Safia Habib

Biochemistry of Nitric Oxide

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 non-selective nitric oxide synthase inhibitor with respect to isoforms of nitric oxide.

Hydroxyl radical modification of collagen type II increases its arthritogenicity and immunogenicity.

Background The 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 Findings Hydroxyl 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/Significance Neo-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.

Peroxynitrite‐modified DNA: a better antigen for systemic lupus erythematosus anti‐DNA autoantibodies

Native human DNA was modified by ONOO− (peroxynitrite), generated by the synergistic action of sodium nitroprusside [Na2Fe(CN)5NO], an NO donor, and pyrogallol, a superoxide donor. The modifications were analysed by UV absorption characteristics, fluorescence emission transitions, nuclease S1 digestibility and melting‐temperature studies. Modified DNA was found to be highly immunogenic, inducing high‐titre immunogen‐specific antibodies in experimental animals. A maximum of 85% inhibition of the antibody binding was observed in competition ELISA with immunogen as inhibitor. SLE (systemic lupus erythematosus) anti‐DNA autoantibodies recognized modified DNA as a better antigen than the native analogue. Modification of native DNA by ONOO−, forming neoepitopes on the molecule, may be one of the factors for the induction of the autoimmune response seen in SLE.

Human DNA damage by the synergistic action of 4-aminobiphenyl and nitric oxide: an immunochemical study.

4‐Aminobiphenyl (4‐ABP), an aromatic amine is a major environmental carcinogen found mainly in cigarette smoke. It has been vastly implicated in mutagenesis and cancer development. In this study, commercially available human placental DNA was exposed to 4‐ABP (1.3 mM) in presence of sodium nitroprusside (SNP; 8 mM) at 37°C for 3 h. The 4‐ABP + SNP‐mediated structural changes in human DNA were studied by ultraviolet, circular dichroism and fluorescence spectroscopy, thermal melting profile, agarose gel electrophoresis, and nuclease S1 digestibility assay. Spectroscopical analysis and melting temperature studies suggest structural perturbations in the DNA as a result of modification. This might be due to generation of single‐stranded regions and destabilization of hydrogen bonds. Modification was also visualized in agarose gel electrophoresis. Furthermore, nuclease S1 digestibility confirmed the generation of single strand breaks. Rabbits challenged with 4‐ABP‐SNP‐modified human DNA‐induced high‐titer immunogen‐specific antibodies, which showed Cross‐reaction with modified/unmodified DNA bases and ss‐DNA in competitive inhibition assay. The immunogen specificity of induced antibodies against 4‐ABP‐SNP‐modified human DNA was further confirmed in gel retardation assay. It may be concluded that induction of anti‐modified DNA antibodies could be due to perturbation in the DNA structure and its subsequent recognition by immunoregulatory cells as a foreign molecule.

Preferential recognition of peroxynitrite modified human DNA by circulating autoantibodies in cancer patients

Peroxynitrite (ONOO(-)) has been vastly implicated in mutagenesis and cancer development. Present study probes the antigenicity of peroxynitrite damaged DNA (ONOO(-)-DNA) in cancer patients. Purified human placental DNA was damaged by the synergistic action of sodium nitroprusside (SNP) and Pyrogallol for 3h at 37 degrees C. Binding characteristics of cancer autoantibodies as well as experimentally induced anti-peroxynitrite-DNA (anti-ONOO(-)-DNA) antibodies were assessed by ELISA and band shift assay. DNA modifications produced single strand breaks, decreased melting temperature (T(m)), hyperchromicity in UV spectrum and decreased fluorescence intensity. The ONOO(-)-DNA induced high titre antibodies in experimental animals. Cancer autoantibodies exhibited enhanced binding with the modified DNA as compared to the native form. Lymphocyte DNA from cancer patients showed appreciable recognition of anti-ONOO(-)-DNA IgG as compared to the DNA from healthy subjects. The peroxynitrite modified DNA presents unique epitopes which may be one of the factors for the autoantibody induction in cancer patients.

Genotoxic effect of N-hydroxy-4-acetylaminobiphenyl on human DNA: implications in bladder cancer.

Background The interaction of environmental chemicals and their metabolites with biological macromolecules can result in cytotoxic and genotoxic effects. 4-Aminobiphenyl (4-ABP) and several other related arylamines have been shown to be causally involved in the induction of human urinary bladder cancers. The genotoxic and the carcinogenic effects of 4-ABP are exhibited only when it is metabolically converted to a reactive electrophile, the aryl nitrenium ions, which subsequently binds to DNA and induce lesions. Although several studies have reported the formation of 4-ABP-DNA adducts, no extensive work has been done to investigate the immunogenicity of 4-ABP-modified DNA and its possible involvement in the generation of antibodies in bladder cancer patients. Methodology/Principal Findings Human DNA was modified by N-hydroxy-4-acetylaminobiphenyl (N-OH-AABP), a reactive metabolite of 4-ABP. Structural perturbations in the N-OH-AABP modified DNA were assessed by ultraviolet, fluorescence, and circular dichroic spectroscopy as well as by agarose gel electrophoresis. Genotoxicity of N-OH-AABP modified DNA was ascertained by comet assay. High performance liquid chromatography (HPLC) analysis of native and modified DNA samples confirmed the formation of N-(deoxyguanosine-8-yl)-4-aminobiphenyl (dG-C8-4ABP) in the N-OH-AABP damaged DNA. The experimentally induced antibodies against N-OH-AABP-modified DNA exhibited much better recognition of the DNA isolated from bladder cancer patients as compared to the DNA obtained from healthy individuals in competitive binding ELISA. Conclusions/Significance This work shows epitope sharing between the DNA isolated from bladder cancer patients and the N-OH-AABP-modified DNA implicating the role of 4-ABP metabolites in the DNA damage and neo-antigenic epitope generation that could lead to the induction of antibodies in bladder cancer patients.

Structural changes in histone H2A by methylglyoxal generate highly immunogenic amorphous aggregates with implications in auto-immune response in cancer

The role of aberrant protein modifications in cancer and its diagnosis have emerged as a promising research field. Nonenzymatic glyco-oxidation of proteins under oxidative stress has been associated with carcinogenesis through advanced glycation end products (AGE)-receptors for advanced glycation end products (RAGE) axis. Modified proteins that are immunogenic and stimulate cellular and humoral immune responses are being studied to develop early detection markers of cancer. This study has probed the structural alternations; leading to the formation of adducts and aggregates, in histone H2A upon in vitro modification by methylglyoxal (MG). The immunogenicity of modified histone H2A and its binding with cancer autoantibodies was also assessed. MG induced lysine side chain modifications, blocking of free amino groups and the formation of condensed cross structures in histone H2A; and its effect was inhibited by carbonyl scavengers. It led to the adduct formation and generation of N-epsilon-(carboxyethyl)lysine (CEL) and its decomposition forms as revealed by Matrix-assisted laser desorption ionization-mass spectrometry, high-performance liquid chromatography and LC-MS. MG-H2A showed amorphous aggregate formation under electron microscopy and altered binding with DNA in circular dichroism studies. The modified histone elicited high titer immunogen-specific antibodies in rabbits when compared with the native, thus pointing toward the generation of neo-epitopes in MG-H2A. The autoantibodies derived from cancer patients exhibited enhanced binding with MG-H2A as compared with the native histone in enzyme-linked immunosorbent assay and gel retardation assay. This reflects sharing of epitopes on MG-H2A and histones in cancer patients. The neo-epitopes on H2A may be responsible for induction and elevated levels of antibodies in cancer patients. Thus, MG-H2A may be considered as potential antigenic candidate for auto-immune response in cancer.

Autoimmune response to AGE modified human DNA: Implications in type 1 diabetes mellitus

Aims Non-enzymatic glycation of DNA both in vivo and in vitro results in generation of free radicals, known as glycoxidation. Glycoxidation leads to structural perturbation of DNA resulting in generation of neo-antigenic epitopes having implication in autoimmune disorders like diabetes mellitus. In this study human placental DNA was glycated with methylglyoxal (MG) and lysine (Lys) in the presence of Cu2+ and its auto-antibody binding was probed in Type 1 diabetes patients. Methods Glycation was carried out by incubating DNA with MG, Lys and Cu2+ for 24 h at 37 °C. Carboxyethyl deoxyguanosine (CEdG) formed in glycation reaction was studied by LC-MS and the pathway for Amadori formation was studied by ESI-MS techniques. Furthermore, binding characteristics of auto-antibodies in diabetes patients were assessed by direct binding, competitive ELISA and band shift assay. Results DNA glycation with MG, Lys and Cu2+ results in the formation of CEdG (marker of DNA glycation) which was confirmed by LC-MS. The intermediate stages of glycation were confirmed by ESI-MS technique. Serum from diabetes patients exhibited enhanced binding and specificity for glycated DNA as compared to native form. Conclusions Glycation of DNA has resulted in structural perturbation causing generation of neo-antigenic epitopes thus recognizing auto-antibodies in diabetes.

Pathophysiological Role of Peroxynitrite Induced DNA Damage in Human Diseases: A Special Focus on Poly(ADP-ribose) Polymerase (PARP)

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.

Preferential recognition of epitopes on AGE–IgG by the autoantibodies in rheumatoid arthritis patients

Incubation of proteins with glucose lead to their non-enzymatic glycation ultimately resulting in the formation of advanced glycation end products (AGEs) in vivo. AGEs alter unique three dimensional structures of various plasma proteins such as IgG. The role of oxidative stress in the pathogenesis of rheumatoid arthritis (RA), a chronic inflammatory autoimmune disease, is well established. In view of this, commercially available human IgG was glycated in vitro with physiological concentration of glucose (5mM) and the possible involvement of glycated IgG (AGE-IgG) in RA was evaluated. The RA patients were divided into two groups on the basis of disease onset with respect to age: group I (early onset: 20-32 years) and group II (late onset: 36-54 years). AGE-IgG and oxidative stress levels were detected in RA patients and normal healthy individuals by nitroblue tetrazolium (NBT) assay and carbonyl content estimation respectively. Binding characteristics and specificity of RA antibodies were analyzed by enzyme-linked immunosorbent assay (ELISA). We observed preferential binding of RA antibodies to AGE-IgG in comparison to native IgG. Band shift assay further substantiated the enhanced recognition of AGE-IgG by RA antibodies. The results suggest that glycation of IgG results in the generation of neo-epitopes, making it a potential immunogen. Our findings project AGE-IgG as one of the factors for induction of circulating RA autoantibodies.