Antonio Perin

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

Abstract 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 not occur in guinea-pig kidney slices, a tissue virtually devoid of alcohol dehydrogenase, and is prevented in liver by pyrazol. Acetaldehyde brings about 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.

Diamine oxidase in relation to diamine and polyamine metabolism

Diamine oxidase catalyzes the oxidative deamination of short chain aliphatic diamines, like putrescine, and histamine. The enzyme is rate-limiting in the terminal catabolism of polyamines, which are endogenous polycations important for cell growth and differentiation. This review examines the behavior of diamine oxidase in mammalian tissues in relation to diamine and polyamine metabolism under physiological and pathological conditions. The role of diamine oxidase in the control of putrescine levels in growing tissues and the known mechanisms responsible for the enzyme expression are also described.

Induction of diamine oxidase activity in rat kidney during compensatory hypertrophy

Diamine oxidase (EC 1.4.3.6) activity, measured as [14C]delta1 -pyrroline formation from [14C]putrescine, was studied in homogenates of rat kidney during compensatory hypertrophy after unilateral nephrectomy. Acetaldehyde and to a lesser degree phenobarbital, at concentrations which did not modify the activity of a preparation of hog kidney diamine oxidase, increased delta1 -pyrroline formation in kidney homogenate, which suggests that aldehyde-metabolizing enzymes present in this tissue may interfere with the yield of delta1 -pyrroline formation and that the use of acetaldehyde may give better information on kidney diamine oxidase activity. Other inhibitors of aldehyde-metabolizing enzymes such as chloral hydrate, disulfiram, and pyrazole cannot be used for diamine oxidase determination since they stimulated or depressed this enzyme activity. In rat kidney undergoing compensatory hypertrophy the levels of putrescine, spermidine, and spermine increased rapidly and were followed by an increase in diamine oxidase activity that presented a first peak on day 2 and a second peak on day 6. The administration of cycloheximide or actinomycin D to nephrectomized rates prevented the increase in diamine oxidase activity. The study of the turnover rate of diamine oxidase with cycloheximide demonstrated that the half-life of this enzyme was about 14 h in normal and hypertrophic kidney. The results suggest that the increase in diamine oxidase activity in renal hypertrophy was due to the synthesis of new enzymes rather than to slowing of its degradation.

Polyamine levels and diamine oxidase activity in hypertrophic heart of spontaneously hypertensive rats and of rats treated with isoproterenol

Polyamine levels and diamine oxidase (EC 1.4.3.6) activity were studied in hypertrophic heart of spontaneously hypertensive rats as well as in the heart of Wistar rats during the development and regression of cardiac hypertrophy induced by isoproterenol administration. In spontaneously hypertensive rats, putrescine content and diamine oxidase activity were higher than those found in normotensive Kyoto-Wistar control rats. During the development of cardiac hypertrophy induced by isoproterenol, there was an increase in polyamine content and diamine oxidase activity. The administration of cycloheximide or actinomycin D prevented the increase in diamine oxidase activity during the first 24 h after isoproterenol administration, demonstrating that the rise in diamine oxidase activity was due to synthesis of new enzyme. Following the cessation of isoproterenol treatment, cardiac hypertrophy regressed and polyamine levels and diamine oxidase activity diminished toward control values. The administration of aminoguanidine to isoproterenol-treated rats caused in the heart an inhibition of diamine oxidase activity that led to an increase in putrescine level beyond the values found in animals given isoproterenol alone. The results suggest that the enhancement of diamine oxidase activity plays a role in the regulation of putrescine level in hypertrophic heart.

Diamine and polyamine oxidase activities in phytohaemagglutinin-induced growth of rat small intestine

The activities of diamine and polyamine oxidases, two enzymes of polyamine catabolism, were studied in hyperplastic growth of rat small intestine induced by phytohaemagglutinin. This growth, evaluated by the elongation of Lieberkühns crypts, was more extensive in the proximal than in the distal parts of the gut. The activity of diamine oxidase was significantly reduced in the proximal (70%), medial (45%) and the distal (25%) parts. The activity of polyamine oxidase was doubled. The concentrations of putrescine, cadaverine and spermidine were significantly elevated in the three intestinal parts studied, whereas those of histamine and spermine were unchanged. It appears that changes in the activities of diamine and polyamine oxidases may contribute to the increased putrescine content, which is necessary to maintain active polyamine turnover for sustaining growth of the gut.

Increased synthesis of N1-acetylspermidine in hepatic preneoplastic nodules and hepatomas

Hepatic preneoplastic nodules and hepatomas obtained in a multistep protocol of rat hepatocarcinogenesis (diethylnitrosamine, 2-acetylaminofluorene, and partial hepatectomy) showed high values of spermidine N1-acetyltransferase activity, the rate-limiting enzyme in polyamine interconversion. Such an increase was associated with the appearance of N1-acetylspermidine, an enhancement in putrescine, and a decline in spermine. Such changes suggest an activation of the interconversion pathway of high into lower polyamines in hepatic preneoplastic nodules and hepatomas.

Studies on the anti-tumour activity of aliphatic aldehydes. V. Preferential inhibition of protein synthesis in normal or neoplastic tissues in relation to molecular structure.

Abstract The in vitro study of kinetics of the incorporation of labelled amino acids into protein of normal and neoplastic tissues has demonstrated that l - erythro -α,β-dihydroxybutyraldehyde ( l - erythro -DHBA), at concentrations which do not appreciably affect cell respiration, inhibits in a preferential way protein synthesis in tumours. In the presence of 5 mM l - erythro -DHBA, the leucine incorporation into protein of a large number of tumours is completely inhibited during the 2 nd hr of incubation. Under the same experimental conditions, the amino acid incor poration rate into protein of various normal tissues decreases as compared to controls, but it is never blocked. A relationship between the rate of protein synthesis and the sensitivity to the aldehyde has been observed in normal tissues but not in tumours. The tumours are affected by the drug independently of their histogenetic origin. l - erythro -DHBA is not the only substance which acts preferentially on tumours. The comparative study of the effect of various 3 - and 4 -carbon aliphatic aldehydes on the in vitro leucine incorporation into protein of rat liver and Yoshida ascites hepatoma has demonstrated that α-hydroxybutyraldehyde, β-hydroxybutyraldehyde, α,β-dihydroxybutyraldehyde and its structural isomers are more active on the hepatoma than on the liver, while propionaldehyde, lactaldehyde, glyceraldehyde, n -butyraldehyde, iso -butyraldehyde, crotonaldehyde, and α-methylglyceraldehyde are more active on the liver than on the hepatoma. A complete inhibition of protein synthesis during the 2 nd hr of incubation has been observed in the rat liver in the presence of 5 mM iso -butyraldehyde. On the whole, this data leads one to conclude that the branching of the carbon chain in the C 4 -aldehydes increases their selective toxicity for the liver, whereas the hydroxylation of the straight chain in the above-mentioned aldehydes gives rise to substances with a preferential action against tumours.

Diamine oxidase activity induction in regenerating rat liver

The synthesis and turnover of diamine oxidase (EC 1.4.3.6) activity was studied in regenerating rat liver after partial hepatectomy using inhibitors of protein and RNA syntheses. The administration to animals of cycloheximide or actinomycin D prevented the increase in diamine oxidase activity normally observed during the first hours after hepatectomy. The study of the turnover rate of diamine oxidase with cycloheximide demonstrated that the half-life of this enzyme was about 15 h in normal and regenerating liver. These results suggest that the rise in diamine oxidase activity in regenerating rat liver was due to the synthesis of new enzyme rather than to a lengthening of its turnover.

Transglutaminase activity in rat liver after acute ethanol administration.

The acute effect of ethanol on hepatic transglutaminase (EC 2.3.2.13) activity and polyamine levels were investigated in the rat. A high dose of ethanol (5 g/kg body weight, given by gastric intubation) caused in homogenate and cytosolic fraction an inhibition of 50-70% from 3 to 24 h which thereafter was reversible. Such a decrease may be in part responsible for the observed enhancement in putrescine and spermidine contents observed at the same times. Pyrazole, an inhibitor of alcohol dehydrogenase, prevented the ethanol-induced reduction in transglutaminase activity. Disulfiram, an inhibitor of aldehyde dehydrogenase, allowed detection of an inhibitory effect on enzyme activity even at a low dose of ethanol (2 g/kg), which per se did not modify transglutaminase activity. The hepatic cytosolic fraction, incubated in the presence of various concentrations of acetaldehyde, showed a dose-dependent inhibition of transglutaminase activity. All of these results suggest that acetaldehyde, the first and toxic metabolite of ethanol, inhibits hepatic transglutaminase activity, probably by its binding to the active thiol site of the enzyme. The reduction in transglutaminase may lead to an alteration of cytoskeleton, since the enzyme is known to be involved in tubuline polymerization and microfilament assembly.

Stimulation of hepatic and renal diamine oxidase activity after acute ethanol administration.

The effect of a single administration of ethanol (2 g/kg body weight) on hepatic and renal diamine oxidase activity was studied in fasted rats. Diamine oxidase activity significantly increased in liver and kidney 6 h after ethanol intubation. Pyrazole (an inhibitor of alcohol dehydrogenase), cycloheximide or actinomycin D (inhibitors of macromolecular syntheses), as well as prior adrenalectomy, prevented the ethanol-induced stimulation of diamine oxidase in the liver, but not in the kidney. The results demonstrated that the enhancement of diamine oxidase activity in the liver was due to an enzyme induction mediated by alcohol metabolism as well as by adrenals. In contrast, the stimulation of diamine oxidase activity in the kidney did not depend on synthesis of new enzyme molecules and was not mediated by ethanol metabolism or adrenal hormones.