In Vitro Assays of Inorganic Arsenic Methylation

Zuzana Drobna1, Miroslav Styblo2, David J. Thomas3

1 Department of Nutrition, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, 2 Center for Environmental Medicine, Asthma, and Lung Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, 3 Pharmacokinetics Branch, Experimental Toxicology Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 4.32
DOI:  10.1002/0471140856.tx0432s42
Online Posting Date:  November, 2009
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Abstract

Inorganic arsenic is extensively metabolized to produce mono‐, di‐, and trimethylated products. The formation of these metabolites produces a variety of intermediates that differ from inorganic arsenic in terms of patterns of distribution and retention and in toxic effects. In order to elucidate the pathway for arsenic methylation, it was necessary to develop a reliable in vitro assay system in which the formation of methylated metabolites could be monitored. Here, we describe an in vitro assay system that uses the postmicrosomal supernatant from rat liver as the source of the enzymatic activity that catalyzes methylation reactions. This system can be used to study the requirements for methylation reactions (e.g., identifying the donor of methyl groups) and for screening of compounds as potential activators or inhibitors of arsenic methylation. Curr. Protoc. Toxicol. 42:4.32.1‐4.32.10. © 2009 by John Wiley & Sons, Inc.

Keywords: arsenic; rat liver; postmicrosomal fraction; methylation reactions; methylated arsenicals

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Inorganic Arsenic Methylation in Rat Liver Cytosol Assay System
  • Support Protocol 1: Preparation of Rat Liver Cytosol as a Source of Methylating Activity
  • Support Protocol 2: Reduction of [73As] Arsenate to [73As] Arsenite for Use as a Radiolabeled Substrate in In Vitro Assays of Arsenic Methylation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Inorganic Arsenic Methylation in Rat Liver Cytosol Assay System

  Materials
  • Rat liver cytosol (see protocol 2), frozen
  • 10 mM AdoMet stock solution (see recipe)
  • S‐Adenosylmethionine, p‐toluenesulfonate salt (Sigma)
  • Arsenous (AsIII) acid, sodium salt (iAsIII) (Sigma)
  • Carrier‐free (>50 Ci per gram As) sodium [73As] arsenate (Los Alamos Meson Production Facility through Oak Ridge National Lab), reduced (see protocol 3)
  • 37°C water bath
  • Small beaker cooled in an ice bath
  • 1.5‐ml polypropylene tubes, capped
  • Additional reagents and equipment for analysis of arsenical metabolites by thin‐layer chromatography (unit 4.33)

Support Protocol 1: Preparation of Rat Liver Cytosol as a Source of Methylating Activity

  Materials
  • Adult male Fischer 344 rats, 8‐ to 10‐weeks‐old (Charles River Laboratory)
  • Phenobarbital (or another appropriate anesthetic)
  • 70% Isopropanol
  • Buffer A (see recipe)
  • Buffer B (see recipe)
  • Dissecting instruments including:
    • Disposable scalpels
    • Operating scissors (straight sharp points, 4.5‐in.)
    • Dissecting scissors (fine sharp points, bent tip, 4.5‐in.)
    • Hemostatic forceps (straight, 5.5‐in.)
    • Tissue forceps (5.5‐in.)
    • Dissecting forceps (fine tip, curved, 4.5‐in.)
    • Clamp (bulldog, straight, 1.5‐in.)
    • Chromic gut suture
  • 20‐G butterfly needle (Becton Dickinson)
  • Glass mortar with loose fitting teflon pestle
  • Refrigerated ultracentrifuge, capable of speeds up to 135,000 × g
  • Pasteur pipet modified with a U‐shaped curve in its tip
  • Graduated cylinder
  • Additional reagents and equipment for anesthetizing the rat (Donovan and Brown, )

Support Protocol 2: Reduction of [73As] Arsenate to [73As] Arsenite for Use as a Radiolabeled Substrate in In Vitro Assays of Arsenic Methylation

  Materials
  • Carrier‐free (>50 Ci per gram As) sodium [73As] arsenate (Los Alamos Meson Production Facility through Oak Ridge National Lab)
  • Reducing solution (see recipe)
  • Small capped tubes
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Figures

Videos

Literature Cited

Literature Cited
   Buchet, J.P. and Lauwerys, R. 1985. Study of inorganic arsenic methylation by rat liver in vitro: Relevance for the interpretation of observations in man. Arch. Toxicol. 57:125‐129.
   Buchet, J.P. and Lauwerys, R. 1987. Study of factors influencing the in vivo methylation of inorganic arsenic in rats. Toxicol. Appl. Pharmacol. 91:65‐74.
   Donovan, J. and Brown, P. 1998. Anesthesia. Curr. Protoc. Immunol. 27:1.4.1‐1.4.5.
   Hirata, M., Mohri, T., Hisanaga, A., and Ishinishi, N. 1989. Conversion of arsenite and arsenate to methylarsenic and dimethylarsenic compounds by homogenates prepared from livers and kidneys of rats and mice. Appl. Organomet. Chem. 3:335‐341.
   Lin, S., Shi, Q., Nix, F.B., Styblo, M., Beck, M.A., Herbin‐Davis, K.M., Hall, L.L., Simeonsson, J.B., and Thomas, D.J. 2002. A novel S‐adenosyl‐L‐methionine:arsenic(III) methyltransferase from rat liver cytosol. J. Biol. Chem. 277:10795‐10803.
   Lu, M., Wang, H., Li, X.F., Lu, X., Cullen, W.R., Arnold, L.L., Cohen, S.M., and Le, X.C. 2004. Evidence of hemoglobin binding to arsenic as a basis for the accumulation of arsenic in rat blood. Chem. Res. Toxicol. 17:1733‐1742.
   Lu, M., Wang, H., Li, X.F., Arnold, L.L., Cohen, S.M., and Le, X.C. 2007. Binding of dimethylarsinous acid to cys‐13alpha of rat hemoglobin is responsible for the retention of arsenic in rat blood. Chem. Res. Toxicol. 20:27‐37.
   Reay, P.F. and Asher, C.J. 1977. Preparation and purification of 74As‐labeled arsenate and arsenite for use in biological experiments. Anal. Biochem. 78:557‐560.
   Smith, M.S., Jones, P.R., and Shirachi, D.Y. 1992. The biomethylation of sodium arsenate by rat liver cytosol determined by mass spectroscopy. Proc. West. Pharmacol. Soc. 35:53‐55.
   Styblo, M. and Thomas, D.J. 1997. Binding of arsenicals to proteins in an in vitro methylation system. Toxicol. Appl. Pharmacol. 147:1‐8.
   Styblo, M., Yamauchi, H., and Thomas, D.J. 1995. Comparative in vitro methylation of trivalent and pentavalent arsenic species. Toxicol. Appl. Pharmacol. 135:172‐178.
   Styblo, M., Delnomdedieu, M., and Thomas, D.J. 1996. Mono‐ and dimethylation of arsenic in rat liver cytosol in vitro. Chem. Biol. Interact. 99:147‐167.
   Waters, S.B., Devesa, V., Del Razo, L.M., Styblo, M., and Thomas, D.J. 2004. Endogenous reductants support the catalytic function of recombinant rat cyt19, an arsenic methyltransferase. Chem. Res. Toxicol. 17:404‐409.
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