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