Skip to main content
SpringerLink
Log in
Book cover

In Vitro Toxicology Systems pp 501–519Cite as

Consideration of Metabolism in In Vitro Cellular Systems

  • Protocol
  • First Online:

  • 1711 Accesses

Part of the book series: Methods in Pharmacology and Toxicology ((MIPT))

Abstract

Toxicokinetic (TK) behavior of most chemicals is determined to a large extent by metabolism (xenobiotic-metabolizing enzymes). If metabolic processes were to be measured in in vitro cellular systems, it is of utmost importance to ensure that they are actually functional or at least incorporated in one way or another and at levels similar to those in vivo.

In this chapter, current and future experimental possibilities to develop and characterize cells capable of metabolism and having other important dispositional characteristics mimicking the in vivo situation are described. Consideration is also given to incorporating this information into tissue models, and some examples are presented, with special reference to the metabolic and disposition competence of the systems. In conclusion, in vitro cellular systems should be developed and validated in a much more detailed way, taking into account the early characterization of metabolism and other important kinetic processes, because the thorough characterization is the prerequisite for the validation and use of cellular systems for pharmacological and toxicological studies.

This is a preview of subscription content, log in via an institution.

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   119.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   159.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   249.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

Notes

  1. 1.

    Mentioned in an abstract of the ISSX International Conference in Istanbul, 2010 and in HepaRG product leaflets of Biopredic and Millipore.

References

  1. Coecke S, Pelkonen O, Batista Leite S et al (2013) Toxicokinetics as a key to the integrated toxicity risk assessment based primarily on non-animal approaches. Toxicol In Vitro 27:1570–1577

    Article  CAS  PubMed  Google Scholar 

  2. Coecke S, Ahr H, Blaauboer BJ et al (2006) Metabolism: a bottleneck in in vitro toxicological test development: the report and recommendations of ECVAM workshop 54. Altern Lab Anim 34:49–84

    CAS  PubMed  Google Scholar 

  3. Pelkonen O, Kapitulnik J, Gundert-Remy U, Boobis AR, Stockis A (2008) Local kinetics and dynamics of Xenobiotics. Crit Rev Toxicol 38:697–720

    Article  CAS  PubMed  Google Scholar 

  4. Park BK, Boobis A, Clarke S (2011) Managing the challenge of chemically reactive metabolites in drug development. Nat Rev Drug Discov 10:292–306

    Article  CAS  PubMed  Google Scholar 

  5. Adler S, Basketter D, Creton S et al (2011) Alternative (non-animal) methods for cosmetics testing: current status and future prospects—2010. Arch Toxicol 85:367–485

    Article  CAS  PubMed  Google Scholar 

  6. Tolonen A, Pelkonen O (2013) Analytical challenges for conducting rapid metabolism characterization for QIVIVE. Toxicology. doi:10.1016/j.tox.2013.08.010

    PubMed  Google Scholar 

  7. Hartung T (2009) Toxicology for the twenty-first century. Nature 460:208–212

    Article  CAS  PubMed  Google Scholar 

  8. Hartung T, Hoffmann S (2009) Food for thought … on in silico methods in toxicology. ALTEX 26:155–166

    PubMed  Google Scholar 

  9. Giacomini KM, Huang SM, Tweedie DJ et al (2010) International transporter consortium. Membrane transporters in drug development. Nat Rev Drug Discov 9:215–236

    Article  CAS  PubMed  Google Scholar 

  10. Wang YM, Ong SS, Chai SC, Chen T (2012) Role of CAR and PXR in xenobiotic sensing and metabolism. Expert Opin Drug Metab Toxicol 8:803–817

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  11. Wienkers LC, Heath TG (2005) Predicting in vivo drug interactions from in vitro drug discovery data. Nat Rev Drug Discov 4:825–833

    Article  CAS  PubMed  Google Scholar 

  12. Zanger UM, Turpeinen M, Klein K et al (2008) Functional pharmacogenetics/genomics of human cytochromes P450 involved in drug biotransformation. Anal Bioanal Chem 392:1093–1108

    Article  CAS  PubMed  Google Scholar 

  13. Gómez-Lechón MJ, Castell JV, Donato MT (2008) An update on metabolism studies using human hepatocytes in primary culture. Expert Opin Drug Metab Toxicol 4:837–854

    Article  PubMed  Google Scholar 

  14. Aninat C, Piton A, Glaise D et al (2006) Expression of cytochromes P450, conjugating enzymes and nuclear receptors in human hepatoma HepaRG cells. Drug Metab Dispos 34:75–83

    Article  CAS  PubMed  Google Scholar 

  15. Kanebratt KP, Andersson TB (2008) Evaluation of HepaRG cells as an in vitro model for human drug metabolism studies. Drug Metab Dispos 36:1444–1452

    Article  CAS  PubMed  Google Scholar 

  16. Turpeinen M, Tolonen A, Chesne C et al (2009) Functional expression, inhibition and induction of cytochrome P450 (CYP) enzymes in human hepatoma HepaRG cells. Toxicol In Vitro 23:748–753

    Article  CAS  PubMed  Google Scholar 

  17. Le Vee M, Jigorel E, Glaise D et al (2006) Functional expression of sinusoidal and canalicular hepatic drug transporters in the differentiated human hepatoma HepaRG cell line. Eur J Pharm Sci 28:109–117

    Article  PubMed  Google Scholar 

  18. Zanelli U, Caradonna NP, Hallifax D et al (2012) Comparison of cryopreserved HepaRG cells with cryopreserved human hepatocytes for prediction of clearance for 26 drugs. Drug Metab Dispos 40:104–110

    Article  CAS  PubMed  Google Scholar 

  19. Kanebratt KP, Andersson TB (2008) HepaRG cells as an in vitro model for evaluation of cytochrome P450 induction in humans. Drug Metab Dispos 36:137–145

    Article  CAS  PubMed  Google Scholar 

  20. Lübberstedt M, Müller-Vieira U, Mayer M et al (2011) HepaRG human hepatic cell line utility as a surrogate for primary human hepatocytes in drug metabolism assessment in vitro. J Pharmacol Toxicol Methods 63:59–68

    Article  PubMed  Google Scholar 

  21. Rogue A, Lambert C, Spire C et al (2012) Interindividual variability in gene expression profiles in human hepatocytes and comparison with HepaRG cells. Drug Metab Dispos 40:151–158

    Article  CAS  PubMed  Google Scholar 

  22. Hoekstra R, Nibourg GA, van der Hoeven TV et al (2013) Phase 1 and phase 2 drug metabolism and bile acid production of HepaRG cells in a bioartificial liver in absence of dimethyl sulfoxide. Drug Metab Dispos 41:562–567

    Article  CAS  PubMed  Google Scholar 

  23. Hart SN, Li Y, Nakamoto K et al (2010) A comparison of whole genome gene expression profiles of HepaRG cells and HepG2 cells to primary human hepatocytes and human liver tissues. Drug Metab Dispos 38:988–994

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  24. Andersson TB, Kanebratt KP, Kenna JG (2012) The HepaRG cell line: a unique in vitro tool for understanding drug metabolism and toxicology in human. Expert Opin Drug Metab Toxicol 8:909–920

    Article  CAS  PubMed  Google Scholar 

  25. McGill MR, Yan HM, Ramachandran A et al (2011) HepaRG cells: a human model to study mechanisms of acetaminophen hepatotoxicity. Hepatology 53:974–982

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  26. Park BK, Pirmohamed M, Kitteringham NR (1995) The role of cytochrome P450 enzymes in hepatic and extrahepatic human drug toxicity. Pharmacol Ther 68:385–424

    Article  CAS  PubMed  Google Scholar 

  27. Ding X, Kaminsky LS (2003) Human extrahepatic cytochromes P450: function in xenobiotic metabolism and tissue-selective chemical toxicity in the respiratory and gastrointestinal tracts. Annu Rev Pharmacol Toxicol 43:149–173

    Article  CAS  PubMed  Google Scholar 

  28. Cencic A, Langerholc T (2010) Functional cell models of the gut and their applications in food microbiology—a review. Int J Food Microbiol 31(141 Suppl 1):S4–S14

    Article  Google Scholar 

  29. Lancerholc T, Maragkoudakis PA, Wollgast J et al (2011) Novel and established intestinal cell line models—an indispensable tool in food science and nutrition. Trends Foof Sci Tech 22:11–20

    Article  Google Scholar 

  30. Hayeshi R, Hilgendorf C, Artursson P et al (2008) Comparison of drug transporter gene expression and functionality in Caco-2 cells from 10 different laboratories. Eur J Pharm Sci 18:383–396

    Article  Google Scholar 

  31. Hubatsch I, Ragnarsson EG, Artursson P (2007) Determination of drug permeability and prediction of drug absorption in Caco-2 monolayers. Nat Protoc 2:2111–2119

    Article  CAS  PubMed  Google Scholar 

  32. Wetmore BA, Wambaugh JF, Ferguson SS et al (2012) Integration of dosimetry, exposure, and high-throughput screening data in chemical toxicity assessment. Toxicol Sci 125:157–174

    Article  CAS  PubMed  Google Scholar 

  33. Korjamo T, Honkakoski P, Toppinen MR et al (2005) Absorption properties and P-glycoprotein activity of modified Caco-2 cell lines. Eur J Pharm Sci 26:266–279

    Article  CAS  PubMed  Google Scholar 

  34. Pelkonen O, Turpeinen M, Hakkola J et al (2013) Preservation, induction or incorporation of metabolism into the in vitro cellular system—views to current opportunities and limitations. Toxicol In Vitro 27:1578–1583

    Article  CAS  PubMed  Google Scholar 

  35. Soldatow VY, LeCluyse EL, Griffith LG et al (2013) In vitro models for liver toxicity testing. Toxicol Res (Camb) 2:23–39

    Article  CAS  Google Scholar 

  36. Ginai M, Elsby R, Hewitt CJ et al (2013) The use of bioreactors as in vitro models in pharmaceutical research. Drug Discov Today 18(19–20):922–935, pii: S1359-6446(13)00162-1

    Article  CAS  PubMed  Google Scholar 

  37. Zeilinger K, Schreiter T, Darnell M et al (2011) Scaling down of a clinical three-dimensional perfusion multicompartment hollow fiber liver bioreactor developed for extracorporeal liver support to an analytical scale device useful for hepatic pharmacological in vitro studies. Tissue Eng Part C Methods 17(5):549–556

    Article  CAS  PubMed  Google Scholar 

  38. Ulvestad M, Darnell M, Molden E et al (2012) Evaluation of organic anion-transporting polypeptide 1B1 and CYP3A4 activities in primary human hepatocytes and HepaRG cells cultured in a dynamic three-dimensional bioreactor system. J Pharmacol Exp Ther 343(1):145–156

    Article  CAS  PubMed  Google Scholar 

  39. Darnell M, Schreiter T, Zeilinger K et al (2011) Cytochrome P450-dependent metabolism in HepaRG cells cultured in a dynamic three-dimensional bioreactor. Drug Metab Dispos 39(7):1131–1138

    Article  CAS  PubMed  Google Scholar 

  40. Nibourg GA, Hoekstra R, van der Hoeven TV et al (2013) Increased hepatic functionality of the human hepatoma cell line HepaRG cultured in the AMC bioreactor. Int J Biochem Cell Biol 45(8):1860–1868

    Article  CAS  PubMed  Google Scholar 

  41. Gunness P, Mueller D, Shevchenko V et al (2013) 3D organotypic cultures of human HepaRG cells: a tool for in vitro toxicity studies. Toxicol Sci 33(1):67–78

    Article  Google Scholar 

  42. Takahashi K, Tanabe K, Ohnuki M et al (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131(5):861–872

    Article  CAS  PubMed  Google Scholar 

  43. Baxter MA, Rowe C, Alder J et al (2010) Generating hepatic cell lineages from pluripotent stem cells for drug toxicity screening. Stem Cell Res 5(1):4–22

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  44. Scott CW, Peters MF, Dragan YP (2013) Human induced pluripotent stem cells and their use in drug discovery for toxicity testing. Toxicol Lett 219:49–58

    Article  CAS  PubMed  Google Scholar 

  45. Jozefczuk J, Prigione A, Chavez L, Adjaye J (2011) Comparative analysis of human embryonic stem cell and induced pluripotent stem cell-derived hepatocyte-like cells reveals current drawbacks and possible strategies for improved differentiation. Stem Cells Dev 20:1259–1275

    Article  CAS  PubMed  Google Scholar 

  46. Kia R, Sison RL, Heslop J et al (2013) Stem cell-derived hepatocytes as a predictive model for drug-induced liver injury: are we there yet? Br J Clin Pharmacol 75(4):885–896

    Article  PubMed Central  PubMed  Google Scholar 

  47. Hakkola J, Tanaka E, Pelkonen O (1998) Developmental expression of cytochrome P450 enzymes in human liver. Pharmacol Toxicol 82:209–217

    Article  CAS  PubMed  Google Scholar 

  48. Takebe T, Sekine K, Enomura M et al (2013) Vascularized and functional human liver from an iPSC-derived organ bud transplant. Nature 499(7459):481–484. doi:10.1038/nature12271

    Article  CAS  PubMed  Google Scholar 

  49. Parkinson A, Ogilvie BW (2010) Biotransformation of xenobiotics. In: Klaassen CD (ed) Casarett and Doull’s toxicology: the basic science of poisons, 7th edn. McGraw-Hill, New York, pp 161–304

    Google Scholar 

  50. Pelkonen O, Tolonen A, Turpeinen M, Uusitalo J (2008) In vitro metabolism in preclinical drug development. In: Cox Gad S (ed) Preclinical development handbook: ADME and biopharmaceutical properties. Wiley, New York, pp 743–774, Chapter 21

    Chapter  Google Scholar 

  51. Pelkonen O, Turpeinen M (2007) In vitro–in vivo extrapolation of hepatic clearance: biological tools, scaling factors, model assumptions and correct concentrations. Xenobiotica 37:1066–1089

    Article  CAS  PubMed  Google Scholar 

  52. Tolonen A, Turpeinen M, Pelkonen O (2009) Liquid chromatography-mass spectrometry in in vitro drug metabolite screening. Drug Discov Today 14:120–133

    Article  CAS  PubMed  Google Scholar 

  53. Pelkonen O, Tolonen A, Korjamo T et al (2009) From known knowns to known unknowns: predicting in vivo drug metabolites. Bioanalysis 1:393–414

    Article  CAS  PubMed  Google Scholar 

  54. Turpeinen M, Ghiciuc C, Orpitoui M et al (2007) Predictive value of animal models for human cytochrome P450 (CYP)-mediated enzymes: a comparative study in vitro. Xenobiotica 37:1367–1377

    Article  CAS  PubMed  Google Scholar 

  55. Pelkonen O, Turpeinen M, Hakkola J et al (2008) Inhibition and induction of human cytochrome P450 enzymes—current status. Arch Toxicol 82:667–715

    Article  CAS  PubMed  Google Scholar 

  56. Hukkanen J (2012) Induction of cytochrome P450 enzymes: a view on human in vivo findings. Expert Rev Clin Pharmacol 5(5):569–585

    Article  CAS  PubMed  Google Scholar 

  57. Johansson I, Ingelman-Sundberg M (2011) Genetic polymorphism and toxicology—with emphasis on cytochrome p450. Toxicol Sci 120(1):1–13

    Article  CAS  PubMed  Google Scholar 

  58. Faucette SR, Hawke RL, Lecluyse EL et al (2000) Validation of bupropion hydroxylation as a selective marker of human cytochrome P450 2B6 catalytic activity. Drug Metab Dispos 28:1222–1230

    CAS  PubMed  Google Scholar 

  59. Hesse LM, Venkatakrishnan K, Court MH et al (2000) CYP2B6 mediates the in vitro hydroxylation of bupropion: potential drug interactions with other antidepressants. Drug Metab Dispos 28:1176–1183

    CAS  PubMed  Google Scholar 

  60. Walsky RL, Obach RS (2004) Validated assays for human cytochrome P450 activities. Drug Metab Dispos 32:647–660

    Article  CAS  PubMed  Google Scholar 

  61. Tolonen A, Petsalo A, Turpeinen M et al (2007) In vitro interaction cocktail assay for nine major cytochrome P450 enzymes with 13 probe reactions and a single LC/MSMS run: analytical validation and testing with monoclonal anti-CYP antibodies. J Mass Spectrom 42:960–966

    Article  CAS  PubMed  Google Scholar 

  62. Alden PG, Plumb RS, Jones MD et al (2010) A rapid ultra-performance liquid chromatography/tandem mass spectrometric methodology for the in vitro analysis of pooled and cocktail cytochrome P450 assays. Rapid Commun Mass Spectrom 24:147–154

    Article  CAS  PubMed  Google Scholar 

  63. Wu X, Wang J, Tan L et al (2012) In vitro ADME profiling using high-throughput rapid-fire mass spectrometry: cytochrome p450 inhibition and metabolic stability assays. J Biomol Screen 17:761–772

    Article  CAS  PubMed  Google Scholar 

  64. An WF, Tolliday NJ (2009) Introduction: cell-based assays for high-throughput screening. Methods Mol Biol 486:1–12

    Article  CAS  PubMed  Google Scholar 

  65. Donato MT, Gómez-Lechón MJ (2013) Fluorescence-based screening of cytochrome P450 activities in intact cells. Methods Mol Biol 987:135–148

    Article  CAS  PubMed  Google Scholar 

  66. Edwards RJ, Boobis AR, Davies DS (2003) A strategy for investigating the CYP superfamily using targeted antibodies is a paradigm for functional genomic studies. Drug Metab Dispos 31:1476–1480

    Article  CAS  PubMed  Google Scholar 

  67. Petushkova NA, Lisitsa AV (2012) Producing a one-dimensional proteomic map for human liver cytochromes p450. Methods Mol Biol 909:63–82

    CAS  PubMed  Google Scholar 

  68. Rae JM, Johnson MD, Lippman ME et al (2001) Rifampin is a selective, pleiotropic inducer of drug metabolism genes in human hepatocytes: studies with cDNA and oligonucleotide expression arrays. J Pharmacol Exp Ther 299:849–857

    CAS  PubMed  Google Scholar 

  69. Anthérieu S, Chesné C, Li R et al (2010) Stable expression, activity, and inducibility of cytochromes P450 in differentiated HepaRG cells. Drug Metab Dispos 38:516–525

    Article  PubMed  Google Scholar 

  70. Zhang T, Zhao M, Pang Y (2012) Recent progress on bioinformatics, functional genomics, and metabolomics research of cytochrome P450 and its impact on drug discovery. Curr Top Med Chem 12:1346–1355

    Article  CAS  PubMed  Google Scholar 

  71. Dopazo J (2013) Genomics and transcriptomics in drug discovery. Drug Discov Today. doi:10.1016/j.drudis.2013.06.003, pii: S1359-6446(13)00166-9

    PubMed  Google Scholar 

  72. Sevior DK, Pelkonen O, Ahokas JT (2012) Hepatocytes: the powerhouse of biotransformation. Int J Biochem Cell Biol 44:257–261

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Pharmacology and Toxicology, University of Oulu, Oulu, Finland

    Olavi Pelkonen

  2. Toxicology, School of Medical Sciences, RMIT-University, Melbourne, VIC, Australia

    Jorma T. Ahokas

  3. Department of Pharmacology and Toxicology, and Medical Research Center Oulu, Oulu University Hospital and University of Oulu, Oulu, Finland

    Jukka Hakkola & Miia Turpeinen

Authors
  1. Olavi Pelkonen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jorma T. Ahokas
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jukka Hakkola
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Miia Turpeinen
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Olavi Pelkonen .

Editor information

Editors and Affiliations

  1. European Commission Joint Research Centre, Ispra, Varese, Italy

    Anna Bal-Price

  2. Innsbruck Medical University, Innsbruck, Austria

    Paul Jennings

Rights and permissions

Reprints and permissions

Copyright information

© 2014 Springer Science+Business Media New York

About this protocol

Cite this protocol

Pelkonen, O., Ahokas, J.T., Hakkola, J., Turpeinen, M. (2014). Consideration of Metabolism in In Vitro Cellular Systems. In: Bal-Price, A., Jennings, P. (eds) In Vitro Toxicology Systems. Methods in Pharmacology and Toxicology. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0521-8_22

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-0521-8_22

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-0520-1

  • Online ISBN: 978-1-4939-0521-8

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation