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Effect of urethan on polyamine and DNA synthesis in the regenerating rat liver
Chem.-Biol. Interactions, 38 (1980) 35--43 © Elsevier/North-HollandScientific Publishers Ltd.
35
EFFECT OF URETHAN ON POLYAMINE AND DNA SYNTHESIS IN THE REGENERATING RAT LIVER
ISAO MATSUI, SHUZO OTANI and SEIJI MORISAWA Department o f Biochemistry, Osaka City University Medical School, Asahimachi, A benoku, Osaka 545 (Japan)
(Received October 30th, 1979) (Revision received March 28th, 1980) (Accepted August 3rd, 1980)
SUMMARY When a single dose of urethan was injected into the peritoneal cavity of rats immediately after partial hepatectomy, DNA synthesis was delayed by 12 h. The induction of omithine decarboxylase which was induced biphasically following partial hepatectomy was also reduced and delayed by 14--15 h by the administration of urethan. S-Adenosylmethionine decarboxylase activity in urethan-treated rat liver at 20 h and 29 h after operation was significantly lower than that of untreated animals. This enzyme activity was shown to increase thereafter, reaching a higher level than in untreated rats at 37--42 h. Hepatic spermidine content changed biphasically in a manner similar to DNA synthesis. These results suggest that the activities of omithine decarboxylase and S-adenosylmethionine decarboxylase may correlate with DNA synthesis and that an increase of spermidine concentration is necessary to DNA synthesis. INTRODUCTION It has been reported that urethan is a potent liver carcinogen especially acting on rapidly proliferating liver cells [1--3]. Regenerating liver of the adult rodent is therefore more susceptible to this carcinogenic action than resting liver [4,5]. Although the detailed molecular mechanism by which this compound induces neoplasia is not completely understood, previous reports have suggested that urethan suppresses the uptake of [3H] thymidine into DNA in partially hepatectomized rats [6,7], because of inhibition of thymidine kinase but not DNA polymerase [6]. In our previous report [8], we studied the effect of urethan on the induction of ornithine decarboxylase in regenerating liver, since the increase of omithine decarboxylase, a rate-limiting enzyme of polyamine biosynthesis, and intracellular levels of polyamines are quickly induced in the early stage of liver regeneration. In these experiments, we showed that although
36 ornithine decarboxylase was induced biphasically by partial hepatectomy, a single intraperitoneal injection of urethan resulted in the reduction of both phases [8]. Concerning the relationship between polyamine and DNA synthesis, some controversial experimental results have been reported; a close correlation of polyamine level and ornithine decarboxylase activity to DNA synthesis has been observed in many experimental system [9--18], although it was suggested that ornithine decarboxylase activity did n o t necessarily correlate with DNA synthesis [ 1 9 - 2 2 ] . In the present report, we have studied further the effect of urethan on DNA synthesis, ornithine decarboxylase and S-adenosylmethionine decarboxylase activities and the polyamine content in regenerating rat liver induced by partial hepatectomy to elucidate whether activation of the polyamine synthetic pathway is linked with the onset of DNA synthesis. MATERIALS AND METHODS Chemicals [6-3H]Thymidine (5 Ci/mmol), DL-[ 1-~4C] ornithine monohydrochloride (59 mCi/mmol) and S-adenosyl-L-[carboxy -~4C] methionine (60 mCi/mmo 1) were purchased from The Radiochemical Centre, Amersham, U.K. Urethan was obtained from Wako Pure Chemicals Co., Osaka, Japan and L~rnithine from Kyowa Hakko Co., Tokyo, Japan. Animals Male Sprague--Dawley rats, weighing about 200 g, were used throughout the experiments. All animals were given food and water ad lihitum until 24 h prior to the operation. Partial hepatectomy (65 -+ 5%) was performed according to the standard procedure of Higgins and Anderson [23] under light ether anaesthesia and sham-operated animals were laparotomized. Urethan was dissolved in 0.9% NaCI solution and injection intraperitoneally at a dosage of 1 mg/g body weight. Preparation o f crude liver extract The liver was removed immediately after sacrifice and homogenized with 4 vols. of 0.25 M sucrose containing 0.01 M Tris--HC1 {pH 7.3) and 0.01 M 2-mercaptoethanol. The homogenates were centrifuged at 105 000 × g for 60 rain and the supernatants were used for enzyme assay. Assay o f enzyme activity Ornithine decarboxylase activity was determined by the release of ~4CO2 from DL-[ 1-14C] ornithine as previously described [8]. The reaction mixture consisted of DL-[1-14C] ornithine {0.25/~Ci), 80 nmol L~rnithine, 0.1 mM EDTA, 0.05 M Tris--HC1 (pH 7.3), 0.05 mM pyridoxal phosphate, 1.0 mM dithiothreitol and enzyme solution in the total volume of 1.0 ml. Incubation was for 60 rain at 37°C in tightly~capped conical flasks containing 0.3 ml
37 of 1 M hyamine hydroxide in the center well. The hyamine hydroxide solution was transfered into a vial containing 5 ml of toluene-based scinl£1lation fluid (0.4% PPO, 0.01% POPOP). The radioactivity was counted in a Packard Tricarb liquid scintillation counting system. S-Adenosylmethionine decarboxylase activity was determined by measuring the release of 14CO2 from S-adenosyl-L-[carboxy-14C]methionine. The assay medium was composed of 80 mM Tris--HC1 (pH 7.3), 10 mM dithiothreitol, 0.04 mM pyridoxal phosphate, 0.25 mM putrescine, 100 nmol 8-adenosyl-L-methionine and S~denosyl-L-[carboxy-~4C]methionine (0.125 /~Ci) in a total volume of 1.0 ml. Incubation was for 60 rain at 37°C. The reactions were linear up to 90 rain. Protein was determined by the m e t h o d of Lowry et al. [24].
Determination of [3H] thymidine uptake to DNA Animals were injected intraperitoneally with 10 /JCi of [3H]thymidine per 200 g body weight and sacrificed 1 h after injection. The liver was excised immediately and washed with ice-cold 0.9% NaC1 solution. Sul> sequently the liver (about 1 g wet weight) was homogenized with 4 volume of cold distilled water. One milliliter of ice-cold 10% trichloroacetic acid was added to 1 ml of the homogenate and centrifuged at 900 × g for 10 rain in the cold. After the resulting pellet was washed with cold ethanol, 2 ml of 5% trichloroacetic acid was added and heated at 90°C for 20 min to hydrolyse DNA. An aliquot of the supematant (1 mi) was p u t into a scintillation vial and 10 ml of the toluene-based scintillation cocktail (0.4% PPO, 0.01% POPOP, 33% Triton X-100) was added prior to determination of radioactivity. [3H]Thymidine incorporation into liver DNA was expressed as cpm/g liver. Determination of polyamine levels The polyamines in liver homogenates were extracted with 5% trichloroacetic acid and analysed by fluorometry after forming l
Effect of urethan on DNA synthesis in regenerating rat liver We have previously reported that the induction of omithine decarboxylase in the early stage of rat liver regeneration was suppressed by administration of urethan immediately after partial hepatectomy [8]. To investigate the relationship between the induction of ornithine decarboxylase and the synthesis of DNA, we measured the incorporation of [3H]thymidine into hepatic DNA, Hwang et al. [6] have reported that DNA synthesis was delayed for 4--5 h by the injection of urethan within one hour of parti'al hepatectomy. We have confirmed these results. However, as shown in Fig. 1, the initial wave of DNA synthesis was delayed by about 12 h in comparison with that of urethan-untreated control rats when urethan was injected at the time of operation. This difference may be due to the body weights of rats employed
38
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, 30 partial
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Fig. 1. Time course o f D N A synthesis following treatment with urethan. Animals received intraperitoneally 1 ml 0.9% NaCI solution containing 1 mg o f urethan per b o d y weight just after partial hepatectomy. Control animals received 1 ml 0.9% NaCI solution just
after partial hepatectomy. [3H]Thymidine incorporation into hepatic DNA fraction was measured as described in Materials and Methods. Each point and vertical bar show the mean and standard error of 3--5 different animals, o, urethan-treated rats; o, control rats.
in our experiments, since the pattern of DNA synthesis in regenerating liver is highly dependent upon their age and weight [26].
Effect o f urethan on the induction o f ornithine decarboxylase Ornithine decarboxylase activity was induced biphasically having peaks at 4 h and 12 h post-operatively and declined by 30 h. This pattern of enzyme induction is similar to t h a t described by H611t~ and J~nne [27] and Gaza et al. [10]. As shown in Fig. 2, the activity of ornithine decarboxylase was suppressed at 16 h and increased biphasically in urethan-treated rats, showing peaks at 18 h and 27 h. Accordingly, the induction o f two peaks of the enzyme was reduced and delayed by 14--15 h in comparison with those in the urethan-untreated control animals.
Effect o f urethan on the activity o f S-adenosylmethionine decarboxylase The effect of urethan on the activity of 8-adenosylmethione decarboxylase required for the synthesis o f spermidine and spermine was studied. Induction of S-adenosylmethionine decarboxylase was delayed by administration o f urethan. In urethan-treated rats, the enzyme activity was m u c h
39
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10 20 Hours after partia[ hepatectomy
30
Fig. 2. Time course of ornithine decarboxylase activity following treatment with urethan. Animals were treated as described in Fig. 1. Ornithine decarboxylase activity was measured as described in Materials and Methods. Each point and vertical bar show the mean and standard error of 3--5 different a n i m a l s , . , urethan-treated rats; o, control rats.
lower than that found in extracts from control rats at 20 h and 29 h after operation (Fig. 3). The activity of the enzyme later rose and was higher than control values at 37 h and 42 h. This period of increasing activity of S-adenosylmethionine decarboxylase was consistent with that o f the initial wave of DNA synthesis of urethan-treated rat liver.
Effect of urethan on intracellularcontents of polyamines Figure 4 shows the changes of polyamine contents in regenerating rat liver 37 h after operation. In urethan.treated rats,putrescine content had increased at 25 h and by 37 h was higher than that in controls. This increase m a y reflect the enhancement of omithine decarboxylase activity. Spermidine content was higher than the ba~l level at 20 h and 37 h in control rats. This increase was also observed at 37 h in urethan-treated rats, b u t at 20 h was very small, showing that spermidine content is increased just before or at the same time as DNA synthesis. However, cellular sperrnine content was relatively constant by 37 h in both urethan-treated and control rats. DISCUSSION
Although it has been shown that omithine decarboxylase is induced in proliferating cells,it is not clear that the induction is an obligatory process of cell proliferation. In the present experiments, we have shown that treat-
40
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Hours after partial hepatectomy Fig. 3. Time course of S-adenosylmethionine decarboxylase activity following treatment with urethan. Animals were treated as described in the legend of Fig. 1. S-Adenosylmethionine decarboxylase activity was measured as described in Materials and Methods. Each point and vertical bar show the mean and standard error of 3 different animals. o, partial hepatectomized rats; e, partial hepatectomiT~d, urethan-treated rats; ~, sham operated rats; A, sham operated, urethan-treated rats.
ment with umthan delayed both ornithine decarboxylase induction and D N A synthesis in a similar fashion. This is consistent with the induction of omithine decexboxylase being responsible for D N A synthesis, although we cannot completely exclude the possibility that induction of omithine decarboxylase is unrelated to D N A synthesis. A close relationship between the increase of intracellular concentration of polyamines and D N A synthesis has been shown in m a n y experimental system [9, 11--18]. In the regenerating liver,Kallio et al. [15] and Wiegand and Pegg [16] suggested that prior accumulation of spermidine m a y be essential for the stimulation of D N A synthesis. However, other reports have suggested that an increase of putrescine, but not spermidine or spermine, is necessary to D N A synthesis of rat liver [28,29]. As shown in Fig. 4, we observed that intracellular concentration of spermidine changed biphasically in a manner similar to D N A synthesis in regenerating liver. In the regenera-
41
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Fig. 4. Time course o f hepatic polyamine content following treatment with urethan. Animals were treated as described in the legend of Fig. 1. Polyamine content was measured as described in Materials and Methods. Each point and vertical bar indicated the mean and standard error of 3--5 different animala Aa, putrescine; *% spermidine; =% spermine in urethan-treated rats (closed) and control rats (open).
ting liver of urethan-treated rats, spermidine concentration was lower at 20 h, but was higher than that of regenerating liver of untreated rats after 30 h when DNA synthesis began in urethan-treated rat liver. 8-Adenosylmethionine decarboxylase activity which is responsible for spermidine synthesis also changed in the regenerating liver of urethan-treated rats. The activity was low when DNA synthesis was suppressed, but increased when DNA synthesis began. In contrast, increase of putrescine concentration did n o t coincide with DNA synthesis. Furthermore, spermine content was relatively constant. These results suggest that increase of omithine decarboxylase activity and of spermidine concentration may correlate with DNA synthesis and that delay of DNA synthesis by urethan administration is caused by suppression of spermidine accumulation resulted from suppression of ornithine decarboxylase and S-adenosylmethionine decarboxylase activity. ACKNOWLEDGEMENTS We wish to thank Dr. W. Wilson, The Wellcome Foundation Ltd., for his
42
helpful advice and Miss M. Masutani for her skilled technical assistance. This study was supported by a grant from the Ministry of Education of Japan. REFERENCES 1 L. Chieco-Bianchi, G. DeBenedictis, G. Tridente and L. Fiere-Donati, Influence of age on susceptibility to liver carcinogenesis and skin initiating action by urethan in Swiss mice, Br. J. Cancer, 17 (1963) 672. 2 K. Klein, Influence o f age on induction with urethan o f hepatomas and other tumours in infant mice, J. Natl. Cancer Inst., 36 (1966) 1111. 3 V.R.C. Kommineni, M. Greenblatt, N. Mihailovich and S.D. Vesselinovitch, The significant o f prenatal age periods and the dose of urethan on the t u m o u r profile in MRC rat, Cancer Res., 30 (1970) 2552. 4 G.E. Grogen, M. Lane, R.A. Liebelt and F.E. Smith, The effect of partial h e p a t e c t o m y on the metabolism of urethan in young adult mice, Cancer Res., 30 (1970) 1806. 5 I.N. Chernozemski and G.P. Warwick, Liver regeneration and induction of hepatomas in B G A F 1 mice by urethan, Cancer Res., 30 (1970) 2685. 6 K.W. Hwang, S.A. Murphree and A.C. Sartorelli, The effect of urethan on the incorporation of thymidine-3H into DNA and the activities of some enzymes required for DNA biosynthesis in rat regenerating liver, Cancer Res., 33 (1973) 2149. 7 P. Rocchi, S. Grilli, A.M. Ferreri and G. Prodi, The effect of urethan on the synthesis o f nucleic acids in the cells o f regenerating rat liver, Z. Krebsforsch., 82 (1974) 277. 8 I. Matsui, S. Otani and S. Morisawa, Effect of urethan on the induction of ornithine decarboxylase in regenerating rat liver, Biochim. Biophys. Acta, 544 (1978) 372. 9 J.E. Kay and A.E. Pegg, Effect of inhibition of spermidine formation on protein and nucleic acid synthesis during l y m p h o c y t e activation, FEBS Lett., 29 (1973) 301. 10 D.J. Gaza, J. Short and I. Lieberman, On the possibility that the prereplicative increase in ornithine decarboxylase activity are related to DNA synthesis in liver, FEBS Lett., 32 (1973) 251. 11 S. Otani, Y. Mizoguchi, I. Matsui and S. Morisawa, Inhibition of DNA synthesis by methylglyoxal bis(guanylhydrazone) during l y m p h o c y t e transformation, Mol. Biol. Rept., 1 (1974) 431. 12 R.H. Fillingame, C.W. Jorstad and D.R. Morris, Increased cellular levels o f spermidine or spermine are required for optimal DNA synthesis in lymphocytes activated by concanavalin A, Proc. Natl. Acad. Sci. U.S.A., 72 (1975) 4042. 13 P.S. Mamont, P. Bohler, P.P. McGann, P. Bey, F. Schuler and C. Tardif, ~-Methylornithine, a p o t e n t competitive inhibitor o f ornithine decarboxylase, blocks proliferation of rat hepatoma cells in culture, Proc. Natl. Acad. Sci. U.S.A., 73 (1976) 1626. 14 H. P~s~ and J. J~/nne, Inhibition of polyamine accumulation and deoxyribonucleic acid synthesis in regenerating rat liver, Biochem. J., 158 (1976) 485. 15 A. Kallio, H. P~s5 and J. J~nne, Inhibition of prereplicative polyamine accumulation in regenerating rat liver, Biochim. Biophys. Acta, 479 (1977) 345. 16 M. Takigawa, H. Inoue, E. Gohda, A. Asada, Y. Takeda and Y. Mori, The role of putrescine in cell proliferation of the skin of mice induced by ethylphenylpropiolate, Exp. MoL Pathol., 27 (1977) 183. 17 L. Wiegand and A.E. Pegg, Effect of inhibitors of S-adenosylmethionine decarboxylase and ornithine decarboxylase on DNA synthesis in rat liver after partial hepatectomy, Biochim. Biophys. Acta, 517 (1978) 169. 18 M.K. Haddox, K.F. Frasier Scott and D.H. Russell, Retinol inhibition of ornithine decarboxylase induction and G 1 progression in chinese hamster ovary cells, Cancer Res., 39 (1979) 4930. 19 U. Lichti, T.J. Slaga, T. Ben, E. Patterson, H. Hennings and S.H. Yuspa, Dissociation o f t u m o r promoter-stimulated ornithine decarboxylase activity and DNA synthesis in mouse epidermis in vivo and in vitro by fluocinolone acetonide, a tumor-promotion inhibitor, Proc. Natl. Acad. Sci. U.S.A., 74 (1977) 3809.
43 20 L Clark-Lewis and A.W. Murray, Tumor promotion and the induction of epidermal ornithine decarboxylase activity in mechanically stimulated mouse skin, Cancer Res., 38 (1978) 494. 21 J.A~ McGowan and N. Fausto, Ornithine decarboxylase activity and the onset of deoxyribonucleic acid synthesis in regenerating liver, Biochem. J., 170 (1978) 123. 22 T.G. O'Brien, M.A. Lewis and L. Diamond, Ornithine decarboxylase activity and DNA synthesis after treatment of cells in culture with 12-o-tetradecanoyl-phorbol13-acetate, Cancer Res., 39 (1979) 4477. 23 J.J. Higgins and R.M. Anderson, Experimental pathology of the liver, Arch. Pathol., 12 (1931) 186. 24 O. H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, Protein measurement with the folin phenol reagent, J. Biol. Chem., 193 (1951) 265. 25 A. Dion and E. Herbst, Polyamine changes during development of Drosophila meganogaster, Ann. N.Y. Acad. Sci., 171 (1970) 723. 26 N.L.R. Bucher, M.N. Swaffield and J.F. DiTroia, The influence of age upon the incorporation of thymidine-2-14C into the DNA of regenerating rat liver, Cancer Res., 24 (1964) 509. 27 E. H~llt~ and J. J~/nne, Ornithine decarboxylase activity and the accumulation of putrescine at early stage of liver regeneration, FEBS Lett., 23 (1972) 117. 28 Y. Kato, H. Nishihara, H. Inoue, E. Gohda and Y. Takeda, Polyamine metabolism and DNA synthesis in regenerating rat liver, J. Biochem., 84 (1978) 1617. 29 T. Kameji, Y. Murakami and S. Hayashi, Effect of diaminobutane and diaminopropane on diet-stimulated polyamine synthesis and cell proliferation in rat liver, J. Biochem., 86 (1979) 191.
35
EFFECT OF URETHAN ON POLYAMINE AND DNA SYNTHESIS IN THE REGENERATING RAT LIVER
ISAO MATSUI, SHUZO OTANI and SEIJI MORISAWA Department o f Biochemistry, Osaka City University Medical School, Asahimachi, A benoku, Osaka 545 (Japan)
(Received October 30th, 1979) (Revision received March 28th, 1980) (Accepted August 3rd, 1980)
SUMMARY When a single dose of urethan was injected into the peritoneal cavity of rats immediately after partial hepatectomy, DNA synthesis was delayed by 12 h. The induction of omithine decarboxylase which was induced biphasically following partial hepatectomy was also reduced and delayed by 14--15 h by the administration of urethan. S-Adenosylmethionine decarboxylase activity in urethan-treated rat liver at 20 h and 29 h after operation was significantly lower than that of untreated animals. This enzyme activity was shown to increase thereafter, reaching a higher level than in untreated rats at 37--42 h. Hepatic spermidine content changed biphasically in a manner similar to DNA synthesis. These results suggest that the activities of omithine decarboxylase and S-adenosylmethionine decarboxylase may correlate with DNA synthesis and that an increase of spermidine concentration is necessary to DNA synthesis. INTRODUCTION It has been reported that urethan is a potent liver carcinogen especially acting on rapidly proliferating liver cells [1--3]. Regenerating liver of the adult rodent is therefore more susceptible to this carcinogenic action than resting liver [4,5]. Although the detailed molecular mechanism by which this compound induces neoplasia is not completely understood, previous reports have suggested that urethan suppresses the uptake of [3H] thymidine into DNA in partially hepatectomized rats [6,7], because of inhibition of thymidine kinase but not DNA polymerase [6]. In our previous report [8], we studied the effect of urethan on the induction of ornithine decarboxylase in regenerating liver, since the increase of omithine decarboxylase, a rate-limiting enzyme of polyamine biosynthesis, and intracellular levels of polyamines are quickly induced in the early stage of liver regeneration. In these experiments, we showed that although
36 ornithine decarboxylase was induced biphasically by partial hepatectomy, a single intraperitoneal injection of urethan resulted in the reduction of both phases [8]. Concerning the relationship between polyamine and DNA synthesis, some controversial experimental results have been reported; a close correlation of polyamine level and ornithine decarboxylase activity to DNA synthesis has been observed in many experimental system [9--18], although it was suggested that ornithine decarboxylase activity did n o t necessarily correlate with DNA synthesis [ 1 9 - 2 2 ] . In the present report, we have studied further the effect of urethan on DNA synthesis, ornithine decarboxylase and S-adenosylmethionine decarboxylase activities and the polyamine content in regenerating rat liver induced by partial hepatectomy to elucidate whether activation of the polyamine synthetic pathway is linked with the onset of DNA synthesis. MATERIALS AND METHODS Chemicals [6-3H]Thymidine (5 Ci/mmol), DL-[ 1-~4C] ornithine monohydrochloride (59 mCi/mmol) and S-adenosyl-L-[carboxy -~4C] methionine (60 mCi/mmo 1) were purchased from The Radiochemical Centre, Amersham, U.K. Urethan was obtained from Wako Pure Chemicals Co., Osaka, Japan and L~rnithine from Kyowa Hakko Co., Tokyo, Japan. Animals Male Sprague--Dawley rats, weighing about 200 g, were used throughout the experiments. All animals were given food and water ad lihitum until 24 h prior to the operation. Partial hepatectomy (65 -+ 5%) was performed according to the standard procedure of Higgins and Anderson [23] under light ether anaesthesia and sham-operated animals were laparotomized. Urethan was dissolved in 0.9% NaCI solution and injection intraperitoneally at a dosage of 1 mg/g body weight. Preparation o f crude liver extract The liver was removed immediately after sacrifice and homogenized with 4 vols. of 0.25 M sucrose containing 0.01 M Tris--HC1 {pH 7.3) and 0.01 M 2-mercaptoethanol. The homogenates were centrifuged at 105 000 × g for 60 rain and the supernatants were used for enzyme assay. Assay o f enzyme activity Ornithine decarboxylase activity was determined by the release of ~4CO2 from DL-[ 1-14C] ornithine as previously described [8]. The reaction mixture consisted of DL-[1-14C] ornithine {0.25/~Ci), 80 nmol L~rnithine, 0.1 mM EDTA, 0.05 M Tris--HC1 (pH 7.3), 0.05 mM pyridoxal phosphate, 1.0 mM dithiothreitol and enzyme solution in the total volume of 1.0 ml. Incubation was for 60 rain at 37°C in tightly~capped conical flasks containing 0.3 ml
37 of 1 M hyamine hydroxide in the center well. The hyamine hydroxide solution was transfered into a vial containing 5 ml of toluene-based scinl£1lation fluid (0.4% PPO, 0.01% POPOP). The radioactivity was counted in a Packard Tricarb liquid scintillation counting system. S-Adenosylmethionine decarboxylase activity was determined by measuring the release of 14CO2 from S-adenosyl-L-[carboxy-14C]methionine. The assay medium was composed of 80 mM Tris--HC1 (pH 7.3), 10 mM dithiothreitol, 0.04 mM pyridoxal phosphate, 0.25 mM putrescine, 100 nmol 8-adenosyl-L-methionine and S~denosyl-L-[carboxy-~4C]methionine (0.125 /~Ci) in a total volume of 1.0 ml. Incubation was for 60 rain at 37°C. The reactions were linear up to 90 rain. Protein was determined by the m e t h o d of Lowry et al. [24].
Determination of [3H] thymidine uptake to DNA Animals were injected intraperitoneally with 10 /JCi of [3H]thymidine per 200 g body weight and sacrificed 1 h after injection. The liver was excised immediately and washed with ice-cold 0.9% NaC1 solution. Sul> sequently the liver (about 1 g wet weight) was homogenized with 4 volume of cold distilled water. One milliliter of ice-cold 10% trichloroacetic acid was added to 1 ml of the homogenate and centrifuged at 900 × g for 10 rain in the cold. After the resulting pellet was washed with cold ethanol, 2 ml of 5% trichloroacetic acid was added and heated at 90°C for 20 min to hydrolyse DNA. An aliquot of the supematant (1 mi) was p u t into a scintillation vial and 10 ml of the toluene-based scintillation cocktail (0.4% PPO, 0.01% POPOP, 33% Triton X-100) was added prior to determination of radioactivity. [3H]Thymidine incorporation into liver DNA was expressed as cpm/g liver. Determination of polyamine levels The polyamines in liver homogenates were extracted with 5% trichloroacetic acid and analysed by fluorometry after forming l
Effect of urethan on DNA synthesis in regenerating rat liver We have previously reported that the induction of omithine decarboxylase in the early stage of rat liver regeneration was suppressed by administration of urethan immediately after partial hepatectomy [8]. To investigate the relationship between the induction of ornithine decarboxylase and the synthesis of DNA, we measured the incorporation of [3H]thymidine into hepatic DNA, Hwang et al. [6] have reported that DNA synthesis was delayed for 4--5 h by the injection of urethan within one hour of parti'al hepatectomy. We have confirmed these results. However, as shown in Fig. 1, the initial wave of DNA synthesis was delayed by about 12 h in comparison with that of urethan-untreated control rats when urethan was injected at the time of operation. This difference may be due to the body weights of rats employed
38
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,m'-
8
.---
~,E2 :6 U
£b ZIz eel
o
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, 20 Hours after
, 30 partial
,-;o
s6
hepatec~omy
Fig. 1. Time course o f D N A synthesis following treatment with urethan. Animals received intraperitoneally 1 ml 0.9% NaCI solution containing 1 mg o f urethan per b o d y weight just after partial hepatectomy. Control animals received 1 ml 0.9% NaCI solution just
after partial hepatectomy. [3H]Thymidine incorporation into hepatic DNA fraction was measured as described in Materials and Methods. Each point and vertical bar show the mean and standard error of 3--5 different animals, o, urethan-treated rats; o, control rats.
in our experiments, since the pattern of DNA synthesis in regenerating liver is highly dependent upon their age and weight [26].
Effect o f urethan on the induction o f ornithine decarboxylase Ornithine decarboxylase activity was induced biphasically having peaks at 4 h and 12 h post-operatively and declined by 30 h. This pattern of enzyme induction is similar to t h a t described by H611t~ and J~nne [27] and Gaza et al. [10]. As shown in Fig. 2, the activity of ornithine decarboxylase was suppressed at 16 h and increased biphasically in urethan-treated rats, showing peaks at 18 h and 27 h. Accordingly, the induction o f two peaks of the enzyme was reduced and delayed by 14--15 h in comparison with those in the urethan-untreated control animals.
Effect o f urethan on the activity o f S-adenosylmethionine decarboxylase The effect of urethan on the activity of 8-adenosylmethione decarboxylase required for the synthesis o f spermidine and spermine was studied. Induction of S-adenosylmethionine decarboxylase was delayed by administration o f urethan. In urethan-treated rats, the enzyme activity was m u c h
39
>.~ 3
X4-, 0 o
eE2
"lO~. el o w
0
0
10 20 Hours after partia[ hepatectomy
30
Fig. 2. Time course of ornithine decarboxylase activity following treatment with urethan. Animals were treated as described in Fig. 1. Ornithine decarboxylase activity was measured as described in Materials and Methods. Each point and vertical bar show the mean and standard error of 3--5 different a n i m a l s , . , urethan-treated rats; o, control rats.
lower than that found in extracts from control rats at 20 h and 29 h after operation (Fig. 3). The activity of the enzyme later rose and was higher than control values at 37 h and 42 h. This period of increasing activity of S-adenosylmethionine decarboxylase was consistent with that o f the initial wave of DNA synthesis of urethan-treated rat liver.
Effect of urethan on intracellularcontents of polyamines Figure 4 shows the changes of polyamine contents in regenerating rat liver 37 h after operation. In urethan.treated rats,putrescine content had increased at 25 h and by 37 h was higher than that in controls. This increase m a y reflect the enhancement of omithine decarboxylase activity. Spermidine content was higher than the ba~l level at 20 h and 37 h in control rats. This increase was also observed at 37 h in urethan-treated rats, b u t at 20 h was very small, showing that spermidine content is increased just before or at the same time as DNA synthesis. However, cellular sperrnine content was relatively constant by 37 h in both urethan-treated and control rats. DISCUSSION
Although it has been shown that omithine decarboxylase is induced in proliferating cells,it is not clear that the induction is an obligatory process of cell proliferation. In the present experiments, we have shown that treat-
40
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U
o m
0.2
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oE
0
2b
3b
sb
Hours after partial hepatectomy Fig. 3. Time course of S-adenosylmethionine decarboxylase activity following treatment with urethan. Animals were treated as described in the legend of Fig. 1. S-Adenosylmethionine decarboxylase activity was measured as described in Materials and Methods. Each point and vertical bar show the mean and standard error of 3 different animals. o, partial hepatectomized rats; e, partial hepatectomiT~d, urethan-treated rats; ~, sham operated rats; A, sham operated, urethan-treated rats.
ment with umthan delayed both ornithine decarboxylase induction and D N A synthesis in a similar fashion. This is consistent with the induction of omithine decexboxylase being responsible for D N A synthesis, although we cannot completely exclude the possibility that induction of omithine decarboxylase is unrelated to D N A synthesis. A close relationship between the increase of intracellular concentration of polyamines and D N A synthesis has been shown in m a n y experimental system [9, 11--18]. In the regenerating liver,Kallio et al. [15] and Wiegand and Pegg [16] suggested that prior accumulation of spermidine m a y be essential for the stimulation of D N A synthesis. However, other reports have suggested that an increase of putrescine, but not spermidine or spermine, is necessary to D N A synthesis of rat liver [28,29]. As shown in Fig. 4, we observed that intracellular concentration of spermidine changed biphasically in a manner similar to D N A synthesis in regenerating liver. In the regenera-
41
7
e- A
•~ .__. .--~5
®-6 .¢: E 3 E '5""
1
0
-Y','5
I
20
I
:,5
3'0
I
35
Hours after partial hepatectomy
Fig. 4. Time course o f hepatic polyamine content following treatment with urethan. Animals were treated as described in the legend of Fig. 1. Polyamine content was measured as described in Materials and Methods. Each point and vertical bar indicated the mean and standard error of 3--5 different animala Aa, putrescine; *% spermidine; =% spermine in urethan-treated rats (closed) and control rats (open).
ting liver of urethan-treated rats, spermidine concentration was lower at 20 h, but was higher than that of regenerating liver of untreated rats after 30 h when DNA synthesis began in urethan-treated rat liver. 8-Adenosylmethionine decarboxylase activity which is responsible for spermidine synthesis also changed in the regenerating liver of urethan-treated rats. The activity was low when DNA synthesis was suppressed, but increased when DNA synthesis began. In contrast, increase of putrescine concentration did n o t coincide with DNA synthesis. Furthermore, spermine content was relatively constant. These results suggest that increase of omithine decarboxylase activity and of spermidine concentration may correlate with DNA synthesis and that delay of DNA synthesis by urethan administration is caused by suppression of spermidine accumulation resulted from suppression of ornithine decarboxylase and S-adenosylmethionine decarboxylase activity. ACKNOWLEDGEMENTS We wish to thank Dr. W. Wilson, The Wellcome Foundation Ltd., for his
42
helpful advice and Miss M. Masutani for her skilled technical assistance. This study was supported by a grant from the Ministry of Education of Japan. REFERENCES 1 L. Chieco-Bianchi, G. DeBenedictis, G. Tridente and L. Fiere-Donati, Influence of age on susceptibility to liver carcinogenesis and skin initiating action by urethan in Swiss mice, Br. J. Cancer, 17 (1963) 672. 2 K. Klein, Influence o f age on induction with urethan o f hepatomas and other tumours in infant mice, J. Natl. Cancer Inst., 36 (1966) 1111. 3 V.R.C. Kommineni, M. Greenblatt, N. Mihailovich and S.D. Vesselinovitch, The significant o f prenatal age periods and the dose of urethan on the t u m o u r profile in MRC rat, Cancer Res., 30 (1970) 2552. 4 G.E. Grogen, M. Lane, R.A. Liebelt and F.E. Smith, The effect of partial h e p a t e c t o m y on the metabolism of urethan in young adult mice, Cancer Res., 30 (1970) 1806. 5 I.N. Chernozemski and G.P. Warwick, Liver regeneration and induction of hepatomas in B G A F 1 mice by urethan, Cancer Res., 30 (1970) 2685. 6 K.W. Hwang, S.A. Murphree and A.C. Sartorelli, The effect of urethan on the incorporation of thymidine-3H into DNA and the activities of some enzymes required for DNA biosynthesis in rat regenerating liver, Cancer Res., 33 (1973) 2149. 7 P. Rocchi, S. Grilli, A.M. Ferreri and G. Prodi, The effect of urethan on the synthesis o f nucleic acids in the cells o f regenerating rat liver, Z. Krebsforsch., 82 (1974) 277. 8 I. Matsui, S. Otani and S. Morisawa, Effect of urethan on the induction of ornithine decarboxylase in regenerating rat liver, Biochim. Biophys. Acta, 544 (1978) 372. 9 J.E. Kay and A.E. Pegg, Effect of inhibition of spermidine formation on protein and nucleic acid synthesis during l y m p h o c y t e activation, FEBS Lett., 29 (1973) 301. 10 D.J. Gaza, J. Short and I. Lieberman, On the possibility that the prereplicative increase in ornithine decarboxylase activity are related to DNA synthesis in liver, FEBS Lett., 32 (1973) 251. 11 S. Otani, Y. Mizoguchi, I. Matsui and S. Morisawa, Inhibition of DNA synthesis by methylglyoxal bis(guanylhydrazone) during l y m p h o c y t e transformation, Mol. Biol. Rept., 1 (1974) 431. 12 R.H. Fillingame, C.W. Jorstad and D.R. Morris, Increased cellular levels o f spermidine or spermine are required for optimal DNA synthesis in lymphocytes activated by concanavalin A, Proc. Natl. Acad. Sci. U.S.A., 72 (1975) 4042. 13 P.S. Mamont, P. Bohler, P.P. McGann, P. Bey, F. Schuler and C. Tardif, ~-Methylornithine, a p o t e n t competitive inhibitor o f ornithine decarboxylase, blocks proliferation of rat hepatoma cells in culture, Proc. Natl. Acad. Sci. U.S.A., 73 (1976) 1626. 14 H. P~s~ and J. J~/nne, Inhibition of polyamine accumulation and deoxyribonucleic acid synthesis in regenerating rat liver, Biochem. J., 158 (1976) 485. 15 A. Kallio, H. P~s5 and J. J~nne, Inhibition of prereplicative polyamine accumulation in regenerating rat liver, Biochim. Biophys. Acta, 479 (1977) 345. 16 M. Takigawa, H. Inoue, E. Gohda, A. Asada, Y. Takeda and Y. Mori, The role of putrescine in cell proliferation of the skin of mice induced by ethylphenylpropiolate, Exp. MoL Pathol., 27 (1977) 183. 17 L. Wiegand and A.E. Pegg, Effect of inhibitors of S-adenosylmethionine decarboxylase and ornithine decarboxylase on DNA synthesis in rat liver after partial hepatectomy, Biochim. Biophys. Acta, 517 (1978) 169. 18 M.K. Haddox, K.F. Frasier Scott and D.H. Russell, Retinol inhibition of ornithine decarboxylase induction and G 1 progression in chinese hamster ovary cells, Cancer Res., 39 (1979) 4930. 19 U. Lichti, T.J. Slaga, T. Ben, E. Patterson, H. Hennings and S.H. Yuspa, Dissociation o f t u m o r promoter-stimulated ornithine decarboxylase activity and DNA synthesis in mouse epidermis in vivo and in vitro by fluocinolone acetonide, a tumor-promotion inhibitor, Proc. Natl. Acad. Sci. U.S.A., 74 (1977) 3809.
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