title: Real-time monitoring of oxygen uptake in hepatic bioreactor shows CYP450-independent mitochondrial toxicity of acetaminophen and amiodarone.
abstract: Prediction of drug-induced toxicity is complicated by the failure of animal models to extrapolate human response, especially during assessment of repeated dose toxicity for cosmetic or chronic drug treatments. In this work, we present a 3D microreactor capable of maintaining metabolically active HepG2/C3A spheroids for over 28 days in vitro under stable oxygen gradients mimicking the in vivo microenvironment. Mitochondrial respiration was monitored using two-frequency phase modulation of phosphorescent microprobes embedded in the tissue. Phase modulation is focus independent and unaffected by cell death or migration. This sensitive measurement of oxygen dynamics revealed important information on the drug mechanism of action and transient subthreshold effects. Specifically, exposure to antiarrhythmic agent, amiodarone, showed that both respiration and the time to onset of mitochondrial damage were dose dependent showing a TC50 of 425 μm. Analysis showed significant induction of both phospholipidosis and microvesicular steatosis during long-term exposure. Importantly, exposure to widely used analgesic, acetaminophen, caused an immediate, reversible, dose-dependent loss of oxygen uptake followed by a slow, irreversible, dose-independent death, with a TC50 of 12.3 mM. Transient loss of mitochondrial respiration was also detected below the threshold of acetaminophen toxicity. The phenomenon was repeated in HeLa cells that lack CYP2E1 and 3A4, and was blocked by preincubation with ascorbate and TMPD. These results mark the importance of tracing toxicity effects over time, suggesting a NAPQI-independent targeting of mitochondrial complex III might be responsible for acetaminophen toxicity in extrahepatic tissues.
authors.affiliation: Fraunhofer Institute for Cell Therapy and Immunology, Branch Bioanalytics and Bioprocesses (Fraunhofer IZI-BB), Potsdam, Germany.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Prill
authors.affiliation: Grass Center for Bioengineering, The Hebrew University of Jerusalem, Jerusalem, Israel.
authors.forename: Danny
authors.initials: D
authors.lastname: Bavli
authors.affiliation: Grass Center for Bioengineering, The Hebrew University of Jerusalem, Jerusalem, Israel.
authors.forename: Gahl
authors.initials: G
authors.lastname: Levy
authors.affiliation: Grass Center for Bioengineering, The Hebrew University of Jerusalem, Jerusalem, Israel.
authors.forename: Elishai
authors.initials: E
authors.lastname: Ezra
authors.affiliation: Colibri Photonics GmbH, Potsdam, Germany.
authors.forename: Elmar
authors.initials: E
authors.lastname: Schmälzlin
authors.affiliation: Fraunhofer Institute for Cell Therapy and Immunology, Branch Bioanalytics and Bioprocesses (Fraunhofer IZI-BB), Potsdam, Germany.
authors.forename: Magnus S
authors.initials: MS
authors.lastname: Jaeger
authors.affiliation: Institute of Toxicology, University of Tuebingen, Tübingen, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Schwarz
authors.affiliation: Fraunhofer Institute for Cell Therapy and Immunology, Branch Bioanalytics and Bioprocesses (Fraunhofer IZI-BB), Potsdam, Germany.
authors.forename: Claus
authors.initials: C
authors.lastname: Duschl
authors.affiliation: Grass Center for Bioengineering, The Hebrew University of Jerusalem, Jerusalem, Israel.
authors.forename: Merav
authors.initials: M
authors.lastname: Cohen
authors.affiliation: Grass Center for Bioengineering, The Hebrew University of Jerusalem, Jerusalem, Israel. ynahmias@cs.huji.ac.il.
authors.forename: Yaakov
authors.initials: Y
authors.lastname: Nahmias
mesh_terms: D000082:Acetaminophen / Q000378:metabolism / Q000633:toxicity*; D065767:Activation, Metabolic; D000638:Amiodarone / Q000378:metabolism / Q000633:toxicity*; D018712:Analgesics, Non-Narcotic / Q000378:metabolism / Q000633:toxicity*; D000889:Anti-Arrhythmia Agents / Q000378:metabolism / Q000633:toxicity*; D015415:Biomarkers / Q000378:metabolism; D019149:Bioreactors; D060833:Cellular Microenvironment; D056486:Chemical and Drug Induced Liver Injury / Q000201:enzymology / Q000209:etiology* / Q000473:pathology; D018920:Coculture Techniques; D019392:Cytochrome P-450 CYP2E1 / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004867:Equipment Design; D056945:Hep G2 Cells; D022781:Hepatocytes / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology; D006801:Humans; D056656:Lab-On-A-Chip Devices; D008930:Mitochondria, Liver / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology; D010101:Oxygen Consumption; D018874:Spheroids, Cellular; D013997:Time Factors
chemical_list: D018712:Analgesics, Non-Narcotic; D000889:Anti-Arrhythmia Agents; D015415:Biomarkers; D000082:Acetaminophen; D019392:Cytochrome P-450 CYP2E1; D000638:Amiodarone
keywords: Acetaminophen; Amiodarone; Bioreactor; Liver on chip; Mitochondria; Oxygen uptake