Drug-Induced Endoplasmic Reticulum and Oxidative Stress Responses Independently Sensitize Toward TNFα-Mediated Hepatotoxicity

Abstract

Drug-induced liver injury (DILI) is an important clinical problem. Here, we used a genomics approach to in detail investigate the hypothesis that critical drug-induced toxicity pathways act in synergy with the pro-inflammatory cytokine tumor necrosis factor α (TNFα) to cause cell death of liver HepG2 cells. Transcriptomics of the cell injury stress response pathways initiated by two hepatoxicants, diclofenac and carbamazepine, revealed the endoplasmic reticulum (ER) stress/translational initiation signaling and nuclear factor-erythroid 2 (NF-E2)-related factor 2 (Nrf2) antioxidant signaling as two major affected pathways, which was similar to that observed for the majority of ∼80 DILI compounds in primary human hepatocytes. Compounds displaying weak or no TNFα synergism, namely ketoconazole, nefazodone, and methotrexate, failed to synchronously induce both pathways. The ER stress induced was primarily related to protein kinase R-like ER kinase (PERK) and activating transcription factor 4 (ATF4) activation and subsequent expression of C/EBP homologous protein (CHOP), which was all independent of TNFα signaling. Identical ATF4 dependent transcriptional programs were observed in primary human hepatocytes as well as primary precision-cut human liver slices. Targeted RNA interference studies revealed that whereas ER stress signaling through inositol-requiring enzyme 1α (IRE1α) and activating transcription factor 6 (ATF6) acted cytoprotective, activation of the ER stress protein kinase PERK and subsequent expression of CHOP was pivotal for the onset of drug/TNFα-induced apoptosis. Whereas inhibition of the Nrf2-dependent adaptive oxidative stress response enhanced the drug/TNFα cytotoxicity, Nrf2 signaling did not affect CHOP expression. Both hepatotoxic drugs enhanced expression of the translational initiation factor EIF4A1, which was essential for CHOP expression and drug/TNFα-mediated cell killing. Our data support a model in which enhanced drug-induced translation initiates PERK-mediated CHOP signaling in an EIF4A1 dependent manner, thereby sensitizing toward caspase-8-dependent TNFα-induced apoptosis.

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

“…A specific activation of the ER stress route through the ATF4 transcriptional activity has also been reported in DCF-treated liver cells (Fredriksson et al, 2014). Our results showed that transcripts levels of ATF4, ATF6, CHOP and GRP78 were all overexpressed in differentiated HepaRG cells exposed to 200µM DCF for 24h.The increases were significant with 200µM for the 4 genes and co-treatment with TNF-α led to a further significant increase.…”

“…Selection of TNF-α concentration (10ng/ml) was based on previous studies (Bachour-El Azzi et al, 2014;Fredriksson et al, 2014) and preliminary experiments on determination of caspase 3 activity and CRP secretion levels. However, except otherwise indicated a co-treatment with DCF and cytokine meant a pre-treatment with the cytokine alone before treatment with DCF/cytokine combination.…”

“…The involvement of the intrinsic apoptotic pathway characterized by disruption of mitochondrial integrity has been demonstrated in various studies (Fredriksson et al, 2011;Gomez-Lechon et al, 2003a), and oxidative and endoplasmic reticulum (ER) stresses have been identified as independent cytotoxic responses to both DCF alone and the combination DCF/TNF-α (Fredriksson et al, 2014). The synergistic effect of DCF/TNF-α co-treatment appeared to occur mostly via activation of the extrinsic apoptotic pathway (Fredriksson et al, 2011).…”

Role of endoplasmic reticulum stress in drug‐induced toxicity

“…2013; Fredriksson et al. 2014), it is still unclear whether this event plays a primary role in diclofenac‐induced cell demise, compared to mitochondrial dysfunction. Moreover, the mechanism whereby diclofenac induces ER stress in hepatocytes is still unknown.…”

Genome-Wide Association Studies for Idiosyncratic Drug-Induced Hepatotoxicity: Looking Back–Looking Forward to Next-Generation Innovation

“…include direct toxicity, mitochondrial dysfunction, metabolic abnormalities, cellular stress, and immune-mediated reactions (Daly, 2010a;Fredriksson et al, 2014;Yuan and Kaplowitz, 2013).…”