Liaskos, Nektarios; (2025) Evaluation of HepG2 Cell Models of Drug-Induced Liver Injury (DILI) using NMR-based Metabonomics and Investigation of the Antioxidant Potential of Plant Extracts. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Drug-induced liver injury (DILI) is a leading cause of acute liver failure and a major cause of death in the US and the UK. DILI is often induced by reactive metabolites that form during the biotransformation of xenobiotic substances in the liver. The primary aim of this project was to develop two in vitro models of DILI in HepG2 monolayers and spheroids using acetaminophen (APAP) and carbon tetrachloride (CCl4) and evaluate them as potential platforms for the discovery of metabolite biomarkers of DILI. Selected plant extracts with high antioxidant capacity were assessed for hepatoprotection in both models. Finally, the cell models were compared to an animal model of DILI, with and without pre-treatment with a phlorotannin extract from Fucus vesiculosus (PT). Dose-response and time-response studies in HepG2 cell monolayers revealed that models of mild (below TD20) and severe DILI (above TD80 or TDmax) could be generated by treatment with 5 mM and 30 mM APAP for 24 hours, and 0.05% and 0.3% (v/v) CCl4 for 6 hours. These dose levels align with previously reported toxic concentrations in HepG2 cells, although direct correlation to in vivo situations is limited due to differences in exposure and metabolism. Antioxidant assays revealed changes in SOD-1 and catalase levels as well as increased malondialdehyde concentrations in media suggesting oxidative stress and lipid peroxidation had been induced. 1H-NMR-based metabonomic analysis of cell lysates and media samples from monolayers treated with APAP and CCl4 revealed changes to the cellular metabolome indicative of potential disruptions in gluconeogenesis and glycolysis, altered TCA cycle activity, and changes in levels of endogenous antioxidants, β-hydroxybutyrate, homocysteine and methionine. HepG2 spheroid models of DILI were created using the same dosing and sample collection schedules as monolayers. When compared to monolayers, HepG2 spheroids showed similar susceptibility to CCl4 based on DILI-related metabolite changes, but lower susceptibility to APAP-induced injury. 1H-NMR-based metabonomic analysis of cell lysate and media samples from the spheroids revealed metabolome changes similar to those observed in HepG2 monolayers, such as altered transsulfuration metabolite levels, including endogenous antioxidants. A total of 45 plant extracts were screened for their phenolic content. Twelve were then selected based on their high phenolic content and their radical scavenging properties confirmed using DPPH and ORAC assays. Pre-treatment with three of the extracts, swamp horsetail, short-fruited willow herb, and PT resulted in improvement of viability in both APAP- and CCl4 treated HepG2 monolayer cells based on the MTT assay. PT was selected for further metabonomic studies since it showed the highest antioxidant capacity across all employed assays. Supplementation with PT 24 hours prior to dosing with APAP and CCl4 showed significantly reduced hepatotoxicity in monolayers. 1H-NMR of cell lysate and media samples from monolayers and spheroids revealed there were differences in the metabolome profile of PT pre-treated cells when compared to groups without the PT supplementation in all APAP and CCl4 dosing groups. In a previous animal model in our laboratory Hanover Wistar rats were treated with 1.2 mg/kg bw CCl4, with and without pre-treatment with 25 mg/kg bw PT for 7 days. Data from this animal study was compared to the findings in the HepG2 cell models. Changes in similar metabolites were identified, particularly oxidative stress and transsulfuration, although the directionality of changes differed between cellular and animal models, potentially reflecting differences in biological complexity, metabolism, and cellular responses. PT pre-treatment in the animals revealed signs of hepatoprotection, such as no increases in glutamate and glutamine, lactate, and taurine levels that were observed to increase in response to CCl4 treatment. In this project, we developed monolayer and spheroid models of DILI using APAP and CCl4. 1H-NMR analysis allowed for characterisation of cellular metabolome changes in mild and severe DILI, but also potential hepatoprotection from the supplementation with PT. Comparison with the animal model showed that pre-treatment with PT resulted in reversal of metabolite changes in response to CCl4 in all three models. This was more prominent in the animal study, highlighting the potential hepatoprotective benefits of this extract.

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