Abstract
Transcriptomics is a powerful tool for high-throughput gene expression profiling. Transcriptome microarray experiments conducted with RNA isolated from hepatocytes after exposure to toxicants enable a deep insight into the molecular mechanisms of hepatotoxicity. This understanding, along with structure–activity relationships underlying hepatotoxicity, will provide a novel strategy to design cost-effective and safer therapeutics. Transcriptomics studies conducted with established hepatotoxic drugs in various in vitro and in vivo hepatotoxicity test systems have contributed to the elucidation of the mechanistic basis of liver insults, which were later on substantiated at the proteomics and metabolomics levels. The present chapter is focused on comprehensive transcriptomics of cultured primary hepatocytes treated with chemicals by applying Affymetrix microarray technology. It also describes the detailed protocol for culturing of hepatocytes, their exposure to toxicants as well as sample collection, including RNA isolation, RNA target preparation and finally the hybridization to gene chips for microarray expression analysis.
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References
Dumortier G, Cabaret W, Stamatiadis L et al (2002) Hepatic tolerance of atypical antipsychotic drugs. Encéphale 28:542–551
Kaplowitz N (2005) Idiosyncratic drug hepatotoxicity. Nat Rev Drug Discov 4:489–499
Need AC, Motulsky AG, Goldstein DB (2005) Priorities and standards in pharmacogenetic research. Nat Genet 37:671–681
Hengstler J, Hammad S, Ghallab A et al (2014) In vitro systems for hepatotoxicity testing. In: Bal-Price A, Jennings P (eds) In vitro toxicology system. Springer, New York, NY, pp 27–44
Atienzar FA, Novik EI, Gerets HH et al (2014) Predictivity of dog co-culture model, primary human hepatocytes and HepG2 cells for the detection of hepatotoxic drugs in humans. Toxicol Appl Pharmacol 275:44–61
Tolosa L, Pinto S, Donato MT et al (2012) Development of a multiparametric cell-based protocol to screen and classify the hepatotoxicity potential of drugs. Toxicol Sci 127:187–198
Soldatow VY, LeCluyse EL, Griffith LG et al (2013) In vitro models for liver toxicity testing. Toxicol Res (Camb) 2:23–39
Meganathan K, Jagtap S, Wagh V et al (2012) Identification of thalidomide-specific transcriptomics and proteomics signatures during differentiation of human embryonic stem cells. Plos ONE 7:e44228
Krug AK, Kolde R, Gaspar JA et al (2013) Human embryonic stem cell-derived test systems for developmental neurotoxicity: a transcriptomics approach. Arch Toxicol 87:123–143
Leist M, Ringwald A, Kolde R et al (2013) Test systems of developmental toxicity: state-of-the art and future perspectives. Arch Toxicol 87:2037–2042
Kienhuis AS, van de Poll MCG, Wortelboer H et al (2009) Parallelogram approach using rat-human in vitro and rat in vivo toxicogenomics predicts acetaminophen-induced hepatotoxicity in humans. Toxicol Sci 107:544–552
Liguori MJ, Anderson MG, Bukofzer S et al (2005) Microarray analysis in human hepatocytes suggests a mechanism for hepatotoxicity induced by trovafloxacin. Hepatology 41:177–186
Parmentier C, Truisi GL, Moenks K et al (2013) Transcriptomic hepatotoxicity signature of chlorpromazine after short- and long-term exposure in primary human sandwich cultures. Drug Metab Dispos 41:1835–1842
Gerets HHJ, Tilmant K, Gerin B et al (2012) Characterization of primary human hepatocytes, HepG2 cells, and HepaRG cells at the mRNA level and CYP activity in response to inducers and their predictivity for the detection of human hepatotoxins. Cell Biol Toxicol 28:69–87
Heijne WHM, Slitt AL, van Bladeren PJ et al (2004) Bromobenzene-induced hepatotoxicity at the transcriptome level. Toxicol Sci 79:411–422
Hirode M, Omura K, Kiyosawa N et al (2009) Gene expression profiling in rat liver treated with various hepatotoxic-compounds inducing coagulopathy. J Toxicol Sci 34:281–293
Kienhuis AS, Bessems JGM, Pennings JLA et al (2011) Application of toxicogenomics in hepatic systems toxicology for risk assessment: acetaminophen as a case study. Toxicol Appl Pharmacol 250:96–107
Reilly TP, Bourdi M, Brady JN et al (2001) Expression profiling of acetaminophen liver toxicity in mice using microarray technology. Biochem Biophys Res Commun 282:321–328
Heinloth AN, Irwin RD, Boorman GA et al (2004) Gene expression profiling of rat livers reveals indicators of potential adverse effects. Toxicol Sci 80:193–202
Suh SK, Jung KK, Jeong YK et al (2006) Gene expression profiling of acetaminophen induced hepatotoxicity in mice. Mol Cell Toxicol 2:236–243
Dalma-Weiszhausz DD, Warrington J, Tanimoto EY et al. (2006) The affymetrix GeneChip (R) platform: an overview. DNA microarrays part A: array platforms and wet-bench protocols methods in Enzymology 410, 3–28
http://www.affymetrix.com/support/mas/index.affx (consulted July 2014)
http://www.partek.com/pgs (consulted July 2014)
http://david.abcc.ncifcrf.gov/ (consulted July 2014)
http://vortex.cs.wayne.edu/projects.htm (consulted July 2014)
Acknowledgements
We thank Margit Henry, Tamara Rotshteyn, and Susan Rohani for the excellent technical support. This work was supported by grants from the European Union (Project ESNATS) and the German Ministry for Research (BMBF, Project SysDT).
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Shinde, V. et al. (2015). Transcriptomics of Hepatocytes Treated with Toxicants for Investigating Molecular Mechanisms Underlying Hepatotoxicity. In: Vinken, M., Rogiers, V. (eds) Protocols in In Vitro Hepatocyte Research. Methods in Molecular Biology, vol 1250. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2074-7_16
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DOI: https://doi.org/10.1007/978-1-4939-2074-7_16
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