Real-time monitoring of oxygen uptake in hepatic bioreactor shows CYP450-independent mitochondrial toxicity of acetaminophen and amiodarone

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• 作者：Sebastian Prill ; Danny Bavli ; Gahl Levy ; Elishai Ezra…
• 刊名：Archives of Toxicology
• 出版年：2016
• 出版时间：May 2016
• 年：2016
• 卷：90
• 期：5
• 页码：1181-1191
• 全文大小：2,780 KB
• 刊物主题：Pharmacology/Toxicology; Occupational Medicine/Industrial Medicine; Environmental Health; Biomedicine general;
• 出版者：Springer Berlin Heidelberg
• ISSN：1432-0738
• 卷排序：90

文摘

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.KeywordsLiver on chipAcetaminophenAmiodaroneMitochondriaOxygen uptakeBioreactor