In vivo effects of amtolmetin guacyl on lipid peroxidation and antioxidant defence systems. Comparison with non-selective and COX-2 selective NSAIDs.

1. The in vivo effects of the non-steroid anti-inflammatory drug (NSAID) amtolmetin guacyl, a pro-drug of the NSAID tolmetin, on lipid peroxidation, glutathione levels and activity of antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase) in rat gastric mucosa, colon mucosa and liver, were compared with the effects of non-selective (indomethacin, diclofenac) and COX-2 selective (celecoxib) NSAIDs. 2. Indomethacin treatment led to an increase in lipid peroxidation, glutathione peroxidase and glucose-6-phosphate dehydrogenase activities and to a decrease in catalase activity and glutathione levels in gastric mucosa. In contrast, amtolmetin guacyl treatment was without effects in gastric and colon mucosa, or liver from control animals. Like amtolmetin guacyl, celecoxib had no effect on the lipid peroxidation, or on enzyme and non-enzyme antioxidant defence systems in gastric mucosa. 3. It is suggested that the lack of pro-oxidant effects in vivo associated with amtolmetin guacyl treatment contribute improved gastric tolerability.

Potential antioxidant activity of celecoxib and amtolmetin guacyl: in vitro studies.

1. In vitro studies of the potential antioxidant activity of the selective cyclo-oxygenase-2 inhibitor celecoxib and the non-steroid anti-inflammatory drug amtolmetin guacyl (AMG) were carried out. The study included experiments on the ability of these drugs to affect some indices of the oxidative stress [lipid peroxidation (LP), activity of antioxidant enzymes, glutathione (GSH) level] in rat stomach and colon mucosa and in liver. 2. Celecoxib and AMG did not change the activity of the enzymes GSH-peroxidase, oxidased glutathione (GSSG)-reductase and glucose-6-phosphate-dehydrogenase, as well as the GSH level in all tissue preparations. An increased superoxide dismutase (SOD) activity and a tendency to a decreased Fe/ascorbic acid-induced LP in stomach and colon mucosa were found, but only in the presence of AMG. 3. In the liver, both celecoxib and AMG decreased spontaneous and Fe/ascorbic acid-induced LP. SOD activity was enhanced only in the presence of AMG. 4. Experiments aimed at studying celecoxib and AMG in free oxygen radical-generating systems were also carried out. AMG and tolmetin (the main metabolite of AMG) inhibited OH*-provoked deoxyribose degradation in a Fenton system. Celecoxib had no effect on free radicals when tested in the same system. 5. In conclusion, the results of the present in vitro studies suggest that AMG and celecoxib possess antioxidant and metal-chelating abilities, which might contribute to their beneficial effects.

In vivo effects of amtolmetin guacyl on lipid peroxidation and antioxidant defence systems in different models of gastrointestinal injury.

1. The in vivo effects of the non-steroid anti-inflammatory drug (NSAID) amtolmetin guacyl (AMG) on lipid peroxidation (LP) and on antioxidant enzyme and non-enzyme defence systems were investigated in models of stomach and colon damages, induced by other NSAIDs, by ethanol or by 2,4,6-trinitrobenzenesulfonic acid (TNBS). 2. Indomethacin increased LP, glutathione peroxidase (GSH-PX) and glucose-6-phosphate dehydrogenase (Glu-6-P-DH) activities and decreased glutathione levels in gastric mucosa. Pretreatment with AMG normalized some of the parameters affected by indomethacin. 3. Treatment of rats with ethanol for 0.5 h led to a decrease in glutathione levels as well as activities of glutathione reductase and Glu-6-P-DH in gastric mucosa. AMG, administered 0.5 h before ethanol, limited the adverse actions of ethanol. 4. Amtolmetin guacyl failed to abolish the TNBS-induced changes in the followed-up parameters in colon mucosa and liver, but additional alterations (as with tolmetin) were not observed. 5. The beneficial profile of AMG in the various experimental models of free radical-induced damage investigated in this study suggests the possibility that this drug might possess antioxidant activity.

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.

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.

Hydroxyl radicals production in the vanadium ions/dialuric acid systems.

1. The effects of vanadium ions on the .OH radical production in the presence of and in the absence of dialuric acid were studied. 2. Dialuric acid enhanced deoxyribose degradation. 3. Vanadium ions and vanadium/EDTA complexes decreased the degradation of deoxyribose in the presence and in the absence of dialuric acid. 4. The question as to whether or not free .OH radicals are formed via reaction of vanadium ions with H2O2 in the presence of dialuric acid is discussed. 5. The results are interpreted with a view to the vanadium ability to decrease the toxic effects of dialuric acid.

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 vitro effects of aspirin on malondialdehyde formation and on activity of antioxidant and some metal-containing enzymes.

The in vitro effects of aspirin in different concentrations on malondialdehyde formation and on the activity of antioxidant and some metal-containing enzymes in rat liver homogenate and erythrocytes were studied. Aspirin showed a biphasic dependence on concentration: low concentrations (to 1.0 mM) stimulated the spontaneously formed malondialdehyde in the liver homogenate and the high concentration (5.0 mM) inhibited it; all aspirin concentrations tested had no effect on the liposomes; 5.0 mM aspirin inhibited the Fe(2+)-induced lipid peroxidation in the liver homogenate but had an opposite effect on the liposomes. Aspirin dose-dependently inhibited nitro-blue tetrazolium reduction and decreased deoxyribose degradation. The higher aspirin concentrations inhibited the activity of antioxidant and some metal-containing enzymes. It is suggested that these aspirin effects are determined by the aspirin-metal complexes formed rather than by aspirin itself.

Lipid peroxidation and antioxidant enzymes in vanadate-treated rats.

Male Wistar rats received an aqueous solution of ammonium metavanadate (AMV) of 0.15 mg/V/ml concentration instead of water for 14 days. The erythrocyte count and haemoglobin level in blood were not changed; the haematocrit index was slightly increased. The spontaneous lipid peroxidation in kidney and liver homogenates was increased. The Fe(II)- or ascorbate-induced lipid peroxidation was more pronounced in the kidney than in the liver. No changes in lipid peroxidation were observed in erythrocytes after AMV treatment. The AMV treatment resulted in a decrease in the activity of the antioxidant enzymes, catalase and glutathione peroxidase in the kidney and liver; the cytosolic Cu,Zn-SOD and mitochondrial Mn-SOD were unchanged. The activity of the enzymes in blood was not changed. The results are discussed with a view to the participation of lipid peroxidation in vanadium toxicity.

In vivo effects of indomethacin on the activity of metal-containing enzymes.

1. Indomethacin injected subcutaneously at a single "ulcerogenic" dose decreased aminooxidase and leucine aminopeptidase activity and did not change alcohol dehydrogenase and ceruloplasmin activity. 2. Indomethacin administered at an oral "therapeutic" dose inhibited aminooxidase activity in small intestinal mucosa but not in liver and did not change leucine aminopeptidase activity in blood, liver and intestinal mucosa; it, however, increased alcohol dehydrogenase and ceruloplasmin activity. 3. The decreased activity of ceruloplasmin and alcohol dehydrogenase by metal deficiency increased after oral indomethacin treatment, reaching the control values when indomethacin was chelated with copper. 4. The results suggest the participation of endogenous metals in the indomethacin effect.

In vivo effects of indomethacin--I. Activity of antioxidant enzymes and lipid peroxidation.

1. The in vivo effects of indomethacin on the activity of antioxidant enzymes and on lipid peroxidation in erythrocytes, liver and small intestines of rats were examined. 2. The activity of the enzymes studied increased or remained unchanged depending on the preparation and model used: treatment with "therapeutic" or "ulcerogenic" dose of indomethacin. 3. Indomethacin inhibited lipid peroxidation in the liver but not in the erythrocytes. 4. The results suggest that the stimulation of antioxidant enzymes, probably through in vivo formed metal complexes, is an alternative mechanism of the antiinflammatory action of indomethacin.

In vivo effects of indomethacin--II. Antioxidant enzymes in metal-deficient rats.

1. The in vivo effects of indomethacin on the activity of antioxidant enzymes in erythrocytes, liver and small intestinal mucosa of rats fed a metal-deficient diet were studied. 2. Metal deficiency led to a significant decrease in the activity of the enzymes studied. 3. Neither with the "ulcerogenic" nor with the "therapeutic" dose of indomethacin significant alterations in the enzyme activity were observed. 4. The oral treatment of metal-deficient rats with a copper complex of indomethacin caused a significant increase in the activity of the enzymes studied. 5. The results suggest the participation of indomethacin in the regulation and redistribution of metals in the organism, which is probably effected through in vivo chelation of endogenous metals.

The effects of aspirin, indomethacin and their copper complexes on phospholipase activity and on lipid peroxidation in rat liver microsomes.

Aspirin and indomethacin decreased the hydrolysis of microsomal phospholipids by exogenous soluble phospholipase A2 and increased lipid peroxidation in rat liver microsomes. Copper chelates of the non-steroidal anti-inflammatory drugs tested more strongly decreased the activity of soluble phospholipase A2 as compared to the ligands. In contrast to the stimulant effects of aspirin and indomethacin these chelates exerted dose-dependent inhibitory effects on enzymatic and non-enzymatic lipid peroxidation. The effects of CuSO4 were similar to those of Cu(II)-aspirin and Cu(II)-indomethacin. Bathocuproine sulfonate, a specific chelator for Cu+, completely prevented the inhibitory effects of copper complexes and of CuSO4 both on lipid peroxidation and on microsomal NADPH-oxidation. Therefore, cupric ions, free or chelated, as well as their reduction to Cu+ by microsomes did not affect the activity of NADPH-dependent cytochrome P-450 reductase. These data are explained by drug-induced changes in the membrane structure as well as by the redox cycling of cupric ions in the copper complexes of aspirin and indomethacin and the subsequent and/or simultaneous interaction of Cu2+/Cu+ with some component(s) of the reaction medium.

In vitro study of paraquat effects on malondialdehyde formation in thymus cells.

The present experiments have shown that paraquat enhanced both O2- production and oxidation of exogenous NADPH in thymus cells by NADPH-dependent enzyme system. The effects of paraquat on malondialdehyde formation were also NADPH-dependent and opposite in the absence and in the presence of ferrous ions: Paraquat inhibited the NADPH-dependent malondialdehyde formation in the absence of Fe2+; it activated this formation in the presence of Fe2+ and had no effect on the nonenzymic Fe2+- and Fe2+-ascorbate-stimulated lipid peroxidation. It is suggested that the activating effect of paraquat on the enzymic NADPH-dependent MDA formation in the presence of Fe2+ is due to the formation of a powerful oxidant product (FeO2)+ or to some Fe2+-paraquat radical complex, acting similarly to the ADP-chelated ferrous ions.

Lipid peroxidation in liver mitochondria from copper-loaded rats.

The amount of phospholipids in liver mitochondria decreased after chronic alimentary copper-loading of rats (40 mg CuSO4 per rat per day in the course of two weeks), while 24 hours after a single intraperitoneal injection of copper (20 mg CuSO4 per kg body weight) it remained unchanged, notwithstanding that both copper treatments highly increased the copper level in mitochondria. Alimentary copper-loading led to a decrease in the relative proportions of the majority of unsaturated fatty acids in mitochondrial phospholipids. Both the spontaneous and Fe2+-induced formation of malonaldehyde were more enhanced in the mitochondria from the two experimental groups as compared to the controls.

Effect of p-chloromercurybenzoate on the MDH-activity of cytosol and rat liver submitochondrial fractions in different buffers.

The effect of p-chloromercurybenzoate (p-CMB) on the MDH-activity of cytosol and submitochondrial fractions (matrix and inner membranes) of rat liver is studied in different buffer systems with identical pH. It is found that p-CMB has two effects on the MDH-activity, depending on the subcellular localization of the enzyme and on the type of buffer used: inhibitory (for the enzyme of the three fractions in HEPES-NaOH and potassium-phosphate buffers) and activating (for the enzyme of the submitochondrial fractions in Tris-HCI buffer). The effect of the SH-reagent on the formation and stability of enzyme-substrate complexes in the different buffers is also studied, as well as the temperature dependence of the p-CMB action on the MDH-activity of the preparations. It is found that the association of m-MDH with the inner mitochondrial membranes does not change its kinetic behaviour. The probable different conformation of the mitochondrial enzyme in the different buffer systems is discussed.

Influence of the temperature on the MDH activity of cytosol and submitochondrial fractions of rat liver in different buffers.

The temperature optimum and the temperature stability of malate dehydrogenase (MDH) in cytosol and submitochondrial fractions (matrix and inner membranes) in the buffers: Tris-HCl, potassium-phosphate and HEPEC-NaOH at pH 7.5, are determined. The temperature optimum of MDH (EC 1.1.1.37) in cytosol does not depend on the type of the buffer used, unlike the optimum in mitochondrial preparations. The mitochondrial enzyme is less resistant to temperature compared to the cytosol preparation. The effect of all three buffers used on the native and on the temperature-changed enzyme in the preparations in preserved the same. However, the presence of the buffer during the temperature action on the MDH-activity in the preparations changes the temperature stability of the enzyme. Changes are assumed in the conformation of the mitochondrial enzyme under the effect of phosphate ions, leading to activation of the native enzyme and to a rise in its temperature stability.