Characterization of Human Glycated DNA modified with Peroxynitrite

Author Name: Aadil Wani, Shaheena Mushtaq, Haseeb Ahsan , *Rizwan Ahmad
Author Email: ahmadriz.biochem@gmail.com

Abstract

Glycation is the non-enzymatic addition or insertion of sugar molecules into DNA, proteins and lipids that occur in biological molecules. The glycation of DNA gives rise to characteristic nucleotide adducts, some of which have been found to increase in oxidative stress. The glycation causes damage to DNA which is associated with mutagenesis, carcinogenesis and is also considered to be a pathogenic factor for diabetes mellitus. The present study was designed to evaluate the influence of glucose on DNA isolated from human blood and its modification with peroxynitrite. Modified, glycated DNA was characterized by UV spectroscopy, melting temperature and electrophoretic profile. Oxidation incurred by glycation of DNA and peroxynitrite was also analyzed by scavenging studies. In the present study peroxynitrite of glycated DNA leads to damage which may be due to nitration, deamination along with the formation of strand break and mutation. The modified DNA showed 18% hyperchromicity effect with the peak shift of approximately 5 nm to higher wavelength which may be due to alteration in normal helical structure and exposure of bases causes marked absorption in the UV range. Agarose gel electrophoresis clearly demonstrates that the instability in β-helix is as a result of peroxynitrite modification of glycated DNA. Additional evidence for the structural perturbation in DNA were further analyzed by thermal denaturation study. Peroxynitrite modification showed significant decrease in Tm which could be attributed to single strand generation.

Keywords

glycated DNA, peroxynitrite, human DNA

Introduction

Glycation is the result of a sugar molecule, such as fructose or glucose binding to a protein, nucleic acids or lipid molecule without the controlling action of an enzyme sometimes called non-enzymatic glycosylation [1, 2]. Enzyme-controlled addition of sugars to protein or lipid molecules is termed as glycosylation [2]. Advanced glycated end products are molecules formed from the non-enzymatic reaction of reducing sugars with free amino group of proteins lipids and nucleic acids [3].
The non-enzymatic reaction between reducing sugar and free amino group on DNA bases results in the formation of glycation and advanced glycation end products (AGEs). The cross linking of genetic material to glucose is one of the fundamental lesions produced in biological systems. The glycated form of DNA is a potent immunogen and may induce harmful immune response. The presence of AGE’s on DNA causes unusual transpositional rearrangements. The AGE’s contributes in the development of arthrosclerosis, kidney, vascular and neurological diseases in both diabetes and the aging process. The glycated substances may also be involved in the pathogenesis of Alzheimer’s disease. Depletion of cellular antioxidant GSH leads to an increase in the binding of glucose derivatives to DNA. DNA can be cross linked with different substances, some of the nucleotide AGE’s are N2-carboxymethyl oxyguanosine, 5-glycolyl deoxycytidine etc [4].
Previously, it was suggested that glycation of DNA resulting in the form of nucleotide AGEs is associated with increase in mutation frequency and cytotoxicity [4]. DNA glycation may contribute to the toxicity of several clinical cytotoxic anti-tumor agents. Over expression of enzymatic anti-glycation defence is associated with multi drug resistance in major classes of tumors. Improved understanding of DNA glycation may give guidance on decreasing the risk of tumor associated with dietary factors [4].
Peroxynitrite is reactive nitrogen species, formed by rapid reaction of highly reactive nitric oxide and superoxide anion (O2-.). Peroxynitrite is a mutagenic agent, with the potential to produce nitration, nitrosation and deamination reaction on DNA bases. Due to the oxidizing property of peroxynitrite, it can damage a wide array of biomolecules in cells including DNA, lipids, proteins which lead to the oxidative stress. Peroxynitrite is a potent initiator of DNA strand breakage which is an obligatory stimulus for activation of nuclear enzyme, poly-ADP ribosyl synthase or polymerase (PARP)
excess activation of PARP can kill cell by depleting NAD+ and energy production which may be one of the mechanism by which cells with excess DNA damage are eliminated (which undergoes necrosis or apoptosis) [5, 6].
To counter the harmful effects of reactive species antioxidant defense mechanism operates to detoxify or scavenge these reactive species. Antioxidants are able to neutralize free radical and render them harmless. It has been suggested that the extent of damage caused by the free radical might be modified by three dietary intervention strategies: a) caloric restriction and thus a depression in free radicals arising due to normal metabolism; b) minimizing the intake of components that increase the free radicals such as poly-unsaturated fatty acids; c) supplementation with one or more antioxidants in diet.
A wide array of enzymatic antioxidants defence exists, including superoxide dismutase, glutathione reductase, catalase etc. The present study is designed to evaluate the influence of glucose on DNA isolated from human blood and its modification with peroxynitrite. Modified, glycated DNA was characterized by UV-spectroscopy, NBT reduction test, hydroxy radical, superoxide radical assay, melting temperature and electrophoretic profile. Oxidation incurred by glycation of DNA and peroxynitrite was also analyzed by scavenging studies.

Conclusion

a) Isolated genomic DNA showed A260/280 ratio of 1.7 and a sharp band on agarose gel electrophoresis.
b) The Glycation of DNA causes formation of nucleotide AGEs leading to the alteration in DNA analyzed by UV spectra and thermal denaturation characteristics.
c) Amadori product content estimation confirmed the AGEs induced modification of DNA.
d) Peroxynitrite induced damage to glycated DNA was analyzed by UV spectra and decrease in melting temperature.
e) Scavenging effect of GSH provide clue for the generation of free radical during glycation.

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