In vitro inhibition of protein synthesis in rat liver as a consequence of ethanol metabolism

101

Biochimica et Biophysica Acta, 366 (1974) 101--108 Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands BBA 98089

IN V I T R O INHIBITION OF P R O T E I N SYNTHESIS IN R A T L I V E R AS A CONSEQUENCE OF E T H A N O L METABOLISM

ANTONIO PERIN, GIUSEPPE SCALABRINO, ANGELA SESSA and ALBA ARNABOLDI

Institute o f General Pathology and C.N.R. Centre for Research in Cell Pathology, University o f Milan, 1-20133 Milan (Italy) (Received March 5th, 1974)

Summary Ethanol depresses the incorporation of amino acids into cell proteins of rat liver slices. This inhibition is nearly concentration independent for alcohol concentrations between 3 and 150 mM, and disappears after removal of ethanol. The effect of ethanol on liver protein synthesis is lower in fasting than in fed rats. The inhibition of protein synthesis does n o t occur in guinea-pig kidney slices, a tissue virtually devoid of alcohol dehydrogenase, and is prevented in liver by pyrazol. Acetaldehyde brings a b o u t a concentration-dependent inhibition of protein synthesis both in rat liver and in guinea-pig kidney. Acetoin, up to 5 mM concentration, does not influence protein synthesis in liver. Sorbitol, which mimics ethanol as regards the shifting of the redox level in the hepatocyte, depresses protein synthesis in liver in relation to its concentration in the medium. Pyruvate partially removes the inhibition of cell protein synthesis induced by ethanol. The data show that the inhibition of cell protein synthesis in rat liver slices by ethanol is a consequence of alcohol metabolism. They also suggest that this inhibition is due to acetaldehyde as well as to the shifting of the redox level in the cell.

Introduction

Data on the effect of ethanol on liver protein synthesis are inconclusive since they are few and sometimes conflicting. Studies on isolated perfused rat liver showed that ethanol does not influence the incorporation of labeled amino acids into cell liver proteins, while it depresses albumin synthesis [1]

102 and impairs the release of lipoproteins [2]. In contrast, no modifications [3] or an increase [4,5] in liver and plasma protein synthesis were observed in ethanol-intoxicated rats. An increased lipoprotein production by the liver was also observed in rats chronically treated with ethanol [6,7]. On the other hand ethanol inhibits the Na ÷- and K+-stimulated ATPase as well as the active transport of amino acids in liver and other tissues [8--13]. Moreover, ethanol inhibits the incorporation of labeled leucine into proteins of hepatic mitochondria membranes both in vitro and in vivo (i.e. in chronic alcohol intoxication) [ 14]. Recently, during the course of studies on the effect of aliphatic compounds on protein synthesis of normal and neoplastic tissues, we observed that ethanol depresses the incorporation of labeled amino acids into cell proteins of rat liver slices. This effect is described in the present paper. Evidence suggesting that the inhibition of protein synthesis is a consequence of ethanol metabolism will be presented. Material and Methods

Chemicals Ethanol and acetaldehyde were purchased from C. Erba, Milan, Italy; D-sorbitol and pyruvate (sodium salt) from E. Merck A.G., Darmstadt, G.F.R.; acetoin from B.D.H., Chemicals Ltd., Poole, England; pyrazol from T. Schuchardt, Munich, G.F.R. L-[1-~4C]Leucine was purchased from C.E.A., Gif-sur-Yvette, France; [~ 4 C2 ] glycine, L-[ 14 C6 ] lysine, L-[ 14 C4 ] threonine from the Radiochemical Centre, Amersham, England. Unlabeled L-leucine, glycine, L-lysine and L-threonine were obtained from Sigma Chemical Co., St. Louis, Mo., U.S.A. Animals Male albino Wistar rats weighing 150--180 g and guinea pigs weighing approx. 250 g fed on a commercial standard diet (Piccioni, Brescia, Italy) and water ad libitum were used. Incubation procedure Tissue slices (8--12 mg dry weight) were incubated in small stoppered flasks in Krebs-Ringer phosphate (final vol. 3.0 ml) containing 1.5 pmoles of labeled amino acid (spec. act. 0.266 Ci/mole) in the absence or presence of the substance under investigation. The gas phase was air. Incubation was carried out in a D u b n o f f metabolic shaker at 38 ° C. At fixed times, slices were homogenized in a mortar with 5% trichloroacetic acid and the precipitated proteins were purified according to Rabinowitz et al. [15]. Scintillation analysis was carried out with hyamine-treated samples to which 10 ml of scintillation fluid (4 g omnifluor in a mixture of 700 ml toluene and 300 ml ethylene glycolmonoethylether) were added. Radioactivity was measured in a Tri-Carb liquid scintillation spectrometer (Model 3365, Packard Instruments Co. Inc.) and corrected to 100% efficiency. On the basis of the radioactivity and of the specific activity, data were expressed as pmoles of amino acid incorporated/g proteins according to Rabinowitz et al. [15]. The significance of the differences between the means was evaluated by the Dunnett test [ 1 6 ] .

103 TABLE I IN V I T R O E F F E C T O F E T H A N O L O N T H E I N C O R P O R A T I O N INTO CELL PROTEINS OF RAT LIVER

OF SOME LABELED AMINO ACIDS

Liver slices ( 8 - - 1 2 m g , d r y w t ) f r o m fed r a t s w e r e i n c u b a t e d at 3 8 ° C in K r e b s - - R i n g e r p h o s p h a t e c o n t a i n i n g 1 . 5 # m o l e s o f l a b e l e d a m i n o a c i d (spec. a c t . 0 . 2 6 6 C i / m o l e ) a n d e t h a n o l as i n d i c a t e d . M e d i u m v o l u m e , 3 ml; gas p h a s e , air. T h e r e s u l t s are e x p r e s s e d as m e a n values _+ S.E.M. w i t h n u m b e r o f o b s e r v a t i o n s in p a r e n t h e s e s . Amino acid

Ethanol concn (mM)

Amino acid incorporation ( p m o l e s / g p r o t e i n s p e r h)

L-Leucine

0 0.5 1 2 3 5 10 50 100 150 0 10 50 100 0 10 50 100 0 10 50 100

3.68 3.31 2.80 2.65 2.58 2.47 2.45 2.21 2.03 1.97 1.12 0.76 0.72 0.71 0.67 0.44 0.46 0.41 0.80 0.53 0.51 0.43

Glycine

L-Lysine

L-Threonine

± 0.10 +_0.15 _+0.18 ± 0.12 ±0.14 ±0.12 ±0.11 +_0.15 +_0.14 _+0.12 _+ 0 . 0 5 +-0.07 +0.05 +-0.04 + 0.03 +-0.02 ± 0.02 _+0.02 +- 0 . 0 3 _+0.04 +-0.02 _+ 0 . 0 2

(8) (8) (8)* (8)** (8)** (8)** (8)** (8)** (8)** (8)** (4) (4)** (4)** (4)** (4) (4)** (4)** (4)** (4) (4)** (4)** (4)**

Inhibition (%)

10 24 28 30 33 34 40 45 46 32 36 37 34 31 39 34 36 46

* P ~0.05.

** P ~ 0 . 0 1 .

Results

Effect of ethanol on protein synthesis of rat liver The results reported in Table I show that ethanol depresses the incorporation of amino acids into cell proteins of rat liver slices. The inhibition of leucine incorporation is a function of ethanol concentration when its concentration is below 3 mM. At concentrations between 3 and 150 mM, the inhibition ranges from 30--46% with respect to the control. Under the same concentrations of ethanol, the inhibition is nearly the same for all tested amino acids. Kinetics experiments (Fig. 1) demonstrate that under our experimental conditions ethanol slows down leucine incorporation, but does not abolish it completely. Moreover, the removal of the drug reverses the inhibition previously established. Data reported in Table II show that the inhibition in fed animals is nearly double that found in fasting rats. Results reported in Table III demonstrate that pyrazol, at a concentration (2 mM) which completely inhibits alcohol dehydrogenase activity of rat liver slices [ 17], prevents the effect of ethanol on protein synthesis.

la

f 0j O

E

o z~

5 JAs.

/ ~ ~A~

Y~ OY~ u c

E.,

g d

0

30

60

90

120

150

180

Time (rain) F i g . 1. I n v i t r o e f f e c t o f e t h a n o l o n t h e k i n e t i c s o f l e u c i n e i n c o r p o r a t i o n i n t o c e l l p r o t e i n s o f r a t l i v e r slices. T h e e x p e r i m e n t a l c o n d i t i o n s w e r e as i n T a b l e I, C u r v e s r e p r e s e n t l e u c i n e i n c o r p o r a t i o n i n t o c e l l proteins in the absence (o '-~) a n d i n t h e p r e s e n c e ( • e) of 10 mM ethanol. At the time indicated by the arrow, some slices were removed from the incubation medium, rinsed with Krebs--Ringer p h o s p h a t e a n d a g a i n i n c u b a t e d i n t h e a b s e n c e o f e t h a n o l (ix . . . . . ~). Points represent values of a typical experiment.

TABLE

II

IN VITRO EFFECT OF ETHANOL ON LEUCINE LIVER OF FED AND FASTING RATS

INCORPORATION

INTO

CELL

PROTEINS

OF

T h e e x p e r i m e n t a l c o n d i t i o n s w e r e as i n T a b l e I. T h e t i m e o f f a s t i n g w a s 1 6 - - 1 8 h. T h e r e s u l t s a r e e x p r e s s e d as m e a n v a l u e s _+ S . E . M . w i t h n u m b e r o f o b s e r v a t i o n s i n p a r e n t h e s e s . Animals

Ethanol concn (mM)

Leucine incorporation (pmoles/g proteins per h)

Fed rats

0 10 50 0 10 50

4.O4 2.57 2.42 2.00 1.70 1.58

Fasting rats

_+0.17 +_0.15 -+ 0 . 1 6 -+ 0 , 2 2 4-_ 0 . 1 5 -+0.18

(6) (6)** (6)** (6) (6)* (6)*

Inhibition (%)

36 40 15 21

* P ~ 0.05. ** P ~ 0.01. TABLE

III

EFFECT OF PYRAZOL ON THE IN VITRO INHIBITION CELL PROTEINS OF RAT LIVER BY ETHANOL

OF

LEUCINE

INCORPORATION

INTO

T h e e x p e r i m e n t a l c o n d i t i o n s w e r e as i n T a b l e I. I n t h e c a s e o f p y r a z o l , s l i c e s w e r e p r e i n c u b a t e d w i t h t h i s substance for 15 rain before leucine or ethanol and leucine addition. Results are values of a typical experiment. Conditions

Leucine incorporation (pmoles/g proteins per h)

Control Ethanol 10 mM Pyrazol 2 mM Pyrazol 2 mM+ ethanol 10 mM

2.10 1.47 2.25 2.26

105 T A B L E IV IN V I T R O E F F E C T O F E T H A N O L A N D A C E T A L D E H Y D E O N L E U C I N E I N C O R P O R A T I O N CELL PROTEINS OF KIDNEY CORTEX SLICES OF GUINEA PIG

INTO

T h e e x p e r i m e n t a l c o n d i t i o n s w e r e as in T a b l e I. T h e r e s u l t s are e x p r e s s e d as m e a n values +_ S.E.M. w i t h n u m b e r o f o b s e r v a t i o n s in p a r e n t h e s e s . Substance

Concentration (raM)

Leucine incorporation ( # m o l e s / g p r o t e i n s p e r h)

Inhibition (%)

Ethanol

0 10 20 40 80 100 0 0.1 1.0 2.0 5.0

1.23 1.30 1.33 1.29 1.24 1.18 0.90 0.84 0.77 0.69 0.55

7 14 2G 39

Acetaldehyde

_+ 0 . 2 7 +_ 0 . 2 4 +_0.31 _+ 0 . 2 4 +_ 0 . 2 4 +_0.22 _+ 0 . 0 2 _+ 0 . 0 3 +_0.02 +_0.04 _+ 0 . 0 4

(5) (5) (5) (5) (5) (5) (5) (5) (5)* (5)** (5)**

* p <0.05.

** P ~ 0 . 0 1 .

TABLE V IN V I T R O E F F E C T O F A C E T A L D E H Y D E , A C E T O I N A N D S O R B I T O L TION INTO CELL PROTEINS OF RAT LIVER

ON LEUCINE INCORPORA-

T h e e x p e r i m e n t a l c o n d i t i o n s w e r e as in T a b l e I. T h e r e s u l t s a r e e x p r e s s e d as m e a n v a l u e s + S.E.M. w i t h n u m b e r o f o b s e r v a t i o n s in p a r e n t h e s e s . Substance

Concentration (raM)

Leucine incorporation ( p m o l e s ] g p r o t e i n s p e r h)

Acetaldehyde

0 0.05 0.1 0.5 1.0 2.0 5.0 0 0.1 1.0 5.0 0 5 10 20 50 100

3.39 2.96 2.76 2.68 2.44 1.68 1.18 2.72 2.54 2.95 2.71 4.15 2.42 1.67 1.29 0.90 0.67

Acetoin

Sorbitol

* P ~0.05.

** P ~ 0 . 0 1 .

_+ 0 . 2 0 _+ 0 . 1 4 _+0.10 +_0.10 _+0.10 _+0.07 +0.03 + 0.06 _+ 0 . 1 4 +0.05 _+ 0 . 0 7 _+ 0 . 3 0 _+0.17 _+0.09 _+0.07 +0.05 + 0.05

(8) (8) (8)* (8)** (8)** (8)** (8)** (4) (4) (4) (4) (5) (5)** (5)** (5)** (5)** (5)**

Inhibition (%)

13 16 21 28 50 65

41 60 69 78 84

106 TABLE

VI

EFFECT OF PYRUVATE ON THE IN VITRO INHIBITION CELL PROTEINS OF RAT LIVER BY ETHANOL

OF LEUCINE

T h e e x p e r i m e n t a l c o n d i t i o n s w e r e as i n T a b l e I. T h e r e s u l t s a r e e x p r e s s e d number of observations in parentheses. Conditions

Leucine incorporation (pmoles/g proteins per h)

Control Ethanol 10 mM Pyruvate 10 mM Ethanol 10 mM + p~cruvate 10 mM

2.74 1.40 3.27 2.01

_ 0.22 +_0.09 4- 0 . 1 6 +_ 0 . 0 6

(5) (5)** (5) (5)*

INCORPORATION

INTO

as m e a n v a l u e s 4-_ S . E . M . w i t h

A%

- 49 + 19 - 27

* P ~0.05. ** P ~ 0.01.

Effect of ethanol and acetaldehyde on protein synthesis o f guinea-pig kidney Data reported in Table IV show that ethanol, at concentrations affecting protein synthesis in liver, does not m o d i f y leucine incorporation into cell proteins of guinea-pig kidney, a tissue virtually devoid of alcohol dehydrogenase [18]. In contrast, acetaldehyde, the principal metabolite of ethanol, brings about an inhibition dependent on its concentration in the medium.

Effect of acetaldehyde, acetoin and cell redox level on protein synthesis of rat liver Results reported in Table V show that acetaldehyde, at concentrations starting from 1 • 10 -4 M, depresses leucine incorporation into cell proteins of rat liver slices. In contrast, acetoin, up to 5 mM concentration, does not m o d i f y the rate of target amino acid incorporation. Sorbitol, which like ethanol brings about a decrease in NAD/NADH ratio in the hepatocyte [19--21], inhibits protein synthesis in relation to its concentration in the medium. Results reported in Table VI show that pyruvate, which raises the NAD/NADH ratio in the hepatocyte [20], removes, by nearly 46%, the inhibition of leucine incorporation due to ethanol. Discussion The results reported in this paper clearly show that ethanol brings about a reversible inhibition of amino acids incorporation into cell proteins of rat liver slices. This finding is in contrast to the above reported observations on isolated perfused rat liver and on animals treated with ethanol. Probably this discrepancy depends on the different experimental conditions. It is well known in fact that aeration and availability of metabolites can profoundly affect the metabolism of ethanol by the liver [22--24]. Our results suggest that the inhibition of protein synthesis is not due to ethanol per se, but is a consequence of ethanol metabolism. In fact, fasting reduces the ethanol inhibition of protein synthesis in liver by nearly 50% in respect of fed animals as was observed by other authors for ethanol oxidation

107 [20] and for alcohol dehydrogenase activity [25]. Moreover, the inhibition of protein synthesis does not occur in guinea-pig kidney, a tissue virtually devoid of alcohol dehydrogenase[18] and is also prevented in liver by pyrazol, a well known inhibitor of this enzyme [ 1 7 , 2 6 ] . The experiments on the effects of the main products deriving from ethanol metabolism on leucine incorporation into proteins show that acetoin, up to 5 mM concentration, has no influence on protein synthesis in liver. In contrast, acetaldehyde seems to play an important role in determining the inhibition of liver protein synthesis. The effect of acetaldehyde is similar to that which we already observed for other aliphatic aldehydes in normal and neoplastic tissues [ 27 ]. The mechanism by which aliphatic aldehydes depress protein-synthesis is still uncertain. A condensation of these substances with cysteine to form thiazolidine-4-carboxylic acids [28] as well as an impairment of the attachment of m R N A to ribosomes [29] have been described. Moreover, preliminary results indicate that acetaldehyde may have also an effect on rat liver aminoacyl-tRNA synthetase (Del Monte, U. and Cini, G., unpublished). The fact that acetaldehyde brings a b o u t an inhibition of protein synthesis both in rat liver and in guinea-pig kidney stresses the importance of this substance in causing damage in tissues of alcoholic subjects. In fact in these patients an increase of acetaldehyde in liver and blood after drinking has been described [ 3 0 - - 3 2 ] . Our results, furthermore, show that not only acetaldehyde, but also the decrease in the N A D / N A D H ratio connected with ethanol oxidation [19,20] play a role in the inhibition of protein synthesis in liver. In fact, sorbitol, which mimics ethanol as regards the shifting of the redox level of the hepatocyte [ 21], depresses protein synthesis in rat liver slices. In contrast, pyruvate, which increases the N A D / N A D H ratio in the liver cell [ 2 0 ] , reduces the inhibitory effect of ethanol on protein synthesis. It must be pointed out that sorbitoJ, unlike ethanol, brings a b o u t in liver an inhibition that is related to its concentration in the medium. Probably this difference is due to the fact that sorbitol dehydrogenase is not a limiting enzyme in sorbitol metabolism [ 3 3 ] , while alcohol dehydrogenase is a rate-limiting step of ethanol oxidation [34,35]. Additional studies are necessary to define the mechanism by which the shifting of the redox level in the cell brings a b o u t a protein synthesis inhibition in liver. Acknowledgements The authors wish to thank Professor E. Ciaranfi (Milan) for his interest and advice, and Professor E. Antonini (Rome) for helpful discussion and criticism. References 1 2 3 4 5 6 7 8

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