The generation of HepG2 transmitochondrial cybrids to reveal the role of mitochondrial genotype in idiosyncratic drug-induced liver injury
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 o...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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eLife Sciences Publications Ltd
2023-06-01
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| Series: | eLife |
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| Online Access: | https://elifesciences.org/articles/78187 |
| _version_ | 1827925045095170048 |
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| author | Amy Louise Ball Carol E Jolly Mark G Lennon Jonathan J Lyon Ana Alfirevic Amy E Chadwick |
| author_facet | Amy Louise Ball Carol E Jolly Mark G Lennon Jonathan J Lyon Ana Alfirevic Amy E Chadwick |
| author_sort | Amy Louise Ball |
| collection | DOAJ |
| description | 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 utilizing ATP assays and extracellular flux analysis.
Results: 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). |
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| issn | 2050-084X |
| language | English |
| last_indexed | 2024-03-13T05:19:33Z |
| publishDate | 2023-06-01 |
| publisher | eLife Sciences Publications Ltd |
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| series | eLife |
| spelling | doaj.art-cac0c9cc70c841ffb77c6bdfa52724f52023-06-15T15:44:52ZengeLife Sciences Publications LtdeLife2050-084X2023-06-011210.7554/eLife.78187The generation of HepG2 transmitochondrial cybrids to reveal the role of mitochondrial genotype in idiosyncratic drug-induced liver injuryAmy Louise Ball0Carol E Jolly1Mark G Lennon2Jonathan J Lyon3Ana Alfirevic4Amy E Chadwick5https://orcid.org/0000-0002-7399-8655Centre for Drug Safety Science, Department of Pharmacology and Therapeutics, University of Liverpool, Liverpool, United KingdomCentre for Drug Safety Science, Department of Pharmacology and Therapeutics, University of Liverpool, Liverpool, United KingdomGSK, BioStatistics, Stevenage, United KingdomGSK, Safety Assessment, Ware, United KingdomThe Wolfson Centre for Personalised Medicine, Department Pharmacology and Therapeutics, University of Liverpool, Liverpool, United KingdomCentre for Drug Safety Science, Department of Pharmacology and Therapeutics, University of Liverpool, Liverpool, United KingdomBackground: 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 utilizing ATP assays and extracellular flux analysis. Results: 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).https://elifesciences.org/articles/78187mitochondriadrug safetymtDNAdrug-induced liver injurytransmitochondrial cybridHepG2 |
| spellingShingle | Amy Louise Ball Carol E Jolly Mark G Lennon Jonathan J Lyon Ana Alfirevic Amy E Chadwick The generation of HepG2 transmitochondrial cybrids to reveal the role of mitochondrial genotype in idiosyncratic drug-induced liver injury eLife mitochondria drug safety mtDNA drug-induced liver injury transmitochondrial cybrid HepG2 |
| title | The generation of HepG2 transmitochondrial cybrids to reveal the role of mitochondrial genotype in idiosyncratic drug-induced liver injury |
| title_full | The generation of HepG2 transmitochondrial cybrids to reveal the role of mitochondrial genotype in idiosyncratic drug-induced liver injury |
| title_fullStr | The generation of HepG2 transmitochondrial cybrids to reveal the role of mitochondrial genotype in idiosyncratic drug-induced liver injury |
| title_full_unstemmed | The generation of HepG2 transmitochondrial cybrids to reveal the role of mitochondrial genotype in idiosyncratic drug-induced liver injury |
| title_short | The generation of HepG2 transmitochondrial cybrids to reveal the role of mitochondrial genotype in idiosyncratic drug-induced liver injury |
| title_sort | generation of hepg2 transmitochondrial cybrids to reveal the role of mitochondrial genotype in idiosyncratic drug induced liver injury |
| topic | mitochondria drug safety mtDNA drug-induced liver injury transmitochondrial cybrid HepG2 |
| url | https://elifesciences.org/articles/78187 |
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