Depletion of S-adenosylmethionine pool and promoter hypermethylation of Arsenite methyltransferase in arsenic-induced skin lesion individuals: A case-control study from West Bengal, India.

Methylation of arsenic compounds in the human body occurs following a series of biochemical reactions in the presence of methyl donor S-adenosylmethionine (SAM) and catalyzed by arsenite methyltransferase (AS3MT). However, the extent and pattern of methylation differs among the arsenic exposed individuals leading to differential susceptibility. The mechanism for such inter-individual difference is enigmatic. In the present case-control study we recruited exposed individuals with and without arsenic induced skin lesion (WSL and WOSL), and an unexposed cohort, each having 120 individuals. Using ELISA, we observed a reduction in SAM levels (p < 0.05) in WSL compared to WOSL. Linear regression analysis revealed a negative correlation between urinary arsenic concentration and SAM concentration between the study groups. qRT-PCR revealed a significant down-regulation (p < 0.01) of key regulatory genes like MTHFR, MTR, MAT2A and MAT2B of SAM biogenesis pathway in WSL cohort. Methylation-specific PCR revealed significant promoter hypermethylation of AS3MT (WSL vs. WOSL: p < 0.01) which resulted in its subsequent transcriptional repression (WSL vs. WOSL: p < 0.001). Linear regression analysis also showed a negative correlation between SAM concentration and percentage of promoter methylation. Taken together, these results indicate that reduction in SAM biogenesis along with a higher utilization of SAM results in a decreased availability of methyl donor. These along with epigenetic down-regulation of AS3MT may be responsible for higher susceptibility in arsenic exposed individuals.

Epigenetic alteration of mismatch repair genes in the population chronically exposed to arsenic in West Bengal, India.

INTRODUCTION: Arsenic exposure and its adverse health outcome, including the association with cancer risk are well established from several studies across the globe. The present study aims to analyze the epigenetic regulation of key mismatch repair (MMR) genes in the arsenic-exposed population. METHOD: A case-control study was conducted involving two hundred twenty four (N=224) arsenic exposed [with skin lesion (WSL=110) and without skin lesion (WOSL=114)] and one hundred and two (N=102) unexposed individuals. The methylation status of key MMR genes i.e. MLH1, MSH2, and PMS2 were analyzed using methylation-specific PCR (MSP). The gene expression was studied by qRTPCR. The expression of H3K36me3, which was earlier reported to be an important regulator of MMR pathway, was assessed using ELISA. RESULTS: Arsenic-exposed individuals showed significant promoter hypermethylation (p < 0.0001) of MLH1 and MSH2 compared to those unexposed with consequent down-regulation in their gene expression [MLH1 (p=0.001) and MSH2 (p<0.05)]. However, no significant association was found in expression and methylation of PMS2 with arsenic exposure. We found significant down-regulation of H3K36me3 in the arsenic-exposed group, most significantly in the WSL group (p<0.0001). The expression of SETD2, the methyltransferase of an H3K36me3 moiety was found to be unaltered in arsenic exposure, suggesting the involvement of other regulatory factors yet to be identified. DISCUSSION: In summary, the epigenetic repression of DNA damage repair genes due to promoter hypermethylation of MLH1 and MSH2 and inefficient recruitment of MMR complex at the site of DNA damage owing to the reduced level of H3K36me3 impairs the mismatch repair pathway that might render the arsenic-exposed individuals more susceptible towards DNA damage and associated cancer risk.

Targeting the 'DNA methylation mark': Analysis of early epigenetic-alterations in children chronically exposed to arsenic.

Chronic exposure to arsenic causes adverse health effects in children. Aberrant epigenetic modifications including altered DNA methylation pattern are one of the major steps towards malignant transformation of cells. Our group has previously identified significant alteration in DNA methylation mark in arsenic exposed adults, affecting major biological pathways. Till date, no information is available exploring the altered DNA methylation mark in telomere regulation and altered mitochondrial functionality in association with DNA damage in arsenic-exposed children. Our study aims in identifying signature epigenetic pattern associated with telomere lengthening, mitochondrial functionality and DNA damage repair in children with special emphasis on DNA methylation. Biological samples (blood and urine) and drinking water were collected from the children aged between 5 and 16 years of arsenic exposed areas (N = 52) of Murshidabad district and unexposed areas (N = 50) of East Midnapur districts, West Bengal, India. Methylation-specific PCR was performed to analyse subtelomeric methylation status and promoter methylation status of target genes. Results revealed altered DNA methylation profile in the exposed children compared to unexposed. Promoter hypermethylation was observed in MLH1 and MSH2 (p < 0.05 and p < 0.001) indicating inefficiency in DNA damage repair. Hypomethylation in mitochondrial D-loop (p < 0.05) and TFAM promoter region (p < 0.05) along with increased mitochondrial DNA copy number among exposed children was also observed. Significant increase in telomere length and region specific subtelomeric hypermethylation (XpYp, p < 0.05) was found. Analysis of S-Adenosyl Methionine (SAM) and 8-oxoDG level revealed significant depletion of SAM (p < 0.001) and elevated oxidative DNA damage (p < 0.001) respectively in arsenic toxicity. Our study identified key methylation patterns in arsenic-exposed children which may act as an early predictive biomarker in the near future. Further in-depth studies involving large sample size and transcriptomic analysis are required for understanding the mechanistic details.

Identification of Biomarkers for Arsenicosis Employing Multiple Kernel Learning Embedded Multiobjective Swarm Intelligence.

Arsenic is a carcinogen, and long-term exposure to it may result in the development of multi-organ disease. Understanding the underlying intricate molecular network of toxicity and carcinogenicity is crucial for identifying a small set of differentially expressed biomarker genes to predict the risk of the exposed population. In this paper, a multiple kernel learning (MKL) embedded multi-objective swarm intelligence technique has been proposed to identify the candidate biomarker genes from the transcriptomic profile of arsenicosis samples. To achieve the optimal classification accuracy along with the minimum number of genes, a multi-objective random spatial local best particle swarm optimization (MO-RSplbestPSO) has been utilized. The proposed MO-RSplbestPSO also guides the multiple kernel learning mechanism which provides data specific classification. The proposed computational framework has been applied to the developed whole genome DNA microarray prepared using blood samples collected from a specific arsenic exposed area of the Indian state of West Bengal. A set of twelve biomarker genes, with four novel genes, are successfully identified for the classification of exposure to arsenic and its subcategories, which can be used as future prognostic biomarkers for screening of arsenic exposed populations. Also, the biological significance of each gene is detailed to delineate the complex molecular networking and mode of toxicity.

Peripheral blood mononuclear cells - Can they provide a clue to the pathogenesis of Graves' Orbitopathy?

PURPOSE: Graves' disease (GD) is an autoimmune disorder affecting primarily the thyroid gland. The most common extrathyroidal manifestation of GD is known as Graves' orbitopathy (GO). Bone marrow-derived fibrocytes represent a subset of monocytes in peripheral blood mononuclear cells (PBMCs), infiltrate the orbital tissues, and contribute to the pathogenesis of GO. Hence objectives of the study included whether the concentration of fibrocytes in peripheral blood was higher in GO, whether TSHR m RNA expression and TSHR surface expression in peripheral blood were higher in GO in comparison to Graves' Disease (GD) and Control subjects. METHODS: The percentage of circulating fibrocytes (FC) along with TSHR on its cell surface (CD 34+, CD 45+, CXCR4+, Collagen 1+, TSHR+) were assessed by flow cytometry of 50 patients with GD and GO and 15 healthy donors (Control). TSHR mRNA expression was measured by q RT PCR. RESULT: The concentration of circulating fibrocytes was significantly higher in GO compared to GD and control [GO 17% vs GD 3% vs control 0.7% (p < 0.05)]. Moreover, these fibrocytes express a significantly higher level of TSHR in GO. This was corroborated by the measure of TSH mRNA; in GD it was 2.3-fold higher and in GO it was 3.9 fold higher than in control, in GO this transcript level was 1.7fold higher than GD (p < 0.05). TSHR+ fibrocytes were significantly positively correlated with CAS (p = 0.004) and negatively correlated with age (p = 0.01) and duration of disease (p = 0.01) in GO. CONCLUSION: This study sheds further light on the pathogenesis of GO.

Recent Advances in Arsenic Research: Significance of Differential Susceptibility and Sustainable Strategies for Mitigation.

Arsenic contamination in drinking water and associated adverse outcomes are one of the major health issues in more than 50 countries worldwide. The scenario is getting even more detrimental with increasing number of affected people and newer sites reported from all over the world. Apart from drinking water, the presence of arsenic has been found in various other dietary sources. Chronic arsenic toxicity affects multiple physiological systems and may cause malignancies leading to death. Exposed individuals, residing in the same area, developed differential dermatological lesion phenotypes and varied susceptibility toward various other arsenic-induced disease risk, even after consuming equivalent amount of arsenic from the similar source, over the same duration of time. Researches so far indicate that differential susceptibility plays an important role in arsenic-induced disease manifestation. In this comprehensive review, we have identified major population-based studies of the last 20 years, indicating possible causes of differential susceptibility emphasizing arsenic methylation capacity, variation in host genome (single nucleotide polymorphism), and individual epigenetic pattern (DNA methylation, histone modification, and miRNA expression). Holistic multidisciplinary strategies need to be implemented with few sustainable yet cost-effective solutions like alternative water source, treatment of arsenic-contaminated water, new adaptations in irrigation system, simple modifications in cooking strategy, and dietary supplementations to combat this menace. Our review focuses on the present perspectives of arsenic research with special emphasis on the probable causes of differential susceptibility toward chronic arsenic toxicity and sustainable remediation strategies.

Epigenetic alteration of mitochondrial biogenesis regulatory genes in arsenic exposed individuals (with and without skin lesions) and in skin cancer tissues: A case control study.

Chronic arsenic toxicity has become a global concern due to its adverse pathophysiological outcome and carcinogenic potential. It is already established that arsenic induced reactive oxygen species alters mitochondrial functionality. Major regulatory genes for mitochondrial biogenesis, i.e., PGC1α, Tfam, NRF1and NRF2 are located in the nucleus. As a result, mitochondria-nucleus crosstalk is crucial for proper mitochondrial function. This previous hypothesis led us to investigateinvolvement of epigenetic alteration behindenhanced mitochondrial biogenesis in chronic arsenic exposure. An extensive case-control study was conducted with 390 study participants (unexposed, exposed without skin lesion, exposed with skin lesion and exposed skin tumour) from highly arsenic exposed areas ofWest Bengal, India. Methylation specific PCRrevealed significant promoter hypomethylation oftwo key biogenesis regulatory genes, PGC1αandTfam in arsenic exposed individuals and also in skin tumour tissues. Linear regression analysis indicated significant negative correlation between urinary arsenic concentration and promoter methylation status. Increased expression of biogenesis regulatory genes wasobtained by quantitative real-time PCR analysis. Moreover, altered mitochondrial fusion-fission regulatory gene expression was also observed in skin tumour tissues. miR663, having tumour suppressor gene like function was known to be epigenetically regulated through mitochondrial retrograde signal. Promoter hypermethylation with significantly decreased expression of miR663 was found in skin cancer tissues compared to non-cancerous control tissue. In conclusion, results indicated crucial role of epigenetic alteration in arsenic induced mitochondrial biogenesis and arsenical skin carcinogenesis for the first time. However, further mechanistic studies are necessary for detailed understanding of mitochondria-nucleus crosstalk in arsenic perturbation.

Hypomethylation of mitochondrial D-loop and ND6 with increased mitochondrial DNA copy number in the arsenic-exposed population.

Groundwater arsenic contamination has become a serious global concern due to its adverse effects on human health. Arsenic-induced reactive oxygen species trigger oxidative stress inside mitochondria, which initiate a cascade of events including altered mitochondrial (mt) membrane potential, uncoupling of electron transport chain, and mtDNA damage. A case-control study was conducted to examine the association between arsenic exposure and differences in mtDNA methylation and to assess the downstream consequences. We recruited 221 arsenic-exposed individuals, including 106 individuals with skin lesions (WSL) and 115 subjects without any skin lesions (WOSL) from the Murshidabad district, West Bengal, India. The unexposed group included 101 individuals from the arsenic unexposed area in East Midnapore. We analyzed the status of mtDNA methylation in D-loop region and ND6 gene by methylation-specific PCR. Gene expression was studied by quantitative real-time PCR. Significant hypomethylation in both D-loop and ND6 was observed with a consequent increase in their target gene expression and higher mtDNA copy number in arsenic-exposed populations compared to controls. Further mechanistic insights regarding mitochondrial epigenetic alteration in arsenic exposure will be of critical importance for the prevention of adverse health effects.