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
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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

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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
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Basic Protocol 1: Mitochondrial Protein Thiol Assessment with Proteomics

  • 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

  • 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

  • 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

  • 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

  • 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
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  •   FigureFigure 14.8.1 Treatment with Angeli's salt, peroxynitrite, and 4‐hydroxynonenal increases protein thiol modifications in liver mitochondria. In this figure representative experiments are presented, which show modification of mitochondrial protein thiols by reactive nitrogen and lipid species using the BIAM labeling technique in combination with one‐dimensional SDS‐PAGE and immunoblotting. Freshly isolated rat liver mitochondria (1.0 mg protein/ml) were incubated with increasing concentrations of the nitroxyl donor Angeli's salt (AS, 0 to 100 µM; A), peroxynitrite (ONOO, 0 to 1000 µM; B), or 4‐hydroxynonenal (4HNE, 0‐200 µM; C) for 15 min at 37°C. After incubations, sample tubes were placed on ice and immediately centrifuged 10 min at 10,000 × g, 4°C. After centrifugation, the supernatant was discarded and mitochondrial pellets were subjected to the BIAM‐labeling protocol included in . The BIAM‐labeled mitochondrial samples (5 or 10 µg protein) were then separated on one‐dimensional SDS‐PAGE gels (10% acrylamide) using standard gel electrophoresis techniques. The extent of labeling was determined by immunoblotting using streptavidin‐HRP (). Note that the left‐side panel show gels with blots on the right‐side panels. It is important to note that these treatments, especially at high concentrations, did not result in protein degradation or loss of protein in samples. More importantly, however, is the loss or decrease in the BIAM signal in mitochondrial samples treated with AS and ONOO, with less of a decrease observed in the 4HNE group. These basic experiments demonstrate that protein thiol groups have been oxidized or modified as a consequence of these treatments. These changes in thiols are detected using immunoblotting where decreased labeling with BIAM is shown by decreased streptavidin‐HRP immunoreactivity (i.e., signal) on the blot.
  •   FigureFigure 14.8.2 Modification of the mitochondrial thiol proteome by chronic alcohol consumption and environmental tobacco smoke. In this figure, representative two‐dimensional immunoblots are shown for BIAM‐labeled mitochondrial proteins. For this experiment, mice were exposed to a control or an ethanol (29% total daily calories) containing diet and either filtered air or low‐level environmental tobacco smoke (ETS, 10 mg/m3 total suspended particulate) for 4 weeks (Bailey et al., ). After exposures, mitochondria were isolated from livers of mice and incubated with the thiol labeling reagent BIAM (50 µM) per . The samples were then subjected to two‐dimensional gel electrophoresis and BIAM labeling was determined by immunoblotting with streptavidin‐HRP (). A decrease in BIAM‐labeling signal indicates oxidation and/or modification of cysteine residues in proteins. Based on this, a preliminary assessment of the mitochondrial thiol proteome shows that co‐exposure to ethanol and ETS results in a significant decrease in thiol labeling of multiple mitochondrial proteins (D, circled area) as compared to control groups (A,C) and the ethanol alone group (B, circled area). It is important to note analysis of the gels showed equal loading of protein and no major loss in protein content in the ethanol + ETS group. Therefore, the decreased signal is due to a loss in protein thiols in response to modification/oxidation of thiols and not simply a change in protein content (gels not shown).
  •   FigureFigure 14.8.3 Liver mitochondria respiration. This figure illustrates a representative experiment showing liver mitochondria consuming oxygen in the presence of the Complex I‐linked oxidizable substrates glutamate/malate. ADP is added at the arrows to stimulate state 3 respiration, i.e., ADP‐dependent respiration. When the ADP is depleted (i.e., converted to ATP), mitochondria revert back to a slower rate of oxygen consumption; state 4 respiration; i.e., ADP‐independent respiration. The change in oxygen concentration over time for state 3 and state 4 respiration (dashed line sections) are shown on the trace (numbers in parentheses). These numbers are used to calculate the respiration rates and the respiratory control ratio as described in .
  •   FigureFigure 14.8.4 Liver mitochondria calcium accumulation and MPT induction. This figure shows a representative experiment of mitochondrial calcium accumulation using the fluorescent dye calcium green 5N (CaG5N) that monitors extra‐mitochondrial calcium. As shown in the figure, with each 50 nmol calcium addition (arrows) there is a brief increase in fluorescence signal followed by a rapid decrease in the signal as calcium is taken up and accumulated into mitochondria. Note that mitochondria have a finite ability to accumulate calcium. When this calcium threshold level is reached, there is a rapid release of calcium, which is indicated by the rapid rise in fluorescence (black line). This rapid release of calcium into the extra‐mitochondrial space is related to formation of the mitochondrial permeability transition (MPT) pore. Note that when mitochondria are pretreated with cyclosporin A (CsA), an inhibitor of the MPT pore, mitochondria are able to accumulate twice as much calcium before the induction of the MPT pore, i.e., rapid increase in fluorescence signal (gray line).
  •   FigureFigure 14.8.5 Liver mitochondrial reactive oxygen species production—effect of antimycin and FCCP. Freshly isolated liver mitochondria were suspended at a concentration of 0.5 mg/ml and incubated with 2 µM H2DCFDA for 60 min in the presence of 0.2 mM succinate. Identical flasks were set up in the presence of antimycin with and without FCCP. The flasks containing the mitochondrial suspension are sealed and aerated with 21% O2 throughout the incubation. After 60 min, the mitochondrial suspensions are transferred to fluorometric cuvettes and the excitation/emission of DCF was read at 488/525 nm, respectively. Note that antimycin, a respiratory inhibitor of Complex III, stimulates ROS production whereas dissipation of the mitochondrial membrane potential with FCCP attenuates ROS production in mitochondria incubated with and without antimycin.


Literature Cited

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