title: 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.
authors.affiliation: Division of Toxicology, Leiden Academic Centre for Drug Research, Leiden University, 2333 CC Leiden The Netherlands.
authors.forename: Lisa
authors.initials: L
authors.lastname: Fredriksson
authors.affiliation: Division of Toxicology, Leiden Academic Centre for Drug Research, Leiden University, 2333 CC Leiden The Netherlands.
authors.forename: Steven
authors.initials: S
authors.lastname: Wink
authors.affiliation: Division of Toxicology, Leiden Academic Centre for Drug Research, Leiden University, 2333 CC Leiden The Netherlands.
authors.forename: Bram
authors.initials: B
authors.lastname: Herpers
authors.affiliation: Division of Toxicology, Leiden Academic Centre for Drug Research, Leiden University, 2333 CC Leiden The Netherlands.
authors.forename: Giulia
authors.initials: G
authors.lastname: Benedetti
authors.affiliation: Division of Pharmacokinetics, Toxicology and Targeting, Department of Pharmacy, University of Groningen, 9713 AV Groningen, The Netherlands.
authors.forename: Mackenzie
authors.initials: M
authors.lastname: Hadi
authors.affiliation: Division of Toxicology, Leiden Academic Centre for Drug Research, Leiden University, 2333 CC Leiden The Netherlands.
authors.forename: Hans
authors.initials: H
authors.lastname: de  Bont
authors.affiliation: Division of Pharmacokinetics, Toxicology and Targeting, Department of Pharmacy, University of Groningen, 9713 AV Groningen, The Netherlands.
authors.forename: Geny
authors.initials: G
authors.lastname: Groothuis
authors.affiliation: The National Institute for Public Health and the Environment (RIVM), 3720 BA Bilthoven, The Netherlands.
authors.forename: Mirjam
authors.initials: M
authors.lastname: Luijten
authors.affiliation: Division of Toxicology, Leiden Academic Centre for Drug Research, Leiden University, 2333 CC Leiden The Netherlands.
authors.forename: Erik
authors.initials: E
authors.lastname: Danen
authors.affiliation: Division of Toxicology, Leiden Academic Centre for Drug Research, Leiden University, 2333 CC Leiden The Netherlands.
authors.forename: Marjo
authors.initials: M
authors.lastname: de  Graauw
authors.affiliation: Division of Toxicology, Leiden Academic Centre for Drug Research, Leiden University, 2333 CC Leiden The Netherlands.
authors.forename: John
authors.initials: J
authors.lastname: Meerman
authors.affiliation: Division of Toxicology, Leiden Academic Centre for Drug Research, Leiden University, 2333 CC Leiden The Netherlands b.water@lacdr.leidenuniv.nl.
authors.forename: Bob
authors.initials: B
authors.lastname: van  de Water
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002220:Carbamazepine / Q000633:toxicity*; D056486:Chemical and Drug Induced Liver Injury / Q000209:etiology* / Q000235:genetics / Q000378:metabolism; D004008:Diclofenac / Q000633:toxicity*; D004357:Drug Synergism; D059865:Endoplasmic Reticulum Stress / Q000187:drug effects; D020869:Gene Expression Profiling; D055106:Genome-Wide Association Study; D056945:Hep G2 Cells; D022781:Hepatocytes / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D008810:Mice, Inbred C57BL; D051267:NF-E2-Related Factor 2 / Q000378:metabolism; D018384:Oxidative Stress / Q000187:drug effects* / Q000276:immunology; D059467:Transcriptome / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism* / Q000633:toxicity
chemical_list: D051267:NF-E2-Related Factor 2; C495635:NFE2L2 protein, human; D014409:Tumor Necrosis Factor-alpha; D004008:Diclofenac; D002220:Carbamazepine
keywords: RNA interference; drug-induced liver injury; high content microscopy; transcriptomics