Cox, Christopher
(2023)
Evaluating the Role of Mitochondrial
Genetics in Adverse-Drug Reactions and the
Application of iPSC-Derived Hepatocytes
for Modelling Drug-Induced Liver Injury.
PhD thesis, University of Liverpool.
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Abstract
Drug-induced liver injury (DILI) remains a major cause of drug attrition during drug development and is associated with a large proportion of drugs that have been withdrawn from the market. Although there are numerous factors associated with DILI, it is becoming clear that genetic variation in drug-metabolising enzymes and transporters (DMETs) can play an important role in its development. It is, therefore, essential that we have sufficient techniques to investigate the genetic basis of DILI and screen for them as early as possible during the drug-development pipeline. Although primary human hepatocytes (PHHs) remain the gold standard for investigating patient-specific DILI, they are difficult to source, require invasive surgery, and rapidly de-differentiate when cultured in vitro, limiting their application for downstream research. Nevertheless, it is now possible to generate induced-pluripotent stem cells (iPSCs) from easily accessible cells, providing a potentially unlimited supply of patient-specific cells that can differentiate into any cell type in the adult human body. In this project, the metabolic capabilities of iPSC-derived hepatocyte-like cells (HLCs) was evaluated to assess their application for investigating metabolism-dependent toxicities, providing a possible alternative to PHHs. Data indicates that Laminin-521, a defined growth matrix, provides a suitable alternative to Matrigel, a routinely used undefined matrix, for differentiation of HLCs. This could help to improve the reproducibility of HLC data. However, HLCs on both substrates showed low or undetectable expression for some important DMETs, for example, CYP3A4 and CYP2E1. At the end of differentiation, the cells also appeared to have a mixed phenotype, with expression of hepatocyte and gastrointestinal markers. Furthermore, they failed to show induction of CYP3A4 via activation of the pregnane X receptor (PxR), in response to rifampicin, a routinely used positive control. Being able to identify drugs that activate nuclear receptors and induce DMETs is an important consideration when assessing a drug's potential for drug-drug interactions and drug-induced toxicities. HLCs differentiated in this study, therefore, have limited application for investigating drug-drug interactions. Further work is needed to improve the phenotype of these cells before they can be used as a suitable alternative to PHHs, for assessing a drug's toxic potential. The influence of mitochondrial genetics on nuclear-encoded DMETs and sensitivity to drug-induced toxicities were also investigated in vitro using a panel of HepG2 transmitochondrial cybrid cell lines, which have the same nuclear genetic background but different mitochondrial genetics / haplogroups. Mitochondrial haplogroups tested in this study included H1c3, H1b, H2a1e1a1, and J1c1e. Data indicated that mitochondrial genetic variation may influence the expression of an important detoxification enzyme UGT1A1, with differential expression across cybrids at the transcript and protein level. This provides a potential mechanism of how mitochondrial genetic variation could contribute to DILI. Following the in vitro assessment of mitochondrial genetic variation, the association between mitochondrial genetics and adverse drug reactions was further investigated using the UK Biobank dataset, which contains phenotype and genotype data for almost 500,000 volunteers. This analysis revealed that individuals with mitochondrial haplogroup X and certain single nucleotide polymorphisms may be more at risk for suffering a statin-related toxicity. These findings could help to stratify patients and reduce the number of statin-related toxicities. It also leads the way for future studies looking at other drug classes.
| Item Type: | Thesis (PhD) |
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| Uncontrolled Keywords: | Genetics, iPSC, Mitochondria |
| Divisions: | Faculty of Health and Life Sciences |
| Depositing User: | Symplectic Admin |
| Date Deposited: | 11 Aug 2023 14:35 |
| Last Modified: | 11 Aug 2023 14:36 |
| DOI: | 10.17638/03171039 |
| Supervisors: |
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| URI: | https://livrepository.liverpool.ac.uk/id/eprint/3171039 |
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