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Updated: Jul 5, 2025
Experimental Protocol for Detecting Mitochondrial Function in Hepatocytes Exposed to Organochlorine Pesticides
Published on: September 16, 2020
The integrated stress response-related expression of CHOP due to mitochondrial toxicity is a warning sign for DILI
Matthijs Vlasveld1, Giulia Callegaro1, Ciarán Fisher2
1Division of Drug Discovery and Safety, Leiden Academic Centre for Drug Research (LACDR), Leiden University, Leiden, The Netherlands.
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.
Insights
Early screening for CHOP expression can predict drug-induced liver injury (DILI) risk. This method identifies compounds with mitochondrial toxicity, aiding drug development decisions and reducing trial failures.
Area of Science:
- Hepatotoxicity and Drug Development
- Mitochondrial Toxicology
- Biomarker Discovery
Background:
- Drug-induced liver injury (DILI) is a significant cause of drug development failure and market withdrawal.
- Early identification of DILI risk is a critical challenge in pharmaceutical research.
- Mitochondrial toxicity is an emerging mechanism underlying DILI.
Purpose of the Study:
- To develop and validate a mechanism-based, weight-of-evidence approach for identifying DILI liabilities.
- To assess the utility of cellular stress responses as early indicators of DILI.
- To investigate the role of mitochondrial dysfunction in drug-induced hepatotoxicity.
Main Methods:
- Screening of 1587 FDA-approved drugs and 378 kinase inhibitors using an imaging-based HepG2 BAC-GFP reporter platform.
- Assessed activation of integrated stress response (CHOP), DNA damage response (P21), and oxidative stress response (SRXN1).
- Utilized whole-genome RNA-sequencing of primary human hepatocytes for transcriptomic evaluation of selected drug classes.
Main Results:
- 389, 219, and 104 drugs induced CHOP-GFP, P21-GFP, and SRXN1-GFP expression, respectively.
- 154 FDA-approved drugs were identified as critical CHOP-GFP inducers.
- Antimycotic and CNS agents showed impaired mitochondrial oxygen consumption and affected mitochondrial processes, NRF2 signaling, and xenobiotic metabolism.
Conclusions:
- Early pre-clinical screening for CHOP expression serves as a potential indicator of mitochondrial toxicity in DILI.
- This screening approach can act as a valuable warning signal in drug development decision-making.
- The findings support the integration of mitochondrial toxicity assessment into early drug safety evaluations.
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