Study of Genetic Susceptibility to Oxidative Stress and DNA Damage Repair Gene in Male Brick Kiln Workers

Abstract

A DNA damaging repair gene, 8-Oxo-Guanine Glycosylase 1 (hOGG1), encodes glycosylase enzyme with involvement of inflammatory physiological pathways, plays crucial roles in protecting the body from cancer progression and oxidative pathologies. DNA glycosylases fix the DNA damage and remove oxidized base (8-Oxo-G) through a mechanism called base excision repair pathway (BER). The repair action of glycosylases is highly sensitive to heavy metals because they have an ability to bind sulphur group of cysteine residues and form dimer shapes to destabilize the original configuration of DNA and proteins. Literature has reported that variations in exon 7, especially transversion of C>G at codon 326 of hOGG1 gene, is responsible for the inactivation of DNA glycosylase repair activity. Heavy metals are one of the major factors for occurrence of DNA base transversions and genetic polymorphism. The brick kiln industry releases heavy metals at appreciably higher concentration. Number of studies reported that heavy metals from the brick kiln industry induces down-regulation of DNA repair genes in occupational workers leading to DNA damage and oxidative stress. They have the potential to alter redox balance by generation of Reactive oxygen species (ROS) inside cells, which causes genotoxic effects inside the body. The present study aims to elucidate the relation of hOGG1 down regulated expression profile with single nucleotide polymorphism in exon 7, at codon 326; locus rs1052133 of hOGG1 gene located at chromosome 3p25. For this purpose, blood samples (n=20) from brick kiln emissions exposed workers were collected. All disease and health related profiles of workers were noted in a pre designed questionnaire. DNA extraction from blood samples were carried out using phenol chloroform method. 1% gel electrophoresis confirmed the extraction of genomic DNA. Primer-3 software https://primer3.ut.ee/ was used to design primers for amplification of hOGG1 gene to analyse variation at exon 7 of hOGG1 gene. The general conditions, including annealing temperature, size of amplicon, concentration of salt and length of primers were optimized. The Ensemble website: http://asia.ensemblr.org/Homo_sapiens/Info/Index provided the reference sequence that was essential for building primers. Specificity of the chosen primers was confirmed through BLAT (Blast Like Alignment Tool) on the UCSC genome browser and the In-Silico PCR tool. Polymerase chain reaction was performed on selected DNA samples of five male workers with more exposure time as well as smoking and prevalence of disease were selected for further analysis. vi Abstract 2% gel electrophoresis confirmed the 679 bp PCR product of amplified gene. PCR purification was performed, and samples were sent for commercial DNA sequencing (sanger sequencing) to Macrogen Korea. Analysis of sanger sequencing results revealed C>G transversion at codon 326 in exon 7 of hOGG1 gene. Furthermore, other sequence variants in this exon were also be identified. In conclusion only 5 samples were analysed. Genomic analysis on large number of samples for hOGG1 gene exon 7 polymorphism is needed to reveal the role of this sequence variant in increasing risk of DNA damage related health issues in other brick kiln workers