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

Gen. Pharmac. Vol. 23, No. 3, pp. 503-507, 1992

0306-3623/92 $5.00 + 0.00 Copyright © 1992 Pergamon Press Ltd

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IN VIVO EFFECTS OF I N D O M E T H A C I N - - I . ACTIVITY OF A N T I O X I D A N T E N Z Y M E S A N D LIPID P E R O X I D A T I O N M. KIRKOVA,* T. KASSABOVAand E. RUSSANOV Institute of Physiology, Bulgarian Academy of Sciences, Acad. G. Bonchev Str., Bldg 23, I 113 Sofa, Bulgaria (Received 20 August 1991)

Abstract--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.

INTRODUCTION Free-radical processes and peroxidation o f many i m p o r t a n t cell structures underlie the pathogenesis o f m a n y diseases (inflammation, atherosclerosis, ischemia, etc.) ( M c C o r d and Roy, 1982). It is k n o w n that natural protectors o f the organism against the cytotoxicity o f oxygen radicals are the cell antioxidants (mainly tocopherol, ascorbate and glutathione) and enzymes eliminating the intermediates o f oxygen reduction (Kellogg and Fridovich, 1975). Some authors suggest that many non-steroidal antiinflammatory drugs (NSAIDs) can act efficiently through prevention o f the cell from free-radicals-induced damage ( M c C o r d and Fridovich, 1978; Hiller et al., 1983; Shelly and Hoff, 1989). The present work was undertaken to examine the in vivo effects o f indomethacin, a typical representative o f N S A I D s , on the activity o f antioxidant enzymes and on lipid peroxidation in erythrocytes, liver and small intestinal mucosa using two experimental models o f drug treatment. MATERIALS AND METHODS Chemicals

Indomethacin was purchased from Sigma; NADPH, reduced glutathione and glutathione reductase from Boehringer; 2-thiobarbituric acid (TBA), riboflavine and H202 from Merck. All other reagents were analytical grade. The solutions were prepared with water redistilled over glass.

duration and path of drug penetration into the organism. The animals were starved 24 hr before sacrification and were killed by exsanguination under light ether anaesthesia. Tissue preparations

Blood was taken by a heparinized syringe through puncture of the left heart ventricle and blood plasma and erythrocytes were obtained after centrifugation at 600 g for 10 min. Erythrocytes were washed twice with 0.9% sodium chloride and were centrifuged under the same conditions. The 5% erythrocyte suspension in saline was lysed through freezing (-20°C) for 24hr and was used for enzyme measurements. The liver perfused with cooled 0.15 M KCI was used for obtaining a 10% homogenate in 0.15 M KC1, mitochondria and 12,000 g postmitochondrial supernatant. Small intestinal mucosa cells were isolated by the method of Hegazy et al. (1983), suspended with 0.15 M KCI, sonicated 3 x 15 sec and stored at -20°C. Enzyme measurements

Catalase (EC 1.11.1.6) was determined according to Aebi (1970). Enzyme activity was expressed as E240/min per mg protein or haemoglobin. Superoxide dismutase (EC 1.15.1.1) activity was determined after Beauchamp and Fridovich (1971) as erythrocytes and mucosa cells were pretreated according to Maral et al. (1977). A unit of SOD activity is the amount of the enzyme giving 50% inhibition of NBT reduction. Glutathione peroxidase activity was measured by the method of Giinzler et al. (1972) with tbutylhydroperoxide as substrate and was expressed in nmol NADPH oxidized/min per mg protein or haemoglobin. Lipid peroxidation

Lipid peroxidation was determined by the amount of the TBA-reacting material formed in fresh biological preparations. Erythrocyte suspension (0.5% according to haemoglobin) in 0.9% sodium chloride-10mM K-PO 4 buffer, pH 7.4, was incubated for 60min at 37°C in the presence and in the absence of 10 mM H202 (Gilbert et al., 1984). Postnuclear liver homogenate or mitochondria (mg protein/ml) in 0.15 M KCI-10 mM K-PO4 buffer, pH 7.4, was incubated for 60 min at 37°C in the presence and in the absence of 0.5 mM sodium ascorbate. Protein content was measured by the method of Lowry et al. (1951). The amount of haemoglobin was determined by Merck test cat. No. 3317.

Animals and treatment

Male Wistar rats, weighing 180-200 g were fed a standard diet and water ad libitum. Two models of in vivo treatment with indomethacin were used: (i) a low "therapeutic" dose (0.71 mg/rat per day) was given orally with tapwater for 6 days (Weser et al., 1980); and (ii) a high "ulcerogenic" dose (40 mg/kg body wt) was injected subcutaneously (Hayden et al., 1978) only once 3 hr before sacriflcation. The models were chosen with a view to study the effect of the dose, *To whom all correspondence should be addressed. 503

504

M. KIRKOVA et al.

activity was enhanced in the liver and intestinal mucosa but not in the erythrocytes. Superoxide dismutase activity increased in the intestinal mucosa only. On single subcutaneous injection of an "ulcerogenic" dose of indomethacin (Fig. 1), we also found an increase of catalase activity in the preparations except for the erythrocytes. Glutathione peroxidase activity in the intestinal mucosa increased, while in the liver and erythrocytes it was not changed. Superoxide dismutase activity was increased both in the intestinal mucosa and in the liver. In vitro experiments were performed to examine the direct effect of indomethacin on the activity of the enzymes studied (Table I). Since indomethacin is thought to be a suitable ligand in metal complexes, its in vitro effect was compared to the effect of an equimolar concentration of EDTA, the latter being a typical metal chelator. Indomethacin (10-4M) exerted no effect on glutathione peroxidase activity in the three preparations and slightly decreased catalase

Statistics The data were assessed for statistical significance using Student's t-test. RESULTS

There are data that indomethacin has a very high "gastric lesion index" (Sacchi et al., 1989). In the present studies indomethacin applied orally at a "therapeutic" dose for 6 days produced no gastric ulceration but decreased intestinal elasticity. Neither visible alterations in the liver nor differences in the haemoglobin level and in the haematocrit (data not shown) were observed with the two in vivo models of indomethacin treatment. Indomethacin administered at an oral "therapeutic" dose for 6 days altered the activity of the antioxidant enzymes studied (Fig. 1). Catalase activity increased in the three biological preparations: small intestinal mucosa (Fig. IA), erythrocytes (Fig. 1B) and liver (Fig. 1C). Glutathione peroxidase

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Fig. 1. Effects of indomethacin on the activity of antioxidant enzymes from small intestinal mucosa (A), erythrocytes (B) and liver postmitochondrial supernatant (C): Co--control group; p.o.--rats treated orally with indomethacin; s.c.--rats injected subcutaneously with indomethacin. Results are mean + SE of 8-10 separate experiments. activity in the erythrocytes and liver. The drug did not either affect superoxide dismutase in the intestinal mucosa and erythrocytes but inhibited it in the liver. Similar was the effect of EDTA. The effect of indomethacin on lipid peroxidation was evaluated by the amount of the TBA-reacting material formed in the presence of an inductor of peroxidation (Fig. 2). The amount of the TBA-reacting material in the erythrocytes with H20: as inductor changed neither after in vivo nor after in vitro indomethacin treatment. EDTA added to the incubation medium had no effect on lipid peroxidation in the erythrocytes in the controls and in the rats treated with indomethacin in vivo (data not shown). After multiple oral administration of a low dose of indomethacin the production of malonedialdehyde with ascorbate as inductor decreased in the liver

homogenate but to a less extent as compared to that observed after single injection of an "ulcerogenic" dose of indomethacin. Malonedialdehyde production in the liver also greatly decreased upon in vitro addition of indomethacin (10 -4 M). The inhibition of lipid peroxidation by the in vitro added EDTA was stronger than 90% in liver homogenates from both controls and rats treated with indomethacin in vivo (data not shown). Similar but less pronounced were the effects of indomethacin on lipid peroxidation in liver mitochondria (data not shown). DISCUSSION

It is known that NSAIDs inhibit prostaglandin synthesis (Vane, 1971; Ferreira et al., 1971; Sacchi

Table 1. In vitro effects of indomethacin on the activity of antioxidant enzymes Preparations

GSH-peroxidase

Superoxide dismutase

3.1 + 0 . 1 4 2.5_+0.19t 2.7_+0.32

74.0-+6.51 70.2___6.36 68.8_+5.22

7.8_+0.42 7.7_+0.82 7.8+0.33

11.5 _+ 0.55 7.9 _+ 0.38t 10.6-+0.56

256.3 + 21.0 253.3 _-+32.2 235.7-+28.4

93.0 ___2.91 52.7 5: 3.06t 58.0-+ 1.60"I"

0.26+_0.012 0.22_+0.018 0.27_+0.016

11.6_+0.98 12.1 _+0.86 11.3_+1.58

Catalase*

Erythroeytes

+ lndomethacin (10 4M) + E D T A (10-4 M) Liver + lndomethacin (10 4 M) + E D T A (10 4M) Small intestinal mucosa

+ Indomethacin (10 4M) + E D T A (10 4M)

10.8±0.33 12.0_+ 1.69 12.2_+0.67

Enzyme activities were expressed as: E240/min per mg prot. (Hb) for catalase; nmol oxidased NADPH/min per mg prot. (Hb) for GSH-peroxidase; U/mg prot. (Hb) for SOD. Each data point represents the mean _+ SE for a minimum of six animals. *Indomethacin and EDTA are in concentration 5.10 -5 M. tStatistically significant differences at P < 0.05.

506

M. KIRKOVAet al.

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Fig. 2. In vivo and in vitro effects of indomethacin on the amount of TBA-reactivematerials in postnuclear rat liver homogenate (A) and erythrocytes (B): Co--control group; p.o.--rats treated orally with indomethacin; s.c.--rats injected subcutaneously with indomethacin. Results are mean + SE of 7-10 separate experiments.

et al., 1989). Many authors suggest that one of the

possible functions of NSAIDs is the elimination of toxic oxygen radicals as 'Oz and OH" which play a role in the inhibition of inflammatory process. Elimination of active oxygen species could be effected either by the drugs themselves (there are data that indomethacin interacts in vitro with ~O2 and O H ' - Bodaness and Chan, 1980; Hiller and Wilson, 1983) or by their in vivo formed metal complexes (De Alvare et al., 1976; Weser et al., 1978). The present results showed that in vivo indomethacin activated the antioxidant enzymes studied. In parallel experiments, using the same models of in vivo treatment, we found that the metal-containing enzymes ceruloplasmin and alcohol dehydrogenase were also activated by indomethacin (unpublished data). If one assumes that indomethacin forms metal complexes in vivo, the activation of the enzymes studied could be explained by some effects of these complexes. Sorenson (1979) has suggested that the induction of superoxide dismutase and lysyloxidase is one of the possible mechanisms of the antiinflammatory and antiulcerous action of the copper complexes of NSAIDs. This suggestion is supported by our observation of a decreased elasticity of the small intestines after oral administration of indomethacin, which is probably due to the inhibited lysyloxidase (a copper-containing enzyme). On the background of indomethacin-activated enzymes with antioxidant action, it is reasonable to expect an inhibition of lipid peroxidation. The strong inhibition of MDA production in the liver by a high dose of indomethacin (an effect observed in in vitro experiments, too) could be explained by indomethacin chelation of endogenous metals. Supporting evidence is the fact that the effect of EDTA on MDA production was similar to that of indomethacin. It should be mentioned that the action of ascorbate used as inductor of peroxidation is mediated by minimal amounts of endogenous

metal ions which have already been chelated by indomethacin or EDTA. Indomethacin decreases MDA formation in the gastric mucosa, too (Nosalova et al., 1989). It is feasible that the effect of indomethacin on the amount of TBA-reacting material is determined by the inhibition of PG synthesis (Nosalova et al., 1989; Sacchi et al., 1989) or by the inhibition of phospholipase activity (Russanov et al., 1986). The unchanged amount of MDA formed after indomethacin as well as the smaller changes in the enzyme activity in the erythrocytes as compared to the mucosa (which is the most vulnerable region of the gastrointestinal wall in ischemia or inflamation-Parks et al., 1983; Younes et al., 1984; Ramage et al., 1988) and liver might be due to the decreased efficient dose of indomethacin as a result of its complexing by serum albumin (Zini et al., 1979). The present results suggest that the stimulation of antioxidant enzymes, probably through in vivo formed metal complexes with indomethacin, is an alternative mechanism of the antiinflammatory action of indomethacin. Further studies are required to support this suggestion.

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In vivo effects of indomethacin--I

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