Measuring Covalent Binding in Hepatotoxicity

Sachin S. Devi1, Prajakta S. Palkar2, Harihara M. Mehendale2

1 Michigan State University, East Lansing, Michigan, 2 The University of Louisiana at Monroe, Monroe, Louisiana
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 14.6
DOI:  10.1002/0471140856.tx1406s32
Online Posting Date:  May, 2007
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Many hepatotoxicants like acetaminophen, chloroform, carbon tetrachloride, halothane, and thioacetamide cause hepatotoxicity through covalent binding of their reactive metabolites to proteins. The covalent binding to proteins may lead to dysfunction of critical proteins such as enzymes, transporters, receptors, and regulatory molecules. Because most reactive metabolites covalently bind to tissue macromolecules and tend to be unstable, they can not be isolated, and direct quantitation of the formation of reactive metabolites is not possible. Measuring their covalent binding to proteins offers a convenient way to estimate the amount of reactive metabolite formation. Such estimates have been used to quantify the bioactivation‐based injury due to such hepatotoxicants. There are various methods by which covalent binding may be measured. This unit describes a protocol in which a radiolabeled compound can be utilized to measure covalent binding. Alternate protocols involve immunoblotting and immunohistochemistry. The time and method of measuring covalent binding play an important role in the evaluation.

Keywords: covalent binding; hepatotoxicity; immunoblotting; immunohistochemistry; radioactivity

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Table of Contents

  • Basic Protocol 1: Measurement of Covalently Bound CCl4 Metabolites to Proteins Using Radiolabeled [14C]Cl4
  • Alternate Protocol 1: Measurement of Covalent Binding by Immunoblotting
  • Alternate Protocol 2: Evaluating Covalent Binding by Immunohistochemistry
  • Reagents and Solutions
  • Commentary
  • Literature Cited
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Basic Protocol 1: Measurement of Covalently Bound CCl4 Metabolites to Proteins Using Radiolabeled [14C]Cl4

  • Rats
  • 14C‐carbon tetrachloride (5 mCi/mmol)
  • Corn oil
  • Liver samples from treated and control animals
  • Saline or 0.9% (w/v) NaCl
  • 0.9 M and 0.6 M trichloroacetic acid (TCA)
  • 80% methanol
  • Scintillation fluid (Scintiverse E, Fisher Scientific)
  • 1 M sodium hydroxide
  • Homogenizer with motor‐driven glass‐Teflon or a glass‐glass homogenizer
  • 15‐ml centrifuge tubes
  • Centrifuge
  • Agitating mixer (Thermolyne)
  • Scintillation counter (Beckman Coulter)
  • Microtiter plate reader
  • Additional reagents and equipment for estimation of total protein concentrations ( appendix 3I)

Alternate Protocol 1: Measurement of Covalent Binding by Immunoblotting

  • Protein sample
  • 10% SDS‐PAGE gel (12 × 14.5–mm, 0.75‐ mm thickness; Bio‐Rad)
  • Transfer buffer (see recipe)
  • Coomassie brilliant blue solution (see recipe)
  • Destaining solution (see recipe)
  • Blocking buffer (see recipe)
  • Methanol
  • Primary antibody specific against protein adduct of interest
  • Tris‐buffered saline (TBS; see recipe) containing 0.05% (w/v) Tween 20 (TBS/Tween; store up to 1 week at room temperature)
  • Secondary antibody: alkaline phosphatase– or horseradish peroxidase–conjugated IgG raised against the host animal used for production of the primary antibody
  • Developing buffer (see recipe)
  • Chemiluminescence‐based detection system: e.g., enhanced chemiluminescence (ECL) Kit (Amersham) or SuperSignal Kit (Pierce Chemical)
  • Trans‐Blot apparatus (Bio‐Rad) including:
    • Fiber pads
    • Transfer tank
    • Cassette gel holder
  • Glass dishes (e.g., baking dishes)
  • Transfer membranes: nitrocellulose (0.2‐ to 0.45‐µm pore size) or polyvinylidene difluoride (PVDF; 0.45‐µm pore size)
  • Whatman no. 1 filter paper
  • Platform shaker or rocker
  • Kodak X‐Omat AR or equivalent 8 × 10–in. (20.32 × 25.4‐cm) X‐ray film
  • Lead autoradiography cassette with intensifying screen
  • Additional reagents and equipment for polyacryladmide gel electrophoresis ( appendix 3F) and autoradiography ( appendix 3D)

Alternate Protocol 2: Evaluating Covalent Binding by Immunohistochemistry

  • Tissue sections (1‐mm thickness) containing protein‐xenobiotic adducts
  • 0.9% (w/v) NaCl
  • 50%, 95%, and 100% ethanol
  • Xylene
  • Tris‐buffered saline (TBS; see recipe)
  • 0.5% (w/v) pepsin in 0.1 N HCl (prepare fresh), prewarmed to 37°C
  • Nonimmune serum from species in which secondary antibody was raised
  • Primary antibody: anti‐APAP
  • TBS containing 0.5% (w/v) bovine serum albumin (BSA; TBS/BSA)
  • AS/AP Universal Rabbit Detection System (Bio‐Can Scientific; or equivalent detection system employing alkaline phosphatase–conjugated anti‐rabbit secondary antibody)
  • Double‐strength (2×) Gill's hematoxylin (Sigma)
  • Ammonium hydroxide
  • Aqueous mounting medium (Crystal Mount from BioMedia or equivalent)
  • Plastic tissue‐processing cassettes
  • Microwave oven with temperature probe
  • Programmable automated tissue processor (e.g., LYNX Automated Tissue Processor)
  • Equipment for paraffin embedding and sectioning
  • Vacuum desiccator
  • Poly‐L‐lysine‐coated glass microscope slides (unit 2.7)
  • 60°C and 80°C drying ovens
  • Coplin jars
  • Glass staining pan with metal slide rack
  • Coverslips
  • Additional reagents and equipment for paraffin embedding (Zeller, )
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Literature Cited

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