Adaptation of rat gastric tissue against indomethacin toxicity

Chemico-Biological Interactions 186 (2010) 82–89

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Adaptation of rat gastric tissue against indomethacin toxicity夽 Beyzagul Polat a , Halis Suleyman a,∗ , Hamit Hakan Alp b a b

Ataturk University, Faculty of Medicine, Department of Pharmacology, 25240 Erzurum, Turkey Ataturk University, Faculty of Medicine, Department of Biochemistry, 25240 Erzurum, Turkey

a r t i c l e

i n f o

Article history: Received 12 December 2009 Received in revised form 18 March 2010 Accepted 23 March 2010 Available online 31 March 2010 Keywords: Gastric adaptation Indomethacin Gastric ulcer Oxidant and antioxidant parameters Oxidative DNA damage Rat

a b s t r a c t Indomethacin is used in the treatment of inflammatory diseases. But the drug toxicity limits its usage. This study investigated whether adaptation occurred after various dosages of repeated (chronic) indomethacin in rats to the gastro-toxic effects of indomethacin. It also examined whether the adaptation was related to oxidant–antioxidant mechanisms and oxidative DNA damage in gastric tissue. To illuminate the adaptation mechanism in the gastric tissue of rats given various dosages of chronic indomethacin, the levels of oxidants and antioxidants (GSH, MDA, NO, SOD and MPO), activities of COX-1 and COX-2 enzymes and oxidative DNA damage (8-OHd Gua/105 Gua) were measured. Results were compared to 25-mg/kg singledose indomethacin group, and the role of oxidant and antioxidant parameters and oxidative DNA damage in the adaptation mechanism was evaluated. The average ulcer areas of gastric tissue of the 0.5-, 1-, 2-, 3-, 4-, and 5-mg/kg dosages of chronic indomethacin given to rats were 19.5 ± 3.7, 12.5 ± 3.3, 10 ± 5.2, 4.5 ± 3.6, 8.6 ± 2.4, and 9.5 ± 2.1 mm2 , respectively. This rate was measured as 21.3 ± 2.6 mm2 in the single-dose indomethacin group. Consequently, after various dosages of repeated (chronic) indomethacin administration in rats, it was observed that a clear adaptation developed against gastric damage and that gastric damage was reduced. The best adaptation was observed in the gastric tissue of the 3-mg/kg chronic indomethacin group. In parallel with the damage reduction, the oxidant parameters (MDA and MPO) and oxidative DNA damage (8-OHd Gua/105 Gua) were reduced, and the antioxidant parameters (GSH, NO and SOD) were increased. There is no relation between COX enzymes and adaptation mechanism. This circumstance shows that not COX-1 and COX-2 enzymes, oxidant and antioxidant parameters may play a role in the adaptation mechanism. © 2010 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Indomethacin, an indole acetic acid derivative non-steroidal anti-inflammatory drug (NSAID), is used in the treatment of inflammatory diseases, such as rheumatoid arthritis, osteoarthritis, gut arthritis, burst, tendinitis, traumatic synovitis, and ankylosing spondylitis [1]. However, the drug’s toxicity is a big problem [2] and limits usage [3]. The most common toxic effect of indomethacin is gastrointestinal tract (GIT) damage; the toxic effects vary from unknown blood loss to ulcer perforation [3,4]. Since indomethacin is an ulcerogenic substance [5], it is frequently used in the development of experimental ulcer models [6,7]. It is well-known that the toxic effect of indomethacin in rat gastric tissue is more severe than that of other NSAIDs [8]. Studies have showed that both anti-inflammatory effect

夽 This work was conducted in Ataturk University, Faculty of Medicine, Department of Pharmacology Laboratories, 25240 Erzurum, Turkey. ∗ Corresponding author. Tel.: +90 442 231 65 58; fax: +90 442 236 09 68. E-mail addresses: [email protected], [email protected] (H. Suleyman). 0009-2797/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2010.03.041

and gastro-toxic effect of NSAIDs is linked to cyclooxygenase (COX) enzyme inhibition [9]. Inhibition of COX-2 is responsible from the therapeutic (anti-inflammatory) effect of NSAIDs and inhibition of COX-1 is responsible from gastrointestinal toxic effect of them [1,10]. It is well-known that indomethacin inhibits both of them. It was reported that this detrimental effect of indomethacin was linked to prostaglandin (PG) synthesis (especially PGE2) inhibition [11]. COX is an enzyme that plays a role in the PG pathway. In light of this idea, we decide to determine the COX-1 and COX-2 enzyme activities as an indicator of PGE2. Many methods were developed for preventing NSAIDs’ toxic effects on GIT. For this reason, nitric oxide releasing NSAIDs (NONSAIDs) have been produced [3]. In addition, some investigations have shown that GIT tissue can adapt to the toxic effects of chronic administered drugs and have supposed that this phenomenon, adaptation, may be useful in the decrease of the toxic effects of drugs [12–14]. In the literature, we found some evidence that human gastric tissue can adapt to the toxic effects of indomethacin [15]. But in animals this evidence is controversial. Kuwayama et al. have shown that rat gastric tissue adapts to 5 mg/kg of indomethacin [16]. But other literature has indicated that in animals there is no adaptation to 5 mg/kg or higher doses of

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indomethacin [14,17,18]. In addition, despite some literature that shows indomethacin adaptation [16], adaptation-dose correlation and mechanisms that play a role in the adaptation phenomenon have not been indicated. The significant difference between the oxidant–antioxidant parameters of damaged and healthy tissue [19–21] means that oxidation may play a role in the adaptation phenomenon. For this reason, in this investigation we aimed to define in which dose, chronic administered indomethacin would developed the best adaptation in rat gastric tissue, and to show the relation between adaptation-dose correlation and oxidant–antioxidant parameters, COX-1/COX-2 enzymes and oxidative DNA damage. 2. Materials and methods 2.1. Chemicals Whole biochemical assay compounds were purchased from SIGMA Chem. Co. (Germany) and MERCK (Germany). In addition, indomethacin was bought from Deva Drugs (Turkey), and thiopental was obtained from IE Ulagay (Turkey). 2.2. Animals A total of 48 male albino Wistar rats weighing 200–220 g were obtained from the Ataturk University Medicinal and Experimental Application and Research Center. The animals were divided into treatment groups before the experimental procedures were initiated. The animals were housed and fed under standard conditions in a laboratory where the temperature was kept at 22 ◦ C. Animal experiments were performed in accordance with the national guidelines for the use and care of laboratory animals and were approved by the local animal care committee of Ataturk University. 2.3. Indomethacin ulcer test In this experiment, the 48 rats were divided into 8 groups, each consisting of 6 rats. The first 6 groups were chronic indomethacin groups, the 7th group was the 25 mg/kg single-dose indomethacin group, and the 8th group consisted of healthy intact rats. Doses of 0.5, 1, 2, 3, 4, and 5 mg/kg/day of indomethacin were administered daily by oral gavage to the first 6 groups for 14 days. During this period, the single-dose indomethacin group and the healthy intact rats were given the same volume of distilled water orally. After the last administration, the rats were fasted for 24 h. At the end of this period, a dose of 25 mg/kg of indomethacin was applied by oral gavage to all groups except the healthy intact rats. Distilled water was administered to the healthy intact rats. Six hours after the administration of indomethacin, all rats were sacrificed using a high dose (50 mg/kg) of thiopental. The stomachs were removed, and the ulcer focus on the gastric surface was assessed macroscopically. Ulcer areas were measured by millimetric paper [22]. Results obtained from the groups given chronic indomethacin were compared to those of the single-dose indomethacin group. After this assessment, the stomachs were transported to the biochemistry laboratory for determination of the oxidant–antioxidant parameters and oxidative DNA damage. 2.4. Biochemical analysis of gastric tissue In this part, 0.2 mg of whole gastric tissue (damaged and healthy parts together) was weighed for each stomach. The samples were homogenized in ice with 2-ml buffers (consisting of 0.5% HDTMAB [0.5% hexa desil tri methyl ammonium bromide] pH 6 potassium phosphate buffer for myeloperoxidase analyze, consisting of 1.15% potassium chloride solution for malondialdehyde analysis and pH

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7.5 phosphate buffer for the other analyses). Then, they were centrifuged at 4 ◦ C, 10,000 rpm for 15 min. The supernatant part was used as the analysis sample. 2.5. Total glutathione (tGSH) analysis The amount of GSH in the total homogenate was measured according to the method of Sedlak and Lindsay with some modifications [23]. The sample was weighed and homogenized in 2 ml of 50 mM Tris–HCl buffer containing 20 mM EDTA and 0.2 mM sucrose at pH 7.5. The homogenate was immediately precipitated with 0.1 ml of 25% trichloroacetic acid, and the precipitate was removed after centrifugation at 4200 rpm for 40 min at 4 ◦ C and the supernatant was used to determine GSH level. 1500 ␮l of measurement buffer (200 mM Tris–HCl buffer containing 0.2 mM EDTA at pH 7.5), 500 ␮l supernatant, 100 ␮l DTNB (10 mM) and 7900 ␮l methanol were added to a tube and vortexed and incubated for 30 min in 37 ◦ C. 5,5-dithiobis (2-nitrobenzoic acid) (DTNB) was used as an chromogen and it formed a yellow-colored complex with SH groups. The absorbance was measured at 412 nm using a spectrophotometer. The standard curve was obtained by using reduced glutathione. 2.6. Superoxide dismutase (SOD) analysis Measurements were performed according to Sun et al. [24]. When xanthine is converted into uric acid by xanthine oxidase, SOD forms. If nitro blue tetrazolium (NBT) is added to this reaction, SOD reacts with NBT and a purple-colored formazan dye occurs. The sample was weighed and homogenized in 2 ml of 20 mM phosphate buffer containing 10 mM EDTA at pH 7.8. The sample was centrifuged at 6000 rpm for 10 min and than the brilliant supernatant was used as assay sample. The measurement mixture containing 2450 ␮l measurement mixture (0.3 mM xanthine, 0.6 mM EDTA, 150 ␮M NBT, 0.4 M Na2 CO3 , 1 g/l bovine serum albumin), 500 ␮l supernatant and 50 ␮l xanthine oxidase (167 U/l) was vortexed. Then it was incubated for 10 min. At the end of the reaction, formazan occurs. The absorbance of the purple-colored formazan was measured at 560 nm. As more of the enzyme exists, the least O2 •− radical that reacts with NBT occurs. 2.7. NO (nitric oxide) analysis Nitric oxide levels were measured by the Griess reaction [25,26]. Nitric oxide measurement is difficult because of its brief half-life. Therefore, nitrate and nitrite levels, which are stable end products of nitric oxide metabolism, were used. The measurement mixture [100 ␮l sample, 100 ␮l NADPH (50 ␮mol/l), 100 ␮l FAD (5 ␮mol/l), 20 ␮l nitrate reductase (200 U/l)] was prepared and incubated for 20 min in 37 ◦ C. Then 25 ␮l ZnSO4 (300 g/l) added to this mixture and by this way deproteinization occurred. This mixture centrifuged for 15 min at 1000 rpm. The supernatant part was used as measurement assay. 100 ␮l Griess reagent and 100 ␮l metaphosphoric acid were added to the supernatant and a deep purple azo compound occurred. The Griess reagent consists of 0.5 g sulfanilamide, 12.5 g phosphoric acid and 0.05 g N-(1-napthyl)ethylenediamine in 500 ml distilled water. The method is based on a two-step process. At the first step, nitrate is converted nitrite by nitrate reductase. At the second step, nitrite reacts with the Griess reagent and at the end of this reaction a deep purple azo compound occurs. The absorbance of this deep purple azo compound was measured at 540 nm wavelength by photometric measurement. This azo chromophore accurately determines nitrite concentration as a marker of NO.

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2.8. Malondialdehyde (MDA) analysis The concentrations of gastric mucosal lipid peroxidation were determined by estimating MDA using the thio barbituric acid test [27]. The rat stomachs were promptly excised and rinsed with cold saline. The corpus mucosa was scraped, weighed, and homogenized in 10 ml of 100 g/l KCl. The homogenate (0.5 ml) was added to a solution containing 0.2 ml of 80 g/l sodium lauryl sulfate, 1.5 ml of 200 g/l acetic acid, 1.5 ml of 8 g/l 2-thiobarbiturate, and 0.3 ml distilled water. The mixture was incubated at 98 ◦ C for 1 h. Upon cooling, 5 ml of n-butanol:pyridine (15:l) was added. The mixture was vortexed for 1 min and centrifuged for 30 min at 4000 rpm. The absorbance of the supernatant was measured at 532 nm. The standard curve was obtained by using 1,1,3,3-tetramethoxypropane. 2.9. Myeloperoxidase (MPO) analysis The activity of MPO in the total homogenate was measured according to the method of Wei and Frenkel with some modifications [28]. The sample was weighed and homogenized in 2 ml of 50 mM phosphate buffer containing 0.5% hexadecyltrimethyl ammonium bromide (HDTMAB) and centrifuged at 3500 rpm for 60 min at 4 ◦ C. The supernatant was used to determine MPO activity using 1,3 ml 4-aminoantipyrine-2% phenol (25 mM) solution. 25 mM 4-aminoantipyrine–2% phenol solution and 0.0005% 1.5 ml H2 O2 were added and equilibrated for 3–4 min. After establishing the basal rate, a 0.2 ml sample suspension was added and quickly mixed. Increases in absorbance at 510 nm for 4 min at 0.1-min intervals were recorded. Absorbance was measured at 412 nm using a spectrophotometer. 2.10. Determination of COX activity For this part of our experiment, COX activity of gastric tissues of rats was measured via a COX activity assay kit (Cayman, Ann Arbor, MI, USA). Gastric tissues were collected and washed thoroughly with ice-cold Tris buffer, pH 7.4, containing 0.16 mg/ml of heparin to remove any red blood cells and clots. A sample of gastric tissue was homogenized in 5 ml of cold buffer (0.1 M Tris–HCl, pH 7.8, containing 1 mM EDTA) per gram of tissue and centrifuged at 10,000 × g for 15 min at 4 ◦ C. Supernatant was removed for assay and stored on ice. Protein concentration in the supernatant was measured by the Bradford method [29]. The COX kit measures the peroxidase activity of COX. The peroxidase activity is assayed colorimetrically by monitoring the appearance of oxidized N,N,N ,N -tetramethylp-phenylenediamine at 590 nm. Results are given as units per milligram of protein for COX-1 and COX-2 activity. 2.11. Isolation of DNA from gastric tissue Gastric tissue was drawn and DNA isolated using Shigenaga et al.’s modified method [30]. Samples (50–200 mg) were homogenized at 4 ◦ C in 1 ml of homogenization buffer (0.1 M NaCl, 30 mM Tris, pH 8.0, 10 mM EDTA, 10 mM 2-mercaptoethanol, 0.5% (v/v) Triton X-100) with six passes of a Teflon-glass homogenizer at 200 rpm. The samples were centrifuged at 4 ◦ C for 10 min at 1000 × g to pellet nuclei. The supernatant was discarded, and the crude nuclear pellet re-suspended and re-homogenized in 1 ml of extraction buffer (0.1 M Tris, pH 8.0, 0.1 M NaCl, 20 mM EDTA) and re-centrifuged as above for 2 min. The washed pellet was resuspended in 300 ␮l of extraction buffer with a wide-orifice 200-␮l Pipetman tip. The re-suspended pellet was subsequently incubated at 65 ◦ C for 1 h with the presence of 0.1 ml of 10% SDS, 40 ␮l proteinase K, and 1.9-ml leukocyte lysis buffer. Then, ammonium acetate was added to the crude DNA sample to give a final concentration of 2.5 mol/l, and centrifuged in a micro-centrifuge for

5 min. The supernatant was removed and mixed with two volumes of ethanol to precipitate the DNA fraction. After centrifugation, the pellet was dried under reduced pressure and dissolved in sterile water. The absorbance of this fraction was measured at 260 and 280 nm. Purification of DNA was determined as A260/280 ratio 1.8. 2.12. DNA hydrolysis with formic acid Approximately 50 mg of DNA was hydrolyzed with 0.5 ml of formic acid (60%, v/v) for 45 min at 150 ◦ C [31]. The tubes were allowed to cool. The contents were then transferred to Pierce microvials, covered with Kleenex tissues cut to size (secured in place using a rubber band), and cooled in liquid nitrogen. Formic acid was then removed by freeze-drying. Before analysis by HPLC, they were re-dissolved in the eluent (final volume 200 ␮l). 2.13. Measurement of 8-hydroxy-2 deoxyguanine (8-OH Gua) with HPLC The amount of 8-OH Gua and guanine (Gua) was measured by using a HPLC system equipped with an electrochemical detector (HP Agilent 1100 module series, E.C.D. HP 1049 A), as described previously [31,32]. The amount of 8-OH Gua and Gua was analyzed on a 250 mm × 4.6 mm Supelco LC-18-S reverse-phase column. The mobile phase was 50 mM potassium phosphate, pH 5.5, with acetonitrile (97 volume acetonitrile and 3 volume potassium phosphate), and the flow rate was 1.0 ml/min. The detector potential was set at −0.80 V for measuring the oxidized base. Gua and 8OH Gua (25 pmol) were used as standards. The 8-OH Gua levels were expressed as the number of 8-OH Gua molecules/105 Gua molecules [33].

2.14. Statistical analyses All data were subjected to one-way ANOVA using SPSS 13.0 software. Differences among groups were attained using the LSD option and significance was declared at p < 0.05. Results are means ± SEM. 3. Results 3.1. Indomethacin ulcer test Macroscopic examinations showed that in the rat stomachs of the 25-mg/kg single-dose indomethacin group and all chronic indomethacin groups, ulcer areas occurred. In these stomachs, different numbers and sizes of ulcer focus were determined. The ulcer focus was composed of mucosal defects that were circular and/or oval shaped and dispersed to all stomach surfaces. The ulcer edges were clear, and a blister was seen on the edge. Hyperemia in the stomachs of the rats was clearer than in those of all the chronic indomethacin groups. As seen in Table 1, the most ulcerous areas were observed in 25-mg/kg single-dose indomethacin group when compared to intact rat group (p < 0.0001). Except 0.5 mg/kg dose (p > 0.05), all chronic indomethacin groups decreased ulcer formation (p < 0.0001) when compared to single-dose indomethacin group. The least ulcerous area and the best adaptation was seen in 3 mg/kg chronic indomethacin group when rats were pretreated with low dose indomethacin (p < 0.0001). The ulcer formation reduced with the dose increase until 3 mg/kg, and then as the dose increased to 4 and 5 mg/kg, ulcer formation increased once more. These results show that except 0.5 mg/kg dose, all doses of chronic indomethacin treatment have protective effect against gastric damage caused by indomethacin.

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Table 1 Effects of different dose chronic indomethacin administration on indomethacin-induced ulcers. Drugs

Dose (mg kg−1 )

N

Ulcer area (mm2 )

Anti-toxic adaptive effect (%)

p

Indomethacin Indomethacin Indomethacin Indomethacin Indomethacin Indomethacin Single-dose indomethacin Intact

0.5 1 2 3 4 5 25 –

6 6 6 6 6 6 6 6

19.5 ± 12.5 ± 10.0 ± 4.5 ± 8.6 ± 9.5 ± 21.3 ± –

9 41 53 79 59 55 – –

>0.05 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 – –

3.7 3.3 5.2 3.6 2.4 2.1 2.6

istration when compared to intact rat group (p < 0.0001). While the 0.5-mg/kg chronic indomethacin intake did not change the levels and the activities of these antioxidants (p > 0.05), the other chronic indomethacin doses (1, 2, 3, 4, and 5 mg/kg) increased these levels compared to single-dose indomethacin group. The biggest increase was shown in the 3-mg/kg dose (p < 0.0001). Until 3 mg/kg dose, the antioxidants increased in a correlated manner to the dose increase, and then as the dose increased, they started to decrease once more. These results show that except 0.5 mg/kg dose, all doses of chronic indomethacin treatment have antioxidant effect against gastric damage caused by indomethacin.

Fig. 1. Effects of different dose chronic indomethacin administration on total glutathione (tGSH) level in indomethacin-induced ulcer model in rat gastric tissue. Results are means ± SEM. N (the number of rats): 6. * Refers p < 0.05 and ** refers p < 0.0001 compared to 25 mg/kg single-dose indomethacin group in LSD test.

3.2. Biochemical analyses 3.2.1. tGSH, SOD, and NO analyses As seen in Figs. 1–3, the levels of tGSH and NO and the activity of SOD were reduced by 25-mg/kg single-dose indomethacin admin-

Fig. 2. Effects of different dose chronic indomethacin administration on superoxide dismutase (SOD) activity in indomethacin-induced ulcer model in rat gastric tissue. Results are means ± SEM. N (the number of rats): 6. * refers p < 0.05 and ** refers p < 0.0001 compared to 25 mg/kg single-dose indomethacin group in LSD test.

3.2.2. MDA and MPO analyses As shown in Figs. 4 and 5, the MDA level and MPO activity in the 25-mg/kg single-dose indomethacin group showed a significant increase compared to the intact rats (p < 0.0001). On the other hand, these oxidants decreased significantly in all chronic indomethacin doses except 0.5-mg/kg chronic indomethacin dose (p > 0.05) when compared to 25-mg/kg indomethacin group. The 3-mg/kg chronic indomethacin dose reduced the activity of these oxidant enzymes the most (p < 0.0001). Until 3 mg/kg dose, the oxidants decreased in a correlated manner to the dose increase, and then as the dose increased, they started to increase once more. These results show that except 0.5 mg/kg dose, all doses of chronic indomethacin treatment have a protective effect against oxidative damage caused by indomethacin. 3.2.3. COX-1 and COX-2 analyses In Table 2, COX-1 activity decreased significantly in the 25-mg/kg single-dose indomethacin group when compared to

Fig. 3. Effects of different dose chronic indomethacin administration on nitric oxide (NO) level in indomethacin-induced ulcer model in rat gastric tissue. Results are means ± SEM. N (the number of rats): 6. * Refers p < 0.05 and ** refers p < 0.0001 compared to 25 mg/kg single-dose indomethacin group in LSD test.

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COX-2 level significantly (p < 0.0001). The other doses did not show a decrease (p > 0.05). 3.2.4. 8-OH Gua molecules/105 Gua molecule analyses As shown in Table 3, The 8-OH Gua/105 Gua level in the 25-mg/kg single-dose indomethacin group increased significantly versus the intact group (p < 0.0001). Oxidative DNA damage in all the chronic indomethacin groups was reduced compared to the intact rat group (p < 0.0001). In the 3-mg/kg dose in which adaptation formed the best, this rate was measured at the lowest level. Until 3 mg/kg dose, the 8-OH Gua/105 Gua level decreased in a correlated manner to the dose increase, and then as the dose increased, it started to increase once more. 4. Discussion

Fig. 4. Effects of different dose chronic indomethacin administration on malondialdehyde (MDA) level in indomethacin-induced ulcer model in rat gastric tissue. Results are means ± SEM. N (the number of rats): 6. ** Refers p < 0.0001 compared to 25 mg/kg single-dose indomethacin group in LSD test.

Fig. 5. Effects of different dose chronic indomethacin administration on myeloperoxidase (MPO) activity in indomethacin-induced ulcer model in rat gastric tissue. Results are means ± SEM. N (the number of rats): 6. * refers p < 0.05 and ** refers p < 0.0001 compared to 25 mg/kg single-dose indomethacin group in LSD test.

intact rat group (p < 0.0001). Chronic indomethacin treatment reduced COX-1 activity significantly except 0.5-, 1- and 2-mg/kg chronic doses (p < 0.0001). COX-2 activity of 25 mg/kg singledose indomethacin reduced versus intact rat group significantly (p < 0.0001). Only 5 mg/kg chronic indomethacin dose decreased

In this study, it was investigated in rat gastric tissue whether an adaptation occurred against the gastro-toxic effect of indomethacin after continued (chronic) administration of this drug in various doses and whether adaptation phenomenon had a relationship with oxidant–antioxidant parameters, COX-1/COX-2 enzymes and oxidative DNA damage. To illuminate the adaptation mechanism, the levels of oxidant–antioxidant parameters (GSH, SOD, NO, MDA and MPO) and oxidative DNA damage [8-hydroxyguanine (8-OH Gua)/105 guanine (Gua)] were measured. It is well-known that indomethacin is used not only as an antiinflammatory drug but also to induce an experimental ulcer model in rats [34,35]. Indomethacin caused clearly observed damage in rat gastric tissue in the 10-, 20-, and 25-mg/kg doses [36,37]. In the highest dose (30 mg/kg), indomethacin caused more severe gastric damage [38]. In this study, the average ulcer areas of the stomachs of the rats administered with chronic indomethacin were reduced significantly in all doses, except the 0.5-mg/kg dose, compared to the stomachs of the rats administered with 25-mg/kg singledose indomethacin. A decrease in ulcerous areas was observed in the 1-mg/kg chronic indomethacin group first, and in the group that received the 3-mg/kg indomethacin dose, the decrease was the largest. Then ulcerous areas started to increase in the 4- and 5-mg/kg doses once more. This result indicates an adaptation formation in these chronic doses. The best adaptation was seen in the rats administered with 3-mg/kg chronic indomethacin. These results were in accordance with those of Kuwayama et al., who reported an adaptation in rat gastric tissue with the 5-mg/kg dose. But in this study, although the rats exhibited the adaptation phenomenon, the authors did not mention any knowledge about which dose of indomethacin caused the most effective adaptation [16]. It is known that gastric tissue has a mucosal damage decrease and even completely prevents various irritants; this phenomenon is called cyto-protection (gastroprotection) [39]. However, contrary to the short-term gastroprotection that formed after short-term ischemia and against mild irritants, there is also mention of a long-term phenomenon called adaptive cyto-protection (gastric adaptation). Gastric adaptation is a resistance increase in gastric mucosa against frequently repeated damage [40]. Contact by a

Table 2 Effects of different dose chronic indomethacin administration on COX-1 and COX-2 enzyme activities in rat gastric tissue. Drugs

Dose (mg kg−1 )

N

COX-1 activity (u/mg protein)

Indomethacin Indomethacin Indomethacin Indomethacin Indomethacin Indomethacin Single-dose indomethacin Intact

0.5 1 2 3 4 5 25 –

6 6 6 6 6 6 6 6

8.8 5.6 5.0 3.8 2.1 1.2 7.1 20.8

± ± ± ± ± ± ± ±

0.60 0.49 0.57 0.46 0.47 0.23 0.60 1.88

p

COX-2 activity (u/mg protein)

>0.05 >0.05 >0.05 <0.0001 <0.0001 <0.0001 – <0.0001

4.6 3.5 2.6 2.3 2.3 1.8 3.7 10.7

± ± ± ± ± ± ± ±

0.66 0.56 0.49 0.49 0.49 0.40 0.49 1.05

p >0.05 >0.05 >0.05 >0.05 >0.05 <0.05 – <0.0001

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Table 3 Effects of different dose chronic indomethacin administration on DNA damage in rat gastric tissue. Drugs

N

Dose (mg kg−1 )

DNA damage 8-OHGua/105 Gua (pmol/l)

Indomethacin Indomethacin Indomethacin Indomethacin Indomethacin Indomethacin Single-dose indomethacin Intact

6 6 6 6 6 6 6 6

0.5 1 2 3 4 5 25 –

1.62 1.08 0.82 0.52 0.93 0.93 3.71 0.28

single-dose of an NSAID with gastric mucosa causes severe mucosal damage, which exhibits many bloody lesions, but mucosal lesions are reduced when the drug is administered continuously; thus, gastric adaptation occurs [41]. In addition, it was also reported that for adaptation formed against NSAIDs’ ulcerogenic toxic effects in gastric tissue, continuous and more long-term drug administration was necessary [42,43]. But in higher doses, it was shown that rats died due to the gastro-toxic effect of the chronic administered indomethacin [17]. In a preliminary study, we also observed that all of the rats that received 10-mg/kg/day indomethacin for 7 days died. It was reported that this detrimental effect of indomethacin was linked to prostaglandin (PG) synthesis (especially PGE2) inhibition at the end of COX enzyme inhibition [11]. In the explanation of NSAIDs’ gastric toxic effect mechanism, the COX theory falls short [44]. Because in some studies, it was shown that although PGE2 synthesis was inhibited significantly by continuous drug administration, a decrease was observed in the mucosal lesion as well [40,45]. In this study we also measured COX enzyme activities as an indicator of PGE2. We found that single-dose indomethacin decreased COX-1 and COX-2 activity when compared to intact group. COX-1 activity was reduced by 3-, 4- and 5-mg/kg chronic indomethacin doses significantly and COX-2 activity was reduced by only 5 mg/kg chronic indomethacin dose. Despite the best adaptation observed and the least damage occurred in 3 mg/kg dose, COX-1 activity of this group was lower than single-dose indomethacin group. This means that other factors, except PG, may play a role in the gastric adaptation mechanism. In light of this idea, to determine whether oxidant–antioxidant parameters had a role or not, we measured GSH, SOD, and NO, MDA, MPO, and 8-OH Gua/105 Gua levels. In an experiment conducted by Naito et al., the role of reactive oxygen species (ROS) was shown in the etiopathogenesis of indomethacin-induced gastric damage [46]. Against these detrimental effects of ROS, in tissues, enzymatic and non-enzymatic defense mechanisms were produced [47–49]. Tissue damage starts with lipid radical formation in the cell membrane. This radical first turns into lipid hydroperoxide, and then the damage is completed by the formation of toxic products, such as aldehyde, alkane, and malondialdehyde [46]. A decrease in ulcer areas due to chronic indomethacin administration means that this may trigger antioxidant activity in gastric tissue. The results have indicated that the average GSH level of the chronic indomethacin groups in all doses, except the 0.5-mg/kg dose, increased versus the 25-mg/kg single-dose indomethacin group. In addition, this increase showed a parallel to the adaptation phenomenon. The higher the GSH level, the less the damage that occurred. The highest level was measured in the 3-mg/kg indomethacin dose. GSH and other antioxidants (i.e., melatonin, vitamins) prevented tissue damage by keeping ROS levels in physiologic concentrations [50,51]. The theory that an antioxidant enzyme, SOD, is a protective factor in indomethacin-induced damage control was supported [52]. SOD shows this effect by converting toxic superoxide radicals into non-toxic hydroxyl peroxide and

± ± ± ± ± ± ± ±

0.63 0.10 0.06 0.10 0.12 0.54 1.31 0.02

p <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 – –

molecular oxygen [53]. Otherwise, NO is an antioxidant that regulates acid and gastric mucus release and mucosal blood flow [54]. NO also prevents peroxidation of membrane lipids [55]. NO and NO-releasing substances, such as glycerin trinitrate and nitroprusside prevent gastric mucosa from damage and accelerate healing [56–58]. This two antioxidant levels was reduced by 25-mg/kg single-dose indomethacin administration. While the 0.5-mg/kg chronic indomethacin intake did not change the levels of SOD and NO, the other chronic indomethacin doses (1, 2, 3, 4, and 5 mg/kg) increased these levels. The largest increase was shown in the 3mg/kg dose. The MDA and MPO levels in the 25-mg/kg single-dose indomethacin group showed a significant increase compared to the intact rats. On the other hand, these levels decreased significantly in all chronic indomethacin doses. The 3-mg/kg chronic indomethacin dose reduced the activity of these oxidant enzymes the most. It is known that toxic oxygen radicals that produce largely in oxidativestress-exposed tissues stimulate lipid peroxidation, which causes MDA formation [59,60]. If MPO activity is high in gastric tissue, an increase in neutrophil infiltration has been shown in this region. For this reason, increased MPO activity has been approved as a messenger of neutrophil infiltration in various gastric injury models [61]. ROS causes various types of DNA damage, which result in mutation [23]. Increase in ROS formation and decrease in antioxidant enzymes lead to oxidative DNA injury [62,63]. Today, approximately one hundred types of oxidative DNA base damage have been described [64]. In recent studies, base damage has been measured frequently as a marker of DNA damage; since Cu++ ions exist at a high percentage in guanine-cytosine-rich areas, guanine is the most oxidative damage exposed base [62,63,65]. For this reason, 8-hydroxy-2-deoxy guanine (8-OH Gua) is one of the most measured substances. 8-OH Gua/105 Gua is accepted and measured as a biomarker of oxidative DNA base damage [66]. We also observed that the 8-OH Gua/105 Gua level in the 25-mg/kg single-dose indomethacin group increased significantly versus the intact group. Oxidative DNA damage in all the chronic indomethacin groups was reduced significantly compared to the intact rat group. In the 3mg/kg dose in which adaptation formed the best, this rate was measured at the lowest level. This means that decrease in oxidative DNA damage might have a role in the chronic indomethacinadministered induced adaptation mechanism. In conclusion, it was observed that after various dose continuous (chronic) indomethacin administrations, a clear adaptation occurred against gastric damage in rats, and the damage diminished. It was also determined that, in the 3-mg/kg chronic indomethacin dose, the best adaptation occurred. Parallel to the decrease in damage formation, it was shown that oxidant parameters (MDA and MPO) and DNA damage (8-OH Gua/105 Gua) decreased and antioxidant parameters (GSH, SOD and NO) increased. This result shows us that in the adaptation mechanism an increase in the antioxidant parameters and a decrease in the oxidant parameters may have a role. The existence of the adaptation phenomenon may be used by clinicians to increase the prevalence of its usage.

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Conflict of interest None of the authors for this study had conflicts of interest, sources of financial support, corporate involvement, patent holdings, etc.

Acknowledgements We want to express our special thanks to Prof. Fatma Gocer, Assoc. Prof. Zekai Halici and Assoc. Prof. Ahmet Hacimuftuoglu and Dr. Elif Cadirci and Dr. Abdulmecit Albayrak for their contribution to this work.

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