DNA Glycation Research Articles

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.

Mutagenesis and Repair Induced by the DNA Advanced Glycation End Product N2-1-(Carboxyethyl)-2′-deoxyguanosine in Human Cells

Glycation of biopolymers by glucose-derived α-oxo-aldehydes such as methylglyoxal (MG) is believed to play a major role in the complex pathologies associated with diabetes and metabolic disease. In contrast to the extensive literature detailing the formation and physiological consequences of protein glycation, there is little information about the corresponding phenomenon for DNA. To assess the potential contribution of DNA glycation to genetic instability, we prepared shuttle vectors containing defined levels of the DNA glycation adduct N(2)-(1-carboxyethyl)-2'-deoxyguanosine (CEdG) and transfected them into isogenic human fibroblasts that differed solely in the capacity to conduct nucleotide excision repair (NER). In the NER-compromised fibroblasts, the induced mutation frequencies increased up to 18-fold relative to background over a range of ∼10-1400 CEdG adducts/10(5) dG, whereas the same substrates transfected into NER-competent cells induced a response that was 5-fold over background at the highest adduct density. The positive linear correlation (R(2) = 0.998) of mutation frequency with increasing CEdG level in NER-defective cells suggested that NER was the primary if not exclusive mechanism for repair of this adduct in human fibroblasts. Consistent with predictions from biochemical studies using CEdG-substituted oligonucleotides, guanine transversions were the predominant mutation resulting from replication of MG-modified plasmids. At high CEdG levels, significant increases in the number of AT → GC transitions were observed exclusively in NER-competent cells (P < 0.0001). This suggested the involvement of an NER-dependent mutagenic process in response to critical levels of DNA damage, possibly mediated by error-prone Y-family polymerases.

Oxidative damage to DNA and lipids: correlation with protein glycation in patients with type 1 diabetes

Diabetic hyperglycemia is associated with increased production of reactive oxygen species (ROS). ROS reacts with DNA resulting in various products, such as 8-hydroxydeoxyguanosine (8-OHdG), that excrete in urine owing to DNA repair processes. Urinary 8-OHdG has been proposed as an indicator of oxidative damage to DNA. This study aimed to evaluate relationship between oxidative damage to DNA and protein glycation in patients with Type 1 diabetes. We measured urinary 8-OHdG level in diabetic patients and healthy subjects and discussed its relationship to glycated hemoglobin (HbA(1c)) and glycated serum protein (GSP) levels. Furthermore plasma malondialdehyde (MDA) level monitored as an important indicator of lipid peroxidation in diabetes. We studied 32 patients with Type 1 diabetes mellitus and compared the measured factors with those of 48 age-matched nondiabetic controls. GSP and MDA were measured bycolorimetric assay. Urinary 8-OHdG measurement was carried out using ELISA. In this study urinary 8-OHdG, HbA(1c), plasma MDA, and GSP levels were progressively higher in diabetics than in control subjects (P<0.05). Furthermore we found significant correlation between urinary 8-OHdG and HbA(1c) (P<0.05) in diabetic group. Correlation between fasting blood sugar and GSP were significant. We also found significant correlation between fasting blood sugar and MDA. This case-control study in young diabetic patients showed increased blood glucose and related metabolic disorders result in oxidative stress and oxidative damage to DNA and lipids. Furthermore oxidative damage to DNA is associated to glycemic control level, whereas lipid peroxidation level was not significantly correlated with glycemic control level. J. Clin. Lab. Anal. 24:72-76, 2010. (c) 2010 Wiley-Liss, Inc.

Mutagenic Potential of DNA Glycation: Miscoding by (R)- and (S)-N2-(1-Carboxyethyl)-2′-deoxyguanosine

Elevated circulating glucose resulting from complications of obesity and metabolic disease can result in the accumulation of advanced glycation end products (AGEs) of proteins, lipids, and DNA. The formation of DNA-AGEs assumes particular importance as these adducts may contribute to genetic instability and elevated cancer risk associated with metabolic disease. The principal DNA-AGE, N(2)-(1-carboxyethyl)-2'-deoxyguanosine (CEdG), is formed as a mixture of R and S isomers at both the polymer and monomer levels. In order to examine the miscoding potential of this adduct, oligonucleotides substituted with (R)- and (S)-CEdG and the corresponding triphosphates (R)- and (S)-CEdGTP were synthesized, and base-pairing preferences for each stereoisomer were examined using steady-state kinetic approaches. Purine dNTPs were preferentially incorporated opposite template CEdG when either the Klenow (Kf(-)) or Thermus aquaticus (Taq) polymerases were used. The Kf(-) polymerase preferentially incorporated dGTP, whereas Taq demonstrated a bias for dATP. Kf(-) incorporated purines opposite the R isomer with greater efficiency, but Taq favored the S isomer. Incorporation of (R)- and (S)-CEdGTP only occurred opposite dC and was catalyzed by Kf(-) with equal efficiencies. Primer extension from a 3'-terminal CEdG was observed only for the R isomer. These data suggest CEdG is the likely adduct responsible for the observed pattern of G transversions induced by exposure to elevated glucose or its alpha-oxoaldehyde decomposition product methylglyoxal. The results imply that CEdG within template DNA and the corresponding triphosphate possess different syn/anti conformations during replication which influence base-pairing preferences. The implications for CEdG-induced mutagenesis in vivo are discussed.

Effect of Acetyl Salicyilic Acid on Glycation and Mutability of Escherichia Coli Chromosomal DNA

ABSTRACTRecently, we have shown that the process of non-enzymatic glycosylation (glycation) affects Escherichia coli chromosomal DNA under physiological conditions in vivo. We have further demonstrated that chromosomal DNA glycation is a source of spontaneous mutations in E. coli. The present study reveals that inhibition of glycation does not necessarily lead to suppression of DNA mutability. We tested the effect of acetyl salicylic acid (ASA) on chromosomal DNA glycation and spontaneous mutagenesis in E. coli. At a concentration of 0.1 mM in the growth medium ASA proved to be non-toxic and inhibits significantly glycation of E. coli chromosomal DNA. At a concentration of 5 mM ASA partially suppressed bacterial growth but had negligible impact on DNA glycation. Most importantly, at both concentrations tested ASA caused an increase in the frequency of spontaneous reversions from Arg− to Arg+ phenotype. In addition, we observed an accelerated hydrolysis of DNA in ASA solutions as compared to that in solutions of hydrochloric acid at the same pH. The latter result suggests a possible interaction of ASA with DNA, which explains the observed genotoxicity of ASA.

Associated links among mtDNA glycation, oxidative stress and colony sectorization in Metarhizium anisopliae

Mycelial colonies of filamentous fungi often deteriorate when maintained on artificial media, and this can take the form of sterile sectors. We previously established that sectorization by the entomopathogenic fungus Metarhizium anisopliae correlates with intracellular accumulation of reactive oxygen species (ROS). In this study we demonstrate that: (1) H 2O 2 increases rates of sectorization; (2) a stable strain of M. anisopliae eliminates intracellular ROS more rapidly than an unstable strain; (3) mitochondrial DNA from sectors undergoes a non-enzymatic glycation of deoxyguanosine that is not shown by genomic DNA; (4) the membrane potential of mitochondria in sector cells is decreased in comparison to wild type cells indicating loss of function; (5) DNA glycation changes the properties of DNA and (6) treating wild type mycelia with H 2O 2 reproduced the glycation pattern shown in sectors. H 2O 2 also reproduced the morphological changes in mitochondria and lipid droplets that occur in sector cells. Fungal sectorization thus displays aging related developmental impairments resulting from oxidative stress, suggesting a new research direction for studies on fungal colony deterioration. Mitochondrial DNA has a very high AT bias. We speculate that reducing the consequences of glycation could provide an adaptive reason for this.

Analysis and biological relevance of advanced glycation end‐products of DNA in eukaryotic cells

Advanced glycation end-products (AGEs) of DNA are formed spontaneously by the reaction of carbonyl compounds such as sugars, methylglyoxal or dihydroxyacetone in vitro and in vivo. Little is known, however, about the biological consequences of DNA AGEs. In this study, a method was developed to determine the parameters that promote DNA glycation in cultured cells. For this purpose, the formation rate of N2-carboxyethyl-2'-deoxyguanosine (CEdG), a major DNA AGE, was measured in cultured hepatic stellate cells by liquid chromatography (LC)-MS/MS. In resting cells, a 1.7-fold increase of CEdG formation rate was observed during 14 days of incubation. To obtain insights into the functional consequences of DNA glycation, CEdG was introduced into a luciferase reporter gene vector and transfected into human embryonic kidney (HEK 293 T) cells. Gene activity was determined by chemiluminescence of the luciferase. Thus, CEdG adducts led to a dose-dependent and highly significant decrease in protein activity, which is caused by loss of functionality of the luciferase in addition to reduced transcription of the gene. When the CEdG-modified vector was transformed into Escherichia coli, a loss of ampicillin resistance was observed in comparison to transformation with the unmodified plasmid. These results indicate that CEdG accumulates in the genomic DNA of resting cells, which could lead to diminished protein activity.

Detection of DNA‐bound advanced glycation end‐products by immunoaffinity chromatography coupled to HPLC‐diode array detection

Sugars and sugar degradation products are formed during food processing, but also endogenously in vivo. In vitro, nucleosides and DNA react readily with these carbonyl compounds during the formation of the two diastereomers of N(2)-carboxyethyl-2'-deoxyguanosine (CEdG(A,B)), leading to a loss of DNA integrity. Only little is known about DNA glycation in vivo and about the influence of nutrition on CEdG formation. In this study, we developed a sensitive method to analyze DNA glycation by HPLC. For this purpose, immunoaffinity chromatography (IAC) using a polyclonal antibody against N(2)-carboxyethylguanine (CEguanine) was coupled to HPLC-DAD. In some samples, peak identity was confirmed by LC-MS/MS. The recovery of CEguanine from the IAC columns was 52.5% +/- 3.6 (n = 4). Thus, it was possible for the first time to detect CEdG(A,B), N(2)-carboxyethylguanosine (CEG(A,B)), and CEguanine in 11 human urine samples. However, due to imprecision of IAC, valid quantification of the adducts could not be achieved. Furthermore, CEdG was also detected in the DNA of cultured human smooth muscle cells (SMCs) and bovine aorta endothelium cells (BAECs). In BAECs, CEdG(A,B) were found by HPLC-DAD and LC-MS/MS after immunoaffinity purification, whereas in SMCs DNA-advanced glycation end-products were only detected with the more sensitive LC-MS/MS method.

Exploring the antioxidant property of bioflavonoid quercetin in preventing DNA glycation: A calorimetric and spectroscopic study

Reducing sugars for example glucose, fructose, etc., and their phosphate derivatives non-enzymatically glycate biological macromolecules (e.g., proteins, DNA and lipids) and is related to the production of free radicals. Here we present a novel study, using differential scanning calorimetry (DSC) along with UV/Vis absorption and photon correlation spectroscopy (PCS), on normal and glycated human placenta DNA and have explored the antioxidant property of the naturally occurring polyhydroxy flavone quercetin (3,3′,4′,5,7-pentahydroxyflavone) in preventing the glycation. The decrease in the absorption intensity of DNA in presence of sugars clearly indicates the existence of sugar molecules between the two bases of a base pair in the duplex DNA molecule. Variations were perceptible in the PCS relaxation profiles of normal and glycated DNA. The melting temperature of placenta DNA was decreased when glycated suggesting a decrease in the structural stability of the double-stranded glycated DNA. Our DSC and PCS data showed, for the first time, that the dramatic changes in the structural properties of glycated DNA can be prevented to a significant extent by adding quercetin. This study provides valuable insights regarding the structure, function, and dynamics of normal and glycated DNA molecules, underlying the manifestation of free radical mediated diseases, and their prevention using therapeutically active naturally occurring flavonoid quercetin.

Evidence for non‐enzymatic glycosylation of Escherichia coli chromosomal DNA

We have recently shown that the process of non-enzymatic glycosylation (glycation) takes place in Escherichia coli under physiological conditions and affects both recombinant and endogenous bacterial proteins. In this study, we further demonstrate that E. coli chromosomal DNA is also subjected to glycation under physiological growth conditions. The E. coli DNA accumulates early glycation (Amadori) products as proven by the nitroblue tetrazolium (NBT) reduction assay. It showed also immunoreactivity to a monoclonal antibody raised against N(in)-(carboxymethyl)lysine and fluorescent properties indicative of modifications with advanced glycation end-products. Two types of fluorophores were detected in the E. coli DNA with excitation maxima at 360 nm and 380 nm and emission maxima at 440 nm and 410 nm. Using the NBT reduction assay, fluorescence spectroscopy and enzyme-linked immunosorbent assay we revealed that glycation adducts accumulate in DNA predominantly in the stationary phase of growth, although they could be detected also in exponential-phase cells. Besides on the growth phase, the extent of DNA glycation depends also on the nutrient broth composition being more extensive in rich media. Thiamine was found to inhibit both DNA glycation and spontaneous point mutations as judged by the decreased rate of the argE3 to Arg(+) reversions in the E. coli strain AB1157.

Protecting the genome: defence against nucleotide glycation and emerging role of glyoxalase I overexpression in multidrug resistance in cancer chemotherapy.

Glycation of nucleotides in DNA forms AGEs (advanced glycation end-products). Nucleotide AGEs are: the imidazopurinone derivative dG-G [3-(2'-deoxyribosyl)-6,7-dihydro-6,7-dihydroxyimidazo[2,3-b]purin-9(8)one], CMdG ( N (2)-carboxymethyldeoxyguanosine) and gdC (5-glycolyldeoxycytidine) derived from glyoxal, dG-MG [6,7-dihydro-6,7-dihydroxy-6-methylimidazo-[2,3-b]purine-9(8)one], dG-MG(2) [ N (2),7-bis-(1-hydroxy-2-oxopropyl)deoxyguanosine] and CEdG [ N (2)-(1-carboxyethyl)deoxyguanosine] derived from methylglyoxal, and dG-3DG [ N (2)-(1-oxo-2,4,5,6-tetrahydroxyhexyl)deoxyguanosine] derived from 3-deoxyglucosone and others. Glyoxal and methylglyoxal induce multi-base deletions, and base-pair substitutions - mostly occurring at G:C sites with G:C-->C:G and G:C-->T:A transversions. Suppression of nucleotide glycation by glyoxalase I and aldehyde reductases and dehydrogenases, and base excision repair, protects and recovers DNA from damaging glycation. The effects of DNA glycation may be most marked in diabetes and uraemia. Mutations arising from DNA glycation may explain the link of non-dietary carbohydrate intake to incidence of colorectal cancer. Overexpression of glyoxalase I was found in drug-resistant tumour cells and may be an example of an undesirable effect of the enzymatic protection against DNA glycation. Experimental overexpression of glyoxalase I conferred resistance to drug-induced apoptosis. Glyoxalase I-mediated drug resistance was found in human leukaemia and lung carcinoma cells. Methylglyoxal-mediated glycation of DNA may contribute to the cytotoxicity of some antitumour agents as a consequence of depletion of NAD(+) by poly(ADP-ribose) polymerase, marked increases in triosephosphate concentration and increased formation of methylglyoxal. S - p -Bromobenzylglutathione cyclopentyl diester is a cell-permeable glyoxalase I inhibitor. It countered drug resistance and was a potent antitumour agent against lung and prostate carcinoma. Glyoxalase I overexpression was also found in invasive ovarian cancer and breast cancer.

Whole-body metabolic rate appears to determine the rate of DNA oxidative damage and glycation involved in aging

While aging has been found to be a multifactorial process, it seems logical that different aging parameters which reflect the deleterious effects of normal basal metabolism should be directly related. Three such putative aging parameters were therefore measured in adult male Fischer 344 rats on three different long-term diets which have been shown to yield different lifespans. It was found that the daily caloric intake per unit organ weight, a measure of whole-body metabolic rate, was directly proportional to: (1) the level of 8-hydroxydeoxyguanosine in skin dermal cells, used as a measure of the rate of DNA oxidative damage; (2) the proportion of hemoglobin that was glycated, used as a measure of the rate of glycation. This appears to be the first evidence suggesting that whole-body metabolic rate plays a role in determining both the rate of DNA oxidative damage and the rate of glycation involved in aging, because whole-body metabolic rate was the only one of these three variables manipulated in the study. The study also found that there were no significant between-group differences in brain, kidney and liver 8-hydroxydeoxyguanosine, suggesting that DNA oxidative damage in non-mitotic and slow-dividing cells is not a reliable linear biomarker of aging.

N2-(1-Carboxyethyl)deoxyguanosine, a Nonenzymatic Glycation Adduct of DNA, Induces Single-Strand Breaks and Increases Mutation Frequencies

N2-(1-Carboxyethyl)deoxyguanosine (CEdG) is a major nonenzymatic glycation product of DNA. The effect of CEdG modification, which was specifically prepared by incubation with dihydroxyacetone, on plasmid DNA topology was evaluated by gel electrophoresis. A time-dependent decrease of supercoiled plasmid–DNA was observed in parallel to the increase of CEdG adducts; the half-life time of the supercoiled plasmid–DNA was estimated to be approximately 16–18 h. CEdG-modified plasmid DNA showed a 25-fold reduced transformation efficiency. When modified DNA was used to transform Escherichia coli cells, a 6-fold increase in mutation frequency was determined by measuring loss of alpha-complementation. For the mutator strain BMH71-18mutS, an 8-fold increase in mutation frequency was observed. Although the exact mechanism of DNA damage is unclear, the occurrence of spontaneous depurination is likely. These findings suggest that a defined DNA glycation reaction can lead to DNA damage in vivo.

Genetic and Environmental Modulations of Chromosomal Stability: Their Roles in Aging and Oncogenesis

The primary objective of this review is to suggest a major role of cytogenetic pathology in the genesis of both the neoplastic phenotype and the senescent phenotype of aging mammalian organisms. It is hypothesized that allelic variation at a number of genetic loci that have the potential to modulate various types of chromosomal mutation could account, in part, for species-specific variations in maximum life-span potentials and the times of onset of characteristic age-related neoplasms. As a corollary to this proposition, attention is directed to the potential importance of environmental clastogens and aneugens as both oncogenic and "gerontogenic" agents. By way of introduction, a series of definitions of our subject material is given (aging, oncogenesis, proliferative homeostasis, point mutation, chromosomal mutation, epimutation, mutagens, clastogens, aneugens, gerontogens). There follows a defense of the view that there is a tight coupling of abnormalities in proliferative homeostasis (including atrophies, hyperplasias, benign neoplasias and malignant neoplasias) with intrinsic biological aging. This view differs from those of other authors who either: 1) believe that, to qualify as a bona fide component of aging, the phenotype in question (in this case, neoplasia) must be "genetically programmed" or 2) insist that the rate of development of the neoplastic phenotype should parallel species-specific Gompertz kinetics for the survival of the population of organisms. After briefly reviewing the classification of chromosomal lesions, we consider evidence for constitutional genetic determinants of chromosomal stability, oncogenesis, and some other aspects of the senescent phenotype, both with respect to interspecific and intraspecific differences. The discussion will include the recent evidence characterizing a human segmental progeroid syndrome (Werner's syndrome) as a deletor mutator strain. A summary of research on environmental chemical clastogens and aneugens is given, showing both discordances and concordances of such assays with assays for point mutagens. An analysis of the literature indicates that there is no statistical evidence for the positive correlations when substances showing effects only at comparatively high concentrations (greater than 1 mM) are excluded from the analysis. Brief mention is also made of the roles of viral agents and ionizing radiation in the genesis of chromosomal mutations. Finally, some possible pathogenetic mechanisms common to chromosomal mutagenicity, oncogenicity and gerontogenicity are considered, including chemical free radicals (active oxygen species), DNA transposition, DNA amplification, DNA glycation, virally induced cell fusion, posttranslational modifications of centromeric and mitotic spindle proteins, and alterations in the metabolism of telomeric DNA.