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Diaplacental carcinogenic effects of 5-azacytidine in NMRI-mice
Cancer Letters, 27 (1985) 81-90 Elsevier Scientific Publishers Ireland Ltd.
DIAPLACENTAL IN NMRI-MICE
CARCINOGENIC
81
EFFECTS OF 5AZACYTIDINE
WOLFGANG SCHMAHLa, ELISABETH GEBER* and WALTER LEHMACHERb *Abteilung fiir Nuklearbiologie Systemforschung, Gesellschaft D-8042 Neuherberg (F.R.G.)
and bMEDIS-Inetitut fiir medizinieche Informatik und fiir Strahlen- und Umweltforschung m.b.H. Miinchen,
(Accepted 15 October 1984) (Revised version received 28 January 1985) (Accepted 21 October 1985)
SUMMARY
5-Azacytidine was applied to NMRI-mice (l-2 mg/kg) either on gestation day 12,14, or 16. In the first case it mainly induced leukemias, while in the latter experiments leukemias, lung adenomas and soft tissue sarcomas represent the main effects. The experiments performed on gestation day 14 led to tumor rates below the spontaneously occurring tumor frequencies in NMRI-mice. There is a clear-cut inverse dose-response relationship in leukemia induction, as the higher dose principally shows a lower degree of effectiveness. This, as well as a reduction of tumor frequency below control levels after application of this drug on day 14, can be explained by an “overkill” effect. The cytotoxic and embryolethal efficacy of the agent thus surpasses the transformation effects at the cellular genome. While a negative correlation between the general embryotoxicity of azacytidine and the simultaneous tumor inducibility is to be observed, there is no correlation at the target organ level between the embryotoxic and the carcinogenic effects.
INTRODUCTION
5-Azacytidine is a base analogue incorporated into cellular DNA during the S-phase of the generation cycle [ 11. It inhibits DNA synthesis, exerts lethality predominantly in the S-phase and thus also inhibits the mitosis rate of cells in culture. Azacytidine is apparently similar to 8-azaguanine in its action, particularly in its capacity to disaggregate polysomes [2]. It has been reported to cause chromosome aberrations in cultured mammalian cells [3,4] and to act as a mutagen in Vicia f&a [5]. Mutagenicity of azacytidine was also seen as a DNA repair-related event in the trpE8 strain of 0304-3835/85/$03.30 0 1985 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
82
Salmonella typhimurium, but not in those Salmonella strains conventionally used for the Ames test [6]. This compound has recently proved to be a valuable tool in experimental cell biology as it leads to an undermethylation of the nuclear DNA [ 71 and thus produces effects on gene regulation activity [ 81. Most of the embryotoxic effects of 5-azacytidine [9] can be deduced from these cellular influences. Azacytidine also induces cell transformation in vitro [4], simultaneously with chromosome changes and retrovirus activation [lo]. As azacytidine was also brought into relationship with some forms of tumors in adult mice [ 11,121, we consequently studied in the present experiments the diaplacental carcinogenic potency of this drug in comparison with the organ-specificities of the acute embryotoxic effects determined in a recent study [ 91. MATERIALS
AND METHODS
The breeding conditions of the NMRI-mice and the experimental procedure were basically the same as described recently [9]. Azacytidine was purchased from Sigma Chemie as a pure, crystalline powder. It was dissolved in sterile saline and used within the following hour. Applications of 5azacytidine to pregnant mice were performed by a single intraperitoneal injection at 0900 h of either saline (controls) or of the azacytidine solution. This occurred either on day 12 p.c., day 14 p.c. or day 16 p.c. On each day 2 doses of azacytidine were used; 1 mg/kg and 2 mg/kg. The number of animals in the different groups is listed in Table 1. The abbreviations for the different experimental groups refer to the azacytidine dose (1 mg/kg = lA, 2 mg/kg = 2A) and the day of application. The offspring which were whelped at regular times were registered and weighed. Stillbirths and neonatal deaths were removed immediately. The observation period lasted for a maximum of 960 days. All animals were autopsied either at this time or after their spontaneous death or when severely ill. As far as necessary for the evaluation, organs were also processed by histological techniques. This material was sectioned at 6 pm and stained with hematoxylin and eosin. The tumor frequency was registered per litter and subsequently the mean percentage + standard error of mean (S.E.M.) was calculated. The experimental results were statistically evaluated by application of the x2-test, the exact Fisher test and the Student test. RESULTS
Neonatal da to The mean litter size was not affected by any treatment (Table 1). The frequency of stillbirth was not significantly changed after application of
53 34 33 37 34 32 35
Controls lA12 2Al2 lA14 2A14 lA16 2A16
12.8 12.5 11.8 12.7 12.8 12.1 13.1
+ 0.7 f 0.7 It 0.6 f 0.7 f 0.7 -k 0.6 50.8
Mean litter size + S.E.M.
99.4 99.1 95.6 98.1 92.9 98.5 97.7
Live offspring (%)
1.41 1.23 1.19 1.17 1.15 1.37 1.34
f 0.07 zt 0.04b * 0.05b” * o.03c +- 0.03= * 0.05 +-0.06a 11.7 19.7 32.2 19.7 42.8 14.1 21.0
GESTATION
677 422 376 463 404 382 424
At birth
572 323 223 349 198 313 309
(293 (165 (113 (178 ( 97 (153 (158
'P < 0.05 compared to controls. bP < 0.01 compared to controls. 'P < 0.001 compared to controls.
+ + + + + + +
279) 158) 110) 171) 101) 160) 151)
DAYS
At the end of the study, total (d + 0)
No. of offspring
ON DIFFERENT
Mortality rate during the weaning period (%)
WITH 5-AZACYTIDINE
Mean offspring weight (g) at birth
DIAPLACENTALLY
Significances as evaluated with the x’-test after Yates correction. dP < 0.05 compared to the corresponding group of the same gestation day. eP < 0.01 compared to the corresponding group of the same gestation day. 'P< 0.001 compared to the corresponding group of the same gestation day.
No. of dams
DATA IN MICE TREATED
Treatment group
NEONATAL
TABLE 1
84
1 mg/kg either on day 12,14, or 16. In contrast, a dose of 2 mg/kg raised the stillbirth rate both on day 12 p.c. (4.1%) and 14 p.c. (7.1%), as well as 16 p.c. (2.3%). The mean birthweight was significantly (P < 0.05; Student test) reduced in ah treatment groups with the exception of lA16; within the 1-mg dosages it was most severely affected after treatment on day 14 p.c. The mortality rates until weaning were significantly (P < 0.05) increased in all groups with the exception of lA16. An additional percentage of animals were lost for evaluation during the course of the study. This was mostly due to sudden deaths and already severe autolysis until the carcasses were found. Each group showed a highly balanced sex distribution. Life span and general tumor frequency (Table 2) Within a distinct treatment group the survival rates did not differ significantly between the 2 sexes. Groups lA12, lA16, and 2A16 showed a quite steep initial decrease of the number of surviving animals. This effect was more pronounced in lA12 and in lA16, in contrast to the corresponding groups (2A12 and 2A16) with the higher dosage of azacytidine. The total tumor incidence in the control animals was 63.1 + 2.8%. In treatment groups lA12,1A16 and 2A16 the tumor incidence was significantly increased to 93.0 f 6.5%, 129.6 T 8.3%, and 109.2 + 4.4%, respectively. The values for groups 2A12, lA14 and 2A14 were not statistically different from the control value. The mean frequency of tumor-bearing animals within the litters amounted in the controls to 52.6 f 2.3% (d) TABLE
2
LIFE SPAN AND TUMOR INCIDENCE IN THE OFFSPRING DIAPLACENTALLY WITH 5-AZACYTIDINE Treatment group
Controls lA12 2A12 lA14 2A14 lA16 2A16
No. of animals (n)
d(293) O(279) d(165) O(158) d(l13) O(110) d( 178) O(171) d( 97) O(101) d(153) O(160) d(158) 9(151)
OF MICE TREATED
400 days
600 days
800 days
Tumorbearing animals (%)
77.7 73.5 59.6 62.0 79.6 80.0 83.7 86.6 83.5 88.1 49.7 46.9 69.0 62.9
48.3 51.2 48.2 51.3 45.1 56.4 56.2 66.1 46.4 45.5. 23.5 19.4 30.4 29.1
26.4 30.8 36.7 39.9 19.5 27.3 27.0 42.7 33.1 21.8 13.7 8.7 12.0 17.9
52.6 51.1 65.4 62.1 46.5 40.4 46.3 40.9 49.6 59.3 72.5 78.1 78.9 73.0
Survival rates (%) after
Index marks for significances, see Table 1.
+ 2.3 + 1.9 + 3.7a f 4.1a +- 4.2*e + 3.8Ke + 4.0’ f 3.7a f 4.2 f 4.8a*e + 3.5’ f 3.0’ + 3.1’ f 3.4=
85
and 51.1 + 1.9% (O), respectively. This value was significantly increased in lA12, lA16 and 2A16, but depressed in 2Al2 and in lA14. These results are also reflected by the number of tumors per affected animals (tumor multiplicity): the control value was 1.22. A significant increase was only observed in lA12-1.45, in group lA16-1.69 and in 2A16-1.43; the values of the other groups remained within the control range. Frequencies of distinct tumors Leukemias and lymphomas (Table 3): The frequency of these tumors was significantly increased in both sexes of animals from group lA12,1A16 TABLE
3
FREQUENCY OF LEUKEMIC DISEASES EXPOSURE WITH 5-AZACYTIDINE Treatment group
No. of animals (n)
% Leukemias and lymphomas
IN MICE OFFSPRING
AFTER
PRENATAL
% Lymphatic leukemia
% Reticulocellsarcoma
% Myeloid leukemia
(no.)
(no.)
(no.)
(no.) Controls
lA12
2Al2
lA14
2A14
lA16
2A16
d( 293)
28.7
+- 2.7
12.8 + 1.9
12.5 + 2.2
3.1 + 0.8
O(279)
(84) 29.3 * 3.4
(38) 14.9 + 2.4
(37) 12.0 -I 2.0
(9) 2.6 + 1.2
d( 165)
49.1
(82) +4.7c
34.7
(41) + 4.4c
(34) 12.0 + 2.4
(7) 2.4 + 1.1
9(158)
(81) 50.8 + 4.2’
30.9
(57) * 3.7c
(20) 17.3 + 3.2
(4) 2.0 -I 1.6
d(113)
(80) 25.0 + 5.2”’
(50) 10.0 f 3.4’
(27) 9.8 + 2.9
(3) 5.3 f 2.4d
Q(ll0)
23.;?!.6cf
lO.lf
(11) 6.1 + 2.4d
7.3 + 2.6a’e
d(178)
(26) 23.6 f4.0
(11) 7.4 f 3.1C
9(171)
(42) 15.4 + 3.4u
(13) 5.8 +- 1.7’
( 7) 11.9 * 1.9 (21) 7.4 dz l.ga
(6)
(8) 4.5 * 1.4 (8) 1.8 * 1.5 (3) -
9.5(::.7cf
(10) 7.3 + 3.7c
Q(lO1)
( 9) 14.0 + 2.7’
( 7) 3.0 * 2.0c
6.9 + 2.4b
d(153)
(14) 63.8 + 6.1’
( 3) 35.4 dz4.8’
( 7) 19.6 + 3.4a
(2) 8.6 * 2.2a
o(160)
60.9
(97) + 6.0’
35.0
(54) i 4.0c
(30) 20.3 + 3.0’
(13) 6.3 f 1.8a
d(158)
41.8
(98) f 3.3’”
27.2
(56) f 3.1’”
(32) 11.5 + 3.6e
(10) 3.8 + 1.2e
9(151)
38.1
(67) +- 4.2”
27.4
(43) f 3.7’”
d(97)
(41)
(57) Index
(11) 2.9’
marks for significances,
see Table
1.
( 2)
2.0 +- 1.5
(6) 3.3 + l.7e (5)
86
(P < 0.001; x2-test) and 2A16 (P < 0.06). In contrast, a significant depression of this tumor rate occurred in lA14 (P < 0.01) and 2A14 animals (P < 0.001); this value was only marginally below the control range in group 2A12. In general, there were no marked sex differences. Leukemia frequency in the lA12 animals was largely due to an increase of lymphatic leukemias. In contrast, all 3 subtypes of leukemia equally contributed to the increase of the total leukemia rate in the group lA16 animals. The decrease of leukemia frequency in the lA14 animals was mainly brought about by a reduced incidence of lymphatic leukemia. All these effects showed no sex dependency. Lung tumors (Table 4a): Neither treatment on day 12, nor on day 14 with 1 mg azacytidine had any effect on the frequency of lung tumors. The apTABLE 4a FREQUENCY OF NON-LEUKEMIC DISEASES IN MICE OFFSPRING AFTER PRENATAL EXPOSURE WITH 5-AZACYTIDINE Treatment group
No. of animals (n)
% Lung tumors (no.)
% Liver tumors (no.)
Controls
d(293)
19.5 +_1.8 (57) 19.1 + 2.5 (53) 18.2 i 3.3 (30) 20.9 f 3.5 (33) 19.6 + 3.6 (22) 20.1 * 5.3 (22) 16.3 f 3.4 (29) 18.4 f 3.6 (31) 47.3 f 5.8”’
4.8 + 1.2 (14) 3.9 +_0.9 (11) 9.2 + 2.1b (15) 3.9 zk1.3 ( 6) 9.9 + 3.8b (11) 8.2 z!z2.8a’d ( 9) 6.7 + 2.1 (12) 11.8 +- 2.3’ (20) 11.3 + 3.8b
42.6(:;.ScJ (43) 53.0 f 5.gc (81) 62.0 f 5.5’ (99) 49.5 + 5.3c (78) 54.3 f 4.6’ (82)
6.9 + 2.4d ( 7) 9.2 f 2.4b (14) 5.1 * 1.5
Q(279) lA12
d(165) Q(158)
2A12
d(113) Q(l10)
lA14
d(178) Q(171)
2A14
d( 97) 9(101)
lA16
d(153) Q( 160)
2Al6
d(158) Q(151)
Index marks for significances, see Table 1.
(11)
( 8)
11.2 -I 2.6b
(18)
3.4 f 1.8 ( 5)
% Soft tissue sarcomas (no.)
0.3 f 0.3 (1) 0.6 zk0.5 (1) 1.9 f 0.8 (3) 0.9 +_0.8 (1) 0.9 _+0.7 (1) 1.1 + 0.6 (2) 1.7 f 0.8 (3) 1.0 f 0.6 (1) 1.0 z!z0.7 (1) 1.9 f l.la (3) 1.2 + 0.8’ (2) 1.8 + l.la (3) 3.2 + l.gad (5)
87 TABLE 4b FREQUENCY OF NON-LEUKEMIC DISEASES IN MICE OFFSPRING AFTER PRENATAL EXPOSURE WITH 5-AZACYTIDINE Treatment group
No. of animals (n)
Controls
d(293) O(279)
lA12
2A12
lA14
d(165) Q(158) d(l13) Q(ll0) d(
178)
Q(171) 2A14
d(97) Q(101)
lA16
d(153) Q(160)
2Al6
d(158) Q(151)
% ovary tumors (no.)
-
13.3 * 2.1 (37) 12.1 f 2.6 (19) 5.4;
% Mammary tumors (no.)
3.6b*e
( 6) -
7.2k1.7 (20) 8.2+ 2.3 (13) 3.6i2.0M ( 4) -
96 Miscellaneous tumors (no.) 0.3 f 0.3 (1) -
1.2 + 0.5
(2) 1.8 + 0.8 (2) 1.7 * 0.5 (3) 0.6 + 0.3 (1) 2.0 f 0.4a
11.1 It 2.7 (19) -
7.0+ 2.3 (12) -
7.9k3.0 ( 6) -
6.9f 2.3 ( 7) -
3.1+1.2a@
6.9 f 2.2b (11) -
5.0f 1.6 ( 6) -
1.2 * o.5a
(2)
10.6 f 3.6 (16)
(3) 1.3 * 0.7*
(2)
5.9 f 1.7 ( 9)
(2)
1.9 +- 0.8’ (3) 0.6 + 0.3 (1)
Index marks for significances, see Table 1.
plication of 2 mg/kg on day 14 resulted in a marked increase (P < 0.001) of the lung tumor frequency in both sexes. Lung tumors were most effectively induced in group lA16 and in 2A16 animals, irrespective of their sex. These values were significantly different from the control values (P < 0.001, x2test). Liver tumors (Table 4a): Liver tumors were generally more frequent in azacytidine treated offspring than in controls. This increase was largely brought about by affected male animals in groups lA12, 2A12, 2A14, lA16 and 2A16 (P < 0.01; x2-test). In the latter groups, with exception of 2A12, the liver tumor incidence in males was also significantly (P < 0.05) higher than the incidence of liver tumors in the corresponding females. A significant (P < 0.001) increase of liver tumor frequency occurred in the female offspring only in group lA14. The latter value also surpassed signifi-
88
cantly the liver tumor frequency of the corresponding male offspring (P < 0.05). Soft tissue sarcomas (Table 4a): In the experimental animals these tumors were mostly situated in the subcutis of the legs and in the perianal region. Their frequency was significantly increased in lA16 and 2A16 animals of both sexes. The 3 cases which were observed in male offspring from group 2A16 were non-metastasizing angiosarcomas of the hindlegs. Other tumors: These are compiled in Table 4b. The miscellaneous tumors comprise cases of Harderian gland adenomas, intestinal tumors and tumors arising from the skin. DISCUSSION
The critical stages for inducing diaplacental carcinogenic effects with 5-azacytidine are inversely parallel to the phase-dependent embryotoxic effects of this compound. On gestation day 12, when mild embryotoxicity could be induced, there was a distinct increase in tumor frequency. The latter was largely due to leukemia induction. On gestation day 14, the time of maximum embryotoxicity, the carcinogenic effects had decreased below the level of spontaneous incidence, whereas a significant tumor response was induced on gestation day 16, a developmental stage without any effect according to the standard embryotoxicity evaluation [ 91. The tumor pattern following azacytidine treatment on day 16 was mainly restricted to leukemia, lung tumors and soft tissue sarcomas. There was also a marked, reverse dose-response relationship of tumor induction, especially in the case of leukemia, the lower dose (1 mg/kg) being significantly more potent than the higher dose (2 mg/kg) of azacytidine. It is reasonable to assume an accentuation of the cytolethal effects with an increasing dose. An eradication either of potentially transformed fetal cells or of fetuses with an increased tumor risk may be the consequence [13,14]. We propose that two different paths, depending on the gestation stage of azacytidine application, lead to leukemias on gestation day 12 and gestation day 16. The one path, during the early stage (day 12 p.c.) of differentiation of the immune system [ 151, is characterized by an almost exclusive induction of lymphatic leukemias, whereas the same treatment on day 16 p.c. apparently provoked a pathogenetic path including all subtypes of leukemia at nearly equal rates. An inexplicable result is the depression of leukemia incidence below the control value after axacytidine application on day 14 p.c. Preliminary studies with the stillbirth and neonatal deaths in groups lA14 and 2A14, however, presented no results which would indicate a selective eradication of those littermates with an excessive risk of leukemia. Azacytidine is known from experiments in adult mice as a potent inducer of leukemia in the AKR strain [ 121 and of lung tumors in the A strain [ 111.
89
The latter results of Stoner (Shimkin model) are obviously confirmed by our results. However, it is quite interesting that lung tumors were induced by azacytidine only on days 14 and 16, but not on day 12. Thus, it may be suggested that a certain degree of cellular differentiation of lung tissue is a necessary requisite to enable carcinogenic transformation. Obviously, the spectrum of cancerogenic effects upon fetal tissues does not differ substantially from those effects in adult animals. In contrast, the teratogenic effects of 5-azacytidine are mainly directed against the developing CNS and the fetal liver [16,17]. Thus, the present results obviously confirm the opinion that the organ-specific site of teratogenic action is strictly different from the preferential sites of cancerogenesis [ 181. Our recent embryotoxicity study on azacytidine [ 91 likewise presented no arguments for an abnormal cellular differentiation in those fetal tissues which proved to be most susceptible to carcinogenesis in the present study (e.g. lung tissue). Consequently, we assume that any specific effects of azacytidine leading to carcinogenesis are only detectable during the postnatal period. REFERENCES 1 Li, L.H., Olin, E.J., Fraser, T.J. and Bhuyan, B.K. (1970) Phase specificity of 5-azacytidine against mammalian cells in tissue culture. Cancer Res., 30, 2770-2775. 2 Cihak, A., Vesely, H. and Sorm, F. (1968) Thymidine kinase and polyribosome distribution in regenerating rat liver following 5-azacytidine. Biochim. Biophys. Acta, 166, 277-286. 3 Tobey, R.A. (1972) Effects of cytosine arabinoside, daunomycin, mithramycin, azacytidine, adriamycin, and camptothecin on mammalian ceU cycle traverse. Cancer Res., 32, 2720-2725. 4 Harrison, J.J., Anisowicz, A., Gadi, I.K., Raffeld, M. and Sager, R. (1983) Azacytidine-induced tumorigenesis of CHEF/18 cells: correlated DNA methylation and chromosome changes. Proc. Natl. Acad. Sci. U.S.A., 80, 6606-610. 5 Fucik, V., Michaelis, A. and Rieger, R. (1970) On the induction of segment-extension and chromatid structural changes in Vicia faba chromosomes after treatment with 5-azacytidine. Mutat. Res., 9, 599-606. 6 Podger, D.M. (1983) Mutagenicity of 5-azacytidine in Salmonella typhimurium. Mutat. Res., 121, l-6. 7 Jones, P.A. and Taylor, S.M. (1980) Cellular differentiation: cytidine analogs and DNA methylation. CeII, 20, 85-93. 8 Lindahl, T. (1981) DNA methylation and control of gene expression. Nature, 290, 363-364. 9 Schmahl, W., TSrSk, P. and Kriegel, H. (1984) Embryotoxicity of 5-azacytidine in mice. I. Phase- and dose specificity studies. Arch. Toxicol., 55, 143-147. 10 Tennant, R.W., Otten, J.A., Myer, F.E..and Rascati, R.J. (1982) Induction of retrovirus gene expression in mouse cells by some chemical mutagens. Cancer Res., 42, 3050-3055. 11 Stoner, G.D., Shimkin, M.B., Kniazeff, A.J., Weisburger, J.H., Weisburger, E.K. and Gori, G.B. (1973) Test for carcinogenicity of food additives and chemotherapeutic agents by the pulmonary tumor response in strain A mice. Cancer Res., 33, 306!+ 3085.
90
12 Vesely, J. and Clhak, A. (1973) High-frequency induction in vivo of mouse leukemia in AKR strain by 5-azacytidine and 5-iodo-2-deoxyuridine. Experientia, 29,11321133. 13 Goerttler, K., Loehrke, H., Hesse, B., Milz, A. and Schweizer, J. (1981) Diaplacental initiation of NMRI mice with 7,12-dimethylbenz(a)anthracene during gestation days 6-20 and postnatal treatment of the F,-generation with the phorbol ester 12-Otetradecanoylphorbol-13.acetate: tumor incidence in organs other than the skin. Carcinogenesis, 2, 1087-1094. 14 Schmahl, W. and Kriegel, H. (1980) Ovary tumors in NMRI mice subjected to fractionated X-irradiation during fetal development. J. Cancer Res. Clin. Oncol., 98, 66-74. 15 Melchers, F. (1979) Murine embryonic B lymphocyte development in the placenta. Nature (London), 277, 219. 16 Seifertova, M., Vesely, J. and Sorm, F. (1968) Effect of 5-azacytidine on developing mouse embryo. Experientla, 24,487-488. 17 Seifertova, M., Vesely, J., Cihak, A. and Sorm, F. (1977) Pycnotic degeneration of ventricular cells in embryonic brain following transplacental exposure to 5-azacytidine. Experientia, 28, 841-842. 18 Druckrey, H. (1973) Specific carcinogenic and teratogenic effects of indirect alkylating methyl and ethyl compounds, and their dependency on stages of ontogenic developments. Xenobiotica, 3, 271-303.
DIAPLACENTAL IN NMRI-MICE
CARCINOGENIC
81
EFFECTS OF 5AZACYTIDINE
WOLFGANG SCHMAHLa, ELISABETH GEBER* and WALTER LEHMACHERb *Abteilung fiir Nuklearbiologie Systemforschung, Gesellschaft D-8042 Neuherberg (F.R.G.)
and bMEDIS-Inetitut fiir medizinieche Informatik und fiir Strahlen- und Umweltforschung m.b.H. Miinchen,
(Accepted 15 October 1984) (Revised version received 28 January 1985) (Accepted 21 October 1985)
SUMMARY
5-Azacytidine was applied to NMRI-mice (l-2 mg/kg) either on gestation day 12,14, or 16. In the first case it mainly induced leukemias, while in the latter experiments leukemias, lung adenomas and soft tissue sarcomas represent the main effects. The experiments performed on gestation day 14 led to tumor rates below the spontaneously occurring tumor frequencies in NMRI-mice. There is a clear-cut inverse dose-response relationship in leukemia induction, as the higher dose principally shows a lower degree of effectiveness. This, as well as a reduction of tumor frequency below control levels after application of this drug on day 14, can be explained by an “overkill” effect. The cytotoxic and embryolethal efficacy of the agent thus surpasses the transformation effects at the cellular genome. While a negative correlation between the general embryotoxicity of azacytidine and the simultaneous tumor inducibility is to be observed, there is no correlation at the target organ level between the embryotoxic and the carcinogenic effects.
INTRODUCTION
5-Azacytidine is a base analogue incorporated into cellular DNA during the S-phase of the generation cycle [ 11. It inhibits DNA synthesis, exerts lethality predominantly in the S-phase and thus also inhibits the mitosis rate of cells in culture. Azacytidine is apparently similar to 8-azaguanine in its action, particularly in its capacity to disaggregate polysomes [2]. It has been reported to cause chromosome aberrations in cultured mammalian cells [3,4] and to act as a mutagen in Vicia f&a [5]. Mutagenicity of azacytidine was also seen as a DNA repair-related event in the trpE8 strain of 0304-3835/85/$03.30 0 1985 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
82
Salmonella typhimurium, but not in those Salmonella strains conventionally used for the Ames test [6]. This compound has recently proved to be a valuable tool in experimental cell biology as it leads to an undermethylation of the nuclear DNA [ 71 and thus produces effects on gene regulation activity [ 81. Most of the embryotoxic effects of 5-azacytidine [9] can be deduced from these cellular influences. Azacytidine also induces cell transformation in vitro [4], simultaneously with chromosome changes and retrovirus activation [lo]. As azacytidine was also brought into relationship with some forms of tumors in adult mice [ 11,121, we consequently studied in the present experiments the diaplacental carcinogenic potency of this drug in comparison with the organ-specificities of the acute embryotoxic effects determined in a recent study [ 91. MATERIALS
AND METHODS
The breeding conditions of the NMRI-mice and the experimental procedure were basically the same as described recently [9]. Azacytidine was purchased from Sigma Chemie as a pure, crystalline powder. It was dissolved in sterile saline and used within the following hour. Applications of 5azacytidine to pregnant mice were performed by a single intraperitoneal injection at 0900 h of either saline (controls) or of the azacytidine solution. This occurred either on day 12 p.c., day 14 p.c. or day 16 p.c. On each day 2 doses of azacytidine were used; 1 mg/kg and 2 mg/kg. The number of animals in the different groups is listed in Table 1. The abbreviations for the different experimental groups refer to the azacytidine dose (1 mg/kg = lA, 2 mg/kg = 2A) and the day of application. The offspring which were whelped at regular times were registered and weighed. Stillbirths and neonatal deaths were removed immediately. The observation period lasted for a maximum of 960 days. All animals were autopsied either at this time or after their spontaneous death or when severely ill. As far as necessary for the evaluation, organs were also processed by histological techniques. This material was sectioned at 6 pm and stained with hematoxylin and eosin. The tumor frequency was registered per litter and subsequently the mean percentage + standard error of mean (S.E.M.) was calculated. The experimental results were statistically evaluated by application of the x2-test, the exact Fisher test and the Student test. RESULTS
Neonatal da to The mean litter size was not affected by any treatment (Table 1). The frequency of stillbirth was not significantly changed after application of
53 34 33 37 34 32 35
Controls lA12 2Al2 lA14 2A14 lA16 2A16
12.8 12.5 11.8 12.7 12.8 12.1 13.1
+ 0.7 f 0.7 It 0.6 f 0.7 f 0.7 -k 0.6 50.8
Mean litter size + S.E.M.
99.4 99.1 95.6 98.1 92.9 98.5 97.7
Live offspring (%)
1.41 1.23 1.19 1.17 1.15 1.37 1.34
f 0.07 zt 0.04b * 0.05b” * o.03c +- 0.03= * 0.05 +-0.06a 11.7 19.7 32.2 19.7 42.8 14.1 21.0
GESTATION
677 422 376 463 404 382 424
At birth
572 323 223 349 198 313 309
(293 (165 (113 (178 ( 97 (153 (158
'P < 0.05 compared to controls. bP < 0.01 compared to controls. 'P < 0.001 compared to controls.
+ + + + + + +
279) 158) 110) 171) 101) 160) 151)
DAYS
At the end of the study, total (d + 0)
No. of offspring
ON DIFFERENT
Mortality rate during the weaning period (%)
WITH 5-AZACYTIDINE
Mean offspring weight (g) at birth
DIAPLACENTALLY
Significances as evaluated with the x’-test after Yates correction. dP < 0.05 compared to the corresponding group of the same gestation day. eP < 0.01 compared to the corresponding group of the same gestation day. 'P< 0.001 compared to the corresponding group of the same gestation day.
No. of dams
DATA IN MICE TREATED
Treatment group
NEONATAL
TABLE 1
84
1 mg/kg either on day 12,14, or 16. In contrast, a dose of 2 mg/kg raised the stillbirth rate both on day 12 p.c. (4.1%) and 14 p.c. (7.1%), as well as 16 p.c. (2.3%). The mean birthweight was significantly (P < 0.05; Student test) reduced in ah treatment groups with the exception of lA16; within the 1-mg dosages it was most severely affected after treatment on day 14 p.c. The mortality rates until weaning were significantly (P < 0.05) increased in all groups with the exception of lA16. An additional percentage of animals were lost for evaluation during the course of the study. This was mostly due to sudden deaths and already severe autolysis until the carcasses were found. Each group showed a highly balanced sex distribution. Life span and general tumor frequency (Table 2) Within a distinct treatment group the survival rates did not differ significantly between the 2 sexes. Groups lA12, lA16, and 2A16 showed a quite steep initial decrease of the number of surviving animals. This effect was more pronounced in lA12 and in lA16, in contrast to the corresponding groups (2A12 and 2A16) with the higher dosage of azacytidine. The total tumor incidence in the control animals was 63.1 + 2.8%. In treatment groups lA12,1A16 and 2A16 the tumor incidence was significantly increased to 93.0 f 6.5%, 129.6 T 8.3%, and 109.2 + 4.4%, respectively. The values for groups 2A12, lA14 and 2A14 were not statistically different from the control value. The mean frequency of tumor-bearing animals within the litters amounted in the controls to 52.6 f 2.3% (d) TABLE
2
LIFE SPAN AND TUMOR INCIDENCE IN THE OFFSPRING DIAPLACENTALLY WITH 5-AZACYTIDINE Treatment group
Controls lA12 2A12 lA14 2A14 lA16 2A16
No. of animals (n)
d(293) O(279) d(165) O(158) d(l13) O(110) d( 178) O(171) d( 97) O(101) d(153) O(160) d(158) 9(151)
OF MICE TREATED
400 days
600 days
800 days
Tumorbearing animals (%)
77.7 73.5 59.6 62.0 79.6 80.0 83.7 86.6 83.5 88.1 49.7 46.9 69.0 62.9
48.3 51.2 48.2 51.3 45.1 56.4 56.2 66.1 46.4 45.5. 23.5 19.4 30.4 29.1
26.4 30.8 36.7 39.9 19.5 27.3 27.0 42.7 33.1 21.8 13.7 8.7 12.0 17.9
52.6 51.1 65.4 62.1 46.5 40.4 46.3 40.9 49.6 59.3 72.5 78.1 78.9 73.0
Survival rates (%) after
Index marks for significances, see Table 1.
+ 2.3 + 1.9 + 3.7a f 4.1a +- 4.2*e + 3.8Ke + 4.0’ f 3.7a f 4.2 f 4.8a*e + 3.5’ f 3.0’ + 3.1’ f 3.4=
85
and 51.1 + 1.9% (O), respectively. This value was significantly increased in lA12, lA16 and 2A16, but depressed in 2Al2 and in lA14. These results are also reflected by the number of tumors per affected animals (tumor multiplicity): the control value was 1.22. A significant increase was only observed in lA12-1.45, in group lA16-1.69 and in 2A16-1.43; the values of the other groups remained within the control range. Frequencies of distinct tumors Leukemias and lymphomas (Table 3): The frequency of these tumors was significantly increased in both sexes of animals from group lA12,1A16 TABLE
3
FREQUENCY OF LEUKEMIC DISEASES EXPOSURE WITH 5-AZACYTIDINE Treatment group
No. of animals (n)
% Leukemias and lymphomas
IN MICE OFFSPRING
AFTER
PRENATAL
% Lymphatic leukemia
% Reticulocellsarcoma
% Myeloid leukemia
(no.)
(no.)
(no.)
(no.) Controls
lA12
2Al2
lA14
2A14
lA16
2A16
d( 293)
28.7
+- 2.7
12.8 + 1.9
12.5 + 2.2
3.1 + 0.8
O(279)
(84) 29.3 * 3.4
(38) 14.9 + 2.4
(37) 12.0 -I 2.0
(9) 2.6 + 1.2
d( 165)
49.1
(82) +4.7c
34.7
(41) + 4.4c
(34) 12.0 + 2.4
(7) 2.4 + 1.1
9(158)
(81) 50.8 + 4.2’
30.9
(57) * 3.7c
(20) 17.3 + 3.2
(4) 2.0 -I 1.6
d(113)
(80) 25.0 + 5.2”’
(50) 10.0 f 3.4’
(27) 9.8 + 2.9
(3) 5.3 f 2.4d
Q(ll0)
23.;?!.6cf
lO.lf
(11) 6.1 + 2.4d
7.3 + 2.6a’e
d(178)
(26) 23.6 f4.0
(11) 7.4 f 3.1C
9(171)
(42) 15.4 + 3.4u
(13) 5.8 +- 1.7’
( 7) 11.9 * 1.9 (21) 7.4 dz l.ga
(6)
(8) 4.5 * 1.4 (8) 1.8 * 1.5 (3) -
9.5(::.7cf
(10) 7.3 + 3.7c
Q(lO1)
( 9) 14.0 + 2.7’
( 7) 3.0 * 2.0c
6.9 + 2.4b
d(153)
(14) 63.8 + 6.1’
( 3) 35.4 dz4.8’
( 7) 19.6 + 3.4a
(2) 8.6 * 2.2a
o(160)
60.9
(97) + 6.0’
35.0
(54) i 4.0c
(30) 20.3 + 3.0’
(13) 6.3 f 1.8a
d(158)
41.8
(98) f 3.3’”
27.2
(56) f 3.1’”
(32) 11.5 + 3.6e
(10) 3.8 + 1.2e
9(151)
38.1
(67) +- 4.2”
27.4
(43) f 3.7’”
d(97)
(41)
(57) Index
(11) 2.9’
marks for significances,
see Table
1.
( 2)
2.0 +- 1.5
(6) 3.3 + l.7e (5)
86
(P < 0.001; x2-test) and 2A16 (P < 0.06). In contrast, a significant depression of this tumor rate occurred in lA14 (P < 0.01) and 2A14 animals (P < 0.001); this value was only marginally below the control range in group 2A12. In general, there were no marked sex differences. Leukemia frequency in the lA12 animals was largely due to an increase of lymphatic leukemias. In contrast, all 3 subtypes of leukemia equally contributed to the increase of the total leukemia rate in the group lA16 animals. The decrease of leukemia frequency in the lA14 animals was mainly brought about by a reduced incidence of lymphatic leukemia. All these effects showed no sex dependency. Lung tumors (Table 4a): Neither treatment on day 12, nor on day 14 with 1 mg azacytidine had any effect on the frequency of lung tumors. The apTABLE 4a FREQUENCY OF NON-LEUKEMIC DISEASES IN MICE OFFSPRING AFTER PRENATAL EXPOSURE WITH 5-AZACYTIDINE Treatment group
No. of animals (n)
% Lung tumors (no.)
% Liver tumors (no.)
Controls
d(293)
19.5 +_1.8 (57) 19.1 + 2.5 (53) 18.2 i 3.3 (30) 20.9 f 3.5 (33) 19.6 + 3.6 (22) 20.1 * 5.3 (22) 16.3 f 3.4 (29) 18.4 f 3.6 (31) 47.3 f 5.8”’
4.8 + 1.2 (14) 3.9 +_0.9 (11) 9.2 + 2.1b (15) 3.9 zk1.3 ( 6) 9.9 + 3.8b (11) 8.2 z!z2.8a’d ( 9) 6.7 + 2.1 (12) 11.8 +- 2.3’ (20) 11.3 + 3.8b
42.6(:;.ScJ (43) 53.0 f 5.gc (81) 62.0 f 5.5’ (99) 49.5 + 5.3c (78) 54.3 f 4.6’ (82)
6.9 + 2.4d ( 7) 9.2 f 2.4b (14) 5.1 * 1.5
Q(279) lA12
d(165) Q(158)
2A12
d(113) Q(l10)
lA14
d(178) Q(171)
2A14
d( 97) 9(101)
lA16
d(153) Q( 160)
2Al6
d(158) Q(151)
Index marks for significances, see Table 1.
(11)
( 8)
11.2 -I 2.6b
(18)
3.4 f 1.8 ( 5)
% Soft tissue sarcomas (no.)
0.3 f 0.3 (1) 0.6 zk0.5 (1) 1.9 f 0.8 (3) 0.9 +_0.8 (1) 0.9 _+0.7 (1) 1.1 + 0.6 (2) 1.7 f 0.8 (3) 1.0 f 0.6 (1) 1.0 z!z0.7 (1) 1.9 f l.la (3) 1.2 + 0.8’ (2) 1.8 + l.la (3) 3.2 + l.gad (5)
87 TABLE 4b FREQUENCY OF NON-LEUKEMIC DISEASES IN MICE OFFSPRING AFTER PRENATAL EXPOSURE WITH 5-AZACYTIDINE Treatment group
No. of animals (n)
Controls
d(293) O(279)
lA12
2A12
lA14
d(165) Q(158) d(l13) Q(ll0) d(
178)
Q(171) 2A14
d(97) Q(101)
lA16
d(153) Q(160)
2Al6
d(158) Q(151)
% ovary tumors (no.)
-
13.3 * 2.1 (37) 12.1 f 2.6 (19) 5.4;
% Mammary tumors (no.)
3.6b*e
( 6) -
7.2k1.7 (20) 8.2+ 2.3 (13) 3.6i2.0M ( 4) -
96 Miscellaneous tumors (no.) 0.3 f 0.3 (1) -
1.2 + 0.5
(2) 1.8 + 0.8 (2) 1.7 * 0.5 (3) 0.6 + 0.3 (1) 2.0 f 0.4a
11.1 It 2.7 (19) -
7.0+ 2.3 (12) -
7.9k3.0 ( 6) -
6.9f 2.3 ( 7) -
3.1+1.2a@
6.9 f 2.2b (11) -
5.0f 1.6 ( 6) -
1.2 * o.5a
(2)
10.6 f 3.6 (16)
(3) 1.3 * 0.7*
(2)
5.9 f 1.7 ( 9)
(2)
1.9 +- 0.8’ (3) 0.6 + 0.3 (1)
Index marks for significances, see Table 1.
plication of 2 mg/kg on day 14 resulted in a marked increase (P < 0.001) of the lung tumor frequency in both sexes. Lung tumors were most effectively induced in group lA16 and in 2A16 animals, irrespective of their sex. These values were significantly different from the control values (P < 0.001, x2test). Liver tumors (Table 4a): Liver tumors were generally more frequent in azacytidine treated offspring than in controls. This increase was largely brought about by affected male animals in groups lA12, 2A12, 2A14, lA16 and 2A16 (P < 0.01; x2-test). In the latter groups, with exception of 2A12, the liver tumor incidence in males was also significantly (P < 0.05) higher than the incidence of liver tumors in the corresponding females. A significant (P < 0.001) increase of liver tumor frequency occurred in the female offspring only in group lA14. The latter value also surpassed signifi-
88
cantly the liver tumor frequency of the corresponding male offspring (P < 0.05). Soft tissue sarcomas (Table 4a): In the experimental animals these tumors were mostly situated in the subcutis of the legs and in the perianal region. Their frequency was significantly increased in lA16 and 2A16 animals of both sexes. The 3 cases which were observed in male offspring from group 2A16 were non-metastasizing angiosarcomas of the hindlegs. Other tumors: These are compiled in Table 4b. The miscellaneous tumors comprise cases of Harderian gland adenomas, intestinal tumors and tumors arising from the skin. DISCUSSION
The critical stages for inducing diaplacental carcinogenic effects with 5-azacytidine are inversely parallel to the phase-dependent embryotoxic effects of this compound. On gestation day 12, when mild embryotoxicity could be induced, there was a distinct increase in tumor frequency. The latter was largely due to leukemia induction. On gestation day 14, the time of maximum embryotoxicity, the carcinogenic effects had decreased below the level of spontaneous incidence, whereas a significant tumor response was induced on gestation day 16, a developmental stage without any effect according to the standard embryotoxicity evaluation [ 91. The tumor pattern following azacytidine treatment on day 16 was mainly restricted to leukemia, lung tumors and soft tissue sarcomas. There was also a marked, reverse dose-response relationship of tumor induction, especially in the case of leukemia, the lower dose (1 mg/kg) being significantly more potent than the higher dose (2 mg/kg) of azacytidine. It is reasonable to assume an accentuation of the cytolethal effects with an increasing dose. An eradication either of potentially transformed fetal cells or of fetuses with an increased tumor risk may be the consequence [13,14]. We propose that two different paths, depending on the gestation stage of azacytidine application, lead to leukemias on gestation day 12 and gestation day 16. The one path, during the early stage (day 12 p.c.) of differentiation of the immune system [ 151, is characterized by an almost exclusive induction of lymphatic leukemias, whereas the same treatment on day 16 p.c. apparently provoked a pathogenetic path including all subtypes of leukemia at nearly equal rates. An inexplicable result is the depression of leukemia incidence below the control value after axacytidine application on day 14 p.c. Preliminary studies with the stillbirth and neonatal deaths in groups lA14 and 2A14, however, presented no results which would indicate a selective eradication of those littermates with an excessive risk of leukemia. Azacytidine is known from experiments in adult mice as a potent inducer of leukemia in the AKR strain [ 121 and of lung tumors in the A strain [ 111.
89
The latter results of Stoner (Shimkin model) are obviously confirmed by our results. However, it is quite interesting that lung tumors were induced by azacytidine only on days 14 and 16, but not on day 12. Thus, it may be suggested that a certain degree of cellular differentiation of lung tissue is a necessary requisite to enable carcinogenic transformation. Obviously, the spectrum of cancerogenic effects upon fetal tissues does not differ substantially from those effects in adult animals. In contrast, the teratogenic effects of 5-azacytidine are mainly directed against the developing CNS and the fetal liver [16,17]. Thus, the present results obviously confirm the opinion that the organ-specific site of teratogenic action is strictly different from the preferential sites of cancerogenesis [ 181. Our recent embryotoxicity study on azacytidine [ 91 likewise presented no arguments for an abnormal cellular differentiation in those fetal tissues which proved to be most susceptible to carcinogenesis in the present study (e.g. lung tissue). Consequently, we assume that any specific effects of azacytidine leading to carcinogenesis are only detectable during the postnatal period. REFERENCES 1 Li, L.H., Olin, E.J., Fraser, T.J. and Bhuyan, B.K. (1970) Phase specificity of 5-azacytidine against mammalian cells in tissue culture. Cancer Res., 30, 2770-2775. 2 Cihak, A., Vesely, H. and Sorm, F. (1968) Thymidine kinase and polyribosome distribution in regenerating rat liver following 5-azacytidine. Biochim. Biophys. Acta, 166, 277-286. 3 Tobey, R.A. (1972) Effects of cytosine arabinoside, daunomycin, mithramycin, azacytidine, adriamycin, and camptothecin on mammalian ceU cycle traverse. Cancer Res., 32, 2720-2725. 4 Harrison, J.J., Anisowicz, A., Gadi, I.K., Raffeld, M. and Sager, R. (1983) Azacytidine-induced tumorigenesis of CHEF/18 cells: correlated DNA methylation and chromosome changes. Proc. Natl. Acad. Sci. U.S.A., 80, 6606-610. 5 Fucik, V., Michaelis, A. and Rieger, R. (1970) On the induction of segment-extension and chromatid structural changes in Vicia faba chromosomes after treatment with 5-azacytidine. Mutat. Res., 9, 599-606. 6 Podger, D.M. (1983) Mutagenicity of 5-azacytidine in Salmonella typhimurium. Mutat. Res., 121, l-6. 7 Jones, P.A. and Taylor, S.M. (1980) Cellular differentiation: cytidine analogs and DNA methylation. CeII, 20, 85-93. 8 Lindahl, T. (1981) DNA methylation and control of gene expression. Nature, 290, 363-364. 9 Schmahl, W., TSrSk, P. and Kriegel, H. (1984) Embryotoxicity of 5-azacytidine in mice. I. Phase- and dose specificity studies. Arch. Toxicol., 55, 143-147. 10 Tennant, R.W., Otten, J.A., Myer, F.E..and Rascati, R.J. (1982) Induction of retrovirus gene expression in mouse cells by some chemical mutagens. Cancer Res., 42, 3050-3055. 11 Stoner, G.D., Shimkin, M.B., Kniazeff, A.J., Weisburger, J.H., Weisburger, E.K. and Gori, G.B. (1973) Test for carcinogenicity of food additives and chemotherapeutic agents by the pulmonary tumor response in strain A mice. Cancer Res., 33, 306!+ 3085.
90
12 Vesely, J. and Clhak, A. (1973) High-frequency induction in vivo of mouse leukemia in AKR strain by 5-azacytidine and 5-iodo-2-deoxyuridine. Experientia, 29,11321133. 13 Goerttler, K., Loehrke, H., Hesse, B., Milz, A. and Schweizer, J. (1981) Diaplacental initiation of NMRI mice with 7,12-dimethylbenz(a)anthracene during gestation days 6-20 and postnatal treatment of the F,-generation with the phorbol ester 12-Otetradecanoylphorbol-13.acetate: tumor incidence in organs other than the skin. Carcinogenesis, 2, 1087-1094. 14 Schmahl, W. and Kriegel, H. (1980) Ovary tumors in NMRI mice subjected to fractionated X-irradiation during fetal development. J. Cancer Res. Clin. Oncol., 98, 66-74. 15 Melchers, F. (1979) Murine embryonic B lymphocyte development in the placenta. Nature (London), 277, 219. 16 Seifertova, M., Vesely, J. and Sorm, F. (1968) Effect of 5-azacytidine on developing mouse embryo. Experientla, 24,487-488. 17 Seifertova, M., Vesely, J., Cihak, A. and Sorm, F. (1977) Pycnotic degeneration of ventricular cells in embryonic brain following transplacental exposure to 5-azacytidine. Experientia, 28, 841-842. 18 Druckrey, H. (1973) Specific carcinogenic and teratogenic effects of indirect alkylating methyl and ethyl compounds, and their dependency on stages of ontogenic developments. Xenobiotica, 3, 271-303.