Nilanjana Banerjee

Analysis of T-cell proliferation and cytokine secretion in the individuals exposed to arsenic

Over six million people in nine districts of West Bengal, India are exposed to very high levels of arsenic primarily through their drinking water. More than 300,000 people showed arsenic-induced skin lesions in these districts. This is regarded as the greatest arsenic calamity in the world. Chronic arsenicosis causes varied dermatological signs ranging from pigmentation changes, hyperkeratosis to non-melanocytic cancer of skin, and also malignancies in different internal organs. Higher incidences of opportunistic infections are found in the arsenic-exposed individuals, indicating that their immune systems may be impaired somehow. We have thus investigated the effect of arsenic on T-cell proliferation and cytokine secretion in 20 individuals with arsenic-induced skin lesions and compared the results with 18 arsenic-unexposed individuals. A marked dose-dependent suppression of Concanavalin A (Con A) induced T-cell proliferation was observed in the arsenic-exposed individuals compared with the unexposed (P < 0.001) individuals. This correlated with a significant decrease in the levels of secreted cytokines by the T cells (TNF-α, IFN-γ, IL2, IL10, IL5, and IL4) in the exposed individuals (P < 0.001). Thus it can be inferred that arsenic exposure can cause immunosuppression in humans.

High arsenic in rice is associated with elevated genotoxic effects in humans

Arsenic in drinking water may cause major deleterious health impacts including death. Although arsenic in rice has recently been demonstrated to be a potential exposure route for humans, there has been to date no direct evidence for the impact of such exposure on human health. Here we show for the first time, through a cohort study in West Bengal, India, involving over 400 human subjects not otherwise significantly exposed to arsenic through drinking water, elevated genotoxic effects, as measured by micronuclei (MN) in urothelial cells, associated with the staple consumption of cooked rice with >200 μg/kg arsenic. Further work is required to determine the applicability to populations with different dietary and genetic characteristics, but with over 3 billion people in the world consuming rice as a staple food and several percent of this rice containing such elevated arsenic concentrations, this study raises considerable concerns over the threat to human health.

Arsenic exposure through drinking water increases the risk of liver and cardiovascular diseases in the population of West Bengal, India

BackgroundArsenic is a natural drinking water contaminant affecting 26 million people in West Bengal, India. Chronic arsenic exposure causes cancer, cardiovascular disease, liver disease, neuropathies and ocular diseases. The aims of the present study were to assess bioindicators of hepatocellular injury as indicated by the levels of liver enzymes, to determine the auto immune status, as indicated by the amounts of anti-nuclear antibodies (ANA) and anti-dsDNA antibodies in their serum, and to predict cardiovascular risk in the arsenic exposed population.MethodsEffect of chronic arsenic exposure on liver was determined by liver function tests. Autoimmune status was measured by measuring ANA and anti-dsDNA in serum. Inflammatory cytokines associated with increased cardiovascular disease risk, IL6, IL8 and MCP-1 were determined.ResultsOur results indicated that serum levels of bilirubin, alanine transaminase, aspartate transaminase, alkaline phosphatase and ANA were increased in the arsenic exposed population. Serum levels of IL6 and IL8 also increased in the arsenic exposed group.ConclusionsChronic arsenic exposure causes liver injury, increases the serum levels of autoimmune markers and imparts increased cardiovascular risk.

Arsenic-induced mitochondrial instability leading to programmed cell death in the exposed individuals.

In West Bengal, India, more than 6 million people in nine districts are exposed to arsenic through drinking water. It is regarded as the greatest arsenic calamity in the world. Arsenic is a well-documented human carcinogen, which does not induce cancer in any other animal model. Interestingly, at lower concentrations, arsenic is known to induce apoptosis in various cancer cell lines in vitro. We have studied apoptosis in human peripheral blood mononuclear cells (PBMC) of 30 arsenic exposed skin lesion individuals by annexin V-FITC staining and compared with 28 unexposed individuals. The percentage of apoptotic cells in individuals with skin lesions was significantly higher (p<0.001) in comparison to unexposed individuals. In the exposed individuals with skin lesions, there were elevated levels of intracellular reactive oxygen species (ROS), mitochondrial membrane permeability and increased cytochrome c release, leading to increased downstream caspase activity. Arsenic-induced DNA damage was confirmed by DNA ladder formation and confocal microscopy. We also observed that chronic arsenic exposure reduced Bcl-2/Bax ratio and also resulted in cell cycle arrest of PBMC in G0/G1 phase. All these observations indicate that mitochondria-mediated pathway may be responsible for arsenic-induced apoptosis.

Polymorphisms in the TNF-α and IL10 Gene Promoters and Risk of Arsenic-Induced Skin Lesions and Other Nondermatological Health Effects

In West Bengal, India, at present, more than 26 million people are exposed to arsenic through drinking water. Among them, only 15-20% manifest arsenic-induced noncancerous, precancerous, and cancerous skin lesions, indicating that genetic variants play important role in arsenic susceptibility. Chronic arsenic exposure has been associated with impairment of immune systems in the exposed individuals. Because cytokines are important immune mediators, alteration in expression of these gene products may lead to arsenic-specific disease manifestations. The aim of the present work was to investigate the association between the TNF-α-308G>A (rs1800629) and IL10 -3575T>A (rs1800890) polymorphisms and arsenic-induced dermatological and nondermatological health outcomes. A case-control study was conducted in West Bengal, India, involving 207 cases with arsenic-induced skin lesions and 190 controls without skin lesions having similar arsenic exposure. The polymorphisms were determined using conventional PCR-sequencing method. ELISA was done to determine the serum levels of the two cytokines tumor necrosis factor α (TNF-α) and interleukin 10 (IL10). Associations between the polymorphisms studied and nondermatological health effects in the study subjects were determined from our epidemiological survey data. Individuals with GA/AA (-308 TNF-α) and TA/AA (-3575 IL10) genotypes were at higher risk of developing arsenic-induced skin lesions, ocular, and respiratory diseases. Also the -308 TNF A allele corresponded to a higher production of TNF-α, and -3575 IL10 A allele corresponded to a lower production of IL10. Thus, the polymorphisms studied impart significant risk toward development of arsenic-induced dermatological and nondermatological health effects in the chronically exposed population of West Bengal, India.

Epigenetic modifications of DAPK and p16 genes contribute to arsenic-induced skin lesions and nondermatological health effects

Over 26 million people in West Bengal, India, are exposed to very high levels of arsenic through drinking water, leading to several deleterious endpoints including cancers. To elucidate the role of promoter methylation in arsenic-induced dermatological and nondermatological health effects, methylation status of p16 and DAPK genes was determined. A case-control study was conducted involving 72 individuals with arsenic-induced skin lesions (cases) and 50 individuals without skin lesions (controls), having similar arsenic exposure through drinking water. Methylation status was determined by bisulfite conversion of genomic DNA and methylation-specific PCR. Expression of the genes was determined by real-time PCR and Western blot analysis. Associations between the promoter methylation status and nondermatological health effects were determined from epidemiological survey data. Significant hypermethylation was found in the promoters of both DAPK and p16 genes in the cases compared with the controls resulting in downregulation of both the genes in the cases. There was a 3.4-fold decrease in the expression of death-associated protein kinase and 2.2-fold decrease in gene expression of p16 in the cases compared to the controls, the lowest expression being in the cancer tissues. Promoter hypermethylation of the genes was also associated with higher risk of developing arsenic-induced skin lesions, peripheral neuropathy, ocular and respiratory diseases. This study for the first time makes an attempt to correlate epigenetic modifications of the tumor suppressor genes with dermatological and nondermatological health outcomes in a population chronically exposed to arsenic.

Arsenic-induced promoter hypomethylation and over-expression of ERCC2 reduces DNA repair capacity in humans by non-disjunction of the ERCC2–Cdk7 complex

Arsenic in drinking water is of critical concern in West Bengal, India, as it results in several physiological symptoms including dermatological lesions and cancers. Impairment of the DNA repair mechanism has been associated with arsenic-induced genetic damage as well as with several cancers. ERCC2 (Excision Repair Cross-Complementing rodent repair, complementation group 2), mediates DNA-repair by interacting with Cdk-activating kinase (CAK) complex, which helps in DNA proof-reading during transcription. Arsenic metabolism alters epigenetic regulation; we tried to elucidate the regulation of ERCC2 in arsenic-exposed humans. Water, urine, nails, hair and blood samples from one hundred and fifty seven exposed and eighty eight unexposed individuals were collected. Dose dependent validation was done in vitro using HepG2 and HEK-293. Arsenic content in the biological samples was higher in the exposed individuals compared with the content in unexposed individuals (p < 0.001). Bisulfite-modified methylation specific PCR showed a significant (p < 0.0001) hypomethylation of the ERCC2 promoter in the arsenic-exposed individuals. Densitometric analysis of immunoblots showed a nearly two-fold increase in expression of ERCC2 in exposed individuals, but there was an enhanced genotoxic insult as measured by micronuclei frequency. Immuno-precipitation and western blotting revealed an increased (p < 0.001) association of Cdk7 with ERCC2 in highly arsenic exposed individuals. The decrease in CAK activity was determined by observing the intensity of Ser(392) phosphorylation in p53, in vitro, which decreased with an increase in arsenic dose. Thus we infer that arsenic biotransformation leads to promoter hypomethylation of ERCC2, which in turn inhibits the normal functioning of the CAK-complex, thus affecting DNA-repair; this effect was highest among the arsenic exposed individuals with dermatological lesions.

Evaluation of the serum catalase and myeloperoxidase activities in chronic arsenic-exposed individuals and concomitant cytogenetic damage

Chronic arsenic exposure through contaminated drinking water is a major environmental health issue. Chronic arsenic exposure is known to exert its toxic effects by a variety of mechanisms, of which generation of reactive oxygen species (ROS) is one of the most important. A high level of ROS, in turn, leads to DNA damage that might ultimately culminate in cancer. In order to keep the level of ROS in balance, an array of enzymes is present, of which catalase (CAT) and myeloperoxidase (MPO) are important members. Hence, in this study, we determined the activities of these two enzymes in the sera and chromosomal aberrations (CA) in peripheral blood lymphocytes in individuals exposed and unexposed to arsenic in drinking water. Arsenic in drinking water and in urine was used as a measure of exposure. Our results show that individuals chronically exposed to arsenic have significantly higher CAT and MPO activities and higher incidence of CA. We found moderate positive correlations between CAT and MPO activities, induction of CA and arsenic in urine and water. These results indicate that chronic arsenic exposure causes higher CAT and MPO activities in serum that correlates with induction of genetic damage. We conclude that the serum levels of these enzymes might be used as biomarkers of early arsenic exposure induced disease much before the classical dermatological symptoms of arsenicosis begin to appear.

Increased microRNA 21 expression contributes to arsenic induced skin lesions, skin cancers and respiratory distress in chronically exposed individuals

More than 26 million people in West Bengal, India, are exposed to arsenic through drinking water, leading to several deleterious endpoints including precancerous and cancerous skin lesions and other non-dermatological health effects. Here, our aim was to identify whether miR21 is associated with such dermatological and non-dermatological health outcomes in chronically exposed humans. A total of 123 subjects from West Bengal were recruited for this study (45 exposed individuals with skin lesions, 38 exposed individuals without skin lesions and 40 unexposed individuals). The miR21 expression patterns in the lymphocytes were studied by quantitative realtime PCR and the effects on downstream targets were validated by Western blotting. Associations between the miR21 expression patterns and non-dermatological health effects were determined from epidemiological survey data. In vitro studies were done with low dose (0.05ppm) of chronic arsenic exposure to HaCaT cells for 15 passages. Interestingly, within the exposed group, the skin lesion individuals showed almost 4.5 fold up-regulation of miR21 compared to the no skin lesion group. The expression of the downstream targets of miR21 (PTEN and PDCD4) varied inversely, while the expression of pAKT and PI3K varied proportionately with its expression levels. Results of in vitro studies showed similar trends. Again miR21 was 2.03 fold up-regulated in the exposed individuals with respiratory diseases compared to the individuals without the same. This study for the first time shows that miR21 plays an important role in contributing to arsenic induced dermatological and non-dermatological health outcomes in an exposed population.

Evaluatıon of health effects, genetıc damage and telomere length ın children exposed to arsenic in West Bengal, İndia

Increasing evidence of arsenic contamination in ground water and its associated adverse health outcomes affects millions of people worldwide. However, arsenic toxicity studies in children have gained impetus very recently due to the non-prominence of the hallmarks of arsenic toxicity i.e skin lesions. We recognized the need to evaluate the status of genetic damage brought about by early life exposure to arsenic in children as measured by micronucleus (MN) assay for three cell types namely buccal mucosa, urothelial cells and lymphocytes. A thorough health checkup and complete haematogram of the study participants was performed to measure overall health effects and changes in the blood profile in children exposed to arsenic through drinking water in West Bengal, India. Since telomere length alteration has been identified as a good indicator of arsenic toxicity in adults, we measured the telomere length of the arsenic exposed and unexposed children. We found that all the three cell types had significantly higher (P < 0.0001) MN frequency in the arsenic exposed children when compared to the unexposed. Blood profiling showed significantly altered neutrophil, eosinophil, lymphocyte and haemoglobin levels in the arsenic exposed children than their unexposed counterparts. Telomere length in the arsenic exposed children was slightly higher than the unexposed. This is a firsthand report of the genetic damage observed in children exposed to arsenic through drinking water in West Bengal, India.