title: Development of a multiparametric cell-based protocol to screen and classify the hepatotoxicity potential of drugs.
abstract: Hepatotoxicity is a major reason for drug nonapprovals and withdrawals. The multiparametric analysis of xenobiotic toxicity at the single cells level using flow cytometry and cellular imaging-based approaches, such as high-content screening (HCS) technology, could play a key role in the detection of toxicity and the classification of compounds based on patterns of cellular injury. This study aimed to develop and validate a practical, reproducible, in vitro multiparametric cell-based protocol to assess those drugs that are potentially hepatotoxic to humans and to suggest their mechanisms of action. The assay was applied to HepG2 human cell line cultured in 96-well plates and exposed to 78 different compounds for 3 and 24 h at a range of concentrations (1-1000μM). After treatments, cells were simultaneously loaded with five fluorescent dyes showing optical compatibility and were then analyzed with the High-Content Screening Station Scan^R (Olympus). By using the new technology of HCS cell parameters associated with nuclear morphology, plasma membrane integrity, mitochondrial function, intracellular calcium concentration, and oxidative stress, indicative of prelethal cytotoxic effects and representative of different mechanisms of toxicity, were measured at the single cells level, which allows high-throughput screening. This strategy appears to identify early and late events in the hepatotoxic process and also suggests the mechanism(s) implicated in the toxicity of compounds to thereby classify them according to their degree of injury (no injury, low, moderate, and high injury).
authors.affiliation: Unidad de Hepatología Experimental, Centro de Investigación, Instituto de Investigación Sanitaria Hospital La Fe, 46009 Valencia, Spain.
authors.forename: Laia
authors.initials: L
authors.lastname: Tolosa
authors.forename: Sandra
authors.initials: S
authors.lastname: Pinto
authors.forename: M Teresa
authors.initials: MT
authors.lastname: Donato
authors.forename: Agustín
authors.initials: A
authors.lastname: Lahoz
authors.forename: José V
authors.initials: JV
authors.lastname: Castell
authors.forename: J Enrique
authors.initials: JE
authors.lastname: O'Connor
authors.forename: M José
authors.initials: MJ
authors.lastname: Gómez-Lechón
mesh_terms: D000826:Animal Testing Alternatives; D020013:Calcium Signaling / Q000187:drug effects; D002463:Cell Membrane Permeability / Q000187:drug effects; D002467:Cell Nucleus / Q000187:drug effects; D056486:Chemical and Drug Induced Liver Injury; D056945:Hep G2 Cells; D022781:Hepatocytes / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D008930:Mitochondria, Liver / Q000187:drug effects; D018384:Oxidative Stress / Q000187:drug effects; D011237:Predictive Value of Tests; D015203:Reproducibility of Results; D018675:Toxicity Tests / Q000379:methods*; D015262:Xenobiotics / Q000145:classification / Q000633:toxicity*
chemical_list: D015262:Xenobiotics