In Vitro Toxicology

Reference work entry

Abstract

In recent years, considerable progress has been made in developing in vitro test systems. In vitro assays are ideal tools for rapid screening with high reproducibility and increased sensitivity, and they are relatively inexpensive, especially compared with in vivo studies. In pharmaceutical industry, there has been a dramatic increase in interest in in vitro toxicology and in the search for in vitro methods for predicting toxicological effects. The industry needs early, rapid, and robust preclinical lead optimization technology screening assays, which allow a lead series of compounds to be ranked for desirable or undesirable characteristics.

Keywords

HepG2 Cell Mitochondrial Permeability Transition Pore H9c2 Cell Primary Hepatocyte Mitochondrial Permeability Transition Pore 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.
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References and Further Reading

  1. Allen JW, Khetani SR, Bhatia SN (2005) In vitro zonation and toxicity in a hepatocytes bioreactor. Toxicol Sci 84:110–119PubMedCrossRefGoogle Scholar
  2. Alley MC, Scudiero DA, Monks A, Hursey ML, Czerwinski MJ, Fine DL et al (1988) Feasibility of drug screening with panels of human tumor cell lines using a microculture tetrazolium assay. Cancer Res 48:589–601PubMedGoogle Scholar
  3. Atterwill CK, Steele CE (1987) In vitro methods in toxicology. Cambridge University Press, CambridgeCrossRefGoogle Scholar
  4. Babich H, Borenfreund E (1992) Neutral red assay for toxicology in vitro. In: Watson RR (ed) In vitro methods of toxicology. CRC Press, Boca Raton, pp 237–251Google Scholar
  5. Bach PH, Vickers AEM, Fisher R, Baumann A, Brittebo E, Carlile DJ et al (1996) The use of tissue slices for pharmacotoxicology studies (The report and recommendations of ECVAM workshop) ATLA 24:893–923Google Scholar
  6. Barile FA (1994) Introduction to in vitro cytotoxicology mechanisms and methods. CRC Press, Boca RatonGoogle Scholar
  7. Bayliss MK, Bell JA, Wilson K, Park GR (1994) 7-Ethoxycoumarin O-deethylase kinetics in isolated rat, dog and human hepatocytes. Xenobiotica 24:231–241PubMedCrossRefGoogle Scholar
  8. Berry MN, Friend DS (1969) High yield preparation of isolated rat liver parenchymal cells. J Cell Biol 43:506–520PubMedCrossRefGoogle Scholar
  9. Bhandari N, Figueroa DJ, Lawarence JW, Gerhold DL (2008) Phospholipidosis assays in HepG2 cells and rat or rhesus hepatocytes using phospholipid probe NBD-PE. Assay Drug Dev Technol 6(3):407–419PubMedCrossRefGoogle Scholar
  10. Blattner JR, He L, Lemasters JJ (2001) Screening assays for the mitochondrial permeability transition using a fluorescence multiwell plate reader. Anal Biochem 295:220–226PubMedCrossRefGoogle Scholar
  11. Boerma M, van der Wees CGC, Wondergem J, van der Laarse A, Persoon M, van Zeeland AA, Mullenders LHF (2002) Separation of neonatal rat ventricular myocytes and non-myocytes by centrifugal elutriation. Pflugers Arch Eur J Physiol 444:452–456CrossRefGoogle Scholar
  12. Boogaard PJ, Mulder GJ, Nagelkerke JF (1989) Isolated proximal tubular cells from rat kidney as an in vitro model for studies on nephrotoxicity. Toxicol Appl Pharmacol 101(135):143Google Scholar
  13. Boom SPA, Gribnau FWJ, Russel FGM (1992) Organic cation transport and cationic drug interactions in freshly isolated proximal tubular cells of the rat. J Pharmacol Exp Ther 263(2):445–450PubMedGoogle Scholar
  14. Bugelski PJ, Atif U, Molton S, Toeg I, Lord PG, Morgan DG (2000) A strategy for primary high throughput cytotoxicity screening in pharmaceutical toxicology. Pharm Res 17(10):1265–1272PubMedCrossRefGoogle Scholar
  15. Coulet M, Eeckhoutte C, Larrieu G, Sutra JF, Hoogenboom LAP, Huveneers-Oorsprong MBM et al (1998) Comparative metabolism of thiabendazole in cultured hepatocytes from rats, rabbits, calves, pigs, and sheep, including the formation of protein-bound residues. J Agric Food Chem 46:742–748PubMedCrossRefGoogle Scholar
  16. de Kanter R et al (2002) Precision-cut organ slices as a tool to Study toxicity and metabolism of xenobiotics with special reference to non-hepatic tissues. Curr Drug Metab 3:39–59PubMedCrossRefGoogle Scholar
  17. Dong M, Novartis, Basel, SwitzerlandGoogle Scholar
  18. Dorato M, Buckley L (2007) Toxicology testing in drug discovery and development. Curr Protoc Toxicol (Suppl 31):19.1.1–19.1.35Google Scholar
  19. Dykens JA, Will Y (2007) The significance of mitochondrial toxicity testing in drug development. Drug Discov Today 12(17/18):777–785PubMedCrossRefGoogle Scholar
  20. Eckl PM, Bresgen N (2003) The cultured primary hepatocytes and its application in toxicology. J Appl Biomed 1:117–126Google Scholar
  21. Ehrich M, Sharova L (2000) In vitro methods for detecting cytotoxicity. Curr Protoc Toxicol (Suppl 3):2.6.1–2.6.27Google Scholar
  22. Ekins S et al (1996) Xenobiotic metabolism in rat, dog, and human precision-cut liver slices, freshly isolated hepatocytes, and vitrified precision-cut liver slices. Drug Metab Dispos 24(9):990–995PubMedGoogle Scholar
  23. Evans SM, Casartelli A, Herreros E, Minnick DT, Day C, George E, Westmoreland C (2001) Development of a high throughput in vitro toxicity screen predictive of high acute in vivo toxic potential. Toxicol In Vitro 15:579–584PubMedCrossRefGoogle Scholar
  24. Fu J, Gao J, Pi R, Liu P (2005) An optimized protocol for culture of cardiomocyte from neonatal rat. Cytotechnology 49:109–116CrossRefGoogle Scholar
  25. Glöckner R et al (2002) In vitro induction of cytochrome P450 2B1- and 3A1-mRNA and enzyme immunostaining in cryopreserved precision-cut rat liver slices. Toxicology 176:187–193PubMedCrossRefGoogle Scholar
  26. Gogvadze V, Orrenius S, Zhivotovsky B (2004) Analysis of mitochondrial dysfunction uring cell death. Curr Protoc Toxicol (Suppl 19):2.10.1–2.10.27Google Scholar
  27. Groneberg DA, Grosse-Siestrup C, Fischer A (2002) In vitro models to study hepatotoxicity. Toxicol Pathol 30(3):394–399PubMedCrossRefGoogle Scholar
  28. Gross CJ, Kramer JA (2007) The role of investigative molecular toxicology in early stage drug development. Expert Opin Drug Saf 2(2):147–159CrossRefGoogle Scholar
  29. Guillouzo A (1998) Liver cell models in in vitro toxicology. Environ Health Perspect 106(suppl 2):511–532PubMedCrossRefGoogle Scholar
  30. Hashemi E et al (1999) Structural and functional integrity of precision-cut liver slices in xenobiotic metabolism: a comparison of the dynamic organ and multiwell plate culture procedures. Xenebiotic 29(1):11–25CrossRefGoogle Scholar
  31. Heiskanen KM et al (1999) Mitochondrial depolarization accompanies cytochrome c release during apoptosis in PC6 cells. J Biol Chem 274:5654–5658PubMedCrossRefGoogle Scholar
  32. Hopwood J, Summers C, Barret G, Jones K, Pognan F, Kenna G (2006) Modulation of bile efflux in rat hepatocytes: a novel method for quantitation of transporter inhibition. Poster presented at SOT annual meeting, The Toxicologist -An official Journal of the Society of Toxicology, 90(1)Google Scholar
  33. Hynes J, Marroquin LD, Ogurtsov VI, Christiansen KN, Stevens GJ, Papkovsky DB, Willl Y (2006) Investigation of drug-induced mitochondrial toxicity using fluorescence-based oxygen-sensitive probes. Toxicol Sci 92(1):186–200PubMedCrossRefGoogle Scholar
  34. Hynes J, Natoli E Jr, Will Y (2009) Fluorescent pH and oxygen probes of the assessment of mitochondrial toxicity in isolated mitochondria and whole cells. Curr Protoc Toxicol (Suppl 40):2.16.1–2.16.22Google Scholar
  35. Jamieson NV, Lindell S, Sundberg R, Southard JH, Belzer FD (1988) Preservation of the canine liver for 24–48 hours using simple cold storage with UW solution. Transplantation 46:517PubMedCrossRefGoogle Scholar
  36. Jewell WT, Miller MG (1999) Comparison of human and rat metabolism of molinate in liver microsomes and slices. Drug Metab Dispos 27(7):842–847PubMedGoogle Scholar
  37. Kidambi S, Yarmush RS, Novik E, Chao P, Yarmush ML, Nahmias Y (2009) Oxygen-mediated enhancement of primary hepatocytes metabolism, functional polarization, gene expression, and drug clearance. Proc Natl Acad Sci 106(37):15714–15719PubMedCrossRefGoogle Scholar
  38. Krumdieck Tissue Slicer, Krumdieck Alabama Research and DevelopmentGoogle Scholar
  39. LeCluyse EL, Bullock PL, Parkinson A, Hochman JH (1996a) Cultured rat hepatocytes. In: Borchardt RT, Smith PL, Wilson G (eds) Models for assessing drug absorption and metabolism. Plenum, New York, pp 121–159Google Scholar
  40. LeCluyse EL, Bullock PL, Parkinson A (1996b) Strategies for restoration and maintenance of normal hepatic structure and function in long term cultures of rat hepatocytes. Adv Drug Deliv Rev 22:133–186CrossRefGoogle Scholar
  41. Louch WE, Sheehan KA, Wolska BM (2011) Methods in cardiomyocyte isolation, culture, and gene transfer. J Mol Cell Cardiol 51(3):288–298PubMedCrossRefGoogle Scholar
  42. Marroquin LD, Hynes J, Dykens JA, Jamieson JD, Will Y (2007) Circumventing the crabtree effect: replacing media glucose with galactose increases susceptibility of HepG2 cells to mitochondrial toxicants. Toxicol Sci 97(2):539–547PubMedCrossRefGoogle Scholar
  43. McKim JM Jr (2010) Building a tiered approach to in vitro predictive toxicity screening: a focus on assays with in vivo relevance. Comb Chem High Throughput Screen 13(2):188–206PubMedCrossRefGoogle Scholar
  44. McKim JM, Wilga PC, Pregenzer JF, Petrella DK (2005) A biochemical approach to in vitro toxicity testing. Pharm Discov Jan:32–36Google Scholar
  45. Mills CO, Rahman K, Coleman R, Elias E (1991) Cholyl-lysylfluorescein: synthesis, biliary excretion in vivo and during single-pass perfusion of isolated perfused rat liver. Biochim Biophys Acta I115:151–156CrossRefGoogle Scholar
  46. Mitcheson JS, Hancox JC, Levi AJ (1998) Cultured adult cardiac myocytes: future applications, culture methods, morphological and electrophysiological properties. Cardiovasc Res 39:280–300PubMedCrossRefGoogle Scholar
  47. Miyamoto S, Matsumoto A, Mori I, Horinouchi A (2009) Toxicol Mech Meth 19(8):477–485Google Scholar
  48. Monteith DK, Morgan RE, Halstead B (2006) In vitro assays and biomarkers for drug-induced phospholipidosis. Expert Opin Drug Metab Toxicol 2(5):687–696PubMedCrossRefGoogle Scholar
  49. Mudra DR, Parkinson A (2001) Preparation of hepatocytes. Curr Protoc Toxicol (Suppl 8):14.2.1–14.2.13Google Scholar
  50. Nanoyama T, Fukuda R (2008) Drug-induced phospholipidosis – pathological aspects and its prediction. J Toxicol Pathol 21:9–24CrossRefGoogle Scholar
  51. Nieminen AL, Saylor AK, Tesfai SA, Herman B, Lemasters JJ (1995) Contribution of the mitochondrial permeability transition to lethal injury after exposure of hepatocytes to t-butylhydroperoxide. Biochem J 307:99–106PubMedGoogle Scholar
  52. NIH Publication No.01-4499 (2001) Report of the international workshop on in vitro methods for assessing systemic toxicityGoogle Scholar
  53. Nioi P, Perry BK, Wang EJ, Gu YZ, Snyder RD (2007) In vitro detection of drug-induced phospholipidosis using gene expression and fluorescent phospholipid-based methodologies. Toxicol Sci 99(1):162–173PubMedCrossRefGoogle Scholar
  54. Nioi P, Pardo IDR, Snyder RD (2008) Monitoring the accumulation of fluorescently labaled phospholipids in cell cultures provides an accurate screen for drugs that induce phospholipidosis. Drug Chem Toxicol 31:5151–5528CrossRefGoogle Scholar
  55. Novic E, Maguire TJ, Chao P, Kc C, Yarmush ML (2009) A microfluidic hepatic coculture platform for cell-based drug metabolism studies. Biochem Pharmacol 10382:1–9Google Scholar
  56. O’Hare S, Atterwill CK (1995) Methods in molecular biology 43: in vitro toxicity testing procedures. Human Press, TotowaCrossRefGoogle Scholar
  57. Padda MS, Sanchez M, Akhtar AJ, Boyer JL (2011) Drug-induced cholestasis. Hepatology 53:1377–1387PubMedCrossRefGoogle Scholar
  58. Pauli-Magnus C, Meier PJ, Stieger B (2010) Genetic determinants of drug-induced cholestasis and interhepatic cholestasis of pregnancy. Semin Liver Dis 30(2):147–159PubMedCrossRefGoogle Scholar
  59. Pereira SL, Ramalho-Santos R, Branco AF, Sardao VA, Oliveira PJ, Carvalho RA (2011) Metabolic remodeling during H9c2 myoblast differentiation: relevance for in vitro toxicity studies. Cardiovasc Toxicol 11:180–190PubMedCrossRefGoogle Scholar
  60. Peters TS (2005) Do preclinical testing strategies help predict human hepatotoxic potentials? Toxicol Pathol 33:146–154PubMedCrossRefGoogle Scholar
  61. Quistorff B, Dich J, Grunnet N (1989) Preparation of isolated rat liver hepatocytes. In: Pollard JW, Walker JM (eds) Methods in molecular biology, vol 5, Animal cell culture. Humana Press, Totowa, pp 151–160Google Scholar
  62. Reasor KJ, Kacew S (2001) Drug-induced phospholipidosis: are there functional consequences? Exp Biol Med 226(9):825–830Google Scholar
  63. Rodrıguez-Enrıquez S, Juarez O, Rodrıguez-Zavala JS, Moreno-Sanchez R (2001) Multisite control of the Crabtree effect in ascites hepatoma cells. Eur J Biochem 268:2512–2519PubMedCrossRefGoogle Scholar
  64. Scaduto RC Jr, Grotyohann LW (1999) Measurement of mitochondrial membrane potential using fluorescent rhodamine derivatives. Biophysical J 76:469–477CrossRefGoogle Scholar
  65. Seddon T, Michelle I, Chnery RJ (1989) Comparitive drug metabolism of diazepam in hepatocytes isolated from man, rat, monkey and dog. Biochem Pharmacol 38:1657–1665PubMedCrossRefGoogle Scholar
  66. Seglen PO (1976) Preparation of isolated rat liver cells. In: Prescott DM (ed) Methods in cell biology, vol 19. Academic, New York, pp 29–83Google Scholar
  67. Schneider WC, Hogeboom GH (1950) Intracellular distribution of enzymes: V. Further studies on the distribution of cytochrome c in rat liver homogenates. J Biol Chem 183:123–128Google Scholar
  68. Shi R, Huang CC, Aronstam RS, Ercal N, Martin A, Huang YW (2009) N-acetylcysteine amide decreases oxidative stress but not cell death induced by doxorubicin in H9c2 cardiomyocytes. BMC Pharmacol 9:7PubMedCrossRefGoogle Scholar
  69. Sohlenius-Sternbeck AK, Floby E, Svedling M, Orzechowski A (2000) High conservation of both phase I and II drug-metabolizing activities in cryopreserved rat liver slices. Xenobiotica 30(9):891–903PubMedCrossRefGoogle Scholar
  70. Taya VKS, Wanga AS, Leowa KY, Onga MMK, Wongb KP, Boelsterli US (2005) Mitochondrial permeability transition as a source of superoxide anion induced by the nitroaromatic drug nimesulide in vitro. Free Radic Biol Med 39:949–959CrossRefGoogle Scholar
  71. Thum T, Borlak J (2000) Isolation and cultivation of Ca2+ tolerant cardiomyocytes from the adult rat: improvements and applications. Xenobiotica 30(11):1063–1077PubMedCrossRefGoogle Scholar
  72. Thum T, Borlak J (2001) Butanedione Monoxime increases the viability and yield of adult cardiomyocytes in primary cultures. Cardiovasc Toxicol 1(1):61–72PubMedCrossRefGoogle Scholar
  73. Tomizawa K, Sugano K, Yamada H, Hri I (2006) Physicochemical and cell-based approach for early screening of phospholipidosis-inducing potential. J Toxicol Sci 31(4):315–324PubMedCrossRefGoogle Scholar
  74. Valente MJ, Henrique R, Costa VL, Jeronimo C, Carvalho F, Bastos ML et al (2011) A rapid and simple procedure for the establishment of human normal and cancer renal primary cell cultures from surgical specimens. PLoS One 6(5):e19337PubMedCrossRefGoogle Scholar
  75. Walum E, Stenberg K, Jenssen D (1990) Understanding cell toxicology: principles and practice, Series in biochemistry and biotechnology. Ellis Horwood, Chichester, p 206Google Scholar
  76. Wang W, Watanabe M, Nakamura T, Kudo Y, Ochi R (1999) Properties and expression of Ca2+-activated K+ channels in H9c2 cells derived from rat ventricle. Am J Physiol 276:H1559–H1566PubMedGoogle Scholar
  77. Waymouth C, Ham RG, Chapple PJ (1981) The growth requirements of vertebrate cells in vitro. Cambridge University Press, CambridgeGoogle Scholar
  78. Will Y, Hynes J, Ogurtsov VI, Papkovsky DB (2006) Analysis of mitochondrial function using phosphorescent oxygen-sensitive probes. Nat Protoc 1(5):1–10Google Scholar
  79. Wolf KK, Vora S, Webster LO, Generaux GT, Polli JW, Brouwer K (2008) Use of rat and human sandwich-cultured hepatocytes and a cassette dosing approach to assess inhibition of bile acis transport. The Toxicologist -An official Journal of the Society of Toxicology, 102(1)Google Scholar
  80. Xu JJ, Diaz D, O’Brien PJ (2004) Application of cytotoxicity assays and pre-lethal mechanistic assays for assessment of human hepatotoxicity potential. Chem Biol Interact 150:115–128PubMedCrossRefGoogle Scholar
  81. Yashikado T, Takada T, Yamamoto T, Yamaji H, Ito K, Santa T et al (2011) Itraconazole-induced cholestasis: involvement of inhibition of bile canalicular phospholipid translocator MDR3/ABCB4. Mol Pharmacol 79:241–250CrossRefGoogle Scholar
  82. Zeilinger K, Sauer IM, Pless G, Strobel C, Rudzitis J, Wang A et al (2002) Three-dimensional co-culture of primary human liver cells in bioreactors for in vitro drug studies: effects of the initial cell quality on the long-term maintenance of hepatocyte-specific functions. Altern Lab Anim 30:525–538PubMedGoogle Scholar
  83. Zhao K, Zhao GM, Wu D, Soong Y, Birk AV, Schiller PW, Szeto HH (2004) Cell-permeable peptide antioxidants targeted to inner mitochondrial membrane inhibit mitochondrial swelling, oxidative cell death, and reperfusion injury. JBC 279(33):34682–34690CrossRefGoogle Scholar
  84. Zhou YY, Wang SQ, Zhu WZ, Chruscinski A, Kobilka BK, Ziman B, Wang S et al (2000) Culture and adenoviral infection of adult mouse cardiac myocytes: methods for cellular genetic physiology. Am J Physiol Heart Circ Physiol 279:H429–H436PubMedGoogle Scholar
  85. Zordoky BNM, El-Kadi AOS (2007) H9c2 cell line is a valuable in vitro model to study the drug metabolizing enzymes in the heart. J Pharmacol Toxicol Methods 56:317–322PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  1. 1.Sanofi Pharma Deutschland GmbHA company of the Sanofi-Aventis Group DSARFrankfurtGermany

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