Low-dose acetaminophen induces early disruption of cell-cell tight junctions in human hepatic cells and mouse liver

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.

Validation of Reference Genes for Gene Expression Studies by RT-qPCR in HepaRG Cells during Toxicity Testing and Disease Modelling

“…The cell culture was carried out according to previously published protocol [9,11,17,19]. HepaRG fully differentiated cells (HPR116-TA08; Cryopreserved HepaRG™ cells; Biopredic Int., Rennes, France) were thawed from liquid nitrogen and plated in Biopredic's proprietary General Purpose Medium (GPS) without dimethyl sulfoxide (DMSO) for up to 2 days.…”

“…Throughout the experimental process, differentiated HepaRG cells were regularly assessed for viability according to previously published procedure [17,19]. The cells were seeded on a 96 well standard tissue culture plate and grown to confluency (day 8).…”

“…Both CPZ and APAP did not compromise HepaRG cell viability at sub toxic concentration. Using total ATP and PrestoBlue assays, the effect of CPZ and APAP on HepaRG cells have already been published from our group [17,19].…”

“…In this study, we have investigated the suitability of 12 commonly used housekeeping genes for use as RT-qPCR normalisers in the fully differentiated HepaRG cell line. The cells were subjected to two different liver hepatotoxins: acetyl-para-aminophenol (APAP), known to affect intercellular tight junctions [17] and chlorpromazine (CPZ), which causes damage resulting in cholestasis [18]. The aim of this study was first, to establish whether expression of 'classical' reference genes in the HepaRG cell line were differentially affected by exposure over the same time period to different hepatotoxins.…”

HepaRG-Progenitor Cell Derived Hepatocytes Cultured in Bioartificial Livers Are Protected from Healthy- and Acute Liver Failure-Plasma Induced Toxicity

“…Due to the scarcity of human ALF plasma there was no opportunity to assess the effect of plasma from ALF patients. In this study we used a model of paracetamol-induced liver failure which is commonly associated with mitotoxicity and disruption of cell tight junctions [33], although also ALF-plasma of other origins has been reported to impair mitochondrial activity to varying degrees [31,34]. However, the detrimental effects of ALF-plasma are likely to vary between etiologies, patients and clinical status, implicating the necessity of close monitoring of biocomponent functionality during therapy.…”