Quantification of Glutathione in Caenorhabditis elegans

Samuel W. Caito1, Michael Aschner1

1 Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 6.18
DOI:  10.1002/0471140856.tx0618s64
Online Posting Date:  May, 2015
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Glutathione (GSH) is the most abundant intracellular thiol with diverse functions from redox signaling, xenobiotic detoxification, and apoptosis. The quantification of GSH is an important measure for redox capacity and oxidative stress. This protocol quantifies total GSH from Caenorhabditis elegans, an emerging model organism for toxicology studies. GSH is measured using the 5,5′‐dithiobis‐(2‐nitrobenzoic acid) (DTNB) cycling method originally created for cell and tissue samples but optimized for whole worm extracts. DTNB reacts with GSH to from a 5′‐thio‐2‐nitrobenzoic acid (TNB) chromophore with maximum absorbance of 412 nm. This method is both rapid and sensitive, making it ideal for studies involving a large number of transgenic nematode strains. © 2015 by John Wiley & Sons, Inc.

Keywords: glutathione; Caenorhabditis elegans

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1:

  Materials
  • Caenorhabditis elegans (30,000‐50,000 L1 stage worms or 20,000 L4/adult worms per sample; Caenorhabditis Genetics Center, University of Minnesota).
  • Sterile water
  • Extraction buffer (see recipe)
  • KPE buffer (see recipe)
  • GSH (reduced form) standards (see recipe)
  • DTNB solution (see recipe)
  • GR solution (see recipe)
  • NADPH solution (see recipe)
  • 15‐ml conical centrifuge tubes (e.g., BD Falcon)
  • Centrifuge
  • Wand sonicator
  • 96‐well microtiter plate
  • Multichannel pipettor
  • Microplate reader (with 412 nm filter)
  • Protein quantification assay kit
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
  Balabanic, D., Rupnik, M., and Klemencic, A.K. 2011. Negative impact of endocrine‐disrupting compounds on human reproductive health. Reprod. Fertil. Dev. 23:403‐416.
  Bornhorst, J., Chakraborty, S., Meyer, S., Lohren, H., Brinkhaus, S.G., Knight, A.L., Caldwell, K.A., Caldwell, G.A., Karst, U., Schwerdtle, T., Bowman, A., and Aschner, M. 2014. The effects of pdr1, djr1.1 and pink1 loss in manganese‐induced toxicity and the role of alpha‐synuclein in C. elegans. Metallomics 6:476‐490.
  Caito, S.W., Valentine, W.M., and Aschner, M. 2013. Dopaminergic neurotoxicity of S‐ethyl N,N‐dipropylthiocarbamate (EPTC), molinate, and S‐methyl‐N,N‐diethylthiocarbamate (MeDETC) in Caenorhabditis elegans. J. Neurochem.127:837‐851.
  Choe, K.P., Przybysz, A.J., and Strange, K. 2009. The WD40 repeat protein WDR‐23 functions with the CUL4/DDB1 ubiquitin ligase to regulate nuclear abundance and activity of SKN‐1 in Caenorhabditis elegans. Mol. Cell Biol. 29:2704‐2715.
  de Cock, M. and van de Bor, M. 2014. Obesogenic effects of endocrine disruptors, what do we know from animal and human studies? Environ. Int.70:15‐24.
  Dickinson, B.C., Tang, Y., Chang, Z., and Chang, C.J. 2011. A nuclear‐localized fluorescent hydrogen peroxide probe for monitoring sirtuin‐mediated oxidative stress responses in vivo. Chem. Biol. 18:943‐948.
  Dusek, P., Roos, P.M., Litwin, T., Schneider, S.A., Flaten, T.P., and Aaseth, J. 2014. The neurotoxicity of iron, copper and manganese in Parkinson's and Wilson's diseases. J. Trace. Elem. Med. Biol.
  Gallo, M., Park, D., and Riddle, D.L. 2011. Increased longevity of some C. elegans mitochondrial mutants explained by activation of an alternative energy‐producing pathway. Mech. Ageing. Dev. 132:515‐518.
  Hartwig, K., Heidler, T., Moch, J., Daniel, H., and Wenzel, U. 2009. Feeding a ROS‐generator to Caenorhabditis elegans leads to increased expression of small heat shock protein HSP‐16.2 and hormesis. Genes Nutr. 4:59‐67.
  Kaletta, T. and Hengartner, M.O. 2006. Finding function in novel targets: C. elegans as a model organism. Nat. Rev. Drug. Discov. 5:387‐398.
  Leiser, S.F., Fletcher, M., Begun, A., and Kaeberlein, M. 2013. Life‐span extension from hypoxia in Caenorhabditis elegans requires both HIF‐1 and DAF‐16 and is antagonized by SKN‐1. J. Gerontol. A Biol. Sci. Med. Sci. 68:1135‐1144.
  Liao, V.H. and Yu, C.W. 2005. Caenorhabditis elegans gcs‐1 confers resistance to arsenic‐induced oxidative stress. Biometals 18:519‐528.
  Link, C.D. and Johnson, C.J. 2002. Reporter transgenes for study of oxidant stress in Caenorhabditis elegans. Methods Enzymol. 353:497‐505.
  McDonald, P.W., Jessen, T., Field, J.R., and Blakely, R.D. 2006. Dopamine signaling architecture in Caenorhabditis elegans. Cell Mol. Neurobiol. 26:593‐618.
  Morse, D. and Rosas, I.O. 2014. Tobacco smoke‐induced lung fibrosis and emphysema. Annu. Rev. Physiol. 76:493‐513.
  Park, S.K. 2013. Electrolyzed‐reduced water increases resistance to oxidative stress, fertility, and lifespan via insulin/IGF‐1‐like signal in C. elegans. Biol. Res. 46:147‐152.
  Rahman, I., Kode, A., and Biswas, S.K. 2007. Assay for quantitative determination of glutathione and glutathione disulfide levels using enzymatic recycling method. Nat. Protoc. 1:3159‐3165.
  Reed, D.J., Babson, J.R., Beatty, P.W., Brodie, A.E., Ellis, W.W., and Potter, D.W. 1980. High‐performance liquid chromatography analysis of nanomole levels of glutathione, glutathione disulfide, and related thiols and disulfides. Anal. Biochem.106:55‐62.
  Yang, W. and Hekimi, S. 2010. A mitochondrial superoxide signal triggers increased longevity in Caenorhabditis elegans. PLoS Biol. 8:e1000556.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library