcoal (Hurlbert, '57; Chojnacki and Matysiak, '71). An aliquot (0.5 ml) of each acid-soluble fraction was passed through a separate small column containing about 200 mg of acid-washed Darco G-60 activated charcoal. The column was eluted with 2 separate washes (0.3 ml) of distilled water. A separate aliquot was assayed directly. The estimated nucleotidespecific activity was determined by calculating the difference between the 2 samples and was expressed as acid-soluble,charcoaladsorbed dpm/g wet weight. To determine availability time, decay constants, 95% confidence limits (CL), and half-lives were calculated as described previously (Packard et al., '73). The specific activity of the DNA fraction was determined by adding an aliquot to 15 ml of scintillation cocktail (Skalko et al., '71) for counting in a Beckman LS-250 scintillation spectrometer. Another aliquot was taken to determine the DNA concentration by the diphenylamine reaction (Schneider, '57). DNA specific activity was expressed as dpm/pg DNA. Retention of [ 3 H ]IdU It has been repeatedly demonstrated that DNA is ordinarily a very stable cellular fraction and that label incorporated into DNA is retained (Fresco and Bendich, '60), although, with successive cell cycles, the specific activity decreases (Taylor et al., '57; Meselsohn and Stahl, '58; Packard et al., '73). To study retention of 3H derived from [3H] IdU in the DNA fraction the concentration of radioactivity (dpmlml) was multiplied by the total volume to yield total dpm/ tissue. This was done for all tissues anallyzed and for all time points (15 min-48 h) after injection of the labeled compound. In 3

Camm Research Institute, Wayne, New Jersey.

159

IdU INCORPORATION INTO MOUSE EMBRYOS

TABLE 1 Nucleotide pool turnover kinetics f o r

[ 3 H ] IdU

[3H] IdU (carrier) 2

13H] IdU (trace) Tissue k'

Liver Kidney Spleen Gut Placenta Embryo

CL

*95%

0.0102 0.0050 0.0130 0.0136 0.0159 0.0304

- 0.0156

- 0.0206 - 0.0221

- 0.0168 - 0.0181 -0.0152

T1,,3

k'

& 95%

- 0.0053 - 0.0120 - 0.0113 - 0.0121 - 0.0090 - 0.0054

44 34 31 41 38 46

CL

0.0042 0.0058 0.0062 0.0084 0.0108 0.0087

131 58 61 57

-

*

77

128

200 pCi/kg; calculation based o n data from 1S120 min except embryo where peak activity occurred at 30 min. 300 mg 200 pCi/kg; calculation based o n data from 15-240 min (kidney, spleen), 30-240 m i n (liver, gut, placenta), and 60-240 min (embryo). Minutes.

+

addition the DNA concentration, as determined by the diphenylamine reaction, was also multiplied by the total volume to yield the total amount of DNA extracted from that tissue. To determine DNA concentrationlembryo or placenta this value was divided by the total number of each of these removed at the beginning of the experiment. Chromatographic analysis of ]H"[ iodouracil ( I U ) To demonstrate that the tritium molecules present in the DNA fraction were derived from [3H] IU the DNA fractions of

i i TIME

24 AFTER INJECTION

40 IHOURS)

Fig. 2 Distribution of a teratogenic dose (200 pCi 300 mg/kg) of [6-3H] IdU i n the DNA fraction of embryos between 1 and 48 h after injection. Values represent the mean f SE of at least 3 experiments. ( . . ) = total 3H activitylembryo; (0- - - - 0)= specific activity.

+

... .

0 4

, 24

0

TIME

AFTER

INJECTION

40 (HOURS)

Fig. 1 Distribution of a trace dose (200 p C l / kg) of [6-3H] IdU in the DNA fraction of embryos between 1 and 48 h after injection. Values repreSE of at least 3 experiments. sent the mean (0 -0 ) = total 3H activitylembryo; (0- - - 0) = specific activity.

*

maternal spleen and embryo taken 1 h after [3H] IdU administration were subjected to base chromatographic analysis on MN-300 thin-layer sheets4 according to methods described previously (Packard et al., '73). To visualize IU, parallel samples were run with carrier IU. In addition thymine (T), JU, and a sample of calf thymus DNA bases prepared in a similar manner were also chromatogrammed. The DNA samples containing [3H] IU were assayed for total radioactivity by eluting regions on the chromatogram corresponding to T, IU,

~

Brinkmann Instruments, Westbury, New York.

160

R. G. SKALKO, D. S. PACKARD, JR

D. A. CANIANO AND R. D. SAX

cytosine (C), and the purines adenine (A) and guanine (G) in 0.1 N HC1. The eluates were then prepared for liquid scintillation spectrometry.

1....

RESULTS

The availability of a trace dose of ["HI IdU, after ip injection, was similar for all tissues analyzed (table 1) with T1/2 values ranging from 31 (spleen) to 46 min (embryo). These findings are in agreement with many previous observations of the turnover of the normal metabolite, [BH]dT, in intact animals (Potter, '59, '74; Cleaver, '67). The administration of a carrier dose (300 mgl kg) of [:'HI IdU resulted in a n increase in the half-life of the precursor in all tissues 2- to 3-fold (table l), a n observation that is consistent with results previously reported for a biologically effective doseof [3H] BrdU in pregnant ICR mice (Packard et al., '73). Significant levels of radioactivity were incorporated into the DNA fraction of all mitotically active tissues. In embryos peak levels occurred 1 4 h after injection of a trace dose of [:'HI IdU (fig. 1). Continued embryo growth resulted in an increase in total DNA content (table 4) and a n eventual decrease in DNA specific activity, although total 3H activity was retained in the DNA fiaction throughout the entire 48 h period.

0

4

8

ZQ

......................

1.-.....................................

t

0

4

8

24

48

TIME AFTER INJECTION ( H O U R S )

Fig. 4 Distribution of a teratogenic dose (200 PCi 300 mg/kg) of [6-3H] IdU in the DNA fraction of the placenta between 1 and 48 h after iMection. Values represent the mean & SE of at least 3 experiments. ( 0 -0 ) = 3H activitylplacenta; (0- - - - 0)= specific activity.

+

When a teratogenic dose of [SH] IdU was administered peak levels of incorporation occurred a t 2-4 h (fig. 2). In this situation, also, loss in specific activity was accompanied by the retention of total activity. A similar pattern was observed in the placenta in both experimental series (figs. 3, 4). Analysis of 3H activity in the DNA fraction of spleen revealed a different distribution

48

TIME AFTER INJECTION (HOURS)

Fig. 3 Distribution of a trace dose (200 pCi/ kg) of [6-3H] IdU in the DNA fraction of the placenta between 1 and 48 h after idection. Values represent the mean f SE of a t least 3 experiments. ( 0 -0 ) = total 3H activity/placenta; (0- - - - 0) = specific activity.

Fig. 5 Distribution of a trace dose (200 WCi/ kg) of [6-3H] IdU in the DNA fraction of the matern a l spleen between 1 and 48 b after injection. Values represent the mean & SE of a t least 3 experiments. ( 0 -0 ) = total 3H activitylspleen; (0 - - - - 0)= specific activity.

161

IdU INCORPORATION INTO MOUSE EMBRYOS

, Lo5 0

4

8

24

TIME

AFTER

48

INJECTION (HOURS)

Fig. 6 Distribution of a teratogenic dose (200 PCi 300 mgikg) of [6-3H] IdU i n the DNA fraction of the maternal spleen between 1 and 48 h after injection. Values represent the mean k S E of at least 3 experiments. ( 0 __ 0 ) = total 3H activitylspleen; (0 - - - - 0 ) = specific activity.

+

from that observed in the other tissues. After 8 h both specific and total "H activity in the DNA fraction declined and this occurred with both the trace (fig. 5) and carrier (fig. 6) doses. This phenomenon also occurred with [3H] d T and [3H] BrdU (Packard et al., '74) in mouse spleen and appears to be related to the hematopoietic activity of this organ during pregnancy (Fowler and Nash, '68). The loss of total activity, therefore, appears to reflect the passage of labeled cells into the circulation. When purine and pyrimidine bases were isolated by thin-layer chromatography the bulk of the radioactivity was found to migrate with carrier 5-IU (table 2). The presence of 3H in thymine may represent either cochromatography of IU and T or dehalogenation of [3H] IdU to deoxyuridine and subsequent conversion to [3H] dT. The former is possible owing to the closeness of the R p of T and IU (Skalko and Packard, '74). Although IdU is highly embryotoxic at the dosage used no effects were observed on the wet weight of embryo, placenta, and spleen (table 3). No effects were observed on the DNA content of embryo and placenta (table 4), although a significant reduction

TABLE 2

Distribution of 3 H derivedfiom

[ 3 H ] IdU

in D N A bases

Percent total activity in Tissue

Spleen

1

Embryo

1

Thymine

Iodouracil

Cytosine

Adenine

Guanine

25.5

73.0

0.5

0.3

65

37.0

63.0

-

-

-

1 Values from DNA base chromatogram to which unlabeled iodouracil was added to each sample a s a marker.

TABLE 3

Tissue wet weights ufter administration of [W] Id U ~

~~

~

~

Time after i q e c t i o n (h)

1

2

4

8

24

48

[3H] IdU

(trace) 1 [3H]IdU (carrier) 2

2 8 . 3 f 5.33 3 3 . 6 t 4.8

Embryo 2 6 . 0 2 3.9 41.5k 9.9 32.4k 4.0 34.0k 2.7

[3H] IdU (trace) [3H] IdU (carrier)

37.0k 4.5 46.0% 4.9

4 3 . 4 k 3.2 43.2-t 3.0

Placenta 41.1k 3.2 41.0k 2.8

38.9% 3.2 47.2% 5.9

5 6 . 6 ~0 . 2 4 9 . 4 2 6.9

76.5210.5 7 2 . 7 t 4.1

[3H] IdU (trace) [3H] IdU (carrier)

290.2k55.9 339.2 f 23.9

341.5k49.7 315.1 k 32.9

Spleen 411.4k62.7 343.2 f 27.3

3 1 9 . l t 3.2 319.5 k 69.3

465.7-97.2 283.1 k 37.6

340.1k30.8 276.8 f 15.2

200 pCi/kg.

* 300 m g + 200 pCi/kg. 3

Wet weighutissue or organ, mean

&

S E (in mg).

44.4k14.4 44.8-10.4

8 1 . 3 2 4.8 74.3217.1

160.6230.6 1 1 5 . 2 2 9.2

162

R. G. SKALKO, D. S . PACKARD, JR., D . A. CANIANO A N D R. D. SAX TABLE 4

Tissue D N A c o n t e n t after administration of [3H] IdU Time after injection (h)

1

2

4

8

24

48

[3H] IdU (trace) 1 [3H] IdU (carrier) 2

192.8 f 24.1 3 175.82 10.1 268.0 f 20.6 225.1 f 23.7

Embryo 218.3 k 30.3 240.0 f 33.2

265.4 2 45.2 302.9 f 38.7

423.9 f 75.9 1062.5 +- 167.8 525.0 f 137.6 867.4 f 42.3

[3H] IdU

(trace) [3H] IdU (carrier)

146.6218.4 260.0 f40.4

178.3f21.6 208.6 t 15.5

Placenta 261.6f99.2 206.6 i 28.1

157.9f14.1 228.7 t 5.2

185.0f 17.4 255.5 t 35.5

[3H] IdU (trace) [3H] IdU (carrier)

7,125 f 992 9,791 f 796

7,750 f 661 9,291 f 1624

Spleen 9,875 t 943 8,516 f 144

1

2

356.2f 41.0 357.5 2 34.9

7,833? 631 10,333f 4834 9,541 2 1332 8,333f 1302 6,416 f 157 3,250f 430

200 pCi/kg. 300 m g 200 pCi/kg.

+

p g DNAltissue or organ, mean -t SE.

was noted in spleen 48 h after injection of 300 mglkg [6-3H] IdU. The possibility that this latter observation reflected a late effect of IdU on DNA replication remains to be determined.

within 48 h of administration (Packard et al., '74). Incorporation of BrdU into DNA does not necessarily result in complete impairment of cell function (Pasztor et al., '73) and not all cells that are exposed to BrdU are similarly affected (Skalko et al., DIS CUSS1 ON '71). Thus, the second possibility, that inThis study has shown that, at a time in corporation of IdU into embryonic DNA may development when mouse embryos are sus- be coincident with, but not the cause of, ceptible to its embryotoxic action, IdU was IdU-induced embryotoxicity, would appear incorporated into the DNA of embryos and to be a reasonable one. However, by using other mitotically active tissues (placenta, methods identical with those described here, maternal spleen). This observation leads Packard et al. ('74) showed that, at the to two important questions which directly same stage of development, a teratogenic bear on the ability of IdU to act as a ter- dose (500 mglkg) of ]H"[ BrdU turned over, atogen. First, is the incorporation into i.e., label was lost from the DNA fraction embryonic DNA causally related to embry- of the embryo. This turnover of label was otoxicity? Second, is the presence of IdU rapid ( T ~ = 1 6h; Skalko and Packard, in embryonic DNA a coincident phenom- '75) and did not occur with trace doses of enon which simply demonstrates that IdU either [3H] dT or ["I BrdU (Packard et al., has gained access to the embryo? '74). The present study, in contrast, has As many studies with the related haloge- shown that a teratogenic dose (300 mglkg) nated nucleoside analogue BrdU have of ["HI IdU did not turn over but was reshown (Wilt and Anderson, '72) attempts tained in the embryonic DNA fraction to answer the first of these questions in the throughout the entire 48 h of the experaffirmative should be approached with con- iment. siderable caution. Previous studies by us If the embryo is the definitive site of ac(Skalko et al., '71; Skalko and Packard, tion of the thymidine analogues then any '73, '74) demonstrated that the biological one of a number of factors could contribute effects of BrdU and IdU are not identical to their differential toxicity. These would when administered to pregnant mice. BrdU include: vehicle-mediated differences; difwas much less toxic in terms of induced ferences in availability time; differences in embryolethality at equivalent dosages and, level of incorporation into embryonic DNA in contrast with the results reported here, or differences in degree of retention in DNA produced significant changes in both wet fraction after incorporation. The i d u e n c e weight and DNA content of the embryos of the vehicle employed could not be conve-

IdU INCORPORATION INTO MOUSE EMBRYOS

niently studied owing to the very low solubility of IdU (Clifton et al., '63). However, if these differences were paramount their influence would most likely be reflected as differences in availability time, in level of incorporation into DNA, or both. However, as this study has shown (table 1), the availability of a teratogenic dose of IdU was comparable to that previously reported for BrdU (Packard et al., '73). In addition the level of incorporation of teratogenic doses of the two analogues into embryonic DNA was also identical (Skalko and Packard, '75). The only consistent difference between BrdU and IdU is that 3H was retained in the DNA fraction throughout the time period studied. The results reported here are consistent with a hypothesis that assumes that the DNA molecule is the definitive intracellular site for the embryotoxic action of IdU. The enhanced toxicity of this compound, therefore, may be related to the retention of IdU at its presumed site of action for a longer period of time than the related analogue BrdU. Association between the retention of a teratogen and its embryotoxic effects has been noted previously. Levine et al. ('68) showed that [ I T ] cortisol is retained by fetuses of the sensitive NJ strain of mice for a longer time than by those of the resistant CBA strain. Similar results have been reported using [3H]triamcinolone acetonide as the teratogen by Zimmerman and Bowen ('72), and Short and Gibson ('74) have suggested that the embryotoxicity of [ IJC] cyclophosphamide in mice may be related to its retention in the nucleic acid fraction of the embryo. Collectively these reports suggest that yet another pharmacolunetic parameter, retention of a compound in situ, must be considered in evaluating its ability to produce embryotoxic effects. ACKNOWLEDGMENTS

We gratefully acknowledge the assistance provided by Allen M. Niles, Carol A. Cooper, Louise L. Skalko, and Don Driscoll during the course of this study. Partial support was provided by a grant from the Nattional Foundation -March of Dimes. LITERATURE CITED Chaube, S., a n d M. L. Murphy 1964 Teratogenic effects of 5-chlorodeoxyuridine o n the rat fetus;

163

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Incorporation of 5-lododeoxyuridine into the DNA of mouse embryos: its relation to embryotoxicity.

Pregnant female ICR mice were administered, ip, either a trace (200 muCi/kg) or teratogenic (200 muCi + 300 mg/kg) dose of [6(-3)H] 5-iododeoxyuridine...
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