Elsevier

Journal of Environmental Sciences

Volume 49, November 2016, Pages 104-112
Journal of Environmental Sciences

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

https://doi.org/10.1016/j.jes.2016.06.018Get rights and content

Abstract

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.

Introduction

Inorganic arsenic (iAs), a potent human carcinogen, is ubiquitous in the environment and accumulates in aquifers naturally and through anthropogenic activities. The ingestion of iAs through contaminated drinking water, most commonly as arsenite (iAsIII) and arsenate (iAsV), has been associated with numerous adverse effects, including peripheral vascular disease, hypertension, and cancer of the lungs, liver, and bladder (Tseng et al., 2007, Wang et al., 2007a, IARC Working Group on the Evaluation of Carcinogenic Risks). A recent National Toxicology Program workshop examining the effects of environmental chemicals on the development of diabetes and obesity concluded that there was sufficient evidence to link iAs exposures to an increased risk of diabetes in populations exposed to high levels of iAs in drinking water (Maull et al., 2012).

The enzyme, arsenic (+ 3 oxidation state) methyltransferase (AS3MT) catalyzes the S-adenosylmethionine (SAM)-dependent methylation of iAs to tri- and pentavalent methylated metabolites (Thomas, 2004). AS3MT mRNA has been found in several human and rodent tissues, including, liver, kidney, urinary bladder, heart, lung, testes, and adrenal gland (Lin et al., 2002). Once ingested, iAs is sequentially methylated by AS3MT producing methylarsonite (MAsIII), methylarsonate (MAsV), dimethylarsinite (DMAsIII), dimethylarsinate (DMAsV), and trimethylarsine oxide (TMAsVO). Growing evidence suggests that the methylated trivalent As (AsIII) species, MAsIII and DMAsIII, produced in the course of iAs metabolism, are more toxic than iAs or their pentavalent counterparts (Thomas et al., 2001, Lin et al., 2001, Drobna et al., 2003, Wang et al., 2007b, Douillet et al., 2013).

Laboratory-based studies have shown that iAs exposure alters glucose homeostasis and several mechanisms regulating glucose metabolism. Specifically, in our studies, exposure to subtoxic concentrations of iAsIII, MAsIII or, DMAsIII inhibited glucose-stimulated insulin secretion by isolated murine pancreatic islets without affecting basal insulin secretion or insulin content and expression, suggesting that AsIII species inhibit insulin transport vesicle packaging or translocation to the plasma membrane (Douillet et al., 2013). In β-cell lines exposed to iAs, an impairment of glucose-stimulated insulin secretion has been associated with reduced insulin expression (Díaz-Villaseñor et al., 2006) alterations in Ca2 + oscillations (Diaz-Villasenor et al., 2008), or with an Nrf2-mediated antioxidant response suppressing endogenous reactive oxygen species (Yen et al., 2007, Fu et al., 2010) that may be required for insulin secretion (Pi and Collins, 2010). In other cell culture models, iAsIII has been shown to inhibit differentiation of adipocytes (Trouba et al., 2000, Wauson et al., 2002) and myotubes (Steffens et al., 2011), the cell types that are involved in glucose utilization in vivo. Moreover, we have shown that AsIII species inhibit insulin signaling and insulin-stimulated glucose uptake in cultured differentiated adipocytes (Paul et al., 2007a, Walton et al., 2004). We have also shown that in C57BL/6 mice exposure to 50 mg/L As as iAsIII in drinking water resulted in impaired glucose tolerance (Paul et al., 2007b, Paul et al., 2011). Notably, mice chronically exposed to iAsIII in combination with high-fat diet produced a unique diabetic phenotype characterized by impaired glucose tolerance in the absence of significant obesity and insulin resistance (Paul et al., 2011), suggesting that the mechanisms underlying As-induced diabetes differ from those responsible for development of the obesity-associated type 2 diabetes.

Genetically altered, C57BL/6 As3mt-knockout (KO) mice have been recently developed and partially characterized (Drobna et al., 2009). When exposed to iAs these mice retained significantly more As than WT mice (Chen et al., 2011, Drobna et al., 2009, Hughes et al., 2010) and exhibited increased sensitivity to iAs toxicity (Yokohira et al., 2010, Yokohira et al., 2011). Chemical analyses have shown that iAs was the predominant species in tissues of As3mt-KO mice exposed to iAs; however, methylated As metabolites were detected in liver and plasma, suggesting the methylation of iAs by other methyltransferases or by intestinal microbiota (Drobna et al., 2009, Naranmandura et al., 2012). The oxidation states of iAs or the methylated As species found in tissues of As3mt-KO mice have never been determined. In spite of this information gap, the As3mt-KO mice have been used as a laboratory model to explore the role of iAs methylation and the contribution of trivalent methylated arsenicals in the development of iAs-induced diseases.

Hydride generation-cryotrapping-atomic absorption spectrometry (HG-CT-AAS) is uniquely suited for the oxidation state specific speciation analysis of As in complex biological matrices. The analysis using HG-CT-AAS does not require sample pretreatment or extraction, thus preserving the methylation state of unstable MAsIII and DMAsIII (Matoušek et al., 2008, Hernández-Zavala et al., 2008, Currier et al., 2011a, Currier et al., 2011b). This method has been successfully used to determine concentrations of the methylated trivalent arsenicals, MAsIII and DMAsIII, in human urine (Del Razo et al., 2001, Del Razo et al., 2011, Valenzuela et al., 2004, Valenzuela et al., 2009), mouse tissues (Currier et al., 2011a, Currier et al., 2011b), in vitro cell cultures (Del Razo et al., 2001, Hernández-Zavala et al., 2008) and in vitro mixtures for methylation of iAs by recombinant AS3MT (Hernández-Zavala et al., 2008, Ding et al., 2012). In this study, we used HG-CT-AAS to characterize the retention of tri- and pentavalent arsenicals in tissues of wild-type (WT) and As3mt-KO C57/BL6 mice after exposure to iAsIII.

Section snippets

Arsenicals

The following pentavalent arsenicals were used for calibration during the HG-CT-AAS analysis: sodium arsenite (NaAsIIIO2) and sodium arsenate (Na2HAsVO4) (both ≥ 99% pure) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Methylarsonic acid, disodium salt (CH3AsVO(ONa)2), and dimethylarsinic acid ((CH3)2AsVO(OH)), both better than 98% pure, were purchased from Chem Service (West Chester, PA, USA). The As content in each of the standards was determined by graphite furnace-AAS (Matoušek et

Water consumption and body weights

The consumption of water for each exposure group and individual body weights were measured weekly throughout the study. Fig. 1 depicts the estimated daily water consumption and the corresponding iAs intake for each exposure group over the 4-week study period. Water intake increased after the first week and then plateaued for the remaining study period except in As3mt-KO mice exposed to 25 mg/L As, which exhibited decreased water consumption in weeks 3 and 4 (Fig. 1a). The As3mt-KO mice exposed

Conclusions

In As3mt-KO mice exposed to 15, 20, 25, or 30 mg/L As, iAsIII is the most prevalent species in liver, pancreas, and adipose tissues. The majority of iAs and methylated As species retained in liver and pancreas of WT mice exposed to 50 mg/L As are in the trivalent form. DMAsV is the most prevalent species retained in skeletal muscle and adipose tissue of WT mice. For tissues critical to glucose homeostasis, doses of 25 and 30 mg/L As as iAsIII will produce in As3mt-KO mice total As levels

Acknowledgments

We would like to dedicate this paper to our good friend and colleague Dr. William Cullen and thank him for his continuous support and advice. This work was supported by NIH grant No. 2 R01 ES010845 to M.S., the UNC Nutrition Obesity Research Center grant no. DK056350, and by NIH grant No. P30ES010126 to the UNC Center for Environmental Health and Susceptibility. The investigation by J.C. was supported by a pre-doctoral traineeship (National Research Service Award T32 ES007126) from the National

References (40)

  • D. Thomas

    Elucidating the pathway for arsenic methylation*1

    Toxicol. Appl. Pharmacol.

    (2004)
  • D.J. Thomas et al.

    The cellular metabolism and systemic toxicity of arsenic

    Toxicol. Appl. Pharmacol.

    (2001)
  • K.J. Trouba et al.

    Sodium arsenite inhibits terminal differentiation of murine C3H 10T1/2 PREADIPOCYTES

    Toxicol. Appl. Pharmacol.

    (2000)
  • O.L. Valenzuela et al.

    Association of AS3MT polymorphisms and the risk of premalignant arsenic skin lesions

    Toxicol. Appl. Pharmacol.

    (2009)
  • F.S. Walton et al.

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

    Toxicol. Appl. Pharmacol.

    (2004)
  • C.-H. Wang et al.

    A review of the epidemiologic literature on the role of environmental arsenic exposure and cardiovascular diseases

    Toxicol. Appl. Pharmacol.

    (2007)
  • T.-C. Wang et al.

    Trivalent arsenicals induce lipid peroxidation, protein carbonylation, and oxidative DNA damage in human urothelial cells

    Mutat. Res. Fundam. Mol. Mech. Mutagen.

    (2007)
  • C. Yen et al.

    The diabetogenic effects of the combination of humic acid and arsenic: in vitro and in vivo studies

    Toxicol. Lett.

    (2007)
  • M. Yokohira et al.

    Severe systemic toxicity and urinary bladder cytotoxicity and regenerative hyperplasia induced by arsenite in arsenic (+ 3 oxidation state) methyltransferase knockout mice. A preliminary report

    Toxicol. Appl. Pharmacol.

    (2010)
  • B. Chen et al.

    Mouse arsenic (+ 3 oxidation state) methyltransferase genotype affects metabolism and tissue dosimetry of arsenicals after Arsenite Administration in Drinking Water

    Toxicol. Sci.

    (2011)
  • Cited by (31)

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

      2022, Toxicology and Applied Pharmacology
      Citation Excerpt :

      Laboratory mice, like humans, methylate iAs to MAs and DMAs metabolites (Vahter, 2002). These metabolites are found in tissues of mice exposed to iAs along with the parent compound (Currier et al., 2016). Therefore, it is practically impossible to determine the extent by which iAs or its toxic methylated metabolites contribute to the adverse effects of iAs exposure in specific tissues.

    • Concentrations of blood and urinary arsenic species and their characteristics in general Korean population

      2022, Environmental Research
      Citation Excerpt :

      One of the reasons for relatively high %DMA in urine and high %iAs in the blood might be due to the rapid clearance of iAs in the body. It is known that the distribution of iAs, MMA, and DMA in the blood and tissues is higher for iAs and lower for DMA compared to urine (Currier et al., 2016; Kenyon et al., 2005). In addition, blood iAs may undergo additional methylation before renal excretion because iAs methylation through As methyltransferase (AS3MT) (Currier et al., 2016; Hughes et al., 2010) encoded by the AS3MT gene, which is abundant in the liver and kidney (Engstrom et al., 2011).

    • A single or short time repeated arsenic oral exposure in mice impacts mRNA expression for signaling and immunity related genes in the gut

      2019, Food and Chemical Toxicology
      Citation Excerpt :

      Additionally, studies have also shown that ileal tissue efficiently metabolize arsenite into pentavalent and trivalent methylated metabolites. Methylation of arsenite (either due to bacterium-induced conversion or due to host factors) is generally viewed as a detoxification reaction because DMAv is the predominant species excreted in urine and methylation appears to protect mice from acute toxicity of arsenite (Currier et al., 2016). There is an exception to this trend in the RD to control comparison, but this gene expression difference could perhaps be explained by long-term exposure to arsenic and its effect on the gastrointestinal homeostasis.

    • Metabolism and disposition of arsenic species from controlled dosing with dimethylarsinic acid (DMA<sup>V</sup>) in adult female CD-1 mice. V. Toxicokinetic studies following oral and intravenous administration

      2019, Food and Chemical Toxicology
      Citation Excerpt :

      Moreover, metabolism of Asi appears central to the toxic effects by producing additional trivalent arsenic intermediates and pentavalent arsenic products (Scheme; Twaddle et al., 2018a; Twaddle et al., 2018b; Twaddle et al., 2019). Indeed, evidence supports roles for both methylation of arsenite by arsenite methyltransferase (As3MT; Dheeman et al., 2014; Currier et al., 2016) and direct reduction of pentavalent arsenic species (Delnomdedieu et al., 1994; Stýblo et al., 1997; Németi and Gregus, 2013; Twaddle et al., 2018a, 2018b, 2019) in the formation and binding of reactive trivalent arsenic species, even in tissues with low As3MT activity (Kobayashi et al., 2007). However, the evidence for metabolic activation of Asi by methylation is seemingly paradoxical, given that As3MT-catalyzed methylation appears to facilitate urinary excretion of the major metabolite, dimethylarsinic acid (DMAV), and decrease acute toxicity in wild-type vs. As3MT-knockout mice (Currier et al., 2016).

    View all citing articles on Scopus
    View full text