Mitochondrial permeability transition in acetaminophen-induced necrosis and apoptosis of cultured mouse hepatocytes

Abstract

Acetaminophen overdose causes massive hepatic failure via mechanisms involving glutathione depletion, oxidative stress, and mitochondrial dysfunction. The ultimate target of acetaminophen causing cell death remains uncertain, and the role of apoptosis in acetaminophen-induced cell killing is still controversial. Our aim was to evaluate the mitochondrial permeability transition (MPT) as a key factor in acetaminophen-induced necrotic and apoptotic killing of primary cultured mouse hepatocytes. After administration of 10 mmol/L acetaminophen, necrotic killing increased to more than 49% and 74%, respectively, after 6 and 16 hours. MPT inhibitors, cyclosporin A (CsA), and NIM811 temporarily decreased necrotic killing after 6 hours to 26%, but cytoprotection was lost after 16 hours. Confocal microscopy revealed mitochondrial depolarization and inner membrane permeabilization approximately 4.5 hours after acetaminophen administration. CsA delayed these changes, indicative of the MPT, to approximately 11 hours after acetaminophen administration. Apoptosis indicated by nuclear changes, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, and caspase-3 activation also increased after acetaminophen administration. Fructose (20 mmol/L, an adenosine triphosphategenerating glycolytic substrate) plus glycine (5 mmol/L, a membrane stabilizing amino acid) prevented nearly all necrotic cell killing but paradoxically increased apoptosis from 37% to 59% after 16 hours. In the presence of fructose plus glycine, CsA decreased apoptosis and delayed but did not prevent the MPT. In conclusion, after acetaminophen a CsA-sensitive MPT occurred after 3 to 6 hours followed by a CsA-insensitive MPT 9 to 16 hours after acetaminophen. The MPT then induces ATP depletion-dependent necrosis or caspase-dependent apoptosis as determined, in part, by ATP availability from glycolysis.

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

“…This is an abrupt increase in permeability of the mitochondrial inner membrane in response to excessive oxidative stress, which causes the leakage of mitochondrial contents up to a size of 1.5 kDa into the cytosol [56]. Induction of the MPT results in a loss of the mitochondrial membrane potential due to dissipation of the proton gradient and accompanying collapse of mitochondrial ATP production, ultimately resulting in cell necrosis [57–60]. It is generally agreed on that the MPT is caused by the opening of a large channel (mitochondrial permeability transition pore), which is composed of various components, including Bax and Bak [61, 62] on the mitochondrial outer membrane and subunits of the ATP synthase on the inner membrane [56].…”

“…It is generally agreed on that the MPT is caused by the opening of a large channel (mitochondrial permeability transition pore), which is composed of various components, including Bax and Bak [61, 62] on the mitochondrial outer membrane and subunits of the ATP synthase on the inner membrane [56]. Additional regulatory components include cyclophilin D [63], modulation of which can influence APAP hepatotoxicity in a dose dependent manner [57, 59, 60]. While inhibition of cyclophilin D provided partial protection in vitro [57] and prevented APAP-induced liver injury at a moderate overdose of 200mg/kg in vivo [59], no protection was seen at a higher APAP dose of 600mg/kg [60].…”

“…Additional regulatory components include cyclophilin D [63], modulation of which can influence APAP hepatotoxicity in a dose dependent manner [57, 59, 60]. While inhibition of cyclophilin D provided partial protection in vitro [57] and prevented APAP-induced liver injury at a moderate overdose of 200mg/kg in vivo [59], no protection was seen at a higher APAP dose of 600mg/kg [60]. This dose-dependent modulation of the MPT was corroborated in vivo by another study using intravital microscopy, where a dose of 150mg/kg APAP resulted in transient activation of JNK and reversible loss of membrane potential without cell death [8], suggesting that activation of the MPT could be restricted to certain mitochondria within cells without propagation to cell death.…”

Highlight report: acetaminophen hepatotoxicity

“…Much effort has been invested to gain a better understanding of the molecular mechanisms of APAP hepatotoxicity (Saito et al 2010;Kon et al 2004;Hwang et al 2015;Sjogren et al 2014;Schyschka et al 2013;Singh et al 2013) and to identify biomarkers of APAP overdose (McGill et al 2014;Beger et al 2015). APAP is known to be metabolically activated to the reactive N-acetyl-p-benzoquinone imine (NAPQI), which forms protein adducts including mitochondrial proteins leading to mitochondrial oxidative stress (Ramachandran et al 2013;Cohen et al 1997).…”

“…As a consequence c-jun N-terminal kinase is translocated to the mitochondria, which enhances generation of reactive oxygen species (Ramachandran et al 2013;Hanawa et al 2008;Saito et al 2010). This may lead to opening of the mitochondrial membrane transition pore and cause a form of cell death which differs from classical apoptosis and has been named 'necroptosis' (Kon et al 2004;Gujral et al 2002;Ni et al 2012). Although cytochrome c is released from the mitochondria, for so far unknown reasons apoptotic cell death is not induced (Gujral et al 2002;Lawson et al 1999;Williams et al 2010).…”