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

Abstract

The role of reactive metabolites and inflammatory stress has been largely evoked in idiosyncratic hepatotoxicity of diclofenac (DCF); however mechanisms remain poorly understood. We aimed to evaluate the influence of liver cell phenotype on the hepatotoxicity of DCF combined or not with TNF-α using differentiated and undifferentiated HepaRG cells, and for comparison, HepG2 cells. Our results demonstrate that after a 24h-treatment metabolizing HepaRG cells were less sensitive to DCF than their undifferentiated non-metabolizing counterparts as shown by lower oxidative and endoplasmic reticulum stress responses and lower activation of caspase 9. Differentiated HepaRG cells were also less sensitive than HepG2 cells. Their lower sensitivity to DCF was related to their high content in glutathione transferases. DCF-induced apoptotic effects were potentiated by TNF-α only in death receptor-expressing differentiated HepaRG and HepG2 cells and were associated with marked activation of caspase 8. TNF-α co-treatment did not aggravate DCF-induced cholestatic features. Altogether, our results demonstrate that (i) lower sensitivity to DCF of differentiated HepaRG cells compared to their non-metabolically active counterparts was related to their high detoxifying capacity, giving support to the higher sensitivity of nonhepatic tissues than liver to this drug; (ii) TNF-α-potentiation of DCF cytotoxicity occurred only in death receptor-expressing cells.

N-acetylcysteine potentiates diclofenac toxicity inSaccharomyces cerevisiae: stronger potentiation in ABC transporter mutant strains

“…DCF induces adverse reactions in some patients (Boelsterli, 2003). Involvement of oxidative stress enzymes and drug metabolizing enzymes in this toxicity was reported in in vitro hepatic and non-hepatic models (Al-Attrache et al, 2016;Fredriksson et al, 2011;van Leeuwen et al, 2011c). DCF was reported to induce oxidative stress, apoptosis, cholestasis by diverse mechanisms (Al-Attrache et al, 2016;Fredriksson et al, 2011;Fredriksson et al, 2014;Maiuri et al, 2015) including deregulation of some ABC and SLC transporters (Al-Attrache et al, 2016), respiratory chain components and PKC signaling (van Leeuwen et al, 2011b).…”

“…DCF adverse effects have been related to different factors including its metabolism into 4′-OH-DCF, 5-OH-DCF, and acyl glucuronides (Bort et al, 1999;Kretz-Rommel and Boelsterli, 1993;Wang et al, 2004). We recently demonstrated that DCF-induced toxicity in hepatic HepaRG cell lines decreases as the cells differentiate and express DCF-metabolizing and detoxifying enzymes (Al-Attrache et al, 2016). An opposite effect was reported in Saccharomyces cerevisiae where heterologous expression of DCF-metabolizing cytochrome P450 potentiates DCF toxicity and reactive oxygen species (ROS) generation (van Leeuwen et al, 2011c).…”

“…NAC and DCF act in synergy as antiinflammatory drugs through cyclooxygenase inhibition (Parasassi et al, 2005), and inhibition of TNF-α secretion (Mulhall et al, 2003). NAC was reported to reduce DCF-induced renal toxicity (Efrati et al, 2007) and ROS-mediated DCF-induced toxicity and apoptosis (Al-Attrache et al, 2016;Inoue et al, 2004). However, NAC has no effect on DCF-methyl phenyl pyridinium potentiated toxicity (Morioka et al, 2004).…”

Pro-inflammatory cytokines enhance dilatation of bile canaliculi caused by cholestatic antibiotics

“…IL-6 and IL-1β but not TNF-α aggravated BC dilatation caused by all cholestatic ATBs in HepaRG hepatocytes. Noticeably, TNF-α was also found to be ineffective on diclofenac-induced BC deformation (Al-Attrache et al, 2016). Importantly, cytokine effects were evaluated in a medium containing 2% bovine serum (i.e.…”

“…At concentrations that have been widely used in in vitro studies, i.e. TNF-α (10 ng/ml), IL-6 (5 ng/ml) and IL-1β (0.5 ng/ml)(Al-Attrache et al, 2016;Bachour-El Azzi et al, 2014;Rubin et al, 2015), none of these cytokines caused any cytotoxicity, as evaluated by caspase-3 activity and MTT assays after 2 daily repeated additions.…”