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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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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Basic Protocol 1: Rainbow Trout Liver S9 Fraction Preparation

  • 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

  • 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

  • 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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Literature Cited

Literature Cited
   Arnot, J.A. and Gobas, F.A.P.C. 2003. A generic QSAR for assessing the bioaccumulation potential of organic chemicals in aquatic food webs. QSAR Comb. Sci. 22:337‐345.
   Arnot, J.A., Mackay, D., and Bonnell, M. 2008. Estimating biotransformation rates in fish from laboratory data. Environ. Toxicol. Chem. 27:341‐351.
   Arnot, J.A., Meylan, W., Tunkel, J., Howard, P.H., Mackay, D., Bonnell, M., and Boethling, R.S. 2009. A quantitative structure‐activity relationship for predicting metabolic biotransformation rates for organic chemicals in fish. Environ. Toxicol. Chem. 28:1168‐1177.
   ASTM (American Society for Testing and Materials). 2007. ASTM E729 Standard Guide for Conducting Acute Toxicity Tests on Test Materials with Fishes, Macroinvertebrates, and Amphibians. ASTM, Philadelphia, Pennsylvania.
   Billard, R. 1992. Reproduction in rainbow trout: Sex differentiation, dynamics of gametogenesis, biology and preservation of gametes. Aquacult. 100:263‐298.
   Bourlier, A. and Billard, R. 1984. Delayed gametogenesis and spawning in rainbow trout (Salmo gairdneri) kept under permanent light during the first and second reproductive cycles. Aquacult. 43:259‐268.
   Buhler, D.R., Miranda, C.L., Deinzer, M.L., Griffin, D.A., and Henderson, M.C. 1997. The regiospecific hydroxylation of lauric acid by rainbow trout (Oncorhynchus mykiss) cytochrome P450s. Drug Metab. Disp. 25:1176‐1183.
   Carpenter, H.M., Fredrickson, L.S., Williams, D.E., Buhler, D.R., and Curtis, L.R. 1990. The effect of thermal acclimation on the activity of arylhydrocarbon hydroxylase in rainbow trout (Oncorhynchus mykiss). Comp. Biochem. Physiol. 97C:127‐132.
   Castle, P.J., Merdink, J.L., Okita, J.R., Wrington, S.A., and Okita, R.T. 1995. Human liver lauric acid hydroxylase activities. Drug Metab. Disp. 23:1037‐1043.
   Choi, M.H., Skipper, P.L., Wishnok, J.S., and Tannenbaum, S.R. 2005. Characterization of testosterone 11B‐hydroxylation catalyzed by human liver microsomal cytochromes P450. Drug Metab. Disp. 33:714‐718.
   Cowan‐Ellsberry, C.E., Dyer, S.D., Erhardt, S., Bernhard, M.J., Roe, A.L., Dowty, M.E., and Weisbrod, A.V. 2008. Approach for extrapolating in vitro metabolism data to refine bioconcentration factor estimates. Chemosphere 70:1804‐1817.
   Dyer, S.D., Bernhard, M.J., Cowan‐Ellsberry, C., Perdu‐Durand, E., Demmerle, S., and Cravedi, J.‐P. 2008. In vitro biotransformation of surfactants in fish. Part I: Linear alkylbenze sulfonate (C12‐LAS) and alcohol ethoxylate (C13EO8). Chemosphere 72:850‐862.
   Förlin, L. and Andersson, T. 1985. Storage conditions of rainbow trout liver cytochrome P‐450 and conjugating enzymes. Comp. Biochem. Physiol. 80B:569‐572.
   Förlin, L. and Haux, C. 1990. Sex differences in hepatic cytochrome P‐450 monoxygenase activities in rainbow trout during an annual reproductive cycle. J. Endocrinol. 124:207‐213.
   Gomez, C.F., Constantine, L., and Huggett, D.B. 2010. The influence of gill and liver metabolism on the predicted bioaconcentration of three pharmaceuticals in fish. Chemosphere 81:1189‐1195.
   Habig, W.H., Pabst, M.J., Fleischner, G., Gatmaitan, Z., Arias, I.M., and Jakoby, W.B. 1974. The identity of glutathione S‐transferase B with ligandin, a major binding protein of liver. Proc. Natl. Acad. Sci. U.S.A. 71:3879‐3882.
   Han, X., Nabb, D.L., Mingoia, R.T., and Yang, C.H. 2007. Determination of xenobiotic intrinsic clearance in freshly isolated hepatocytes from rainbow trout (Oncorhynchus mykiss) and rat and its application in bioaccumulation assessment. Environ. Sci. Technol. 41:3269‐3276.
   Han, X., Mingoia, R.T., Nabb, D.L., Yang, C.H., Snajdr, S.L., and Hoke, R.A. 2008. Xenobiotic intrinsic clearance in freshly isolated hepatocytes from rainbow trout (Oncorhynchus mykiss): Determination of trout hepatocellularity, optimization of cell concentrations and comparison of serum and serum‐free incubations. Aquat. Toxicol. 89:11‐17.
   Han, X., Nabb, D.L., Yang, C.‐H., Snajdr, S.I., and Mingoia, R.T. 2009. Liver microsomes and S9 from rainbow trout (Oncorhynchus mykiss): Comparison of basal‐level enzyme activities with rat and determination of xenobiotic intrinsic clearance in support of bioaccumulation assessment. Environ. Toxicol. Chem. 28:481‐488.
   Hänninen, O., Aito, A., and Hartiala, K. 1968. Gastrointestinal distribution of glucuronide synthesis and the relevant enzymes in the rat. Scand. J. Gastroenterol. 3:461‐464.
   Hansson, T. and Gustafsson, J.Å. 1981. Sex differences in hepatic in vitro metabolism of 4‐androstene‐3,17‐dione in rainbow trout, Salmo gairdneri. Gen. Comp. Endocr. 44:181‐188.
   Iwatsubo, T., Hirota, N., Ooie, T., Suzuki, H., Shimada, N., Chiba, K., Ishizaki, T., Green, C.E., Tyson, C.A., and Sugiyama, Y. 1997. Prediction of in vivo drug metabolism in the human liver from in vitro metabolism data. Pharmacol. Ther. 73:147‐171.
   Karr, S.W., Reinert, R.E., and Wade, A.E. 1985. The effects of temperature on the cytochrome P‐450 system of thermally acclimated bluegill. Comp. Biochem. Physiol. 80C:135‐139.
   Lindström‐Seppä, P. and Hänninen, O. 1988. Sampling and storage conditions of rainbow trout liver affects monooxygenase and conjugation enzymes. Comp. Biochem. Physiol. 89C:221‐224.
   Mano, Y., Usui, T., and Kamimura, H. 2005. In vitro inhibitory effects on non‐steroidal antiinflamatory drugs on UDPglucunosyltransferase 1A1‐catalysed estradiol 3beta‐glucuronidation in human liver microsomes. Biopharm. Drug Disp. 26:35‐39.
   Mekenyan, O.G., Dimitrov, S.D., Pavlov, T.S., and Veith, G.D. 2005. POPs: A QSAR system for developing categories for persistent, bioaccumulative and toxic chemicals and their metabolites. SAR QSAR Environ. Res. 16:103‐133.
   Nichols, J.W., Schultz, I.R., and Fitzsimmons, P.N. 2006. In vitro‐in vivo extrapolation of quantitative hepatic biotransformation data for fish. I. A review of methods, and strategies for incorporating intrinsic clearance estimates into chemical kinetic models. Aquat. Toxicol. 78:74‐90.
   Nichols, J.W., Erhardt, S., Dyer, S., James, M., Moore, M., Plotzke, K., Segner, H., Schultz, I., Thomas, K., Vasiluk, L., and Weisbrod, A. 2007. Use of in vitro absorption, distribution, metabolism, and excretion (ADME) data in bioaccumulation assessments for fish. Hum. Ecol. Risk Assess. 13:1164‐1191.
   Nichols, J.W., Bonnell, M., Dimitrov, S.D., Escher, B.I., Han, X., and Kramer, N.I. 2009. Bioaccumulation assessment using predictive approaches. Integ. Environ. Manage. Assess. 5:577‐597.
   Obach, R.S., Baxter, J.G., Liston, T.E., Silber, B.M., Jones, B.C., MacIntyre, F., Rance, D.J., and Wastall, P. 1997. The prediction of human pharmacokinetic parameters from preclinical and in vitro metabolism data. J. Pharmacol. Exp. Ther. 283‐46‐58.
   OECD (Organization for Economic Co‐operation and Development). 1996. OECD Guidelines for the Testing of Chemicals. Section 3, 305, Bioconcentration: Flow‐through fish test. Organization for Economic Co‐operation and Development, Paris, France.
   Schlenk, D., Celander, M., Gallagher, E.P., George, S., James, M., Kullman, S.W., van den Hurk, P., and Willet, K. 2008. Biotransformation in fishes. In The Toxicology of Fishes (R.T. Di Giulio, and D.E. Hinton, eds.). pp. 153‐234. CRC Press, Boca Raton, Florida.
   Shultz, I.R. and Hayton, W.L. 1999. Interspecies scaling of the bioaccumulation of lipophilic xenobiotics in fish: An example using trifluralin. Environ. Toxicol. Chem. 18:1440‐1449.
   Smeets, J.M.W., Wamsteker, J., Roth, B., Everaarts, J., and van der Berg, M. 2002. Cytochrome P4501A induction and testosterone hydroxylation in cultured hepatocytes of four fish species. Chemosphere 46:163‐172.
   Stegeman, J.J. and Chevion, M. 1980. Sex differences in cytochrome P‐450 and mixed function oxygenase activity in gonadally mature trout. Biochem. Pharmacol. 29:553‐558.
   Van Cantfort, J., Goujon, F.M., and Gielen, J.E. 1979. Benzo[a]pyrene metabolism in rat fetal hepatocytes cutlture. Improved methodology and effect of substrate concentration. Chem. Biol. Interact. 28:147‐160.
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