Toxicogenomics Insight to Arsenic Exposure Through Drinking Water in Rural Settings of Eastern Punjab, Pakistan

Abstract

Background: Arsenic (As) contamination in groundwater is a global public health concern responsible for numerous adverse health outcomes among millions of people. Despite significant population being exposed to As through drinking water, it has been so far a neglected area of environmental health research in Pakistan. Health risks-related responses differ between As-exposed populations due to inter-individual variability in metabolizing As and variability in genetic predisposition. However, till date, no studies have been conducted including multiple SNPs involved in As metabolism and disease risk using a linear mixed effect model approach to analyze peripheral blood transcriptomics and epigenomics results. Moreover, impact of As exposure on various blood biochemical parameters has also not been evaluated in Pakistani exposed individuals. Objectives: A systematic study was designed to investigate the impact of differential As exposure on various biochemical parameters in blood and genome-wide alterations comprising transcriptomic and epigenomic responses in blood due to As exposure. As a most novels aspect, the impact of important SNPs related to As exposure on the transcriptome and methylome was investigated among rural As-exposed individuals from As endemic area of Pakistan being differentially exposed to As through drinking water, thereby using a linear mixed effect model approach taking urinary As levels as exposure biomarker. Moreover, the influence of DNA methylation on gene expression among As-exposed individuals was also evaluated suggesting epigenetic mechanisms controlling the gene expression of As exposed subjects. Methods: Blood parameters comprising complete blood count, serum SGPT, SGOT, bilirubin and random glucose levels were measured in freshly taken samples along with acquiring demographic information from respondents. Transcriptome levels were measured using Agilent 8x60K expression arrays. Genotyping (GSTM1 and GSTT1) was performed using multiplex PCR while important genetic risk factors SNPs (As3MT, DNMT1a, ERCC2, EGFR and MTHFR) were measured using the TaqMan method. An integrated genetic risk factor for each respondent was calculated by assigning a specific value to the measured genotypes based on known risk allele numbers. Genome-wide DNA methylation was investigated using MeDIP in combination with NimbleGen 2.1M Deluxe Promotor arrays. Generated data from transcriptomics and epigenomics experiments was subjected to bioinformatics analysis in order to find significantly associated pathways related to As exposure and report novel genes xvi not reported to be associated with As previously. Pathway analysis of identified gene targets from transcriptomics and epigenomics data was performed using ConsensusPathDB. Results: Different blood parameters associated with cardiovascular disease, liver toxicity and diabetes were found elevated among the differentially As-exposed subjects. Results from transcriptomics data revealed important signaling, growth factor, cancer and other disease related pathways known to be associated with increased As exposure levels. In addition, upon implementing integrative SNPs-based genetic risk factor, pathways associated with an increased risk of NAFLD and diabetes appeared significantly enhanced by down-regulation of genes NDUFV3, IKBKB, IL6R, ADIPOR1, PPARA, OGT and FOXO1. Epigenome-wide findings suggested that out of nine generated linear mixed models, Model 1 comprising the integrated genetic risk factor disclosed biochemical pathways including muscle contraction, cardio-vascular diseases, ATR signaling, GPCR signaling, methionine metabolism and chromatin modification in association with hypo- and hyper-methylated gene targets. Since various models were compared in study, a unique pathway direct P53 effector was found associated with the individual DNMT1a polymorphism due to hyper-methylation of CSE1L and TRRAP. Most importantly, current study provides the first evidence of As-associated DNA methylation in relation with expression of genes (ATR, ATF7IP, TPM3, UBE2J2) which are all involved in important cellular functions. Conclusion: This doctoral thesis reports the first comprehensive study applying state-of-the art bioinformatics approaches to address the impact of multiple SNPs on genome-wide data comprising gene expression and DNA methylation thereby suggesting interindividual variability in adverse molecular responses among subjects exposed to drinking water As contamination in Pakistan. Results provide first evidence of various gene expression targets associated with the development of known As-related diseases. Furthermore, results also revealed that integrating SNPs data with methylome data generates a more refined and representative transcriptome and epigenome profile and discloses better insight in disease risks of As-exposed individuals and may be used as an important variable of interest in future studies addressing genome-wide alterations associated with As exposure. Key Words: Arsenic, Pakistan, Urine, Blood, Transcriptomics, Epigenomics, Risk Factor, SNPs, Pathways