Uzma Shahab

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.

Glycoxidation of biological macromolecules: A critical approach to halt the menace of glycation

Glycation is the result of covalent bonding of a free amino group of biological macromolecules with a reducing sugar, which results in the formation of a Schiff base that undergoes rearrangement, dehydration and cyclization to form a more stable Amadori product. The final products of nonenzymatic glycation of biomacromolecules like DNA, proteins and lipids are known as advanced glycation end products (AGEs). AGEs may be generated rapidly or over long times stimulated by distinct triggering mechanisms, thereby accounting for their roles in multiple settings and disease states. Both Schiff base and Amadori glycation products generate free radicals resulting in decline of antioxidant defense mechanisms and can damage cellular organelles and enzymes. This critical review primarily focuses on the mechanistic insight of glycation and the most probable route for the formation of glycation products and their therapeutic interventions. Furthermore, the prevention of glycation reaction using therapeutic drugs such as metformin, pyridoxamine and aminoguanidine (AG) are discussed with special emphasis on the novel concept of the bioconjugation of these drugs like, AG with gold nanoparticles (GNPs). At or above 10 mM concentration, AG is found to be toxic and therefore has serious health concerns, and the study warrants doing this novel bioconjugation of AG with GNPs. This approach might increase the efficacy of the AG at a reduced concentration with low or no toxicity. Using the concept of synthesis of GNPs with abovementioned drugs, it is assumed that toxicity of various drugs which are used at high doses can be minimized more effectively.

Inhibitory Effect of Metformin and Pyridoxamine in the Formation of Early, Intermediate and Advanced Glycation End-Products

Background Non-enzymatic glycation is the addition of free carbonyl group of reducing sugar to the free amino groups of proteins, resulting in the formation of a Schiff base and an Amadori product. Dihydroxyacetone (DHA) is one of the carbonyl species which reacts rapidly with the free amino groups of proteins to form advanced glycation end products (AGEs). The highly reactive dihydroxyacetone phosphate is a derivative of dihydroxyacetone (DHA), and a product of glycolysis, having potential glycating effects to form AGEs. The formation of AGEs results in the generation of free radicals which play an important role in the pathophysiology of aging and diabetic complications. While the formation of DHA-AGEs has been demonstrated previously, no extensive studies have been performed to assess the inhibition of AGE inhibitors at all the three stages of glycation (early, intermediate and late) using metformin (MF) and pyridoxamine (PM) as a novel inhibitor. Methodology/Principal Findings In this study we report glycation of human serum albumin (HSA) & its characterization by various spectroscopic techniques. Furthermore, inhibition of glycation products at all the stages of glycation was also studied. Spectroscopic analysis suggests structural perturbations in the HSA as a result of modification which might be due to generation of free radicals and formation of AGEs. Conclusion The inhibition in the formation of glycation reaction reveals that Pyridoxamine is a better antiglycating agent than Metformin at all stages of the glycation (early, intermediate and late stages).

Bio-physical characterization of ribose induced glycation: a mechanistic study on DNA perturbations.

The non-enzymatic addition of carbonyl group of sugar moiety to the amino group of DNA macromolecule leads to the formation of early glycation products (Amadori products) which undergoes rearrangement, cyclization and dehydration to form advanced glycation end products (AGEs). While the formation of glucose derived glycated DNA has been previously demonstrated, however no extensive studies have been performed to assess the glycation of DNA using D-ribose as glycating agent. D-ribose, an important monosaccharide, is a highly reactive pentose sugar which results in the rapid formation of AGEs. To the best of our knowledge, this is the first study to characterize the d-ribose-induced changes in calf thymus DNA, as well as calf thymus DNA Amadori and calf thymus DNA-AGEs. The main objective of the study is to investigate the non-enzymatic glycation of calf thymus DNA by using different concentrations of d-ribose at increasing time period. The obtained Amadori products, AGEs were characterized with respect to the extent of DNA strand break and base modifications. Additionally, their nitroblue tetrazolium (NBT) reduction assay, absorbance, agarose gel electrophoresis, fluorescence, circular dichroism (CD) and thermal denaturation (Tm) characteristics were extensively studied. We found significant changes in the modification of DNA and in AGE-specific fluorescence, using different concentration of modifiers (D-ribose). The results provide the mechanistic insight of D-ribose induced glycation in calf thymus DNA. The bio-availability of D-ribose makes this carbonyl species quite reactive and damaging, therefore having direct implication in diabetes. This is the preliminary study done on D-ribose glycation and warrants further study to probe the role ribose-DNA glycation in different disease state including diabetes.

Studies on glycation of human low density lipoprotein: A functional insight into physico-chemical analysis

Glycation of biomolecules leads to the establishment of advanced glycation end products (AGEs), which have significant role in the pathophysiology of diabetes leading to disorders, such as atherosclerosis. Ribose appears to be the most reactive among the naturally occurring sugars and contribute significantly to the glycation reactions in vivo, however, no report has been published to discuss D-ribose induced glycation of low density lipoprotein (LDL). The present study initially focuses on the glycation of commercially available LDL using different concentrations of D-ribose for varied incubation time intervals. The structural perturbations induced in LDL were analyzed by UV-vis, fluorescence & circular dichroism spectroscopy, and thermal denaturation studies. The ketoamine moieties, carbonyl content and HMF content were also quantitated in native and glycated LDL. We report structural perturbations, increased carbonyl content, ketoamine moieties and HMF content in D-ribose induced glycated LDL (LDL-AGEs) as compared to native analogue. These results provide evidence that LDL-AGEs could interfere in normal physiological functions and might contribute in the initiation of diabetes induced atherosclerosis and other secondary cardiovascular problems in diabetic patients. Though ours is a preliminary study, we are sure it would open the topic for further studies.

Immunogenicity of DNA-advanced glycation end product fashioned through glyoxal and arginine in the presence of Fe3+: Its potential role in prompt recognition of diabetes mellitus auto-antibodies

Glyoxal, methylglyoxal and 3-deoxyglucosones are reactive dicarbonyl compounds, which transform free amino groups of proteins and lipoproteins macromolecule into advanced glycation end-products (AGEs). AGEs play a significant role in the pathophysiology of aging and diabetic complications because of their genotoxic effect. Glyoxal also reacts with free amino group of nucleic acids resulting in the formation of DNA-AGEs. The present study reports the genotoxicity and immunogenicity of AGEs formed by Glyoxal-Arginine-Fe(3+) (G-Arg-Fe(3+)) system as a glycating agent. Immunogenicity of native and G-Arg-Fe(3+)-DNA was probed in female rabbits. Immunofluorescence suggests the presence of immune complex deposition in the kidney section of immunized rabbits. Spectroscopic analysis and melting temperature indicates the structural modification in the human DNA. The modified human DNA is found to be highly immunogenic, whereas unmodified form was simply non-immunogenic. This study shows the presence of auto-antibodies against G-Arg-Fe(3+) modified human DNA in the sera of diabetes type 1 and in few cases type 2 patients due to secondary complications of nephropathy. The glyco-oxidative lesions have also been detected in the lymphocyte DNA isolated from patients having type 1 and type 2 diabetes. The results show structural perturbations generating new epitopes in G-Arg-Fe(3+)-DNA rendering it pretty immunogenic.

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.

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.

Recent advances in detection of AGEs: Immunochemical, bioanalytical and biochemical approaches

Advanced glycation end products (AGEs) are a cohort of heterogeneous compounds that are formed after the nonenzymatic glycation of proteins, lipids and nucleic acids. Accumulation of AGEs in the body is implicated in various pathophysiological conditions like diabetes, cardiovascular diseases and atherosclerosis. Numerous studies have reported the connecting link between AGEs and the various complications associated with diseases. Hence, detection and measurement of AGEs becomes centrally important to understand and manage the menace created by AGEs inside the body. In recent years, an increasing number of immunotechniques as well as bioanalytical techniques have been developed to efficiently measure the levels of AGEs, but most of them are still far away from being clinically consistent, as relative disparity and ambiguity masks their standardization. This article is designed to critically review the recent advances and the emerging techniques for detection of AGEs. It is an attempt to summarize the major techniques that exist currently for the detection of AGEs both qualitatively and quantitatively. This review primarily focuses on the detection and quantification of AGEs which are formed in vivo. Immunochemical approach though costly but most effective and accurate method to measure the level of AGEs. Literature review suggests that detection of autoantibody targeting AGEs is a promising way that can be utilized for detection of AGEs. Future research efforts should be dedicated to develop this method in order to push forward the clinical applications of detection of AGEs.