Ornithine decarboxylase induction and polyamine levels in the kidney of estradiol-treated castrated male rats

Life Sciences, Vol. 26, pp. 689-698 Printed in the U.S,A.

Pergamon Pres;

ORNITHINE DECARBOXYLASEINDUCTION AND POLYAMINE LEVELS IN THE KIDNEY OF ESTRADIOL-TREATED CASTRATEDMALE RATS Hajime Nawata, Richard S. Yamamoto and Lionel A. Poirier Nutrition and Metabolism Section, Laboratory of Carcinogen Metabolism, DCCP, NCI, NIH, Bethesda, Maryland 20205 (Received in final form January 3, 1980)

SU!',¢rIARY

Ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase (SAMDC), and thymidine kinase (TK) activities and polyamine concentrations on the kidneys of male castrated rats were studied following sc injection of estradiol. Estradiol caused an l l - f o l d increase in ODC activity 24 hours after administration. SAMDCactivity doubled but TK activity decreased by two-thirds 2 days after estradiol treatment. The concentrations of polyamines, especially putrescine, showed sharp elevations 2 days following estradiol treatment, l day after the peak of ODC activity. The increase in ODC activity was suppressed by cycloheximide and by actinomycin D. Estradiol and diethylstilbestrol (DES), but not progesterone increased ODC activity. Estradiol suppressed ODC activities of l i v e r , thymus, adrenal glands, testes and prostate. A specific estradiol-binding protein was demonstrated in.~he rat kidney. The dissociation constant (Kd) was 1.64 x lO-! M and numbers of binding sites were 31 fmoles/mg protein. Correlation between the binding of estradiol to the cytosol protein and elevation of ODC by estradiol was observed. Ornithine decarboxylase (E C 4.1.I.17) (ODC) appears to be the rate limiting enzyme in the biosynthesis of polyamines, and its increase in activity correlates well with cell growth (1). Many hormones have been shown to increase ODC activity in appropriate target tissues (2). Administration of estradiol and diethylstilbestrol (DES) was shown to increase ODC and S-adenosylmethionine decarboxylase (E C 4. I. I. 50) (SAMDC) activities as well as to increase polyamine levels in such target organs of estrogens as the oviduct of chicks (3) and the uterus of rats (4). Although the details of estrogen action on the kidney have not been extensively studied, a specific high-affinity estrogen-binding protein was demonstrated in the kidney of the male golden Syrian hamster, which develops kidney tumors upon the administration of estrogen (5, 6). Estrogen administration enhances the activities of mouse renal arginase (9) and rat renal ornithine aminotransferase (lO). Estrogen-binding proteins have also been found in the kidneys of mice (7) and rats (8). The intracellular levels of thymidine kinase (E C 2. 7. I. 21) (TK) have been related to the rate of cell proliferation in tumors and in hormonally stimulated tissues ( l l , 12). Therefore, as the kidney seems to be a target organ of estrogen, i t is of interest to determine the estrogen effect on ODC and polyamine synthesis and to correlate the estradiol binding a f f i n i t y with ODC induction in the kidney. 0024-3205/80/090689-10502.00/0 Copyright (c) 1980 Pergamon Press Ltd

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MATERIALS AND METHODS Chemicals and ~rmones: DL-[I-14C] o r n i t h i n e monohydrochloride (SA, 45.0 mCi/mmol), [ I ~ ] " C ] carboxy-labelled adenosyl-L-methionine (SA, 52.3 mCi/nmlol), and [ 2 , 4 , 6 , 7 - H] e s t r a d i o l (SA, llO Ci/mmol) were purchased from New England Nuclear. E s t r a d i o l , other hormones, polyamines, dansyl c h l o r i d e , ATP and Norit A were purchased from Sigma Chemical Company. S i l i c a gel plates were purchased from Analtech, Inc. Whatmann DEAE c e l l u l o s e paper (DE 81) was obtained from Fisher Chemical Company. Dextran was purchased from Pharmacia. Animals and Hormone Administration: Male F344/cr rats (40-50 g) were obtained from the DCT Animal Program of NCI. A f t e r a r r i v a l at t h i s laboratory they were housed in hanging p l a s t i c cages (6 rats/cage) and received food (NIH 07 Chow) and water ad l i b i t u m f o r 7 days p r i o r to f u r t h e r treatnlent. The animals were then injected with hormones or were castrated and maintained f o r another 7 days p r i o r to hormone treatment. Since renal ODC has diurnal rhythm (13), f o r experiments l a s t i n g 24 hours or more, the animals were injected between 9:00 and I0:00 a. m. and s a c r i f i c e d at 24-hour i n t e r v a l s . Preparation of Tissue Extracts: The rats were k i l l e d by decapitation; the kidneys were r a p i d l y excised; t h e i r capsules were immediately removed; and the kidneys were placed on ice. The r i g h t kidney was homogenized in 5 vol of an ice-cold solution containing 0.25 M sucrose, 0.01M sodium phosphate (pH 7.0), 0.2 mM pyridoxal phosphate, 0.5 mM d i t h i o t h r e i t o l and O.l mM EDTA. One ml of the homogenate was used f o r the analysis of t o t a l p r o t e i n , RNA and DNA. The rest of the homogenate was centrifuged at 105,000 x g at 4°C f o r 60 min and the supernatant was used f o r the ODC and SAMDC assays. The l e f t kindey was cut along the l o n g i t u d i n a l axis. One-half of the kidney was homogenized in 5 vol of an ice-cold solution containing 50 mM Tris-HCl (pH 7.5), 0.25 M sucrose and 3 mM CaCl2 and centrifuged at 105,000 x g at 4°C f o r 60 min. The soluble f r a c t i o n was used f o r the assay of TK. The other h a l f of the l e f t kidney was homogenized in cold 0.2 M HClO4 and cent r i f u g e d at I0,000 x g. The supernatant f r a c t i o n was used f o r polyamine analysis. ODC Assay: ODC was assayed by modification of the method of Russell and Snyder (14). O n e - f i f t h ml of enzJ~e solution was preincubated in a water bath f o r 5 min at 37°C with 0.4 ml of assay mixture consisting of 25 mM sodium phosphate (pH 7.0), 0.I mM pyridoxal phosphate, 0.5 mM d i t h i o t h r e i t o l and 0.I mM EDTA. One-tenth ml of s u b s t ~ t e solution containing 0.049 ~moles of L - o r n i t h i n e and 0.005 ~moles of D L - [ ~ C ] o r n i t h i n e was then added, and the tubes were capped with serum stoppers equipped with polypropylene center wells (Kontes). A f t e r a l-hour incubation, the enzyme reaction was stopped by the i n j e c t i o n of 0.2 ml of 2 M HCIO4. O n e - f i f t h ml of NCS s o l u b i l i z e r (Amersham) was then injected into the center w e l l . Incubation was continued f o r another 60 min to allow the absorption of the released CO2 by the NCS. The center well including the paper and NCS were then transferred to counting v i a l s with I0 ml of toluene-based s c i n t i l l a t o r and counted in a Beckman (Model No LS 9000) s c i n t i l l a t i o n counter. The enzyme a c t i v i t y was expressed as pmoles CO2 liberated/mg protein/30 or 60 min. Renal ODC was r e l a t i v e l y unstable even in the presence of d i t h i o t h r e i t o l and pyridoxal phosphate. When stored f o r 3 days at -20°C, cytosol ODC l o s t 40% of i t s original activity. Thus the enzyme a c t i v i t y was assayed w i t h i n 6 hours of preparation of the supernatant.

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SAMDC Assay: SAMDCa c t i v i t y was assayed by a modification of the procedure of Pegg and Williams-Ashman (15). One-fifth ml of supernatant was preincubated in a shaker-water bath for 5 min at 37°C with 0.2 ml of the assay mixture of IO0 ~ sodium phosphate (pH 7.1), O.l mM pyridoxal phosphate, 5 mM d i t h i o t h r e i t o l , O.l mM EDT#.and 2.5 mM putrescine. One-tenth ml solution containing 53.8 nmoles of [ l - ' 4 C ] SAM (Specific a c t i v i t y , 1.93 mCi/mmole) was then added. After 30 min the reaction was stopped as in the ODC assay. The incubation was continued for another 60 min to trap CO2. Enzymea c t i v i t y was expressed as pmoles CO2 liberated/ mg protein/30 min. SAMDCa c t i v i t y was greatest in solutions containing putrescine concentrations greater than 0.25 mM. TK Assay: TK was assayed by the DEAE-cellulose paper disk method of Bresnick (16). The reaction mixture contained 0.2 ml of the enzyme preparation, 5 mM ATP, 5 mMMgCl2, and 80 mM Tris HCI (pH 7.5) in a $gtal volume of 0.5 ml. The reaction was started by adding 3.7 nmoles of ['~C] thymidine (Specific a c t i v i t y , 26.1 mCi/mmole). The mixture was incubated at 37°C for 30 min. The reaction was terminated by immersing the tubes in a boiling water bath for 2 min. The precipitates were removed by centrifugation and 0.2 mlaliquots of the supernatants were applied to DEAE-cellulose paper squares (3 x 3 cm) and dried at room temperature. The dried squares were immersed for lO min in a tray containing l him ammonium formate, then the papers were washed in d i s t i l l e d water for another lO min. This procedure was repeated 3 times. Finally the papers were immersed in 95% ethanol and dried at 8O°C. The counting of radioactivity was made by directly immersing the dried paper in a counting vial with IO ml of toluene-based s c i n t i l l a t i o n f l u i d . The specific a c t i v i t y of TK was expressed as nmoles dTMP formed/mg protein/3O min. Polyamine Determination: Polyamineswere determined by the modification of Seiler's dansyl procedure previously described by Wyatt et. al. (17). Thawed HCIO4 extracts (0.2 ml) were incubated at room temperature~h 0.4 ml dansyl chloride (30 mg/ml acetone) and 19.5 mg NapCOR for 16 hours in the dark. Excess dansyl chloride was eliminated by the-addition of O.l ml proline (lO0 mg/ml water) and after 30 min at room temperature, acetone was evaporated from the reaction mixture with N?. The dansylamide derivatives of the amines were dissolved with 0.5 ml of benzene. Ten pl from the benzene extract were applied to 400 ~M layers of s i l i c a gel on 20 x 20 cm glass plates. Separation of dansylamide derivatives of spermine, spermidine and putrescine was achieved by chromatographic development with ethylacetate-cyclohexane (2/3, v/v) at room temperature. The plates were run twice. Dansyl derivatives were visualized on the plate with u l t r a v i o l e t l i g h t (350 nm), and the areas corresponding to the fluorescent spots were scraped off and extracted with dioxane. Fluorescence of the extract was measured in a spectrofluorometer at 337 and 485 nm for activation and emission, respectively. Determination of Specific Bindin 9 of [3H]Estradiol: Rat kidneys were homogenized in 3 vol of ice cold TED buffer (O.Ol M Tris-HCl, O.OOl5 M EDTA, 0.005 M d i t h i o t h r e i t o l , and IO% glycerol, pH 7.4 at 4°C) using a Potter glass homogenizer. The homogenates were centrifuged at IO5,0OO x g for 60 min at 4°C, and the cytosol was removed carefully to avoid the floating layer of fat. Aliquots of cytosol (with more than l mg/ml of protein concentration) were incubated for 18 - 20 hours at O°C with ~n equal volume of TED buffer containing the appropriate concentration of [aH] estradiol both with and without a lO0-fold excess of nonradioactive DES. At the end of incubation a slurry of dextran-coated charcoal consisting of 0.5% acid-washed Norit A and 0.05% dextran in TED buffer was added to cytosol incubations. After

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15 min at O°C, the samples were centrifuged at 800 x g for lO min, and the radioactivity in the supernatant was determined. Nonspecific binding was determined by measuring the radioactivity not adsorbed to dextran-coated charcoal incubation containing a lO0-fold unlabelled DES. This3valuewas subtracted from the binding obtained for the incubation with [ H] estradio] only. The difference between the two represents specific [~H] estradiol bound to high-affinity, low-capacity binding sites. Analysis of nucleic acids and protein: The homogenates were fractionated by Schneider's procedure (18). DNA was then determined with the diphenylamine reaction and RNA with the orcinol method as described by Ashwell (19). Protein was determined by the Lowry method (20). RESULTS Dose-Response to Estradio] on the Stimulation of ODC in Castrated Male Rats: Twenty-four hours after the injection of various concentrations of estradiol into the castrated male rats, the rats were k i l l e d and ODC a c t i v i t i e s of kidneys were determined. A linear dose-response curve was obtained with doses varying between 0 and 50 ~g of estradio]. Maximal response was obtained at the doses of 50 to 500 ~g of estradiol. The Effect of Estradiol on ODC~ SAMDC and TK levels: The results of the activ i t i e s of ODC, SAMDCand TK are shown in Fig. 1; The kinetics of the increase in ODC a c t i v i t y in rat kidney following a single sc injection of estradio] showed a steep rise in ODC a c t i v i t y . The peak of ODC a c t i v i t y occurred 24 hours after estradio] injection. Estradiol caused an ] l - f o l d increase in the ODC a c t i v i t y . After 24 hours ODC a c t i v i t y gradually decreased. SAMDCactiv i t y doubled 2 days after treatment. On the other hand TK a c t i v i t y decreased sharply to one-third of its i n i t i a l level 2 days after estradiol treatment. '

t

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2

2:

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2

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TABLE I I HORMONAL SPECIFICITY ON STIMULATION OF ODC AND ON ESTRADIOL BINDING PROTEIN IN THE KIDNEYS OF CASTRATED MALE RATS. ODC a c t i v i t ya Drug Control

(pmoles C02/mg protein/60 min) 444 + 54

17B-Estradiol

4123 + 74c

17~-Estradiol

795 + 95d

[3H]estradiol binding b (fmoles/mg protein) 14.33 4.33c I0.43

Estrone

I064 + 209c

5.56c

Estriol

2983 + 409c

5.60c

Diethylstilbestrol

3585 + 270c

4.61 c

Progesterone

559 + 84

14.05

a. Each drug in corn oil (500 ~g/lOOg BW) was injected sc. Rats were k i l l e d 24 hours after injection. Results were expressd as mean + S. E. The numbers of rats are 12 in control and 4 in h~rmone-treated ~oup. b. Renal cytosol in TED buffer was incubated with [~H]estradiol (final concentration 2.5 nM) in the presence or abscence of a lO0-~old excess of cold competitors at O°C for 20 hours. The bound form of [~H]estradiol was separated by dextran coated charcoal, c. p < 0.005. d. 0.005 < p < 0.02. Polyamine Concentrations in the Kidneys of Castrated Male Rats Treated with Estradiol: The concentrations of polyamines in the kidneys of castrated male rats at various times following single sc injection of estradiol are shown in Fig. 2. There was a gradual decrease in putrescine concentration for 24 hours after estradiol treatment. Then 2 days after estradiol treatment a sharp increase in putrescine concentrations occurred (from 41.3 + 8.0 to 75.1 + 2.4 nmoles/g kidney) and followed by gradual decline. The cha-~ges seen in ~he renal concentrations of spermidine and spermine were similar but smaller than those observed for putrescine. The renal levels of spermidine O, l , and 2 days following estradio] injection were 349.8 + 9.8, 269.8 + 28.2 and 405.7 + 16.2 nmoles/g kidney, respectively; the c-orresponding ~alues for spermin-e were 616.2 ~ 20.4, 452.4 + 57.8 and 647.7 + 16.9 nmoles/g kidney (Fig. 2). Inhibition by Cycloheximide and Actinomycin D on the Stimulation of Renal ODC Levels by Estradiol: The effect of inhibitors of protein synthesis and RNA synthesis on the estradiol-stimulated increase in ODC a c t i v i t y is shown in Table I I I o Cycloheximide completely prevented the rise in ODC a c t i v i t y caused by estradiol. A single injection of actinomycin D at the time of the injection of estradiol inhibited 66% of the rise in ODC a c t i v i t y . Repeated injections of actinomycin D inhibited 91% of the rise in ODC a c t i v i t y following estradiol treatment.

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ODC Induction in E2-Treated Rat Kidneys

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and TK a c t i v i t i e s were assayed as described in Materials and Methods. Each point represents the mean + S. E. of 4 rats. Open c i r c l e , ODC a c t i v i t y , closed c i r c l e , SAMDCactivTty, open triangle, TK a c t i v i t y . Effect of Estradiol on the Weight and RNA and DNA Contents of Rat kidney: Kidney weight, RNA and DNA contents were determined 0, 6, 12, 24, 48 and 72 hours after sc injection of estradiol. Kidneyweight (mg/lO0 g body weight) increased about If% 12 and 24 hours after estradiol treatment (p < 0.05). The RNA content of kidney increased from 487.6 + 32.2 to 590.2 + 29.2 ~g/kidney (p < 0.001) 24 hours after estradiol t r e a t ~ n t , while renal-protein and DNA contents did not change s i g n i f i c a n t l y 24 hours after estradiol injection. Repeated injections of estradiol produced similar responses to those following a single injection of the hormone.

Organ and Hormone S p e c i f i c ! t y to the Stimulation of ODC A c t i v i t y by Estradiol: The effect of estradiol on the ODC a c t i v i t i e s in other organs was examined (Table I ) . Twenty-four hours a f t e r the sc injection of e s t r a d i o l , ODC a c t i v i t y decreased in l i v e r , thymus, adrenal glands, testes and prostate from 50% to 95%. Among the organs examined only the kidney had increased ODC a c t i v i t y following estradiol treatment. Several sex hormones were examined to determine the s p e c i f i c i t y of the hormones. Twenty-four hours a f t e r sc injections of 6 female sex hormones, estradiol and DES produced 8-9 fold increases in renal ODC a c t i v i t y . The nonestrogenic isomer of e s t r a d i o l , 17~-estradiol, and progesterone were weakly active, while e s t r i o l and estrone led to approximately 5-fold increases in renal ODC a c t i v i t y (Table I I ) .

TABLE I EFFECTS OF ESTRADIOL ON THE STIMULATION OF ODC IN VARIOUS ORGANS IN INTACT MALE RATS ODC (pmoles C02/mg protein/60 min)

% of Control

Organ Control Liver

Estradiol-treated

10.3 +

1.5

Kidney

520.3 ~

62.0

Adrenal

178.4 +

42.8

9.3 +

1.2

5

Testis

141.1 +

15.1

97.5 +

13.5

69

1731.6+ 310.5

183.8 +

12.5

lO

202.0 ~

63.5

55

Prostate Thymus

366.1 ~

45.8

5.6 +

0.2

54

5567.2 ~ 134.0

1070

Estradiol in corn oil (500 ~g/lOOg BW) was injected sc. The rats were k i l l e d 24 hours later. The results are expressed as mean ~ S. E. The numbers of rats were 8 in control and 4 in estradiol-treated groups.

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ODC Induction in E2-Treated Rat Kidneys

695

Bindin 9 to Cytosol Protein by [3H] Estradiol: Equilibrium curves of [3H] estradiol to renal cytosol were graphed in Fig. 3~ A Scatchard plot is shown in the insert. From the slope a Kd of 1.65 x lO-ju M was calculated. From the intercept on the abscissa, i t can be estimated that 31 fmoles of estradiol/mg protein are specifically bound. Thus a specific binding protein with high a f f i n i t y for estradiol exists fn the renal cytosol of F344 rats. Specificity of Steroid to Estradiol Bindin 9 Protein: In a study of binding specificity, renal cytosol was incubated with [ H] estradiol in the presence or absence of a lO0-fold excess of various ynlabeled steroids. As shown in Table I I , estradiol and DES competed with [~H]estradiol for renal3cytosol most strongly. Estrone and estriol also competed s l i g h t l y with [ H]estradiol for cytosol binding sites. On the other hand,_progesterone and 17~-estradio] interfered only a l i t t l e with the binding of [3H]estradiol. i

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FIG. 2 Polyamine concentrations in the kidneys of castrated male rats after a single sc injection of estradiol (500 ~g/lOOg BW). Polyamineswere determined as described in Materials and Methods. Each point represents the mean + S. E. of 4 rats. Closed circle, putrescine, open circle, spermidine, open-triangle, spermine.

Castrated male rats recieved one ip injection of actinomycin D (150 ~g/lO0 g BW) at the time of estradiol injection (500 ~g/lO0 g BW) or recieved one ip injection of cycloheximide (200 ~g/lOOg BW) 21 hours after estradiol injection. All rats were k i l l e d 24 hours after estradiol injection. ODC a c t i v i t y was determined as described in Materials and Methods. Results are represented as mean~ S. E. of 4 rats.

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ODC Induction in E2-Treated Rat Kidneys

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TABLE I l l INHIBITION OF ODC STIMULAION BY INHIBITORS OF PROTEIN SYNTHESIS

ODC a c t i v i t y (pmoles C02/m9 protein/60 min)

Treatment None

725 + 89

Estradiol

7840 + 395

Estradiol + cycloheximide

170 +

16

Estradiol + actinoymcin D

3169 + 252

Estradiol + 3 Injections of actinomycin D

1352 + 210

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FIG. 3 Equilibrium binding of [3H]estradiol to the rat renal cytosol. Aliquots of cytosol (3.1 mg pro~ein/ml) were incubated at O°C for 20 hours with indicated concentrations of [JH]estradio]. Parallel incubations with an added excess of unlabeled estradiol were carried out to determine nonspecific binding. The binding data represent specifically bound estradiol. The insert represents a Scatchard plot of the same data. DISCUSSION

Previous studies have identified specific estrogen-binding protein in estrogen-sensitive tissue such as the uterus (21, 22). In this target organ estradiol also induced ODC a c t i v i t y and polyamine synthesis (23, 24). Rat kidney has been considered a nontarget organ of estradiol. With the exceptions of arginase (9) and ornithine aminotransferase (lO), few studies have been published on the control of renal enzymes by estradiol. In this study specific estradiol binding protein was found in the renal cytosol of rats.

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ODC Induction in E2-Treated Rat Kidneys

697

The current concept on the biochemical action of estrogen in its target tissue is that estradiol enters the cell, forms a complex with its receptor in cytoplasm, and is then translocated into the nucleus to affect RNA and protein synthesis. Therefore, the interaction of estradiol with its receptor proposed for the uterus may also be applicable to the kidney. In the present study estradiol increased ODC activity and polyamine levels in the kidney of castrated male rats. The stimulation of ODC levels was hormone and organ-specific. Estradiol and DES had the most potent effect on the stimulation of ODC activity, while 17~-estradiol and progesterone had l i t t l e effect. Among 6 organs examined in the present study only the kidney was sensitive to the sitmulation of ODC by estradiol. Estrad~ol and DES stimulated ODC levels and they also successfully competed with [JH]estradiol for the binding protein. On the other hand, 17~-estradiol and progesterone, which affected ODC activities slightly, interfered very l i t t l e with the binding of [~H]estradiol. Therefore, an inverse correlation exists between the a b i l i t y of estrogen to induce ODC activity and its a b i l i t y to compete with [~H]estradiol for the binding protein. This study supports the concept that binding to a cytoplasmic receptor protein by estradiol is a prerequisite for its a b i l i t y to induce ODC. The suppression by cycloheximide and by actinomycin D of the adaptive response of ODC to estradiol is consistent with a short h a l f - l i f e and very rapid turnover for ODC (26). It appears that increases in ODC activities reflect de novo enzyme synthesis and that such synthesis requires DNA-dependent RNA syn~ thesis. The increase in kidney weights and RNA contents following estradiol treatment parallels the stimulating effect of polyamines on RNA and protein synthesis and accumulation (27, 28). Hormones such as GH (29), prolactin (30), hydrocortisone (29), LH (31) and human chorionic gonadotropin (31) rapidly and maximally increased ODC activity in the target organs within 4 hours after treatment. However, uterus (21) as well as kidney are unique in that estradiol increased ODC activity only slightly by 4 hours, but maximally at 24 hours after treatment. The kinetics of the stimulation of ODC and polyamine levels by estradiol indicates that ODC appears to be the rate limiting enzyme for the formation of polyamines in rat kidney. The inability of repeated treatment with estradiol to maintain high renal levels of ODC resembled the inability of GH to maintain high hepatic levels of ODC (29) and of LH to maintain high ovarian levels of ODC (32). This phenomenon may represent the normal hormonal regulation of ODC stimulation in the hormone target organs. An unexpected observation was the suppression of renal TK by estradiol. Previous studies had shown an increase in TK activity prior to DNA synthesis in the adrenal cortex of ACTH-treated animals (33) and an increase in DNA synthesis of rat uterus 24 hours after injection with estradiol (8). However, dissociation of the simulation of ODC and TK by prolactin has been reported in rat l i v e r (34). Estradiol and DES are known to induce adenocarcinoma in the kidney of Syrian golden hamsters (6). Increased levels of ODC have been associated with the promotion stage of tumorigenesis (35). Therefore, the evidence of ODC induction and polyamine synthesis in the kidney by estradiol may present a good tool to aid in studying the mechanism of estrogen carcinogenesis. ACKNOWLEDGEMENT We are grateful to Ms. Susan Rector for excellent technical assistance and Ms. Willette Oliver for excellent secretarial assistance.

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