Neo-epitopes on crotonaldehyde modified DNA preferably recognize circulating autoantibodies in cancer patients.

DNA damage is one of the leading causes of various pathological conditions including carcinogenesis. Crotonaldehyde is a 4-carbon unsaturated bifunctional aldehyde which is found ubiquitously and produced both exogenously and endogenously. It reacts with deoxyguanosine and form adducts with DNA. These adducts were detected and found involved in tumor formation in rats treated with crotonaldehyde. In the present study, structural changes in DNA by crotonaldehyde were evaluated by Fourier transform infrared (FTIR) spectroscopy, differential scanning colorimetry (DSC), dynamic light scattering (DLS), high-performance liquid chromatography (HPLC), and atomic force microscopy (AFM). Enhanced binding was observed in cancer autoantibodies with the DNA modified by crotonaldehyde than the native counterpart. Immunological studies revealed enhanced binding of cancer autoantibodies with crotonaldehyde modified DNA, compared to the native form. Furthermore, lymphocyte DNA isolated from cancer patients demonstrated considerable recognition of anti-Cro-DNA IgG as compared to the DNA from healthy individuals. Therefore, we suggest that crotonaldehyde modified DNA presents unique epitopes, that may trigger autoantibody induction in cancer patients.

Peroxynitrite modified DNA presents better epitopes for anti-DNA autoantibodies in diabetes type 1 patients.

Peroxynitrite (ONOO(-)), formed by the reaction between nitric oxide (NO) and superoxide (O2(-)), has been implicated in the etiology of numerous disease processes. Peroxynitrite interacts with DNA via direct oxidative reactions or via indirect radical-mediated mechanism. It can inflict both oxidative and nitrosative damages on DNA bases, generating abasic sites, resulting in the single strand breaks. Plasmid pUC 18 isolated from Escherichiacoli was modified with peroxynitrite, generated by quenched flow process. Modifications incurred in plasmid DNA were characterized by ultraviolet and fluorescence spectroscopy, circular dichroism, HPLC and melting temperature studies. Binding characteristics and specificity of antibodies from diabetes patients were analyzed by direct binding and inhibition ELISA. Peroxynitrite modification of pUC 18 plasmid resulted in the formation of strand breaks and base modification. The major compound formed when peroxynitrite reacted with DNA was 8-nitroguanine, a specific marker for peroxynitrite induced DNA damage in inflamed tissues. The concentration of 8-nitroguanine was found to be 3.8 µM. Sera from diabetes type 1 patients from different age groups were studied for their binding to native and peroxynitrite modified plasmid. Direct binding and competitive-inhibition ELISA results showed higher recognition of peroxynitrite modified plasmid, as compared to the native form, by auto-antibodies present in diabetes patients. The preferential recognition of modified plasmid by diabetes autoantibodies was further reiterated by gel shift assay. Experimentally induced anti-peroxynitrite-modified plasmid IgG was used as a probe to detect nitrosative lesions in the DNA isolated from diabetes patients.

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.

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.

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.

Levels of anti-fructose-modified HSA antibodies correlate with disease status in diabetic subjects.

OBJECTIVE: This study aimed to assess the changes induced in HSA upon fructose-modification and to use the modified protein as an antigen for studying the presence of antibodies in diabetic patients. Further, magnitude of oxidative stress was also assessed. METHODS: HSA was modified with fructose, changes induced were studied by DSC measurements and near-UV CD. The binding characteristics of antibodies in the sera of diabetes patients to native and modified-HSA was assessed by ELISA and band shift assay. The oxidative stress in these patients was studied by carbonyl content estimation, FRAP assay and TBARS determination RESULTS: DSC revealed that fructose modified-HSA was more thermostable than its native form. Changes in tertiary structure of fructose-modified HSA were seen in near-UV CD. Patient studies showed that fructose-modified HSA acts as a potent immunogen compared to its native form and the levels of antibodies against fructose-modified HSA served as a parameter for tracking the glycemic control and oxidative stress parameters (carbonyl content, FRAP value and MDA level) in diabetic patients. CONCLUSIONS: Fructose-modification of HSA causes perturbations in its structure and function, thereby, making the protein antigenic besides decreasing its antioxidant capacity. This study suggests that fructose-modified-HSA is an important contributor in diabetic pathophysiology.

Role of peroxynitrite induced structural changes on H2B histone by physicochemical method.

Histones are small highly conserved cationic proteins which bind DNA and remain confined in the nucleus. These histones are quite vulnerable to oxidizing and nitrating agents. Peroxynitrite is a powerful oxidant and nitrating agent present in the biological system. In this study, peroxynitrite-induced nitration and oxidation of H2B was assessed by various physicochemical techniques. The carbonyl content and dityrosine were markedly elevated in peroxynitrite-modified H2B histone as compared to the native histone. Cross-linking of H2B was evident on polyacrylamide gel electrophoresis. 3-Nitrotyrosine was present only in peroxynitrite-modified H2B revealed by HPLC. The results showed that peroxynitrite-mediated nitration and oxidation in H2B histone exhibited hyperchromicity, decrease of tyrosine fluorescence accompanied by increase in ANS-binding specific fluorescence, loss of ß-sheet structure, appearance of new peak in FT-IR, increase in melting temperature and also loss of α-helix to produce a partially folded structure in comparison to intrinsically disordered structure of native H2B histone. We concluded that the H2B histone, a constituent of core histones, is highly sensitive to peroxynitrite and can adopt different structures under nitrosative and oxidative stress in order to protect the packaged DNA from the deleterious insult of peroxynitrite.

Immunological studies on peroxynitrite modified human DNA.

Peroxynitrite (ONOO(-)) is a strong and potent oxidizing and nitrating agent, formed by rapid reaction of two highly reactive, nitric oxide and superoxide anion. The action of peroxynitrite generated by synergistic action of diethylamine NONOate (a nitric oxide donor) and 1,4-hydroquinone (a superoxide donor), on human placental DNA was monitored by ultraviolet and fluorescence spectroscopy, melting temperature studies, S1 nuclease digestibility and alkaline agarose electrophoresis. The peroxynitrite modified human DNA (ONOO(-)-DNA) was found to be highly immunogenic in rabbits inducing high titre immunogen specific antibodies. However, the induced antibodies exhibited appreciable cross-reactivity with various polynucleotides and nucleic acids. The data demonstrate that the antibodies, though cross-reactive, preferentially bind ONOO(-)-modified epitopes on DNA. Visual detection of immune complex formation with native and ONOO(-)-DNA reiterated preferential binding with modified human DNA. DNA modified by ONOO(-) presents unique epitopes which may be one of the factors for the induction of autoantibodies in cancer patients.

Preferential recognition of peroxynitrite-modified human serum albumin by circulating autoantibodies in cancer.

Peroxynitrite is a potent oxidizing and nitrating agent and has in vivo existence. Several studies have shown the damaging role of this molecule in biological system. Human serum albumin (HSA), being most abundant plasma protein, is easily targeted by different oxidizing and nitrating agents. Free radicals increase the onset of different cancers as evident by several researchers. In the present study, structural perturbations in HSA by peroxynitrite were observed by MALDI-MS, DSC and DLS. Immunological studies showed enhanced binding of peroxynitrite-modified HSA with cancer autoantibodies, compared to the native protein. A decline in the antioxidant property of peroxynitrite-modified HSA was also observed. Therefore, we may conclude that peroxynitrite exposure results in structural alteration and hence generation of neo-epitopes in HSA molecule along with the decrease in its antioxidant property. The possible role of peroxynitrite-modified HSA in carcinogenesis has been discussed.

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.

Studies on peroxynitrite-modified H1 histone: implications in systemic lupus erythematosus.

Peroxynitrite is a powerful nitrating and oxidizing molecule and capable of modifying proteins' structure. Hyper-nitration of tyrosine residues has been seen in various pathological states, including autoimmune disorders like systemic lupus erythematosus (SLE) and rheumatoid arthritis. SLE, a chronic autoimmune disease, is primarily characterized by increased levels of autoantibodies, predominantly against ds-DNA. However, the initial antigenic stimulus for the disease etiopathogenesis has remained elusive. Carbonyl and nitrotyrosine have been extensively used as a biomarker of oxidative and nitrosative stress. In this study, commercially available H1 histone was exposed to increasing concentrations of peroxynitrite for 30 min. The peroxynitrite-mediated structural changes in histone were studied by ultraviolet & fluorescence spectroscopy, CD, HPLC, 1-anilinonaphthalene-8-sulfonic acid binding and polyacrylamide gel electrophoresis. Analysis of results revealed that carbonyl and nitrotyrosine contents were significantly increased in peroxynitrite-modified H1 compared to native H1. In experimental animal, peroxynitrite-modified H1 induced high titre antibodies as compared to native H1, and the immunogenicity was found to be directly proportional to nitrotyrosine content. Further, the induced antibodies showed specificity for the immunogen and appreciable cross-reactions with tyrosine rich nitrated proteins. Formation of high molecular weight immune complex with retarded mobility further supports the specificity of induced anti-peroxynitrite-modified H1 antibodies for the immunogen. Binding of SLE anti-DNA autoantibodies with peroxynitrite-modified H1 was analyzed by direct binding and competition ELISA. The data show preferential binding of SLE autoantibodies to peroxynitrite-modified H1 as compared to native H1 histone and native DNA. The results point towards the possible role of peroxynitrite-modified H1 histone in SLE etiopathogenesis.

Peroxynitrite modified DNA may be an antigenic trigger for antibodies in various cancers of gynecologic origin.

This study aimed towards probing the role of peroxynitrite damaged human DNA (ONOO(-)-DNA) in the induction of circulating antibodies in certain cancers of gynecologic origin. We have compared the binding specificity of DNA isolated from the lymphocytes of cancer patients with that of the experimentally modified DNA. Also, the induced anti-ONOO(-)-DNA antibodies have been used to probe oxidative damage in the DNA isolated from cancer patients. Human placental DNA was modified with peroxynitrite (ONOO(-)) and analyzed by ultraviolet (UV) and fluorescence spectroscopy, gel electrophoresis, thermal denaturation profile, etc. Antibodies against modified DNA were induced in experimental animals. Specific binding of the antibodies was evaluated by ELISA and band shift assay. 91 cancer patients were selected and grouped according to the type of cancer. Specific binding characteristics of circulating autoantibodies (IgG) were determined by competitive-inhibition ELISA, using different inhibitors. Maximum inhibition of antibody activity by ONOO(-)-DNA reflected specific recognition of modified epitopes by cancer IgG. This shows generation of neo-epitopes on DNA, upon modification with ONOO(-), that are recognized by cancer IgG. Our results indicate epitope sharing between the DNA isolated from cancer patients and the in-vitro modified ONOO(-)-DNA. The possible role of nitrosative stress in the gynecologic oncology has been discussed.

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.

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.

Physicochemical analysis of structural changes in DNA modified with glucose.

Reactions of reducing sugars with free amino groups of proteins can form advanced glycation end products (AGEs). While the formation of nucleoside AGEs has been studied in detail, no extensive work has been carried out to assess DNA Amadori and DNA advanced glycation end products. In this study, we report biophysical/chemical characterization of glucose-induced changes in DNA, as well as DNA Amadori and DNA advanced glycation end products. Glucose treated DNA exhibited hyperchromicity, decrease in melting temperature, and enhanced emission intensity in a time dependent manner. Formation of DNA Amadori product and DNA advanced glycation end products, mainly CEdG (N²-carboxyethyl-2'-deoxyguanosine), were the major outcome of the study.

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-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.

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.