Abstract
The identification of biomarkers for toxicity is becoming increasingly important for drug discovery and development. This chapter describes the preparation and utilization of primary rat hepatocytes as a cellular model of steatosis. A protocol is presented for dosing the cells with the steatosis-inducing compound amiodarone, along with the conduction of assays for measuring lipid accumulation and mitochondrial function. A differential solubility extraction procedure is also presented, which can be used for proteomic profiling analysis.
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References
Kramer JA, Sagartz JE, Morris DL (2007) The application of discovery toxicology and pathology towards the design of safer pharmaceutical lead candidates. Nat Rev Drug Discov 6(8):636–649
Weiler S, Merz M, Kullak-Ublick GA (2015) Drug-induced liver injury: the dawn of biomarkers? F1000Prime Rep 7:34. https://doi.org/10.12703/P7-34
Kullak-Ublick GA, Andrade RJ, Merz M, End P, Benesic A, Gerbes AL et al (2017) Drug-induced liver injury: recent advances in diagnosis and risk assessment. Gut 66(6):1154–1164
Mortishire-Smith RJ, Skiles GL, Lawrence JW, Spence S, Nicholls AW, Johnson B et al (2004) Use of metabonomics to identify impaired fatty acid metabolism as the mechanism of a drug-induced toxicity. Chem Res Toxicol 17(2):165–173
Meneses-Lorente G, Guest PC, Lawrence J, Muniappa N, Knowles MR, Skynner HA et al (2004) A proteomic investigation of drug-induced steatosis in rat liver. Chem Res Toxicol 17(5):605–612
Berson A, De Beco V, Lettéron P, Robin MA, Moreau C, El Kahwaji J et al (1998) Steatohepatitis-inducing drugs cause mitochondrial dysfunction and lipid peroxidation in rat hepatocytes. Gastroenterology 114(4):764–774
Strom SC, Jirtle RL, Jones RS, Novicki DL, Rosenberg MR, Novotny A, Irons G et al (1982) Isolation, culture, and transplantation of human hepatocytes. J Natl Cancer Inst 68(5):771–778
Stoddart MJ (2011) Cell viability assays: introduction. Methods Mol Biol 740:1–6
McMillian MK, Grant ER, Zhong Z, Parker JB, Li L, Zivin RA et al (2001) Nile Red binding to HepG2 cells: an improved assay for in vitro studies of hepatosteatosis. In Vitr Mol Toxicol 14(3):177–190
Gerlier D, Thomasset N (1986) Use of MTT colorimetric assay to measure cell activation. J Immunol Methods 94(1–2):57–63
Molloy MP, Herbert BR, Walsh BJ, Tyler MI, Traini M, Sanchez JC et al (1998) Extraction of membrane proteins by differential solubilization for separation using two-dimensional gel electrophoresis. Electrophoresis 19(5):837–844
Meneses-Lorente G, Watt A, Salim K, Gaskell SJ, Muniappa N, Lawrence J et al (2006) Identification of early proteomic markers for hepatic steatosis. Chem Res Toxicol 19(8):986–998
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Guest, P.C. (2019). The Use of Primary Hepatocytes in Assessment of Drug Safety and Toxicity. In: Guest, P. (eds) Pre-Clinical Models. Methods in Molecular Biology, vol 1916. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8994-2_27
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DOI: https://doi.org/10.1007/978-1-4939-8994-2_27
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Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-8993-5
Online ISBN: 978-1-4939-8994-2
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