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Arsenic (As) is a toxic metalloid. Inorganic arsenic (iAs) is a form of As commonly found in drinking water and in some foods. Overwhelming evidence suggests that people chronically exposed to iAs are at risk of developing cancer or cardiovascular, neurological, and metabolic diseases. Although the mechanisms underlying iAs-associated illness remain poorly characterized, a growing body of literature raises the possibility that microRNAs (miRNAs), post-transcriptional gene suppressors, may serve as mediators and/or early indicators of the pathologies associated with iAs exposure. To characterize the circulating miRNA profiles of individuals chronically exposed to iAs, samples of plasma were collected from 109 healthy residents of the city of Zimapán and the Lagunera area in Mexico, the regions with historically high exposures to iAs in drinking water. These plasma samples were analyzed for small RNAs using high-throughput sequencing and for iAs and its methylated metabolites. Associations between plasma levels of arsenic species and miRNAs were evaluated. Six circulating miRNAs (miRs-423-5p, -142-5p -2, -423-5p +1, -320c-1, -320c-2, and -454-5p), two of which have been previously linked to cardiovascular disease and diabetes (miRs-423-5p, -454-5p), were found to be significantly correlated with plasma MAs. No miRNAs were associated with plasma iAs or DMAs after correction for multiple testing. These miRNAs may represent mechanistic links between iAs exposure and disease or serve as markers of disease risks associated with this exposure.

There is strong epidemiologic evidence linking chronic exposure to inorganic arsenic (iAs) to myriad adverse health effects, including cancer of the bladder. We set out to identify DNA methylation patterns associated with arsenic and its metabolites in exfoliated urothelial cells (EUCs) that originate primarily from the urinary bladder, one of the targets of arsenic-induced carcinogenesis. Genome-wide, gene-specific promoter DNA methylation levels were assessed in EUCs from 46 residents of Chihuahua, Mexico, and the relationship was examined between promoter methylation profiles and the intracellular concentrations of total arsenic and arsenic species. A set of 49 differentially methylated genes was identified with increased promoter methylation associated with EUC tAs, iAs, and/or monomethylated As (MMAs) enriched for their roles in metabolic disease and cancer. Notably, no genes had differential methylation associated with EUC dimethylated As (DMAs), suggesting that DMAs may influence DNA methylation-mediated urothelial cell responses to a lesser extent than iAs or MMAs. Further analysis showed that 22 of the 49 arsenic-associated genes (45%) are also differentially methylated in bladder cancer tissue identified using The Cancer Genome Atlas repository. Both the arsenic- and cancer-associated genes are enriched for the binding sites of common transcription factors known to play roles in carcinogenesis, demonstrating a novel potential mechanistic link between iAs exposure and bladder cancer.

Glutathione S-transferases, including GST-T1 and GST-M1, are known to be involved in the phase II detoxification pathways for xenobiotics as well as in the metabolism of endogenous compounds. Polymorphisms in these genes have been linked to an increased susceptibility to carcinogenesis and associated with risk factors that predispose to certain inflammatory diseases. In addition, GST-T1 and GST-M1 null genotypes have been shown to be responsible for interindividual variations in the metabolism of arsenic, a known human carcinogen. To assess the specific GST genotypes in the Mexican population chronically exposed to arsenic, we have developed a multiplex High Resolution Melting PCR (HRM-PCR) analysis using a LightCycler480 instrument. This method is based on analysis of the PCR product melting curve that discriminates PCR products according to their lengths and base sequences. Three pairs of primers that specifically recognize GST-T1, GST-M1, and β-globin, an internal control, to produce amplicons of different length were designed and combined with LightCycler480 High Resolution Melting Master Mix containing ResoLight, a completely saturating DNA dye. Data collected from melting curve analysis were evaluated using LightCycler480 software to determine specific melting temperatures of individual melting curves representing target genes. Using this newly developed multiplex HRM-PCR analysis, we evaluated GST-T1 and GST-M1 genotypes in 504 DNA samples isolated from the blood of individuals residing in Zimapan, Lagunera, and Chihuahua regions in Mexico. We found that the Zimapan and Lagunera populations have similar GST-T1 and GST-M1 genotype frequencies which differ from those of the Chihuahua population. In addition, 14 individuals have been identified as carriers of the double null genotype, i.e., null genotypes in both GST-T1 and GST-M1 genes. Although this procedure does not distinguish between biallelic (+/+) and monoallelic (+/-) genotypes, it can be used in an automated workflow as a simple, sensitive, and time and money saving procedure for rapid identification of the GST-T1 and GST-M1 positive or null genotypes.

Inorganic arsenic (iAs) is an environmental toxicant currently poisoning millions of people worldwide, and chronically exposed individuals are susceptible to arsenicosis or arsenic poisoning. Using a state-of-the-art technique to map the methylomes of our study subjects, we identified a large interactome of hypermethylated genes that are enriched for their involvement in arsenic-associated diseases, such as cancer, heart disease, and diabetes. Notably, we have uncovered an arsenic-induced tumor suppressorome, a complex of 17 tumor suppressors known to be silenced in human cancers. This finding represents a pivotal clue in unraveling a possible epigenetic mode of arsenic-induced disease.

The postulated scheme for the metabolism of inorganic As involves alternating steps of oxidative methylation and of reduction of As from the pentavalent to the trivalent oxidation state, producing methylated compounds containing AsIII that are highly reactive and toxic. S-Adenosyl-L-methionine:AsIII methyltransferase purified from rat liver catalyzes production of methyl and dimethyl arsenicals from inorganic As. This protein is encoded by the cyt19 gene orthologous with cyt19 genes in mouse and human. The reductants dithiothreitol or tris(2-carboxylethyl)phosphine support catalysis by recombinant rat cyt19 (rrcyt19). Coupled systems containing an endogenous reductant (thioredoxin/thioredoxin reductase/NADPH, glutaredoxin/glutathione/glutathione reductase/NADPH, or lipoic acid/thioredoxin reductase/NADPH) support inorganic As methylation by rrcyt19. Although glutathione alone does not support rrcyt19's catalytic function, its addition to reaction mixtures containing other reductants increases the rate of As methylation. Aurothioglucose, an inhibitor of thioredoxin reductase, reduces the rate of As methylation by rrcyt19 in thioredoxin-supported reactions. Addition of guinea pig liver cytosol, a poor source of endogenous As methyltransferase activity, to reaction mixtures containing rrcyt19 shows that endogenous reductants in cytosol support the enzyme's activity. Methylated compounds containing either AsIII or AsV are detected in reaction mixtures containing rrcyt19, suggesting that cycling of As between oxidation states is a component of the pathway producing methylated arsenicals. This enzyme may use endogenous reductants to reduce pentavalent arsenicals to trivalency as a prerequisite for utilization as substrates for methylation reactions. Thus, cyt19 appears to possess both AsIII methyltransferase and AsV reductase activities.

Thioredoxin reductase (TR), an NADPH-dependent flavoenzyme that catalyzes the reduction of many disulfide-containing substrates, plays an important role in the cellular response to oxidative stress. Trivalent arsenicals, especially methyl As that contains trivalent arsenic (MAs(III)), are potent noncompetitive inhibitors of TR purified from mouse liver. Because MAs(III) is produced in the biomethylation of As, it was postulated that the extent of inhibition of TR in cultured rat hepatocytes would correlate with the intracellular concentration of methyl As. Exposure of cultured hepatocytes to inorganic As(III) (iAs(III)), MAs(III), or aurothioglucose (ATG, a competitive inhibitor of TR activity) for 30 min caused a concentration-dependent reduction in TR activity. The estimated IC(50) was >>100 microM for iAs(III), approximately 10 microM for ATG, and approximately 3 microM for MAs(III). In hepatocytes exposed to 1 microM MAs(III) for up to 24 h, the inhibition of TR activity was maximal ( approximately 40%) after exposure for 15 min. After exposure for 3 h [when most MAs(III) has been converted to dimethyl As (DMAs)], TR activity in these cells had returned to control levels. Notably, exposure of the cell to 50 microM DMAs(III) did not affect TR activity. In hepatocytes exposed to 10 microM iAs(III) for up to 24 h, the inhibition of TR activity was progressive; at 24 h, activity was reduced approximately 35%. Following exposure to iAs(III) or MAs(III), the extent of inhibition of TR activity correlated strongly with the intracellular concentration of MAs. Taken together, these results suggest that arsenicals formed in the course of cellular metabolism of As are potent inhibitors of TR activity. In particular, MAs(III), an intermediate in the metabolic pathway, is an especially potent inhibitor of TR. Hence, the capacity of cells to produce or consume the intermediates in the pathway for As methylation may be an important determinant of susceptibility to the toxic effects of As.

The liver is considered a major site for methylation of inorganic arsenic (iAs). However, there is little data on the capacity of human liver to methylate iAs. This work examined the metabolism of arsenite (iAs(III)), arsenate (iAs(V)), methylarsine oxide (MAs(III)O), methylarsonic acid (MAs(V)), dimethylarsinous acid (DMAs(III)), and dimethylarsinic acid (DMAs(V)) in primary cultures of normal human hepatocytes. Primary rat hepatocytes were used as methylating controls. iAs(III) and MAs(III)O were metabolized more extensively than iAs(V) and MAs(V) by either cell type. Neither human nor rat hepatocytes metabolized DMAs(III) or DMAs(V). Methylation of iAs(III) by human hepatocytes yielded methylarsenic (MAs) and dimethylarsenic (DMAs) species; MAs(III)O was converted to DMAs. The total methylation yield (MAs and DMAs) increased over the range of 0.1 to 4 microM iAs(III). However, DMAs production was inhibited by iAs(III) in a concentration-dependent manner, and the DMAs/MAs ratio decreased. iAs(III) (10 and 20 microM) inhibited both methylation reactions. Inhibition of DMAs synthesis resulted in accumulation of iAs and MAs in human hepatocytes, suggesting that dimethylation is required for iAs clearance from cells. Methylation capacities of human hepatocytes obtained from four donors ranged from 3.1 to 35.7 pmol of iAs(III) per 10(6) cells per hour and were substantially lower than the methylation capacity of rat hepatocytes (387 pmol of iAs(III) per 10(6) cells per hour). The maximal methylation rates for either rat or human hepatocytes were attained between 0.4 and 4 microM iAs(III). In summary, (i) human hepatocytes methylate iAs, (ii) the capacities for iAs methylation vary among individuals and are saturable, and (iii) moderate concentrations of iAs inhibit DMAs synthesis, resulting in an accumulation of iAs and MAs in cells.