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Increased cerebellar thymidine kinase and DNA during early postnatal development of diethylstilbestrol-treated rats
EXPERIMENTAL
NEUROUMY
75,289-298
(1982)
Increased Cerebellar Thymidine Kinase and DNA during Early Postnatal Development of DiethylstilbestrolTreated Rats M. LITTERIA’ Neuroendocrine Research Laboratory (1 SIB), Veterans Administration North Chicago, Illinois 60064
Medical Center,
Received February 3. 1981; revision received September 18. 1981 Thymidine kinase activity and DNA content were determined in the cerebellum after the administration of diethylstilbestrol (DES) to neonatal female rats. Rats were injected S.C. with either 30 pg DES or vehicle 48 h after birth. DNA and thymidine kinase were measured in the cerebellum at 3, 7, 10, 18, 21, 26, and 60 days of age. Thymidine kinase activity and DNA content in the DES-treated littermates were significantly increased between ages 3 to 21 days and ages 3 to 26 days, respectively. The data suggest diethylstilbestrol-induced relationships between thymidine kinase activity and DNA synthesis during the period of rapid cerebellar development.
INTRODUCTION Diethylstilbestrol (DES), a nonsteroid synthetic estrogen, was frequently prescribed between 1940 and the early 1970s for the treatment of threatened abortion and other complications of pregnancy (13). An association has been firmly established between the in utero exposure to DES and the subsequent but infrequent development of clear-cell adenocarcinoma of the vagina and cervix in female offspring (13). In addition to the common occurrence of structural and nonneoplastic abnormalities of the genital tract (13, 15), several studies suggest that daughters exposed to DES in Abbreviation: DES-diethylstilbestrol. ’ I thank C. G. Popoff for excellent technical assistance; Dr. M. Breen and H. G. Weinstein, Research-in-Aging Laboratory, for their critical review of the manuscript; R. Heinz, Researchin-Aging Laboratory, for editorial assistance; and S. Grimm for typing the manuscript. This work was supported by the Veterans Administration. 289 0014-4886/82/020289-10$02.OQ/O Copyri&t0 1982 by Academic FWs, Inc. Au rigbtt of repmdttti
in my form rcmvcd.
290
M.
LITTERIA
utero have an increased incidence of anovulation, infertility, and fetal wastage (14, 39). Anovulatory sterility is similarly induced in the offspring of pregnant rodents treated with either DES or the natural estrogens (12, 32, 34, 35) and in adult rats treated neonatally with these hormones (10, 20). In the latter animals, postpuberal sterility is due to estrogen-induced alterations in hypothalamic and limbic structures regulating cyclic ovulation (2). The effects of the natural estrogens on the developing central nervous system are not limited to brain regions traditionally associated with the regulation of cyclic ovulation, i.e., certain hypothalamic and limbic structures. Thus, injection of estradiol benzoate into neonatal rats during the sexually critical period of brain differentiation induced long-term postpubertal alterations in the incorporation of [ 3H]lysine into proteins of specific neurons of the hypothalamus (26), limbic and paralimbic structures (2 1), cerebral cortex (27), and cerebellar cortex (25). Moreover, neonatally administered estradiol benzoate (23) and testosterone propionate (24) induced dissimilar changes in cerebellar thymidine kinase activity during early postnatal development. Incorporation of preformed thymidine into DNA (the “salvage” pathway) is dependent on a sequence of phosphorylations through thymidine mono-, di-, and triphosphate (37). The activity of thymidine kinase, the enzyme converting thymidine to the monophosphate, is consistently elevated in diverse proliferating tissues, including the cerebellum of the neonatal rat (37, 42). In the cerebellum of the rat, thymidine kinase activity and the incorporation of thymidine into DNA are both maximal approximately 6 days after birth (41, 46). The rapid growth that characterizes the cerebellum of the rat during the first 3 postnatal weeks is due to the proliferation of granule cells in the external granular layer (1). This layer disappears at approximately 21 days after birth due to the migration of its cells to the molecular and internal granular layers (1). To our knowledge, the effects of DES on the developing brain have not been examined. It therefore seemed relevant to determine if the administration of DES to neonatal female rats is associated with changes in cerebellar thymidine kinase activity and DNA content. MATERIALS
AND
METHODS
Litters were bred from 3- to 6-month-old Sprague-Dawley rats (Madison, Wis.) exposed to a 14: 10 h light-dark schedule and fed Purina Rat Chow and water ad libitum. Litter size was reduced to eight pups within 14 h of birth and the day of birth is designated as day 0. Forty-eight hours after birth female littermates, paired according to body weights, were in-
DES
AND
CEREBELLAR
THYMIDINE
KINASE
291
jetted subcutaneously with either 30 pg DES dissolved in 0.05 ml sesame oil or with an equivalent volume of vehicle. Rats were decapitated at 3, 7, 10, 18, 21, 26, or 60 days of age and the cerebellum rapidly removed, .weighed, and homogenized at 2°C. The cerebellum from each of two control or two DES-treated rats was combined at 3 days of age to provide sufficient material for all of the analyses, whereas only one cerebellum was required at the older ages. All experiments were scheduled at 0800 h to avoid possible circadian variations in enzyme activity and DNA synthesis. A 10% crude homogenate (w/v) was prepared in ice-cold medium consisting of 0.25 M sucrose, 0.004 M MgC&, and 0.02 M Tris-HCI buffer, pH 7.4 (46). Samples (0.05 ml) were removed for the extraction and determination of DNA (5). The remaining homogenate was centrifuged 60 min at 105,000 g at 2°C and the resultant supernatant served as the enzyme preparation (46). Protein content was measured in 0.02-ml samples of this supernatant (28). Thymidine kinase activity was measured according to previously detailed methodology (24). Briefly, the enzyme reaction mixture contained, in a final volume of 0.25 ml: 0.05 M Tris-HCl buffer (JIH 8), 5 mM adenosine triphosphate, 2.5 mM MgC&, 19.8 pM methyl[ 3H]thymidine (sp act 2 Ci/mmol), and 0.02 ml enzyme supernatant (0.033 to 0.051 mg protein). After incubating 10 min at 37”C, the reaction tubes were boiled 3 min, chilled in ice, and centrifuged 30 min at 1600 g and 2°C. Duplicate 0.02-ml samples of the supernatants were spread over the surface of 23-mm diethylaminoethyl (DEAE8 1) cellulose disks. The dried disks were placed into individual scintillation vials and 5 ml 0.001 M ammonium formate was added. The vials were agitated 5 min in a Dubnoff shaker and the formate discarded. This was repeated four times each with ammonium formate and distilled water, and two times with absolute alcohol.This washing procedure removed 99.7% of the unreacted [3H]thymidine from the disks. The phosphorylated derivatives of [3H]thymidine were then eluted from the disks by adding 1 ml 0.1 M HCl/ 0.2 M KC1 to the vials and shaking 15 min ( 16). Counts per minute were determined in a Packard Tri-Carb Scintillation Spectrometer after the addition of 10 ml scintillation cocktail (Dimilume TM-30) to the vials. Specific activity of thymidine kinase is expressed as (picomoles [3H]thymidine phosphorylated per minute per milligram protein) X 10-l. Significance of differences between control and DES-treated littermates was determined by the two-tailed Student’s t test for paired data; P 5 0.05 is considered significant. RESULTS Daily vaginal smears recorded between 45 and 60 days of age demonstrated persistent cornification (anovulation) in 87% of the DES-treated
292
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rats and normal 5-day estrous cycles in their littermate controls. Ovarian and uterine weights recorded for the treated rats at 26 and 60 days of age were significantly decreased below those of their controls. Thus, at age 26 days, the mean + SE for ovarian and uterine wet weights (mg) for control and treated rats were, respectively, 33.10 +- 0.31 vs. 25.58 + 0.67, P < 0.001; and 50.98 -t 1.50 vs. 36.57 + 0.92, P c 0.001. Similarly, at age 60 days, these values were 101.22 + 3.49 vs. 62.77 + 3.32, P < 0.001, for ovarian weights; and 303.27 + 7.71 vs. 246.58 f 7.52, P < 0.001, for uterine weights. Body weights of the DES-treated rats were significantly heavier than their controls at ages 3 to 10 days; however, there were no differences in the wet weights of the cerebellum (Table 1) or whole brain at any age. There were no differences in the protein content of the enzyme supernatant between control and DES-treated rats at any age. Thymidine kinase activity and DNA content were significantly elevated in the cerebellum of DES-treated rats between ages 3 to 21 days and ages 3 to 26 days, respectively (Table 1). Thymidine kinase activity-total cerebellar DNA ratios were not significantly different between treated and control rats in any age group (Table 1). There were no differences in either cerebellar thymidine kinase activity or DNA content in the 60-day-old control rats during proestrus, estrus, and diestrus; therefore, these data were combined. In agreement with previous work, enzyme activity of both control and treated rats was maximum at age 7 days (23, 24) and rapidly declined to the low values observed in the adult (42, 46). The in vitro effect of DES was determined by adding the estrogen to the enzyme assay medium in a final concentration of either 1 X lop5 or 1 X 10m6M. DES did not have any effect on thymidine kinase activity in cerebellar preparations from either 3- or 18-day-old control female rats. These results suggest that the stimulatory effect of DES per se on thymidine kinase is not mediated by direct activation of the enzyme. However, the possibility still exists that a metabolic product of DES (19) activates the enzyme. In admixture experiments, equal volumes of the 10% crude homogenates prepared from the cerebella of either 3- or 18-day-old DES-treated rats and their control littermates were mixed. Supernatants were prepared as described above. The activity of the enzyme in the mixtures was equal to the sum of the activities of the individual preparations. These data suggest that the changes induced in the activity of the enzyme in the cerebellum of DES-treated rats are not due to the presence of endogenous activators or inhibitors in the enzyme preparations. DISCUSSION Administration of DES to pregnant rodents is followed by its accumulation in both the maternal and fetal brain (8, 35). Moreover, DES injected
DES AND CEREBELLAR
THYMIDINE
KINASE
293
directly into neonatal rats binds to the estrogen receptors that are localized in specific regions of the brain (3 1,40). DES and its estrogenic metabolites also bind to the cytoplasmic estrogen receptor in tissues such as the oviduct and uterus (7, 17, 19,43). Translocation of the cytoplasmic DES-receptor complex is followed by transcription of specific genes and the synthesis of new proteins in the uterus and oviduct (7, 17, 19,43). Although differences between the mechanism of action of estrogen receptors in brain and uterus have been reported (18) the demonstration of putative estrogen receptors in the rodent cerebellum (9) does provide a biochemical mechanism for the action of DES in this structure. Thus, the increase in thymidine kinase activity and the related increase in cerebellar DNA content during the first 3 postnatal weeks could represent DES-induced changes in genetic expression which result in either an increase in enzyme synthesis, a decrease in enzyme degradation, or modification of a proenzyme to an active form. The results of the in vitro and admixture experiments provide some support for our suggestion that the stimulatory action of DES and/or its metabolites ( 19) on cerebellar thymidine kinase activity is due to the effects of the estrogen at the level of transcription and/or translation. The activity of thymidine kinase and the content of DNA in the cerebellum of the DES-treated littermates were significantly increased between ages 3 to 21 days and ages 3 to 26 days, respectively (Table 1). The observation that the thymidine kinase activity-total cerebellar DNA ratios (Table 1) were not significantly different between control and treated rats in any age group suggests a direct causal relationship between the increase in cerebellar thymidine kinase activity and DNA content after neonatal treatment with DES. The activity of thymidine kinase is specifically related to the synthesis of DNA during the premitotic S phase of the cell cycle (4). Thus, at least part of the increase of cerebellar DNA content in the treated rats must be attributed to DES-induced replication of genomic DNA. The latter could reflect an actual increase in cell number (neurons and/or glia) and/ or the occurrence of polyploidy. In this regard it should be noted that DES is a mitogenic agent (38) and that it also induced chromosomal polyploidization and nondisjunction in embryo cells (3). Postnatal neurogenesis in the cerebellum terminates approximately 3 weeks after birth (1); thus the transient inductive effect of DES on thymidine kinase and DNA during this period coincides with the availability of dividing cells susceptible to the mitogenic agent. Failure to detect the mitogenic effect of DES after age 26 days (Table 1) is due to the cessation of neurogenesis in the older cerebellum. Although speculative, part of the elevated DNA content in the cerebellum of the DES-treated rats may reflect an increase in DNA repair
60
C T
C T
C T
c T
191.3 + 2.3 (13) 193.8 k 2.6 (13)
71.2 + 0.8 (12) 70.4 f 1.2 (12)
50.9 * I.2 (12) 51.2 + 1.3 (12)
40.0 * 0.4 (IS) 39.9 + 0.4 (15)
22.4 k 0.3 (15) 23.3 + 0.2 (IS)
15.4+0.3 (ll)d 16.5 + 0.3 (II)
C T
C T
6.6 + 0.3 (IS)‘ 8.8 + 0.2 (18)
C’ T
Body weight (g)
in parentheses. phosphorylatcd/min/mg
245.15 + 2.12 (13) 246.31 f 1.62 (13)
195.23 zk 1.83 (12) 197.37 ? 2.17 (12)
179.41 2 2.91 (12) 181.78 f 2.87 (12)
165.82 2 1.84 (15) 163.80f 1.65 (15)
77.54 k 1.20 (15) 78.07 + 2.24 (15)
45.63 + 1.40 (II) 47.83 k I.14 (II)
18.32 + 0.68 (18) 18.92 + 0.61 (18)
Wet weight (mg)
’ Each value is the mean t SE for the number of determinations indicated ’ Spxitic activity of thymidine kinase is expressed as (pmol [‘H]thymidine ‘C-control; T-treated with DES. dP a 0.001. c P 5; 0.005. ‘P d 0.02. g P L 0.025. * P L 0.05.
26
21
I8
IO
3
Age (days)
TABLE
Effects of Diethylstilbestrol
.
(,ag)
X IO-‘.
+ 27.97 (13) + 30.97 (13)
+ 17.69 (12)’ ? 28.35 (12)
+ 43.84 (12)d f 31.83 (12)
_+ 17.97 (14)d + 15.90 (14)
+ 13.50 (15)d + 16.28 (15)
protein)
1429.15 1452.00
1421.50 1478.50
1404.33 1548.83
1288.43 1423.43
690.33 803.33
348.09 + II.21 (Il)d 406.91 k 9.00 (I I )
184.78 f 11.20 (9)d 221.54 f II.75 (9)
Total DNA
Cerebellum
(DES) on Female Rats”
1
I.78 k 0.04 (13) 1.73 + 0.05 (13)
1.76 + 0.04 (12) 1.84 k 0.03 (12)
2.42 f 0. I3 ( 12)’ 2.81 + 0.14 (12)
4.61 k 0.27 (15)d 5.49 t 0.21 (15)
58.10 t 1.54 (15)d 70.23 + 1.29 (15)
69.84 + I.88 (ll)d 84.27f 2.39(11)
35.51 f 2.09 (9) 45.15 + 2.51 (9)
Specific activity of thymidine kinase x 10-I b
+ 7.59 _+ 6.71
1.25 f 0.03 1.20 f 0.04
I.24 f 0.03 1.25 f 0.02
1.73 f 0.09 1.82 k 0.08
3.56 f 0.23 3.87 2 0.17
84.88 f 3.26 87.37 + 2.61
202.31 207.88
194.32 + 9.58 204.97 + 7.73
(Thymidine kinase/DNA) x 101
F
!Z
K
DES AND CEREBELLAR
THYMIDINE
KINASE
295
and/or synthesis of metabolic DNA (6, 33). Thus, DES-induced damage to genomic DNA could accelerate DNA repair and/or promote an increase in the synthesis of metabolic DNA related to gene amplification. The decline of cerebellar DNA to control values in the DES-treated rats between ages 26 to 60 days (Table 1) may reflect the catabolism of excess metabolic DNA in differentiated cells (6, 36) and the diminished ability of nondividing cells to engage in DNA repair (6). The role of DES in neuronal death is currently unknown. Therefore, the possibility cannot be excluded that the increase in cerebellar thymidine kinase activity and DNA content in the treated rats is at least partially due to the inhibition of cell death during early postnatal development. Alternatively, other possible mechanisms include: (i) A nongenomic or activational effect (30) of DES and/or its metabolites. For example, DES could have produced changes in the transport and therefore intracellular availability of thymidine and/or other metabolites in the cerebellum. In this regard, administration of estradiol benzoate to neonatal rats enhanced the transport of a small metabolically inert amino acid (cu-aminoisobutyric acid) into specific brain regions during early postnatal development (22). (ii) Primary DES-induced activational and/or genomic changes in other brain structures could secondarily influence the metabolism of cerebellar neurons via multiple interconnections between these structures and the cerebellum. (iii) Our results could be secondary to either other currently undefined DES-induced changes in the cerebellum or to possible differences in the hormonal profile of the treated rats. Neither previous work nor the data presented in this study are sufficient to distinguish between the alternatives that have been discussed. We recently reported a sexual dimorphism in the activity of cerebellar thymidine kinase as well as differences in the response of the enzyme to neonatally administered testosterone propionate and estradiol benzoate (23,24). Similarly, the develop,mental pattern of changes in enzyme activity that follow injection with DES differed from that observed in female rats treated with either estradiol benzoate or testosterone propionate [Table 1, (23, 24)]. For example, thymidine kinase activity in the cerebellum of rats treated neonatally with estradiol benzoate was (i) greater than that of their control littermates at 3 and 4 days of age, (ii) the same as the controls at age 6 days, (iii) lower than the controls between 7 and 12 days of age, and (iv) the same as control values by 15 days of age (23). The changes induced in enzyme activity by each of the three hormones were associated with parallel changes in cerebellar DNA content [Table 1, (23, 24)]. Possible mechanisms of action of testosterone propionate and estradiol benzoate on thymidine kinase activity and DNA content in the developing cerebellum have been previously detailed (23, 24). It is not surprising to
296
M. LITTERIA
find differences in the response of cerebellar thymidine kinase to the sex steroids because these hormones frequently have dissimilar effects on the same enzyme in the brain [see (29) for review]. Moreover, opposite effects of testosterone and estradiol on thymidine kinase activity in the anterior pituitary of immature male rats have been reported (44). Differences in the response of cerebellar thymidine kinase to DES and estradiol may include the following factors: (i) The quantity of estrogen that was administered must be considered. Pharmacologic concentrations of estradiol benzoate (23) and DES (see Materials and Methods) were used in each study. However, a much lower quantity of DES was injected because, in contrast to estradiol (21, 23, 25-27), an identical quantity of DES (and its excretion products) would sterilize the littermate controls (11). (ii) The dissimilar effects of the two estrogens on the enzyme may be mediated by different metabolites of DES and estradiol rather than by the hormones per se. (iii) The clearance of DES and estradiol and/or their effective metabolites from the brain may differ. (iv) DES and estradiol (and/or their effective metabolites) may differentially affect the various isoenzymes of cerebellar thymidine kinase (24, 45). To summarize: Although the mechanisms are unknown, the data presented in this study definitively link the increase in thymidine kinase activity and DNA content in the developing cerebellum to the administration of DES. The latent neoplastic potential of DES on differentiating cells (13, 15) seems to merit additional studies of this estrogen on the developing brain. REFERENCES 1. ALTMAN, J. 1970. Postnatal neurogenesis and the problem of neural plasticity. Pages 197-237 in W. A. HIMWICH, Ed., Developmental Neurobiology. Thomas, Springfield, Ill. 2. ARAI, Y., AND T. KUSAMA. 1968. Effect of neonatal treatment with estrone on hypothalamic neurons and regulation of gonadotrophin secretion. Neuroendocrinology 3: 107-l 14. 3. BARRETT, J. C., A. WONG, AND J. A. MCLACHLAN. 1981. Diethylstilbestrol induces neoplastic transformation without measurable gene mutation at two loci. Science 212: 1402- 1404. 4. BASERGA, R. 1981. The cell cycle. N. Engl. J. Med. 304: 453-459. 5. BURTON, K. 1956. A study of the conditions and mechanism of the diphenylamine reaction for the calorimetric estimation of deoxyribonucleic acid. Biochem. J. 62: 3 15-323. 6. CAMERON, I. L., AND E. K. ADRIAN, JR. 1979. Unstable nuclear DNA in hypoglossal neurons of adult mice. Cytobios 25: 85-92. 7. CHAN, L., AND B. W. O’MALLEY. 1976. Mechanism of action of the sex steroid hormones. N. Engl. J. Med. 294: 1372-1381. 8. FISCHER, L. J., J. L. WEISSINGER, D. E. RICKERT, AND K. L. HINTZE. 1976. Studies on the biological disposition of diethylstilbestrol in rats and humans. J. Toxicol. Environ. Health 1: 587-605.
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KINASE
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NEUROUMY
75,289-298
(1982)
Increased Cerebellar Thymidine Kinase and DNA during Early Postnatal Development of DiethylstilbestrolTreated Rats M. LITTERIA’ Neuroendocrine Research Laboratory (1 SIB), Veterans Administration North Chicago, Illinois 60064
Medical Center,
Received February 3. 1981; revision received September 18. 1981 Thymidine kinase activity and DNA content were determined in the cerebellum after the administration of diethylstilbestrol (DES) to neonatal female rats. Rats were injected S.C. with either 30 pg DES or vehicle 48 h after birth. DNA and thymidine kinase were measured in the cerebellum at 3, 7, 10, 18, 21, 26, and 60 days of age. Thymidine kinase activity and DNA content in the DES-treated littermates were significantly increased between ages 3 to 21 days and ages 3 to 26 days, respectively. The data suggest diethylstilbestrol-induced relationships between thymidine kinase activity and DNA synthesis during the period of rapid cerebellar development.
INTRODUCTION Diethylstilbestrol (DES), a nonsteroid synthetic estrogen, was frequently prescribed between 1940 and the early 1970s for the treatment of threatened abortion and other complications of pregnancy (13). An association has been firmly established between the in utero exposure to DES and the subsequent but infrequent development of clear-cell adenocarcinoma of the vagina and cervix in female offspring (13). In addition to the common occurrence of structural and nonneoplastic abnormalities of the genital tract (13, 15), several studies suggest that daughters exposed to DES in Abbreviation: DES-diethylstilbestrol. ’ I thank C. G. Popoff for excellent technical assistance; Dr. M. Breen and H. G. Weinstein, Research-in-Aging Laboratory, for their critical review of the manuscript; R. Heinz, Researchin-Aging Laboratory, for editorial assistance; and S. Grimm for typing the manuscript. This work was supported by the Veterans Administration. 289 0014-4886/82/020289-10$02.OQ/O Copyri&t0 1982 by Academic FWs, Inc. Au rigbtt of repmdttti
in my form rcmvcd.
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utero have an increased incidence of anovulation, infertility, and fetal wastage (14, 39). Anovulatory sterility is similarly induced in the offspring of pregnant rodents treated with either DES or the natural estrogens (12, 32, 34, 35) and in adult rats treated neonatally with these hormones (10, 20). In the latter animals, postpuberal sterility is due to estrogen-induced alterations in hypothalamic and limbic structures regulating cyclic ovulation (2). The effects of the natural estrogens on the developing central nervous system are not limited to brain regions traditionally associated with the regulation of cyclic ovulation, i.e., certain hypothalamic and limbic structures. Thus, injection of estradiol benzoate into neonatal rats during the sexually critical period of brain differentiation induced long-term postpubertal alterations in the incorporation of [ 3H]lysine into proteins of specific neurons of the hypothalamus (26), limbic and paralimbic structures (2 1), cerebral cortex (27), and cerebellar cortex (25). Moreover, neonatally administered estradiol benzoate (23) and testosterone propionate (24) induced dissimilar changes in cerebellar thymidine kinase activity during early postnatal development. Incorporation of preformed thymidine into DNA (the “salvage” pathway) is dependent on a sequence of phosphorylations through thymidine mono-, di-, and triphosphate (37). The activity of thymidine kinase, the enzyme converting thymidine to the monophosphate, is consistently elevated in diverse proliferating tissues, including the cerebellum of the neonatal rat (37, 42). In the cerebellum of the rat, thymidine kinase activity and the incorporation of thymidine into DNA are both maximal approximately 6 days after birth (41, 46). The rapid growth that characterizes the cerebellum of the rat during the first 3 postnatal weeks is due to the proliferation of granule cells in the external granular layer (1). This layer disappears at approximately 21 days after birth due to the migration of its cells to the molecular and internal granular layers (1). To our knowledge, the effects of DES on the developing brain have not been examined. It therefore seemed relevant to determine if the administration of DES to neonatal female rats is associated with changes in cerebellar thymidine kinase activity and DNA content. MATERIALS
AND
METHODS
Litters were bred from 3- to 6-month-old Sprague-Dawley rats (Madison, Wis.) exposed to a 14: 10 h light-dark schedule and fed Purina Rat Chow and water ad libitum. Litter size was reduced to eight pups within 14 h of birth and the day of birth is designated as day 0. Forty-eight hours after birth female littermates, paired according to body weights, were in-
DES
AND
CEREBELLAR
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291
jetted subcutaneously with either 30 pg DES dissolved in 0.05 ml sesame oil or with an equivalent volume of vehicle. Rats were decapitated at 3, 7, 10, 18, 21, 26, or 60 days of age and the cerebellum rapidly removed, .weighed, and homogenized at 2°C. The cerebellum from each of two control or two DES-treated rats was combined at 3 days of age to provide sufficient material for all of the analyses, whereas only one cerebellum was required at the older ages. All experiments were scheduled at 0800 h to avoid possible circadian variations in enzyme activity and DNA synthesis. A 10% crude homogenate (w/v) was prepared in ice-cold medium consisting of 0.25 M sucrose, 0.004 M MgC&, and 0.02 M Tris-HCI buffer, pH 7.4 (46). Samples (0.05 ml) were removed for the extraction and determination of DNA (5). The remaining homogenate was centrifuged 60 min at 105,000 g at 2°C and the resultant supernatant served as the enzyme preparation (46). Protein content was measured in 0.02-ml samples of this supernatant (28). Thymidine kinase activity was measured according to previously detailed methodology (24). Briefly, the enzyme reaction mixture contained, in a final volume of 0.25 ml: 0.05 M Tris-HCl buffer (JIH 8), 5 mM adenosine triphosphate, 2.5 mM MgC&, 19.8 pM methyl[ 3H]thymidine (sp act 2 Ci/mmol), and 0.02 ml enzyme supernatant (0.033 to 0.051 mg protein). After incubating 10 min at 37”C, the reaction tubes were boiled 3 min, chilled in ice, and centrifuged 30 min at 1600 g and 2°C. Duplicate 0.02-ml samples of the supernatants were spread over the surface of 23-mm diethylaminoethyl (DEAE8 1) cellulose disks. The dried disks were placed into individual scintillation vials and 5 ml 0.001 M ammonium formate was added. The vials were agitated 5 min in a Dubnoff shaker and the formate discarded. This was repeated four times each with ammonium formate and distilled water, and two times with absolute alcohol.This washing procedure removed 99.7% of the unreacted [3H]thymidine from the disks. The phosphorylated derivatives of [3H]thymidine were then eluted from the disks by adding 1 ml 0.1 M HCl/ 0.2 M KC1 to the vials and shaking 15 min ( 16). Counts per minute were determined in a Packard Tri-Carb Scintillation Spectrometer after the addition of 10 ml scintillation cocktail (Dimilume TM-30) to the vials. Specific activity of thymidine kinase is expressed as (picomoles [3H]thymidine phosphorylated per minute per milligram protein) X 10-l. Significance of differences between control and DES-treated littermates was determined by the two-tailed Student’s t test for paired data; P 5 0.05 is considered significant. RESULTS Daily vaginal smears recorded between 45 and 60 days of age demonstrated persistent cornification (anovulation) in 87% of the DES-treated
292
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rats and normal 5-day estrous cycles in their littermate controls. Ovarian and uterine weights recorded for the treated rats at 26 and 60 days of age were significantly decreased below those of their controls. Thus, at age 26 days, the mean + SE for ovarian and uterine wet weights (mg) for control and treated rats were, respectively, 33.10 +- 0.31 vs. 25.58 + 0.67, P < 0.001; and 50.98 -t 1.50 vs. 36.57 + 0.92, P c 0.001. Similarly, at age 60 days, these values were 101.22 + 3.49 vs. 62.77 + 3.32, P < 0.001, for ovarian weights; and 303.27 + 7.71 vs. 246.58 f 7.52, P < 0.001, for uterine weights. Body weights of the DES-treated rats were significantly heavier than their controls at ages 3 to 10 days; however, there were no differences in the wet weights of the cerebellum (Table 1) or whole brain at any age. There were no differences in the protein content of the enzyme supernatant between control and DES-treated rats at any age. Thymidine kinase activity and DNA content were significantly elevated in the cerebellum of DES-treated rats between ages 3 to 21 days and ages 3 to 26 days, respectively (Table 1). Thymidine kinase activity-total cerebellar DNA ratios were not significantly different between treated and control rats in any age group (Table 1). There were no differences in either cerebellar thymidine kinase activity or DNA content in the 60-day-old control rats during proestrus, estrus, and diestrus; therefore, these data were combined. In agreement with previous work, enzyme activity of both control and treated rats was maximum at age 7 days (23, 24) and rapidly declined to the low values observed in the adult (42, 46). The in vitro effect of DES was determined by adding the estrogen to the enzyme assay medium in a final concentration of either 1 X lop5 or 1 X 10m6M. DES did not have any effect on thymidine kinase activity in cerebellar preparations from either 3- or 18-day-old control female rats. These results suggest that the stimulatory effect of DES per se on thymidine kinase is not mediated by direct activation of the enzyme. However, the possibility still exists that a metabolic product of DES (19) activates the enzyme. In admixture experiments, equal volumes of the 10% crude homogenates prepared from the cerebella of either 3- or 18-day-old DES-treated rats and their control littermates were mixed. Supernatants were prepared as described above. The activity of the enzyme in the mixtures was equal to the sum of the activities of the individual preparations. These data suggest that the changes induced in the activity of the enzyme in the cerebellum of DES-treated rats are not due to the presence of endogenous activators or inhibitors in the enzyme preparations. DISCUSSION Administration of DES to pregnant rodents is followed by its accumulation in both the maternal and fetal brain (8, 35). Moreover, DES injected
DES AND CEREBELLAR
THYMIDINE
KINASE
293
directly into neonatal rats binds to the estrogen receptors that are localized in specific regions of the brain (3 1,40). DES and its estrogenic metabolites also bind to the cytoplasmic estrogen receptor in tissues such as the oviduct and uterus (7, 17, 19,43). Translocation of the cytoplasmic DES-receptor complex is followed by transcription of specific genes and the synthesis of new proteins in the uterus and oviduct (7, 17, 19,43). Although differences between the mechanism of action of estrogen receptors in brain and uterus have been reported (18) the demonstration of putative estrogen receptors in the rodent cerebellum (9) does provide a biochemical mechanism for the action of DES in this structure. Thus, the increase in thymidine kinase activity and the related increase in cerebellar DNA content during the first 3 postnatal weeks could represent DES-induced changes in genetic expression which result in either an increase in enzyme synthesis, a decrease in enzyme degradation, or modification of a proenzyme to an active form. The results of the in vitro and admixture experiments provide some support for our suggestion that the stimulatory action of DES and/or its metabolites ( 19) on cerebellar thymidine kinase activity is due to the effects of the estrogen at the level of transcription and/or translation. The activity of thymidine kinase and the content of DNA in the cerebellum of the DES-treated littermates were significantly increased between ages 3 to 21 days and ages 3 to 26 days, respectively (Table 1). The observation that the thymidine kinase activity-total cerebellar DNA ratios (Table 1) were not significantly different between control and treated rats in any age group suggests a direct causal relationship between the increase in cerebellar thymidine kinase activity and DNA content after neonatal treatment with DES. The activity of thymidine kinase is specifically related to the synthesis of DNA during the premitotic S phase of the cell cycle (4). Thus, at least part of the increase of cerebellar DNA content in the treated rats must be attributed to DES-induced replication of genomic DNA. The latter could reflect an actual increase in cell number (neurons and/or glia) and/ or the occurrence of polyploidy. In this regard it should be noted that DES is a mitogenic agent (38) and that it also induced chromosomal polyploidization and nondisjunction in embryo cells (3). Postnatal neurogenesis in the cerebellum terminates approximately 3 weeks after birth (1); thus the transient inductive effect of DES on thymidine kinase and DNA during this period coincides with the availability of dividing cells susceptible to the mitogenic agent. Failure to detect the mitogenic effect of DES after age 26 days (Table 1) is due to the cessation of neurogenesis in the older cerebellum. Although speculative, part of the elevated DNA content in the cerebellum of the DES-treated rats may reflect an increase in DNA repair
60
C T
C T
C T
c T
191.3 + 2.3 (13) 193.8 k 2.6 (13)
71.2 + 0.8 (12) 70.4 f 1.2 (12)
50.9 * I.2 (12) 51.2 + 1.3 (12)
40.0 * 0.4 (IS) 39.9 + 0.4 (15)
22.4 k 0.3 (15) 23.3 + 0.2 (IS)
15.4+0.3 (ll)d 16.5 + 0.3 (II)
C T
C T
6.6 + 0.3 (IS)‘ 8.8 + 0.2 (18)
C’ T
Body weight (g)
in parentheses. phosphorylatcd/min/mg
245.15 + 2.12 (13) 246.31 f 1.62 (13)
195.23 zk 1.83 (12) 197.37 ? 2.17 (12)
179.41 2 2.91 (12) 181.78 f 2.87 (12)
165.82 2 1.84 (15) 163.80f 1.65 (15)
77.54 k 1.20 (15) 78.07 + 2.24 (15)
45.63 + 1.40 (II) 47.83 k I.14 (II)
18.32 + 0.68 (18) 18.92 + 0.61 (18)
Wet weight (mg)
’ Each value is the mean t SE for the number of determinations indicated ’ Spxitic activity of thymidine kinase is expressed as (pmol [‘H]thymidine ‘C-control; T-treated with DES. dP a 0.001. c P 5; 0.005. ‘P d 0.02. g P L 0.025. * P L 0.05.
26
21
I8
IO
3
Age (days)
TABLE
Effects of Diethylstilbestrol
.
(,ag)
X IO-‘.
+ 27.97 (13) + 30.97 (13)
+ 17.69 (12)’ ? 28.35 (12)
+ 43.84 (12)d f 31.83 (12)
_+ 17.97 (14)d + 15.90 (14)
+ 13.50 (15)d + 16.28 (15)
protein)
1429.15 1452.00
1421.50 1478.50
1404.33 1548.83
1288.43 1423.43
690.33 803.33
348.09 + II.21 (Il)d 406.91 k 9.00 (I I )
184.78 f 11.20 (9)d 221.54 f II.75 (9)
Total DNA
Cerebellum
(DES) on Female Rats”
1
I.78 k 0.04 (13) 1.73 + 0.05 (13)
1.76 + 0.04 (12) 1.84 k 0.03 (12)
2.42 f 0. I3 ( 12)’ 2.81 + 0.14 (12)
4.61 k 0.27 (15)d 5.49 t 0.21 (15)
58.10 t 1.54 (15)d 70.23 + 1.29 (15)
69.84 + I.88 (ll)d 84.27f 2.39(11)
35.51 f 2.09 (9) 45.15 + 2.51 (9)
Specific activity of thymidine kinase x 10-I b
+ 7.59 _+ 6.71
1.25 f 0.03 1.20 f 0.04
I.24 f 0.03 1.25 f 0.02
1.73 f 0.09 1.82 k 0.08
3.56 f 0.23 3.87 2 0.17
84.88 f 3.26 87.37 + 2.61
202.31 207.88
194.32 + 9.58 204.97 + 7.73
(Thymidine kinase/DNA) x 101
F
!Z
K
DES AND CEREBELLAR
THYMIDINE
KINASE
295
and/or synthesis of metabolic DNA (6, 33). Thus, DES-induced damage to genomic DNA could accelerate DNA repair and/or promote an increase in the synthesis of metabolic DNA related to gene amplification. The decline of cerebellar DNA to control values in the DES-treated rats between ages 26 to 60 days (Table 1) may reflect the catabolism of excess metabolic DNA in differentiated cells (6, 36) and the diminished ability of nondividing cells to engage in DNA repair (6). The role of DES in neuronal death is currently unknown. Therefore, the possibility cannot be excluded that the increase in cerebellar thymidine kinase activity and DNA content in the treated rats is at least partially due to the inhibition of cell death during early postnatal development. Alternatively, other possible mechanisms include: (i) A nongenomic or activational effect (30) of DES and/or its metabolites. For example, DES could have produced changes in the transport and therefore intracellular availability of thymidine and/or other metabolites in the cerebellum. In this regard, administration of estradiol benzoate to neonatal rats enhanced the transport of a small metabolically inert amino acid (cu-aminoisobutyric acid) into specific brain regions during early postnatal development (22). (ii) Primary DES-induced activational and/or genomic changes in other brain structures could secondarily influence the metabolism of cerebellar neurons via multiple interconnections between these structures and the cerebellum. (iii) Our results could be secondary to either other currently undefined DES-induced changes in the cerebellum or to possible differences in the hormonal profile of the treated rats. Neither previous work nor the data presented in this study are sufficient to distinguish between the alternatives that have been discussed. We recently reported a sexual dimorphism in the activity of cerebellar thymidine kinase as well as differences in the response of the enzyme to neonatally administered testosterone propionate and estradiol benzoate (23,24). Similarly, the develop,mental pattern of changes in enzyme activity that follow injection with DES differed from that observed in female rats treated with either estradiol benzoate or testosterone propionate [Table 1, (23, 24)]. For example, thymidine kinase activity in the cerebellum of rats treated neonatally with estradiol benzoate was (i) greater than that of their control littermates at 3 and 4 days of age, (ii) the same as the controls at age 6 days, (iii) lower than the controls between 7 and 12 days of age, and (iv) the same as control values by 15 days of age (23). The changes induced in enzyme activity by each of the three hormones were associated with parallel changes in cerebellar DNA content [Table 1, (23, 24)]. Possible mechanisms of action of testosterone propionate and estradiol benzoate on thymidine kinase activity and DNA content in the developing cerebellum have been previously detailed (23, 24). It is not surprising to
296
M. LITTERIA
find differences in the response of cerebellar thymidine kinase to the sex steroids because these hormones frequently have dissimilar effects on the same enzyme in the brain [see (29) for review]. Moreover, opposite effects of testosterone and estradiol on thymidine kinase activity in the anterior pituitary of immature male rats have been reported (44). Differences in the response of cerebellar thymidine kinase to DES and estradiol may include the following factors: (i) The quantity of estrogen that was administered must be considered. Pharmacologic concentrations of estradiol benzoate (23) and DES (see Materials and Methods) were used in each study. However, a much lower quantity of DES was injected because, in contrast to estradiol (21, 23, 25-27), an identical quantity of DES (and its excretion products) would sterilize the littermate controls (11). (ii) The dissimilar effects of the two estrogens on the enzyme may be mediated by different metabolites of DES and estradiol rather than by the hormones per se. (iii) The clearance of DES and estradiol and/or their effective metabolites from the brain may differ. (iv) DES and estradiol (and/or their effective metabolites) may differentially affect the various isoenzymes of cerebellar thymidine kinase (24, 45). To summarize: Although the mechanisms are unknown, the data presented in this study definitively link the increase in thymidine kinase activity and DNA content in the developing cerebellum to the administration of DES. The latent neoplastic potential of DES on differentiating cells (13, 15) seems to merit additional studies of this estrogen on the developing brain. REFERENCES 1. ALTMAN, J. 1970. Postnatal neurogenesis and the problem of neural plasticity. Pages 197-237 in W. A. HIMWICH, Ed., Developmental Neurobiology. Thomas, Springfield, Ill. 2. ARAI, Y., AND T. KUSAMA. 1968. Effect of neonatal treatment with estrone on hypothalamic neurons and regulation of gonadotrophin secretion. Neuroendocrinology 3: 107-l 14. 3. BARRETT, J. C., A. WONG, AND J. A. MCLACHLAN. 1981. Diethylstilbestrol induces neoplastic transformation without measurable gene mutation at two loci. Science 212: 1402- 1404. 4. BASERGA, R. 1981. The cell cycle. N. Engl. J. Med. 304: 453-459. 5. BURTON, K. 1956. A study of the conditions and mechanism of the diphenylamine reaction for the calorimetric estimation of deoxyribonucleic acid. Biochem. J. 62: 3 15-323. 6. CAMERON, I. L., AND E. K. ADRIAN, JR. 1979. Unstable nuclear DNA in hypoglossal neurons of adult mice. Cytobios 25: 85-92. 7. CHAN, L., AND B. W. O’MALLEY. 1976. Mechanism of action of the sex steroid hormones. N. Engl. J. Med. 294: 1372-1381. 8. FISCHER, L. J., J. L. WEISSINGER, D. E. RICKERT, AND K. L. HINTZE. 1976. Studies on the biological disposition of diethylstilbestrol in rats and humans. J. Toxicol. Environ. Health 1: 587-605.
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