Abstract
Drug-induced liver injury (DILI) is the most prevalent adversity encountered in drug development and clinical settings leading to urgent needs to understand the underlying mechanisms. In this study, we have systematically investigated the dynamics of the activation of cellular stress response pathways and cell death outcomes upon exposure of a panel of liver toxicants using live cell imaging of fluorescent reporter cell lines. We established a comprehensive temporal dynamic response profile of a large set of BAC-GFP HepG2 cell lines representing the following components of stress signaling: i) unfolded protein response (UPR) [ATF4, XBP1, BIP and CHOP]; ii) oxidative stress [NRF2, SRXN1, HMOX1]; iii) DNA damage [P53, P21, BTG2, MDM2]; and iv) NF-κB pathway [A20, ICAM1]. We quantified the single cell GFP expression as a surrogate for endogenous protein expression using live cell imaging over > 60 h upon exposure to 14 DILI compounds at multiple concentrations. Using logic-based ordinary differential equation (Logic-ODE), we modelled the dynamic profiles of the different stress responses and extracted specific descriptors potentially predicting the progressive outcomes. We identified the activation of ATF4-CHOP axis of the UPR as the key pathway showing the highest correlation with cell death upon DILI compound perturbation. Knocking down main components of the UPR provided partial protection from compound-induced cytotoxicity, indicating a complex interplay among UPR components as well as other stress pathways. Our results suggest that a systematic analysis of the temporal dynamics of ATF4-CHOP axis activation can support the identification of DILI risk for new candidate drugs.
Highlights
Drug-induced liver injury (DILI) is one of the most frequently encountered drug-related toxicities in drug development and clinical usage [2,6]
The occurrence of cytotoxic events in DILI has been linked to the activation of specific cellular stress response pathways [34] including: inflammatory response (IR) pathways medi ated by the NF-κB transcription factor [20,37], oxidative stress response (OSR) pathways mediated by nuclear translocation of NRF2 (NFE2L2) [9], DNA damage response (DDR) pathways mediated by TP53 activa tion [8,18], and the unfolded protein response (UPR) pathway mediated by ATF4, ATF6, and XBP1 transcriptional programs [10,21]
We used a panel of fluorescently tagged protein HepG2 reporter cell lines covering 4 different stress response pathways: i) oxidative stress response (OSR) [HMOX1, SRXN1, NRF2]; ii) unfolded protein response (UPR) [BIP, XBP1, CHOP, ATF4]; iii) DNA damage response (DDR) [BTG2, P21, MDM2, P53]; and iv) inflammation response (IR) [ICAM1, A20]
Summary
Drug-induced liver injury (DILI) is one of the most frequently encountered drug-related toxicities in drug development and clinical usage [2,6]. Various key events are involved in DILI, including the release of toxic metabolites that damages neighboring. A major critical key event during liver toxicity is the onset of cell death of hepatocytes. The occurrence of cytotoxic events in DILI has been linked to the activation of specific cellular stress response pathways [34] including: inflammatory response (IR) pathways medi ated by the NF-κB transcription factor [20,37], oxidative stress response (OSR) pathways mediated by nuclear translocation of NRF2 (NFE2L2) [9], DNA damage response (DDR) pathways mediated by TP53 activa tion [8,18], and the unfolded protein response (UPR) pathway mediated by ATF4, ATF6, and XBP1 transcriptional programs [10,21]. Under standing the dynamics and amplitude of the activation and interaction of these cellular stress response pathways in hepatic cells in relation to protection against or onset of cytotoxicity could provide better insights in critical determinants underlying DILI
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