Activation of the Nrf2 response by intrinsic hepatotoxic drugs correlates with suppression of NF-κB activation and sensitizes toward TNFα-induced cytotoxicity.

Bram Herpers,Hans De Bont,Harry Vrieling,Lisa Fredriksson,Bob Van De Water,Zi Di,Steven Wink,Giel Hendriks

doi:10.1007/s00204-015-1536-3

Abstract

Drug-induced liver injury (DILI) is an important problem both in the clinic and in the development of new safer medicines. Two pivotal adaptation and survival responses to adverse drug reactions are oxidative stress and cytokine signaling based on the activation of the transcription factors Nrf2 and NF-κB, respectively. Here, we systematically investigated Nrf2 and NF-κB signaling upon DILI-related drug exposure. Transcriptomics analyses of 90 DILI compounds in primary human hepatocytes revealed that a strong Nrf2 activation is associated with a suppression of endogenous NF-κB activity. These responses were translated into quantitative high-content live-cell imaging of induction of a selective Nrf2 target, GFP-tagged Srxn1, and the altered nuclear translocation dynamics of a subunit of NF-κB, GFP-tagged p65, upon TNFR signaling induced by TNFα using HepG2 cells. Strong activation of GFP-Srxn1 expression by DILI compounds typically correlated with suppression of NF-κB nuclear translocation, yet reversely, activation of NF-κB by TNFα did not affect the Nrf2 response. DILI compounds that provided strong Nrf2 activation, including diclofenac, carbamazepine and ketoconazole, sensitized toward TNFα-mediated cytotoxicity. This was related to an adaptive primary protective response of Nrf2, since loss of Nrf2 enhanced this cytotoxic synergy with TNFα, while KEAP1 downregulation was cytoprotective. These data indicate that both Nrf2 and NF-κB signaling may be pivotal in the regulation of DILI. We propose that the NF-κB-inhibiting effects that coincide with a strong Nrf2 stress response likely sensitize liver cells to pro-apoptotic signaling cascades induced by intrinsic cytotoxic pro-inflammatory cytokines.Electronic supplementary materialThe online version of this article (doi:10.1007/s00204-015-1536-3) contains supplementary material, which is available to authorized users.

Highlights

Drug safety issues that lead to drug-induced liver injury (DILI) are the major reason for drug-related hospitalizations and drug withdrawals
We demonstrated that for diclofenac (DCF), the synergy with TNFα to kill hepatocytes is directly related to inhibition of NF-κB nuclear translocation and activation and that inhibition of NF-κB signaling sensitizes toward cytotoxicity caused by DCF (Fredriksson et al 2011)
We focused on the interplay of two pivotal cellular stress response signaling pathways in DILI: TNFαmediated NF-κB signaling and chemical stress-induced Nrf2 activation

Summary

Introduction

Drug safety issues that lead to drug-induced liver injury (DILI) are the major reason for drug-related hospitalizations and drug withdrawals. We investigated the transcriptional response to 90 DILI-associated drugs as well as several cytokines/growth factors in primary human hepatocytes (PHH) at multiple concentrations and time points, based on publicly available data (Uehara et al 2010). To translate these findings to high-throughput approaches, we established novel GFP-based reporter cell lines amenable for highcontent high-throughput live-cell imaging to quantitatively assess Nrf and NF-κB activation on a cell-to-cell basis.

Materials and methods

Results

A BAC‐Srxn1‐GFP HepG2 cell line reports xenobiotic‐mediated Nrf2 activation

Discussion