Nitric oxide (NO) a free radical having both cytoprotective as well as tumor promoting agent is formed from L-arginine by converting it to L-citrulline via nitric oxide synthase enzymes. The reaction product of nitric oxide with superoxide generates potent oxidizing agent, peroxynitrite which is the main mediator of tissue and cellular injury. Peroxynitrite is reactive towards many biomolecules which includes amino acids, nucleic acid bases; metal containing compounds, etc. NO metabolites may play a key role in mediating many of the genotoxic/ carcinogenic effects as DNA damage, protein or lipid modification, etc. The basic reactions of nitric oxide can be divided as direct effect of the radical where it alone plays a role in either damaging or protecting the cell milieu and an indirect effect in which the byproducts of nitric oxide formed by convergence of two independent radical generating pathways play the role in biological reactions which mainly involve oxidative and nitrosative stress. Nitric oxide is also capable of directly interacting with mitochondria through inhibition of respiration or by permeability transition. Reaction of nitric oxide with metal ions include its direct interaction with the metals or with oxo complexes thereby reducing them to lower valent state. Excessive production of nitric oxide can be studied by inhibiting the synthetic pathway of nitric oxide using both selective or specific nitric oxide synthase inhibitor or nonselective nitric oxide synthase inhibitor with respect to isoforms of nitric oxide.Keywords Nitric oxide Á Nitric oxide synthase Á Nitric oxide inhibitors Á Generation of nitric oxide Á Cancer Á Systemic lupus erythematosus Á Direct and indirect effect of nitric oxide Á Effect of nitric oxide on mitochondria

The available data suggest that among cellular constituents, proteins are the major target for oxidation primarily because of their quantity and high rate of interactions with ROS. Proteins are susceptible to ROS modifications of amino acid side chains which alter protein structure. Among the amino acids, Cysteine (Cys) is more prone to oxidation by ROS because of its high nucleophilic property. The reactivity of Cys with ROS is due to the presence of thiol group. In the oxidised form, Cys forms disulfide bond, which are primary covalent cross-link found in proteins, and which stabilize the native conformation of a protein. Indirect evidence suggests that thiol modifications by ROS may be involved in neurodegenerative disorders, but the significance and precise extent of the contributions are poorly understood. Here, we review the role of oxidized Cys in different pathological consequences and its biochemistry may increase the research in the discovery of new therapies. The purpose of this review is to re-examine the role and biochemistry of oxidised Cys residues.

Advanced glycation end-products (AGEs) are known to be mutagenic, diabetogenic and vascular disease risk factors. Methylglyoxal (MG) is a dicarbonyl species that reacts with biological macromolecule (proteins, DNA and lipids) to give AGEs. Nonenzymatic glycation of MG with lysine (Lys) in the presence of copper (Cu(2+)) is reported to generate reactive oxygen species (ROS) capable of causing DNA damage. We show that DNA modification in MG-Lys-Cu(2+) system results in the generation of strand breaks, base modification, hyperchromicity and increased fluorescence intensity. Superoxide generation in the MG-Lys system was found to be significantly higher when compared with that in the MG and Lys alone. Moreover, d-penicillamine and pyridoxal phosphate significantly inhibited the formation of glycation products. The presence of a major DNA glycation adduct, N(2)-carboxyethyl-2'-deoxyguanosine (CEdG), was detected by high performance liquid chromatography (HPLC) and confirmed by nuclear magnetic resonance (NMR). As reported earlier, modified DNA (MG-Lys-Cu(2+)-DNA) was highly immunogenic in experimental animals. Furthermore, induced anti-MG-Lys-Cu(2+)-DNA antibodies were effective probe for detecting glycoxidative lesions in human genomic DNA of type I diabetes patients. Our results clearly imply that interaction of MG-Lys and Cu(2+) leads to the formation of AGEs and also the production of potent ROS, capable of causing DNA damage, thereby playing an important role in diabetes mellitus.

Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease characterized by circulating and tissue fixed autoantibodies reactive with self-antigens, including nucleic acid and other nuclear components. The pathways by which these autoantibodies act as a pathogenic factor remain elusive. Present study has investigated the role of estrogens in SLE etiopathogenesis. Estrogen-modified DNA [4-OHE(2)-Cu(II)-DNA] showed single- and double-strand breaks, hyperchromicity, decrease in Tm, and modification of bases. The 4-OHE(2)-Cu(II)-DNA exhibited increased binding with naturally occurring anti-DNA autoantibodies as compared to the unmodified native form (P < 0.001) as assessed by ELISA, quantitative precipitin titration, and gel retardation assay. The relative affinity of anti-DNA antibodies for modified and native DNA was in the order of 2.1 x 10(-7) M and 1.3 x 10(-6) M, respectively. The data suggested that DNA modified with 4-OHE(2) and Cu(II) may be one of the factors for the induction of circulating anti-DNA autoantibodies in SLE.

Peroxynitrite is formed in biological systems when nitric oxide and superoxide rapidly interact at near equimolar ratio. Peroxynitrite, though not a free radical by chemical nature, is a powerful oxidant which reacts with proteins, DNA and lipids. These reactions trigger a wide array of cellular responses ranging from subtle modulations of cell signaling to overwhelming oxidative injury, committing cells to necrosis or apoptosis. The present review outlines the various peroxynitrite-induced DNA modifications with special mention to the formation of 8-nitroguanine and 8-oxoguanine as well as the induction of DNA single strand breakage. Low concentrations of peroxynitrite cause apoptotic death, whereas higher concentrations cause necrosis with cellular energetics (ATP and NAD(+)) serving as control between the two modes of cell death. DNA damage induced by peroxynitrite triggers the activation of DNA repair systems. A DNA nick sensing enzyme, poly(ADP-ribose) polymerase-1 (PARP-1) becomes activated upon detecting DNA breakage and it cleaves NAD(+) into nicotinamide and ADP-ribose and polymerizes the latter on nuclear acceptor proteins. Over-activation of PARP induced by peroxynitrite consumes NAD(+) and consequently ATP decreases, culminating in cell dysfunction, apoptosis or necrosis. This mechanism has been implicated in the pathogenesis of various diseases like diabetes, cardiovascular diseases and neurodegenerative diseases. In this review, we have discussed the cytotoxic effects (apoptosis and necrosis) of peroxynitrite in the etiology of the mentioned diseases, focusing on the role of PARP in DNA repair in presence of peroxynitrite.

BackgroundThe oxidation of proteins by endogenously generated free radicals causes structural modifications in the molecules that lead to generation of neo-antigenic epitopes that have implications in various autoimmune disorders, including rheumatoid arthritis (RA). Collagen induced arthritis (CIA) in rodents (rats and mice) is an accepted experimental model for RA.Methodology/Principal FindingsHydroxyl radicals were generated by the Fenton reaction. Collagen type II (CII) was modified by •OH radical (CII-OH) and analysed by ultraviolet-visible (UV-VIS), fluorescence and circular dichroism (CD) spectroscopy. The immunogenicity of native and modified CII was checked in female Lewis rats and specificity of the induced antibodies was ascertained by enzyme linked immunosorbent assay (ELISA). The extent of CIA was evaluated by visual inspection. We also estimated the oxidative and inflammatory markers in the sera of immunized rats. A slight change in the triple helical structure of CII as well as fragmentation was observed after hydroxyl radical modification. The modified CII was found to be highly arthritogenic and immunogenic as compared to the native form. The CII-OH immunized rats exhibited increased oxidative stress and inflammation as compared to the CII immunized rats in the control group.Conclusions/SignificanceNeo-antigenic epitopes were generated on •OH modified CII which rendered it highly immunogenic and arthritogenic as compared to the unmodified form. Since the rodent CIA model shares many features with human RA, these results illuminate the role of free radicals in human RA.

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