Abstract
Dysfunction of cell-cell tight junction (TJ) adhesions is a major feature in the pathogenesis of various diseases. Liver TJs preserve cellular polarity by delimiting functional bile-canalicular structures, forming the blood-biliary barrier. In acetaminophen-hepatotoxicity, the mechanism by which tissue cohesion and polarity are affected remains unclear. Here, we demonstrate that acetaminophen, even at low-dose, disrupts the integrity of TJ and cell-matrix adhesions, with indicators of cellular stress with liver injury in the human hepatic HepaRG cell line, and primary hepatocytes. In mouse liver, at human-equivalence (therapeutic) doses, dose-dependent loss of intercellular hepatic TJ-associated ZO-1 protein expression was evident with progressive clinical signs of liver injury. Temporal, dose-dependent and specific disruption of the TJ-associated ZO-1 and cytoskeletal-F-actin proteins, correlated with modulation of hepatic ultrastructure. Real-time impedance biosensing verified in vitro early, dose-dependent quantitative decreases in TJ and cell-substrate adhesions. Whereas treatment with NAPQI, the reactive metabolite of acetaminophen, or the PKCα-activator and TJ-disruptor phorbol-12-myristate-13-acetate, similarly reduced TJ integrity, which may implicate oxidative stress and the PKC pathway in TJ destabilization. These findings are relevant to the clinical presentation of acetaminophen-hepatotoxicity and may inform future mechanistic studies to identify specific molecular targets and pathways that may be altered in acetaminophen-induced hepatic depolarization.
Highlights
Species-mediated effects on TJ-associated F-actin distribution in human hepatic HepaRG cells[2,3], whilst the tumour-promoter, phorbol ester, a specific protein kinase C-alpha (PKCα) activator, reduces TJ integrity and ablates cell polarity in HepG2 cells[4,5]
We demonstrated that serum from patients with acetaminophen (APAP)-induced acute liver failure (ALF), when applied to primary human hepatocytes (PHHs), cause a loss of cellular adhesion, hepatocyte detachment with actin-cytoskeletal disruption preceding apoptosis, via a β1-integrin pathway[6,7]
APAP toxicity is caused by formation of the highly reactive metabolite N-acetyl-p-benzo-quinoneimine (NAPQI), via cytochrome P450 metabolism; followed by glutathione and ATP depletion, mitochondrial dysfunction, and oxidative stress; which culminates in necrotic cell death through physical disruption of cellular integrity[8] and liver cytoarchitecture[9]
Summary
Species-mediated effects on TJ-associated F-actin distribution in human hepatic HepaRG cells[2,3], whilst the tumour-promoter, phorbol ester, a specific protein kinase C-alpha (PKCα) activator, reduces TJ integrity and ablates cell polarity in HepG2 cells[4,5]. We demonstrated that serum from patients with acetaminophen (APAP)-induced acute liver failure (ALF), when applied to primary human hepatocytes (PHHs), cause a loss of cellular adhesion, hepatocyte detachment with actin-cytoskeletal disruption preceding apoptosis, via a β1-integrin pathway[6,7]. HepaRGs, a bipotent human hepatic cell line, are increasingly regarded as a surrogate to PHHs, providing an excellent model system for exploring mechanisms of APAP hepatotoxicity and cellular polarity[18,20,21]. HepaRG cells form a stable (4 weeks) hepatic co-culture, of hepatocyte-/and cholangiocyte-like cells with highly differentiated, functional morphology. They provide a physiologically-relevant in vitro liver model, with intact drug metabolism, and functional polarity with bile canalicular structures, delineated by junctional complexes
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