Knockout of arsenic (+3 oxidation state) methyltransferase results in sex-dependent changes in phosphatidylcholine metabolism in mice

Arsenic (+3 oxidation state) methyltransferase is the key enzyme in the methylation pathway for inorganic arsenic. We have recently shown that As3mt knockout (KO) has a profound effect on metabolomic profiles in mice. Phosphatidylcholine species (PCs) were the largest group of metabolites altered in both plasma and urine. The present study used targeted analysis to investigate the KO-associated changes in PC profiles in the liver, the site of PC synthesis. Results show that As3mt KO has a systemic effect on PC metabolism and that this effect is sex dependent

Oxidation state specific analysis of arsenic species in tissues of wild-type and arsenic (+ 3 oxidation state) methyltransferase-knockout mice

Arsenic methyltransferase (As3mt) catalyzes the conversion of inorganic arsenic (iAs) to its methylated metabolites, including toxic methylarsonite (MAsIII) and dimethylarsinite (DMAsIII). Knockout (KO) of As3mt was shown to reduce the capacity to methylate iAs in mice. However, no data are available on the oxidation states of As species in tissues of these mice. Here, we compare the oxidation states of As species in tissues of male C57BL/6 As3mt-KO and wild-type (WT) mice exposed to arsenite (iAsIII) in drinking water. WT mice were exposed to 50 mg/L As and As3mt-KO mice that cannot tolerate 50 mg/L As were exposed to 0, 15, 20, 25 or 30 mg/L As. iAsIII accounted for 53% to 74% of total As in liver, pancreas, adipose, lung, heart, and kidney of As3mt-KO mice; tri- and pentavalent methylated arsenicals did not exceed 10% of total As. Tissues of WT mice retained iAs and methylated arsenicals: iAsIII, MAsIII and DMAsIII represented 55%–68% of the total As in the liver, pancreas, and brain. High levels of methylated species, particularly MAsIII, were found in the intestine of WT, but not As3mt-KO mice, suggesting that intestinal bacteria are not a major source of methylated As. Blood of WT mice contained significantly higher levels of As than blood of As3mt-KO mice. This study is the first to determine oxidation states of As species in tissues of As3mt-KO mice. Results will help to design studies using WT and As3mt-KO mice to examine the role of iAs methylation in adverse effects of iAs exposure

shRNA Silencing of AS3MT Expression Minimizes Arsenic Methylation Capacity of HepG2 Cells

Several methyltransferases have been shown to catalyze the oxidative methylation of inorganic arsenic (iAs) in mammalian species. However, the relative contributions of these enzymes to the overall capacity of cells to methylate iAs have not been characterized. Arsenic (+3 oxidation state) methyltransferase (AS3MT) that is expressed in rat and human hepatocytes catalyzes the conversion of iAs, yielding methylated metabolites that contain arsenic in +3 or +5 oxidation states. This study used short hairpin RNA (shRNA) to knock down AS3MT expression in human hepatocellular carcinoma (HepG2) cells. In a stable clonal HepG2/A cell line, AS3MT mRNA and protein levels were reduced by 83 and 88%, respectively. In comparison, the capacity to methylate iAs decreased only by 70%. These data suggest that AS3MT is the major enzyme in this pathway, although an AS3MT-independent process may contribute to iAs methylation in human hepatic cells

Speciation Analysis of Arsenic by Selective Hydride Generation-Cryotrapping-Atomic Fluorescence Spectrometry with Flame-in-Gas-Shield Atomizer: Achieving Extremely Low Detection Limits with Inexpensive Instrumentation

This work describes the method of a selective hydride generation-cryotrapping (HG-CT) coupled to an extremely sensitive but simple in-house assembled and designed atomic fluorescence spectrometry (AFS) instrument for determination of toxicologically important As species. Here, an advanced flame-in-gas-shield atomizer (FIGS) was interfaced to HG-CT and its performance was compared to a standard miniature diffusion flame (MDF) atomizer. A significant improvement both in sensitivity and baseline noise was found that was reflected in improved (4 times) limits of detection (LODs). The yielded LODs with the FIGS atomizer were 0.44, 0.74, 0.15, 0.17 and 0.67 ng L–1 for arsenite, total inorganic, mono-, dimethylated As and trimethylarsine oxide, respectively. Moreover, the sensitivities with FIGS and MDF were equal for all As species, allowing for the possibility of single species standardization with arsenate standard for accurate quantification of all other As species. The accuracy of HG-CT-AFS with FIGS was verified by speciation analysis in two samples of bottled drinking water and certified reference materials, NRC CASS-5 (nearshore seawater) and SLRS-5 (river water) that contain traces of methylated As species. As speciation was in agreement with results previously reported and sums of all quantified species corresponded with the certified total As. The feasibility of HG-CT-AFS with FIGS was also demonstrated by the speciation analysis in microsamples of exfoliated bladder epithelial cells isolated from human urine. The results for the sums of trivalent and pentavalent As species corresponded well with the reference results obtained by HG-CT-ICPMS (inductively coupled plasma mass spectrometry)

Direct analysis and stability of methylated trivalent arsenic metabolites in cells and tissues

Chronic ingestion of water containing inorganic arsenic (iAs) has been linked to a variety of adverse health effects, including cancer, hypertension and diabetes. Current evidence suggests that the toxic methylated trivalent metabolites of iAs, methylarsonous acid (MAs) and dimethylarsinous acid (DMAsIII) play a key role in the etiology of these diseases. Both MAs and DMAsIII have been detected in urine of subjects exposed to iAs. However, the rapid oxidation of DMAsIII and, to a lesser extent, MAsIII in oxygen-rich environments leads to difficulties in the analysis of these metabolites in samples of urine collected in population studies. Results of our previous work indicate that MAsIII and DMAsIII are relatively stable in a reducing cellular environment and can be quantified in cells and tissues. In the present study, we used the oxidation state-specific hydride generation-cryotrapping-atomic absorption spectroscopy (HG-CT-AAS) to examine the presence and stability of these trivalent metabolites in the liver of mice and in UROtsa/F35 cells exposed to iAs. Tri- and pentavalent metabolites of iAs were analyzed directly (without chemical extraction or digestion). Liver homogenates prepared in cold deionized water and cell culture medium and lysates were stored at either 0 °C or −80 °C for up to 22 days. Both MAsIII and DMAsIII were stable in homogenates stored at −80 °C. In contrast, DMAsIII in homogenates stored at 0 °C began to oxidize to its pentavalent counterpart after 1 day; MAsIII remained stable for at least 3 weeks under these conditions. MAsIII and DMAsIII generated in UROtsa/F35 cultures were stable for 3 weeks when culture media and cell lysates were stored at −80 °C. These results suggest that samples of cells and tissues represent suitable material for the quantitative, oxidation state-specific analysis of As in laboratory and population studies examining the metabolism or toxic effects of this metalloid

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

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 [73As]arsenite (iAsIII; 0.3, 0.9, 3.0, 9.0 or 30 nmol As/mg protein) for 24 hours and radiolabeled metabolites were analyzed in cells and culture media. Hepatocytes from all six species methylated iAsIII to methylarsenic (MAs) and dimethylarsenic (DMAs). Notably, dog, rat and monkey hepatocytes were considerably more efficient methylators of iAsIII than mouse, rabbit or human hepatocytes. The low efficiency of mouse, rabbit and human hepatocytes to methylate iAsIII was associated with inhibition of DMAs production by moderate concentrations of iAsIII 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

Methylated trivalent arsenicals are potent inhibitors of glucose stimulated insulin secretion by murine pancreatic islets

Epidemiologic evidence has linked chronic exposure to inorganic arsenic (iAs) with an increased prevalence of diabetes mellitus. Laboratory studies have identified several mechanisms by which iAs can impair glucose homeostasis. We have previously shown that micromolar concentrations of arsenite (iAsIII) or its methylated trivalent metabolites, methylarsonite (MAsIII) and dimethylarsinite (DMAsIII), inhibit the insulin-activated signal transduction pathway, resulting in insulin resistance in adipocytes. Our present study examined effects of the trivalent arsenicals on insulin secretion by intact pancreatic islets isolated from C57BL/6 mice. We found that 48-hour exposures to low subtoxic concentrations of iAsIII, MAsIII or DMAsIII inhibited glucose-stimulated insulin secretion (GSIS), but not basal insulin secretion. MAsIII and DMAsIII were more potent than iAsIII as GSIS inhibitors with estimated IC50≤0.1 μM. The exposures had little or no effects on insulin content of the islets or on insulin expression, suggesting that trivalent arsenicals interfere with mechanisms regulating packaging of the insulin transport vesicles or with translocation of these vesicles to the plasma membrane. Notably, the inhibition of GSIS by iAsIII, MAsIII or DMAsIII could be reversed by a 24-hour incubation of the islets in arsenic-free medium. These results suggest that the insulin producing pancreatic β-cells are among the targets for iAs exposure and that the inhibition of GSIS by low concentrations of the methylated metabolites of iAs may be the key mechanism of iAs-induced diabetes

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

Differential sensitivities of bone marrow, spleen and thymus to genotoxicity induced by environmentally relevant concentrations of arsenite

It is known in humans and mouse models, that drinking water exposures to arsenite (As+3) leads to immunotoxicity. Previously, our group showed that certain types of immune cells are extremely sensitive to arsenic induced genotoxicity. In order to see if cells from different immune organs have differential sensitivities to As+3, and if the sensitivities correlate with the intracellular concentrations of arsenic species, male C57BL/6J mice were dosed with 0, 100 and 500 ppb As+3 via drinking water for 30 d. Oxidation State Specific Hydride Generation-Cryotrapping- Inductively Coupled Plasma- Mass Spectrometry (HG- CT- ICP- MS) was applied to analyze the intracellular arsenic species and concentrations in bone marrow, spleen and thymus cells isolated from the exposed mice. A dose-dependent increase in intracellular monomethylarsonous acid (MMA+3) was observed in both bone marrow and thymus cells, but not spleen cells. The total arsenic and MMA+3 levels were correlated with an increase in DNA damage in bone marrow and thymus cells. An in vitro treatment of 5, 50 and 500 nM As+3 and MMA+3 revealed that bone marrow cells are most sensitive to As+3 treatment, and MMA+3 is more genotoxic than As+3. These results suggest that the differential sensitivities of the three immune organs to As+3 exposure are due to the different intracellular arsenic species and concentrations, and that MMA+3 may play a critical role in immunotoxicity