Protection Against Peroxynitrite Dependent Tyrosine Nitration and A1

Annals ofthe Rheumatic Diseases 1996; 55: 383-387 383

Protection against peroxynitrite dependent Ann Rheum Dis: first published as 10.1136/ard.55.6.383 on 1 June 1996. Downloaded from tyrosine nitration and a1-antiproteinase inactivation by some anti-inflammatory drugs and by the antibiotic tetracycline

Matthew Whiteman, Harparkash Kaur, Barry Halliwell

Abstract physiological pH to give a range of noxious Objective-To examine in vitro the ability products with reactivities resembling those of of several drugs to protect against del- hydroxyl radicals (OH), nitrogen dioxide eterious effects of peroxynitrite, a cyto- (NO2%), and nitronium ion (NO2+).8 toxic agent formed by reaction of nitric Addition of peroxynitrite to tissues and oxide with superoxide radical, that may be biological fluids leads to nitration of aromatic generated in the rheumatoid joint and amino acid residues, and the presence of these could cause joint damage. may be a 'marker' of peroxynitrite mediated Methods-The ability of several drugs to (NO" dependent) damage in vivo.6 9-11 Indeed, protect against such possible toxic actions nitrotyrosine is present in plasma and synovial ofperoxynitrite as inactivation of Gl-anti- fluid from patients with RA, but it was not proteinase and nitration of tyrosine was detected in healthy control subjects.'2 Nitration evaluated. of tyrosine residues on proteins may interfere Results-Most non-steroidal anti-inflam- with cell signal transduction by tyrosine matory drugs were moderately (indo- phosphorylation/dephosphorylation.'3 Peroxy- methacin, diclofenac, naproxen, tolmetin) nitrite also inactivates otl-antiproteinase, the or only weakly (sulindac, ibuprofen, auro- major inhibitor of serine proteases (such as thioglucose, flurbiprofen, sulphasalazine, elastase) in human body fluids,7 14 and con- salicylate, penicillamine disulphide) ef- siderable inactivation of otl-antiproteinase has fective in preventing tyrosine nitration been shown to occur in the inflamed rheuma- and a,-antiproteinase inactivation by toid joint.'5 peroxynitrite, but 5-aminosalicylate and RA is treated by the use of a number

penicillamine were much more effective, of drugs, including non-steroidal anti- http://ard.bmj.com/ as was the antibiotic tetracycline (but not inflammatory drugs (NSAIDs) such as aspirin ampicillin). Phenylbutazone and flu- and indomethacin,.and slower acting 'disease fenamic acid protected effectively against modifying' drugs, such as penicillamine and tyrosine nitration, but could not be tested sulphasalazine, the mechanism of action of in the a,-antiproteinase system. The anal- which is not always clear. gesic paracetamol was highly protective in We have examined the ability of a range of

both assay systems. drugs used in the treatment of RA to protect on September 26, 2021 by guest. Protected copyright. Conclusion-Many drugs used in the against damage by ONOO-. Two measures of treatment of rheumatoid arthritis are un- damage that may be relevant to events in vivo likely to act by scavenging peroxynitrite. were used: ability of the drug to prevent The feasibility ofperoxynitrite scavenging inactivation of ca1-antiproteinase by ONOO-, as a mechanism of penicillamine, and ability of the drug to inhibit the nitration 5-aminosalicylate, and paracetamol of tyrosine by ONOO-. action in vivo is discussed.

(Ann Rheum Dis 1996; 55: 383-387) Materials and methods REAGENTS N-succinyl (ala)3-p-nitroanilide (SANA), cata- In chronic inflammatory diseases such as rheu- lase (type C40), elastase (E0258), al-anti- matoid arthritis (RA) and inflammatory bowel proteinase (A9024), DL-tyrosine, and the anti- disease, there is known to be overproduction of inflammatory drugs were from Sigma oxygen derived species such as superoxide Chemical Corp, Poole, Dorset, UK. Solutions King's College London, radical, O2*.1 There is also considerable evi- of sulindac, paracetamol, ibuprofen, diclo- Pharmacology Group, dence that production of nitric oxide (NO") is fenac, ampicillin, and tetracycline were made Manresa Road, increased-for example from measurements of up in distilled water; those of indomethacin, London SW3 6LX, United Kingdom increased nitric oxide synthase activity and tolmetin, flurbiprofen, sulphasalazine, sulpha- M Whiteman demonstration of increased concentrations of pyridine, and piroxicam were made up in water H Kaur nitrite/nitrate in body fluids from patients with with minimum potassium hydroxide added to B Halliwell RA.2A Nitric oxide reacts with a very high rate ensure solution. Salicylate, 5-aminosalicylate, Correspondence to: B Halliwell. constant with 02-- to give peroxynitrite aurothioglucose, penicillamine, and penicilla- Accepted for publication (ONOO-).5 6 Peroxynitrite can be directly mine disulphide were dissolved in 500 mmol/l 6 February 1996 cytotoxic' 7 and it may also decompose at potassium dihydrogen phosphate-dipotassium 3.8434Whiteman, Kaur, Haliwell

hydrogen phosphate (KH2PO4-K2HPO4) saline, pH 7-4 (140 mmol/l sodium chloride, buffer pH 7-4; phenylbutazone was dissolved 2-7 mmol/l potassium chloride, 16 mmol/I in 0 4% (w/v) sodium carbonate. Solutions disodium hydrogen phosphate, 2-9 mmol/l Ann Rheum Dis: first published as 10.1136/ard.55.6.383 on 1 June 1996. Downloaded from were made up freshly every day and diluted KH2PO4) to a concentration of 4 mg/ml and with distilled water to obtain the required drug elastase was dissolved in the same buffer to a concentrations. final concentration of 5 mg/ml. The volume of ot,-antiproteinase required to inhibit elastase by 80-90% (typically 60-70 gl) was added to PEROXYNITRITE SYNTHESIS6 buffer (500 mmol/I K2HPO4-KH2PO4 pH 7A4), Five millilitres of an acidic solution (0-6 mol/l with or without 0-1 ml of compound to be hydrochloric acid) of hydrogen peroxide tested, to give a volume of 0 945 ml, and (H202) 0 7 mol/l was mixed with 5 ml of incubated in a water bath at 37°C for 15 potassium nitrite 0-6 mol/l on ice for one minutes, when peroxynitrite (typically 5 j.l) second and the reaction quenched with 5 ml was added to give a final concentration of 0 5 of ice cold sodium hydroxide 1-2 moll. The mmol/l. The sample was vortexed for 10 stock was then frozen overnight (-20°C) and seconds and incubated for five minutes, the top layer of the solution collected for the elastase (usually 50 ,ul) was added and the experiment. Concentrations of stock ONOO- sample further incubated at 37°C for 15 min- were redetermined before each experiment utes, followed by addition of 2-0 ml of buffer. using a molar absorption coefficient of 1670 Then after 15 minutes, 0 1 ml of elastase (moll)-1 cm-' at 302 nm.6 substrate (SANA) was added and the rate of reaction followed at 410 nm for 30 seconds.

MEASUREMENT OF TYROSINE NITRATION DL-Tyrosine solutions were made up to a final Results concentration of 10 mmolI by dissolving the INACTIVATION OF ctl-ANTIPROTEINASE: required amount in 8 ml of water with 250 ,l PROTECTION BY DRUGS of 10% (w/v) potassium hydroxide followed by As expected,7 14 addition of ONOO- to al-anti- 250 pl of 5% phosphoric acid and dilution to proteinase led to inactivation of the ability of 10 ml by the addition of 1-5 ml of water. otl-antiproteinase to inhibit elastase. The in- Tyrosine solution 0 1 ml together with 0 1 ml activation was complete within five minutes at of a solution of the compound to be tested was pH 7*4 (data not shown). added to a plastic test tube containing 0 795 The extent of inactivation increased with ml of buffer (500 mmol/l K2HPO4-KH2PO4 ONOO- concentration and a five minute incu- pH 7 4) and incubated in a water bath at 37°C bation time with 0 5 mmol/l ONOO- was for 15 minutes. After this time peroxynitrite selected for further studies. Investigation of the (typically 5 gl) was added to a final concen- possibility that other constituents of the tration of 1 mmol/I and the tubes were vortexed ONOO- solution (such as NO3- and H202, http://ard.bmj.com/ for 15 seconds and incubated for a further 15 which can be present in ONOO- preparations6) minutes. The pH was measured after the were involved in the inactivation demonstrated addition of peroxynitrite and found to be that catalase (final concentration 103 U/ml) between 7-4 and 7-5. had no effect on inactivation of otl-anti- Formation of 3-nitrotyrosine was measured proteinase by ONOO-, and that, when the by high performance liquid chromatography ONOO- solution was added to buffer and (HPLC), rather than spectrophotometrically, to incubated for five minutes at 37°C before the on September 26, 2021 by guest. Protected copyright. avoid interference as a result of the generation addition of aol-antiproteinase, the resulting of chromogen from the drugs. Measurement of 'decomposed ONOO-' solution (which still 3-nitrotyrosine was performed essentially as contains all the other contaminants6) had no described previously8 using a Spherisorb 5 ,um effect on oxt-antiproteinase (data not shown). ODS2 C18 column (25 cm X 4-6 mm) (HPLC Figure 1 summarises the ability of a range of Technology, Wellington House, Cheshire, UK) drugs to protect ac,-antiproteinase against in- with a guard column C,8 cartridge (Hibar, activation by ONOO-. There was wide vari- BDH, Poole, UK). The eluent was 500 mmolI ation in the protective effects seen. Significant K2HPO4-KH2PO4 pH 3-01, with 20% meth- protection was observed with penicillamine, anol (v/v) at a flow rate of 1 ml min-' through paracetamol, indomethacin, 5-aminosalicylate, a Polymer Laboratories pump (Essex Road, diclofenac, naproxen, and tolmetin, but flurbi- Church Stretten, Shropshire, UK); the ultra- profen, salicylate, sulphasalazine, sulindac, violet detector was set at 274 mm (sensitivity aurothioglucose, ibuprofen, and penicillamine 0-02). The 3-nitrotyrosine detected was con- disulphide were weakly effective. Sulpha- firmed by spiking with standards. Peak heights pyridine had no protective effect: indeed, it of tyrosine and 3-nitrotyrosine were measured seemed to aggravate inactivation of otl-anti- and concentrations calculated from a standard proteinase. We also tested the action of tetra- curve. cycline, as this antibiotic has previously been found to exert antioxidant activity in vitro,'7 and found it to be a good inhibitor of damage PREVENTION OF Ct1-ANTIPROTEINASE to otl-antiproteinase by ONOO-, whereas INACTIVATION ampicillin was not (fig 1). Elastase and al-antiproteinase were measured Several drugs underwent changes in colour essentially as described previously.'6 al-Anti- after the addition of ONOO-. Diclofenac and proteinase was dissolved in phosphate buffered paracetamol developed yellow chromogens. Ability ofdrugs to protect against peroxynitnite 385

Inhibition of elastase (%) All experiments were accompanied by 0 20 40 60 80 100 controls'16 to ensure that the drugs tested had

no effect on the assay procedures used to Ann Rheum Dis: first published as 10.1136/ard.55.6.383 on 1 June 1996. Downloaded from Elastase A measure oal-antiproteinase-that is, they did Elast. + a-AP B not directly inhibit elastase, nor did they inter- Elast.+ ONOO--1-AP C fere with inhibition of elastase by oxl-anti- Ampicillin proteinase. These controls were negative for 5-NH2 salicylate the drugs listed here, except that naproxen Aurothioglucose inhibited elastase activity slightly: this was Diclofenac corrected for using appropriate controls. Flurbiprofen Piroxicam 10 mmol/l and phenylbutazone 1 0 Ibuprofen mmol/l markedly inhibited elastase, while Indomethacin flufenamic acid 1-0 mmol/l inhibited the action of al-antiproteinase; data for these drugs are Naproxen not presented. Addition of the drugs listed in Paracetamol figure 1 to the reaction mixtures after five Penicillamine minutes ofincubation ofoal-antiproteinase with Pen. disulphide ONOO- had no effect-that is, the drugs could Salicylate not reactivate cKl-antiproteinase after it had Sulindac been inactivated by ONOO-. Sulphapyridine Figure 2 shows the concentration dependence Sulphasalazine of the protective action of those drugs shown to Tetracycline be most effective in the 'screen' (fig 1). Most Tolmetin protective drugs were active at concentrations of 100 pmol/1-much less than the concentration Figure 1 'Screen' ofanti-inflammatory drugs (final of ONOO- used (0 5 mmol/l). However, even at concentration 0 5 mmol/l) for the ability to protect ct,-antiproteinase against inactivation by peroxynitrite. Bar 1 mmol/l concentrations, most drugs were A represents the activity ofelastase (zero inhibition): unable to protect al-antiproteinase completely: addition ofot,-antiproteinase inhibited the elastase (bar B), the most effective were penicillamine and but treatment of the ax,-antiproteinase with peroxynitrite tetracycline. (ONOO9 (final concentration 0-5 mmol/l) decreased this inhibitory effect (bar C). Drugs able to scavenge ONOOC protect the ot,-antiproteinase and result in less elastase activity. Results are mean, SEM (n = 4). NITRATION OF TYROSINE BY ONOO: Elast. = Elastase; a,-AP= a,-antiproteinase; 5-NH2 salicylate = 5-aminosalicylate. PREVENTION BY DRUGS None of the drugs tested coeluted with nitrotyrosine on HPLC or otherwise interfered with The yellow colour of tetracycline deepened to the HPLC analysis.

orange/yellow after the addition of ONOO- Figure 3 summarises the ability of the various http://ard.bmj.com/ and became yellow/brown during the incu- anti-inflammatory drugs to inhibit the nitration bation. 5-Aminosalicylate developed a purple/ of tyrosine to 3-nitrotyrosine by exposure to pink colour and purple precipitate after ONOO- at pH 74.4611 18 19 Salicylate, tolmetin, ONOO- addition, and this darkened to a sulphasalazine, sulphapyridine, ampicillin, ibu- purple/brown colour during the incubation. profen, sulindac, penicillamine disulphide, Phenylbutazone produced a slightly cloudy naproxen, aurothioglucose, and flurbiprofen

solution after the addition of ONOO-. None of were fairly weak inhibitors, whereas paraceta- on September 26, 2021 by guest. Protected copyright. these colour changes interfered with spectro- mol, flufenamic acid, piroxicam, diclofenac, photometric measurement of elastase activity. tetracycline, 5-aminosalicylate, phenylbutazone, penicillamine, and indomethacin were more effective. Indeed, paracetamol, 5-amino- 90- Untreated a1-AP salicylate, and penicillamine prevented nitration completely at a concentration equimolar to that ^ 80- of ONOO-. Figure 4 shows the concentration dependence of inhibition by the drugs found to , 70- be most effective in the first screen (fig 3). Effects were seen at concentrations of 100-200 a) 60- prmol/, compared with 1 mmol/l ONOO-. 0 .° 50- .0 Discussion - 40- Peroxynitrite generation in vivo has been implicated in a wide range of human diseases, including atherosclerosis,20 lung disease,'0 ONOO- treated a,-AP neurodegenerative disorders,4 and inflamma- OV I~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~I.I tory bowel disease.2' Agents able to protect 0.0 0.2 04 0.6 0.8 1.0 against ONOO- dependent damage should Concentration (mmol/1) therefore be therapeutically useful. The Figure 2 Concentration dependence ofthe protection given by drugs (each added to give reported presence of nitrotyrosine in serum thefinal concentration stated) against the inactivation of oa,-antiproteinase by peroxynitrite. and synovial fluid from patients with RA,'2 The concentration ofperoxynitrite (ONOO-) was kept at 0 5 mmolJI. Results are mean, SEM (n = 4). A = Penicillamine; V = tetracycline; * = naproxen; * = 5-aminosalicylate; combined with the information that synovium A = paracetamol; 0 = indomethacin; l= diclofenac. from RA joints immunostains with antibodies 38638 hiteman, Kaur, Halliwell

3-NO2 tyrosine formed (gmol/l) scavenging is unlikely to contribute to their 0 20 40 60 80 therapeutic actions at the plasma concen-

trations achieved during routine treatment. Ann Rheum Dis: first published as 10.1136/ard.55.6.383 on 1 June 1996. Downloaded from Control Sulphasalazine did not protect well, but its Ampicillin metabolite 5-aminosalicylate was very protec- 5-NH2 salicylate tive. Aminosalicylate might therefore be Aurothioglucose capable of scavenging ONOO- when sulphasalazine is used to treat inflammatory bowel Diclofenac 2 Flufenamic acid diseases, but the action of sulphasalazine in Flurbiprofen RA is unlikely to be the result of ONOO- scavenging, as neither sulphasalazine nor Ibuprofen sulphapyridine exerted significant protective Indomethacin effects. In contrast, penicillamine was a power- Naproxen ful protective agent. Paracetamol Penicillamine and phenylbutazone were also Penicillamine powerfully protective against nitration of tyro- Pen. disulphide sine by peroxynitrite (phenylbutazone could Phenylbutazone not be tested in the oel-antiproteinase system). Piroxicam The disulphide form ofpenicillamine is the one Salicylate usually administered to patients, but it is Sulindac believed to be converted to the dithiol form in Sulphapyridine vivo. Althaus et al22 have also shown that Sulphasalazine penicillamine reacts with ONOO-. The Tetracycline reaction presumably involves the -SH group, as Tolmetin the disulphide was poorly effective. Although, in general, drugs good at inhibiting tyrosine Figure 3 Preventiion ofperoxynitrite dependent tyrosine nitration are also good at protecting oa,-anti- nitration to 3-nitrotyrosine (3-NO2 tyrosine) by drugs proteinase, there is no absolute comparability present in the react:ion mixtures at I mmol/l. DL-Tyrosine between the two assays. This is probably 1 mmol/l was incub7ated with peroxynitrite (ONOO-) 1 mmol/lfor 15 mi,nutes at 37°C. Data are mean, SEM because nitration of tyrosine is a complex (n = 4). 5-Aminosealicylate (5-NH2salicylate), reaction sequence; inhibitory compounds can paracetamol, andpenicillamine inhibited nitration act not only by scavenging ONOO-, but also completely at the 1 by quenching the ONOO- derived nitrating species, the tyrosine radical intermediates in directed againist proteins containing nitro- the nitration pathway, or both."8 tyrosine (J BBeckman, personal communi- It was interesting to note that the analgesic cation) is good evidence that ONOO- is gener- paracetamol was also very effective in pre- ated in the rrheumatoid joint in vivo. As venting tyrosine nitration and otl-anti- http://ard.bmj.com/ inactivation of aol-antiproteinase is known to proteinase activation by ONOO-, as was the occur in RA,15 it is relevant to examine the antibiotic tetracycline (whereas ampicillin had ability ofvariotuis compounds to protect against little effect). This further supports our previous this process. Most NSAIDs, which are argument that not all the biological actions of generally und&erstood to decrease pain and tetracycline are necessarily attributable to its swelling in RA but not inhibit the progression antibacterial action.'7 Our data show that a of joint damalge, protected only moderately possible mechanism of action of certain anti- on September 26, 2021 by guest. Protected copyright. against inactivation, suggesting that ONOO- inflammatory drugs is the scavenging of ONOO-, especially as drugs such as penicillamine were effective at very low drug/ONOO- molar ratios. Our studies were, of course, in vitro and do Z-. not prove that such mechanisms of action 0 E occur in vivo. Many constituents of human body fluids, such as albumin and ascorbate, - also react with ONOO-.'9 We are currently searching for specific end products of the 0 reaction of ONOO- with the most effective drugs: the ability to demonstrate such end 0 products in patients with RA would provide _ direct evidence for the reaction of these drugs 0 * with ONOO- in vivo. The findings of the Z present study have shown us which drugs are ) worth further examination in this context.

1.0 We are grateful to the Arthritis and Rheumatism Council for 0 0.2 0.4 0.6 0.8 research support, including provision of a studentship for M W. Concentration (mmol/1) Figure 4 Concentration dependent prevention ofperoxynitrite dependent tyrosine nitration I Halliwell B. Oxygen radicals, nitric oxide and human to in the reaction mixtures to the inflammatory joint disease. Ann Rheum Dis 1995; 54: 3-nitrotyrosine (3-NO2 tyrosine) by drugs present final qz 505-10. concentrations stated. The concentration ofperoxynitrite (ONQO-) was kept at 1 mmo& 2 Palmer R M J, Hickery M S, Charles I G, Moncada S, Results are mean, SEM (n = 4). O = Piroxicam; V = tetracycline; O = diclofenac; Bayliss M T. Induction of nitric oxide synthase in human * =flufenamic acid; * = phenylbutazone; 0 = indomethacin; A = paracetamol; chondrocytes. Biochem Biophys Res Commun 1993; 193: A = penicillamine; * = 5-aminosalicylate. 398-405. Ability ofdrugs to protect against peroxynitrite 387

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