Enrico Gravela

Studies on fatty liver with isolated hepatocytes: II. The action of carbon tetrachloride on lipid peroxidation, protein, and triglyceride synthesis and secretion

Abstract This report describes some effects of poisoning with carbon tetrachloride on hepatocytes in single cell suspension. In isolated liver cells as well as “ in vivo ” CCl 4 stimulates lipid peroxidation, inhibits both protein synthesis and protein and lipoprotein secretion and induces fat accumulation within the cells. As the action of CCl 4 on lipid peroxidation, our data confirm that its increase induced by CCl 4 depends on the metabolism of this drug by the NADPH-cytochrome P 450 enzyme system. Furthermore, data reported here suggest that the onset of the CCl 4 -induced decrease of lipoprotein secretion is due to a derangement of the secretory pathway.

Studies on fatty liver with isolated hepatocytes: I. The action of colchicine, phalloidin, cytochalasin B, and cycloheximide on protein and triglyceride synthesis and secretion

Abstract This report describes the effect of four different drugs on triglyceride and protein secretion in single cell suspension of isolated hepatocytes. Colchicine, phalloidin, and cycloheximide, through different mechanisms, impair lipoprotein secretion and induce an accumulation of triglycerides within the liver cell. Cytochalasin B, however, has no effect on the above processes. All four drugs reduce the secretion of proteins by hepatocytes. This change results either from a block in protein synthesis, as in the cases of cycloheximide and cytochalasin B, or from an interference with the secretory mechanism, as in the case of colchicine. Some data indicate that phalloidin acts in both ways.

Effects of 4-Hydroxynonenal, A Product of Lipid Peroxidation, on Cell Proliferation and Ornithine Decarboxylase Activity

4-hydroxynonenal (HNE) is one of the major breakdown products of cellular lipid peroxidation. Its effects on proliferation, ornithine decarboxylase (ODC) activity and DNA synthesis have been investigated in leukemic cell lines. The cells were incubated for 1 hour with different aldehyde concentrations, then washed and resuspended in medium with fresh foetal calf serum. HNE concentrations ranging from 10(-5) to 10(-6) M significantly inhibited ODC activity when induced by addition of fresh foetal calf serum both in K562 and HL-60 cells. 3H-Thymidine incorporation in K562 cells was also inhibited from 6 to 12 hours after the treatment. The same HNE concentrations did not inhibit ODC activity when added to cytosol, thus a direct action on the enzyme can be excluded. Moreover, HNE did not affect the half-life of ODC, so that a specific effect on ODC synthesis may be supposed. These data indicate a reduction of proliferative capacity of the cells and are consistent with the possibility that HNE, at concentrations close to those found in normal cells, plays a role in the control of cell proliferation.

Initiation of free radical reactions and hepatotoxicity in rats poisoned with carbon tetrachloride or bromotrichloromethane

Morphological studies [1, 2] have shown that bromotrichloromethane behaves as a more dangerous hepatotoxin than carbon tetrachloride.The present investigation extends those preliminary findings and offers new biochemical evidences for the hypothesis suggested by the relationship between the toxicity and the level of the bond dissociation energy. The electron paramagnetic resonance signal and the UV-spectrum of double bond shifting in microsomal lipids show that CBrCl3 administration induces a more rapid and extensive free radical reaction in liver than CCl4. More severe pathological phenomena are coincident with a more profound pro-oxidant effect of bromotrichloromethane. In fact, 0.26 mmol of CBrCl3 prove lethal after having caused massive fatty liver and necrosis, while an equimolecular amount of CCl4 induces a lower and transient hepatic triglyceride accumulation and necrosis. Under the same experimental conditions, the lethal dose of carbon tetrachloride is twenty times higher (5.16 mmol per 100 g body weight). CCl4-poisoned rats develop a remarkable dissociation of liver polysomes. In animals given an equivalent dose of CBrCl3 these damages occur within a shorter lag period and are more severe. Moreover, the recovery takes place much later than when carbon tetrachloride is used.A clear inverse relationship exists between the level of bond dissociation energy of these two halogenoalkanes (respectively, 49 and 68 kcal mol−1, for CBrCl3 and CCl4) and their potency to stimulate free radical reactions and to produce liver injury.

Studies on the mechanisms of ornithine decarboxylase in vitro inactivation.

Hydrocortisone-induced rat liver ornithine decarboxylase appears quite stable in the soluble fraction of the homogenate incubated at 37 degrees C. In contrast, the incubation of the whole homogenate causes a rapid loss of activity. The ornithine decarboxylase-inactivating capacity appears mainly bound to microsomes. Lysosomes seem to play a role only after the microsome-induced inactivation. Different reducing agents (dithiothreitol, NADPH, NADH, GSH) are effective both in preventing and in reversing ornithine decarboxylase inactivation. NADPH is peculiar in that it can reactivate the enzyme at very low concentrations. Oxidized glutathione potentiates the inactivating effect of microsomes. On the basis of present results it is suggested that ornithine decarboxylase may be reversibly inactivated through microsome-catalyzed formation of mixed or enzyme-enzyme disulfides and that NADPH plays a crucial role in ornithine decarboxylase reactivation, probably by cytosolic reductase(s).

On the mechanism of fatty liver in white phosphorus-poisoned rats.

Abstract Phosphorus-poisoned rats showed an increase in the level of hepatic triglycerides, while pretreatment of these intoxicated animals with propyl gallate or glutathione lowered these values to normal levels. In contrast N,N′ -diphenyl- p -phenylenediamine failed to affect the damage produced by phosphorus. Phenobarbital, given prior to phosphorus, did not cause a further increase in triglyceride accumulation in liver. There was no evidence of lipid peroxidation in any group of animals, as measured by diene conjugation absorption. Furthermore, pretreatment with phenobarbital failed to cause lipid peroxidation in the poisoned animals. A disaggregation of the liver polysome was observed in the liver of phosphorus-treated rats, while poisoned animals pretreated with propyl gallate or glutathione showed a polysomal pattern similar to that of untreated rats. Finally, N,N′ -diphenyl- p -phenylenediamine did not cause any change in the disaggregation of the polysomes induced by phosphorus.

Further observation on the effects of 3-methylcholanthrene and phenobarbital on carbon tetrachloride hepatotoxicity.

Abstract The behavior of PB and 3-MC, inducers of the enzymatic chain of the hepatic DMES, was studied in relation to their effects upon CCl 4 -hepatotoxicity. The secretion of triglycerides from liver into the plasma compartment was inhibited in CCl 4 -poisoned rats, as well, in the animals pretreated with PB and 3-MC. The lipid peroxidative phenomena (diene conjugation absorption), evidenced in rats intoxicated with the halogenated hydrocarbon, were enhanced by pretreatment with PB, while 3-MC, in this respect, had no effect. At 60 min after poisoning, the severe polysomal damage was not modified either by 3-MC or PB, while the recovery process (25 hr of intoxication) was at its highest in rats treated with CCl 4 alone, slower in 3-MC or PB pretreated rats. Cell necrosis, as measured by S-GOT and S-GPT, was protected in CCl 4 -intoxicated rats by pretreating the animals with 3-MC.

Studies on fatty liver with isolated hepatocytes: III. Cumene hydroperoxide-induced change of several cell functions

Isolated rat hepatocytes have been treated with cumene hydroperoxide at concentrations not inducing irreversible cell damage. Under these experimental conditions cells show an enhanced lipid peroxidation, a decrease of glucose-6-phosphatase activity and of cytochrome P-450 content, and a stimulation of aminopyrene demethylation. Furthermore, the hepatocyte incorporation of amino acids is slightly but significantly reduced by the tested compound. Finally, because of the inhibitory effect of cumene hydroperoxide on cell lipoprotein but not on protein secretion, a mechanism of damage acting at the level of the assembly and maturation of lipoprotein micelles is postulated.

Studies on lipid peroxidation using whole liver cells: Influence of damaged cells on the prooxidant effect of ADP-Fe3+ and CCl4

An increase in the number of damaged cells produces an enhancement of lipid peroxidation induced by ADP-Fe3+ or CCl4 ‘in vitro’ on hepatocytes in single cell suspension.

Protection by glutathione and propyl gallate on the impaired in vitro amino acid incorporation into liver microsomal protein of CCl4-poisoned rats

Abstract The endogenous mRNA directed amino acid incorporation into rat liver microsomes in vitro is lowered as soon as 30 min after CCl4 poisoning; at the same time a strong decrease in rat liver polysomes appears. Previous treatment with the watersoluble antioxidants glutathione and propyl gallate completely prevents these changes in polysome activity and structure. These results seem to indicate a direct relationship between lipoperoxidation and alteration in protein synthesis.