Kiran Dixit

Genotoxicity and immunogenicity of DNA-advanced glycation end products formed by methylglyoxal and lysine in presence of Cu2+.

The highly reactive electrophile, methylglyoxal (MG), a break down product of carbohydrates, is a major environmental mutagen having potential genotoxic effects. Previous studies have suggested the reaction of MG with free amino groups of proteins forming advanced glycation end products (AGEs). This results in the generation of free radicals which play an important role in pathophysiology of aging and diabetic complications. MG also reacts with free amino group of nucleic acids resulting in the formation of DNA-AGEs. While the formation of nucleoside AGEs has been demonstrated previously, no extensive studies have been performed to assess the genotoxicity and immunogenicity of DNA-AGEs. In this study we report both the genotoxicity and immunogenicity of AGEs formed by MG-Lys-Cu(2+) system. Genotoxicity of the experimentally generated AGEs was confirmed by comet-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. Immunogenicity of native and MG-Lys-Cu(2+)-DNA was probed in female rabbits. The modified DNA was highly immunogenic eliciting high titre immunogen specific antibodies, while the unmodified form was almost non-immunogenic. The results show structural perturbations in MG-Lys-Cu(2+)-DNA generating new epitopes that render the molecule immunogenic.

Glycated human DNA is a preferred antigen for anti-DNA antibodies in diabetic patients

AIMS Glycation of proteins and DNA, results in the generation of free radicals causing structural modification of biomacromolecule. This leads to the generation of neo-antigenic epitopes having implication in diabetes mellitus. In this study, human placental DNA was glycated with fructose and its binding was probed with the serum antibodies from type 1 and 2 diabetes patients. METHODS Glycation was carried out by incubating DNA (10 μg/ml) with fructose (25 mM) for 5 days at 37°C. The induced structural changes in DNA were studied by spectroscopic techniques, thermal denaturation studies and agarose gel electrophoresis. Furthermore, binding characteristics of autoantibodies in diabetes (type 1 and 2) patients were assessed by direct binding and competitive ELISA. RESULTS DNA glycation with fructose resulted in single strand breaks, hyperchromicity in UV spectrum and increased fluorescence intensity. Thermal denaturation studies demonstrated the unstacking of bases and early onset of duplex unwinding. Type 1 diabetes patients exhibited enhanced binding with glycated DNA as compared to native form, while for type 2 diabetes only those with secondary complications (Nephropathy) showed higher binding. CONCLUSIONS Glycation of DNA has resulted in structural perturbation causing generation of neo-antigenic epitopes that are better antigens for antibodies in diabetes patients.

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.

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.

Antigen binding characteristics of antibodies induced against nitric oxide modified plasmid DNA

Nitric oxide (NO) generated by the reduction of sodium nitrite with sodium dithionite caused damage to plasmid Bluescript DNA leading to strand breaks and base modifications. The NO-plasmid DNA was highly immunogenic in rabbits. The antibody activity was inhibited to the extent of 86% with the immunogen as inhibitor, indicating the induction of immunogen specific antibodies. However, delineating the antigenic specificity of anti-NO-plasmid DNA antibodies by competition ELISA, multiple cross-reactivity was observed. The antibodies recognised B-, A- and allied conformations. The visual detection of immune complex formation with native and NO-plasmid DNA reiterated preferential binding with modified plasmid DNA. DNA modified by nitric oxide presents unique epitopes which may be one of the factors in antigen-driven autoimmune response in systemic lupus erythematosus.

Acquired immunogenicity of human DNA damaged by N‐hydroxy‐N‐acetyl‐4‐aminobiphenyl

4‐Aminobiphenyl, a known carcinogen, has many environmental sources like cigarette smoke, industrial waste, and so forth. It can be metabolized to form a potent mutagen, N‐hydroxy‐N‐acetyl‐4‐aminobiphenyl (N‐OH‐AABP) that undergoes further processing to form electrophilic nitrenium ions which interact with DNA‐forming covalent adducts, thereby exerting genotoxic effects. While the mutagenicity of N‐OH‐AABP has been amply reported, no extensive studies have been performed to assess the immunogenicity of N‐OH‐AABP‐modified DNA. In this study, human placental DNA was modified with N‐OH‐AABP, and the structural perturbations in the DNA molecule were evaluated by ultraviolet spectroscopy and nuclease S1 digestion. Native and N‐OH‐AABP‐modified DNA were used as antigens for immunizing female rabbits. The modified DNA was found to be highly immunogenic, eliciting high titer immunogen‐specific antibodies, while the native form was almost nonimmunogenic. The induced antibodies exhibited wide range of heterogeneity in recognizing various nucleic acid conformers and DNA bases. We also detected deposits of immune complex in glomerular basement membrane in rabbits immunized with N‐OH‐AABP‐DNA. Possible role of N‐OH‐AABP‐DNA in the induction of antibodies in cancer patients and the related consequences have been discussed.

Peroxynitrite-induced modification of H2A histone presents epitopes which are strongly bound by human anti-DNA autoantibodies: Role of peroxynitrite-modified-H2A in SLE induction and progression

Peroxynitrite is a potent oxidant and nitrating agent and has in vivo existence. It is a powerful proinflammatory substance and may increase vascular permeability in inflamed tissues. Systemic lupus erythematosus (SLE) is an autoimmune inflammatory disease of unknown etiology. Since its discovery, numerous self- and non-self, nuclear, and cytoplasmic antigens have been suggested as stimuli for SLE initiation, but the exact trigger is yet to be identified. In this study, an attempt has been made to investigate the binding characteristics of SLE anti-DNA autoantibodies to native DNA and native and peroxynitrite-modified H2A histone to explore the possible role of modified protein antigen(s) in SLE initiation and progression. The nuclear protein (H2A histone) was modified by peroxynitrite synthesized in our laboratory. The peroxynitrite-modified H2A revealed generation of nitrotyrosine, dityrosine, and carbonyls when subjected to investigation by physicochemical methods. Binding characteristics and specificity of SLE anti-DNA antibodies were analyzed by direct binding and inhibition enzyme-linked immunosorbent assay. The data show preferential binding of SLE autoantibodies to peroxynitrite-modified H2A histone in comparison with native H2A histone or native DNA. A band shift assay further substantiated the enhanced recognition of peroxynitirite-modified H2A histone by anti-DNA autoantibodies. The results suggest that peroxynitrite modification of self-antigen(s) can generate neoepitopes capable of inducing SLE characteristic autoantibodies. The preferential binding of peroxynitrite-modified H2A histone by SLE anti-DNA antibodies points out the likely role of oxidatively modified and nitrated H2A histone in the initiation/progression of SLE. Moreover, oxidatively modified and nitrated nuclear protein antigen, rather than nucleic acid antigens, appear to be more suitable as a trigger for SLE.

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.

Physicochemical studies on peroxynitrite-modified H3 histone

Histones are DNA protective proteins and may adopt different structures under nitrosative stress. Peroxynitrite is a powerful oxidant and nitrating agent and has in vivo existence. In this communication, we report effect of peroxynitrite-mediated oxidation and nitration on the structure of calf thymus H3 histone. Fine details of peroxynitrite-modified H3 histone were worked out by UV, fluorescence, circular dichroism and Fourier-transformed infrared spectroscopy and polyacrylamide gel. The results revealed that peroxynitrite-mediated nitration and oxidation in H3 histone produced partially folded structure in comparison to the intrinsically disordered structure of native H3 histone. It may be concluded that the H3 histone, constituent of core histones, is highly sensitive to peroxynitrite and can adopt different structures under nitrosative stress in order to protect the packaged DNA from the deleterious insult of peroxynitrite.

Polydeoxyribonucleotide C photoconjugated with lysine or arginine present unique epitopes for human anti-DNA autoantibodies☆

Studies have been carried out to synthesize and characterize the photoconjugates between positively charged amino acids (lysine and arginine) and the polydeoxyribonucleotide C [poly(dC)]. Poly(dC) was covalently crosslinked with lysine or arginine under ultraviolet light. Both lysine and arginine were found covalently photoconjugated to poly(dC), resulting in the formation of photoadduct. Photoaddition of lysine or arginine to poly(dC) rendered them thermodynamically more stable than their native form. A strong recognition of photoadducts was observed with anti-DNA autoantobodies found in the sera of systemic lupus erythematosus (SLE) patients. Poly(dC)-lysine was recognized more strongly than poly(dC)-argine photoadduct. Poly(dC)-lysine photoadduct appears to provide an immunodominant epitope for SLE autoantibody recognition. The result suggests for the possible involvement of these photoadducts as a potential trigger for anti-DNA autoantibody production.