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.

Risk Assessment of Psychotropic Drugs on Mitochondrial Function Using In Vitro Assays.

Mitochondria are potential targets responsible for some drug- and xenobiotic-induced organ toxicities. However, molecular mechanisms of drug-induced mitochondrial toxicities are mostly unknown. Here, multiple in vitro assays were used to investigate the effects of 22 psychotropic drugs on mitochondrial function. The acute extracellular flux assay identified inhibitors of the electron transport chain (ETC), i.e., aripiprazole, phenytoin, and fluoxetine, an uncoupler (reserpine), substrate inhibitors (quetiapine, carbamazepine, buspirone, and tianeptine), and cytotoxic compounds (chlorpromazine and valproic acid) in HepG2 cells. Using permeabilized HepG2 cells revealed minimum effective concentrations of 66.3, 6730, 44.5, and 72.1 µM for the inhibition of complex-I-linked respiration for quetiapine, valproic acid, buspirone, and fluoxetine, respectively. Assessing complex-II-linked respiration in isolated rat liver mitochondria revealed haloperidol is an ETC inhibitor, chlorpromazine is an uncoupler in basal respiration and an ETC inhibitor under uncoupled respiration (IC50 = 135 µM), while olanzapine causes a mild dissipation of the membrane potential at 50 µM. This research elucidates some mechanisms of drug toxicity and provides some insight into their safety profile for clinical drug decisions.

New approach methods (NAMs) supporting read-across: Two neurotoxicity AOP-based IATA case studies

Read-across approaches are considered key in moving away from in vivo animal testing towards addressing data-gaps using new approach methods (NAMs). Ample successful examples are still required to substantiate this strategy. Here we present and discuss the learnings from two OECD IATA endorsed read-across case studies. They involve two classes of pesticides ­ rotenoids and strobilurins ­ each having a defined mode-of-action that is assessed for its neurological hazard by means of an AOP-based testing strategy coupled to toxicokinetic simulations of human tissue concentrations. The endpoint in question is potential mitochondrial respiratory chain mediated neurotoxicity, specifically through inhibition of complex I or III. An AOP linking inhibition of mitochondrial respiratory chain complex I to the degeneration of dopaminergic neurons formed the basis for both cases but was deployed in two different regulatory contexts. The two cases also exemplify several different read-across concepts: analogue versus category approach, consolidated versus putative AOP, positive versus negative prediction (i.e., neurotoxicity versus low potential for neurotoxicity), and structural versus biological similarity. We applied a range of NAMs to explore the toxicodynamic properties of the compounds, e.g., in silico docking as well as in vitro assays and readouts ­ including transcriptomics ­ in various cell systems, all anchored to the relevant AOPs. Interestingly, although some of the data addressing certain elements of the read-across were associated with high uncertainty, their impact on the overall read-across conclusion remained limited. Coupled to the elaborate regulatory review that the two cases underwent, we propose some generic learnings of AOP-based testing strategies supporting read-across.

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.

A combined in vitro approach to improve the prediction of mitochondrial toxicants.

Drug induced mitochondrial dysfunction has been implicated in organ toxicity and the withdrawal of drugs or black box warnings limiting their use. The development of highly specific and sensitive in vitro assays in early drug development would assist in detecting compounds which affect mitochondrial function. Here we report the combination of two in vitro assays for the detection of drug induced mitochondrial toxicity. The first assay measures cytotoxicity after 24h incubation of test compound in either glucose or galactose conditioned media (Glu/Gal assay). Compounds with a greater than 3-fold toxicity in galactose media compared to glucose media imply mitochondrial toxicity. The second assay measures mitochondrial respiration, glycolysis and a reserve capacity with mechanistic responses observed within one hour following exposure to test compound. In order to assess these assays a total of 72 known drugs and chemicals were used. Dose-response data was normalised to 100× Cmax giving a specificity, sensitivity and accuracy of 100%, 81% and 92% respectively for this combined approach.

Multiple compound-related adverse properties contribute to liver injury caused by endothelin receptor antagonists.

Drug-induced liver injury has been observed in patients treated with the endothelin receptor antagonists sitaxentan and bosentan, but not following treatment with ambrisentan. The aim of our studies was to assess the possible role of multiple contributory mechanisms in this clinically relevant toxicity. Inhibition of the bile salt export pump (BSEP) and multidrug resistance-associated protein 2 was quantified using membrane vesicle assays. Inhibition of mitochondrial respiration in human liver-derived HuH-7 cells was determined using a Seahorse XF(e96) analyzer. Cytochrome P450 (P450)-independent and P450-mediated cell toxicity was assessed using transfected SV40-T-antigen-immortalized human liver epithelial (THLE) cell lines. Exposure-adjusted assay ratios were calculated by dividing the maximum human drug plasma concentrations by the IC50 or EC50 values obtained in vitro. Covalent binding (CVB) of radiolabeled drugs to human hepatocytes was quantified, and CVB body burdens were calculated by adjusting CVB values for fractional drug turnover in vitro and daily therapeutic dose. Sitaxentan exhibited positive exposure-adjusted signals in all five in vitro assays and a high CVB body burden. Bosentan exhibited a positive exposure-adjusted signal in one assay (BSEP inhibition) and a moderate CVB body burden. Ambrisentan exhibited no positive exposure-adjusted assay signals and a low CVB body burden. These data indicate that multiple mechanisms contribute to the rare, but potentially severe liver injury caused by sitaxentan in humans; provide a plausible rationale for the markedly lower propensity of bosentan to cause liver injury; and highlight the relative safety of ambrisentan.

In vitro exploration of potential mechanisms of toxicity of the human hepatotoxic drug fenclozic acid.

The carboxylic acid NSAID fenclozic acid exhibited an excellent preclinical safety profile and promising clinical efficacy, yet was withdrawn from clinical development in 1971 due to hepatotoxicity observed in clinical trials. A variety of modern in vitro approaches have been used to explore potential underlying mechanisms. Covalent binding studies were undertaken with [(14)C]-fenclozic acid to investigate the possible role of reactive metabolites. Time-dependent covalent binding to protein was observed in NADPH-supplemented liver microsomes, although no metabolites were detected in these incubations or in reactive metabolite trapping experiments. In human hepatocytes, covalent binding was observed at lower levels than in microsomes and a minor uncharacterizable metabolite was also observed. In addition, covalent binding was observed in incubations undertaken with dog and rat hepatocytes, where a taurine conjugate of the drug was detected. Although an acyl glucuronide metabolite was detected when liver microsomes from human, rat and dog were supplemented with UDPGA, there was no detectable UDPGA-dependent covalent binding. No effects were observed when fenclozic acid was assessed for P450-dependent and P450-independent cytotoxicity to THLE cell lines, time-dependent inhibition of five major human cytochrome P450 enzymes, inhibition of the biliary efflux transporters BSEP and MRP2 or mitochondrial toxicity to THLE or HepG2 cells. These data suggest that Phase 1 bioactivation plays a role in the hepatotoxicity of fenclozic acid and highlight the unique insight into mechanisms of human drug toxicity that can be provided by investigations of biotransformation and covalent binding to proteins.

Cell based approaches for evaluation of drug-induced liver injury.

An improved understanding of mechanisms that underlie drug-induced liver injury (DILI) is required to enable design of drugs that have minimal potential to cause this adverse reaction in man. Available evidence suggests DILI arises in susceptible patients because of an imbalance between chemical insults (which are an inherent property of certain drugs and/or their metabolites) and the ability of the liver to mount compensatory/adaptive responses. In vivo safety testing in pre-clinical species ensures that drugs which enter clinical trials do not cause reproducible and dose-dependent liver injury in man, but is of limited value for exploration of underlying mechanisms and does not assess potential to cause rare idiosyncratic DILI. This review highlights the value that can be gained from in vitro studies using cultured hepatocytes and also hepatocyte-derived cell lines transfected with individual human cytochrome P450 (CYP450) isoforms. We have evaluated a range of mechanisms and endpoints (cell necrosis, mitochondrial injury, inhibition of biliary transporters and metabolite-mediated toxicity) using these model systems. Our data indicate that multiple mechanisms are likely to be involved in development of idiosyncratic DILI in man caused by numerous drugs, e.g. the anticonvulsant chlorpromazine.