Use this URL to cite or link to this record in EThOS:
Title: Towards improved models for the study of the multi-mechanistic toxicity of drug-induced liver injury
Author: Penman, Sophie
ISNI:       0000 0005 0288 1638
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2020
Availability of Full Text:
Access from EThOS:
Access from Institution:
The detection of adverse drug reactions (ADRs) post-market poses a significant concern for patient health and the pharmaceutical industry given the high cost of drug development and the potential for fatalities. The liver is one of the most reported cases of toxicity and therefore, the detection of drug-induced liver injury (DILI) preclinically is imperative. Whilst rare, cases of DILI that are idiosyncratic are the most problematic as they are characterised by a complex dose-related relationship, lack of predictability from the pharmacology of the drug and interindividual variation. Notably, it is acknowledged that the toxicity of idiosyncratic DILI is multi-mechanistic. Advancements in the field have identified many in vitro assays to evaluate the potential for a compound to cause DILI however, if these are not conducted in an appropriate model, the results can lack in vivo applicability. HepG2 cells are the most common cell line used during preclinical DILI screening however, their utility is limited for certain mechanisms associated with DILI. If limitations of the models are not accounted for, toxicity can be missed, over-estimated or bear little relevance to the toxicity seen in humans. Therefore, the aim of this research was to assess the utility of different hepatic models for their appropriateness in studying mechanisms of toxicity associated with DILI. Biliary transporter alterations and mitochondrial dysfunction are frequently reported to be implicated in DILI and so the utility of HepG2 and HepaRG cells for studying these mechanisms was evaluated. Whilst it is acknowledged that DILI arises due to a combination of drug-related mechanisms, individual susceptibility factors are also involved, but fail to be incorporated into preclinical models. The final aim of this thesis was to use HepG2 transmitochondrial cybrids to assess the effect of mitochondrial DNA (mtDNA) variation upon susceptibility to mitochondrial dysfunction with a compound associated with idiosyncratic DILI. Initial investigations revealed that HepaRG cells possess a more suitable phenotype for assessments of transporter regulation and mitochondrial dysfunction than HepG2 cells. Following this confirmation, the ability of bile acid (BA) mixtures to cause transporter toxicity and mitochondrial dysfunction were evaluated. It was identified that pathological concentrations of BA mixtures caused a temporal reduction in the activity and expression of key biliary transporters. Subsequent experiments investigated the mitotoxic potential of BAs in isolated mitochondria and HepaRG whole cells. Taken collectively, alterations in mitochondrial membrane potential (MMP), ATP content in galactose media and extracellular flux analysis did not reveal mitochondrial dysfunction as a mechanism of BAinduced toxicity. Finally, an mtDNA haplogroup-toxicity association study using haplogroups B, H and J was conducted to assess differential toxicity to tolcapone. A significant difference in susceptibility to tolcapone-induced mitochondrial toxicity was detected between haplogroups following 2 hours dosing. However, extended dosing regimens of 24 hours resulted in a reversal in susceptibility to toxicity. Studies of mitochondrial dynamics and biogenesis revealed that there are complex molecular pathways governing mitochondrial protection and susceptibility to toxicity dependent on mtDNA haplogroup. Specifically, differences in mtDNA copy number, which was used as a surrogate marker for biogenesis, was identified to be temporally different amongst the haplogroups. To conclude, it is likely that idiosyncratic DILI occurs due to a combination of mechanisms in conjunction with personal susceptibility factors. However, failure to employ the correct model can lead to the generation of data that lacks in vivo applicability. Following the use of appropriate, physiologically relevant preclinical models, this research identified biliary transporter dysfunction as a mechanism of BA-induced toxicity but not mitochondrial dysfunction. Additionally, HepG2 transmitochondrial cybrids were identified as a novel model for assessing the role of mtDNA variation and its contributions towards idiosyncratic DILI. Ultimately, the use of the most appropriate and physiologically relevant models is likely to improve the predictivity of DILI screening, leading to improvements in drug safety.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral