Harparkash Kaur

Evidence for nitric oxide‐mediated oxidative damage in chronic inflammation Nitrotyrosine in serum and synovial fluid from rheumatoid patients

Reaction of nitric oxide (NO•) with superoxide radical generates peroxynitrite, which can decompose to products that nitrate aromatic amino acids. Such nitro‐aromatics may be ‘markers’ of NO•‐dependent oxidative damage. Blood serum and synovial fluid from patients with the inflammatory joint disease rheumatoid arthritis contain 3‐nitrotyrosine. By contrast, body fluids from normal subjects and patients with osteoarthritis contain no detectable 3‐nitrotyrosine; much lower levels were found in serum from patients in the early stages of rheumatoid arthritis. This is evidence that NO• plays a role in joint damage in rheumatoid arthritis.

Aromatic hydroxylation and nitration of phenylalanine and tyrosine by peroxynitrite: Evidence for hydroxyl radical production from peroxynitrite

Peroxynitrite is a highly reactive species, generated from Superoxide and nitric oxide. Some effects of peroxynitrite are ascribed to the molecule itself, but decomposition products of the protonated form, peroxynitrous acid, may account for much of its reactivity in biological systems. Suggested products include highly‐reactive hydroxyl radicals, but thermodynamic calculations have been used to claim that free hydroxyl radicals cannot be formed from peroxynitrite. We utilized aromatic hydroxylation of phenylalanine as a specific detector of hydroxyl radicals, and found that incubation of phenylalanine with peroxynitrite leads to a small amount of p‐, m‐ and o‐tyrosine, specific products of attack by this radical. Products of nitration of phenylalanine and tyrosine were also detected, as was dityrosine. Peroxynitrite decomposition generates several reactive species, including some that can nitrate aromatic rings. Formation of nitro‐aromatic compounds may be a useful marker of peroxynitrite generation in biological systems.

Intense oxidative DNA damage promoted by L-dopa and its metabolites. Implications for neurodegenerative disease.

Oxidative DNA damage can cause mutation and cell death. We show that l‐DOPA, dopamine and 3‐O‐methyl‐DOPA cause extensive oxidative DNA damage in the presence of H2O2 and traces of copper ions. 8‐Hydroxyguanine is the major product. Iron ions were much less effective and manganese ions did not catalyse DNA damage. We propose that copper ion release, in the presence of l‐DOPA and its metabolites, may be an important mechanism of neurotoxicity, e.g. in Parkinsons disease and amyotrophic lateral sclerosis.

Interaction of bilirubin and biliverdin with reactive nitrogen species

Bilirubin (BR) and biliverdin (BV), two metabolites produced during haem degradation by haem oxygenase, possess strong antioxidant activities toward peroxyl radical, hydroxyl radical and hydrogen peroxide. Considering the importance attributed to nitric oxide (NO) and its congeners in the control of physiological and pathophysiological processes, we examined the interaction of BR and BV with NO and NO‐related species in vitro. Exposure of BR and BV to agents that release NO or nitroxyl resulted in a concentration‐ and time‐dependent loss of BR and BV, as assessed by high performance liquid chromatography. Peroxynitrite, a strong oxidant derived from the reaction of NO with superoxide anion, also showed high reactivity toward BR and BV. The extent of BR and BV consumption largely depended on the NO species being analysed and on the half‐lives of the pharmacological compounds considered. Of major importance, BR and BV decomposition occurred also in the presence of pure NO under anaerobic conditions, confirming the ability of bile pigments to scavenge the gaseous free radical. Increasing concentrations of thiols prevented BR consumption by nitroxyl, indicating that bile pigments and thiol groups can compete and/or synergise the cellular defence against NO‐related species. In view of the high inducibility of haem oxygenase‐1 by NO‐releasing agents in different cell types, the present findings highlight novel anti‐nitrosative characteristics of BR and BV suggesting a potential function for bile pigments against the damaging effects of uncontrolled NO production.

Peroxynitrite-Dependent Aromatic Hydroxylation and Nitration of Salicylate and Phenylalanine. Is Hydroxyl Radical Involved?

There is considerable dispute about whether the hydroxylating ability of peroxynitrite (ONOO-)- derived species involves hydroxyl radicals (OH.). This was investigated by using salicylate and phenylalanine, attack of OH. upon which leads to the formation of 2,3- and 2,5-dihydroxybenzoates, and o- m- and p- tyrosines respectively. On addition of ONOO- to salicylate, characteristic products of hydroxylation (and nitration) were observed in decreasing amounts with rise in pH, although added products of hydroxylation of salicylate were not recovered quantitatively at pH 8.5, suggesting further oxidation of these products and underestimating of hydroxylation at alkaline pH. Hydroxylation products decreased in the presence of several OH. scavengers, especially formate, to extents similar to those obtained when hydroxylation was achieved by a mixture of iron salts, H2O2 and ascorbate. However, OH. scavengers also inhibited formation of salicylate nitration products. Ortho, p- and m-tyrosines as well as nitration products were also observed when ONOO- was added to phenylalanine. The amount of these products again decreased at high pH and were decreased by addition of OH. scavengers. We conclude that although comparison with Fenton systems suggests OH. formation, simple homolytic fission of peroxynitrous acid (ONOOH) to OH. and NO2. would not explain why OH. scavengers inhibit formation of nitration products.

Protection against peroxynitrite dependent tyrosine nitration and alpha 1-antiproteinase inactivation by some anti-inflammatory drugs and by the antibiotic tetracycline.

OBJECTIVE: To examine in vitro the ability of several drugs to protect against deleterious effects of peroxynitrite, a cytotoxic agent formed by reaction of nitric oxide with superoxide radical, that may be generated in the rheumatoid joint and could cause joint damage. METHODS: The ability of several drugs to protect against such possible toxic actions of peroxynitrite as inactivation of alpha 1-antiproteinase and nitration of tyrosine was evaluated. RESULTS: Most non-steroidal anti-inflammatory drugs were moderately (indomethacin, diclofenac, naproxen, tolmetin) or only weakly (sulindac, ibuprofen, aurothioglucose, flurbiprofen, sulphasalazine, salicylate, penicillamine disulphide) effective in preventing tyrosine nitration and alpha 1-antiproteinase inactivation by peroxynitrite, but 5-aminosalicylate and penicillamine were much more effective, as was the antibiotic tetracycline (but not ampicillin). Phenylbutazone and flufenamic acid protected effectively against tyrosine nitration, but could not be tested in the alpha 1-antiproteinase system. The analgesic paracetamol was highly protective in both assay systems. CONCLUSION: Many drugs used in the treatment of rheumatoid arthritis are unlikely to act by scavenging peroxynitrite. The feasibility of peroxynitrite scavenging as a mechanism of penicillamine, 5-aminosalicylate, and paracetamol action in vivo is discussed.

Evolving measurements of radical products — how much do they tell us about inflammation?

It is now widely accepted that reactive oxygen species (ROS) such as superoxide radical (O2 ·-), hydrogen peroxide (H2O2) and hydroxyl radical (•OH) reactive chlorine species (RCS) such as hypochlorous acid (HOCl) and reactive nitrogen species (RNS) such as nitrogen monoxide (nitric oxide, NO>•) and oxoperoxonitrate (peroxynitrite, ONOO-) contribute to considerable tissue injury during chronic inflamation (reviewed in [1]).