Margarita Kirkova

Effects of proteasome inhibitor, MG132, on proteasome activity and oxidative status of rat liver.

In vivo effects of N‐benzyloxycarbonyl (Cbz)‐Leu–Leu‐leucinal (MG132) on chymotryptic‐like (ChT‐L), tryptic‐like, and post‐glutamyl peptide hydrolytic‐like proteasome activities, protein oxidation, lipid peroxidation (LP), glutathione (GSH) level, as well as on the activity of antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH‐Px), and glutathione‐reductase) in the rat liver were studied. The possibility of MG132 provoking the formation of free oxygen radicals was also assayed in primary hepatocytes. The following results were obtained: (1) In vivo, MG132 did not change the spontaneous LP, but increased Fe‐induced LP and the amount of oxidized proteins; it decreased the GSH level in liver. From the proteasome activities studied in liver cytosol only ChT‐L activity was significantly decreased after MG132 administration. Furthermore, MG132 increased antioxidant enzyme activities of SOD, CAT, and GSH‐Px. (2) In vitro, MG132 increased free radical oxygen species in hepatocytes; this effect disappeared in the presence of CAT or mannitol. In conclusion, since nowadays proteasome inhibitors are entering into the swing of laboratory and clinical practice, the present data could provide useful information for MG132 action. Consequently, future in vivo experiments with MG132 could highlight the possibility of its use at different pathological conditions. Copyright

Effects of cimetidine and its metal complexes on nitroblue tetrazolium and ferricytochrome c reduction by superoxide radicals

The effects of cimetidine, a potent histamine-H2-receptor antagonist and a good -OH scavenger, on the kinetics of ferricytochrome c and NBT reduction by superoxide anions were studied. The drug dose-dependently inhibited ferricytochrome c and NBT reduction by O2- radicals, generated either in xanthine oxidase system or photochemically or directly by KO2. The inhibitory effect of cimetidine remained unchanged in the presence of catalase or mannitol. Cimetidine and its complexes with Cu(II) and Fe(III) ions inhibited ferricytochrome c and NBT reduction even when metal chelators were added to the reaction medium. The results suggest the reaction of cimetidine with O2-radicals.

Lipid peroxidation and antioxidant enzyme activity in aspirin-treated rats.

1. Malondialdehyde formation and antioxidant enzyme activity after oral or intraperitoneal treatment of rats with various doses of aspirin was studied. 2. Aspirin, orally, had no effect on spontaneous, Fe(II)- or Fe(II)/ascorbate-induced malondialdehyde formation in liver homogenates; orally, ascorbate-induced malondialdehyde production was inhibited but only after 5-day treatment with 500 mg/kg aspirin; after intraperitoneal injection, the drug inhibited ascorbate- and Fe(II)/ascorbate-induced production of malondialdehyde. 3. Aspirin had no effect on malondialdehyde formation in erythrocytes, irrespective of the dose and route of drug administration. 4. Aspirin increased glutathione peroxidase activity in liver after 5-day treatment with an oral dose of 500 mg/kg and decreased enzyme activity in both liver and erythrocytes, 24 hr after a single injection of the same dose. 5. Aspirin, in vivo slightly affected lipid peroxidation and antioxidant enzyme activity.

In vivo effects of pentoxifylline on enzyme and non-enzyme antioxidant levels in rat liver after carrageenan-induced paw inflammation

The present study aimed to investigate the effects of pentoxifylline (PTX) on the carrageenan (CG)‐induced paw oedema and on the endogenous levels of cell enzyme and non‐enzyme antioxidants in rat liver, 4 and 24 h after CG injection. PTX (50 mg kg−1, i.p.), administered 30 min before CG, decreased the paw oedema, 2–4 h after CG administration. The drug protected CG‐induced decrease of glutathione (non‐enzyme antioxidant) and had no effect on CG‐unchanged activities of superoxide dismutase, glutathione peroxidase (enzyme antioxidants) and glucose‐6‐phosphate dehydrogenase (enzyme, important for the activity of GSH‐conjugated antioxidant enzymes). The drug showed a good antioxidant capacity in chemical systems, generating reactive oxygen species. The present results suggest that the antioxidant activity of PTX might contribute to its beneficial effects in liver injuries. Copyright

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.

Effects of desipramine on the antioxidant status in rat tissues at carrageenan-induced paw inflammation

The pathogenesis of many diseases and different pathological conditions, including inflammation, is associated with excess production of reactive oxygen species (ROS). The present study aimed to investigate the effects of the antidepressant desipramine (DES) on carrageenan (CG)‐induced inflammation, as well as on the endogenous levels of cell enzyme and non‐enzyme antioxidants in rat liver and spleen, 4 and 24 h after CG injection. The intra‐plantar CG injection into the right hind paw resulted in a time‐dependent increase in the paw volume; the maximum of CG‐induced edema peak was in 2–4 h. A single DES dose of 20 mg·kg−1, administered 30 min before CG, had no effect on paw edema, whereas the higher drug dose used (50 mg·kg−1) suppressed the edematous response to CG. The latter drug dose protected CG‐induced decrease of glutathione (non‐enzyme antioxidant) in the liver; it did not affect CG‐unchanged activities of superoxide dismutase, glutathione peroxidase (enzyme antioxidants) and glucose‐6‐phosphate dehydrogenase (enzyme, important for the activity of glutathione‐conjugated antioxidant enzymes) in both liver and spleen. The drug showed an efficient antioxidant capacity in ROS‐generating chemical systems; it was higher than that of fluoxetine (another type of antidepressant). The present results suggest that the good antioxidant activity of DES might contribute to its beneficial effects in liver injuries. Copyright

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 CuSO4/rat/daily with the drinking water for 2 weeks) and acute (20 mg/kg CuSO4, 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.

In vivo effects of N/OFQ(1–13)NH2 and its structural analogue [ORN9]N/OFQ(1–13)NH2 on carrageenan-induced inflammation: rat-paw oedema and antioxidant status

The effects of nociceptin(1–13)NH2 (N/OFQ(1–13)NH2) and its structural analogue [Orn9]N/OFQ(1–13)NH2 on acute carrageenan (CG)-induced peripheral inflammation and paw antioxidant status were studied. CG was injected intraplantarly in the right hind paw of rats and the volume of the inflamed paw was measured each 30 min for a period of 4h. When administered simultaneously with CG, N/OFQ(1–13)NH2 decreased the paw volume, whereas if injected 15 min before CG it had no effect. [Orn9]N/OFQ(1–13)NH2 produced the opposite effects at the same time-intervals of its administration. We also investigated whether these neuropeptides influence CG-induced changes in cell antioxidant system, especially at the 4th hour of CG administration. CG alone decreased the glutathione level and superoxide dismutase activity, as measured in post-nuclear homogenate of the inflamed paw. However, CG injection increased glutathione peroxidase and glucose-6-phospate dehydrogenase activities, while the activity of glutathione reductase was unchanged. The peptides themselves did not change all measured parameters. Moreover, neither N/OFQ(1–13)NH2 nor [Orn9]N/OFQ(1–13)NH2 modified CG-induced changes in the antioxidant status, regardless of the time of their injection (simultaneously or 15 min before CG). The present results suggest that N/OFQ(1–13)NH2 and [Orn9]N/OFQ(1–13)NH2 most likely affect the neuronal inflammation, rather than act as pro- or antioxidants.

In Vitro Effects of Alloxan–Vanadium Combination on Lipid Peroxidation and on Antioxidant Enzyme Activity

1. The in vitro effects of alloxan, dialuric acid and vanadium ions, alone or in combination, on lipid peroxidation and on antioxidant enzyme activity in rat liver and kidney were studied. 2. Unlike alloxan, alloxan-glutathione (GSH) and dialuric acid increased lipid peroxidation, which could be explained by the decreased activity of catalase and GSH peroxidase during incubation. 3. Vanadium(IV) ions increased the amount of thiobarbituric acid-reacting substances, but neither vanadium(IV) nor vanadium(V) changed the enzyme activity. 4. The combination of vanadium ions and alloxan-GSH or dialuric acid had no additive effect on lipid peroxidation. Vanadium ions decreased the dialuric acid-induced inhibition of catalase activity. 5. The present results suggest the therapeutic value of vanadium as an antidiabetic agent.

Study of the cytotoxicity and antioxidant capacity of N/OFQ(1–13)NH2 and its structural analogues

The effect of side-chain shortening of N/OFQ(1-13)NH(2) at position 9 ([Orn(9)]N/OFQ(1-13)NH(2), [Dab(9)]N/OFQ(1-13)NH(2), [Dap(9)]N/OFQ(1-13)NH(2)) was studied regarding potential toxicity and antioxidant capacity. Staurosporine- and H2O2-induced damage of SH-SY5Y neuroblastoma cells was not changed in the presence of N/OFQ(1-13)NH(2) and [Orn(9)]N/OFQ(1-13)NH(2), but was strongly enhanced in the presence of [Dab(9)]N/OFQ(1-13)NH(2) and [Dap(9)]N/OFQ(1-13)NH(2). Moreover, treatment of cells with the latter two analogues alone led to cell injury. Neuropeptide-dependent differences in the viability of SH-SY5Y cells were also observed, i.e., a cytoprotective effect was observed only in the presence of N/OFQ(1-13)NH(2) and [Orn(9)]N/OFQ(1-13)NH(2). Compared to [Dab(9)]N/OFQ(1-13)NH(2) and [Dap(9)]N/OFQ(1-13)NH(2), the effects of N/OFQ(1-13)NH(2) and [Orn(9)]N/OFQ(1-13)NH(2) were more beneficial in systems generating free oxygen radicals (O(2)(-) and .OH), as well as on the antioxidant status of rat brain and liver. Taken together, our findings show that N/OFQ(1-13)NH(2) and its structural analogue [Orn(9)]N/OFQ(1-13)NH(2) possess more favorable profiles than the other two nociceptin (N/OFQ) analogues. The present results suggest that shortening of the side-chain of N/OFQ(1-13)NH(2) might increase cell damage and reduce the viability of SH-SY5Y neuroblastoma cells. Moreover, such alterations may lead to changes in free-oxygen generating systems and in antioxidant status in animal tissues.