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Biochimica et Biophysica Acta, 544 (1978) 372--380 © Elsevier/North-Holland Biomedical Press
BBA 28736
EFFECT OF URETHAN ON THE INDUCTION OF ORNITHINE DECARBOXYLASE IN REGENERATING RAT LIVER
ISAO MATSUI, SHUZO OTANI and SEIJI MORISAWA
Department of Biochemistry, Osaka City University Medical School, Asahimachi, Abenoku, Osaka 545 (Japan) (Received April 25th, 1978)
Summary The effect of urethan on the induction of ornithine decarboxylase in the early stage of the regeneration of rat liver was studied. The induced activity of ornithine decarboxylase was suppressed by administration of urethan immediately after partial hepatectomy. Although ornithine decarboxylase was induced biphasicaUy by partial hepatectomy, a single intraperitoneal injection of urethan resulted in the reduction of both phases. However, the ornithine decarboxylase activity induced by glucocorticoids and growth hormone was not suppressed by urethan. The increased level of 3',5'-cyclic adenosine monophosphate induced by partial h e p a t e c t o m y was also reduced by urethan and this suppression was proportional to the suppression of ornithine decarboxylase activity. Reversal of the urethan-induced suppression of ornithine decarboxylase by administration of dibutyryl 3',5'-cyclic adenosine monophosphate was also observed.
Introduction Accumulating evidence indicates that rapidly proliferating rat liver cells are more susceptible to the carcinogenic action of urethan than are resting cells [1,2], although the molecular mechanism by which urethan induces neoplasia has not been elucidated. To elucidate urethan carcinogenesis, it is important to study the effect of urethan on biochemical changes induced by partial hepatectomy. Urethan suppresses the incorporation of [3H]thymidine into DNA [3,4], which is due to the inhibition of thymidine kinase activity, but not due to that of DNA polymerase [3]. It has been reported that the treatment of urethan caused 50--55% inhibition of the uptake of [3H]orotic acid into nuclear RNA and heterogenous RNA [5]. The mechanism of inhibition of RNA synthesis by urethan is not yet clear as neither template activity for RNA syn-
373 thesis of chromatin [6,7], nor the activities of R N A polymerase I and II were depressed [6]. We studied the effect of urethan on the change of ornithine decarboxylase (EC 4.1.1.17), since the increases of ornithine decarboxylase activity and intracellular levels of polyamines are quickly induced in this early stage [8--13]. Furthermore, we studied the effect of urethan on intracellular levels of 3',5'cyclic adenosine monophosphate (cyclic AMP), which also increased in regenerating liver and studied the relationship between cyclic AMP and ornithine decarboxylase induction, as the involvement of cyclic AMP in the induction of ornithine decarboxylase was observed in several papers [14--22], b u t n o t in others [23--28]. Materials and Methods
Chemicals. Urethan was purchased from Wako Pure Chemicals Co. Osaka, Japan. N6,O2'-Dibutyryl cyclic AMP, dexamethasone acetate and cortisone acetate came from Sigma Chemical Co. St. Louis, Mo. Bovine somatotropin was obtained from Miles Laboratories Inc., Kankakee, Ill. DL-[1-14C]Ornithine (specific activity 59 mCi/mmol) was recieved form The Radiochemical Centre, Amersham, U.K. and L-ornithine from K y o w a Hakko Co. T o k y o , Japan. Animals. Male Sprague-Dawley rats, weighing about 200 g, were used in all experiments. All animals were given food and water ad libitum until 24 h prior to the operation which was performed between 9 a.m. and noon. Partial hepat e c t o m y (65 +_5%) was carried o u t according to the standard procedure of Higgins and Anderson [29] under light ether anaesthesia and sham-operated animals were laparotomized. Urethan was dissolved in 0.9% NaC1 solution and injected intraperitoneally at a dosage of 1 mg/g b o d y weight. Dexamethasone and cortisone suspended in 0.9% NaC1 solution and growth hormone dissolved in 0.9% NaC1 solution were injected intraperitoneally into rats. Dibutyryl cyclic AMP was dissolved in 0.5 ml 0.9% NaC1 solution and was simultaneously injected together with urethan. Control animals received an equal volume of 0.9% NaC1 solution. Assay of ornithine decarboxylase activity. After the animals were killed, each liver was excised immediately and washed with ice-cold 0.9% NaC1 solution. Subsequently the liver was put in an ice-cold basin followed by homogenization in a Teflon homogenizer with 4 vols. of 0.25 M sucrose solution containing 0.01 M Tris-HC1 buffer (pH 7.3) and 0.01 M 2-mercaptoethanol. The homogenate was centrifuged at 105 000 × g for 30 min and 0.1 ml of the supernatant was used for enzyme assay. The enzyme activity was measured by the release of 14CO2 from DL-[1-14C]ornithine. The reaction mixture contained DL-[1~4C]ornithine (0.5 u Ci), 80 nmol L-ornithine, 0.1 mM EDTA, 0.05 mM pyridoxal phosphate, 1.0 mM dithiothreitol, 0.05 M Tris-HC1 (pH 7.3) and enzyme solution (0.1 ml). Incubation was for 60 min at 37°C in tightly-capped conical flasks containing 0.3 ml of 1 M hyamine hydroxide in the center well. The reaction was terminated by the addition of 0.5 ml 50% trichloroacetic acid solution. The 14CO2 released was absorbed into hyamine hydroxide during additional 60 min incubation at 37°C. The reactions were linear up to 90 min. The radioactivity contained in hyamine hydroxide was counted in a Packard Tricarb
374 liquid scintillation counting system. Protein was determined by the method of Lowry et al. [30]. Assay of cyclic AMP concentration. Liver samples were fixed and extracted by the method of MacManus et al. [31]. The liver was immediately weighed and homogenized with 4 vols. ice-cold 5% trichloroacetic acid at full speed for 2 min in a Teflon homogenizer and then centrifuged at 2500 rev./min for 10 min. The fixation and extraction m e t h o d with trichloroacetic acid was compared to fixation by immersion in liquid nitrogen and no difference could be found. The supernatant was extracted with 8 ml of ethyl ether 5 times and made up to 12 ml following neutralization with 0.1 M NaOH solution. This solution (50 pl) was used for the assay of cyclic AMP. The concentration of cyclic AMP was measured by the binding protein assay using a kit supplied by The Radiochemical Centre, Amersham. Results
The effect of urethan on the induction of ornithine decarboxylase activity in regenerating rat liver. The effect of urethan on the induction of ornithine decarboxylase activity in regenerating rat liver is shown in Table I. Ornithine decarboxylase activity 4 h after partial h e p a t e c t o m y was much reduced by urethan injection. The incorporation of [3H]thymidine into the DNA fraction of regenerating liver from urethan-treated rats was reduced to 54% compared with untreated animals when estimated 21 h after operation (data not shown). Table II shows that the suppression of ornithine decarboxylase activity by urethan 4 h after partial h e p a t e c t o m y is dose dependent. Although ornithine decarboxylase activity increased biphasically after partial h e p a t e c t o m y and peaks were observed post-operatively at 4 and 12 h as reported by HSltt~i and Jgnne [12] and Gaza et al. [32], a single administration of urethan caused an equally marked suppression of both ornithine decarboxylase induction phases. These results are shown in Fig. 1. In order to exclude the possibility that urethan acts directly on the enzyme to suppress its activity, ornithine decarboxylase activity was assayed in the presence of urethan at various concentrations up to 0.1 M in vitro. However,
TABLE I EFFECT OF INJECTION OF URETHAN ON ORNITHINE DECARBOXYLASE OPERATION
ACTIVITY 4 h AFTER
A n i m a l s r e c e i v e d i n t r a p e r i t o n e a l l y 1 m l 0 . 9 % NaC1 s o l u t i o n c o n t a i n i n g 1 m g u r e t h a n per g b o d y w e i g h t just after partial h e p a t e c t o m y . C o n t r o l animals r e c e i v e d 1 m l 0 . 9 % NaC1 s o l u t i o n j u s t a f t e r p a r t i a l hePat e c t o m y o r s h a m o p e r a t i o n . Ornithine d e c a r b o x y l a s e activity w a s m e a s u r e d as d e s c r i b e d in the t e x t . The results are t h e m e a n _+ S.E. o f 4 d i f f e r e n t a n i m a l s . S h a m , s h a m - o p e r a t e d rats; P H , p a r t i a l l y h e p t e c t o m i z e d rats. Animal
Injection
Ornithine decarboxylase activity ( p m o l CO 2 r e l e a s e d / m g p r o t e i n per h)
Sham PH PH
0 . 9 % NaC1 s o l u t i o n 0 . 9 % NaC1 s o l u t i o n Urethan
28 + 7 3252 + 414 288 + 138
375 TABLE
n
DOSE-DEPENDENT LOWING URETHAN
SUPPRESSION OF HEPATIC ADMINISTRATION
ORNITHINE
DECARBOXYLASE
ACTIVITY
FOL-
A n i m a l s received intraperitoneally 1 m l 0 . 9 % N a C l s o l u t i o n containing the indicated q u a n t i t y o f urethan p e r g b o d y weight just after partial h e p a t e c t o m y . Ornithine d e c a r b o x y l a s e activity w a s m e a s u r e d 4 h after partial h e p a t e c t o m y . T h e results are the m e a n +_ S . E . o f 4 different animals. U r e t h a n injected (rag)
Ornithine d e e a r b o x y l a s e activity ( p m o l C O 2 released/mg protein p e r h )
0
3249 2749 1272 267
0.25 0.5 1.0
+ 151 -+ 4 0 1 -+ 2 1 1 -+ 1 0 0
no significant inhibition of enzyme activity was observed at any concentration of urethan (data not shown). It is suggested that the suppressive action of urethan is a pretranslational event by the finding that urethan did not depress the rate of incorporation of [3H]leucine into nascent protein [33]. The effect of urethan on ornithine decarboxylase activity induced by glucocorticoids and growth hormone. To determine whether the suppressive effect of urethan on ornithine decarboxylase induction in regenerating rat liver is a specific phenomenon, the effect of urethan on ornithine decarboxylase activity elevated in intact animals by glucocorticoids and growth hormone was studied. Canellakis and Theoharides [22] reported that the enhancement of ornithine decarboxylase activity by dexamethasone in cultured rat hepatoma cells was suppressed by actinomycin D and Russell et al. [34] also showed that actinomycin D almost totally suppressed the enhanced ornithine decarboxylase activ-
u~ U C ~O."
OO
y~ 0
,
,
,
4 8 12 Hours offer p~tiol heDotectorny
,
16
F i g . 1. T i m e c o u r s e o f ornithine d e c a r b o x y l a s e inhibition f o l l o w i n g t r e a t m e n t w i t h urethan. A n i m a l s received intraperitoneally 1 m l 0 . 9 % NaC1 s o l u t i o n c o n t a i n i n g 1 m g o f u r e t h a n p e r g b o d y weight just after partial h e p a t e c t o m y . Control animals received I m l 0 . 9 % N a C l s o l u t i o n just after partial hepatect o m y . Z e r o time animals were n o t subjected to any operative procedure. Brackets indicate S . E . o f the m e a n o f 3 different animals, o o, c o n t r o l r a t s ; • e , urethan-treated rats.
376 TABLE III EFFECT OF URETHAN ON ORNITHINE CORTICOIDS AND GROWTH HORMONE
DECARBOXYLASE
ACTIVITY INDUCED
BY G L U C O -
D r u g s w e r e a d m i n i s t e r e d i n t r a p e r i t o n e a U y 4 h b e f o r e t h e r a t s w e r e killed at t h e f o l l o w i n g d o s e s p e r 1 0 0 g b o d y w e i g h t : c o r t i s o n e a c e t a t e , 3 mg~ d e x a m e t h a s o n e a c e t a t e , 5 m g ; g r o w t h h o r m o n e , 1 m g ; u r e t h a n , 1 0 0 m g . I n e x p e r i m e n t 1, u r e t h a n w a s i n j e c t e d j u s t a f t e r t h e a d m i n i s t r a t i o n o f g l u c o c o r t i c o i d s a n d g r o w t h h o r m o n e a n d , in e x p e r i m e n t 2, u r e t h a n w a s g i v e n 30 r a i n b e f o r e t h e a d m i n i s t r a t i o n o f t h e s e d r u g s . All anim a l s w e r e g i v e n f o o d a n d w a t e r ad l i b i t u m u n t i l 24 h p r i o r to t h e a d m i n i s t r a t i o n o f d r u g s . T h e r e s u l t s are t h e m e a n ± S.E. o f 4 d i f f e r e n t a n i m a l s . Injected
Ornithine decaxboxylase activity ( p m o l CO 2 r e l e a s e d / r a g p r o t e i n p e r h )
%
Expt. 1 0 . 9 % NaC1 s o l u t i o n Cortisone Cortisone + urethan Dexamethasone Dexamethasone + urethan Growth hormone Growth hormone + urethan
8 786 686 1035 1223 2282 2233
± 4 ± 132 ± 132 -+ 97 _+ 2 6 3 ± 456 -+ 1 3 4
-100 87 100 118 100 98
Expt. 2 Dexamethasone Dexamethasone + urethan
1230 ~ 337 1 1 5 8 ± 82
100 94
ity 4 h after the administration of growth hormone into rats. Therefore, the increased activity of ornithine decarboxylase by glucocorticoids and growth hormone is due to the synthesis of new messenger RNA for the enzyme. Urethan was injected intraperitoneally at 30 min before or just after the administration of glucocorticoids or growth hormone. Ornithine decarboxylase activity was measured 4 h after injection of these hormones, because of the previous findings showing that this is the time of maximum ornithine decarboxylase stimulation [ 3 4 - - 3 7 ] . Table III shows that the increase in ornithine decarboxylase activity on treatment of intact rats with glucocorticoids or growth hormone is not suppressed. In addition, the effect of dexamethasone on orniTABLE IV INDUCTION OF ORNITHINE DECARBOXYLASE
ACTIVITY WITH GLUCOCORTICOID
U r e t h a n ( 1 0 0 m g / 1 0 0 g) a n d d e x a m e t h a s o n e a c e t a t e (5 m g / 1 0 0 g) w e r e i n j e c t e d i n t r a p e r i t o n e a l l y j u s t a f t e r p a r t i a l h e p a t e c t o m y or s h a m o p e r a t i o n . O r n i t h i n e d e c a r b o x y l a s e a c t i v i t y w a s m e a s u r e d 4 h a f t e r t h e o p e r a t i o n . T h e r e s u l t s are t h e m e a n _+ S.E. o f 4 o r 5 d i f f e r e n t a n i m a l s . P H , p a r t i a l l y - h e p a t e c t o m i z e d r a t s ; S h a m , s h a m - o p e r a t e d rats. Animals
Injected
Ornithine decarboxylase activity ( p m o l CO 2 r e l e a s e d / m g p r o t e i n p e r h )
PH PH PH
0 . 9 % NaC1 s o l u t i o n Urethan Urethan + dexamethasone
2 3 1 6 -+ 1 6 3 5 5 2 +- 1 0 7 1470 + 286
PH PH
0.9% NaCI solution Dexarnethasone
2 7 9 4 +- 2 5 2 3 9 1 0 +- 4 5 5
Sham Sham
0 . 9 % NaC1 s o l u t i o n Dexamethasone
12 -+ 3 1267 + 47
377
>e 1.4
03
d
u
~~J 1 0 0-
i
©
I
1 2 3 Hours a f t e r operation
I
4
Fig. 2. T i m e c o u r s e o f cyclic AMP levels in p a r t i a l l y h e p a t e c t o m i z e d r a t liver. A n i m a l s r e c e i v e d i n t r a p e r i t o n e a l l y 1 m l 0.9% NaC1 s o l u t i o n c o n t a i n i n g 1 m g u r e t h a n p e r g b o d y w e i g h t just a f t e r p a r t i a l h e p a t e c f o r a y . C o n t r o l a n i m a l s r e c e i v e d 1 m l 0.9% NaC1 s o l u t i o n just a f t e r p a r t i a l h e p a t e c t o m y a n d s h a m o p e r a tion. Zero t i m e a n i m a l s w e r e n o t s u b j e c t e d t o a n y o p e r a t i v e p r o c e d u r e . B r a c k e t s i n d i c a t e S.E. of t h e m e a n o f 4 d i f f e r e n t animals, o o, c o n t r o l rats; • • , u r e t h a n - t r e a t e d rats; ~ ~, s h a m o p e r a t e d rats.
thine decarboxylase activity in regenerating rat liver was studied. As shown in Table IV, the enzyme activity was also induced in urethan-treated rats by administration of dexamethasone. The effect of urethan on intracellular level of cyclic AMP. The effect of urethan on cyclic AMP concentration in regenerating liver was studied, since Russell and Stambrook [18] reported that the increase in ornithine decarTABLE V E F F E C T O F U R E T H A N O N H E P A T I C C Y C L I C AMP C O N C E N T R A T I O N A n i m a l s r e c e i v e d i n t r a p e r i t o n e a l l y 1 m l 0.9% NaC1 s o l u t i o n c o n t a i n i n g t h e i n d i c a t e d q u a n t i t y o f u r e t h a n p e r g b o d y w e i g h t just a f t e r p a r t i a l h e p a t e c t o m y o r h o r m o n e i n j e c t i o n . T h e h e p a t i c cyclic AMP c o n t e n t 3 h a f t e r t h e o p e r a t i o n or t h e i n j e c t i o n was m e a s u r e d as d e s c r i b e d in t h e t e x t . T h e results are t h e m e a n ±S.E. o f 4 d i f f e r e n t a n i m a l s . S h a m , s h a m - o p e r a t e d rats; PH, p a r t i a l l y - h e p a t e c t o m i z e d rats; I n t a c t , i n t a c t rats. Animals
Hormones
Sham PH PH PH PH
Urethan (mg)
Cyclic AMP ( p m o l / g w e t liver)
0 0 0.25 0.5 1.0
928 1403 1301 1114 1015
-+ 32 + 68 _+ 5 _* 6 8 t 85
Intact Intact Intact
0.9% NaC1 s o l u t i o n growth hormone growth hormone
0 0 1.0
9 0 0 -+ 8 8 9 0 -+ 56 9 1 8 -+ 4 8
Intact Intact Intact
0.9% NaC1 s o l u t i o n cortisone cortisone
0 0 1.0
9 5 2 ± 11 9 5 4 _+4 6 9 8 0 • 53
* P