Assessment of Mitochondrial Dysfunction Arising from Treatment with Hepatotoxicants

Adrienne L. King1, Shannon M. Bailey1

1 University of Alabama at Birmingham, Birmingham, Alabama
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 14.8
DOI:  10.1002/0471140856.tx1408s44
Online Posting Date:  May, 2010
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Mitochondrial dysfunction from toxicants is recognized as a causative factor in the development of numerous liver diseases including steatohepatitis, cirrhosis, and cancer. Toxicant‐mediated damage to mitochondria result in depressed ATP production, inability to maintain proper cellular calcium homeostasis, and increased reactive oxygen species production. These disruptions contribute to hepatocellular death and lead to liver pathology. Herein, we describe a series of basic and advanced methodologies that can be incorporated into research projects aimed to understand the role of mitochondrial dysfunction in toxicant‐induced hepatotoxicity. Protocols are provided for isolation of liver mitochondria, assessment of respiratory function, measurement of mitochondrial calcium uptake, and reactive oxygen species production, as well as characterization of the mitochondrial protein thiol proteome using 2D gel electrophoresis. Data obtained from these methods can be integrated into a logical and mechanistic framework to advance understanding of the role of mitochondrial dysfunction in the pathogenesis of toxicant‐induced liver diseases. Curr. Protoc. Toxicol. 44:14.8.1‐14.8.29. © 2010 by John Wiley & Sons, Inc.

Keywords: mitochondria; hepatotoxicity; proteomics

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

Table of Contents

  • Introduction
  • Basic Protocol 1: Mitochondrial Protein Thiol Assessment with Proteomics
  • Support Protocol 1: Isolation of Liver Mitochondria
  • Basic Protocol 2: Measurement of Mitochondrial Respiration
  • Basic Protocol 3: Measurement of Mitochondrial Calcium Accumulation
  • Basic Protocol 4: Measurement of Mitochondrial Reactive Oxygen Species
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Mitochondrial Protein Thiol Assessment with Proteomics

  Materials
  • Freshly isolated liver mitochondria (see protocol 2)
  • Bio‐Rad protein assay kit (Bio‐Rad, cat. no. 500‐0006)
  • Ice
  • 10 M Tris buffer (pH 8.5) containing 1% (w/v) Triton X‐100
  • Protease inhibitor cocktail (Sigma, cat. no. P8340)
  • Biotin‐conjugated iodoacetamide (BIAM; Invitrogen, cat. no. B1591)
  • Dimethylformamide
  • 2‐mercaptoethanol
  • Rehydration buffer for IEF gel strips (see recipe)
  • Dithiothreitol (DTT) stock solution (1 M DTT dissolved in water; stored at −20°C in 50‐µl aliquots)
  • Ampholines Electrophoresis Reagent (e.g., Sigma, cat. no. A5174, pH between 3 and 10) or equivalent carrier ampholines or ampholytes
  • Tributylphosphine (Bio‐Rad, cat. no. 163‐201)
  • Equilibration buffer (see recipe)
  • Agarose solution: ultrapure, low‐melting‐temperature agarose solution (1.0% w/v agarose in 1× SDS‐PAGE running buffer)
  • 1× SDS‐PAGE buffer (see recipe)
  • 1.5‐mm gel plates of previously prepared acrylamide resolving gel (see reciperecipes for separating and stacking gels)
  • Molecular weight markers
  • Coomassie blue or SYPRO Ruby
  • Transfer buffer (see recipe)
  • Blocking buffer: 1% (w/v) BSA in 1× TBS‐T (filter‐sterilize into bottles and store at 4°C)
  • 10× TBS‐T stock solution for washing blots (see recipe)
  • Streptavidin horseradish peroxidase conjugate (GE Healthcare, cat. no. RPN1231V)
  • SuperSignal west pico chemiluminescent substrate (Pierce, cat. no. 34080)
  • 50%, 50 mM NH 4HCO 3/50% acetonitrile solution
  • Promega Gold trypsin
  • 0.1% (v/v) formic acid
  • α‐cyano‐4‐hydroxycinnamic acid matrix
  • 1.5‐ml microcentrifuge tubes
  • Ice buckets
  • Standard laboratory vortex
  • Aluminum foil
  • Two‐dimensional IEF gel electrophoresis equipment including:
    • IEF gel electrophoresis apparatus (e.g., Invitrogen ZOOM IPG runner, cat. no. ZM0001)
    • Invitrogen ZOOM strips (cat. no. ZM0011, pH 3‐10)
    • ZOOM IPG Runner Cassettes (Invitrogen, cat. no. ZM0003)
    • ZOOM Dual Power Supply (Invitrogen, cat. no. ZP10002)
  • Forceps
  • 15‐ml conical tubes
  • Rotating shaker
  • Two‐dimensional SDS‐PAGE apparatus (e.g., Bio‐Rad Mini‐PROTEAN system)
  • Transfer membrane: nitrocellulose or PVDF
  • Electroblotting apparatus
  • X‐ray film or imaging instrument (e.g., Bio‐Rad Fluor‐S Imager or ChemiDoc XRS) compatible with chemiluminescent detection methods
  • PD‐Quest Image Analysis software (Bio‐Rad Laboratories)
  • Savant SpeedVac
  • C18 ZipTips (Millipore)
  • MALDI‐TOF target plates
  • Voyager De‐Pro mass spectrometer
  • Voyager Explorer software
  • Additional reagents and equipment for isolating liver mitochondria ( protocol 2)

Support Protocol 1: Isolation of Liver Mitochondria

  Materials
  • Isolation buffer (see recipe)
  • Rat, e.g., Sprague‐Dawley, 200‐250 g body weight will have on average an 8‐10 g liver
  • Protease inhibitors (see recipe)
  • Bio‐Rad protein assay kit (Bio‐Rad, cat. no. 500‐0006)
  • 100‐ and 250‐ml beakers
  • Tweezers
  • Scissors
  • 50‐ml glass homogenizer with serrated‐bottom Teflon pestle
  • Motor‐driven homogenizer/mixer (Fisher Dyna‐Mix, cat. no. 14‐498‐45A) or comparable drill press
  • Appropriate‐size centrifuge tubes (e.g., Sorvall centrifuge tube, 50 ml, cat. no. 03146)
  • Standard laboratory centrifuge
  • Glass rods
  • Smooth‐bottom Teflon pestle to fit a 50‐ml glass homogenizer
  • 15‐ml glass homogenizer
  • Smooth‐bottom Teflon pestle to fit a 15‐ml glass homogenizer
  • 10‐ml graduated cylinder
  • Standard spectrophotometric cuvettes for protein determination
  • UV‐visible spectrophotometer
  • Additional reagents and equipment for euthanasia (Donovan and Brown, )

Basic Protocol 2: Measurement of Mitochondrial Respiration

  Materials
  • S16 electrode cleaning kit (Hansatech Instruments) containing:
    • No. 1 coarse electrode disc polish
    • No. 2 fine electrode disc polish
  • 50% saturated KCl electrolyte solution (Fisher Scientific)
  • Air‐saturated H 2O
  • Sodium hydrosulfate
  • HEPES respiration buffer (see recipe)
  • Glutamate‐Malate solution (see recipe)
  • Freshly isolated liver mitochondria (see protocol 2)
  • ADP for respiration measurements: 0.027 M ADP in 0.067 M NaPO 4 buffer, pH 6.8 (see recipe for NaPO 4 buffer)
  • 70% (w/v) ethanol
  • Succinate (1 M solution, pH 7.2 with 10 N KOH and store at 4°C)
  • Rotenone (1 mM in 95% ethanol)
  • Circulating water bath
  • S1 oxygen electrode disc (Hansatech, cat. no. S1)
  • Pasteur pipets, glass
  • Scissors
  • Cigarette paper (Rizla)
  • Teflon membrane (Hansatech, cat. no. S4)
  • Forceps
  • Small and large O‐rings (Hansatech, cat. no. S5)
  • O‐ring membrane applicator (Hansatech, cat. no. A2)
  • Liquid‐phase electrode chamber (Hansatech, cat. no. DW1)
  • Oxygraph controlling unit with Oxygraph software (Hansatech, cat. no. OXYG1)
  • Small stir bar
  • Spatulas
  • 25‐µl syringe (Hamilton, cat. no. 80230) with extended length needle (Hamilton, special order 22S 3.6‐in. point style 2)

Basic Protocol 3: Measurement of Mitochondrial Calcium Accumulation

  Materials
  • Bio‐Rad protein assay kit (Bio‐Rad, cat. no. 500‐0006)
  • HEPES respiration buffer without EDTA (see recipe)
  • Succinate (1 M solution, pH 7.2 with 10 N KOH and store at 4°C)
  • Rotenone (1 mM in 95% ethanol)
  • 0.027 M ADP in 0.067 M NaPO 4 buffer, pH 6.8 (see recipe for NaPO 4 buffer)
  • Oligomycin: 1 mg/m in ethanol (Sigma, cat. no. O4876)
  • Calcium green 5N dye (CaG5N; see recipe) 10 mM calcium chloride dihydrate
  • 1 mM Cyclosporin A (Alexis Biochemical, cat. no. L15684) in ethanol (store in the freezer)
  • Perkin Elmer LS 55 Fluorescence spectrometer or comparable instrument
  • Re‐circulating water bath
  • 4.5‐ml four‐sided clear cuvettes
  • Additional reagents and equipment for isolating liver mitochondria ( protocol 2)

Basic Protocol 4: Measurement of Mitochondrial Reactive Oxygen Species

  Materials
  • Freshly isolated liver mitochondrial suspension (see protocol 2)
  • Bio‐Rad protein assay kit (Bio‐Rad, cat. no. 500‐0006)
  • HEPES respiration buffer (see recipe)
  • 2′,7′‐dichlorodihydrofluorescein diacetate (H 2DCFDA; Invitrogen)
  • 1 M succinate solution (see recipe)
  • Antimycin A: 10 mM solution in DMSO (make fresh for each experiment)
  • Carbonylcyanide‐p‐trifluoromethoxyphenylhydrazone (FCCP): 1 mM solution in ethanol (store up to 1 year at 4°C)
  • 21% O 2/74% N 2/5% CO 2 gas mixture
  • 50‐ml Erlenmeyer flasks
  • Rubber stoppers
  • Shaking water bath set at 37°C
  • PerkinElmer LS 55 Fluorescence spectrometer or comparable instrument
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Andringa, K.K., Bajt, M.L., Jaeschke, H., and Bailey, S.M. 2008. Mitochondrial protein thiol modifications in acetaminophen hepatotoxicity: Effect on HMG‐CoA synthase. Toxicol. Lett. 177:188‐197.
   Andringa, K.K, King, A., and Bailey, S. 2009. Blue native‐gel electrophoresis proteomics. Methods Mol. Biol. 519:241‐258.
   Bailey, S.M. 2003. A review of the role of reactive oxygen and nitrogen species in alcohol‐induced mitochondrial dysfunction. Free Radic. Res. 37:585‐596.
   Bailey, S.M. and Cunningham, C.C. 1998. Acute and chronic ethanol increases reactive oxygen species generation and decreases viability in fresh, isolated rat hepatocytes. Hepatology 28:1318‐1326.
   Bailey, S.M., Andringa, K.K., Landar, A., and Darley‐Usmar, V.M. 2008. Proteomic approaches to identify and characterize alterations to the mitochondrial proteome in alcoholic liver disease. Methods Mol. Biol. 447:369‐380.
   Bailey, S.M., Mantena, S.K., Millender‐Swain, T., Cakir, Y., Jhala, N.C., Chhieng, D., Pinkerton, K.E., and Ballinger, S.W. 2009. Ethanol and tobacco smoke increase hepatic steatosis and hypoxia in the hypercholesterolemic apoE‐/‐ mouse: Implications for a “multi‐hit” hypothesis of fatty liver disease. Free Radic. Biol. Med. 46:928‐938.
   Cooper, C.E., Patel, R.P., Brookes, P.S., and Darley‐Usmar, V.M. 2002. Nanotransducers in cellular redox signaling: Modification of thiols by reactive oxygen and nitrogen species. Trends Biochem. Sci. 27:489‐492.
   Darley‐Usmar, V.M., Capaldi, R.A., Takamiya, S., Millet, F., Wilson, M.T., Malatesta, F., and Sarti, P. 1987. In Mitochondria: A Practical Approach. (V.M. Darley‐Usmar, D. Rickwood and M.T. Wilson, eds.). pp 113‐152. IRL Press, Oxford.
   Dikalov, S., Griendling, K.K., and Harrison, D.G. 2007. Measurement of reactive oxygen species in cardiovascular studies. Hypertension 49:717‐727.
   Donovan, J. and Brown, P. 2006. Euthanasia. Curr. Protoc. Immunol. 73:1.8.1‐1.8.4.
   Gutierrez, J., Ballinger, S.W., Darley‐Usmar, V.M., and Landar, A. 2006. Free radicals, mitochondria, and oxidized lipids: The emerging role in signal transduction in vascular cells. Circ. Res. 99:924‐932.
   Jou, J., Choi, S.S., and Diehl, A.M. 2008. Mechanisms of disease progression in nonalcoholic fatty liver disease. Semin. Liver Dis. 28:370‐379.
   Kim, J.R., Yoon, H.W., Kwon, K.S., Lee, S.R., and Rhee, S.G. 2000. Identification of proteins containing cysteine residues that are sensitive to oxidation by hydrogen peroxide at neutral pH. Anal. Biochem. 283:214‐221.
   Landar, A., Oh, J.Y., Giles, N.M., Isom, A., Kirk, M., Barnes, S., and Darley‐Usmar, V.M. 2006. A sensitive method for the quantitative measurement of protein thiol modification in response to oxidative stress. Free Radic. Biol. Med. 40:459‐468.
   Lemasters, J.J., Qian, T., He, L., Kim, J.S., Elmore, S.P., Cascio, W.E., and Brenner, D.A. 2002. Role of mitochondrial inner membrane permeabilization in necrotic cell death, apoptosis, and autophagy. Antioxid. Redox. Signal 4:769‐781.
   Mantena, S.K., King, A.L., Andringa, K.K., Eccleston, H.B., and Bailey, S.M. 2008. Mitochondrial dysfunction and oxidative stress in the pathogenesis of alcohol‐ and obesity‐induced fatty liver diseases. Free Radic. Biol. Med. 44:1259‐1272.
   McStay, G.P., Clarke, S.J., and Halestrap, A.P. 2002. Role of critical thiol groups on the matrix surface of the adenine nucleotide translocase in the mechanism of the mitochondrial permeability transition pore. Biochem. J. 367:541‐548.
   Radi, R. 2004. Nitric oxide, oxidants, and protein tyrosine nitration. Proc. Natl. Acad. Sci. U.S.A. 101:4003‐4008.
   Tampo, Y., Kotamraju, S., Chitambar, C.R., Kalivendi, S.V., Keszler, A., Joseph, J., and Kalyanaraman, B. 2003. Oxidative stress‐induced iron signaling is responsible for peroxide‐dependent oxidation of dichlorodihydrofluorescein in endothelial cells: Role of transferrin receptor‐dependent iron uptake in apoptosis. Circ. Res. 92:56‐63.
   Tarpey, M.M. and Fridovich, I. 2001. Methods of detection of vascular reactive species: Nitric oxide, superoxide, hydrogen peroxide, and peroxynitrite. Circ. Res. 89:224‐236.
   Venkatraman, A., Landar, A., Davis, A.J., Ulasova, E., Page, G., Murphy, M.P., Darley‐Usmar, V., and Bailey, S.M. 2004. Oxidative modification of hepatic mitochondria protein thiols: effect of chronic alcohol consumption. Am. J. Physiol. Gastrointest. Liver Physiol. 286:G521‐G527.
   Wittig, I., Carrozzo, R., Santorelli, F.M., and Schagger, H. 2007. Functional assays in high‐resolution clear native gels to quantify mitochondrial complexes in human biopsies and cell lines. Electrophoresis 28:3811‐3820.
   Young, T.A., Cunningham, C.C., and Bailey, S.M. 2002. Reactive oxygen species production by the mitochondrial respiratory chain in isolated rat hepatocytes and liver mitochondria: studies using myxothiazol. Arch. Biochem. Biophys. 405:65‐72.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library