Sex-specific transgenerational effects of preconception exposure to arsenite: metabolic phenotypes of C57BL/6 offspring.

Chronic exposure to inorganic arsenic (iAs) has been linked to diabetes in both humans and mice, but the role of iAs exposure prior to conception and its transgenerational effects are understudied. The present study investigated transgenerational effects of preconception iAs exposure in C57BL/6J mice, focusing on metabolic phenotypes of G1 and G2 offspring. Body composition and diabetes indicators, including fasting blood glucose, fasting plasma insulin, glucose tolerance, and indicators of insulin resistance and beta cell function, were examined in both generations. The results suggest that the preconception iAs exposure in the parental (G0) generation induced diabetic phenotypes in G1 and G2 offspring in a sex-dependent manner. G1 females from iAs-exposed parents developed insulin resistance while no significant effects were found in G1 males. In the G2 generation, insulin resistance was observed only in males from iAs-exposed grandparents and was associated with higher bodyweights and adiposity. Similar trends were observed in G2 females from iAs-exposed grandparents, but these did not reach statistical significance. Thus, preconception iAs exposure altered metabolic phenotype across two generations of mouse offspring. Future research will investigate the molecular mechanisms underlying these transgenerational effects, including epigenomic and transcriptomic profiles of germ cells and tissues from G0, G1 and G2 generations.

An interaction of inorganic arsenic exposure with body weight and composition on type 2 diabetes indicators in Diversity Outbred mice.

Type 2 diabetes (T2D) is a complex metabolic disorder with no cure and high morbidity. Exposure to inorganic arsenic (iAs), a ubiquitous environmental contaminant, is associated with increased T2D risk. Despite growing evidence linking iAs exposure to T2D, the factors underlying inter-individual differences in susceptibility remain unclear. This study examined the interaction between chronic iAs exposure and body composition in a cohort of 75 Diversity Outbred mice. The study design mimics that of an exposed human population where the genetic diversity of the mice provides the variation in response, in contrast to a design that includes untreated mice. Male mice were exposed to iAs in drinking water (100 ppb) for 26 weeks. Metabolic indicators used as diabetes surrogates included fasting blood glucose and plasma insulin (FBG, FPI), blood glucose and plasma insulin 15 min after glucose challenge (BG15, PI15), homeostatic model assessment for [Formula: see text]-cell function and insulin resistance (HOMA-B, HOMA-IR), and insulinogenic index. Body composition was determined using magnetic resonance imaging, and the concentrations of iAs and its methylated metabolites were measured in liver and urine. Associations between cumulative iAs consumption and FPI, PI15, HOMA-B, and HOMA-IR manifested as significant interactions between iAs and body weight/composition. Arsenic speciation analyses in liver and urine suggest little variation in the mice's ability to metabolize iAs. The observed interactions accord with current research aiming to disentangle the effects of multiple complex factors on T2D risk, highlighting the need for further research on iAs metabolism and its consequences in genetically diverse mouse strains.

Ex vivo exposures to arsenite and its methylated trivalent metabolites alter gene transcription in mouse sperm cells.

We have previously reported that preconception exposure to iAs may contribute to the development of diabetes in mouse offspring by altering gene expressions in paternal sperm. However, the individual contributions of iAs and its methylated metabolites, monomethylated arsenic (MAs) and dimethylated arsenic (DMAs), to changes in the sperm transcriptome could not be determined because all three As species are present in sperm after in vivo iAs exposure. The goal of the present study was to assess As species-specific effects using an ex vivo model. We exposed freshly isolated mouse sperm to either 0.1 or 1 µM arsenite (iAsIII) or the methylated trivalent arsenicals, MAsIII and DMAsIII, and used RNA-sequencing to identify differentially expressed genes, enriched pathways, and associated protein networks. For all arsenicals tested, the exposures to 0.1 µM concentrations had greater effects on gene expression than 1 µM exposures. Transcription factor AP-1 and B cell receptor complexes were the most significantly enriched pathways in sperm exposed to 0.1 µM iAsIII. The Mre11 complex and Antigen processing were top pathways targeted by exposure to 0.1 µM MAsIII and DMAsIII, respectively. While there was no overlap between gene transcripts altered by ex vivo exposures in the present study and those altered by in vivo exposure in our prior work, several pathways were shared, including PI3K-Akt signaling, Focal adhesion, and Extracellular matrix receptor interaction pathways. Notably, the protein networks associated with these pathways included those with known roles in diabetes. This study is the first to assess the As species-specific effects on sperm transcriptome, linking these effects to the diabetogenic effects of iAs exposure.

Arsenic 3 methyltransferase (AS3MT) automethylates on cysteine residues in vitro.

Arsenic toxicity is a global concern to human health causing increased incidences of cancer, bronchopulmonary, and cardiovascular diseases. In human and mouse, inorganic arsenic (iAs) is metabolized in a series of methylation steps catalyzed by arsenic (3) methyltransferase (AS3MT), forming methylated arsenite (MAsIII), dimethylarsenite (DMAIII) and the volatile trimethylarsine (TMA). The methylation of arsenic is coordinated by four conserved cysteines proposed to participate in catalysis, namely C33, C62, C157, and C207 in mouse AS3MT. The current model consists of AS3MT methylating iAs in the presence of the cofactor S-adenosyl-L-methionine (SAM), and the formation of intramolecular disulfide bonds following the reduction of MAsV to MAsIII. In the presence of endogenous reductants, these disulfide bonds are reduced, the enzyme re-generates, and the second round of methylation ensues. Using in vitro methylation assays, we find that AS3MT undergoes an initial automethylation step in the absence of iAs. This automethylation is enhanced by glutathione (GSH) and dithiothreitol (DTT), suggesting that reduced cysteines accept methyl groups from SAM to form S-methylcysteines. Following the addition of iAs, automethylation of AS3MT is decreased. Furthermore, using a Flag-AS3MT immunoprecipitation coupled to MS/MS, we identify both C33 and C62 as acceptors of the methyl group in vivo. Site-directed mutagenesis (C to A) revealed that three of the previously described cysteines were required for AS3MT automethylation. In vitro experiments show that automethylated AS3MT can methylate iAs in the presence of SAM. Thus, we propose that automethylated may represent an active conformation of AS3MT.

Sex-dependent effects of preconception exposure to arsenite on gene transcription in parental germ cells and on transcriptomic profiles and diabetic phenotype of offspring.

Chronic exposure to inorganic arsenic (iAs) has been linked to diabetic phenotypes in both humans and mice. However, diabetogenic effects of iAs exposure during specific developmental windows have never been systematically studied. We have previously shown that in mice, combined preconception and in utero exposures to iAs resulted in impaired glucose homeostasis in male offspring. The goal of the present study was to determine if preconception exposure alone can contribute to this outcome. We have examined metabolic phenotypes in male and female offspring from dams and sires that were exposed to iAs in drinking water (0 or 200 µg As/L) for 10 weeks prior to mating. The effects of iAs exposure on gene expression profiles in parental germ cells, and pancreatic islets and livers from offspring were assessed using RNA sequencing. We found that iAs exposure significantly altered transcript levels of genes, including diabetes-related genes, in the sperm of sires. Notably, some of the same gene transcripts and the associated pathways were also altered in the liver of the offspring. The exposure had a more subtle effect on gene expression in maternal oocytes and in pancreatic islets of the offspring. In female offspring, the preconception exposure was associated with increased adiposity, but lower blood glucose after fasting and after glucose challenge. HOMA-IR, the indicator of insulin resistance, was also lower. In contrast, the preconception exposure had no effects on blood glucose measures in male offspring. However, males from parents exposed to iAs had higher plasma insulin after glucose challenge and higher insulinogenic index than control offspring, indicating a greater requirement for insulin to maintain glucose homeostasis. Our results suggest that preconception exposure may contribute to the development of diabetic phenotype in male offspring, possibly mediated through germ cell-associated inheritance. Future research can investigate role of epigenetics in this phenomenon. The paradoxical outcomes in female offspring, suggesting a protective effect of the preconception exposure, warrant further investigation.

Metabolism of Inorganic Arsenic in Mice Lacking Genes Encoding GST-P, GST-M, and GST-T.

To investigate the role of glutathione transferases (GSTs) in the metabolism of inorganic arsenic (iAs), we compared the disposition of iAs and its metabolites in wild-type mice and mice lacking genes encoding GST-P, -M and -T after exposure to 100 ppb iAs in drinking water. We found no differences between the two genotypes in the concentrations of total arsenic or arsenic species in urine, liver, and kidneys. No genotype-dependent differences were found in proportions of arsenicals in the tissues, and only small differences were observed in the urine. Thus, under these conditions, GST-P, -M and -T did not play a significant role in iAs metabolism in mice.

Exposure to inorganic arsenic and its methylated metabolites alters metabolomics profiles in INS-1 832/13 insulinoma cells and isolated pancreatic islets.

Inorganic arsenic (iAs) is an environmental diabetogen, but mechanisms underlying its diabetogenic effects are poorly understood. Exposures to arsenite (iAsIII) and its methylated metabolites, methylarsonite (MAsIII) and dimethylarsinite (DMAsIII), have been shown to inhibit glucose-stimulated insulin secretion (GSIS) in pancreatic ß-cells and isolated pancreatic islets. GSIS is regulated by complex mechanisms. Increase in ATP production through metabolism of glucose and other substrates is the ultimate trigger for GSIS in ß-cells. In the present study, we used metabolomics to identify metabolites and pathways perturbed in cultured INS-1 832/13 rat insulinoma cells and isolated murine pancreatic islets by exposures to iAsIII, MAsIII and DMAsIII. We found that the exposures perturbed multiple metabolites, which were enriched primarily in the pathways of amino acid, carbohydrate, phospholipid and carnitine metabolism. However, the effects of arsenicals in INS-1 832/13 cells differed from those in the islets and were exposure specific with very few overlaps between the three arsenicals. In INS-1 832/13 cells, all three arsenicals decreased succinate, a metabolite of Krebs cycle, which provides substrates for ATP synthesis in mitochondria. Acetylcarnitine was decreased consistently by exposures to arsenicals in both the cells and the islets. Acetylcarnitine is usually found in equilibrium with acetyl-CoA, which is the central metabolite in the catabolism of macronutrients and the key substrate for Krebs cycle. It is also thought to play an antioxidant function in mitochondria. Thus, while each of the three trivalent arsenicals perturbed specific metabolic pathways, which may or may not be associated with GSIS, all three arsenicals appeared to impair mechanisms that support ATP production or antioxidant defense in mitochondria. These results suggest that impaired ATP production and/or mitochondrial dysfunction caused by oxidative stress may be the mechanisms underlying the inhibition of GSIS in ß-cells exposed to trivalent arsenicals.

Effects of Preconception and in Utero Inorganic Arsenic Exposure on the Metabolic Phenotype of Genetically Diverse Collaborative Cross Mice.

In humans and mice, in utero exposure to inorganic arsenic (iAs) is associated with adverse health outcomes later in life. The contribution of preconception exposure to the adverse outcomes in offspring has never been studied. Here combined in utero and postnatal exposures produce insulin resistance in two collaborative cross strains. Furthermore, combined preconception and in utero exposure resulted in increased birth weight and developed insulin resistance in one strain. Thus, preconception exposure to arsenic may contribute to the metabolic disorders later in life, but the susceptibility to the effects of this exposure is determined, at least in part, by genetics.

Arsenite and its trivalent methylated metabolites inhibit glucose-stimulated calcium influx and insulin secretion in murine pancreatic islets.

Chronic exposure to inorganic arsenic (iAs), a common drinking water and food contaminant, has been associated with an increased risk of type 2 diabetes in population studies worldwide. Several mechanisms underlying the diabetogenic effects of iAs have been proposed through laboratory investigations. We have previously shown that exposure to arsenite (iAs(III)) or its methylated trivalent metabolites, methylarsonite (MAs(III)) and dimethylarsinite (DMAs(III)), inhibits glucose-stimulated insulin secretion (GSIS) in pancreatic islets, without significant effects on insulin expression or insulin content. The goal of the present study was to determine if iAs(III) and/or its metabolites inhibit Ca2+ influx, an essential mechanism that regulates the release of insulin from ß cells in response to glucose. We found that in vitro exposures for 48 h to non-cytotoxic concentrations of iAs(III), MAs(III), and DMAs(III) impaired Ca2+ influx in isolated murine pancreatic islets stimulated with glucose. MAs(III) and DMAs(III) were more potent inhibitors of Ca2+ influx than iAs(III). These arsenicals also inhibited Ca2+ influx and GSIS in islets treated with depolarizing levels of potassium chloride in the absence of glucose. Treatment with Bay K8644, a Cav1.2 channel agonist, did not restore insulin secretion in arsenical-exposed islets. Tolbutamide, a KATP channel blocker, prevented inhibition of insulin secretion in MAs(III)- and DMAs(III)-exposed islets, but only marginally in islets exposed to iAs(III). Our findings suggest that iAs(III), MAs(III), and DMAs(III) inhibit glucose-stimulated Ca2+ influx in pancreatic islets, possibly by interfering with KATP and/or Cav1.2 channel function. Notably, the mechanisms underlying inhibition of GSIS by iAs(III) may differ from those of its trivalent methylated metabolites.

Differential metabolism of inorganic arsenic in mice from genetically diverse Collaborative Cross strains.

Mice have been frequently used to study the adverse effects of inorganic arsenic (iAs) exposure in laboratory settings. Like humans, mice metabolize iAs to monomethyl-As (MAs) and dimethyl-As (DMAs) metabolites. However, mice metabolize iAs more efficiently than humans, which may explain why some of the effects of iAs reported in humans have been difficult to reproduce in mice. In the present study, we searched for mouse strains in which iAs metabolism resembles that in humans. We examined iAs metabolism in male mice from 12 genetically diverse Collaborative Cross (CC) strains that were exposed to arsenite in drinking water (0.1 or 50 ppm) for 2 weeks. Concentrations of iAs and its metabolites were measured in urine and livers. Significant differences in total As concentration and in proportions of total As represented by iAs, MAs, and DMAs were observed between the strains. These differences were more pronounced in livers, particularly in mice exposed to 50 ppm iAs. In livers, large variations among the strains were found in percentage of iAs (15-48%), MAs (11-29%), and DMAs (29-66%). In contrast, DMAs represented 96-99% of total As in urine in all strains regardless of exposure. Notably, the percentages of As species in urine did not correlate with total As concentration in liver, suggesting that the urinary profiles were not representative of the internal exposure. In livers of mice exposed to 50 ppm, but not to 0.1 ppm iAs, As3mt expression correlated with percent of iAs and DMAs. No correlations were found between As3mt expression and the proportions of As species in urine regardless of exposure level. Although we did not find yet a CC strain in which proportions of As species in urine would match those reported in humans (typically 10-30% iAs, 10-20% MAs, 60-70% DMAs), CC strains characterized by low %DMAs in livers after exposure to 50 ppm iAs (suggesting inefficient iAs methylation) could be better models for studies aiming to reproduce effects of iAs described in humans.

Circulating miRNAs Associated with Arsenic Exposure.

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.

Prenatal arsenic exposure and dietary folate and methylcobalamin supplementation alter the metabolic phenotype of C57BL/6J mice in a sex-specific manner.

Inorganic arsenic (iAs) is an established environmental diabetogen. The link between iAs exposure and diabetes is supported by evidence from adult human cohorts and adult laboratory animals. The contribution of prenatal iAs exposure to the development of diabetes and underlying mechanisms are understudied. The role of factors that modulate iAs metabolism and toxicity in adults and their potential to influence diabetogenic effects of prenatal iAs exposure are also unclear. The goal of this study was to determine if prenatal exposure to iAs impairs glucose metabolism in mice and if maternal supplementation with folate and methylcobalamin (B12) can modify this outcome. C57BL/6J dams were exposed to iAs in drinking water (0, 100, and 1000 µg As/L) and fed a folate/B12 adequate or supplemented diet from before mating to birth of offspring. After birth, dams and offspring drank deionized water and were fed the folate/B12 adequate diet. The metabolic phenotype of offspring was assessed over the course of 14 weeks. Male offspring from iAs-exposed dams fed the folate/B12-adequate diet developed fasting hyperglycemia and insulin resistance. Maternal folate/B12 supplementation rescued this phenotype but had only marginal effects on iAs metabolism in dams. The diabetogenic effects of prenatal iAs exposure in male offspring were not associated with changes in global DNA methylation in the liver. Only minimal effects of prenatal iAs exposure or maternal supplementation were observed in female offspring. These results suggest that prenatal iAs exposure impairs glucose metabolism in a sex-specific manner and that maternal folate/B12 supplementation may improve the metabolic phenotype in offspring. Further studies are needed to identify the mechanisms underlying these effects.

Direct Speciation Analysis of Arsenic in Whole Blood and Blood Plasma at Low Exposure Levels by Hydride Generation-Cryotrapping-Inductively Coupled Plasma Mass Spectrometry.

A method for analysis of toxicologically important arsenic species in blood plasma and whole blood by selective hydride generation with cryotrapping (HG-CT) coupled either to atomic absorption spectrometry (AAS) with a quartz multiatomizer or to inductively coupled plasma mass spectrometry (ICPMS) has been validated. Sample preparation, which involved only 5 times dilution with addition of Triton X-100, Antifoam B, and l-cysteine, suppressed excessive foaming in a hydride generator. Calibration slopes for whole blood and blood plasma spiked with arsenate, monomethylarsonate, and dimethylarsinate at 0.25-1 µg L-1 As and 0.025-0.1 µg L-1 As for AAS and ICPMS detection, respectively, did not differ from slopes in aqueous solutions. HG-CT-AAS was used to analyze samples with elevated levels of arsenic species-blood plasma from patients treated with arsenic trioxide for acute promyelocytic leukemia and whole blood from mice fed an arsenic-containing diet. A good agreement between results of the direct analysis and analysis after mild digestion in phosphoric acid proved the good efficiency of the direct HG-CT procedure for the arsenic species in these types of biological samples. In the next step, plasma and whole blood from healthy donors that were spiked with the plasma from leukemia patients at levels of 0.15-0.4 µg L-1 As were analyzed by direct HG-CT-ICPMS. Good recoveries for all species even at these low levels (88-104%) were obtained. Limits of detection in blood and plasma were 0.014 µg L-1 for inorganic arsenic and below 0.002 µg L-1 As for methylated arsenic species. Thus, the ultrasensitive direct HG-CT-ICPMS method is uniquely suited for analyses of blood plasma and whole blood from individuals at low exposure levels.

Neonatal Metabolomic Profiles Related to Prenatal Arsenic Exposure.

Prenatal inorganic arsenic (iAs) exposure is associated with health effects evident at birth and later in life. An understanding of the relationship between prenatal iAs exposure and alterations in the neonatal metabolome could reveal critical molecular modifications, potentially underpinning disease etiologies. In this study, nuclear magnetic resonance (NMR) spectroscopy-based metabolomic analysis was used to identify metabolites in neonate cord serum associated with prenatal iAs exposure in participants from the Biomarkers of Exposure to ARsenic (BEAR) pregnancy cohort, in Gómez Palacio, Mexico. Through multivariable linear regression, ten cord serum metabolites were identified as significantly associated with total urinary iAs and/or iAs metabolites, measured as %iAs, %monomethylated arsenicals (MMAs), and %dimethylated arsenicals (DMAs). A total of 17 metabolites were identified as significantly associated with total iAs and/or iAs metabolites in cord serum. These metabolites are indicative of changes in important biochemical pathways such as vitamin metabolism, the citric acid (TCA) cycle, and amino acid metabolism. These data highlight that maternal biotransformation of iAs and neonatal levels of iAs and its metabolites are associated with differences in neonate cord metabolomic profiles. The results demonstrate the potential utility of metabolites as biomarkers/indicators of in utero environmental exposure.

Exposures to arsenite and methylarsonite produce insulin resistance and impair insulin-dependent glycogen metabolism in hepatocytes.

Environmental exposure to inorganic arsenic (iAs) has been shown to disturb glucose homeostasis, leading to diabetes. Previous laboratory studies have suggested several mechanisms that may underlie the diabetogenic effects of iAs exposure, including (i) inhibition of insulin signaling (leading to insulin resistance) in glucose metabolizing peripheral tissues, (ii) inhibition of insulin secretion by pancreatic ß cells, and (iii) dysregulation of the methylation or expression of genes involved in maintenance of glucose or insulin metabolism and function. Published studies have also shown that acute or chronic iAs exposures may result in depletion of hepatic glycogen stores. However, effects of iAs on pathways and mechanisms that regulate glycogen metabolism in the liver have never been studied. The present study examined glycogen metabolism in primary murine hepatocytes exposed in vitro to arsenite (iAs3+) or its methylated metabolite, methylarsonite (MAs3+). The results show that 4-h exposures to iAs3+ and MAs3+ at concentrations as low as 0.5 and 0.2 µM, respectively, decreased glycogen content in insulin-stimulated hepatocytes by inhibiting insulin-dependent activation of glycogen synthase (GS) and by inducing activity of glycogen phosphorylase (GP). Further investigation revealed that both iAs3+ and MAs3+ inhibit insulin-dependent phosphorylation of protein kinase B/Akt, one of the mechanisms involved in the regulation of GS and GP by insulin. Thus, inhibition of insulin signaling (i.e., insulin resistance) is likely responsible for the dysregulation of glycogen metabolism in hepatocytes exposed to iAs3+ and MAs3+. This study provides novel information about the mechanisms by which iAs exposure impairs glucose homeostasis, pointing to hepatic metabolism of glycogen as one of the targets.

Knockout of arsenic (+3 oxidation state) methyltransferase is associated with adverse metabolic phenotype in mice: the role of sex and arsenic exposure.

Susceptibility to toxic effects of inorganic arsenic (iAs) depends, in part, on efficiency of iAs methylation by arsenic (+3 oxidation state) methyltransferase (AS3MT). As3mt-knockout (KO) mice that cannot efficiently methylate iAs represent an ideal model to study the association between iAs metabolism and adverse effects of iAs exposure, including effects on metabolic phenotype. The present study compared measures of glucose metabolism, insulin resistance and obesity in male and female wild-type (WT) and As3mt-KO mice during a 24-week exposure to iAs in drinking water (0.1 or 1 mg As/L) and in control WT and As3mt-KO mice drinking deionized water. Results show that effects of iAs exposure on fasting blood glucose (FBG) and glucose tolerance in either WT or KO mice were relatively minor and varied during the exposure. The major effects were associated with As3mt KO. Both male and female control KO mice had higher body mass with higher percentage of fat than their respective WT controls. However, only male KO mice were insulin resistant as indicated by high FBG, and high plasma insulin at fasting state and 15 min after glucose challenge. Exposure to iAs increased fat mass and insulin resistance in both male and female KO mice, but had no significant effects on body composition or insulin resistance in WT mice. These data suggest that As3mt KO is associated with an adverse metabolic phenotype that is characterized by obesity and insulin resistance, and that the extent of the impairment depends on sex and exposure to iAs, including exposure to iAs from mouse diet.

Metabolomic profiles of arsenic (+3 oxidation state) methyltransferase knockout mice: effect of sex and arsenic exposure.

Arsenic (+3 oxidation state) methyltransferase (As3mt) is the key enzyme in the pathway for methylation of inorganic arsenic (iAs). Altered As3mt expression and AS3MT polymorphism have been linked to changes in iAs metabolism and in susceptibility to iAs toxicity in laboratory models and in humans. As3mt-knockout mice have been used to study the association between iAs metabolism and adverse effects of iAs exposure. However, little is known about systemic changes in metabolism of these mice and how these changes lead to their increased susceptibility to iAs toxicity. Here, we compared plasma and urinary metabolomes of male and female wild-type (WT) and As3mt-KO (KO) C57BL/6 mice and examined metabolomic shifts associated with iAs exposure in drinking water. Surprisingly, exposure to 1 ppm As elicited only small changes in the metabolite profiles of either WT or KO mice. In contrast, comparisons of KO mice with WT mice revealed significant differences in plasma and urinary metabolites associated with lipid (phosphatidylcholines, cytidine, acyl-carnitine), amino acid (hippuric acid, acetylglycine, urea), and carbohydrate (L-sorbose, galactonic acid, gluconic acid) metabolism. Notably, most of these differences were sex specific. Sex-specific differences were also found between WT and KO mice in plasma triglyceride and lipoprotein cholesterol levels. Some of the differentially changed metabolites (phosphatidylcholines, carnosine, and sarcosine) are substrates or products of reactions catalyzed by other methyltransferases. These results suggest that As3mt KO alters major metabolic pathways in a sex-specific manner, independent of iAs treatment, and that As3mt may be involved in other cellular processes beyond iAs methylation.

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 50mg/L As and As3mt-KO mice that cannot tolerate 50mg/L As were exposed to 0, 15, 20, 25 or 30mg/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.

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 (iAs(III)) or its methylated trivalent metabolites, methylarsonite (MAs(III)) and dimethylarsinite (DMAs(III)), 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 iAs(III), MAs(III) or DMAs(III) inhibited glucose-stimulated insulin secretion (GSIS), but not basal insulin secretion. MAs(III) and DMAs(III) were more potent than iAs(III) as GSIS inhibitors with estimated IC(50)≤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 iAs(III), MAs(III) or DMAs(III) could be reversed by a 24-hour incubation of the islets in arsenic-free medium. These results suggest that the insulin producing pancreatic ß-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.