Embryotoxicity of Transplacental ly and Intraamniotically Administered 6-Azauridine in Mice M. DOSTAL AND R. JEL~NEK Institute of Experimental Medicine, Czechoslovak Academy ofsciences, Prague, Czechoslovakia
ABSTRACT Embryotoxic effects were compared of intramuscularly (im) and intraamniotically (ia) administered 6-azauridine (Riboazauracil Spofa) in random-bred mice H-Velaz. Effects of single doses (0.25 mg, 2.5 mg, 25.0 mg and 250.0 mg for im and 0.0025 mg, 0.025 mg, 0.25 mg and 2.5 mg for ia administration) on days 11, 12,13 and 14 were evaluated as a sum of dead fetuses and fetuses with cleft lip and/or palate, fetuses with limb deformities and fetuses with deformities constituting the syndrome of caudal regression (hypoplasia of the caudal part of the trunk, absent tail, short tail, curled tail). Considering the sensitivity peaks of the morphogenetic processes which were observed, the doseresponse relationships, the transformation of the teratogenic to a lethal effect and critical period extension with increasing doses, i t was found that the effects of ia and im administered 6-azauridine did not differ. It was concluded that ia administered 6-azauridine had direct effect on embryonic morphogenetic processes and that this, too, was the essential mechanism of embryotoxicity of im administered 6-azauridine. The value of the intraamniotic technique for establishing the direct embryotoxic effect is discussed. Teratologists frequently consider it necessary to analyze the direct interactions of teratogens with embryonic morphogenetic processes. However, opinions differ as to what experimental technique should be used. Staples (‘75), for example, states that “the intraamniotic application is not suitable, as the procedure itself induces cleft palate and only can be performed relatively late in pregnancy.” The present paper aims at: (1) comparing direct and transplacental effects of 6-azauridine, (2) emphasizing that if fine enough technique is applied no danger of nonspecifically induced effects arises (Dostal, ’711, and (3) demonstrating that intraamniotic application has distinct value for determining direct effects of teratogens. MATERIALS AND METHODS
Random-bred albino mice (H-Velaz) were kept in rooms without artificial light and fed DOS 4 (Velaz) diet and water ad libitum. The females were mated overnight and the day of the vaginal plug was considered as being day 1 of pregnancy. 6-azauridine (Riboazauracil Spofa) was administered intramuscularly TERATOLOGY (1979) 19: 143-148.
(im) in single doses of 0.25 mg, 2.5 mg, 25.0 mg and 250.0 mg in 0.1 ml of distilled water on days 11,12,13 or 14. The dose of 250.0 mg was administered in 1.0 ml of distilled water. Three females were used for each day and dose. The intraamniotic injections (ia) were performed from day 9 to day 15 of pregnancy (for method see Dostal, ’71). 6-azauridine (6AzUrd) was injected in doses of 0.0025 mg, 0.025 mg, 0.25 mg and 2.5 mg in 2 microliters of distilled water. Offspring of 5 females were used for each day and dose. In each litter 5 fetuses were injected with 6-AzUrd, the remaining fetuses serving as intact controls. The females were killed on day 17 of pregnancy by cervical dislocation, the fetuses were removed from the fetal membranes, weighed and examined under a dissecting microscope. Embryotoxicity was evaluated as a sum of dead fetuses (including resorptions) and fetuses with malformed limbs (including the presence of hemorrhagic blisters), fetuses with defects constituting the syndrome of caudal regression (marked hypoplasia of the caudal part of the fetus, absent tail, short tail, Received Aug. 22, ’77. Accepted Aug. 18,‘78.
143
144
M. DOSTAL AND R. JELINEK TABLE 1
Effects of im administration of 6-azauridine Done of 6-azauridine ~~
0.25 mg
2.5 mg
D
M
Day
N
DfM
%
%
N
D+M
11 12 13 14
53
2 1
3.8 2.0
0
21 2
0
0
0
1
0
4
32 25 20 34
50
56 25
0
D
M
%
%
N
D+M
6.3 63.3 4.0 4.2
26
26
31
0 0
32 18
31 0
0 0
250.0 mg
25.0 mg
0 0
0
D
M
%
%
34.6 100.0 0 100.0 0 0 0 0
D
M
N
DtM
%
%
31 28 25 32
31 28 22 24
100.0 3.6 0 0
0
100.0 88.0 75.0
N, number of fetuses; D, dead fetuses; M, malformed fetuses. The percent of malformed fetuses is derived from living fetuses.
TABLE 2
Effects of ia administration of 0.0025mg of 6-azauridine Experimental fetuses Day
9 10 11
12 13 14 15
N
D+M
25 25 25 25 25 25 25
5 9 24 0 1 1
2
D
M
%
%
16.0 4.8 24.0 15.8 20.0 95.0 0 0 4.0 0 0 4.0 8.0 0
Control fetuses
N
DfM
24 20 27 32 14 23 16
2 5 9 4 2 2 0
D
M
%
%
8.3 0 0 25.0 22.2 14.3 12.5 0 14.3 0 4.3 4.5 0
0
N.numberoffetuses; D, deadfetuses; M, malformdfetuses. The percentof malformed fetusesis derivedfrom1ivingfetuses.Control fetuses are the non-injected fetuses from experimental litters.
curled tail) and fetuses with cleft lip and/or palate. The x 2 test and for small marginal frequencies Fisher’s test were used for statistical evaluation of the results. RESULTS
The total number of fetuses and the percentage of dead and malformed fetuses (from survivors) are given in table 1. Of the four doses used the first dose resulting in considerable effect was 2.5 mg. When administered on day 11 it induced 63.3%malformed fetuses. The 25.0 mg dose caused an increase in the frequency of malformed fetuses on day 11 and had embryotoxic effects on day 12 as well. The application of 250.0 mg resulted in increased lethality (consequently decreasing the number of malformed fetuses) and further extension of the critical period-this dose was effective even on days 13 and 14. The effects of ia administered dose of 0.0025 mg 6-AzUrd from day 9 to day 15 of pregnancy is shown in table2. Peak sensitivity to this dose of 6AzUrd was reached on day 11 when the inci-
dence of malformed fetuses (95.0%)differed significantly (P < 0.05) from that of dead fetuses in intact controls (14.4%).The proportion of affected fetuses on days 9 and 10 did not differ significantly (P > 0.05) from that of dead fetuses in intact controls and virtually no effect was observed with this dose on days 12, 13,14 and 15. The results obtained with effective doses on days 12, 13, 14 and 15 are summarized in table 3. All three doses (0.025 mg, 0.25 mg and 2.5 mg) had embryotoxic effects when applied on days 12, 13 and 14, the effects on day 12 being dose-related for both teratogenicity and lethality. Embryotoxicity of the 0.25 mg and 2.5 mg doses was lower on day 13 than on day 12. A similar decrease for lethality was found on day 14. The medium and highest doses, however, showed high teratogenic effects due to the incidence of fetuses with cleft palate. On day 15 even the highest dose (2.5 mg) had no visible significant effects. The relative frequencies of fetuses with cleft palate induced by ia and im administration of 6-AzUrd is given in table 4. The effects of both ia and im administration are identical with regard to the occurrence of two peaks of sensitivity, namely on days 11 and 14 after ia administration and on days 11 and 13 after im administration (the difference between 88.0% fetuses with cleft palate observed on day 13 and 75.0%on day 14 is nonsignificant). The incidence of fetuses with malformed limbs after ia and im administration is shown in table 5. Peak sensitivity to im administered 6-AzUrd was found on day 11 with the dose of 25.0 mg and on day 12 with the 250.0 mg dose (due to its high lethal effect on day 11). After ia treatment the limb malformations were induced on days 11 and 12 and the maximal sensitivity was found on day 11. The occur-
145
EMBRYOTOXICITY OF 6-AZAURIDINE TABLE 3
Effects of ia administration of 6-azauridine Dose of 6-azauridine 0.25 mg
0.025 mg
2.5 mg ~
E
c
E E
14 15
M
D
M
D
M
N
DiM
%
%
N
D+M
%
%
N
DiM
%
%
25 25 25 19 25 16 25 16
9 2 13 3 7 2 0 0
12.0 8.0 28.0 15.8 0 6.3 0 0
27.3 0 33.3 0 28.0 6.7 0 0
25 16 25 24 25 20 25 21
20 2 10 2 21 2 3
40.0 0 20.0 8.3 20.0 5.0 0 4.8
66.7 12.5 25.0 0 80.0 5.3 12.0 0
25 11 25 17 25 18 25 25
25 6 23 5 23
84.0 18.2 64.0 11.8 24.0 27.8 12.0 4.0
100.0 44.0 77.8 20.0 89.5 46.2 4.5 4.2
Day
l3
D
1
11
4 2
N, number of fetuses; D. dead fetuees; M.malformed fetuses. The percent of malformed fetuses is derived from living fetuses.
TABLE 4
The percent of the fetuses with cleft palate (% CP) from dead and malformed fetuses f D after ia and im administration of 6-azauridine
+ M)
Dose of 6-azauridine
Day
12
; :;
13
i?
11
14
:
im in
0.25 mg 0.0025 mg
im ia
2.5 mg 0.025 mg
im ia
25.0 mg 0.25 mg
im ia
250.0mg 2.5 mg
D+M
%CP
DiM
%CP
D+M
%CP
D+M
%CP
51 20 49 25
0 35.0 0 0 0 0 0 0
30
-
-
0
17
70.6
-
-
0
-
24 22 20 18 34 25
0 9.1 0 11.1 0 28.0
31 15 32 20 18 20
0 6.7 0 20.0 0 80.0
27 4 25 9 32 19
55.6 0 88.0 66.7 75.0 84.7
56
24 25 25
-
0
TABLE 5
The percent of the fetuses with limb deformities f%limbs) from dead and malformed fetuses f D + M ) after ia and im adminktration of 6-azauridine Dose of 6-azauridine
Day
12
; :;
13
i;
11
14
ir
im ia
0.26 mg 0.0025 mg
im ia
2.5 mg 0.025 mg
im ia
25.0 mg 0.25 mg
im ia
260.0mg 2.5 mg
D+M
%limbs
D+M
%limbs
D+M
%limbs
DiM
%limbs
51 20
0 75.0 0 0 0 0
30
-
6.7
-
17
100.0
-
-
-
24 22 20 18 34 25
0 9.1 0 16.7 0 0
31 15 32 20 18 20
38.7 66.7 0 5.0 0 0
21 4 25 9 32
96.3 (25.0) 0 0 0 0
49
25 56 24 25 25
0 0
-
0
19
0
146
M. DOSTAL AND R. JELINEK TABLE 6
The percent of the fetuses exhibiting various forms of the syndrome of caudal regression (% SCR) from dead and malformed fetuses (D+MI after ia and im administration of 6-azauridine Dose of 6-azauridine
Day ~
12
; ;
13
;i
11
14
;r
im ia
0.25mg 0.0025mg
im ia
2.5 mg 0.025 mg
im ia
25.0 mg 0.25 mg
im ia
250.0mg 2.5 mg
D+M
%SCR
D+M
%SCR
D+M
%SCR
D+M
%SCR
0
30
-
63.3
17
-
100.0
-
-
-
24 22 20 18 34 25
4.2 27.3 0 16.7 0 0
31 15 32 20 18 20
100.0 46.7 0 10.0 0 20.0
27 4 25 9 32 19
100.0 (100.0) 24.0 44.0 3.1 5.3
51 20 49 25 56 24 25 25
80.0 0 0
0 0 0 0
-
0
0
rence of the limb malformations on day 13 is tion of orotidine 5-phosphate decarboxylase not dose-related and has to be considered as with the resultant blocked use of orotidine 5-phosphate in pyrimidine base synthesis being accidental. Similar conclusions were drawn when an- (Handschumacher et al., '62; Skoda, '75). The alyzing the occurrence of malformations con- embryotoxic effects of 6-AzUrd were examstituting the syndrome of caudal regression. ined in a number of studies on chick embryos After both ia and im administration the peak (Grafnetterova e t al., '66; Jelinek, '71, '72; sensitivity appeared on day 11. Increasing Rychter and Jelinek, '71, '72; Rychterova et doses resulted in transforming the teratogenic al., '731, mice (Saunders et al., '61; Vorherr, into a lethal effect and also in inducing the '70; Yoshihara and Dagg, '67), rats (Gutova, teratogenic effect on day 12 and after ia ad- '68; Gutova et al., '71a), rabbits (Saksena and ministration even on day 13. The specificity of Chaudhury, '70) and monkeys (Vanwagenen the teratogenic effects of ia injected 6-AzUrd et al., '70). on day 1 3 is also confirmed by the high mortalBecause of its pharmacokinetic characterisity rate. On the other hand, the incidence of tics 6-AzUrd appears to be an ideal model ter24.0%of affected fetuses after im administra- atogen. 6-AzUrd is not further metabolized tion of 250.0 mg of 6-AzUrd on day 13 is non- following initial phosphorylation, is freely dissignificant assuming that no affected fetus tributed in body fluids and is rapidly elimiwould occur in the control group. nated from mammalian organisms. In clinical Summarizing the overall embryotoxic ef- studies 75% of the administered 6-AzUrd was fects and the results of the analysis of inci- excreted within 4 hours (Handschumacher e t dence of particular malformations we can con- al., '62). The transplacental passage of 6clude that the embryonic processes studied AzUrd was demonstrated in rats (Gutova, '68, react to ia and im administration of 6-AzUrd Gutova et al., '71b) and mice (Raika et al., '66; in a similar way. They also exhibit such char- Jelinek et al., '77). acteristics of teratogen-morphogenetic procIn our experiments the doses for the intraess interaction as the dose-response relation- amniotic injections were inferred from the ships, the transformation of the teratogenic embryotoxic dose for the chick embryo (Jeinto a lethal effect and the extension of the linek, unpublished data). The doses for im adcritical period in dependence on increasing ministration were increased by two orders doses. since i t was found that after the ia administration of labeled 6-AzUrd the radioactivity of DISCUSSION fetuses had amounted to about 1%of the Azapyrimidine nucleoside 6-azauridine, dis- injected radioactivity (Jelinek et al., '77). A covered in 1957 (Handschumacher, '57; Skoda similar level of labeled cortisol intake in et al., '57) interferes with the metabolism of mouse fetuses was found by Dostal and Musil pyrimidine nucleotides (Skoda, '75). The basic ('74,'77). As concerns the nonspecific teratomechanism of its action consists in the inhibi- genic effect of t h e intraamniotic injection
EMBRYOTOXICITY OF 6-AZAURIDINE
(induction of cleft palate-Trasler et al., '561, we have proved that it can be eliminated by using fine glass micropipettes (Dostal, '71). The intraamniotic technique has been in routine use in our laboratory; so that the dose-response relationship was tested rather than using control groups with the 6-AzUrd solvent. The dose-response relation is clearly visible in the experimental groups from day 12 to day 14; the lowest dose is without effect. On days 9,lO and 11 the procedure is complicated by reduced transparency of the uterus and fetal membranes. The level of nonspecific lethal effect is somewhat higher than on later stages, but in the majority of experimental groups it does not exceed 20%of the injected fetuses. This level of nonspecific effect does not, moreover, significantly differ from mortality in intact controls (table 2). The same comparison on days 12, 13 and 14 shows that with the 0.25 mg and 0.025 mg doses, the embryotoxic effects are limited to the injected fetuses. However, both the injected and intact control fetuses are affected by the 2.5 mg dose. Here is the possibility that 6-AzUrd either spreads within the uterus or that its transplacental recirculation occurs through the maternal organism to the whole litter (the total dose for one litter is 5 x 2.5 mg). The second explanation seems to be more probable, namely because a single 12.5 mg dose administered to a pregnant female is teratogenic and the yolk sac may be a barrier to 6-AzUrd. This follows from experiments in which 0.05 ml of 3.75%6-AzUrd injected between the uterine wall and fetal membranes to all the fetuses in one uterine horn of the rat on day 12 of pregnancy had no effect (Gutova, '68). A similar level of sensitivity to im administered 6-AzUrd as in our experiments was found by Yoshihara and Dagg ('67). They reported that a n intraperitoneal injection of 2 micromoles of 6-AzUrd to females of C57BL/ 10 mice induced polydactylous hind feet in approximately 20%of fetuses. The results of the intraamniotic administration of 6-AzUrd confirmed t h e direct teratogenic effect of 6-AzUrd on the fetuses. The similarity of the effects of 6-AzUrd administered ia and im testifies that 6-AzUrd given to pregnant female acts primarily by directly affecting the fetus. Nevertheless, Gutova ('68) observed the accumulation of 6-AzUrd in placentae and believed t h a t a high 6-AzUrd concentration could damage placental functions.
147
The same situation was found by JiFiEka et al. ('65). Jelinek et al. ('77) analyzed the dynamics of this accumulation and its dose dependence and concluded that the phenomenon had two aspects. It evidently impeded a rapid increase in the teratogen level in the fetus. However, in these circumstances, the placental morphogenetic processes could be exposed to much higher levels of the teratogen than the embryonic morphogenetic processes. The question arises whether such a situation can negatively affect the development of the embryo and whether it can occur even when a teratogen is administered ia. Dostal and Musil ('74, '77) studied radioactivity distribution in the fetoplacental unit after ia administration of labeled cortisol and found that the radioactivity retained in the placentae was only 2-3 times higher than that retained in the fetuses. These results, however, should be completed with experiments using higher concentrations of the teratogen in the amniotic fluid. The comparisons of the incidences of individual malformations in experiments with ia and im administration have proved that the same morphogenetic processes may be influenced in the same developmental periods. A more detailed analysis of individual types of malformations (e.g., those of limbs) could not be made owing to the insufficient quantity of material. The decreasing sensitivity of the fetuses with the increasing stage of pregnancy is obviously the consequence of the differing sensitivity of particular morphogenetic processes to the net inhibition of pyrimidine base synthesis which remains at approximately the same level when comparable doses are administered on days 6 and 15 of pregnancy to mice (Raska et al., '66). On the basis of the results we have obtained the view appears justified that the intraamniotic injection technique is useful in teratology. This technique is suitable for proving or excluding the possibility of the direct effect of drugs on the fetus and may be used not only for analytical purposes but also as a rapid screening technique (Dostal, '77). The value of establishing the direct embryotoxicity of a drug in the context of screening systems is another question. LITERATURE CITED Dostd, M. 1971 Morphogenesis of cleft palate induced by exogenous factors. 111. Intraamniotic application of hydrocortisone in mice. Teratology, 4: 63-68. 1977 Intraamniotic administration of drugs in
148
M. DOSTAL AND R. J E L ~ N E K
mammals. Proceedings of the Third Symposium on toxicological testingfor safetyof new drugs, Prague, 1976, in press. Dostal, M., and J. Musil 1974 Kinetics of 3H-cortisol injected into amniotic sac of the mouse fetus. Teratology, 10: 307-308(A). 1977 Kinetics of ~ o r t i s o l - l , 2 - ~administered H intraamniotically. Folia Biologica (Prague),23: 212-219. Grafnetterova, J., E. Grossi, R. Fumagalli, P. Morganti and D. Grafnetter 1966 Effects of 6-azauridine on the developing chick embryo. Neoplasma (Bratislava), 13: 251-258. Gutova, M. 1968 KdEinku 6-azauridinu v prenatalnim obdobi -+oje. Thesis, Charles University, Prague. Gutova, M., J. Elis, and H. Raikova 1971a Teratogenic effect of 6-azauridine in rats. Teratology, 4: 287-294. 1971b Transfer of 6-azauridine through the placental barrier in the rat. Neoplasma (Bratislava), 28: 529-531. Handschumacher, R. E. 1957 Metabolites of 6-azauracil formed by Streptococcus faecalis. Federation Proc., 16: 191-198. Handschumacher, R. E., P. Calabresi, A. D. Welch, V. H. Bono, Jr., H. J. Fallon and E. Frei 1962 111. Summary of current information on 6-azauridine. Cancer Chemother. Rep., 21: 1-18. Jelinek, R., Z. Rychter and V. Seichert 1970 Syndrome of caudal regression in the chick embryo. Folia Morphol. (Prague), 18: 125-137. Jelinek, R., Z. Rychter and E. Klika 1971 Syndrome of caudal regression and dysrhaphic malformation of the spinal cord. Folia Morphol. (Prague), 19: 58-70. Jelinek, R., R. Tykva, and M. Seifertova 1977 6-azauridine intake following intraamniotic application in mice. Folia Morphol. (Prague), in press. Jirieka, J., K. Smetana, I. Janku, J. Elis and J. Novotny 1965 Studies on 6-azauridine and 6-azacytidine. I. Toxicity studies of 6-azauridine and 6-azacytidine in mice. Biochem. Pharmacol., 14: 1517-1523. Raika, K., M. S. Zedeck and A. D. Welch 1966 Relationship between the metabolic effects and the pregnancy-inter-
rupting property of 6-azauridine in mice. Biochem. Pharmacol., 2136-2138. Rychter, Z., and R. Jelinek 1971 0 zpilsobu hodnoceni S i n k u teratogenniho agens (6-azauridinu) na pozdnich vjvojovych stadiich. cs. Fysiologie, 20: 527-540. 1972 Z m h y v postupu vaskularisace komorove svaloviny srdeEni u zarodku kuiete p pfisubeni ruznych davek 6-azauridinu. 6 .Fyziologie, 21: 51-57. Ryehterova, V., Z. Rychter and R. Jelinek 1973 The effect of Bazauridine on the ventricular myocardium of chick embryos (contribution to the teratogenic action of drugs). Teratology, 8: 235(A). Saksena, S. K., and R. R. Chaudhury 1970 The antifertility Part 11. In rabeffect of 2',3',5',-tri-O-acety1-6-azauridine. bits. Indian J. Med. Res., 58: 374-376. Saunders, M. A., B. P. Wiesner and J. Yudkin 1961 Control of fertility by 6-azauridine. Nature, 189: 1015-1016. Skoda, J. 1975 Azapyrimidine Nucleosides. In: Handbook of Experimental Pharmacology. 0. Eichler. A. Farah, H. Herken, A. D. Welch, &. Volume XXXVIIII2, A. C. Sartorelli and D. G. Johns, eds. Springer Verlag Berlin-Heidelberg-New York, pp. 348-372. Soda, J., V. F. Hess, and F. Sorm 1957 The biosynthesis of 6-azauracil riboside by Eacherichia coli growing i n t h e presence of 6-azauracil. Experientia (Basel), 13: 150-151. Staples, R. E. 1975 Potential of direct application techniques for detection of teratogens. In: New Approaches to the Evaluation of Abnormal Development. 11. Symposium on Prenatal Development. D. Neubert and H. J. Merker, eds. Berlin, pp. 71-81. Trader, D. G., B. E. Walker and F. C. Fraser 1956 Congenital malformations produced by amniotic s a c puncture. Science, 124: 439. Vanwagenen, G., R. C. DeConti, R. E. Handschumacher, and E. M. Wade 1970 Abortifacient and teratogenic effects of triacetyl-6-azauridine in the monkey. Am. J. Obst. Gynaecol., 108: 272-281. Vorherr, H. 1970 The mode of interruption of pregnancy by 6-azauridine in mice and rats. Biochem. Pharmacol., 19: 1001-1006. Yoshihara, H., and C. P. Dagg 1967 Teratogenicityof 6-azauridine in inbred mice. Anat. Rec., 157: 345(A).
-
ABSTRACT Embryotoxic effects were compared of intramuscularly (im) and intraamniotically (ia) administered 6-azauridine (Riboazauracil Spofa) in random-bred mice H-Velaz. Effects of single doses (0.25 mg, 2.5 mg, 25.0 mg and 250.0 mg for im and 0.0025 mg, 0.025 mg, 0.25 mg and 2.5 mg for ia administration) on days 11, 12,13 and 14 were evaluated as a sum of dead fetuses and fetuses with cleft lip and/or palate, fetuses with limb deformities and fetuses with deformities constituting the syndrome of caudal regression (hypoplasia of the caudal part of the trunk, absent tail, short tail, curled tail). Considering the sensitivity peaks of the morphogenetic processes which were observed, the doseresponse relationships, the transformation of the teratogenic to a lethal effect and critical period extension with increasing doses, i t was found that the effects of ia and im administered 6-azauridine did not differ. It was concluded that ia administered 6-azauridine had direct effect on embryonic morphogenetic processes and that this, too, was the essential mechanism of embryotoxicity of im administered 6-azauridine. The value of the intraamniotic technique for establishing the direct embryotoxic effect is discussed. Teratologists frequently consider it necessary to analyze the direct interactions of teratogens with embryonic morphogenetic processes. However, opinions differ as to what experimental technique should be used. Staples (‘75), for example, states that “the intraamniotic application is not suitable, as the procedure itself induces cleft palate and only can be performed relatively late in pregnancy.” The present paper aims at: (1) comparing direct and transplacental effects of 6-azauridine, (2) emphasizing that if fine enough technique is applied no danger of nonspecifically induced effects arises (Dostal, ’711, and (3) demonstrating that intraamniotic application has distinct value for determining direct effects of teratogens. MATERIALS AND METHODS
Random-bred albino mice (H-Velaz) were kept in rooms without artificial light and fed DOS 4 (Velaz) diet and water ad libitum. The females were mated overnight and the day of the vaginal plug was considered as being day 1 of pregnancy. 6-azauridine (Riboazauracil Spofa) was administered intramuscularly TERATOLOGY (1979) 19: 143-148.
(im) in single doses of 0.25 mg, 2.5 mg, 25.0 mg and 250.0 mg in 0.1 ml of distilled water on days 11,12,13 or 14. The dose of 250.0 mg was administered in 1.0 ml of distilled water. Three females were used for each day and dose. The intraamniotic injections (ia) were performed from day 9 to day 15 of pregnancy (for method see Dostal, ’71). 6-azauridine (6AzUrd) was injected in doses of 0.0025 mg, 0.025 mg, 0.25 mg and 2.5 mg in 2 microliters of distilled water. Offspring of 5 females were used for each day and dose. In each litter 5 fetuses were injected with 6-AzUrd, the remaining fetuses serving as intact controls. The females were killed on day 17 of pregnancy by cervical dislocation, the fetuses were removed from the fetal membranes, weighed and examined under a dissecting microscope. Embryotoxicity was evaluated as a sum of dead fetuses (including resorptions) and fetuses with malformed limbs (including the presence of hemorrhagic blisters), fetuses with defects constituting the syndrome of caudal regression (marked hypoplasia of the caudal part of the fetus, absent tail, short tail, Received Aug. 22, ’77. Accepted Aug. 18,‘78.
143
144
M. DOSTAL AND R. JELINEK TABLE 1
Effects of im administration of 6-azauridine Done of 6-azauridine ~~
0.25 mg
2.5 mg
D
M
Day
N
DfM
%
%
N
D+M
11 12 13 14
53
2 1
3.8 2.0
0
21 2
0
0
0
1
0
4
32 25 20 34
50
56 25
0
D
M
%
%
N
D+M
6.3 63.3 4.0 4.2
26
26
31
0 0
32 18
31 0
0 0
250.0 mg
25.0 mg
0 0
0
D
M
%
%
34.6 100.0 0 100.0 0 0 0 0
D
M
N
DtM
%
%
31 28 25 32
31 28 22 24
100.0 3.6 0 0
0
100.0 88.0 75.0
N, number of fetuses; D, dead fetuses; M, malformed fetuses. The percent of malformed fetuses is derived from living fetuses.
TABLE 2
Effects of ia administration of 0.0025mg of 6-azauridine Experimental fetuses Day
9 10 11
12 13 14 15
N
D+M
25 25 25 25 25 25 25
5 9 24 0 1 1
2
D
M
%
%
16.0 4.8 24.0 15.8 20.0 95.0 0 0 4.0 0 0 4.0 8.0 0
Control fetuses
N
DfM
24 20 27 32 14 23 16
2 5 9 4 2 2 0
D
M
%
%
8.3 0 0 25.0 22.2 14.3 12.5 0 14.3 0 4.3 4.5 0
0
N.numberoffetuses; D, deadfetuses; M, malformdfetuses. The percentof malformed fetusesis derivedfrom1ivingfetuses.Control fetuses are the non-injected fetuses from experimental litters.
curled tail) and fetuses with cleft lip and/or palate. The x 2 test and for small marginal frequencies Fisher’s test were used for statistical evaluation of the results. RESULTS
The total number of fetuses and the percentage of dead and malformed fetuses (from survivors) are given in table 1. Of the four doses used the first dose resulting in considerable effect was 2.5 mg. When administered on day 11 it induced 63.3%malformed fetuses. The 25.0 mg dose caused an increase in the frequency of malformed fetuses on day 11 and had embryotoxic effects on day 12 as well. The application of 250.0 mg resulted in increased lethality (consequently decreasing the number of malformed fetuses) and further extension of the critical period-this dose was effective even on days 13 and 14. The effects of ia administered dose of 0.0025 mg 6-AzUrd from day 9 to day 15 of pregnancy is shown in table2. Peak sensitivity to this dose of 6AzUrd was reached on day 11 when the inci-
dence of malformed fetuses (95.0%)differed significantly (P < 0.05) from that of dead fetuses in intact controls (14.4%).The proportion of affected fetuses on days 9 and 10 did not differ significantly (P > 0.05) from that of dead fetuses in intact controls and virtually no effect was observed with this dose on days 12, 13,14 and 15. The results obtained with effective doses on days 12, 13, 14 and 15 are summarized in table 3. All three doses (0.025 mg, 0.25 mg and 2.5 mg) had embryotoxic effects when applied on days 12, 13 and 14, the effects on day 12 being dose-related for both teratogenicity and lethality. Embryotoxicity of the 0.25 mg and 2.5 mg doses was lower on day 13 than on day 12. A similar decrease for lethality was found on day 14. The medium and highest doses, however, showed high teratogenic effects due to the incidence of fetuses with cleft palate. On day 15 even the highest dose (2.5 mg) had no visible significant effects. The relative frequencies of fetuses with cleft palate induced by ia and im administration of 6-AzUrd is given in table 4. The effects of both ia and im administration are identical with regard to the occurrence of two peaks of sensitivity, namely on days 11 and 14 after ia administration and on days 11 and 13 after im administration (the difference between 88.0% fetuses with cleft palate observed on day 13 and 75.0%on day 14 is nonsignificant). The incidence of fetuses with malformed limbs after ia and im administration is shown in table 5. Peak sensitivity to im administered 6-AzUrd was found on day 11 with the dose of 25.0 mg and on day 12 with the 250.0 mg dose (due to its high lethal effect on day 11). After ia treatment the limb malformations were induced on days 11 and 12 and the maximal sensitivity was found on day 11. The occur-
145
EMBRYOTOXICITY OF 6-AZAURIDINE TABLE 3
Effects of ia administration of 6-azauridine Dose of 6-azauridine 0.25 mg
0.025 mg
2.5 mg ~
E
c
E E
14 15
M
D
M
D
M
N
DiM
%
%
N
D+M
%
%
N
DiM
%
%
25 25 25 19 25 16 25 16
9 2 13 3 7 2 0 0
12.0 8.0 28.0 15.8 0 6.3 0 0
27.3 0 33.3 0 28.0 6.7 0 0
25 16 25 24 25 20 25 21
20 2 10 2 21 2 3
40.0 0 20.0 8.3 20.0 5.0 0 4.8
66.7 12.5 25.0 0 80.0 5.3 12.0 0
25 11 25 17 25 18 25 25
25 6 23 5 23
84.0 18.2 64.0 11.8 24.0 27.8 12.0 4.0
100.0 44.0 77.8 20.0 89.5 46.2 4.5 4.2
Day
l3
D
1
11
4 2
N, number of fetuses; D. dead fetuees; M.malformed fetuses. The percent of malformed fetuses is derived from living fetuses.
TABLE 4
The percent of the fetuses with cleft palate (% CP) from dead and malformed fetuses f D after ia and im administration of 6-azauridine
+ M)
Dose of 6-azauridine
Day
12
; :;
13
i?
11
14
:
im in
0.25 mg 0.0025 mg
im ia
2.5 mg 0.025 mg
im ia
25.0 mg 0.25 mg
im ia
250.0mg 2.5 mg
D+M
%CP
DiM
%CP
D+M
%CP
D+M
%CP
51 20 49 25
0 35.0 0 0 0 0 0 0
30
-
-
0
17
70.6
-
-
0
-
24 22 20 18 34 25
0 9.1 0 11.1 0 28.0
31 15 32 20 18 20
0 6.7 0 20.0 0 80.0
27 4 25 9 32 19
55.6 0 88.0 66.7 75.0 84.7
56
24 25 25
-
0
TABLE 5
The percent of the fetuses with limb deformities f%limbs) from dead and malformed fetuses f D + M ) after ia and im adminktration of 6-azauridine Dose of 6-azauridine
Day
12
; :;
13
i;
11
14
ir
im ia
0.26 mg 0.0025 mg
im ia
2.5 mg 0.025 mg
im ia
25.0 mg 0.25 mg
im ia
260.0mg 2.5 mg
D+M
%limbs
D+M
%limbs
D+M
%limbs
DiM
%limbs
51 20
0 75.0 0 0 0 0
30
-
6.7
-
17
100.0
-
-
-
24 22 20 18 34 25
0 9.1 0 16.7 0 0
31 15 32 20 18 20
38.7 66.7 0 5.0 0 0
21 4 25 9 32
96.3 (25.0) 0 0 0 0
49
25 56 24 25 25
0 0
-
0
19
0
146
M. DOSTAL AND R. JELINEK TABLE 6
The percent of the fetuses exhibiting various forms of the syndrome of caudal regression (% SCR) from dead and malformed fetuses (D+MI after ia and im administration of 6-azauridine Dose of 6-azauridine
Day ~
12
; ;
13
;i
11
14
;r
im ia
0.25mg 0.0025mg
im ia
2.5 mg 0.025 mg
im ia
25.0 mg 0.25 mg
im ia
250.0mg 2.5 mg
D+M
%SCR
D+M
%SCR
D+M
%SCR
D+M
%SCR
0
30
-
63.3
17
-
100.0
-
-
-
24 22 20 18 34 25
4.2 27.3 0 16.7 0 0
31 15 32 20 18 20
100.0 46.7 0 10.0 0 20.0
27 4 25 9 32 19
100.0 (100.0) 24.0 44.0 3.1 5.3
51 20 49 25 56 24 25 25
80.0 0 0
0 0 0 0
-
0
0
rence of the limb malformations on day 13 is tion of orotidine 5-phosphate decarboxylase not dose-related and has to be considered as with the resultant blocked use of orotidine 5-phosphate in pyrimidine base synthesis being accidental. Similar conclusions were drawn when an- (Handschumacher et al., '62; Skoda, '75). The alyzing the occurrence of malformations con- embryotoxic effects of 6-AzUrd were examstituting the syndrome of caudal regression. ined in a number of studies on chick embryos After both ia and im administration the peak (Grafnetterova e t al., '66; Jelinek, '71, '72; sensitivity appeared on day 11. Increasing Rychter and Jelinek, '71, '72; Rychterova et doses resulted in transforming the teratogenic al., '731, mice (Saunders et al., '61; Vorherr, into a lethal effect and also in inducing the '70; Yoshihara and Dagg, '67), rats (Gutova, teratogenic effect on day 12 and after ia ad- '68; Gutova et al., '71a), rabbits (Saksena and ministration even on day 13. The specificity of Chaudhury, '70) and monkeys (Vanwagenen the teratogenic effects of ia injected 6-AzUrd et al., '70). on day 1 3 is also confirmed by the high mortalBecause of its pharmacokinetic characterisity rate. On the other hand, the incidence of tics 6-AzUrd appears to be an ideal model ter24.0%of affected fetuses after im administra- atogen. 6-AzUrd is not further metabolized tion of 250.0 mg of 6-AzUrd on day 13 is non- following initial phosphorylation, is freely dissignificant assuming that no affected fetus tributed in body fluids and is rapidly elimiwould occur in the control group. nated from mammalian organisms. In clinical Summarizing the overall embryotoxic ef- studies 75% of the administered 6-AzUrd was fects and the results of the analysis of inci- excreted within 4 hours (Handschumacher e t dence of particular malformations we can con- al., '62). The transplacental passage of 6clude that the embryonic processes studied AzUrd was demonstrated in rats (Gutova, '68, react to ia and im administration of 6-AzUrd Gutova et al., '71b) and mice (Raika et al., '66; in a similar way. They also exhibit such char- Jelinek et al., '77). acteristics of teratogen-morphogenetic procIn our experiments the doses for the intraess interaction as the dose-response relation- amniotic injections were inferred from the ships, the transformation of the teratogenic embryotoxic dose for the chick embryo (Jeinto a lethal effect and the extension of the linek, unpublished data). The doses for im adcritical period in dependence on increasing ministration were increased by two orders doses. since i t was found that after the ia administration of labeled 6-AzUrd the radioactivity of DISCUSSION fetuses had amounted to about 1%of the Azapyrimidine nucleoside 6-azauridine, dis- injected radioactivity (Jelinek et al., '77). A covered in 1957 (Handschumacher, '57; Skoda similar level of labeled cortisol intake in et al., '57) interferes with the metabolism of mouse fetuses was found by Dostal and Musil pyrimidine nucleotides (Skoda, '75). The basic ('74,'77). As concerns the nonspecific teratomechanism of its action consists in the inhibi- genic effect of t h e intraamniotic injection
EMBRYOTOXICITY OF 6-AZAURIDINE
(induction of cleft palate-Trasler et al., '561, we have proved that it can be eliminated by using fine glass micropipettes (Dostal, '71). The intraamniotic technique has been in routine use in our laboratory; so that the dose-response relationship was tested rather than using control groups with the 6-AzUrd solvent. The dose-response relation is clearly visible in the experimental groups from day 12 to day 14; the lowest dose is without effect. On days 9,lO and 11 the procedure is complicated by reduced transparency of the uterus and fetal membranes. The level of nonspecific lethal effect is somewhat higher than on later stages, but in the majority of experimental groups it does not exceed 20%of the injected fetuses. This level of nonspecific effect does not, moreover, significantly differ from mortality in intact controls (table 2). The same comparison on days 12, 13 and 14 shows that with the 0.25 mg and 0.025 mg doses, the embryotoxic effects are limited to the injected fetuses. However, both the injected and intact control fetuses are affected by the 2.5 mg dose. Here is the possibility that 6-AzUrd either spreads within the uterus or that its transplacental recirculation occurs through the maternal organism to the whole litter (the total dose for one litter is 5 x 2.5 mg). The second explanation seems to be more probable, namely because a single 12.5 mg dose administered to a pregnant female is teratogenic and the yolk sac may be a barrier to 6-AzUrd. This follows from experiments in which 0.05 ml of 3.75%6-AzUrd injected between the uterine wall and fetal membranes to all the fetuses in one uterine horn of the rat on day 12 of pregnancy had no effect (Gutova, '68). A similar level of sensitivity to im administered 6-AzUrd as in our experiments was found by Yoshihara and Dagg ('67). They reported that a n intraperitoneal injection of 2 micromoles of 6-AzUrd to females of C57BL/ 10 mice induced polydactylous hind feet in approximately 20%of fetuses. The results of the intraamniotic administration of 6-AzUrd confirmed t h e direct teratogenic effect of 6-AzUrd on the fetuses. The similarity of the effects of 6-AzUrd administered ia and im testifies that 6-AzUrd given to pregnant female acts primarily by directly affecting the fetus. Nevertheless, Gutova ('68) observed the accumulation of 6-AzUrd in placentae and believed t h a t a high 6-AzUrd concentration could damage placental functions.
147
The same situation was found by JiFiEka et al. ('65). Jelinek et al. ('77) analyzed the dynamics of this accumulation and its dose dependence and concluded that the phenomenon had two aspects. It evidently impeded a rapid increase in the teratogen level in the fetus. However, in these circumstances, the placental morphogenetic processes could be exposed to much higher levels of the teratogen than the embryonic morphogenetic processes. The question arises whether such a situation can negatively affect the development of the embryo and whether it can occur even when a teratogen is administered ia. Dostal and Musil ('74, '77) studied radioactivity distribution in the fetoplacental unit after ia administration of labeled cortisol and found that the radioactivity retained in the placentae was only 2-3 times higher than that retained in the fetuses. These results, however, should be completed with experiments using higher concentrations of the teratogen in the amniotic fluid. The comparisons of the incidences of individual malformations in experiments with ia and im administration have proved that the same morphogenetic processes may be influenced in the same developmental periods. A more detailed analysis of individual types of malformations (e.g., those of limbs) could not be made owing to the insufficient quantity of material. The decreasing sensitivity of the fetuses with the increasing stage of pregnancy is obviously the consequence of the differing sensitivity of particular morphogenetic processes to the net inhibition of pyrimidine base synthesis which remains at approximately the same level when comparable doses are administered on days 6 and 15 of pregnancy to mice (Raska et al., '66). On the basis of the results we have obtained the view appears justified that the intraamniotic injection technique is useful in teratology. This technique is suitable for proving or excluding the possibility of the direct effect of drugs on the fetus and may be used not only for analytical purposes but also as a rapid screening technique (Dostal, '77). The value of establishing the direct embryotoxicity of a drug in the context of screening systems is another question. LITERATURE CITED Dostd, M. 1971 Morphogenesis of cleft palate induced by exogenous factors. 111. Intraamniotic application of hydrocortisone in mice. Teratology, 4: 63-68. 1977 Intraamniotic administration of drugs in
148
M. DOSTAL AND R. J E L ~ N E K
mammals. Proceedings of the Third Symposium on toxicological testingfor safetyof new drugs, Prague, 1976, in press. Dostal, M., and J. Musil 1974 Kinetics of 3H-cortisol injected into amniotic sac of the mouse fetus. Teratology, 10: 307-308(A). 1977 Kinetics of ~ o r t i s o l - l , 2 - ~administered H intraamniotically. Folia Biologica (Prague),23: 212-219. Grafnetterova, J., E. Grossi, R. Fumagalli, P. Morganti and D. Grafnetter 1966 Effects of 6-azauridine on the developing chick embryo. Neoplasma (Bratislava), 13: 251-258. Gutova, M. 1968 KdEinku 6-azauridinu v prenatalnim obdobi -+oje. Thesis, Charles University, Prague. Gutova, M., J. Elis, and H. Raikova 1971a Teratogenic effect of 6-azauridine in rats. Teratology, 4: 287-294. 1971b Transfer of 6-azauridine through the placental barrier in the rat. Neoplasma (Bratislava), 28: 529-531. Handschumacher, R. E. 1957 Metabolites of 6-azauracil formed by Streptococcus faecalis. Federation Proc., 16: 191-198. Handschumacher, R. E., P. Calabresi, A. D. Welch, V. H. Bono, Jr., H. J. Fallon and E. Frei 1962 111. Summary of current information on 6-azauridine. Cancer Chemother. Rep., 21: 1-18. Jelinek, R., Z. Rychter and V. Seichert 1970 Syndrome of caudal regression in the chick embryo. Folia Morphol. (Prague), 18: 125-137. Jelinek, R., Z. Rychter and E. Klika 1971 Syndrome of caudal regression and dysrhaphic malformation of the spinal cord. Folia Morphol. (Prague), 19: 58-70. Jelinek, R., R. Tykva, and M. Seifertova 1977 6-azauridine intake following intraamniotic application in mice. Folia Morphol. (Prague), in press. Jirieka, J., K. Smetana, I. Janku, J. Elis and J. Novotny 1965 Studies on 6-azauridine and 6-azacytidine. I. Toxicity studies of 6-azauridine and 6-azacytidine in mice. Biochem. Pharmacol., 14: 1517-1523. Raika, K., M. S. Zedeck and A. D. Welch 1966 Relationship between the metabolic effects and the pregnancy-inter-
rupting property of 6-azauridine in mice. Biochem. Pharmacol., 2136-2138. Rychter, Z., and R. Jelinek 1971 0 zpilsobu hodnoceni S i n k u teratogenniho agens (6-azauridinu) na pozdnich vjvojovych stadiich. cs. Fysiologie, 20: 527-540. 1972 Z m h y v postupu vaskularisace komorove svaloviny srdeEni u zarodku kuiete p pfisubeni ruznych davek 6-azauridinu. 6 .Fyziologie, 21: 51-57. Ryehterova, V., Z. Rychter and R. Jelinek 1973 The effect of Bazauridine on the ventricular myocardium of chick embryos (contribution to the teratogenic action of drugs). Teratology, 8: 235(A). Saksena, S. K., and R. R. Chaudhury 1970 The antifertility Part 11. In rabeffect of 2',3',5',-tri-O-acety1-6-azauridine. bits. Indian J. Med. Res., 58: 374-376. Saunders, M. A., B. P. Wiesner and J. Yudkin 1961 Control of fertility by 6-azauridine. Nature, 189: 1015-1016. Skoda, J. 1975 Azapyrimidine Nucleosides. In: Handbook of Experimental Pharmacology. 0. Eichler. A. Farah, H. Herken, A. D. Welch, &. Volume XXXVIIII2, A. C. Sartorelli and D. G. Johns, eds. Springer Verlag Berlin-Heidelberg-New York, pp. 348-372. Soda, J., V. F. Hess, and F. Sorm 1957 The biosynthesis of 6-azauracil riboside by Eacherichia coli growing i n t h e presence of 6-azauracil. Experientia (Basel), 13: 150-151. Staples, R. E. 1975 Potential of direct application techniques for detection of teratogens. In: New Approaches to the Evaluation of Abnormal Development. 11. Symposium on Prenatal Development. D. Neubert and H. J. Merker, eds. Berlin, pp. 71-81. Trader, D. G., B. E. Walker and F. C. Fraser 1956 Congenital malformations produced by amniotic s a c puncture. Science, 124: 439. Vanwagenen, G., R. C. DeConti, R. E. Handschumacher, and E. M. Wade 1970 Abortifacient and teratogenic effects of triacetyl-6-azauridine in the monkey. Am. J. Obst. Gynaecol., 108: 272-281. Vorherr, H. 1970 The mode of interruption of pregnancy by 6-azauridine in mice and rats. Biochem. Pharmacol., 19: 1001-1006. Yoshihara, H., and C. P. Dagg 1967 Teratogenicityof 6-azauridine in inbred mice. Anat. Rec., 157: 345(A).
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