The generation of HepG2 transmitochondrial cybrids to reveal the role of mitochondrial genotype in idiosyncratic drug-induced liver injury: a translational in vitro study
- University of Liverpool, United Kingdom
- GlaxoSmithKline, United Kingdom
Abstract
Background: Evidence supports an important link between mitochondrial DNA (mtDNA) variation and adverse drug reactions such as idiosyncratic drug-induced liver injury (iDILI). Here, we describe the generation of HepG2-derived transmitochondrial cybrids, to investigate the impact of mtDNA variation on mitochondrial (dys)function and susceptibility to iDILI. This study created 10 cybrid cell lines, each containing distinct mitochondrial genotypes of haplogroup H or haplogroup J backgrounds.
Methods: HepG2 cells were depleted of mtDNA to make rho zero cells, before the introduction of known mitochondrial genotypes using platelets from healthy volunteers (n=10), thus generating 10 transmitochondrial cybrid cell lines. The mitochondrial function of each was assessed at basal state and following treatment with compounds associated with iDILI; flutamide, 2-hydroxyflutamide, and tolcapone, and their less toxic counterparts bicalutamide and entacapone utilising ATP assays and extracellular flux analysis.
Findings: Whilst only slight variations in basal mitochondrial function were observed between haplogroups H and J, haplogroup-specific responses were observed to the mitotoxic drugs. Haplogroup J showed increased susceptibility to inhibition by flutamide, 2-hydroxyflutamide and tolcapone, via effects on selected mitochondrial complexes (I and II), and an uncoupling of the respiratory chain.
Conclusions: This study demonstrates that HepG2 transmitochondrial cybrids can be created to contain the mitochondrial genotype of any individual of interest. This provides a practical and reproducible system to investigate the cellular consequences of variation in the mitochondrial genome, against a constant nuclear background. Additionally, the results show that inter-individual variation in mitochondrial haplogroup may be a factor in determining sensitivity to mitochondrial toxicants.
Funding: This work was supported by the Centre for Drug Safety Science supported by the Medical Research Council, United Kingdom (Grant Number G0700654); and GlaxoSmithKline as part of an MRC-CASE studentship (grant number MR/L006758/1).
Data availability
Source Data files have been provided for Figures 2, 3, 4, 5, 6 and supplementary figures s1, s3, s4, s5, s6
Article and author information
Author details
Funding
Medical Research Council (Centre for Drug Safety Science,G0700654)
- Carol Elizabeth Jolly
- Amy E Chadwick
GlaxoSmithKline (MRC-CASE studentship,grant number MR/L006758/1))
- Amy Louise Ball
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Human subjects: This work with human material in this project was approved by the North West of England Research Ethics Committee and all participants gave written informed consent and consent to publish. All procedures were in accordance with the ethical standards of the North West of England Research Ethics Committee (Cell Archive of HLA Typed Healthy Volunteers (HLA), CRN ID 7787, IRAS ID: 15623) with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
Copyright
© 2023, Ball et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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The generation of HepG2 transmitochondrial cybrids to reveal the role of mitochondrial genotype in idiosyncratic drug-induced liver injury: a translational in vitro study
eLife 12:e78187.
https://doi.org/10.7554/eLife.78187
