Skip to main content
SpringerLink
Log in

The Use of Hepatocytes to Investigate Drug Toxicity

  • Protocol
  • First Online:
Hepatocytes

Part of the book series: Methods in Molecular Biology ((MIMB,volume 640))

Abstract

The liver is very active in metabolizing foreign compounds and the major target for toxicity caused by drugs. Hepatotoxicity may be the result of the drug itself or, more frequently, a result of the bioactivation process and the production of reactive metabolites. Prioritization of compounds based on human hepatotoxicity potential is currently a key unmet need in drug discovery, as it can become a major problem for several lead compounds in later stages of the drug discovery pipeline. Therefore, evaluation of potential hepatotoxicity represents a critical step in the development of new drugs. Cultured hepatocytes are increasingly used by the pharmaceutical industry for the screening of hepatotoxic potential of new molecules. Hepatocytes in culture retain hepatic key functions and constitute a valuable tool to identify chemically induced cellular damage. Their use has notably contributed to the understanding of mechanisms responsible for hepatotoxicity (disruption of cellular energy status, alteration of Ca2+ homeostasis, inhibition of transport systems, metabolic activation, oxidative stress, covalent binding, etc.). Assessment of current cytotoxicity and hepatic-specific biochemical effects is limited by the inability to measure a wide spectrum of potential mechanistic changes involved in the drug-induced toxic injury. A convenient selection of endpoints allows a multiparametric evaluation of drug toxicity. In this regard, cytomic, proteomic, toxicogenomic and metabonomic approaches help to define patterns of hepatotoxicity for early identification of potential adverse effects of the drug to the liver.

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

Access this chapter

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 139.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 179.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 219.99
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

Institutional subscriptions

References

  1. Gunawan, B.K. and Kaplowitz, N. (2007) Mechanisms of drug-induced liver disease. Clin. Liver Dis. 11, 459–475.

    PubMed  Google Scholar 

  2. Kaplowitz, N. (2005) Idiosyncratic drug hepatotoxicity. Nat. Rev. Drug Discov. 4, 489–499.

    PubMed  CAS  Google Scholar 

  3. Lee, W.M. (2003) Drug-induced hepatotoxicity. N. Engl. J. Med. 349, 474–485.

    PubMed  CAS  Google Scholar 

  4. Hussaini, S.H. and Farrington, E.A. (2007) Idiosyncratic drug-induced liver injury: an overview. Expert Opin. Drug Saf. 6, 673–684.

    PubMed  CAS  Google Scholar 

  5. Abboud, G. and Kaplowitz, N. (2007) Drug-induced liver injury. Drug Saf. 30, 277–294.

    PubMed  CAS  Google Scholar 

  6. Gómez-Lechón, M.J., Donato, M.T., Castell, J.V., and Jover, R. (2003) Human hepatocytes as a tool for studying toxicity and drug metabolism. Curr. Drug Metab. 4, 292–312.

    PubMed  Google Scholar 

  7. Dambach, D.M., Andrews, B.A., and Moulin, F. (2005) New technologies and screening strategies for hepatotoxicity: use of in vitro models. Toxicol. Pathol. 33, 17–26.

    PubMed  CAS  Google Scholar 

  8. Valet, G. (2005) Cytomics, the human cytome project and systems biology: top-down resolution of the molecular biocomplexity of organisms by single cell analysis. Cell Prolif. 38, 171–174.

    PubMed  CAS  Google Scholar 

  9. Park, K., Williams, D.P., Naisbitt, D.J., Kitteringham, N.R., and Pirmohamed, M. (2005) Investigation of toxic metabolites during drug development. Toxicol. Appl. Pharmacol. 207, 425–434.

    PubMed  Google Scholar 

  10. Walgren, J.L., Mitchell, M.D., and Thompson, D.C. (2005) Role of metabolism in drug-induced idiosyncratic hepatotoxicity. Crit. Rev. Toxicol. 35, 325–361.

    PubMed  CAS  Google Scholar 

  11. Navarro, V.J. and Senior, J.R. (2006) Drug-related hepatotoxicity. N. Engl. J. Med. 354, 731–739.

    PubMed  CAS  Google Scholar 

  12. Sanderson, J.P., Naisbitt, D.J., and Park, B.K. (2006) Role of bioactivation in drug-induced hypersensitivity reactions. AAPS J. 8, E55–E64.

    PubMed  CAS  Google Scholar 

  13. Norris, W., Paredes, A.H., and Lewis, J.H. (2008) Drug-induced liver injury in 2007. Curr. Opin. Gastroenterol. 24, 287–297.

    PubMed  CAS  Google Scholar 

  14. Ulrich, R.G., Bacon, J.A., Brass, E.P., Cramer, C.T., Petrella, D.K., and Sun, E.L. (2001) Metabolic, idiosyncratic toxicity of drugs: overview of the hepatic toxicity induced by the anxiolytic, panadiplon. Chem. Biol. Interact. 134, 251–270.

    PubMed  CAS  Google Scholar 

  15. Castell, J.V. and Castell, M. (2006) Allergic hepatitis induced by drugs. Curr. Opin. Allergy Clin. Immunol. 6, 258–265.

    PubMed  CAS  Google Scholar 

  16. Tang, W. (2007) Drug metabolite profiling and elucidation of drug-induced hepatotoxicity. Expert Opin. Drug Metab. Toxicol. 3, 407–420.

    PubMed  CAS  Google Scholar 

  17. Kalgutkar, A.S. and Soglia, J.R. (2005) Minimising the potential for metabolic activation in drug discovery. Expert Opin. Drug Metab. Toxicol. 1, 91–142.

    PubMed  CAS  Google Scholar 

  18. Ioannides, C. and Lewis, D.F. (2004) Cytochromes P450 in the bioactivation of chemicals. Curr. Top. Med. Chem. 4, 1767–1788.

    PubMed  CAS  Google Scholar 

  19. 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., 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. Rev. 39, 159–234.

    PubMed  CAS  Google Scholar 

  20. Bakke, O.M., Manocchia, M., de Abajo, F., Kaitin, K.I., and Lasagna, L. (1995). Drug safety discontinuations in the United Kingdom, the United States, and Spain from 1974 through 1993: a regulatory perspective. Clin. Pharmacol. Ther. 58, 108–117.

    PubMed  CAS  Google Scholar 

  21. Zimmerman, H.J. (1994) Hepatic injury associated with nonsteroidal anti-inflammatory drugs, in Nonsteroidal Anti-Inflammatory Drugs: Mechanisms and Clinical Uses, 2nd ed., Marcel Dekker, Inc., New York, NY.

    Google Scholar 

  22. Grillo, M.P., and Benet, L.Z. (2002) Studies on the reactivity of clofibryl-S-acyl-CoA thioester with glutathione in vitro. Drug Metab. Dispos. 30, 55–62.

    PubMed  CAS  Google Scholar 

  23. Sidenius, U., Skonberg, C., Olsen, J., and Hansen, S.H. (2004). In vitro reactivity of carboxylic acid-CoA thioesters with glutathione. Chem. Res. Toxicol. 17, 75–81.

    PubMed  CAS  Google Scholar 

  24. Jaeschke, H., Gores, G.J., Cederbaum, A.I., Hinson, J.A., Pessayre, D., and Lemasters, J.J. (2002) Mechanisms of hepatotoxicity. Toxicol. Sci. 65, 166–176.

    PubMed  CAS  Google Scholar 

  25. Cullen, J.M. (2005) Mechanistic classification of liver injury. Toxicol. Pathol. 33, 6–8

    PubMed  CAS  Google Scholar 

  26. Pumford, N.R., Halmes, N.C., and Hinson, J.A. (1997) Covalent binding of xenobiotics to specific proteins in the liver. Drug Metab. Rev. 29, 39–57.

    PubMed  CAS  Google Scholar 

  27. Shin, N.Y., Liu, Q., Stamer, S.L., and Liebler, D.C. (2007) Protein targets of reactive electrophiles in human liver microsomes. Chem. Res. Toxicol. 20, 859–867.

    PubMed  CAS  Google Scholar 

  28. Druckova, A., Mernaugh, R.L., Ham, A.J., and Marnett, L.J. (2007) Identification of the protein targets of the reactive metabolite of teucrin A in vivo in the rat. Chem. Res. Toxicol. 20, 1393–1408.

    PubMed  CAS  Google Scholar 

  29. Reddy, M.V., Storer, R.D., Laws, G.M., Armstrong, M.J., Barnum, J.E., Gara, J.P., McKnight, C.G., Skopek, T.R., Sina, J.F., DeLuca, J.G., and Galloway, S.M. (2002) Genotoxicity of naturally occurring indole compounds: correlation between covalent DNA binding and other genotoxicity tests. Environ. Mol. Mutagen. 40, 1–17.

    PubMed  CAS  Google Scholar 

  30. Boelsterli, U.A. and Lim, P.L. (2007) Mitochondrial abnormalities – a link to idiosyncratic drug hepatotoxicity? Toxicol. Appl. Pharmacol. 220, 92–107.

    PubMed  CAS  Google Scholar 

  31. Kaas, G. (2006) Mitochondrial involvement in drug-induced hepatic injury. Chem. Biol. Interact. 163, 145–159.

    Google Scholar 

  32. Kaplowitz, N. (2004) Drug-induced liver injury. Clin. Infect. Dis. 38, S44–S48.

    PubMed  Google Scholar 

  33. López, P.M., Gómez-Lechón, M.J., and Castell, J.V. (1991) Role of glucose, insulin, and glucagon in glycogen mobilization in human hepatocytes. Diabetes 40, 263–268.

    Google Scholar 

  34. Yang, R., Cao, L., Gasa, R., Brady, M.J., Sherry, A.D., and Newgard, C.B. (2002) Glycogen-targeting subunits and glucokinase differentially affect pathways of glycogen metabolism and their regulation in hepatocytes. J. Biol. Chem. 277, 1514–1523.

    PubMed  CAS  Google Scholar 

  35. Futamura, M., Hosaka, H., Kadotani, A., Shimazaki, H., Sasaki, K., Ohyama, S., Nishimura, T., Eiki, J., and Nagata, Y. (2006) An allosteric activator of glucokinase impairs the interaction of glucokinase and glucokinase regulatory protein and regulates glucose metabolism. J. Biol. Chem. 281, 37668–37674.

    PubMed  CAS  Google Scholar 

  36. Gómez-Lechón, M.J., Donato, M.T., Castell, J.V., and Jover, R. (2004) Human hepatocytes in primary culture: the choice to investigate drug metabolism in man. Curr. Drug Metab. 5, 443–462.

    PubMed  Google Scholar 

  37. Gebhardt, R., Hengstler, J.G., Müller, D., Glöckner, R., Buenning, P., Laube, B., Schmelzer, E., Ullrich, M., Utesch, D., Hewitt, N., Ringel, M., Hilz, B.R., Bader, A., Langsch, A., Koose, T., Burger, H.J., Maas, J., and Oesch, F. (2008) New hepatocyte in vitro systems for drug metabolism: metabolic capacity and recommendations for application in basic research and drug development, standard operation procedures. Drug Metab. Rev. 35, 145–213.

    Google Scholar 

  38. Gómez-Lechón, M.J., Lahoz, A., Jiménez, N., Castell, J.V., and Donato, M.T. (2006) Cryopreservation of rat, dog and human hepatocytes: influence of preculture and cryoprotectants on recovery, cytochrome P450 activities and induction upon thawing. Xenobiotica 36, 457–472.

    PubMed  Google Scholar 

  39. Hewitt, N.J., de Kanter, R., and LeCluyse, E. (2007) Induction of drug metabolizing enzymes: a survey of in vitro methodologies and interpretations used in the pharmaceutical industry – do they comply with FDA recommendations? Chem. Biol. Interact. 168, 51–65.

    PubMed  CAS  Google Scholar 

  40. Rodríguez-Antona, C., Donato, M.T., Boobis, A., Edwards, R.J., Watts, P.S., Castell, J.V., and Gómez-Lechón, M.J. (2002) Cytochrome P450 expression in human hepatocytes and hepatoma cell lines: molecular mechanisms that determine lower expression in cultured cells. Xenobiotica 32, 505–520.

    PubMed  Google Scholar 

  41. Richert, L., Liguori, M.J., Abadie, C., Heyd, B., Mantion, G., Halkic, N., and Waring, J.F. (2006) Gene expression in human hepatocytes in suspension after isolation is similar to the liver of origin, is not affected by hepatocyte cold storage and cryopreservation, but is strongly changed after hepatocyte plating. Drug Metab. Dispos. 34, 870–879.

    PubMed  CAS  Google Scholar 

  42. Hamilton, G.A., Jolley, S.L., Gilbert, D., Coon, D.J., Barros, S., and LeCluyse, E.L. (2001) Regulation of cell morphology and cytochrome P450 expression in human hepatocytes by extracellular matrix and cell–cell interactions. Cell Tissue Res. 306, 85–99.

    PubMed  CAS  Google Scholar 

  43. Guillouzo, A. (1998) Liver cell models in in vitro toxicology. Environ. Health Perspect. 106 Suppl 2, 511–532.

    PubMed  CAS  Google Scholar 

  44. Farkas, D. and Tannenbaum, S.R. (2005) In vitro methods to study chemically-induced hepatotoxicity: a literature review. Curr. Drug Metab. 6, 111–125.

    PubMed  CAS  Google Scholar 

  45. Donato, M.T., Gómez-Lechón, M.J., Jover, R., Nakamura, T., and Castell, J.V. (1998) Human hepatocyte growth factor down-regulates the expression of cytochrome P450 isozymes in human hepatocytes in primary culture. J. Pharmacol. Exp. Ther. 284, 760–767.

    PubMed  CAS  Google Scholar 

  46. Gómez-Lechón, M.J., Jover, R., Donato, T., Ponsoda, X., Rodríguez, C., Stenzel, K.G., Klocke, R., Paul, D., Guillén, I., Bort, R., and Castell, J.V. (1998) Long-term expression of differentiated functions in hepatocytes cultured in three-dimensional collagen matrix. J. Cell Physiol. 177, 553–562.

    PubMed  Google Scholar 

  47. Bi, Y.A., Kazolias, D., and Duignan, D.B. (2006) Use of cryopreserved human hepatocytes in sandwich culture to measure hepatobiliary transport. Drug Metab. Dispos. 34, 1658–1665.

    PubMed  CAS  Google Scholar 

  48. Tuschl, G., Lauer, B., and Mueller, S.O. (2008) Primary hepatocytes as a model to analyze species-specific toxicity and drug metabolism. Expert Opin. Drug Metab. Toxicol. 4, 855–870.

    PubMed  CAS  Google Scholar 

  49. Moridani, M.Y., Cheon, S.S., Khan, S., and O’Brien, P.J. (2003) Metabolic activation of 3-hydroxyanisole by isolated rat hepatocytes. Chem. Biol. Interact. 142, 317–333.

    PubMed  CAS  Google Scholar 

  50. Gómez-Lechón, M.J., Montoya, A., López, P., Donato, T., Larrauri, A., and Castell, J.V. (1988) The potential use of cultured hepatocytes in predicting the hepatotoxicity of xenobiotics. Xenobiotica 18, 725–735.

    PubMed  Google Scholar 

  51. Berson, A., De Beco, V., Lettéron, P., Robin, M.A., Moreau, C., El Kahwaji, J., Verthier, N., Feldmann, G., Fromenty, B., and Pessayre, D. (1998) Steatohepatitis-inducing drugs cause mitochondrial dysfunction and lipid peroxidation in rat hepatocytes. Gastroenterology 114, 764–774.

    PubMed  CAS  Google Scholar 

  52. Modrianský, M., Ulrichová, J., Bachleda, P., Anzenbacher, P., Anzenbacherová, E., Walterová, D., and Simánek, V. (2000) Human hepatocyte – a model for toxicological studies. Functional and biochemical characterization. Gen. Physiol. Biophys. 19(2), 223–235.

    PubMed  Google Scholar 

  53. Dvorak, Z., Ulrichova, J., Pichard-Garcia, L., Modriansky, M., and Maurel, P. (2002) Comparative effect of colchicine and colchiceine on cytotoxicity and CYP gene expression in primary human hepatocytes. Toxicol. In Vitro 16, 219–227.

    PubMed  CAS  Google Scholar 

  54. Ponsoda, X., Bort, R., Jover, R., Gómez-Lechón, M.J., and Castell, J.V. (1995) Molecular mechanism of diclofenac hepatotoxicity: association of cell injury with oxidative metabolism and decrease in ATP levels. Toxicol. In Vitro 9, 439–444.

    PubMed  CAS  Google Scholar 

  55. Kemp, D.C. and Brouwer, K.L. (2004) Viability assessment in sandwich-cultured rat hepatocytes after xenobiotic exposure. Toxicol. In Vitro 18, 869–877.

    PubMed  CAS  Google Scholar 

  56. Donato, M.T., Goethals, F., Gómez-Lechón, M.J., Deboyser, D., De Coster, I., Roberfroid, M., and Castell, J.V. (1992) Toxicity of the antitumoral drug Datelliptium in hepatic cells: use of models in vitro for the prediction of toxicity in vivo. Toxicol. In Vitro 6, 295–302.

    PubMed  CAS  Google Scholar 

  57. Shen, C., Cheng, X., Li, D., and Meng, Q. (2009) Investigation of rifampicin-induced hepatotoxicity in rat hepatocytes maintained in gel entrapment culture. Cell Biol. Toxicol. 25, 265–274.

    Google Scholar 

  58. Bouaïcha, N. and Maatouk, I. (2004) Microcystin-LR and nodularin induce intracellular glutathione alteration, reactive oxygen species production and lipid peroxidation in primary cultured rat hepatocytes. Toxicol. Lett. 148, 53–63.

    PubMed  Google Scholar 

  59. Siraki, A.G., Chan, T.S., and O’Brien, P.J. (2004) Application of quantitative structure-toxicity relationships for the comparison of the cytotoxicity of 14 p-benzoquinone congeners in primary cultured rat hepatocytes versus PC12 cells. Toxicol. Sci. 81, 148–159.

    PubMed  CAS  Google Scholar 

  60. Hynes, J., Hill, R., and Papkovsky, D.B. (2006) The use of a fluorescence-based oxygen uptake assay in the analysis of cytotoxicity. Toxicol. In Vitro 20, 785–792.

    PubMed  CAS  Google Scholar 

  61. Runnegar, M.T., Xie, C., Snider, B.B., Wallace, G.A., Weinreb, S.M., and Kuhlenkamp, J. (2002) In vitro hepatotoxicity of the cyanobacterial alkaloid cylindrospermopsin and related synthetic analogues. Toxicol. Sci. 67(1), 81–87.

    PubMed  CAS  Google Scholar 

  62. Coulet, M., Eeckhoutte, C., Larrieu, G., Sutra, J.F., Alvinerie, M., Macé, K., Pfeifer, A., Zucco, F., Stammati, A.L., De Angelis, I., Vignoli, A.L., and Galtier, P. (2000) Evidence for cytochrome P4501A2-mediated protein covalent binding of thiabendazole and for its passive intestinal transport: use of human and rabbit derived cells. Chem. Biol. Interact. 127, 109–124.

    PubMed  CAS  Google Scholar 

  63. Gil, M.L., Ramirez, M.C., Terencio, M.C., and Castell, J.V. (1995) Immunochemical detection of protein adducts in cultured human hepatocytes exposed to diclofenac. Biochim. Biophys. Acta 1272, 140–146.

    PubMed  Google Scholar 

  64. Feng, G. and Kaplowitz, N. (2002) Mechanism of staurosporine-induced apoptosis in murine hepatocytes. Am. J. Physiol. Gastrointest. Liver Physiol. 282, G825–G834.

    PubMed  CAS  Google Scholar 

  65. Conde de la Rosa, L., Schoemaker, M.H., Vrenken, T.E., Buist-Homan, M., Havinga, R., Jansen, P.L., and Moshage, H. (2006) Superoxide anions and hydrogen peroxide induce hepatocyte death by different mechanisms: involvement of JNK and ERK MAP kinases. J. Hepatol. 44, 918–929.

    Google Scholar 

  66. Pusl, T., Wild, N., Vennegeerts, T., Wimmer, R., Göke, B., Brand, S., and Rust, C. (2008) Free fatty acids sensitize hepatocytes to bile acid-induced apoptosis. Biochem. Biophys. Res. Commun. 371, 441–445.

    PubMed  CAS  Google Scholar 

  67. Igoudjil, A., Massart, J., Begriche, K., Descatoire, V., Robin, M.A., and Fromenty, B. (2008) High concentrations of stavudine impair fatty acid oxidation without depleting mitochondrial DNA in cultured rat hepatocytes. Toxicol. In Vitro 22, 887–898.

    PubMed  CAS  Google Scholar 

  68. Amacher, D.E. and Martin, B.A. (1997) Tetracycline-induced steatosis in primary canine hepatocyte cultures. Fundam. Appl. Toxicol. 40, 256–263.

    PubMed  CAS  Google Scholar 

  69. Mingoia, R.T., Nabb, D.L., Yang, C.H., and Han, X. (2007) Primary culture of rat hepatocytes in 96-well plates: effects of extracellular matrix configuration on cytochrome P450 enzyme activity and inducibility, and its application in in vitro cytotoxicity screening. Toxicol. In Vitro 21, 165–173.

    PubMed  CAS  Google Scholar 

  70. Guidance document of using in vitro data to estimate in vivo starting doses for acute toxicity. ICCVAM and NICEATM, 2001, NIH publication Nº:01-4500. http://www.epa.gov/chemrtk/pubs/general/nih2001b.pdf.

  71. Schoonen, W.G., de Roos, J.A., Westerink, W.M., and Débiton, E. (2005) Cytotoxic effects of 110 reference compounds on HepG2 cells and for 60 compounds on HeLa, ECC-1 and CHO cells. II mechanistic assays on NAD(P)H, ATP and DNA contents. Toxicol. In Vitro 19, 491–503.

    PubMed  CAS  Google Scholar 

  72. Li, P.A. (2005) Preclinical in vitro screening assays for drug-like properties. Drug Discov. Today Technol. 2, 179–185.

    CAS  Google Scholar 

  73. Bort, R., Ponsoda, X., Jover, R., Gómez-Lechón, M.J., and Castell, J.V. (1999) Diclofenac toxicity to hepatocytes: a role for drug metabolism in cell toxicity. J. Pharmacol. Exp. Ther. 288(1), 65–72.

    PubMed  CAS  Google Scholar 

  74. Jemnitz, K., Veres, Z., Monostory, K., Kóbori, L., and Vereczkey, L. (2008) Interspecies differences in acetaminophen sensitivity of human, rat, and mouse primary hepatocytes. Toxicol. In Vitro 22, 961–967.

    PubMed  CAS  Google Scholar 

  75. Gómez-Lechón, M.J., Castell, J.V., and Donato, M.T. (2008) An update on metabolism studies using human hepatocytes in primary culture. Expert Opin. Drug Metab. Toxicol. 4, 837–854.

    PubMed  Google Scholar 

  76. Xu, J.J., Diaz, D., and O’Brien, P.J. (2004) Applications of cytotoxicity assays and pre-lethal mechanistic assays for assessment of human hepatotoxicity potential. Chem. Biol. Interact. 150, 115–128.

    PubMed  CAS  Google Scholar 

  77. Gómez-Lechón, M.J., Donato, M.T., and Castell, J.V. (1997) Use of cultured hepatocytes to investigate drug metabolism and toxicity. In Vitro Toxicol. 10, 63–70.

    Google Scholar 

  78. Qu, B., Li, Q.T., Wong, K.P., Tan, T.M., and Halliwell, B. (2001) Mechanism of clofibrate hepatotoxicity: mitochondrial damage and oxidative stress in hepatocytes. Free Radic. Biol. Med. 31, 659–669.

    PubMed  CAS  Google Scholar 

  79. Höschele, D. (2006) Cell culture models for the investigation of NRTI-induced mitochondrial toxicity. Relevance for the prediction of clinical toxicity. Toxicol. In Vitro 20, 535–546.

    PubMed  Google Scholar 

  80. Nioi, P., Perry, B.K., Wang, E.J., Gu, Y.Z., and Snyder, R.D. (2007) In vitro detection of drug-induced phospholipidosis using gene expression and fluorescent phospholipid based methodologies. Toxicol. Sci. 99, 162–173.

    PubMed  CAS  Google Scholar 

  81. Rohacova, J., Marin, M.L., Martínez-Romero, A., O’Connor, J.E., Gomez-Lechon, M.J., Donato, M.T., Castell, J.V., and Miranda, M.A. (2008) Photophysical characterization and flow cytometry applications of cholylamidofluorescein, a fluorescent bile acid scaffold. Photochem. Photobiol. Sci. 7, 860–866.

    PubMed  CAS  Google Scholar 

  82. Xu, J., Ma, M., and Purcell, W.M. (2003) Characterisation of some cytotoxic endpoints using rat liver and HepG2 spheroids as in vitro models and their application in hepatotoxicity studies. I. Glucose metabolism and enzyme release as cytotoxic markers. Toxicol. Appl. Pharmacol. 189, 100–111.

    PubMed  CAS  Google Scholar 

  83. Guillouzo, A., Morel, F., Langouët, S., Maheo, K., and Rissel, M. (1997) Use of hepatocyte cultures for the study of hepatotoxic compounds. J. Hepatol. 26, 73–80.

    PubMed  CAS  Google Scholar 

  84. Milkiewicz, P., Roma, M.G., Elias, E., and Coleman, R. (2002) Pathobiology and experimental therapeutics in hepatocellular cholestasis: lessons from the hepatocyte couplet model. Clin. Sci. (Lond.) 102, 603–614.

    CAS  Google Scholar 

  85. Huerta, S., Goulet, E.J., Huerta-Yepez, S., and Livingston, E.H. (2007) Screening and detection of apoptosis. J. Surg. Res. 139, 143–156.

    PubMed  CAS  Google Scholar 

  86. Gómez-Lechón, M.J., O’Connor, E., Castell, J.V., and Jover, R. (2002) Sensitive markers used to identify compounds that trigger apoptosis in cultured hepatocytes. Toxicol. Sci. 65, 299–308.

    PubMed  Google Scholar 

  87. Gómez-Lechón, M.J., O’Connor, J.E., Lahoz, A., Castell, J.V., and Donato, M.T. (2008) Identification of apoptotic drugs: multiparametric evaluation in cultured hepatocytes. Curr. Med. Chem. 15, 2071–2085.

    PubMed  Google Scholar 

  88. Riley, R.J. and Kenna, J.G. (2004) Cellular models for ADMET predictions and evaluation of drug-drug interactions. Curr. Opin. Drug Discov. Dev. 7, 86–99.

    CAS  Google Scholar 

  89. Wang, A.G., Xia, T., Yuan, J., Yu, R.A., Yang, K.D., Chen, X.M., Qu, W., and Waalkes, M.P. (2004) Effects of phenobarbital on metabolism and toxicity of diclofenac sodium in rat hepatocytes in vitro. Food Chem. Toxicol. 42, 1647–1613.

    Google Scholar 

  90. Dvorák, Z., Zdarilová, A., Sperlíková, L., Anzenbacherová, E., Simánek, V., and Ulrichová, J. (2006) Cytotoxicity of sanguinarine in primary rat hepatocytes is attenuated by dioxin and phenobarbital. Toxicol. Lett. 165(3), 282–288.

    PubMed  Google Scholar 

  91. Shimada, M., Liu, L., Nussler, N., Jonas, S., Langrehr, J.M., Ogawa, T., Kaminishi, M., Neuhaus, P., and Nussler, A.K. (2006) Human hepatocytes are protected from ethanol-induced cytotoxicity by DADS via CYP2E1 inhibition. Toxicol. Lett. 163, 242–249.

    PubMed  CAS  Google Scholar 

  92. Ishihara, Y., Shiba, D., and Shimamoto, N. (2006) Enhancement of DMNQ-induced hepatocyte toxicity by cytochrome P450 inhibition. Toxicol. Appl. Pharmacol. 214, 109–117.

    PubMed  CAS  Google Scholar 

  93. Takakusa, H., Masumoto, H., Mitsuru, A., Okazaki, O., and Sudo, K. (2008) Markers of electrophilic stress caused by chemically reactive metabolites in human hepatocytes. Drug Metab. Dispos. 36, 816–823.

    PubMed  CAS  Google Scholar 

  94. Chan, K., Jensen, N.S., Silber, P.M., and O’Brien, P.J. (2007) Structure-activity relationships for halobenzene induced cytotoxicity in rat and human hepatocytes. Chem. Biol. Interact. 165, 165–174.

    PubMed  CAS  Google Scholar 

  95. National HumanGenome Research Institute. An overview of the Human Genome Project (2009). Available at: http://www.genome.gov/12011238.

  96. Harrigan, G.G. (2006) Metabolomics: a ‘systems’ contribution to pharmaceutical discovery and drug development. Drug Discovery World. Available at: http://www.ddw–online.com/data/pdfs/metabolomics.pdf.

  97. Harrison, P.M., Kumar, A., Lang, N., Snyder, M., and Gerst, M. (2002) A question of size: the eukaryotic proteome and the problems in defining it. Nucleic Acids Res. 30, 1083–1090.

    PubMed  CAS  Google Scholar 

  98. Yang, Y., Blomme, E.A., and Waring, J.F. (2004) Toxicogenomics in drug discovery: from preclinical studies to clinical trials. Chem. Biol. Interact. 150, 71–85.

    PubMed  CAS  Google Scholar 

  99. Uehara, T., Kiyosawa, N., Hirode, M., Omura, K., Shimizu, T., Ono, A., Mizukawa, Y., Miyagishima, T., Nagao, T., and Urushidani, T. (2008) Gene expression profiling of methapyrilene-induced hepatotoxicity in rat. J. Toxicol. Sci. 33, 37–50.

    PubMed  CAS  Google Scholar 

  100. Thum, T. and Borlak, J. (2008) Detection of early signals of hepatotoxicity by gene expression profiling studies with cultures of metabolically competent human hepatocytes. Arch. Toxicol. 82, 89–101.

    PubMed  CAS  Google Scholar 

  101. Hamadeh, H.K., Bushel, P.R., Jayadev, S., DiSorbo, O., Bennett, L., Li, L., Tennant, R., Stoll, R., Barrett, J.C., Paules, R.S., Blanchard, K., and Afshari, C.A. (2002) Prediction of compound signature using high density gene expression profiling. Toxicol. Sci. 67, 232–240.

    PubMed  CAS  Google Scholar 

  102. Hengstler, J.G., Brulport, M., Schormann, W., Bauer, A., Hermes, M., Nussler, A.K., Fandrich, F., Ruhnke, M., Ungefroren, H., Griffin, L., Bockamp, E., Oesch, F., and von Mach, M.A. (2006) Generation of human hepatocytes by stem cell technology: definition of the hepatocyte. Expert Opin. Drug Metab. Toxicol. 1, 61–74.

    Google Scholar 

  103. Barros, S.A. and Martin, R.B. (2008) Predictive toxicogenomics in preclinical discovery. Methods Mol. Biol. 460, 89–112.

    PubMed  CAS  Google Scholar 

  104. Tamura, K., Ono, A., Miyagishima, T., Nagao, T., and Urushidani, T. (2006) Profiling of gene expression in rat liver and rat primary cultured hepatocytes treated with peroxisome proliferators. J. Toxicol. Sci. 31, 471–490.

    PubMed  CAS  Google Scholar 

  105. Kikkawa, R., Fujikawa, M., Yamamoto, T., Hamada, Y., Yamada, H., and Horii, I. (2006) In vivo hepatotoxicity study of rats in comparison with in vitro hepatotoxicity screening system. J. Toxicol. Sci. 31, 23–34.

    PubMed  CAS  Google Scholar 

  106. Sawada, H., Taniguchi, K., and Takami, K. (2006) Improved toxicogenomic screening for drug-induced phospholipidosis using a multiplexed quantitative gene expression ArrayPlate assay. Toxicol. In Vitro 20, 1506–1513.

    PubMed  CAS  Google Scholar 

  107. Ning, B., Dial, S., Sun, Y., Wang, J., Yang, J., and Guo, L. (2008) Systematic and simultaneous gene profiling of 84 drug-metabolizing genes in primary human hepatocytes. J. Biomol. Screen. 13, 194–201.

    PubMed  CAS  Google Scholar 

  108. Scherf, U., Ross, D.T., Waltham, M., Smith, L.H., Lee, J.K., Tanabe, L., Kohn, K.W., Reinhold, W.C., Myers, T.G., Andrews, D.T., Scudiero, D.A., Eisen, M.B., Sausville, E.A., Pommier, Y., Botstein, D., Brown, P.O., and Weinstein, J.N. (2000) A gene expression database for the molecular pharmacology of cancer. Nat. Genet. 24, 236–244.

    PubMed  CAS  Google Scholar 

  109. Waring, J.F., Ciurlionis, R., Jolly, R.A., Heindel, M., and Ulrich, R.G. (2001) Microarray analysis of hepatotoxins in vitro reveals a correlation between gene expression profiles and mechanisms of toxicity. Toxicol. Lett. 120, 359–368.

    PubMed  CAS  Google Scholar 

  110. de Longueville, F., Atienzar, F.A., Marcq, L., Dufrane, S., Evrard, S., Wouters, L., Leroux, F., Bertholet, V., Gerin, B., Whomsley, R., Arnould, T., Remacle, J., and Canning, M. (2003) Use of a low-density microarray for studying gene expression patterns induced by hepatotoxicants on primary cultures of rat hepatocytes. Toxicol. Sci. 75, 378–392.

    PubMed  Google Scholar 

  111. Kanduc, D., Mittelman, A., Serpico, R., Sinigaglia, E., Sinha, A.A., Natale, C., Santacroce, R., Di Corcia, M.G., Lucchese, A., Dini, L., Pani, P., Santacroce, S., Simona, S., Bucci, R., and Farber, E. (2002) Cell death: apoptosis versus necrosis. Int. J. Oncol. 21, 165–170.

    PubMed  CAS  Google Scholar 

  112. Baker, T.K., Carfagna, M.A., Gao, H., Dow, E.R., Li, Q., Searfoss, G.H., and Ryan, T.P. (2001) Temporal gene expression analysis of monolayer cultured rat hepatocytes. Chem. Res. Toxicol. 14, 1218–1231.

    PubMed  CAS  Google Scholar 

  113. Tuschl, G. and Mueller, S.O. (2006) Effects of cell culture conditions on primary rat hepatocytes-cell morphology and differential gene expression. Toxicology 218, 205–215.

    PubMed  CAS  Google Scholar 

  114. Mutlib, A., Jiang, P., Atherton, J., Obert, L., Kostrubsky, S., Madore, S., and Nelson, S. (2006) Identification of potential genomic biomarkers of hepatotoxicity caused by reactive metabolites of N-methylformamide: application of stable isotope labeled compounds in toxicogenomic studies. Chem. Res. Toxicol. 19, 1270–1283.

    PubMed  CAS  Google Scholar 

  115. Kienhuis, A.S., Wortelboer, H.M., Hoflack, J.C., Moonen, E.J., Kleinjans, J.C., van Ommen, B., van Delft, J.H., and Stierum, R.H. (2006) Comparison of coumarin-induced toxicity between sandwich-cultured primary rat hepatocytes and rats in vivo: a toxicogenomics approach. Drug Metab. Dispos. 34, 2083–2090.

    PubMed  CAS  Google Scholar 

  116. Ringel, M., Oesch, F., Gerl, M., Klebach, M., Quint, M., Bader, A., Böttger, T., and Hengstler, J.G. (2002) Permissive and suppressive effects of dexamethasone on enzyme induction in hepatocyte co-cultures. Xenobiotica 32, 653–666.

    PubMed  CAS  Google Scholar 

  117. Ringel, M., von Mach, M.A., Santos, R., Feilen, P.J., Brulport, M., Hermes, M., Bauer, A.W., Schormann, W., Tanner, B., Schön, M.R., Oesch, F., and Hengstler, J.G. (2005) Hepatocytes cultured in alginate microspheres: an optimized technique to study enzyme induction. Toxicology 206, 153–167.

    PubMed  CAS  Google Scholar 

  118. Beekman, J.M., Boess, F., Hildebrand, H., Kalkuhl, A., and Suter, L. (2006) Gene expression analysis of the hepatotoxicant methapyrilene in primary rat hepatocytes: an interlaboratory study. Environ. Health Perspect. 114, 92–99.

    PubMed  CAS  Google Scholar 

  119. Horii, I. and Yamada, H. (2007) In vitro hepatotoxicity testing in the early phase of drug discovery. AATEX 14, 437–441.

    Google Scholar 

  120. Beigel, J., Fella, K., Kramer, P.J., Kroeger, M., and Hewitt, P. (2008) Genomics and proteomics analysis of cultured primary rat hepatocytes. Toxicol. In Vitro 22, 171–181.

    PubMed  CAS  Google Scholar 

  121. Collins, B.C., Clarke, A., Kitteringham, N.R., Gallagher, W.M., and Pennington, S.R. (2007) Use of proteomics for the discovery of early markers of drug toxicity. Expert Opin. Drug Metab. Toxicol. 3, 689–704.

    PubMed  CAS  Google Scholar 

  122. Fella, K., Glückmann, M., Hellmann, J., Karas, M., Kramer, P.J., and Kröger, M. (2005) Use of two-dimensional gel electrophoresis in predictive toxicology: identification of potential early protein biomarkers in chemically induced hepatocarcinogenesis. Proteomics 5, 1914–1927.

    PubMed  CAS  Google Scholar 

  123. Ruepp, S.U., Tonge, R.P., Shaw, J., Wallis, N., and Pognan, F. (2002) Genomics and proteomics analysis of acetaminophen toxicity in mouse liver. Toxicol. Sci. 65, 135–150.

    PubMed  CAS  Google Scholar 

  124. Kikkawa, R., Yamamoto, T., Fukushima, T., Yamada, H., and Horii, I. (2005) Investigation of a hepatotoxicity screening system in primary cell cultures – “what biomarkers would need to be addressed to estimate toxicity in conventional and new approaches?” J. Toxicol. Sci. 30, 61–72.

    PubMed  CAS  Google Scholar 

  125. Yamamoto, T., Kikkawa, R., Yamada, H., and Horii, I. (2005) Identification of oxidative stress-related proteins for predictive screening of hepatotoxicity using a proteomic approach. J. Toxicol. Sci. 30, 213–227.

    PubMed  CAS  Google Scholar 

  126. Yamamoto, T., Kikkawa, R., Yamada, H., and Horii, I. (2006) Investigation of proteomic biomarkers in in vivo hepatotoxicity study of rat liver: toxicity differentiation in hepatotoxicants. J. Toxicol. Sci. 31, 49–60.

    PubMed  CAS  Google Scholar 

  127. Meneses-Lorente, G., Watt, A., Salim, K., Gaskell, S.J., Muniappa, N., Lawrence, J., and Guest, P.C. (2006) Identification of early proteomic markers for hepatic steatosis. Chem. Res. Toxicol. 19, 986–998.

    PubMed  CAS  Google Scholar 

  128. Gao, A.J., Garulacan, L., Storm, S.M., Hefta, S.A., Opiteck, G.J., Lin, J.H., Moulin, F., and Dambach, D.M. (2004) Identification of in vitro protein biomarkers of idiosyncratic liver toxicity. Toxicol. In vitro 18, 533–541.

    PubMed  CAS  Google Scholar 

  129. Witzmann, F.A., Clack, J.W., Geiss, K., Hussain, S., Juhl, M.J., Rice, C.M., and Wang, C. (2002) Proteomic evaluation of cell preparation methods in primary hepatocyte cell culture. Electrophoresis 23, 2223–2232.

    PubMed  CAS  Google Scholar 

  130. Vangala, S. and Tonelli, A. (2007) Biomarkers, metabonomics, and drug development: can inborn errors of metabolism help in understanding drug toxicity? AAPS J. 9, E284–E297.

    PubMed  CAS  Google Scholar 

  131. Keun, H.C. (2006) Metabonomic modeling of drug toxicity. Pharmacol. Ther. 109, 92–106.

    PubMed  CAS  Google Scholar 

  132. Robertson, D.G., Reily, M.D., and Baker, J.D. (2007) Metabonomics in pharmaceutical discovery and development. J. Proteome Res. 6(2), 526–539.

    PubMed  CAS  Google Scholar 

  133. Lindon, J.C. and Nicholson, J.K. (2008) Spectroscopic and statistical techniques for information recovery in metabonomics and metabolomics. Annu. Rev. Anal. Chem. 1, 45–69.

    CAS  Google Scholar 

  134. Lehman-MacKeenan, L.D. and Car, B.D. (2004) Metabonomics: application in predictive and mechanistic toxicology. Toxicol. Pathol. 32, 94–95.

    Google Scholar 

  135. Ellis, D.I., Dunn, W.B., Griffin, J.L., Allwood, J.W., and Goodacre, R. (2007) Metabolic fingerprinting as a diagnostic tool. Pharmacogenomics 8, 1243–1266.

    PubMed  CAS  Google Scholar 

  136. Delaney, J., Neville, W.A., Swain, A., Miles, A., Leonard, M.S., and Waterfield, C.J. (2004) Phenylacetylglycine, a putative biomarker of phospholipidosis: its origins and relevance to phospholipid accumulation using amiodarone treated rats as a model. Biomarkers 9, 271–290.

    PubMed  CAS  Google Scholar 

  137. Mortishire-Smith, R.J., Skiles, G.L., Lawrence, J.W., Spence, S., Nicholls, A.W., Johnson, B.A., and Nicholson, J.K. (2004) Use of metabonomics to identify impaired fatty acid metabolism as the mechanism of a drug-induced toxicity. Chem. Res. Toxicol. 17, 165–173.

    PubMed  CAS  Google Scholar 

  138. Halegoua-De Marzio, D. and Navarro, V.J. (2008) Drug-induced hepatotoxicity in humans. Curr. Opin. Drug Discov. Dev. 11, 53–59.

    CAS  Google Scholar 

  139. Herrera, G., Diaz, L., Martinez-Romero, A., Gomes, A., Villamón, E., Callaghan, R.C., and O’Connor, J.E. (2007) Cytomix: a multiparametric, dynamic approach to cell research. Toxicol. In Vitro 21, 176–182.

    PubMed  CAS  Google Scholar 

  140. Gomase, V.S. and Tagore, S. (2008) Cytomics. Curr. Drug Metab. 9, 263–266.

    PubMed  CAS  Google Scholar 

  141. O’Connor, J.E., Callaghan, R.C., Escudero, M., Herrera, G., Martínez, A., Monteiro, M.C., and Montolíu, H. (2001) The relevance of flow cytometry for biochemical analysis. IUBMB Life 51, 231–239.

    PubMed  Google Scholar 

  142. Amos, W.B. and White, J.G. (2003) How the confocal laser scanning microscope entered biological research. Biol. Cell 95, 335–342.

    PubMed  CAS  Google Scholar 

  143. O’Brien, P.J., Irwin, W., Diaz, D., Howard-Cofield, E., Krejsa, C.M., Slaughter, M.R., Gao, B., Kaludercic, N., Angeline, A., Bernardi, P., Brain, P., and Hougham, C. (2006) High concordance of drug-induced human hepatotoxicity with in vitro cytotoxicity measured in a novel cell-based model using high content screening. Arch. Toxicol. 80, 580–604.

    PubMed  Google Scholar 

  144. Abraham, V.C., Towne, D.L., Waring, J.F., Warrior, U., and Burns, D.J. (2008) Application of a high-content multiparameter cytotoxicity assay to prioritize compounds based on toxicity potential in humans. J. Biomol. Screen. 13, 527–537.

    PubMed  CAS  Google Scholar 

  145. Martínez-Romero, A., Alvarez-Barrientos, A., Callaghan, R.C., Coecke, S., Arza, E., Nieto, R., Prieto, P., Torralbo, P. and O’Connor, J.E. (2004) Role of CYP2D6-dependent metabolism in the cytotoxicity of mianserin and imipramin. Cytometry 69A, 48–49.

    Google Scholar 

  146. Morelli, J.K., Buehrle, M., Pognan, F., Barone, L.R., Fieles, W., and Ciaccio, P.J. (2006) Validation of an in vitro screen for phospholipidosis using a high-content biology platform. Cell Biol. Toxicol. 22, 15–27.

    PubMed  CAS  Google Scholar 

Download references

Acknowledgements

The authors acknowledge the financial support from the ALIVE Foundation, and the European Commission, grants LSHB-CT-2004-504761, LSHB-CT-2004-512051 and LSSB-CT-2005-037499.

Author information

Authors and Affiliations

  1. Unidad de Hepatología Experimental, Centro de Investigación, Hospital La Fe, Valencia, Spain

    María José Gómez-Lechón, José V. Castell & María Teresa Donato

  2. CIBERHEPAD, FIS, Valencia, Spain

    María José Gómez-Lechón, José V. Castell & María Teresa Donato

  3. Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad de Valencia, Valencia, Spain

    José V. Castell & María Teresa Donato

Authors
  1. María José Gómez-Lechón
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. José V. Castell
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. María Teresa Donato
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to María José Gómez-Lechón .

Editor information

Editors and Affiliations

  1. INSERM U632, Labo. Physiopathologie Hépatique, Université Montpellier I, rte. Mende 1919, Montpellier, 34293, France

    Patrick Maurel

Rights and permissions

Reprints and permissions

Copyright information

© 2010 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Gómez-Lechón, M.J., Castell, J.V., Donato, M.T. (2010). The Use of Hepatocytes to Investigate Drug Toxicity. In: Maurel, P. (eds) Hepatocytes. Methods in Molecular Biology, vol 640. Humana Press. https://doi.org/10.1007/978-1-60761-688-7_21

Download citation

  • DOI: https://doi.org/10.1007/978-1-60761-688-7_21

  • Published:

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-60761-687-0

  • Online ISBN: 978-1-60761-688-7

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation