Hydrogen Peroxide Scavenging Activity by Non-Steroidal Anti-Inflammatory Drugs

Life Sciences 76 (2005) 2841–2848 www.elsevier.com/locate/lifescie

Hydrogen peroxide scavenging activity by non-steroidal anti-inflammatory drugs

David Costa, Ana Gomes, Salette Reis, Jose´ L.F.C. Lima, Eduarda Fernandes*

REQUIMTE, Departamento de Quı´mica-Fı´sica, Faculdade de Farma´cia, Universidade do Porto, Rua Anı´bal Cunha, 164, 4099-030 Porto, Portugal Received 3 September 2004; accepted 27 October 2004

Abstract

Hydrogen peroxide (H2O2) has been shown to be formed during inflammatory processes and is implicated in its pathophysiology. Thus, a putative scavenging activity against this reactive oxygen specie (ROS) by anti- inflammatory drugs may be of great therapeutical value. The present study was undertaken to evaluate the scavenging activity for H2O2 by several non-steroidal anti-inflammatory drugs (NSAIDs), namely indomethacin, acemetacin, etodolac, tolmetin, ketorolac, oxaprozin, sulindac and its metabolites sulindac sulfide and sulindac sulfone. The H2O2 scavenging assay was performed by measuring H2O2-elicited lucigenin chemiluminescence using a microplate reader. The specificity of the method was confirmed by the use of catalase, which completely prevented the H2O2-induced lucigenin chemiluminescence. The endogenous antioxidants melatonin and reduced glutathione (GSH) were used as positive controls. The obtained results demonstrated that all the studied NSAIDs display H2O2 scavenging activity, although in different extents. The ranking order of potency found was sulindac sulfone N sulindac sulfide N GSH N sulindac N indomethacin N acemetacin N etodolac N oxaprozin N ketorolac c melatonin N tolmetin. D 2005 Elsevier Inc. All rights reserved.

Keywords: NSAIDs; Hydrogen peroxide scavenging activity; Microplate chemiluminescence assay

* Corresponding author. Tel.: +351222078968; fax: +351222004427. E-mail address: [email protected] (E. Fernandes).

Introduction

During the course of inflammatory processes, hydrogen peroxide (H2O2) may be generated in large amounts. This occurrence may be due to the activation of mast cells, macrophages, eosinophils, and d neutrophils, which generate superoxide radical (O2 ), predominantly via NADPH oxidase (Barnes, d 1990; Hampton et al., 1998). The O2 is then rapidly converted into H2O2 by superoxide dismutase d (SOD). H2O2 can easily penetrate the membranes of surrounding cells, whereas O2 usually cannot. H2O2 is not an inherently reactive compound. However, H2O2 can be converted into highly reactive and deleterious products: (i) H2O2 may undergo transition-metal-dependent conversion into the extremely deleterious hydroxyl radical (HOd )(Halliwell and Gutteridge, 1985); (ii) myeloperoxidase, a polimorphonuclear neutrophil (PMN) characteristic enzyme generates hypochlorous acid (HOCl) from H2O2 and chloride ions (Winterbourn, 1985). Membrane lipids are particularly susceptible to oxidation by HOd , not only due to their high polyunsaturated fatty acid content (De Groot and Noll, 1987; Schinella et al., 2002), but also due to their association in the cell membrane with enzymatic and non-enzymatic systems able to generate ROS (Schinella et al., 2002). A single HOd can result in the formation of many molecules of lipid hydroperoxides in the cell membrane, which may severely disrupt its function, and lead to cell death. ROS, via lipid peroxidation, causes the release of arachidonic acid from membrane phospholipids and may thus increase the formation of prostaglandins and leucotrienes (Hemler et al., 1979; Taylor et al., 1983). Lipid peroxidation may also alter protein structure, thus altering antigenicity, which may provoke immune responses (Barnes, 1990). The HOCl plays an important role in the bactericidal function (Hampton et al., 1998). However, when produced in excess, HOCl is also highly reactive toward a range of biological substrates and may cause deleterious effects in the surrounding tissue (Hampton et al., 1998; Pullar et al., 1999). Non-steroidal anti-inflammatory drugs (NSAIDs) are the most important therapeutical agents used for the treatment of inflammatory processes. Among them, indomethacin, acemetacin, etodolac, tolmetin, ketorolac, oxaprozin, and sulindac have been widely used, due their inhibitory activity against cycloxygenase (COX) enzymes that catalyse the formation of prostaglandin precursors from arachidonic acid (Fernandes et al., 2004). However, taking into account the above-mentioned rationale, it may be postulated that a putative scavenging activity against H2O2 by anti-inflammatory drugs may also be of great therapeutical value. Thus, the aim of the present study was to evaluate the scavenging activity for H2O2 by the NSAIDs indomethacin, acemetacin, etodolac, tolmetin, ketorolac, oxaprozin, sulindac and its metabolites sulindac sulfide and sulindac sulfone. The endogenous antioxidants catalase, melatonin and reduced glutathione (GSH) were used as positive controls of the assay system.

Methods

Chemicals

Indomethacin, acemetacin, etodolac, tolmetin, ketorolac, sulindac, 30% hydrogen peroxide, reduced glutathione (GSH), melatonin, and lucigenin, were obtained from Sigma Chemical Co. (St. Louis, USA). Oxaprozin was a gift from Helsinn Healthcare SA, Switzerland. Sulindac sulfide and sulindac sulfone were a gift from Merck Research Laboratories, USA. All the other reagents were of analytical grade. D. Costa et al. / Life Sciences 76 (2005) 2841–2848 2843

H2O2 scavenging assay

The H2O2 scavenging activity was measured using a chemiluminescence (CL) methodology, by monitoring the H2O2-induced oxidation of lucigenin, accordingly to a previously described procedure (Chang et al., 2002; Jeng et al., 2002), with modifications. Reaction mixtures contained, in a final volume of 250 AL, the following reagents at the indicated final concentrations: 50 mM Tris buffer, pH 7.4, 3 mM lucigenin, and tested compounds, at various concentrations (0–5000 AM) and 2% H2O2. Ketorolac, oxaprozin, sulindac, sulindac sulfone, and tolmetin were dissolved in the Tris buffer. Acemetacin, indomethacin, etodolac and sulindac sulfide were dissolved in DMSO. Under the present experimental conditions, DMSO did not interfere with the assay. The assays were performed at ambient temperature. Blank values were measured in the absence of H2O2. The endogenous antioxidants melatonin and GSH were also assayed under the same conditions, for comparison. Catalase was used as a positive control to confirm H2O2 specificity. H2O2-elicited lucigenin oxidation results in a CL signal (Fig. 1) that was detected using a microplate reader (BIO-TEK, Synergy HT). The resulting CL was monitored for 5 minutes. Each study corresponds to four experiments, performed in triplicate.

CH3 N

O* CH N-Methylacridone CH3 3 (excited state) N N +

H O 2 2 O O Photon (hυ)

+ N N CH CH3 3

CH3 Lucigenin Lucigenin Dioxetane N

O N-Methylacridone (ground state)

Fig. 1. Schematic illustration of the reaction pathway leading to H2O2-induced lucigenin-chemiluminescence (adapted from Rakicioglu et al., 2001). 2844 D. Costa et al. / Life Sciences 76 (2005) 2841–2848

100

(% inhibition) Sulindac Sulindac sulfide 25 Sulindac sulfone GSH -induced lucigenin chemiluminescence 2 O 2 H 0 0 1000 2000 3000 4000 5000 Conc. (µM)

Fig. 2. H2O2 scavenging activity of sulindac, sulindac sulfide, sulindac sulfone and GSH, represented as a percentual decrease of H2O2-induced lucigenin chemiluminescence (% inhibition). Each point represents the values obtained from four experiments, performed in triplicate (Mean F SEM).

The effects are expressed as the percentual inhibition of the H2O2-induced lucigenin oxidation:

Percentual Inhibition of CLsample CLblank The H2O2 induced ¼ 100 100 CLcontrol CLblank Lucigenin Oxidation

Results

Fig. 2 and Fig. 3 show the results obtained in the H2O2 scavenging assay. The obtained results demonstrated that all the studied NSAIDs and positive controls prevent H2O2-elicited lucigenin oxidation in a concentration dependent manner, although in different extents. The ranking order of potency found was sulindac sulfone N sulindac sulfide N GSH N sulindac N indomethacin N acemetacin N etodolac N oxaprozin N ketorolac c melatonin N tolmetin (IC50s of 941 F 60, 1177 F 13, 1384 F 75, 1476 F 50, 1781 F 129, 2028 F 93, 2224 F 28, 2680 F 195, N5000, N5000, N5000 AM (Mean F SEM) respectively) (Table 1). The specificity of the method was confirmed by the use of catalase, which completely prevented the H2O2-induced lucigenin chemiluminescence (IC50 = 0.050 F 0.005 U/ml).

Discussion

The present results showed that the studied NSAIDs exhibit scavenging activity for H2O2, although with a variable potency. This effect is reported for the first time in the present study for these NSAIDs. Importantly, some reports from the literature indicating H2O2 scavenging activity when the horseradish D. Costa et al. / Life Sciences 76 (2005) 2841–2848 2845

100 Indomethacin Oxaprozin Ketorol ac 75 Etodolac Acemetacin Tolmetin Melatonin 50 (% inhibition)

25 -induced lucigeninchemiluminescence 2 O 2 H 0 0 1000 2000 3000 4000 5000 Conc. (µM)

Fig. 3. H2O2 scavenging activity of indomethacin, acemetacin, etodolac, tolmetin, ketorolac, oxaproxin and melatonin, represented as a percentual decrease of H2O2-induced lucigenin chemiluminescence (% inhibition). Each point represents the values obtained from four experiments, performed in triplicate (Mean F SEM). peroxidase assay is applied may give misleading results due to the inhibitory effect on this enzyme displayed for some of the tested drugs (Parij and Ne`ve, 1996). Thus, when enzymatic systems are involved in such studies due attention should be always given to the possible enzyme inhibitory effects of the compounds under study. The present assay system circumvented such methodological shortcoming. When compared to the endogenous compounds GSH and melatonin, the potency of the studied NSAIDs was similar or even higher, as it was demonstrated for sulindac and its metabolites. Of note, sulindac is not considered as a therapeutical drug per se, but rather as a pro-drug, which is converted in vivo, into to the metabolites sulindac sulfide (responsible for the anti-inflammatory effect) and sulindac sulfone (Fernandes et al., 2003). Acemetacin was another NSAID found to have high H2O2 scavenging

Table 1 Hydrogen peroxide scavenging activity (IC50, Mean F SEM, n = 4) of the tested compounds

Tested compound IC50 (AM) Sulindac sulfide 1177F13 Sulindac sulfone 941F60 Sulindac 1476F50 Indomethacin 1781F129 Acematacin 2028F93 Etodolac 2224F28 Oxaprozin 2680F195 Ketorolac N5000 Tolmetin N5000 Melatonin N5000 GSH 1384F75 2846 D. Costa et al. / Life Sciences 76 (2005) 2841–2848 activity. However, one should bear in mind that in vivo, acemetacin is rapidly converted into indomethacin (Jones et al., 1991), which under the present assay conditions has a lower but still effective activity. The obtained results also indicate that the H2O2-scavenging effect will hardly contribute for ketorolac and tolmetin therapeutic outcome, due to their low activities. In accordance with the present results, Wang and Jiao, 2000, have also reported a weak direct H2O2- scavenging effect of GSH. Nevertheless, in vivo, as it is well known, this endogenous antioxidant participates in the rapid metabolism of cellular H2O2 as the co-factor of the enzyme glutathione peroxidase. On the other hand, Gulcin et al., 2003 reported a strong direct H2O2-scavenging effect of melatonin (83% at 260 AM). In this case, the low amount of H2O2 used (40 mM) comparatively to the concentration used in the present study (about 600 mM), may explain the reported high effectiveness of melatonin. The H2O2-scavenging activity observed in the present study should not be assumed as the total capacity of these drugs to counteract oxidative stress. In fact, many other reactive species are known to be produced during inflammatory processes. Among the various reactive species that may be produced d d d in excess during the inflammatory processes, the ROS peroxyl radical (ROO ), HO ,O2 ,H2O2, and HOCl as well as the reactive nitrogen species (RNS) nitric oxide (d NO) and peroxynitrite anion (ONOO) also play important roles in their pathophysiologies, which makes them potential targets for the chemotherapy of inflammation (Dedon and Tannenbaum, 2004). It was previously shown that d d d sulindac and sulindac sulfone exhibit a strong scavenging activity against O2 ,HO, NO, and d d d ONOO , while sulindac sulfide exhibits a potent scavenging activity over HOCl, O2 ,HO , NO, and ONOO (Fernandes et al., 2003 and references therein). Concerning the other NSAIDs assayed in the present study, it was also previously demonstrated that tolmetin, ketorolac, and oxaprozin are not active d against O2 , while acemetacin, indomethacin and etodolac are highly effective against this radical, exhibiting a concentration dependent effect (Fernandes et al., 2004 and references therein). Oxaprozin was the less active scavenger for HOd , being indomethacin, acemetacin, etodolac, tolmetin, and ketorolac shown to be highly active against this ROS. The scavenging effect against HOCl was not observed for indomethacin, acemetacin, etodolac, tolmetin, ketorolac and oxaprozin, while ROOd was effectively scavenged by etodolac, with indomethacin, acemetacin, tolmetin, ketorolac and oxaprozin much less active. d NO, and ONOO were also effectively scavenged by etodolac, indomethacin, acemetacin, etodolac, tolmetin, ketorolac and oxaprozin (Fernandes et al., 2004). Although the effects here described occur at relatively high concentrations as compared to those in plasma and synovial fluid of healthy humans receiving the drugs in pre-clinical tests (Netter et al., 1989; Urquhart, 1991), some NSAIDs were reported to accumulate in inflamed tissues, leading for example, to concentrations in the synovial fluid about 3 to 8 times higher than in control joints (Graf et al., 1975). It must also be taken into account that the scavenging effect of antioxidants is highly dependent on the amount of reactive species being produced (Valenta˜o et al., 2002). Thus, it is possible that much lower concentrations of NSAIDs are active in vivo. Indeed, indomethacin was already shown to prevent H2O2- induced erythrocytic membrane lipid peroxidation at therapeutic concentrations (Orhan and Sahin, 2001), in spite of the high concentrations needed in vitro for this NSAID. In conclusion, the present study, performed for the NSAIDs sulindac, sulindac sulfide, sulindac sulfone, indomethacin, acemetacin, tolmetin, etodolac, ketorolac and oxaprozin showed, for the first time, that these NSAIDs exhibit scavenging activity for H2O2. It may be postulated that the present results concerning H2O2 scavenging activity plus the ROS and RNS scavenging effects previously reported, indicate that these effects may contribute for the anti-inflammatory activity of the studied D. Costa et al. / Life Sciences 76 (2005) 2841–2848 2847

NSAIDs, sulindac and its metabolites as well as acemetacin and indomethacin being the most promising compounds in this respect.

Acknowledgements

The authors greatly acknowledge FCT and FEDER financial support for the project POCTI/FCB/ 47186/2002. David Costa acknowledges FCT and FSE for his PhD grant (SFRH/BD/10483/2002).

References

Barnes, P.J., 1990. Reactive oxygen species and airway inflammation. Free Radical Biology and Medicine 9, 235–243. Chang, M.C., Uang, B.J., Wu, H.L., Lee, J.J., Hahn, L.J., Jeng, J.H., 2002. Inducing the cell cycle arrest and apoptosis of oral KB carcinoma cells by hydroxychavicol: roles of glutathione and reactive oxygen species. British Journal of Pharmacology 135, 619–630. Dedon, P.C., Tannenbaum, S.R., 2004. Reactive nitrogen species in the chemical biology of inflammation. Archives of Biochemistry and Biophysics 423, 12–22. De Groot, H., Noll, T., 1987. The role of physiological oxygen partial pressures in lipid peroxidation. Theoretical considerations and experimental evidence. Chemistry and Physics of Lipids 44, 209–226. Fernandes, E., Toste, S.A., Lima, J.L.F.C., Reis, S., 2003. The metabolism of sulindac enhances its scavenging activity against reactive oxygen and nitrogen species. Free Radical Biology and Medicine 35, 1008–1017. Fernandes, E., Costa, D., Toste, S.A., Lima, J.L.F.C., Reis, S., 2004. In vitro scavenging activity for reactive oxygen and nitrogen species by nonsteroidal anti-inflammatory indole, pyrrole, and oxazole derivative drugs. Free Radical Biology and Medicine 37, 1895–1905. Graf, P., Glatt, M., Brune, K., 1975. Acidic nonsteroid anti-inflammatory drugs accumulating in inflamed tissue. Experientia 31, 951–953. Gulcin, I., Buyukokuroglu, M.E., Kufrevioglu, O.I., 2003. Metal chelating and hydrogen peroxide scavenging effects of melatonin. Journal of Pineal Research 34, 278–281. Halliwell, B., Gutteridge, J.M., 1985. The importance of free radicals and catalytic metal ions in human diseases. Molecular Aspects of Medicine 8, 89–193. Hampton, M.B., Kettle, A.J., Winterbourn, C.C., 1998. Inside the neutrophil phagosome: oxidants, myeloperoxidase, and bacterial killing. Blood 92, 3007–3017. Hemler, M.E., Cook, H.W., Lands, W.E.M., 1979. Prostaglandin biosynthesis can be triggered by lipid peroxides. Archives of Biochemistry and Biophysics 173, 340–345. Jeng, J.H., Chen, S.Y., Liao, C.H., Tung, Y.Y., Lin, B.R., Hahn, L.J., Chang, M.C., 2002. Modulation of platelet aggregation by areca nut and betel leaf ingredients: roles of reactive oxygen species and cyclooxygenase. Free Radical Biology and Medicine 32, 860–871. Jones, R.W., Collins, A.J., Notarianni, L.J., Sedman, E., 1991. The comparative pharmacokinetics of acemetacin in young subjects and elderly patients. British Journal of Clinical Pharmacology 31, 543–545. Netter, P., Bannwarth, B., Royer-Morrot, M.J., 1989. Recent findings on the pharmacokinetics of non-steroidal anti- inflammatory drugs in synovial fluid. Clinical Pharmacokinetics 17, 145–162. Orhan, H., Sahin, G., 2001. In vitro effects of NSAIDs and paracetamol on oxidative stress-related parameters of human erythrocytes. Experimental and Toxicologic Pathology 53, 133–140. Parij, N., Ne`ve, J., 1996. Nonsteroidal antiinflammatory drugs interact with horseradish peroxidase in an in vitro assay system for hydrogen peroxide scavenging. European Journal of Pharmacology 311, 259–264. Pullar, J.M., Winterbourn, C.C., Vissers, M.C., 1999. Loss of GSH and thiol enzymes in endothelial cells exposed to sublethal concentrations of hypochlorous acid. American Journal of Physiology 277, H1505–H1512. Rakicioglu, Y., Schulman, J.M., Schulman, S.G., 2001. Applications of chemiluminescence in organic analysis. In: Garcı´a-Campan˜a, A.M., Baeyens, W.R.G. (Eds.), Chemiluminescence in Analytical Chemistry. Macel Dekker, Inc., New York, pp. 105–122. 2848 D. Costa et al. / Life Sciences 76 (2005) 2841–2848

Schinella, G.R., Tournier, H.A., Prieto, J.M., Buschiazzo, P.M., Rı´os, J.L., 2002. Antioxidant activity of anti-inflammatory plant extracts. Life Sciences 70, 1023–1033. Taylor, L., Menconi, M.J., Polgar, P., 1983. The participation of hydroperoxides and oxygen radicals in the control of prostaglandin synthesis. Journal of Biological Chemistry 258, 6855–6857. Urquhart, E., 1991. A comparison of synovial fluid concentrations of non-steroidal anti-inflammatory drugs with their in vitro activity. Agents Actions 32, 261–265. Valenta˜o, P., Fernandes, E., Carvalho, F., Andrade, P.B., Seabra, R.M., Bastos, M.L., 2002. Antioxidative properties of cardoon (Cynara cardunculus L.) infusion against superoxide radical, hydroxyl radical, and hypochlorous acid. Journal of Agricultural and Food Chemistry 50, 4989–4993. Wang, S.Y., Jiao, H., 2000. Scavenging capacity of berry crops on superoxide radicals, hydrogen peroxide, hydroxyl radicals, and singlet oxygen. Journal of Agricultural and Food Chemistry 48, 5677–5684. Winterbourn, C.C., 1985. Comparative reactivities of various biological compounds with myeloperoxidase-hydrogen peroxide- chloride, and similarity of the oxidant to hypochlorite. Biochimica et Biophysica Acta 840, 204–210.