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.
Metrics
-
- 946
- views
-
- 137
- downloads
-
- 0
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
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
Further reading
-
- Medicine
- Neuroscience
Neurodegenerative diseases are age-related disorders characterized by the cerebral accumulation of amyloidogenic proteins, and cellular senescence underlies their pathogenesis. Thus, it is necessary for preventing these diseases to remove toxic proteins, repair damaged neurons, and suppress cellular senescence. As a source for such prophylactic agents, we selected zizyphi spinosi semen (ZSS), a medicinal herb used in traditional Chinese medicine. Oral administration of ZSS hot water extract ameliorated Aβ and tau pathology and cognitive impairment in mouse models of Alzheimer’s disease and frontotemporal dementia. Non-extracted ZSS simple crush powder showed stronger effects than the extract and improved α-synuclein pathology and cognitive/motor function in Parkinson’s disease model mice. Furthermore, when administered to normal aged mice, the ZSS powder suppressed cellular senescence, reduced DNA oxidation, promoted brain-derived neurotrophic factor expression and neurogenesis, and enhanced cognition to levels similar to those in young mice. The quantity of known active ingredients of ZSS, jujuboside A, jujuboside B, and spinosin was not proportional to the nootropic activity of ZSS. These results suggest that ZSS simple crush powder is a promising dietary material for the prevention of neurodegenerative diseases and brain aging.
-
- Medicine
Inherited retinal degenerations (IRDs) constitute a group of clinically and genetically diverse vision-impairing disorders. Retinitis pigmentosa (RP), the most common form of IRD, is characterized by gradual dysfunction and degeneration of rod photoreceptors, followed by the loss of cone photoreceptors. Recently, we identified reserpine as a lead molecule for maintaining rod survival in mouse and human retinal organoids as well as in the rd16 mouse, which phenocopy Leber congenital amaurosis caused by mutations in the cilia-centrosomal gene CEP290 (Chen et al., 2023). Here, we show the therapeutic potential of reserpine in a rhodopsin P23H rat model of autosomal dominant RP. At postnatal day (P) 68, when males and females are analyzed together, the reserpine-treated rats exhibit higher rod-derived scotopic b-wave amplitudes compared to the controls with little or no change in scotopic a-wave or cone-derived photopic b-wave. Interestingly, the reserpine-treated female rats display enhanced scotopic a- and b-waves and photopic b-wave responses at P68, along with a better contrast threshold and increased outer nuclear layer thickness. The female rats demonstrate better preservation of both rod and cone photoreceptors following reserpine treatment. Retinal transcriptome analysis reveals sex-specific responses to reserpine, with significant upregulation of phototransduction genes and proteostasis-related pathways, and notably, genes associated with stress response. This study builds upon our previously reported results reaffirming the potential of reserpine for gene-agnostic treatment of IRDs and emphasizes the importance of biological sex in retinal disease research and therapy development.
