Mechanisms of acetaminophen-induced cell death in primary human hepatocytes

Abstract

Acetaminophen (APAP) overdose is the most prevalent cause of drug-induced liver injury in western countries. Numerous studies have been conducted to investigate the mechanisms of injury after APAP overdose in various animal models; however, the importance of these mechanisms for humans remains unclear. Here we investigated APAP hepatotoxicity using freshly isolated primary human hepatocytes (PHH) from either donor livers or liver resections. PHH were exposed to 5mM, 10mM or 20mM APAP over a period of 48 hours and multiple parameters were assessed. APAP dose-dependently induced significant hepatocyte necrosis starting from 24h, which correlated with the clinical onset of human liver injury after APAP overdose. Interestingly, cellular glutathione was depleted rapidly during the first 3h. APAP also resulted in early formation of APAP-protein adducts (measured in whole cell lysate and in mitochondria) and mitochondrial dysfunction, indicated by the loss of mitochondrial membrane potential after 12h. Furthermore, APAP time-dependently triggered c-Jun N-terminal kinase (JNK) activation in the cytosol and translocation of phospho-JNK to the mitochondria. Both co-treatment and post-treatment (3h) with the JNK inhibitor SP600125 reduced JNK activation and significantly attenuated cell death at 24h and 48h after APAP. The clinical antidote N-acetylcysteine offered almost complete protection even if administered 6 hours after APAP and a partial protection when given at 15h. Conclusion: These data highlight important mechanistic events in APAP toxicity in PHH and indicate a critical role of JNK in the progression of injury after APAP in humans. The JNK pathway may represent a therapeutic target in the clinic.

Serine 574 phosphorylation alters transcriptional programming of FOXO3 by selectively enhancing apoptotic gene expression

“…Cells were isolated using a multi-step collagenase procedure as described in detail previously. 36 Media consisted of Williams' Medium E (Life Technologies, Grand Island, NY, USA) supplemented with L-glutamine (2 mM) (Life Technologies), HEPES (10 mM), insulin (10 − 7 M), dexamethasone (10 − 7 M), penicillin (100 U/ml), streptomycin (100 μg/ml) and amphotericin B (0.25 μg/ml). The hepatocytes were brought to a concentration of 0.5 × 10 6 cells/ml in Williams' Medium E, as described above, plus 5% bovine calf serum.…”

Noncoding RNAs as therapeutics for acetaminophen-induced liver injury

“…Currently, primary human hepatocytes are difficult to attain, expensive, and of very limited supply. While some newer cell lines, such as HepaRG cells recapitulate more of the facets of primary human hepatocytes (4,(21)(22)(23), they still only express a relatively small degree of hepatocyte functional genes such as drug metabolizing enzymes and xenobiotic transporters (24). Human ESCs are highly proliferative, have an indefinite life span, and could thus be used to generate on a routine basis new and useful human hepatocyte-like cells that fairly accurately resemble primary human hepatocytes.…”

“…Critical to the generation of new drugs is the continued development of our understanding of APAP-induced ALF in human patients. While a number of recent papers from our group and others have made considerable advances in understanding mechanisms of toxicity in patients and human hepatocytes (2)(3)(4)(5), there is still a lack of actionable therapeutic targets. Liver transplantation is the gold standard procedure for treating late-stage ALF but this operation is expensive and comes with additional costs of lifelong anti-rejection medication.…”

Mitochondrial dysfunction as a mechanism of drug-induced hepatotoxicity: current understanding and future perspectives

“…This indicates that acylcarnitine levels in plasma may indeed reflect mitochondrial dysfunction, not cell death. Interestingly, acylcarnitine levels did not significantly increase during APAP hepatotoxicity in patients despite the evidence that mitochondrial dysfunction is critical for APAP-induced liver injury in patients [164, 268]. However, it was shown that treatment of mice with NAC, which does not only support GSH synthesis but also mitochondrial energetics [18], strongly reduced the increase in acylcarnitine levels in mice [266].…”