Effect of copper intoxication on rat liver proteasome activity: relationship with oxidative stress.

Copper toxicity is associated with formation of reactive oxygen species, which are capable to oxidize proteins. The selective removal of the latter by the 20S proteasome is considered an essential part of the cell antioxidant defense system. The aim of the present study was to investigate whether peptidase activities of rat liver proteasomes were affected by chronic (40 mg CuSO(4)/rat/daily with the drinking water for 2 weeks) and acute (20 mg/kg CuSO(4), s.c.) copper treatment. To evaluate the role of proteasome, its inhibitor MG132 was also used. The degree of copper-induced oxidative stress (OS), established by measuring lipid peroxidation, protein oxidation, and cellular glutathione level, as well as activities of antioxidant enzymes--catalase, superoxide dismutase, and gultathionine peroxidase, depended on the mode of copper administration. Chronic copper administration (mild oxidative stress) did not affect proteasome activities, whereas acute copper treatment (severe oxidative stress) caused a decline in chymotryptic- and tryptic-like activities. The treatment of copper-loaded animals with MG132 did not change copper-induced alterations in the tested indices, except an additional increase in protein oxidation and inhibition of glutathionine peroxidase activity. The results suggested that the in vivo copper-induced oxidative stress was associated with changes in the catalytic activity of proteasome.

Effect of MG132 on proteasome activity and prooxidant/antioxidant status of rat liver subjected to ischemia/reperfusion injury.

AIM: Previous studies have shown that proteasome inhibitors exerted protective effects against ischemia/reperfusion injury (IRI) of brain, heart, kidney and intestine. The aim of the present study was to investigate: (i) whether the proteasome inhibitor MG132 protects rat liver against IRI; and (ii) whether MG132 modulates prooxidant/antioxidant status of rat liver subjected to warm IRI. METHODS: The left lateral and medial lobes (approximately 70% of the total liver volume) of livers of male Wistar rats were subjected to 30-min ischemia followed by 60-min reperfusion. Lactate dehydrogenase (LDH), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) levels were measured in the plasma. Proteasome chymotryptic-like (ChT-L) activity, levels of thiobarbituric acid-reactive substances (TBARS), protein carbonyls (PC) and glutathione (GSH), as well as superoxidase dismutase (SOD), catalase (CAT), glutathionine peroxidase and glutathionine reductase activities were measured in liver fractions. RESULTS: Thirty-min ischemia followed by 60-min reperfusion increased liver TBARS and PC, CAT and SOD activities, but decreased GSH level. Ischemia/reperfusion-induced oxidative stress was exacerbated in mitochondria, indicating that these organelles are the preferential target of IRI. Plasma LDH and AST levels were decreased by MG132 during both ischemia and reperfusion, while ALT values were decreased only after 30 min of reperfusion. MG132 did not significantly affect liver TBARS and GSH levels, but it increased PC and decreased ChT-L activity; the activities of CAT and SOD were also decreased. CONCLUSIONS: MG132 exerts a protective effect during the early phase of reperfusion and it modulates prooxidant/antioxidant status of rat liver subjected to warm IRI.

In vitro effects of alloxan/copper combinations on lipid peroxidation, protein oxidation and antioxidant enzymes.

The in vitro effects of alloxan and the product of its reduction dialuric acid (alone or in combination with copper ions) on lipid peroxidation, carbonyl content, GSH level and antioxidant enzyme activities in rat liver and kidney have been studied. The effects of Cu2+/alloxan and Cu2+/dialuric acid were compared with those of Fe3+/alloxan and Fe3+/dialuric acid. Unlike alloxan, dialuric acid increased liver and kidney lipid peroxidation; similar effects were registered in the presence of Fe3+. In the presence of Cu2+/dialuric acid, the lipid peroxidation was strongly inhibited and vice versa--the liver protein oxidation was increased. Alloxan and dialuric acid, as well as their combinations with Fe3+ had no effect on the total GSH level. Both substances did not affect the Cu2+-induced changes in GSH level, glucose-6-phosphate dehydrogenase and gluthatione reductase activities. In contrast, Cu2+ had no effect on dialuric-acid induced changes in gluthatione peroxidase and superoxide dismutase activities. The present in vitro results, concerning the metal dependence of the effects of alloxan and dialuric acid, are a premise for in vivo study of alloxan effects in metal-loaded animals.

In vitro effects of CB1 receptor ligands on lipid peroxidation and antioxidant defense systems in the rat brain.

The in vitro effects of arachidonyl-2-chloroethylamide (ACEA; a selective CB(1)-receptor agonist) and N-piperidin-l-yl)-5-(4-chlorophenyl)-1-(2,4-cochlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR141716A; a selective CB(1)-receptor antagonist) on lipid peroxidation (spontaneous and Fe (2+)-induced), total glutathione (GSH)-level and activity of antioxidant enzymes (superoxide dismutase, glutathione peroxidase and glutathione reductase) in the rat brain were studied. The effects of these CB(1)-induced), total glutathione (GSH)-level and activity of antioxidant enzymes (superoxide dismutase, glutathione peroxidase and glutathione reductase) in the rat brain were studied. The effects of these CB(1)-ligands in Fenton system (generating *OH radicals) were also examined. The cannabinoids did not change the total GSH-level and were without effects on the activity of antioxidant enzymes in the rat brain. These results proved a lack of in vitro pro-oxidant activity of the CB(1)-receptor ligands, as well as a lack of direct effects on GSH and enzyme molecules. ACEAand SR141716A were without effect on spontaneous lipid peroxidation, but decreased the Fe(2+)-induced brain lipid peroxidation and OH-provoked deoxyribose degradation in Fenton system. It can be suggested that the tested cannabinoids possess a metal-chelating activity, which might contribute to an antioxidant activity. The data, obtained in this study offer a background for investigation of the in vivo effects of these CB(1)-receptor ligands on antioxidant defense systems in the brain of healthy animals and animals, previously subjected to oxidative stress.

In vivo effects of CB1 receptor ligands on lipid peroxidation and antioxidant defense systems in the rat brain of healthy and ethanol-treated rats.

In vivo experiments were conducted to study the effects of N-(piperidin-l-yl)-5-(4-chlorophenyl)-1-(2,4-cochlo-rophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR141716A; a potent and selective CB(1)-receptor antagonist) and ara-chidonyl-2-chloroethylamide (ACEA; a selective CB(1)-receptor agonist) on spontaneous lipid peroxidation, glutathione (GSH) level and activities of antioxidant enzymes in rat tissues. Single doses of SR141716A(3 mg/kg, ip) and ACEA(10 mg/kg, ip) had no effect on all indices, studied in the brain, except for a decrease in GSH level by 10 mg/kg of SR141716A. The effects of repeated administration of the CB(1)-receptor ligands (3 mg/kg, ip, once daily for 2 days) on the above indices in the brain and liver of control and ethanol-treated animals were also studied. Two weeks after ethanol exposure, the rats lost weight (by 41%), which correlated with their decreased water and food consumption (by 52% and 33%, respectively). The time of ethanol action was not sufficient to change the biochemical parameters in the brain, except for the lipid peroxidation. However, a decrease in GSH level and superoxide dismutase activity, as well as an increase in lipid peroxidation and glucose-6-phosphate dehydrogenase activity were registered in the liver. The repeated administration of CB(1) receptor ligands restored some of ethanol-induced changes. The present results suggested lack of pro-oxidant activity and potential antioxidant ability of the studied CB(1) receptor ligands, which might contribute to their beneficial effects.