Assessment of Metabolic Stability Using the Rainbow Trout (Oncorhynchus mykiss) Liver S9 Fraction

Karla Johanning1, Gregg Hancock2, Beate Escher3, Adebayo Adekola4, Mary Jo Bernhard5, Christina Cowan‐Ellsberry6, Jeanne Domoradzki7, Scott Dyer5, Curtis Eickhoff8, Michelle Embry9, Susan Erhardt10, Patrick Fitzsimmons11, Marlies Halder12, James Hill13, Dustin Holden14, Rebecca Johnson15, Sibylle Rutishauser16, Helmut Segner17, Irvin Schultz18, John Nichols11

1 KJohanning Consultancy, Pura Vida Connections LLC, Austin, Texas, 2 Waterborne Environmental Inc., Leesburg, Virginia, 3 National Research Centre for Environmental Toxicology (Entox), The University of Queensland, Brisbane, Australia, 4 BASF, Florham Park, New Jersey, 5 Miami Valley Laboratory, The Procter & Gamble Company, Cincinnati, Ohio, 6 CE2 Consulting, LLC, Cincinnati, Ohio, 7 Dow Corning Corporation, Auburn, Michigan, 8 MAXAM Analytics, Burnaby, British Columbia, Canada, 9 ILSI Health and Environmental Sciences Institute, Washington, D.C., 10 Department of Entomology, Michigan State University, East Lansing, Michigan, 11 Mid‐Continent Ecology Division, U.S. Environmental Protection Agency, Duluth, Minnesota, 12 European Commission, Joint Research Centre, Institute for Health and Consumer Protection, Validation of Alternative Methods, Ispra, Italy, 13 Spot on Sciences, Manor, Texas, 14 Department of Chemistry and Biochemistry, University of Texas at Austin, Austin, Texas, 15 Dell Inc., Round Rock, Texas, 16 Institute of Pharmacology and Toxicology, University of Zurich, Zurich, Switzerland, 17 Centre for Fish and Wildlife Health, University of Bern, Bern, Switzerland, 18 Marine Sciences Lab, Battelle Pacific Northwest National Laboratory, Sequim, Washington
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 14.10
DOI:  10.1002/0471140856.tx1410s53
Online Posting Date:  August, 2012
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Abstract

Standard protocols are given for assessing metabolic stability in rainbow trout using the liver S9 fraction. These protocols describe the isolation of S9 fractions from trout livers, evaluation of metabolic stability using a substrate depletion approach, and expression of the result as in vivo intrinsic clearance. Additional guidance is provided on the care and handling of test animals, design and interpretation of preliminary studies, and development of analytical methods. Although initially developed to predict metabolism impacts on chemical accumulation by fish, these procedures can be used to support a broad range of scientific and risk assessment activities including evaluation of emerging chemical contaminants and improved interpretation of toxicity testing results. These protocols have been designed for rainbow trout and can be adapted to other species as long as species‐specific considerations are modified accordingly (e.g., fish maintenance and incubation mixture temperature). Rainbow trout is a cold‐water species. Protocols for other species (e.g., carp, a warm‐water species) can be developed based on these procedures as long as the specific considerations are taken into account. Curr. Protoc. Toxicol. 53:14.10.1‐14.10.28. © 2012 by John Wiley & Sons, Inc.

Keywords: liver S9 fraction; rainbow trout; metabolism; in vitro assay; fish; metabolism; CYP450; phase I and II metabolism enzymes

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Rainbow Trout Liver S9 Fraction Preparation
  • Basic Protocol 2: In Vitro Determination of Metabolic Stability and Extrapolation to In Vivo Intrinsic Clearance
  • Support Protocol 1: Heat‐Denatured or Inactive Rainbow Trout Liver S9 Fraction Preparation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Rainbow Trout Liver S9 Fraction Preparation

  Materials
  • Rainbow trout (1 to 1.5 years old; 400 to 600 g body weight)
  • Tricaine methanesulphonate (MS‐222; see recipe)
  • NaHCO 3
  • Clearing buffer (solution A, see recipe)
  • Homogenization buffer (solution B, see recipe), ice cold
  • Liquid nitrogen
  • Fish net
  • 10‐liter plastic bucket
  • Fish knife
  • Paper towel or absorbent paper
  • Digital balances for 1‐ to 100‐g and 100‐ to 3000‐g quantities
  • Surgical scissors and forceps
  • Silk suture material (4/0; Roboz, cat. no. SUT‐15‐2)
  • Safety‐winged infusion needle set, 23‐G × 3/ 4‐in. (VWR, cat. no. 14229‐297)
  • 30‐ml disposable plastic syringes
  • 6‐cm glass petri dishes, pre‐chilled
  • Analytical balance (for milligram quantities)
  • 50‐, 150‐ and 250‐ml glass beakers
  • 30‐ml Wheaton Potter‐Elvehjem mortar with Teflon pestle (VWR, cat. no. 62400‐788), ice cold
  • Multi‐speed bench‐top drill press (e.g., Ryobi DP102L)
  • 50‐ml round‐bottom centrifuge tubes (e.g., Nalgene 50‐ml round‐bottom polypropylene copolymer centrifuge tubes; VWR, cat. no. 21010‐829)
  • Two‐pan balance
  • Refrigerated centrifuge (e.g., Beckman J2‐21 or J2‐MC centrifuge equipped with a fixed‐angle JA‐17 or JA‐10 rotor)
  • Pasteur pipets
  • 1.8‐ml working volume cryogenic storage tubes (e.g., Thermo Scientific Nunc; Cole‐Palmer, cat. no. EW‐03755‐10)
  • Plastic freezer bags

Basic Protocol 2: In Vitro Determination of Metabolic Stability and Extrapolation to In Vivo Intrinsic Clearance

  Materials
  • Rainbow trout liver S9 fraction (active and heat‐denatured S9 fractions, see protocol 3; prepared and frozen as described in protocol 1)
  • Commercial protein assay (e.g., Pierce BCA Protein Assay; Thermo Scientific, cat. no. 23227)
  • 100 mM potassium phosphate buffer (solution C; see recipe)
  • Nicotinamide adenine dinucleotide 2′‐phosphate, tetrasodium salt (NADPH; solution F; see recipe)
  • Uridine 5′‐diphosphoglucuronic acid, trisodium salt (UDPGA; solution G; see recipe)
  • L‐Glutathione (GSH; solution H; see recipe)
  • 3′‐Phosphoadenosine 5′‐phosphosulfate (PAPS; solution I; see recipe)
  • Test compound
  • Spiking and extraction solvents appropriate for test compound of interest (HPLC‐grade or better; see recipe)
  • Solution E: alamethicin in 2.5% methanol/97.5% solution C (see recipe)
  • 96‐well flat‐bottom plates (e.g., Thermo Scientific, Nunc; VWR, cat. no. 269620)
  • Microplate reader with UV‐visible spectrophotometer (e.g., Molecular Devices THERMOmax microplate reader)
  • pH meter (e.g., Accumet AB15+ and BioBasic pH/mV/°C meter, Fisher Scientific, cat. no. 13‐636‐AB15P)
  • Glass inserts for 96 deep‐well format (e.g., Hirschmann glass inserts, VWR, cat. no. 89022‐288) or alternative gas chromatography amber glass test tubes target DP T/S septa vials (National Scientific, cat. no. C400‐2W)
  • Parafilm
  • Shaking water bath (e.g., Lab Companion 17‐liter reciprocal shaking water bath; Cole Palmer, cat. no. EW‐12054‐00)
  • Circulating chiller for water bath (e.g., 6‐liter refrigerated circulating bath; Cole Palmer, cat. no. EW‐12108‐00)
  • Holder for 96 glass inserts (e.g., VWR, cat. no. 89022‐294)
  • 25‐ml Erlenmeyer flask
  • Vortex mixer (e.g., Thermo Scientific MaxiMix/vortex mixer, cat. no. 12‐815‐50)
  • 250‐ml beaker
  • Eppendorf Repeater Plus pipettor (e.g., Eppendorf, cat. no. 022260201)
  • Combitips for Repeater Plus pipettor (e.g., 0.2‐ml volume, Eppendorf cat. no. 022266004)
  • Thermo Scientific Nunc 96‐well cap mat (e.g., Fisher Scientific, cat. no. 12‐565‐559)
  • MultiTube vortex (e.g., Fisher Scientific, cat. no. 02‐215‐452), optional

Support Protocol 1: Heat‐Denatured or Inactive Rainbow Trout Liver S9 Fraction Preparation

  Materials
  • Rainbow trout liver S9 fraction with known protein content
  • Glass container or 16 × 100‐mm borosilicate tube with caps
  • 250‐ml glass beaker
  • Hot plate or Bunsen burner
  • Floating test tubes racks (e.g., Fisher, cat no. 14‐127‐45)
  • 15‐ml Wheaton Tenbroeck hand‐held tissue homogenizer (VWR, cat. no. 62400‐530)
  • 1.8‐ml working volume cryogenic storage tubes (e.g., Thermo Scientific Nunc; Cole‐Palmer, cat. no. EW‐03755‐10)
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Figures

Videos

Literature Cited

Literature Cited
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Supplementary Material