Methylation of arsenic by recombinant human wild-type arsenic (+3 oxidation state) methyltransferase and its methionine 287 threonine (M287T) polymorph: Role of glutathione.

Arsenic (+3 oxidation state) methyltransferase (AS3MT) is the key enzyme in the pathway for methylation of arsenicals. A common polymorphism in the AS3MT gene that replaces a threonyl residue in position 287 with a methionyl residue (AS3MT/M287T) occurs at a frequency of about 10% among populations worldwide. Here, we compared catalytic properties of recombinant human wild-type (wt) AS3MT and AS3MT/M287T in reaction mixtures containing S-adenosylmethionine, arsenite (iAs(III)) or methylarsonous acid (MAs(III)) as substrates and endogenous or synthetic reductants, including glutathione (GSH), a thioredoxin reductase (TR)/thioredoxin (Trx)/NADPH reducing system, or tris (2-carboxyethyl) phosphine hydrochloride (TCEP). With either TR/Trx/NADPH or TCEP, wtAS3MT or AS3MT/M287T catalyzed conversion of iAs(III) to MAs(III), methylarsonic acid (MAs(V)), dimethylarsinous acid (DMAs(III)), and dimethylarsinic acid (DMAs(V)); MAs(III) was converted to DMAs(III) and DMAs(V). Although neither enzyme required GSH to support methylation of iAs(III) or MAs(III), addition of 1mM GSH decreased K(m) and increased V(max) estimates for either substrate in reaction mixtures containing TR/Trx/NADPH. Without GSH, V(max) and K(m) values were significantly lower for AS3MT/M287T than for wtAS3MT. In the presence of 1mM GSH, significantly more DMAs(III) was produced from iAs(III) in reactions catalyzed by the M287T variant than in wtAS3MT-catalyzed reactions. Thus, 1mM GSH modulates AS3MT activity, increasing both methylation rates and yield of DMAs(III). AS3MT genotype exemplified by differences in regulation of wtAS3MT and AS3MT/M287T-catalyzed reactions by GSH may contribute to differences in the phenotype for arsenic methylation and, ultimately, to differences in the disease susceptibility in individuals chronically exposed to inorganic arsenic.

Interspecies differences in metabolism of arsenic by cultured primary hepatocytes.

Biomethylation is the major pathway for the metabolism of inorganic arsenic (iAs) in many mammalian species, including the human. However, significant interspecies differences have been reported in the rate of in vivo metabolism of iAs and in yields of iAs metabolites found in urine. Liver is considered the primary site for the methylation of iAs and arsenic (+3 oxidation state) methyltransferase (As3mt) is the key enzyme in this pathway. Thus, the As3mt-catalyzed methylation of iAs in the liver determines in part the rate and the pattern of iAs metabolism in various species. We examined kinetics and concentration-response patterns for iAs methylation by cultured primary hepatocytes derived from human, rat, mice, dog, rabbit, and rhesus monkey. Hepatocytes were exposed to [(73)As]arsenite (iAs(III); 0.3, 0.9, 3.0, 9.0 or 30 nmol As/mg protein) for 24 h and radiolabeled metabolites were analyzed in cells and culture media. Hepatocytes from all six species methylated iAs(III) to methylarsenic (MAs) and dimethylarsenic (DMAs). Notably, dog, rat and monkey hepatocytes were considerably more efficient methylators of iAs(III) than mouse, rabbit or human hepatocytes. The low efficiency of mouse, rabbit and human hepatocytes to methylate iAs(III) was associated with inhibition of DMAs production by moderate concentrations of iAs(III) and with retention of iAs and MAs in cells. No significant correlations were found between the rate of iAs methylation and the thioredoxin reductase activity or glutathione concentration, two factors that modulate the activity of recombinant As3mt. No associations between the rates of iAs methylation and As3mt protein structures were found for the six species examined. Immunoblot analyses indicate that the superior arsenic methylation capacities of dog, rat and monkey hepatocytes examined in this study may be associated with a higher As3mt expression. However, factors other than As3mt expression may also contribute to the interspecies differences in the hepatocyte capacity to methylate iAs.

Metabolism of arsenic in human liver: the role of membrane transporters.

Metabolism of inorganic arsenic (iAs) is one of the key factors determining the character of adverse effects associated with exposure to iAs. Results of previous studies indicate that liver plays a primary role in iAs metabolism. This paper reviews these results and presents new data that link the capacity of human hepatocytes to metabolize iAs to the expression of specific membrane transporters. Here, we examined relationship between the expression of potential arsenic transporters (AQP9, GLUT2, P-gp, MRP1, MRP2, and MRP3) and the production and cellular retention of iAs and its methylated metabolites in primary cultures of human hepatocytes exposed for 24 h to subtoxic concentrations of arsenite. Our results show that the retention of iAs and methylarsenic metabolites (MAs) by hepatocytes exposed to sub-micromolar concentrations of arsenite correlates negatively with MRP2 expression. A positive correlation was found between MRP2 expression and the production of dimethylarsenic metabolites (DMAs), specifically, the concentration of DMAs in culture media. After exposures to high micromolar concentrations of arsenite which almost completely inhibited MAs and DMAs production, a positive correlation was found between the expression of GLUT2 and cellular retention of iAs and MAs. MRP3, AQP9, or P-gp expression had no effect on the production or distribution of iAs, MAs, or DMAs, regardless of the exposure level. Hepatocytes from seven donors used in this study did not contain detectable amounts of MRP1 protein. These data suggest that MRP2 plays an important role in the efflux of DMAs, thus, regulating kinetics of the methylation reactions and accumulation of iAs and MAs by human hepatocytes. The membrane transport of iAs by high-capacity GLUT2 transporters is not a rate-limiting step for the metabolism of arsenite at low exposure level, but may play a key role in accumulation of iAs after acute exposures which inhibit iAs methylation.

Activation of superoxide dismutase in selenium-deficient mice infected with influenza virus.

Selenium (Se) deficiency is associated with decreased activities of Se-dependent antioxidant enzymes, glutathione peroxidase (GPx) and thioredoxin reductase (TR), and with changes in the cellular redox status. We have previously shown that host Se deficiency is responsible for increased virulence of influenza virus in mice due to changes in the viral genome. The present study examines the antioxidant defense systems in the lung and liver of Se-deficient and Se-adequate mice infected with influenza A/Bangkok/1/79. Results show that neither Se status nor infection changed glutathione (GSH) concentration in the lung. Hepatic GSH concentration was lower in Se-deficient mice, but increased significantly day 5 post infection. No significant differences due to Se status or influenza infection were found in catalase activities. As expected, Se deficiency was associated with significant decreases in GPx and TR activities in both lung and liver. GPx activity increased in the lungs and decreased in the liver of Se-adequate mice in response to infection. Both Se deficiency and influenza infection had profound effects on the activity of superoxide dismutase (SOD). The hepatic SOD activity was higher in Se-deficient than Se-adequate mice before infection. However, following influenza infection, hepatic SOD activity in Se-adequate mice gradually increased. Influenza infection was associated with a significant increase of SOD activity in the lungs of Se-deficient, but not Se-adequate mice. The maximum of SOD activity coincided with the peak of pathogenesis in infected lungs. These data suggest that SOD activation in the lung and liver may be a part of a compensatory response to Se deficiency and/or influenza infection. However, SOD activation that leads to increased production of H(2)O(2) may also contribute to pathogenesis and to influenza virus mutation in lungs of Se-deficient mice.

Characterization of the impaired glucose homeostasis produced in C57BL/6 mice by chronic exposure to arsenic and high-fat diet.

BACKGROUND: Type 2 diabetes is characterized by glucose intolerance and insulin resistance. Obesity is the leading cause of type 2 diabetes. Growing evidence suggests that chronic exposure to inorganic arsenic (iAs) also produces symptoms consistent with diabetes. Thus, iAs exposure may further increase the risk of diabetes in obese individuals. OBJECTIVES: Our goal was to characterize diabetogenic effects of iAs exposure and high-fat diet (HFD) in weaned C57BL/6 mice. METHODS: Mice were fed HFD or low-fat diet (LFD) while exposed to iAs in drinking water (25 or 50 ppm As) for 20 weeks; control HFD and LFD mice drank deionized water. Body mass and adiposity were monitored throughout the study. We measured glucose and insulin levels in fasting blood and in blood collected during oral glucose tolerance tests (OGTT) to evaluate the diabetogenic effects of the treatment. RESULTS: Control mice fed HFD accumulated more fat, had higher fasting blood glucose, and were more insulin resistant than were control LFD mice. However, these diabetes indicators decreased with iAs intake in a dose-dependent manner. OGTT showed impaired glucose tolerance for both control and iAs-treated HFD mice compared with respective LFD mice. Notably, glucose intolerance was more pronounced in HFD mice treated with iAs despite a significant decrease in adiposity, fasting blood glucose, and insulin resistance. CONCLUSIONS: Our data suggest that iAs exposure acts synergistically with HFD-induced obesity in producing glucose intolerance. However, mechanisms of the diabetogenic effects of iAs exposure may differ from the mechanisms associated with the obesity-induced type 2 diabetes.

Environmental arsenic as a disruptor of insulin signaling.

Previous laboratory studies have shown that exposures to inorganic As (iAs) disrupt insulin production or glucose metabolism in cellular and animal models. Epidemiological evidence has also linked chronic human exposures to iAs to an increased risk of diabetes mellitus, a metabolic disease characterized by impaired glucose tolerance and insulin resistance. We have recently shown that arsenite and its methylated metabolites inhibit insulin-stimulated glucose uptake in cultured adipocytes by disrupting insulin-activated signal transduction pathway and preventing insulin-dependent translocation of GLUT4 transporters to the plasma membrane. Here, we present results of follow-up studies using male C57BL/6 mice chronically exposed to arsenite (1 to 50 ppm As) or to its metabolite methylarsonite (0.1 to 5 ppm As) in drinking water for 8 weeks. Results of these studies show that only the exposure to arsenite at the highest level of 50 ppm As produces symptoms attributable to impaired glucose tolerance. Notably, tissue concentrations of iAs and its methylated metabolites in pancreas and in major glucose metabolizing tissues in mice in this exposure group were comparable to the concentrations of total As reported in livers of Bangladeshi residents exposed to much lower concentrations of iAs in drinking water. These results suggest that because mice clear iAs and its metabolites more rapidly than humans, much higher exposure levels may be needed in mouse studies to produce the diabetogenic effects of iAs commonly found in human populations exposed to iAs from environmental sources.

Examination of the effects of arsenic on glucose homeostasis in cell culture and animal studies: development of a mouse model for arsenic-induced diabetes.

Previous epidemiologic studies found increased prevalences of type 2 diabetes mellitus in populations exposed to high levels of inorganic arsenic (iAs) in drinking water. Although results of epidemiologic studies in low-exposure areas or occupational settings have been inconclusive, laboratory research has shown that exposures to iAs can produce effects that are consistent with type 2 diabetes. The current paper reviews the results of laboratory studies that examined the effects of iAs on glucose metabolism and describes new experiments in which the diabetogenic effects of iAs exposure were reproduced in a mouse model. Here, weanling male C57BL/6 mice drank deionized water with or without the addition of arsenite (25 or 50 ppm As) for 8 weeks. Intraperitoneal glucose tolerance tests revealed impaired glucose tolerance in mice exposed to 50 ppm As, but not to 25 ppm As. Exposure to 25 and 50 ppm As in drinking-water resulted in proportional increases in the concentration of iAs and its metabolites in the liver and in organs targeted by type 2 diabetes, including pancreas, skeletal muscle and adipose tissue. Dimethylarsenic was the predominant form of As in the tissues of mice in both 25 and 50 ppm groups. Notably, the average concentration of total speciated arsenic in livers from mice in the 50 ppm group was comparable to the highest concentration of total arsenic reported in the livers of Bangladeshi residents who had consumed water with an order of magnitude lower level of iAs. These data suggest that mice are less susceptible than humans to the diabetogenic effects of chronic exposure to iAs due to a more efficient clearance of iAs or its metabolites from target tissues.

Selenium compounds modulate the activity of recombinant rat AsIII-methyltransferase and the methylation of arsenite by rat and human hepatocytes.

Formation of methylated metabolites is a critical step in the metabolism of inorganic arsenic or selenium. We have previously shown that under conditions of a concurrent exposure sodium selenite inhibits methylation of arsenite by cultured rat hepatocytes. Here, we compare the effects of sodium selenite and mono-, di-, and trimethylated selenium compounds on the methylation of arsenite by purified recombinant rat As(III)-methyltransferase (Cyt19) and by primary rat and human hepatocytes. Among these compounds, sodium selenite was the most potent inhibitor of the methylation of arsenite by the recombinant enzyme (K(i) = 1.4 microM) and by cultured cells. In both systems, methylseleninic acid was an order of magnitude less potent an inhibitor (K(i) = 19.4 microM) than was sodium selenite. Dimethylselenoxide and trimethylselenonium iodide were weak activators of recombinant As(III)-methyltransferase activity but were weak inhibitors of arsenite methylation in hepatocytes. These data suggest that selenite, rather than its methylated metabolites, is responsible for inhibition of arsenite methylation in cultured hepatocytes and that inhibition may involve direct interactions between selenite and As(III)-methyltransferase.

Inhibition of insulin-dependent glucose uptake by trivalent arsenicals: possible mechanism of arsenic-induced diabetes.

Chronic exposures to inorganic arsenic (iAs) have been associated with increased incidence of noninsulin (type-2)-dependent diabetes mellitus. Although mechanisms by which iAs induces diabetes have not been identified, the clinical symptoms of the disease indicate that iAs or its metabolites interfere with insulin-stimulated signal transduction pathway or with critical steps in glucose metabolism. We have examined effects of iAs and methylated arsenicals that contain trivalent or pentavalent arsenic on glucose uptake by 3T3-L1 adipocytes. Treatment with inorganic and methylated pentavalent arsenicals (up to 1 mM) had little or no effect on either basal or insulin-stimulated glucose uptake. In contrast, trivalent arsenicals, arsenite (iAs(III)), methylarsine oxide (MAs(III)O), and iododimethylarsine (DMAs(III)O) inhibited insulin-stimulated glucose uptake in a concentration-dependent manner. Subtoxic concentrations of iAs(III) (20 microM), MAs(III)O (1 microM), or DMAs(III)I (2 microM) decreased insulin-stimulated glucose uptake by 35-45%. Basal glucose uptake was significantly inhibited only by cytotoxic concentrations of iAs(III) or MAs(III)O. Examination of the components of the insulin-stimulated signal transduction pathway showed that all trivalent arsenicals suppressed expression and possibly phosphorylation of protein kinase B (PKB/Akt). The concentration of an insulin-responsive glucose transporter (GLUT4) was significantly lower in the membrane region of 3T3-L1 adipocytes treated with trivalent arsenicals as compared with untreated cells. These results suggest that trivalent arsenicals inhibit insulin-stimulated glucose uptake by interfering with the PKB/Akt-dependent mobilization of GLUT4 transporters in adipocytes. This mechanism may be, in part, responsible for the development of type-2 diabetes in individuals chronically exposed to iAs.

Interindividual variation in the metabolism of arsenic in cultured primary human hepatocytes.

Liver is a prime site for conversion of inorganic arsenic (iAs) to methylated metabolites, including methylarsenicals (MAs) and dimethylarsenicals (DMAs). To assess interindividual variation in the capacity of liver to metabolize iAs, we examined the metabolic fate of arsenite (iAs(III)) in normal primary human hepatocytes obtained from eight donors and cultured under standard conditions. Methylation rates, yields, and distribution of arsenicals were determined for hepatocytes exposed to 0.3-30 nmol of iAs(III)/mg of protein for 24 h. Although the accumulation of arsenic (As) by cells was a linear function of the initial concentration of iAs(III) in culture, the concentration of As retained in cells varied several fold among donors. DMAs was the major methylated metabolite found in cultures exposed to low concentrations of iAs(III); at higher concentrations, MAs was always predominant. Maximal rates for methylation of iAs(III) were usually attained at 3 or 9 nmol of iAs(III)/mg of protein and varied about 7-fold among donors. For most donors, the methylation rate decreased at the highest iAs(III) concentrations. MAs was the major methylated metabolite retained in cells regardless of exposure level. DMAs was the major methylated metabolite found in medium. The interindividual differences in rates for iAs(III) methylation were not strictly associated with variations in basal mRNA levels for cyt19, an As-methyltransferase. Analysis of the coding sequence of cyt19 identified one heterozygote with Met287Thr mutation in a single allele. Thus, genetic polymorphism of cyt19 along with other cellular factors is likely responsible for interindividual differences in the capacity of primary human hepatocytes to retain and metabolize iAs(III).