ONOO"

The results of the lymphocyte experiments with N02 and ONOO- are given in Tables 14.4 and 14.5. The major finding is that cells that are treated with the (3-CAR in addition to vitamins E and C in vivo and exposed to N02 show the cell staining of 6.0% whereas, without the antioxidants, the cell staining was 61.4%. That is, the presence of all three of the antioxidants leads to a protection factor (PF) of 10.2. the protection by (3-CAR alone gave a PF of only 2.0, for a-tocopherol alone it was 1.8 and for ascorbic acid 1.2. 

Lymphocyte Membrane Protection by Antioxidants against ONOO-Cell Membrane Destruction Is Shown by Cell Staining with Eosin... 

The second major finding is that cell protection was also observed against the peroxynitrite anion. Thus, in vivo, the staining increased from 5.2% with the three antioxidants to 43.3% without the antioxidants (giving a PF of 8.3). For the in vitro experiments, the corresponding cell staining was 7.3% and 59.5%, that is, a PF against ONOO- of 8.2 as shown in Table 14.5. 

Hence, for both of the oxidants, N02 and ONOO-, a marked synergism in cell protection by the antioxidant combination of P-CAR with vitamins E and C was observed for both in vivo and in vitro experiments, although the synergistic effect was more pronounced in protection from N02 . 

At physiological pH, ONOO- protonates to peroxynitrous acid (ONOOH) which disappears within a few seconds, the end product being largely nitrate. The chemistry of peroxynitrite/peroxynitrous acid is extremely complex, although addition of ONOO to cells and tissues leads to oxidation and nitration of proteins, DNA and lipids with a reactivity that is comparable to that of hydroxyl radicals. 

Protein tyrosine residues constitute key targets for peroxynitrite-mediated nitrations. Attack of various free radicals (ONOO-, N02 ) upon tyrosine generates 3-nitrotyrosine, which can be measured immunologically or by GC/MS or HPLC techniques. The detection of 3-nitrotyrosine was considered a biomarker of peroxynitrite action in vivo. Similarly, attack of HOC1 and HOBr on tyrosine generates chlorotyro-sine and bromotyrosine, respectively, both of which are measured most accurately by GC-MS. 

Peroxynitrite (ONOO-) is a cytotoxic reactive species that is formed by the reaction of nitric oxide and superoxide. Methods for measuring the scavenging capacity of peroxynitrite usually depend on either the inhibition of tyrosine nitration or the inhibition of dihydrorhodamine 123 (DHR) oxidation to rhodamine 123 (MacDonalds-Wicks and... 

ONOO- + C02 => ONOOCOO ONOOCOO- + tyrosine => dityrosine + 3-nitrotyrosine... 

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. 

NO may react with superoxide to yield the highly reactive peroxynitrite, ONOO-. Superoxide may also be converted into H202 and the reactive hydroxyl radical, OH. In this way excessive activation of glutamate receptors leads to oxidative damage. The calcium influx has a major effect on mitochondria and causes them to depolarize and swell. This leads to a pore being formed in the outer mitochondrial membrane, which allows the escape of cytochrome c and procaspases from the mitochondria into the cytosol. Cytochrome c activates the caspase cascade, which leads to apoptotic cell death (Ch. 35). 

The superoxide anion (O2 ) exhibits numerous physiological toxic effects including endothelial cell damage, increased microvascular permeability, formation of chemotactic factors such as leukotriene B4, recruitment of neutrophils at sites of inflammation, lipid peroxidation and oxidation, release of cytokines, DNA singlestrand damage, and formation of peroxynitrite anion (ONOO-), a potent cytotoxic and proinflammatory molecule generated according to equation 7.210 ... 

Other postulated routes (Jourd heuil et al., 2003) to RSNO formation include the reaction between NO and 02 to yield N02 via a second-order reaction. NO and thiolate anion, RS, react giving rise to thiyl radical, (RS ) [e]. RS then reacts with NO to yield RSNO [f]. The reaction between RS and RS- can also be the source of non-enzymatic generation of superoxide anion (02 ) [g], [h]. 02 reacts with NO to produce peroxynitrite (ONOO ) [i] (Szabo, 2003). Thiols react with ONOOH to form RSNOs [k] (van der Vliet et al.,1998). 

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