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Title: An evaluation of in vitro models for the assessment of mitochondrial toxicity within Drug Induced Liver Injury (DILI)
Author: Douglas, O.
ISNI:       0000 0004 6496 6306
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2017
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Drug Induced Liver Injury (DILI) is of major concern to both clinicians and the pharmaceutical industry due to the attrition of lead compounds during preclinical development and the incidence of hepatotoxicity and/or the withdrawal of drugs post marketing. It has been hypothesised that drug-induced mitochondrial dysfunction (DIMD) could be a causative factor of DILI. The nucleoside analogue fialuridine (FIAU) was withdrawn from use following a clinical trial in 1993 in which 5 patients died of hepatic failure. Subsequent research has shown that FIAU induces toxicity via mitochondrial DNA damage. However, current in vitro screening methods do not identify these compounds as toxic and thus this poses a problem to pre-clinical safety assessments. Three methods of metabolic modification, utilising glucose or galactose supplemented media and 2DG were compared to define and monitor mitochondrial toxicity induced by FIAU, and positive control adefovir (ADEF), in HepG2 (human hepatocellular carcinoma) cells. The structural isomer of fialuridine, (FIAU 2’ epimer) was included as a negative control. Mitochondrial toxicity could not be demonstrated in HepG2 cells over a 7-day period by any of these drugs. The HepaRG line is evident to be more hepatocyte-like than HepG2 cells and therefore can overcome the limitations of the widely used HepG2 cells. It is hypothesised that their increased primary human hepatocyte (PHH) like phenotypic characteristics may be more suitable for drug toxicity studies. Here, the utility of HepaRG based models in the detection of mitotoxicants was compared with previous research using a HepG2 model. Bioenergetic phenotyping revealed that the HepaRG line is less metabolically active when compared to HepG2 cells. HepaRG cells have the capacity to undergo metabolic modification using a short term glucose/galactose switch method and thus detect compounds with mitochondrial liabilities (EC50-ATPglu/EC50-ATPgal >2). Following the assessment of 12 hepatotoxins it was concluded that the HepaRG line offered no increased sensitivity for the detection of mitotoxicants compared with HepG2 cells. However, their stability in culture over extended periods (1 to 4 weeks) may be advantageous for the study of delayed toxicity. Therefore, HepaRG cells were further utilised in the assessment of FIAU induced mitochondrial toxicity, using an acute metabolic screen. Mitochondrial toxicity in the absence of cell death was demonstrated following 2-week drug incubation. The measurement of cellular respiration (using Seahorse technology) demonstrated a dose-dependent decrease in mitochondrial respiration in the absence of a decrease in mitochondrial mass. Further studies demonstrated significant drug-dependent decreases in the expression of nuclear encoded complex II, mitochondria-encoded complex IV, plus a decrease in mitochondrial DNA (mtDNA). The studies have demonstrated that the HepaRG cell model is a suitable model for the study of mitochondrial toxicity induced by nucleoside antivirals with evidence indicating that the mechanism of action via effects upon mitochondrial DNA matches the clinical mechanism of hepatotoxicity. Further studies utilising HepaRG were performed to assess the chemical and molecular pathways of toxicity induced by paracetamol. The studies provided evidence that HepaRG cells have the metabolic capacity for the turnover of parent compound to the toxic metabolite NAPQI. Furthermore, acute metabolic screening demonstrated that following short term incubations the parent compound paracetamol contains a mitochondrial liability in the absence of NAPQI-induced GSH depletion. The studies within this thesis have highlighted that the HepaRG line is suitable for the detection of mitotoxicants in which toxicity is delayed or which is mediated via CYP P450 catalysed-metabolites. Additionally, the studies provide much evidence as to the power of in vitro screening models in providing fine and detailed mechanistic information.
Supervisor: Chadwick, A. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral