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Title: Generation and validation of transmitochondrial cybrid and platelet models to investigate the effect of mitochondrial genotype upon drug-induced liver injury
Author: Ball, A. L.
ISNI:       0000 0004 7970 4392
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2019
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Idiosyncratic drug-induced liver injury (DILI) is a major concern for patient health and the pharmaceutical industry. Idiosyncratic DILI is characterised by a complex dose-response relationship, lack of predictivity from the primary pharmacology of a drug and significant interindividual variability, all of which contribute towards the limited preclinical prediction of these reactions. Drug-induced mitochondrial dysfunction is implicated in the onset of DILI, supported by the fact that 50 % of drugs with a black box warning for hepatotoxicity also contain a mitochondrial liability. Mitochondria are also a source of the interindividual variation which underpins idiosyncratic DILI; mitochondria contain multiple copies of their own genome, mitochondrial DNA (mtDNA), which has been found to induce changes in mitochondrial function. Currently, there is limited incorporation of interindividual variation into preclinical models of mitochondrial dysfunction and DILI. This may contribute towards drug attrition. The overall aim of this research was therefore to generate and characterise a personalised in vitro hepatic model which can be used for the preclinical assessment of the effect of mtDNA variation upon mitochondrial function and susceptibility to dysfunction induced by compounds associated with idiosyncratic DILI. HepG2 transmitochondrial cybrids, an in vitro hepatic model, were generated for the first time by the fusion of platelets from volunteers of known mitochondrial genotype, with HepG2 rho zero cells (devoid of mtDNA). This cell line was selected as HepG2 cells are the most commonly used cell line during preclinical hepatotoxicity testing, therefore the personalisation of this model has great utility. Following the assessment of HepG2 cell mitochondrial dysfunction induced by test compounds associated with idiosyncratic DILI (flutamide and tolcapone) alongside non-hepatotoxic structural analogues (bicalutamide and entacapone) and metabolites (2-hydroxyflutamide), 384 volunteers were recruited for whole mitochondrial genome sequencing. From this cohort, 30 participants from 4 common UK mitochondrial haplogroups (H, J, T, and U) were recruited to donate platelets for mitochondrial (dys)function testing using extracellular flux analysis. Subsequently, HepG2 transmitochondrial cybrids were generated from volunteers of haplogroups H and J prior to the assessment of basal mitochondrial function and compound-induced dysfunction. Taken together, extracellular flux analysis, ATP content assays and studies of mitochondrial dynamics in cybrids indicated a differential susceptibility to mitochondrial dysfunction dependent on mitochondrial haplogroup. Specifically, haplogroup J conferred an increase in susceptibility to mitochondrial perturbations associated with tolcapone whereas haplogroup H appeared to confer a degree of protection. To conclude, HepG2 transmitochondrial cybrids are able to provide preclinical representation of the mtDNA variation which may contribute towards the idiosyncrasy of some cases of DILI. The identification of mitochondrial haplogroups associated with differential susceptibility to mitochondrial dysfunction bodes well for future study plans which aim to differentiate individuals at the subhaplogroup level. When considered alongside other factors implicated in the onset of idiosyncratic DILI, continued work in this area has potential future clinical utility in the identification of patients whom can be safely and effectively treated with compounds such as tolcapone, which are currently withdrawn due to the risk of idiosyncratic DILI.
Supervisor: Chadwick, Amy ; Alfirevic, Ana ; Lyon, Jonathan Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral