Determination of Metabolic Stability Using Cryopreserved Hepatocytes from Rainbow Trout (Oncorhynchus mykiss)

Kellie A. Fay1, Diane L. Nabb2, Robert T. Mingoia2, Ina Bischof3, John W. Nichols1, Helmut Segner4, Karla Johanning5, Xing Han2

1 ORD/NHEERL/Mid‐Continent Ecology Division, U.S. EPA, Duluth, Minnesota, 2 DuPont Haskell Global Centers for Health and Environmental Sciences, Newark, Delaware, 3 Fraunhofer Institute for Molecular Biology and Applied Ecology, Schmallenberg, 4 Centre for Fish and Wildlife Health, University of Bern, Bern, 5 KJ Scientific LCC, Texas Life Sciences Collaboration Center, Georgetown, Texas
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 4.42
DOI:  10.1002/0471140856.tx0442s65
Online Posting Date:  August, 2015
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Trout provide a relatively easy source of hepatocytes that can be cryopreserved and used for a range of applications including toxicity testing and determination of intrinsic clearance. Standard protocols for isolating, cryopreserving, and thawing rainbow trout hepatocytes are described, along with procedures for using fresh or cryopreserved hepatocytes to assess metabolic stability of xenobiotics in fish by means of a substrate depletion approach. Variations on these methods, troubleshooting tips, and directions for use of extrapolation factors to express results in terms of in vivo intrinsic clearance are included. These protocols have been developed for rainbow trout, but can be adapted to other fish species with appropriate considerations. © 2015 by John Wiley & Sons, Inc.

Keywords: rainbow trout; metabolic stability; biotransformation; in vitro assay; fish; hepatocytes; substrate depletion

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

Table of Contents

  • Introduction
  • Basic Protocol 1: Rainbow Trout Hepatocyte Isolation
  • Basic Protocol 2: Hepatocyte Cryopreservation
  • Basic Protocol 3: Thawing Cryopreserved Trout Hepatocytes
  • Basic Protocol 4: In Vitro Determination of Metabolic Stability and Extrapolation to In Vivo Intrinsic Clearance
  • Alternate Protocol 1: Controlled Rate Hepatocyte Cryopreservation
  • Alternate Protocol 2: In Vitro Determination of Metabolic Stability using Individual Vials for Each Time Point
  • Support Protocol 1: Cell Staining and Counting
  • Support Protocol 2: Heat‐Inactivating Cells for Use as a Negative Control
  • Support Protocol 3: Time‐Staggering
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Rainbow Trout Hepatocyte Isolation

  Materials
  • Sexually immature rainbow trout (200 to 500 g)
  • Buffer I (see recipe)
  • Buffer II (see recipe)
  • Buffer III (see recipe)
  • 70% (v/v) ethanol
  • Tricaine methanesulfonate (MS‐222; Western Chemical, Inc) anesthetic (see recipe)
  • 90% isotonic Percoll in Dulbecco's phosphate buffered saline (DPBS) (see recipe)
  • Leibovitz L‐15 medium (L‐15; Life Technologies, cat.no. 21083), pH 7.8 at 12°C
  • 10‐gallon buckets or tanks for transferring fish
  • Perfusion apparatus (Fig. ) including:
    • Recirculating water bath capable of chilling water to 12°C
    • Peristaltic pump
    • Pump tubing
    • Water‐jacketed glass coil condenser (45 × 260–mm or similar)
    • Water‐jacketed glass bubble trap with stopcock
    • Surgical platform with catch basin for blood and perfusate (or tray lined with paper towels), optional
  • Surgical instruments:
    • 3 × 3–in. weigh boats
    • Forceps
    • Large and small sharp surgical scissors
    • 21‐G × ¾ safety winged infusion set (butterfly catheter)
    • Micro‐bulldog clamps (Harvard Apparatus, cat. no. NP 52‐3258) or sutures
  • 100‐μm nylon mesh
  • 150‐ml tall glass beaker
  • Fish net
  • Digital balance (1 to 2000 g)
  • 50‐ml conical centrifuge tubes
  • Refrigerated centrifuge
  • Serological pipets
  • Additional reagents and equipment for counting cells (see protocol 7)

Basic Protocol 2: Hepatocyte Cryopreservation

  Materials
  • Isolated primary trout hepatocytes (see protocol 1)
  • Cryopreservation buffer (see recipe)
  • Cryopreservation buffer with 12% DMSO (see recipe)
  • Cryopreservation buffer with 16% DMSO (see recipe)
  • Liquid nitrogen
  • 50‐ml centrifuge tubes
  • Refrigerated centrifuge
  • 1.8‐ml cryogenic vials
  • Cryogenic container
NOTE: Keep cells and media on ice throughout entire procedure unless specifically stated otherwise.

Basic Protocol 3: Thawing Cryopreserved Trout Hepatocytes

  Materials
  • Cryogenic vials containing 1.5 ml cryopreserved hepatocytes at 10 × 106 cells/ml (see protocol 2)
  • Recovery medium (see recipe)
  • Leibovitz‐15 (L‐15) with glutamine, without phenol red, pH 7.8, ice cold
  • 50‐ml centrifuge tubes
  • Room temperature water bath
  • Refrigerated centrifuge
  • Serological pipets

Basic Protocol 4: In Vitro Determination of Metabolic Stability and Extrapolation to In Vivo Intrinsic Clearance

  Materials
  • Test chemical
  • Reference chemical
  • Stopping solution (organic solvent or acid) containing internal standard, if appropriate
  • Leibovitz‐15 (L‐15) with glutamine, without phenol red, pH 7.8, ice cold
  • Sample incubation equipment:
    • Shaking water bath with chiller
    • Shaking incubator with heating and cooling functions
    • Thermomixer block with shaking capabilities
  • 7‐ml glass vials with caps (e.g., scintillation)
  • Vial rack
  • 1.5‐ml microcentrifuge tubes
  • Timer
  • Refrigerated microcentrifuge
  • Multi‐tube vortex mixer
  • HPLC/GC sample vials

Alternate Protocol 1: Controlled Rate Hepatocyte Cryopreservation

  Materials
  • Cryogenic vials containing 1.5 ml of trout hepatocyte suspension (10 × 106 cells/ml; see protocol 2)
  • Liquid nitrogen
  • Cryogenic controlled‐rate freezer, 4°C

Alternate Protocol 2: In Vitro Determination of Metabolic Stability using Individual Vials for Each Time Point

  Materials
  • Cell suspension
  • Test chemical
  • Stopping solution
  • Shaking water bath or chilled incubator
  • 1.5‐ml or larger glass reaction vials (e.g., gas chromatography amber glass DP T/S septa vials; National Scientific, cat. no. C400‐2W)
  • Vial holder (e.g., VWR, cat. no. 89022‐294)
  • Refrigerated centrifuge
  • HPLC/GC sample vials

Support Protocol 1: Cell Staining and Counting

  Materials
  • Cell suspension
  • Leibovitz‐15 (L‐15)
  • 0.04% trypan blue solution (available commercially in 0.81% NaCl and 0.06% K 2HPO 4)
  • 1‐ml microcentrifuge tubes
  • Hemacytometer (improved Neubauer) and coverslips
  • Microscope

Support Protocol 2: Heat‐Inactivating Cells for Use as a Negative Control

  Materials
  • Cell suspension
  • Leibovitz‐15 (L‐15)
  • Heat‐safe vessel (glass)
  • Hotplate
  • Beaker
  • Graduated cylinder
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
  American Society for Testing and Materials (ASTM). 1992. E729‐88a. Standard guide for conducting acute toxicity tests with fishes, macro invertebrates, and amphibians. 1992 Annual Book of ASTM Standards, Section 11: Water and Environmental Technology. 11.04: 19103‐1187.
  Bailey, G., Selivonchick, D., and Hendricks, J. 1987. Initiation, promotion and inhibition of carcinogenesis in rainbow trout. Environ. Health Perspect. 171:147‐153.
  Bains, O.S. and Kennedy, C.J. 2005. Alterations in respiration rate of isolated rainbow trout hepatocytes exposed to the P‐glycoprotein substrate rhodamine 123. Toxicology 214:87‐98.
  Baksi, S.M. and Frazier, J.M. 1990. Isolated fish hepatocytes—Model systems for toxicology research. Aquat. Toxicol. 16:229‐256.
  Berry, M., Grivell, A., Grivell, M., and Phillips, J. 1997. Isolated hepatocytes—Past, present and future. Cell Biol. Toxicol. 13:223‐233.
  Billard, R. and Escaffre, A. 1975. Identification of spermatogenesis stages in the rainbow trout based on gonad morphology and spermiation (in French). Bull. Fr. de Pisciculture 256:111‐118.
  Blazer, V. 2002. Histopathological assessment of gonadal tissue in wild fishes. Fish Physiol. Biochem. 26:85‐1201.
  Boaru, D.A., Dragoş, N., and Schirmer, K. 2006. Microcystin‐LR induced cellular effects in mammalian and fish primary hepatocyte cultures and cell lines: A comparative study. Toxicology 218:134‐148.
  Braunbeck, T. and Segner, H. 2000. Isolation and cultivation of teleost hepatocytes. In The Hepatocyte Review (M. Berry and A. Edwards, eds.) pp. 49‐72. Kluwer Academic Publishers, Dordrecht, Netherlands.
  Brown, H.S., Griffin, M., and Houston, J.B. 2007. Evaluation of cryopreserved human hepatocytes as an alternative in vitro system to microsomes for the prediction of metabolic clearance. Drug Metab. Disposition 35:293‐301.
  Carlile, D., Zomorodi, K., and Houston, J.B. 1997. Scaling factors to relate drug metabolic clearance in hepatic microsomes, isolated hepatocytes, and the intact liver. Drug Metab. Dispos. 25:903‐911.
  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 alkylbenzene sulfonate (C12‐LAS) and alcohol ethoxylate (C13EO8). Chemosphere 72:850‐862.
  Ellesat, K.S., Tollefsen, K.‐E., Åsberg, A., Thomas, K.V., and Hylland, K. 2010. Cytotoxicity of atorvastatin and simvastatin on primary rainbow trout (Oncorhynchus mykiss) hepatocytes. Toxicol In Vitro 24:1610‐1618.
  Fay, K.A., Fitzsimmons, P.N., Hoffman, A.D., and Nichols, J.W. 2014a. Optimizing the use of rainbow trout hepatocytes for bioaccumulation assessments with fish. Xenobiotica 44:345‐351.
  Fay, K.A., Mingoia, R.T., Goeritz, I., Nabb, D.L., Hoffman, A.D., Ferrell, B.D., Peterson, H.M., Nichols, J.W., Segner, H., and Han, X. 2014b. Intra‐ and interlaboratory reliability of a cryopreserved trout hepatocyte assay for the prediction of chemical bioaccumulation potential. Environ. Sci. Technol. 48:8170‐8178.
  Finne, E.F., Cooper, G.A., Koop, B.F., Hylland, K., and Tollefsen, K.E. 2007. Toxicogenomic responses in rainbow trout (Oncorhynchus mykiss) hepatocytes exposed to model chemicals and a synthetic mixture. Aquat. Toxicol. 81:293‐303.
  Freshney, R. 1993. Culture of Animal Cells: A Manual of Basic Technique. 3rd ed. Wiley‐Liss, New York.
  Gagné, F., André, C., Turcotte, P., Gagnon, C., Sherry, J., and Talbot, A. 2013. A comparative toxicogenomic investigation of oil sand water and processed water in rainbow trout hepatocytes. Arch. Environ. Contam. Toxicol. 65:309‐323.
  Gomez, J.M., Mourot, B., Fostier, A., and Le Gac, F. 1999. Growth hormone receptors in ovary and liver during gametogenesis in female rainbow trout (Oncorhynchus mykiss). J. Reprod. Fertil. 115:275‐285.
  Gomez‐Lechon, M., Donato, M., Castell, J., and Jover, R. 2003. Human hepatocytes as a tool for studying toxicity and drug metabolism. Curr. Drug Metab. 4:292‐312.
  Han, X., Nabb, D., Mingoia, R., 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.
  Hengstler, J.G., Utesch, D.T., Steinberg, P., Platt, K.L., Diener, B., Ringler, M., Swales, N., Fischer, T., Biefang, K., Gerl, M., Bottger, T., and Oesch, F. 2000. Cryopreserved primary hepatocytes as a constantly available in vitro model for the evaluation of human and animal drug metabolism and enzyme induction. Drug Metabol. Rev. 32:81‐118.
  Hewitt, N.J., Gómez Lechón, M.J., Houston, J.B., Hallifax, D., Brown, H.S., Maurel, P., Kenna, J.G., Gustavsson, L., Lohmann, C., Skonberg, C., Guillouzo, A., Tuschl, G., Li, A.P., LeCluyse, E., Groothuis, G.M.M., and Hengstler, J.G. 2007. Primary hepatocytes: Current understanding of the regulation of metabolic enzymes and transporter proteins, and pharmaceutical practice for the use of hepatocytes in metabolism, enzyme induction, transporter, clearance, and hepatotoxicity studies. Drug Metab. Revi. 39:159‐234.
  Hildebrand, J., Bains, O., Lee, D., and Kenneday, C. 2009. Functional and energetic characterization of P‐gp‐mediated doxorubicin transport in rainbow trout (Oncorhynchus mykiss) hepatocytes. Comp. Biochem. Physiol. C Toxicol. Pharmacol. 149:65‐72.
  Hodgson, E., Wallace, A.D., Shah, R.R., Choi, K., and Joo, H. 2014. Human variation and risk assessment: Microarray and other studies utilizing human hepatocytes and human liver subcellular preparations. J. Biochem. Mol. Toxicol. 28:1‐10.
  Houston, J.B. 1994. Utility of in vitro drug metabolism data in predicting in vivo metabolic clearance. Biochem. Pharmacol. 47:1469‐1479.
  Hultman, M.T., Rundberget, J.T., and Tollefsen, K.E. 2015. Evaluation of the sensitivity, responsiveness and reproducibility of primary rainbow trout hepatocyte vitellogenin expression as a screening assay for estrogen mimics. Aquat. Toxicol. 159:233‐244.
  Ings, J.S., George, N., Peter, M.C.S., Servos, M.R., and Vijayan, M.M. 2012. Venlafaxine and atenolol disrupt epinephrine‐stimulated glucose production in rainbow trout hepatocytes. Aquat. Toxicol. 106‐107:48‐55.
  Ito, K. and Houston, J.B. 2005. Prediction of Human Drug Clearance from in Vitro and Preclinical Data Using Physiologically Based and Empirical Approaches. Pharmaceutical Research 22: 103‐112.
  Johanning, K., Hancock, G., Escher, B., Adekola, A., Bernhard, M.J., Cowan‐Ellsberry, C., Domoradzki, J., Dyer, S., Eickhoff, C., Embry, M., Erhardt, S., Fitzsimmons, P., Halder, M., Hill, J., Holden, D., Johnson, R., Rutishauser, S., Segner, H., Schultz, I., and Nichols, J. 2012. Assessment of Metabolic Stability Using the Rainbow Trout (Oncorhynchus mykiss) Liver S9 Fraction. Curr. Protoc. Toxicol. 14.10.11‐14.10.28. doi: 10.1002/0471140856.tx1410s53.
  Lacaze, E., Devaux, A., Bruneau, A., Bony, S., Sherry, J., and Gagné, F. 2014. Genotoxic potential of several naphthenic acids and a synthetic oil sands process‐affected water in rainbow trout (Oncorhynchus mykiss). Aquat. Toxicol. 152:291‐299.
  Laue, H., Gfeller, H., Jenner, K.J., Nichols, J.W., Kern, S., and Natsch, A. 2014. Predicting the bioconcentration of fragrance ingredients by rainbow trout using measured rates of in vitro intrinsic clearance. Environ. Sci. Technol. 48:9486‐9495.
  Laville, N., Aıt‐Aıssa, S., Gomez, E., Casellas, C., and Porcher, J.M. 2004. Effects of human pharmaceuticals on cytotoxicity, EROD activity and ROS production in fish hepatocytes. Toxicology 196:41‐55.
  Le Gac, F., Thomas, J.L., Mourot, B., and Loir, M. 2001. In vivo and in vitro effects of prochloraz and nonylphenol ethoxylates on trout spermatogenesis. Aquat. Toxicol. 53:187‐200.
  Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J. 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193:265‐275.
  Markell, L.K., Mingoia, R.T., Peterson, H.M., Yao, J., Waters, S.M., Finn, J.P., Nabb, D.L., and Han, X. 2014. Endocrine disruption screening by protein and gene expression of vitellogenin in freshly isolated and cryopreserved rainbow trout hepatocytes. Chem. Res. Toxicol. 27:1450‐1457.
  Massarsky, A., Labarre, J., Trudeau, V.L., and Moon, T.W. 2014. Silver nanoparticles stimulate glycogenolysis in rainbow trout (Oncorhynchus mykiss) hepatocytes. Aquat. Toxicol. 147:68‐75.
  Mingoia, R.T., Glover, K.P., Nabb, D.L., Yang, C.‐H., Snajdr, S.I., and Han, X. 2010. Cryopreserved hepatocytes from rainbow trout (Oncorhynchus mykiss): A validation study to support their application in bioaccumulation assessment. Environ. Sci. Technol. 44:3052‐3058.
  Mommsen, T., Moon, T.M., and Walsh, P. 1994. Hepatocytes: Isolation, maintenance and utilization. In Biochemistry and Molecular Biology of Fishes, vol. 3 (P. Hochachka and T. Mommsen, eds.) pp. 355‐372. Elsevier, Amsterdam.
  Mudra, D.R. and Parkinson, A. 2001. Preparation of hepatocytes. Curr. Protoc. Toxicol. 14.2.1‐14.2.13. doi: 10.1002/0471140856.tx1402s08
  Navas, J.M. and Segner, H. 2000. Antiestrogenicity of β‐naphthoflavone and PAHs in cultured rainbow trout hepatocytes: Evidence for a role of the arylhydrocarbon receptor. Aquat. Toxicol. 51:79‐92.
  Navas, J.M. and Segner, H. 2006. Vitellogenin synthesis in primary cultures of fish liver cells as endpoint for in vitro screening of the (anti)estrogenic activity of chemical substances. Aquat. Toxicol. 80:1‐22.
  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.
  Nordell, P., Svanberg, P., Bird, J., and Grime, K. 2013. Predicting metabolic clearance for drugs that are actively transported into hepatocytes: Incubational binding as a consequence of in vitro hepatocyte concentration is a key factor. Drug Metab. Dispos. 41:836‐843.
  Obach, R.S. 1999. Prediction of human clearance of twenty‐nine drugs from hepatic microsomal intrinsic clearance data: An examination of in vitro half‐life approach and nonspecific binding to microsomes. Drug Metab. Dispos. 27:1350‐1359.
  Pesonen, M. and Andersson, T.B. 1997. Fish primary hepatocyte culture; An important model for xenobiotic metabolism and toxicity studies. Aquat. Toxicol. 37:253‐267.
  Piper, R.G., McElwain, I.B., Orme, L.E., McCraren, J.P., Fowler, L.G., and Leonard, J.R. 1982. Fish Hatchery Management. U.S. Department of Interior, Fish and Wildlife Service. Washington, D.C.
  Polakof, S., Panserat, S., Craig, P.M., Martyres, D.J., Plagnes‐Juan, E., Savari, S., Aris‐Brosou, S., and Moon, T.M. 2011. The metabolic consequences of hepatic AMP‐kinase phosphorylation in rainbow trout. PLoS One 6:e20228.
  Sathiyaa, R., Campbell, T., and Vijayan, M.M. 2001. Cortisol modulates HSP90 mRNA expression in primary cultures of trout hepatocytes. Comp. Biochem. Physiol. B. Biochem. Mol. Biol. 129:679‐685.
  Seglen, P. 1976. Preparation of isolated rat liver cells. Methods Cell Biol. 13:29‐83.
  Segner, H. 1998. Isolation and primary culture of teleost hepatocytes. Comp. Biochem. Physiol. 120:71‐81.
  Segner, H. and Cravedi, J.P. 2001. Metabolic activity in primary cultures of fish hepatocytes. Altern. Lab. Anim. 29:251‐257.
  Sovadinová, I., Liedtke, A., and Schirmer, K. 2014. Effects of clofibric acid alone and in combination with 17β‐estradiol on mRNA abundance in primary hepatocytes isolated from rainbow trout. Toxicol. In Vitro 28:1106‐1116.
  Sturm, A., Ziemann, C., Hirsch‐Ernst, K.I., and Segner, H. 2001. Expression and functional activity of P‐glycoprotein in cultured hepatocytes from Oncorhynchus mykiss. Am. J. Physiol. Regul. Integr. Comp. Physiol. 281:R1119‐R1126.
  Tollefsen, K.E., Eikvar, S., Finne, E.F., Fogelberg, O., and Gregersen, I.K. 2008. Estrogenicity of alkylphenols and alkylated non‐phenolics in a rainbow trout (Oncorhynchus mykiss) primary hepatocyte culture. Ecotoxicol. Environ. Saf. 71:370‐383.
  Tyler, C.R., Sumpter, J.P., and Witthames, P.R. 1990. The dynamics of oocyte growth during vitellogenesis in the rainbow trout (Oncorhynchus mykiss). Biol. Reprod. 43:202‐209.
  Zaja, R., Munić, V., Klobučar, R.S., Ambriović‐Ristov, A., and Smital, T. 2008. Cloning and molecular characterization of apical efflux transporters (ABCB1, ABCB11 and ABCC2) in rainbow trout (Oncorhynchus mykiss) hepatocytes. Aquat. Toxicol. 90:322‐332.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library