The integrated stress response-related expression of CHOP due to mitochondrial toxicity is a warning sign for DILI liability.

BACKGROUND AND AIMS: Drug-induced liver injury (DILI) is one of the most frequent reasons for failure of drugs in clinical trials or market withdrawal. Early assessment of DILI risk remains a major challenge during drug development. Here, we present a mechanism-based weight-of-evidence approach able to identify certain candidate compounds with DILI liabilities due to mitochondrial toxicity. METHODS: A total of 1587 FDA-approved drugs and 378 kinase inhibitors were screened for cellular stress response activation associated with DILI using an imaging-based HepG2 BAC-GFP reporter platform including the integrated stress response (CHOP), DNA damage response (P21) and oxidative stress response (SRXN1). RESULTS: In total 389, 219 and 104 drugs were able to induce CHOP-GFP, P21-GFP and SRXN1-GFP expression at 50 µM respectively. Concentration response analysis identified 154 FDA-approved drugs as critical CHOP-GFP inducers. Based on predicted and observed (pre-)clinical DILI liabilities of these drugs, nine antimycotic drugs (e.g. butoconazole, miconazole, tioconazole) and 13 central nervous system (CNS) agents (e.g. duloxetine, fluoxetine) were selected for transcriptomic evaluation using whole-genome RNA-sequencing of primary human hepatocytes. Gene network analysis uncovered mitochondrial processes, NRF2 signalling and xenobiotic metabolism as most affected by the antimycotic drugs and CNS agents. Both the selected antimycotics and CNS agents caused impairment of mitochondrial oxygen consumption in both HepG2 and primary human hepatocytes. CONCLUSIONS: Together, the results suggest that early pre-clinical screening for CHOP expression could indicate liability of mitochondrial toxicity in the context of DILI, and, therefore, could serve as an important warning signal to consider during decision-making in drug development.

Systematic screening identifies ABCG2 as critical factor underlying synergy of kinase inhibitors with transcriptional CDK inhibitors.

BACKGROUND: Triple-negative breast cancer (TNBC) is a subtype of breast cancer with limited treatment options and poor clinical prognosis. Inhibitors of transcriptional CDKs are currently under thorough investigation for application in the treatment of multiple cancer types, including breast cancer. These studies have raised interest in combining these inhibitors, including CDK12/13 inhibitor THZ531, with a variety of other anti-cancer agents. However, the full scope of these potential synergistic interactions of transcriptional CDK inhibitors with kinase inhibitors has not been systematically investigated. Moreover, the mechanisms behind these previously described synergistic interactions remain largely elusive. METHODS: Kinase inhibitor combination screenings were performed to identify kinase inhibitors that synergize with CDK7 inhibitor THZ1 and CDK12/13 inhibitor THZ531 in TNBC cell lines. CRISPR-Cas9 knockout screening and transcriptomic evaluation of resistant versus sensitive cell lines were performed to identify genes critical for THZ531 resistance. RNA sequencing analysis after treatment with individual and combined synergistic treatments was performed to gain further insights into the mechanism of this synergy. Kinase inhibitor screening in combination with visualization of ABCG2-substrate pheophorbide A was used to identify kinase inhibitors that inhibit ABCG2. Multiple transcriptional CDK inhibitors were evaluated to extend the significance of the found mechanism to other transcriptional CDK inhibitors. RESULTS: We show that a very high number of tyrosine kinase inhibitors synergize with the CDK12/13 inhibitor THZ531. Yet, we identified the multidrug transporter ABCG2 as key determinant of THZ531 resistance in TNBC cells. Mechanistically, we demonstrate that most synergistic kinase inhibitors block ABCG2 function, thereby sensitizing cells to transcriptional CDK inhibitors, including THZ531. Accordingly, these kinase inhibitors potentiate the effects of THZ531, disrupting gene expression and increasing intronic polyadenylation. CONCLUSION: Overall, this study demonstrates the critical role of ABCG2 in limiting the efficacy of transcriptional CDK inhibitors and identifies multiple kinase inhibitors that disrupt ABCG2 transporter function and thereby synergize with these CDK inhibitors. These findings therefore further facilitate the development of new (combination) therapies targeting transcriptional CDKs and highlight the importance of evaluating the role of ABC transporters in synergistic drug-drug interactions in general.

Differential response of luminal and basal breast cancer cells to acute and chronic hypoxia.

Hypoxia is linked to disease progression and poor prognosis in several cancers, including breast cancer. Cancer cells can encounter acute, chronic, and/or intermittent periods of oxygen deprivation and it is poorly understood how the different breast cancer subtypes respond to such hypoxia regimes. Here, we assessed the response of representative cell lines for the luminal and basal A subtype to acute (24 h) and chronic hypoxia (5 days). High throughput targeted transcriptomics analysis showed that HIF-related pathways are significantly activated in both subtypes. Indeed, HIF1⍺ nuclear accumulation and activation of the HIF1⍺ target gene CA9 were comparable. Based on the number of differentially expressed genes: (i) 5 days of exposure to hypoxia induced a more profound transcriptional reprogramming than 24 h, and (ii) basal A cells were less affected by acute and chronic hypoxia as compared to luminal cells. Hypoxia-regulated gene networks were identified of which hub genes were associated with worse survival in breast cancer patients. Notably, while chronic hypoxia altered the regulation of the cell cycle in both cell lines, it induced two distinct adaptation programs in these subtypes. Mainly genes controlling central carbon metabolism were affected in the luminal cells whereas genes controlling the cytoskeleton were affected in the basal A cells. In agreement, in response to chronic hypoxia, lactate secretion was more prominently increased in the luminal cell lines which were associated with the upregulation of the GAPDH glycolytic enzyme. This was not observed in the basal A cell lines. In contrast, basal A cells displayed enhanced cell migration associated with more F-actin stress fibers whereas luminal cells did not. Altogether, these data show distinct responses to acute and chronic hypoxia that differ considerably between luminal and basal A cells. This differential adaptation is expected to play a role in the progression of these different breast cancer subtypes.

An Adverse Outcome Pathway Network for Chemically Induced Oxidative Stress Leading to (Non)genotoxic Carcinogenesis.

Nongenotoxic (NGTX) carcinogens induce cancer via other mechanisms than direct DNA damage. A recognized mode of action for NGTX carcinogens is induction of oxidative stress, a state in which the amount of oxidants in a cell exceeds its antioxidant capacity, leading to regenerative proliferation. Currently, carcinogenicity assessment of environmental chemicals primarily relies on genetic toxicity end points. Since NGTX carcinogens lack genotoxic potential, these chemicals may remain undetected in such evaluations. To enhance the predictivity of test strategies for carcinogenicity assessment, a shift toward mechanism-based approaches is required. Here, we present an adverse outcome pathway (AOP) network for chemically induced oxidative stress leading to (NGTX) carcinogenesis. To develop this AOP network, we first investigated the role of oxidative stress in the various cancer hallmarks. Next, possible mechanisms for chemical induction of oxidative stress and the biological effects of oxidative damage to macromolecules were considered. This resulted in an AOP network, of which associated uncertainties were explored. Ultimately, development of AOP networks relevant for carcinogenesis in humans will aid the transition to a mechanism-based, human relevant carcinogenicity assessment that involves a substantially lower number of laboratory animals.

Model-based translation of DNA damage signaling dynamics across cell types.

Interindividual variability in DNA damage response (DDR) dynamics may evoke differences in susceptibility to cancer. However, pathway dynamics are often studied in cell lines as alternative to primary cells, disregarding variability. To compare DDR dynamics in the cell line HepG2 with primary human hepatocytes (PHHs), we developed a HepG2-based computational model that describes the dynamics of DDR regulator p53 and targets MDM2, p21 and BTG2. We used this model to generate simulations of virtual PHHs and compared the results to those for PHH donor samples. Correlations between baseline p53 and p21 or BTG2 mRNA expression in the absence and presence of DNA damage for HepG2-derived virtual samples matched the moderately positive correlations observed for 50 PHH donor samples, but not the negative correlations between p53 and its inhibitor MDM2. Model parameter manipulation that affected p53 or MDM2 dynamics was not sufficient to accurately explain the negative correlation between these genes. Thus, extrapolation from HepG2 to PHH can be done for some DDR elements, yet our analysis also reveals a knowledge gap within p53 pathway regulation, which makes such extrapolation inaccurate for the regulator MDM2. This illustrates the relevance of studying pathway dynamics in addition to gene expression comparisons to allow reliable translation of cellular responses from cell lines to primary cells. Overall, with our approach we show that dynamical modeling can be used to improve our understanding of the sources of interindividual variability of pathway dynamics.

High-content high-throughput imaging reveals distinct connections between mitochondrial morphology and functionality for OXPHOS complex I, III, and V inhibitors.

Cells can adjust their mitochondrial morphology by altering the balance between mitochondrial fission and fusion to adapt to stressful conditions. The connection between a chemical perturbation, changes in mitochondrial function, and altered mitochondrial morphology is not well understood. Here, we made use of high-throughput high-content confocal microscopy to assess the effects of distinct classes of oxidative phosphorylation (OXPHOS) complex inhibitors on mitochondrial parameters in a concentration and time resolved manner. Mitochondrial morphology phenotypes were clustered based on machine learning algorithms and mitochondrial integrity patterns were mapped. In parallel, changes in mitochondrial membrane potential (MMP), mitochondrial and cellular ATP levels, and viability were microscopically assessed. We found that inhibition of MMP, mitochondrial ATP production, and oxygen consumption rate (OCR) using sublethal concentrations of complex I and III inhibitors did not trigger mitochondrial fragmentation. Instead, complex V inhibitors that suppressed ATP and OCR but increased MMP provoked a more fragmented mitochondrial morphology. In agreement, complex V but not complex I or III inhibitors triggered proteolytic cleavage of the mitochondrial fusion protein, OPA1. The relation between increased MMP and fragmentation did not extend beyond OXPHOS complex inhibitors: increasing MMP by blocking the mPTP pore did not lead to OPA1 cleavage or mitochondrial fragmentation and the OXPHOS uncoupler FCCP was associated with OPA1 cleavage and MMP reduction. Altogether, our findings connect vital mitochondrial functions and phenotypes in a high-throughput high-content confocal microscopy approach that help understanding of chemical-induced toxicity caused by OXPHOS complex perturbing chemicals.

Transcriptional landscape of mitochondrial electron transport chain inhibition in renal cells.

Analysis of the transcriptomic alterations upon chemical challenge, provides in depth mechanistic information on the compound's toxic mode of action, by revealing specific pathway activation and other transcriptional modulations. Mapping changes in cellular behaviour to chemical insult, facilitates the characterisation of chemical hazard. In this study, we assessed the transcriptional landscape of mitochondrial impairment through the inhibition of the electron transport chain (ETC) in a human renal proximal tubular cell line (RPTEC/TERT1). We identified the unfolded protein response pathway (UPR), particularly the PERK/ATF4 branch as a common cellular response across ETC I, II and III inhibitions. This finding and the specific genes elaborated may aid the identification of mitochondrial liabilities of chemicals in both legacy data and prospective transcriptomic studies.

Transcriptomic-based evaluation of trichloroethylene glutathione and cysteine conjugates demonstrate phenotype-dependent stress responses in a panel of human in vitro models.

Environmental or occupational exposure of humans to trichloroethylene (TCE) has been associated with different extrahepatic toxic effects, including nephrotoxicity and neurotoxicity. Bioactivation of TCE via the glutathione (GSH) conjugation pathway has been proposed as underlying mechanism, although only few mechanistic studies have used cell models of human origin. In this study, six human derived cell models were evaluated as in vitro models representing potential target tissues of TCE-conjugates: RPTEC/TERT1 (kidney), HepaRG (liver), HUVEC/TERT2 (vascular endothelial), LUHMES (neuronal, dopaminergic), human induced pluripotent stem cells (hiPSC) derived peripheral neurons (UKN5) and hiPSC-derived differentiated brain cortical cultures containing all subtypes of neurons and astrocytes (BCC42). A high throughput transcriptomic screening, utilizing mRNA templated oligo-sequencing (TempO-Seq), was used to study transcriptomic effects after exposure to TCE-conjugates. Cells were exposed to a wide range of concentrations of S-(1,2-trans-dichlorovinyl)glutathione (1,2-DCVG), S-(1,2-trans-dichlorovinyl)-L-cysteine (1,2-DCVC), S-(2,2-dichlorovinyl)glutathione (2,2-DCVG), and S-(2,2-dichlorovinyl)-L-cysteine (2,2-DCVC). 1,2-DCVC caused stress responses belonging to the Nrf2 pathway and Unfolded protein response in all the tested models but to different extents. The renal model was the most sensitive model to both 1,2-DCVC and 1,2-DCVG, with an early Nrf2-response at 3 µM and hundreds of differentially expressed genes at higher concentrations. Exposure to 2,2-DCVG and 2,2-DCVC also resulted in the upregulation of Nrf2 pathway genes in RPTEC/TERT1 although at higher concentrations. Of the three neuronal models, both the LUHMES and BCC42 showed significant Nrf2-responses and at higher concentration UPR-responses, supporting recent hypotheses that 1,2-DCVC may be involved in neurotoxic effects of TCE. The cell models with the highest expression of γ-glutamyltransferase (GGT) enzymes, showed cellular responses to both 1,2-DCVG and 1,2-DCVC. Little to no effects were found in the neuronal models from 1,2-DCVG exposure due to their low GGT-expression. This study expands our knowledge on tissue specificity of TCE S-conjugates and emphasizes the value of human cell models together with transcriptomics for such mechanistic studies.

Towards an advanced testing strategy for genotoxicity using image-based 2D and 3D HepG2 DNA damage response fluorescent protein reporters.

In vitro assessment of mutagenicity is an essential component in the chemical risk assessment. Given the diverse modes of action by which chemicals can induce DNA damage, it is essential that these in vitro assays are carefully evaluated for their possibilities and limitations. In this study, we used a fluorescent protein HepG2 reporter test system in combination with high content imaging. To measure induction of the DNA damage response (DDR), we used three different green fluorescent protein reporters for p53 pathway activation. These allowed for accurate quantification of p53, p21 and BTG2 (BTG anti-proliferation factor 2) protein expression and cell viability parameters at a single cell or spheroid resolution. The reporter lines were cultured as 2D monolayers and as 3D spheroids. Furthermore, liver maturity and cytochrome P450 enzyme expression were increased by culturing in an amino acid-rich (AAGLY) medium. We found that culture conditions that support a sustained proliferative state (2D culturing with normal DMEM medium) give superior sensitivity when genotoxic compounds are tested that do not require metabolisation and of which the mutagenic mode of action is dependent on replication. For compounds, which are metabolically converted to mutagenic metabolites, more differentiated HepG2 DDR reporters (e.g. 3D cultures) showed a higher sensitivity. This study stratifies how different culture methods of HepG2 DDR reporter cells can influence the sensitivity towards diverse genotoxicants and how this provides opportunities for a tiered genotoxicity testing strategy.

Stimulation of de novo glutathione synthesis by nitrofurantoin for enhanced resilience of hepatocytes.

Toxicity is not only a function of damage mechanisms, but is also determined by cellular resilience factors. Glutathione has been reported as essential element to counteract negative influences. The present work hence pursued the question how intracellular glutathione can be elevated transiently to render cells more resistant toward harmful conditions. The antibiotic nitrofurantoin (NFT) was identified to stimulate de novo synthesis of glutathione in the human hepatoma cell line, HepG2, and in primary human hepatocytes. In intact cells, activation of NFT yielded a radical anion, which subsequently initiated nuclear-factor-erythroid 2-related-factor-2 (Nrf2)-dependent induction of glutamate cysteine ligase (GCL). Application of siRNA-based intervention approaches confirmed the involvement of the Nrf2-GCL axis in the observed elevation of intracellular glutathione levels. Quantitative activation of Nrf2 by NFT, and the subsequent rise in glutathione, were similar as observed with the potent experimental Nrf2 activator diethyl maleate. The elevation of glutathione levels, observed even 48 h after withdrawal of NFT, rendered cells resistant to different stressors such as the mitochondrial inhibitor rotenone, the redox cycler paraquat, the proteasome inhibitors MG-132 or bortezomib, or high concentrations of NFT. Repurpose of the antibiotic NFT as activator of Nrf2 could thus be a promising strategy for a transient and targeted activation of the endogenous antioxidant machinery. Graphical abstract.

Mapping the cellular response to electron transport chain inhibitors reveals selective signaling networks triggered by mitochondrial perturbation.

Mitochondrial perturbation is a key event in chemical-induced organ toxicities that is incompletely understood. Here, we studied how electron transport chain (ETC) complex I, II, or III (CI, CII and CIII) inhibitors affect mitochondrial functionality, stress response activation, and cell viability using a combination of high-content imaging and TempO-Seq in HepG2 hepatocyte cells. CI and CIII inhibitors perturbed mitochondrial membrane potential (MMP) and mitochondrial and cellular ATP levels in a concentration- and time-dependent fashion and, under conditions preventing a switch to glycolysis attenuated cell viability, whereas CII inhibitors had no effect. TempO-Seq analysis of changes in mRNA expression pointed to a shared cellular response to CI and CIII inhibition. First, to define specific ETC inhibition responses, a gene set responsive toward ETC inhibition (and not to genotoxic, oxidative, or endoplasmic reticulum stress) was identified using targeted TempO-Seq in HepG2. Silencing of one of these genes, NOS3, exacerbated the impact of CI and CIII inhibitors on cell viability, indicating its functional implication in cellular responses to mitochondrial stress. Then by monitoring dynamic responses to ETC inhibition using a HepG2 GFP reporter panel for different classes of stress response pathways and applying pathway and gene network analysis to TempO-Seq data, we looked for downstream cellular events of ETC inhibition and identified the amino acid response (AAR) as being triggered in HepG2 by ETC inhibition. Through in silico approaches we provide evidence indicating that a similar AAR is associated with exposure to mitochondrial toxicants in primary human hepatocytes. Altogether, we (i) unravel quantitative, time- and concentration-resolved cellular responses to mitochondrial perturbation, (ii) identify a gene set associated with adaptation to exposure to active ETC inhibitors, and (iii) show that ER stress and an AAR accompany ETC inhibition in HepG2 and primary hepatocytes.

In Vitro Three-Dimensional Liver Models for Nanomaterial DNA Damage Assessment.

Whilst the liver possesses the ability to repair and restore sections of damaged tissue following acute injury, prolonged exposure to engineered nanomaterials (ENM) may induce repetitive injury leading to chronic liver disease. Screening ENM cytotoxicity using 3D liver models has recently been performed, but a significant challenge has been the application of such in vitro models for evaluating ENM associated genotoxicity; a vital component of regulatory human health risk assessment. This review considers the benefits, limitations, and adaptations of specific in vitro approaches to assess DNA damage in the liver, whilst identifying critical advancements required to support a multitude of biochemical endpoints, focusing on nano(geno)toxicology (e.g., secondary genotoxicity, DNA damage, and repair following prolonged or repeated exposures).

The human hepatocyte TXG-MAPr: gene co-expression network modules to support mechanism-based risk assessment.

Mechanism-based risk assessment is urged to advance and fully permeate into current safety assessment practices, possibly at early phases of drug safety testing. Toxicogenomics is a promising source of mechanisms-revealing data, but interpretative analysis tools specific for the testing systems (e.g. hepatocytes) are lacking. In this study, we present the TXG-MAPr webtool (available at https://txg-mapr.eu/WGCNA_PHH/TGGATEs_PHH/ ), an R-Shiny-based implementation of weighted gene co-expression network analysis (WGCNA) obtained from the Primary Human Hepatocytes (PHH) TG-GATEs dataset. The 398 gene co-expression networks (modules) were annotated with functional information (pathway enrichment, transcription factor) to reveal their mechanistic interpretation. Several well-known stress response pathways were captured in the modules, were perturbed by specific stressors and showed preservation in rat systems (rat primary hepatocytes and rat in vivo liver), with the exception of DNA damage and oxidative stress responses. A subset of 87 well-annotated and preserved modules was used to evaluate mechanisms of toxicity of endoplasmic reticulum (ER) stress and oxidative stress inducers, including cyclosporine A, tunicamycin and acetaminophen. In addition, module responses can be calculated from external datasets obtained with different hepatocyte cells and platforms, including targeted RNA-seq data, therefore, imputing biological responses from a limited gene set. As another application, donors' sensitivity towards tunicamycin was investigated with the TXG-MAPr, identifying higher basal level of intrinsic immune response in donors with pre-existing liver pathology. In conclusion, we demonstrated that gene co-expression analysis coupled to an interactive visualization environment, the TXG-MAPr, is a promising approach to achieve mechanistic relevant, cross-species and cross-platform evaluation of toxicogenomic data.

Fluorescent tagging of endogenous Heme oxygenase-1 in human induced pluripotent stem cells for high content imaging of oxidative stress in various differentiated lineages.

Tagging of endogenous stress response genes can provide valuable in vitro models for chemical safety assessment. Here, we present the generation and application of a fluorescent human induced pluripotent stem cell (hiPSC) reporter line for Heme oxygenase-1 (HMOX1), which is considered a sensitive and reliable biomarker for the oxidative stress response. CRISPR/Cas9 technology was used to insert an enhanced green fluorescent protein (eGFP) at the C-terminal end of the endogenous HMOX1 gene. Individual clones were selected and extensively characterized to confirm precise editing and retained stem cell properties. Bardoxolone-methyl (CDDO-Me) induced oxidative stress caused similarly increased expression of both the wild-type and eGFP-tagged HMOX1 at the mRNA and protein level. Fluorescently tagged hiPSC-derived proximal tubule-like, hepatocyte-like, cardiomyocyte-like and neuron-like progenies were treated with CDDO-Me (5.62-1000 nM) or diethyl maleate (5.62-1000 µM) for 24 h and 72 h. Multi-lineage oxidative stress responses were assessed through transcriptomics analysis, and HMOX1-eGFP reporter expression was carefully monitored using live-cell confocal imaging. We found that eGFP intensity increased in a dose-dependent manner with dynamics varying amongst lineages and stressors. Point of departure modelling further captured the specific lineage sensitivities towards oxidative stress. We anticipate that the newly developed HMOX1 hiPSC reporter will become a valuable tool in understanding and quantifying critical target organ cell-specific oxidative stress responses induced by (newly developed) chemical entities.

Neurotoxicity and underlying cellular changes of 21 mitochondrial respiratory chain inhibitors.

Inhibition of complex I of the mitochondrial respiratory chain (cI) by rotenone and methyl-phenylpyridinium (MPP +) leads to the degeneration of dopaminergic neurons in man and rodents. To formally describe this mechanism of toxicity, an adverse outcome pathway (AOP:3) has been developed that implies that any inhibitor of cI, or possibly of other parts of the respiratory chain, would have the potential to trigger parkinsonian motor deficits. We used here 21 pesticides, all of which are described in the literature as mitochondrial inhibitors, to study the general applicability of AOP:3 or of in vitro assays that are assessing its activation. Five cI, three complex II (cII), and five complex III (cIII) inhibitors were characterized in detail in human dopaminergic neuronal cell cultures. The NeuriTox assay, examining neurite damage in LUHMES cells, was used as in vitro proxy of the adverse outcome (AO), i.e., of dopaminergic neurodegeneration. This test provided data on whether test compounds were unspecific cytotoxicants or specifically neurotoxic, and it yielded potency data with respect to neurite degeneration. The pesticide panel was also examined in assays for the sequential key events (KE) leading to the AO, i.e., mitochondrial respiratory chain inhibition, mitochondrial dysfunction, and disturbed proteostasis. Data from KE assays were compared to the NeuriTox data (AO). The cII-inhibitory pesticides tested here did not appear to trigger the AOP:3 at all. Some of the cI/cIII inhibitors showed a consistent AOP activation response in all assays, while others did not. In general, there was a clear hierarchy of assay sensitivity: changes of gene expression (biomarker of neuronal stress) correlated well with NeuriTox data; mitochondrial failure (measured both by a mitochondrial membrane potential-sensitive dye and a respirometric assay) was about 10-260 times more sensitive than neurite damage (AO); cI/cIII activity was sometimes affected at > 1000 times lower concentrations than the neurites. These data suggest that the use of AOP:3 for hazard assessment has a number of caveats: (i) specific parkinsonian neurodegeneration cannot be easily predicted from assays of mitochondrial dysfunction; (ii) deriving a point-of-departure for risk assessment from early KE assays may overestimate toxicant potency.

Comparing in vitro human liver models to in vivo human liver using RNA-Seq.

The liver plays an important role in xenobiotic metabolism and represents a primary target for toxic substances. Many different in vitro cell models have been developed in the past decades. In this study, we used RNA-sequencing (RNA-Seq) to analyze the following human in vitro liver cell models in comparison to human liver tissue: cancer-derived cell lines (HepG2, HepaRG 3D), induced pluripotent stem cell-derived hepatocyte-like cells (iPSC-HLCs), cancerous human liver-derived assays (hPCLiS, human precision cut liver slices), non-cancerous human liver-derived assays (PHH, primary human hepatocytes) and 3D liver microtissues. First, using CellNet, we analyzed whether these liver in vitro cell models were indeed classified as liver, based on their baseline expression profile and gene regulatory networks (GRN). More comprehensive analyses using non-differentially expressed genes (non-DEGs) and differential transcript usage (DTU) were applied to assess the coverage for important liver pathways. Through different analyses, we noticed that 3D liver microtissues exhibited a high similarity with in vivo liver, in terms of CellNet (C/T score: 0.98), non-DEGs (10,363) and pathway coverage (highest for 19 out of 20 liver specific pathways shown) at the beginning of the incubation period (0 h) followed by a decrease during long-term incubation for 168 and 336 h. PHH also showed a high degree of similarity with human liver tissue and allowed stable conditions for a short-term cultivation period of 24 h. Using the same metrics, HepG2 cells illustrated the lowest similarity (C/T: 0.51, non-DEGs: 5623, and pathways coverage: least for 7 out of 20) with human liver tissue. The HepG2 are widely used in hepatotoxicity studies, however, due to their lower similarity, they should be used with caution. HepaRG models, iPSC-HLCs, and hPCLiS ranged clearly behind microtissues and PHH but showed higher similarity to human liver tissue than HepG2 cells. In conclusion, this study offers a resource of RNA-Seq data of several biological replicates of human liver cell models in vitro compared to human liver tissue.

The hepatotoxic fluoroquinolone trovafloxacin disturbs TNF- and LPS-induced p65 nuclear translocation in vivo and in vitro.

Idiosyncratic drug-induced liver injury (IDILI) is a severe disease that cannot be detected during drug development. It has been shown that hepatotoxicity of some compounds associated with IDILI becomes apparent when these are combined in vivo and in vitro with LPS or TNF. Among these compounds trovafloxacin (TVX) induced apoptosis in the liver and increased pro-inflammatory cytokines in mice exposed to LPS/TNF. The hepatocyte survival and the cytokine release after TNF/LPS stimulation relies on a pulsatile activation of NF-κB. We set out to evaluate the dynamic activation of NF-κB in response to TVX + TNF or LPS models, both in mouse and human cells. Remarkably, TVX prolonged the first translocation of NF-κB induced by TNF both in vivo and in vitro. The prolonged p65 translocation caused by TVX was associated with an increased phosphorylation of IKK and MAPKs and accumulation of inhibitors of NF-κB such as IκBα and A20 in HepG2. Coherently, TVX suppressed further TNF-induced NF-κB translocations in HepG2 leading to decreased transcription of ICAM-1 and inhibitors of apoptosis. TVX prolonged LPS-induced NF-κB translocation in RAW264.7 macrophages increasing the secretion of TNF. In summary, this study presents new, relevant insights into the mechanism of TVX-induced liver injury underlining the resemblance between mouse and human models. In this study we convincingly show that regularly used toxicity models provide a coherent view of relevant pathways for IDILI. We propose that assessment of the kinetics of activation of NF-κB and MAPKs is an appropriate tool for the identification of hepatotoxic compounds during drug development.

Quantitative phosphoproteomics to unravel the cellular response to chemical stressors with different modes of action.

Damage to cellular macromolecules and organelles by chemical exposure evokes activation of various stress response pathways. To what extent different chemical stressors activate common and stressor-specific pathways is largely unknown. Here, we used quantitative phosphoproteomics to compare the signaling events induced by four stressors with different modes of action: the DNA damaging agent: cisplatin (CDDP), the topoisomerase II inhibitor: etoposide (ETO), the pro-oxidant: diethyl maleate (DEM) and the immunosuppressant: cyclosporine A (CsA) administered at an equitoxic dose to mouse embryonic stem cells. We observed major differences between the stressors in the number and identity of responsive phosphosites and the amplitude of phosphorylation. Kinase motif and pathway analyses indicated that the DNA damage response (DDR) activation by CDDP occurs predominantly through the replication-stress-related Atr kinase, whereas ETO triggers the DDR through Atr as well as the DNA double-strand-break-associated Atm kinase. CsA shares with ETO activation of CK2 kinase. Congruent with their known modes of action, CsA-mediated signaling is related to down-regulation of pathways that control hematopoietic differentiation and immunity, whereas oxidative stress is the most prominent initiator of DEM-modulated stress signaling. This study shows that even at equitoxic doses, different stressors induce distinctive and complex phosphorylation signaling cascades.

Correction to: Multiparametric assessment of mitochondrial respiratory inhibition in HepG2 and RPTEC/TERT1 cells using a panel of mitochondrial targeting agrochemicals.

In the original publication of the article.

Multiparametric assessment of mitochondrial respiratory inhibition in HepG2 and RPTEC/TERT1 cells using a panel of mitochondrial targeting agrochemicals.

Evidence is mounting for the central role of mitochondrial dysfunction in several pathologies including metabolic diseases, accelerated ageing, neurodegenerative diseases and in certain xenobiotic-induced organ toxicity. Assessing mitochondrial perturbations is not trivial and the outcomes of such investigations are dependent on the cell types used and assays employed. Here we systematically investigated the effect of electron transport chain (ETC) inhibitors on multiple mitochondrial-related parameters in two human cell types, HepG2 and RPTEC/TERT1. Cells were exposed to a broad range of concentrations of 20 ETC-inhibiting agrochemicals and capsaicin, consisting of inhibitors of NADH dehydrogenase (Complex I, CI), succinate dehydrogenase (Complex II, CII) and cytochrome bc1 complex (Complex III, CIII). A battery of tests was utilised, including viability assays, lactate production, mitochondrial membrane potential (MMP) and the Seahorse bioanalyser, which simultaneously measures extracellular acidification rate [ECAR] and oxygen consumption rate [OCR]. CI inhibitors caused a potent decrease in OCR, decreased mitochondrial membrane potential, increased ECAR and increased lactate production in both cell types. Twenty-fourhour exposure to CI inhibitors decreased viability of RPTEC/TERT1 cells and 3D spheroid-cultured HepG2 cells in the presence of glucose. CI inhibitors decreased 2D HepG2 viability only in the absence of glucose. CII inhibitors had no notable effects in intact cells up to 10 µM. CIII inhibitors had similar effects to the CI inhibitors. Antimycin A was the most potent CIII inhibitor, with activity in the nanomolar range. The proposed CIII inhibitor cyazofamid demonstrated a mitochondrial uncoupling signal in both cell types. The study presents a comprehensive example of a mitochondrial assessment workflow and establishes measurable key events of ETC inhibition.