Oxidative stress plays a major role in chlorpromazine-induced cholestasis in human HepaRG cells

Abstract

Drugs induce cholestasis by diverse and still poorly understood mechanisms in humans. Early hepatic effects of chlorpromazine (CPZ), a neuroleptic drug known for years to induce intrahepatic cholestasis, were investigated using the differentiated human hepatoma HepaRG cells. Generation of reactive oxygen species (ROS) was detected as early as 15 minutes after CPZ treatment and was associated with altered mitochondrial membrane potential and disruption of the pericanalicular distribution of F-actin. Inhibition of [ 3 H]-taurocholic acid efflux was observed after 30 minutes and was mostly prevented by N-acetyl cysteine (NAC) cotreatment, indicating a major role of oxidative stress in CPZ-induced bile acid (BA) accumulation. Moreover, 24-hour treatment with CPZ decreased messenger RNA (mRNA) expression of the two main canalicular bile transporters, bile salt export pump (BSEP) and multidrug resistance protein 3 (MDR3). Additional CPZ effects included inhibition of Na 1 -dependent taurocholic cotransporting polypeptide (NTCP) expression and activity, multidrug resistance-associated protein 4 (MRP4) overexpression and CYP8B1 inhibition that are involved in BA uptake, basolateral transport, and BA synthesis, respectively. These latter events likely represent hepatoprotective responses which aim to reduce intrahepatic accumulation of toxic BA. Compared to CPZ effects, overloading of HepaRG cells with high concentrations of cholic and chenodeoxycholic acids induced a delayed oxidative stress and, similarly, after 24 hours it down-regulated BSEP and MDR3 in parallel to a decrease of NTCP and CYP8B1 and an increase of MRP4. By contrast, low BA concentrations up-regulated BSEP and MDR3 in the absence of oxidative stress. Conclusion: These data provide evidence that, among other mechanisms, oxidative stress plays a major role as both a primary causal and an aggravating factor in the early CPZ-induced intrahepatic cholestasis in human hepatocytes. (HEPATOLOGY 2013;57:1518-1529

Biokinetics of chlorpromazine in primary rat and human hepatocytes and human HepaRG cells after repeated exposure

“…Indeed, the accumulation of CPZ inside the cells was relatively important in primary rat hepatocytes exposed to 20 lM CPZ and in HepaRG cells exposed to 15 lM CPZ. Chlorpromazine has been shown to induce phospholipidosis in vivo (Anderson and Borlak, 2006;Tavoloni and Boyer, 1980) and in vitro (Anthérieu et al, 2013;Bachour-El Azzi et al, 2014) and to bind to lamellar bodies that are formed during phospholipidosis (Joshi et al, 1989). In our experiments, lamellar bodies were observed after the 14-day repeated exposure of HepaRG cells to 15 lM CPZ and of primary rat hepatocytes to 20 lM of CPZ (not shown).…”

A dynamic mathematical model of bile acid clearance in HepaRG cells

“…This means that the time-scales of inhibition and recovery as well as the extent of inhibition during and after CsA treatment can be uniquely determined from the time-course experiments for the three membrane containing transporters. CPZ which is an inhibitor of cellular uptake and canalicular export but not sinusoidal export (Antherieu, et al, 2013) was found to act on TCA clearance rates more slowly when compared to CsA. In agreement, by parameter estimation we could show that inhibition by CPZ is irreversible, the two reaction parameters corresponding to reversibility being compatible with zero.…”

“…In agreement, by parameter estimation we could show that inhibition by CPZ is irreversible, the two reaction parameters corresponding to reversibility being compatible with zero. Indeed, experimental studies showed that the cholestatic mechanism of CPZ is indirect and slower, depending on generation of reactive oxygen species that lead to irreversible bile flow inhibition (Antherieu, et al, 2013). This…”

“…A validation of the model was further achieved by use of two prototypical cholestatic drugs, cyclosporine A (CsA) and chlorpromazine (CPZ), previously shown to exhibit differences in the mechanisms of apical and basolateral transporter inhibition (Antherieu et al, 2013;Sharanek et al, 2014) and their effect on BC dynamics (Burbank, et al 2016).…”

Differential sensitivity of metabolically competent and non-competent HepaRG cells to apoptosis induced by diclofenac combined or not with TNF-α

“…After 2h incubation cells were washed and then scraped in 0.1N NaOH. The remaining radiolabeled substrate was measured through scintillation counting to determine TA efflux (Antherieu et al, 2013).…”