Free radical peroxynitrite formation

This mode of superoxide-dependent free radical-mediated damaging activity remains an important one although the nature of the generated reactive species (free hydroxyl radicals or perferryl, or ferryl ions) is still obscure. However, after the discovery of the fact that many cells produce nitric oxide in relatively large amounts (see below), it became clear that there is another and possibly a more portent mechanism of superoxide-induced free radical damage, namely, the formation of highly reactive peroxynitrite. 

Reaction of nitric oxide with superoxide is undoubtedly the most important reaction of nitric oxide, resulting in the formation of peroxynitrite, one of the main reactive species in free radical-mediated damaging processes. This reaction is a diffusion-controlled one, with the rate constant (which has been measured by many workers, see, for example, Ref. [41]), of about 2 x 109 1 mol-1 s-1. Goldstein and Czapski [41] also measured the rate constant for Reaction (11) ... 

On the other hand, in accord with the free radical mechanism peroxynitrite is dissociated into free radicals, which are supposed to be genuine reactive species. Although free radical mechanism was proposed as early as in 1970 [111], for some time it was not considered to be a reliable one because a great confusion ensued during the next two decades because of misinterpretations of inconclusive experiments, sometimes stimulated by improper thermodynamic estimations [85]. The latest experimental data supported its reliability [107-109]. Among them, the formation of dityrosine in the reaction with tyrosine and 15N chemically induced dynamic nuclear polarization (CIDNP) in the NMR spectra of the products of peroxynitrite reactions are probably the most convincing evidences (see below). 

Probably, the most convincing proof of free radical mechanism of peroxynitrite reactions is the formation of dityrosine [117,118]. It has been suggested [118] that the nitric dioxide radical is responsible for the formation of both 3-nitrotyrosine and dityrosine (Figure 21.1), however, hydroxyl radicals (which were identified in this system by ESR spectroscopy [119]) may also participate in this process. Pfeiffer et al. [118] proposed that dityrosine is predominantly formed at low fluxes of superoxide and nitric oxide, which corresponds to in vivo conditions, however, this observation was not confirmed by Sawa et al. [117],... 

Peroxynitrite reacts with heme proteins such as prostacycline synthase (PGI2), microperoxidase, and the heme thiolate protein P450 to form a ferryl nitrogen dioxide complex as an intermediate [120]. Peroxynitrite also reacts with acetaldehyde with the rate constant of 680 1 mol 1 s" 1 forming a hypothetical adduct, which is decomposed into acetate, formate, and methyl radicals [121]. The oxidation of NADH and NADPH by peroxynitrite most certainly occurs by free radical mechanism [122,123], Kirsch and de Groot [122] concluded that peroxynitrite oxidized NADH by a one-electron transfer mechanism to form NAD and superoxide ... 

Other very convincing evidences for free radical-mediated mechanism of decomposition and reactions of peroxynitrite and nitrosoperoxocarboxylate were demonstrated by Lehnig [140] with the use of CIDNP technique. This technique is based on the effects observed exclusively for the products of free radical reactions their NMR spectra exhibit emission characterizing a radical pathway of their formation. Lehnig has found the enhanced emission in the 15N NMR spectra of N03- formed during the decomposition of both peroxynitrite and nitrosoperoxocarboxylate. This fact indicates that N03- was formed from radical pairs [ N02, H0 ] and [ N02, C03 ]. Emission was also observed in the reaction of both nitrogen compounds with tyrosine supposedly due to the formation of radical pair [ N02, tyrosyl ]. 

Simultaneous generation of nitric oxide and superoxide by NO synthases results in the formation of peroxynitrite. As the reaction between these free radicals proceeds with a diffusion-controlled rate (Chapter 21), it is surprising that it is possible to detect experimentally both superoxide and NO during NO synthase catalysis. However, Pou et al. [147] pointed out that the reason is the fact that superoxide and nitric oxide are generated consecutively at the same heme iron site. Therefore, after superoxide production NO synthase must cycle twice before NO production. Correspondingly, there is enough time for superoxide to diffuse from the enzyme and react with other biomolecules. 

In addition to nitric oxide, superoxide, and peroxynitrite, NO synthases are able to generate secondary free radicals because similar to cytochrome P-450 reductase, the reductase domain can transfer an electron from the heme to a xenobiotic. Thus it has been found [158,159] that neuronal NO synthase NOS I catalyzed the formation of CH3CH(OH) radical from ethanol. It was suggested that the perferryl complex of NOS I is responsible for the formation of such secondary radicals. Miller [160] also demonstrated that 1,3-dinitrobenzene mediated the formation of superoxide by nNOS. It was proposed that the enhancement of superoxide production in the presence of 1,3-dinitrobenzene converted nNOS into peroxynitrite-produced synthase and may be a mechanism of neurotoxicity of certain nitro compounds. 

It should be noted that Reaction (4) is not a one-stage process.) Both free radical N02 and highly reactive peroxynitrite are the initiators of lipid peroxidation although the elementary stages of initiation by these compounds are not fully understood. (Crow et al. [45] suggested that trans-ONOO is protonated into trans peroxynitrous acid, which is isomerized into the unstable cis form. The latter is easily decomposed to form hydroxyl radical.) Another possible mechanism of prooxidant activity of nitric oxide is the modification of unsaturated fatty acids and lipids through the formation of active nitrated lipid derivatives. 

High antioxidative activity carvedilol has been shown in isolated rat heart mitochondria [297] and in the protection against myocardial injury in postischemic rat hearts [281]. Carvedilol also preserved tissue GSL content and diminished peroxynitrite-induced tissue injury in hypercholesterolemic rabbits [298]. Habon et al. [299] showed that carvedilol significantly decreased the ischemia-reperfusion-stimulated free radical formation and lipid peroxidation in rat hearts. Very small I50 values have been obtained for the metabolite of carvedilol SB 211475 in the iron-ascorbate-initiated lipid peroxidation of brain homogenate (0.28 pmol D1), mouse macrophage-stimulated LDL oxidation (0.043 pmol I 1), the hydroxyl-initiated lipid peroxidation of bovine pulmonary artery endothelial cells (0.15 pmol U1), the cell damage measured by LDL release (0.16 pmol l-1), and the promotion of cell survival (0.13 pmol l-1) [300]. SB 211475 also inhibited superoxide production by PMA-stimulated human neutrophils. 

Allopurinol also inhibits reperfusion injury. This injury occurs when organs that either have been transplanted or have had their usual blood perfusion blocked are reperfused with blood or an appropriate buffer solution. The cause of this injury is local formation of free radicals, such as the superoxide anion, the hydroxyl free radical, or peroxynitrite. These substances are strong oxidants and are quite damaging to tissues. 

The stable free radical nitric oxide (NO) has an important role as a biological messenger. The reaction of NO with superoxide (O2 ) forms the powerful oxidant peroxynitrite (ONOO ), and a mechanism for the reaction of ONOO resulting in the abstraction of H from C—H bonds is shown (equations 109, 110). The formation of HO from the spontaneous decomposition of peroxynitrite, and of COJ radicals from CO2 catalyzed decomposition of peroxynitrite, have been demonstrated. ... 

The precise mechanism by which NO causes glutamase neurotoxicity is unknown. Calcium must be required because of the requirement for NMDA- and glutamate-induced NO formation in brain tissue (Garthwaite etal., 1988). Although both NMDA-receptor agonists and sodium nitroprusside induce specific neurotoxicity as well as cyclic GMP formation in brain tissue (Dawson et al., 1991), it is unlikely that cyclic GMP is the ultimate cause of the neurotoxicity. Instead, NO is most likely involved in producing target cell death. One possible mechanistic pathway is that locally synthesized NO and superoxide anion react with each other to yield peroxynitrite anion (Beckman et al., 1990), which can destroy cell membranes either directly via interaction with cellular thiols (Radi et al., 1991) or indirectly via decomposition to hydroxyl and other free radicals (Beckman et al., 1990). 

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