Rat HMGCoA reductase activation in thioacetamide-induced liver injury is related to an increased reactive oxygen species content.

BACKGROUND/AIMS: In thioacetamide-induced liver injury a modification of isoprenoid content and an increase of reactive oxygen species has been described. We have examined how reactive oxygen species influence the 3-hydroxy-3-methylglutaryl coenzyme A reductase, the rate limiting enzyme of the isoprenoid biosynthetic pathway, to verify if changes of that enzyme activity are involved in the changed lipid composition of the liver. METHODS: In chronic and acute thioacetamide-treated rat liver we measured the reactive oxygen species content, the activation state and K(M), the level and degradation rate of the hepatic reductase, its short term regulatory enzymes and the liver lipid profile. RESULTS: In thioacetamide-treated rat liver, the reactive oxygen species content is high and the reductase is fully activated with no modifications in its K(M) and its short term regulatory enzymes. The reductase level is reduced in chronic thioacetamide treated rats and its degradation rate is altered. CONCLUSIONS: The data show a relationship between reactive oxygen species production and altered 3-hydroxy-3-methylglutaryl coenzyme A reductase activity. It is suggested that reducing the levels of reactive oxygen species may improve the altered lipid profile found in liver injury.

Chronic high doses of thioacetamide followed by vitamin A modify dolichol, dolichol isoprenoids, and retinol content in rat liver cells.

Our line of researches follows the hypothesis that dolichol and retinol metabolism might be interrelated and involved in liver fibrosis. To this end, in this study rats were subjected to chronic treatment with thioacetamide (TAA) (300 mg/L liquid diet) for 1 and 2 months and, after liver damage had occurred, supplemented with vitamin A before sacrifice. Dolichol, dolichol isoprene units, and retinol content were determined in isolated parenchymal and sinusoidal liver cells (hepatic stellate cells; Kupffer cells; sinusoidal endothelial cells). Dolichol increased in hepatocytes after TAA treatment, with or without vitamin A. Dolichol decreased in the other cells. Retinol in general decreased. In hepatocytes, retinol decreased only on normal nutrition, while the vitamin A load was taken up normally. The percentages of dolichol isoprene units (Dol-16 to Dol-20, in rats) confirm that Dol-18, which was not modified in percentage by TAA on normal nutrition, did not increase after vitamin A, as it did in control cells (7-12%). The behavior of Dol-18 was similar in all the cells studied. Vitamin A might reveal a latent damage produced by TAA on dolichol homologues. These data support previous hypotheses that the action of TAA depends on the administration modality, the dosage, and the diet, and that Dol-18 might have different functions and compartmentalization in the cells. Furthermore, the results support the hypothesis that dolichol chain length might be interrelated with retinol metabolism, perhaps through their metabolites.

Effect of a load of Vitamin A after acute thioacetamide intoxication on dolichol, dolichol isoprenoids and retinol content in isolated rat liver cells.

This study examines how treatment with a single dose of thioacetamide, a known experimental hepatotoxin, alters the content of dolichol, dolichol isoprene units and retinol in isolated rat parenchymal and non-parenchymal liver cells at different times and when the animals are supplemented with Vitamin A. Thioacetamide (300 mg/kg i.p.) was administered in a single injection to rats, sacrificed at intervals of 0.5, 1, 2, 3, 4, 15 and 30 days. Rats supplemented, following thioacetamide, with Vitamin A, 3 days before sacrifice showed increased mortality and cellular necrosis on the third and fourth days. Parameters indicating tissue necrosis returned to normal values in surviving animals. Dolichol and retinol content showed a variable, reversible decrease, with normal levels being restored in 15-30 days. After Vitamin A, dolichol content only in hepatic stellate cells (HSC) was lower then the controls 3 and 4 days after thioacetamide treatment, in parallel with the decrease of retinol storage. The percentage of dolichol-18 is not modified by thioacetamide alone. When supplemented with Vitamin A the percentage of dolichol-18 always decreased after thioacetamide, showing that damage was still present. Mechanisms that might be operative in liver cells are briefly discussed. This approach would provide an indication to investigate how the length of the dolichol chain is determined.

Association of thioacetamide and ethanol treatment: dolichol and retinol in isolated rat liver cells.

Our aim was to study the distribution of dolichol, dolichol isoprenoids, and retinol in hepatocytes, Kupffer, sinusoidal endothelial and two subfractions of hepatic stellate cells, --Ito-1 and Ito-2--, after chronic treatment of rats for 2 and 4 months with a low dosage of thioacetamide associated with ethanol. Each type of cell responded differently to the two hepatotoxins. Overall, ethanol rarely affected the action of thioacetamide. Some new information emerges with regard to these hepatotoxins in comparison with the effects exerted by each of the drugs separately: treatment with thioacetamide plus ethanol determined an early decrease in dolichol in Kupffer cells (about 13% and 50% after 2 and 4 months, respectively). Moreover, after liver damage, a load of vitamin A evidenced altered percentages of the form of dolichol with eighteen isoprene units; these percentages were modified by all treatments in all cell types. The results confirm that dolichol is the preferred target of oxidative stress and suggest a relationship between dolichol and retinol metabolisms, and a possible new role of dolichol precursors, of prenyltransferases and of retinol metabolites in liver pathology.

Chronic ethanol treatment: dolichol and retinol distribution in isolated rat liver cells.

The aim of this study was to use chronic ethanol intoxication for 2 and 4 months as a means of studying the distribution of dolichol and retinol in isolated rat liver parenchymal cells, Kupffer cells, sinusoidal endothelial cells, and two subfractions of hepatic stellate cells: Ito 1 and Ito 2. Dolichol and retinol were studied in two batches of rats: on normal nutrition and after a load of vitamin A given 3 d before sacrifice. New observations reported are: (i) on normal nutrition, after 2 months of treatment, dolichol in HC seems to be the first target of chronic ethanol, while retinol is the first target in hepatic stellate cells; (ii) the various types of liver cells are differently affected by chronic ethanol, which highlights the importance of studying each type of sinusoidal cell; (iii) a load of vitamin A given when the damage has already occurred restores dolichol content in HC while retinol decreases; and, (iv) a link between dolichol and vitamin A metabolism might be supposed after the load of vitamin A: the percentage distribution of dolichol with 18 isoprene units (Dolichol -18) increases in all the control cells but decreases after chronic ethanol treatment. A different role of this dolichol and/or a different compartmentalization within the cell need to be further investigated.

Dolichol content in isolated sinusoidal liver cells after in vivo chronic treatment with thioacetamide.

The content of dolichol, an isoprenoid present in all biological membranes, was determined in isolated sinusoidal liver cells after treatment of rats for 2 and 4 months with a low dosage of the hepatotoxin thioacetamide. The significant decrease in dolichol observed in hepatocytes after 2 months might be explained by peroxidation of the isoprenoid. At the same time point, retinol was retained, and decreased only after 4 months of treatment. After 4 months of treatment therefore both lipids decreased. In a subfraction of hepatic stellate cells, Ito-1 cells, the main storage site of vitamin A, dolichol decreased significantly only after 4 months. A remarkable difference from hepatocytes is that in Ito-1 cells retinol content significantly decreased after 2 months of treatment. In another subfraction, Ito-2 cells, the content of the two isoprenoids decreased in parallel. This heterogeneous subfraction might represent those transitional hepatic stellate cells that, while losing retinol, are in the process of differentiating into myofibroblasts secreting extracellular matrix components. In Kupffer cells and sinusoidal endothelial cells, impairment of dolichol might be observed later, only after 4 months of treatment, while retinol decreases uniformly over time. Starting after two months of treatment, the decrease of dolichol and the increase of retinol in hepatocytes, at the same time as retinol decreases in hepatic stellate cells, might be taken as an early index of incipient liver injury due to thioacetamide. This hypothesis is discussed with regard to a role of dolichol in the modulation of membrane fluidity for intracellular and intercellular retinol transport.

Influence of chronic ethanol consumption on toxic effects of 1,2-dichloroethane: glycolipoprotein retention and impairment of dolichol concentration in rat liver microsomes and Golgi apparatus.

Our previous investigations demonstrated that 1,2-dichloroethane (DCE) and chronic ethanol treatment separately are able to impair glycoprotein metabolism and secretion, and reduce dolichol concentration in liver membranes. The purpose of this study was to investigate whether chronic ethanol consumption can induce potentiation of rat liver damage due to DCE haloalkane used in several chemical processes and in agriculture. Rats were given 36% of their total energy as ethanol in the Lieber-DeCarli liquid diet for 8 weeks (CH group). The pair-fed control group received an isocaloric amount of dextrine-maltose (PF group). "In vitro" experiments: the DCE (6.5 mM) treatment of isolated hepatocytes from CH rats enhanced glycoprotein retention and further reduced glycoprotein secretion and 14C-glucosamine incorporation compared to the hepatocytes from CH or from PF and DCE treated rats. "In vivo" experiments: a marked decrease of dolichol concentration in microsomes (in which dolichyl phosphate is rate-limiting for the initial glycosylation of protein) and in Golgi membranes (in which total dolichol is very important for membrane permeability, fluidity and vesicle fusion) was observed in CH rats acutely treated with 628 mg/kg bw of DCE (CH+DCE) compared with CH or PF+DCE treated rats. These data suggest that chronic ethanol consumption increases DCE liver toxicity by affecting protein glycosylation processes and impairing glycolipoprotein secretion, with a concomitant retention at the level of the Golgi apparatus.