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Drug-induced liver injury is one of the leading causes of acute liver failure and is a serious barrier to drug development. Furthermore, it is a frequent adverse reaction to the antimicrobials used to treat tuberculosis, resulting in reduced treatment effectiveness, incomplete treatment, relapse and the development of antimicrobial resistance. The aim of the studies in this thesis were threefold, to investigate biomarkers of liver injury in patients receiving anti-tuberculosis medication, to investigate miRNA changes in zebrafish larvae due to drug-induced liver injury, and to develop a novel point of care diagnostic test for miR-122, a miRNA biomarker of liver injury. The novel biomarkers, microRNA-122 (miR-122) and cytokeratin-18 (K18) have been found to be diagnostic and prognostic in patients who overdose on paracetamol. A key question is, do these biomarkers retain their efficacy in the presence of infection? The circulating concentrations of miR-122 and K18 were defined in the following populations: healthy volunteers, active tuberculosis patients, latent tuberculosis patients, patients with non-tuberculous mycobacterial infection and HIV-tuberculosis coinfected patients. Circulating concentrations of miR-122 and K18 were not significantly different across these groups, indicating infection does not affect concentrations of miR-122 and K18. Furthermore, concentrations of miR-122 and K18 did not rise upon starting treatment. There was a significant correlation between the gold standard marker alanine aminotransferase (ALT) and both miR-122 and K18 demonstrating these biomarkers rise with elevations in ALT. In two individuals who developed drug-induced liver injury, miR-122 and K18 rose with ALT, suggesting these biomarkers have diagnostic potential for anti-tuberculosis drug-induced liver injury. To reduce the need for rodent studies a zebrafish larvae model of liver injury was developed. Reducing the number of rodent studies carried out reduces experimental costs and the use of zebrafish larvae enables rapid high-throughput experiments. Furthermore, using zebrafish larvae is in line with the "3Rs" (reduce, refine, replace) approach of animal use in science, with the replacement of rodents, a higher-order mammal, with the lower-order zebrafish larvae. Zebrafish larvae exposed to isoniazid and pyrazinamide developed liver toxicity as determined by mortality, fluorescent imaging and histology. In order to investigate pathways altered in anti-tuberculosis drug-induced liver injury and identify potential novel biomarkers small RNA sequencing was undertaken. Pathways were altered in anti-tuberculosis drug induced liver injury, including those associated with the metabolism of xenobiotics. Building on previous work by my group, zebrafish larvae were exposed to triptolide, a Chinese herbal medicine which induces liver injury. A method was developed to collect specific cell populations, hepatocytes and immune cells, from transgenic zebrafish larvae using fluorescence activated cell sorting (FACS). The miRNA changes in isolated cells populations were determined using qRT-PCR, and in whole fish using small RNA sequencing. Novel miRNA biomarkers of liver injury were identified from the small RNA sequencing data and translated in plasma samples from patients with anti-tuberculosis drug-induced liver injury. Two of these miRNAs rose with ALT in liver injury. A point of care test is needed for drug-induced liver injury, suitable for use in resource poor settings. A novel RNA toehold switch sensor was combined with a fluorescent and colorimetric outputs to provide a quantitative response to miR-122 concentrations. Toehold switches with a range of structures were developed and tested. Switches demonstrated varying levels of sensitivity and specificity for miR-122.

Fulminant hepatitis B (FHBV) is a rare but severe outcome of acute hepatitis B virus infection. The severe liver injury is attributed to an exaggerated immune response directed at clearing virus by causing lysis of infected hepatocytes. Multiple factors could contribute to severity of the disease. Our study has shown that although there are no specific variant(s) linked to severity, there are clusters of variants in the cis-acting regulatory elements which can upregulate replication of FHBV strains. The most novel concept to come out of this study is that, most of these variants are not unique to FHBV sequences but they are significant when they occur in the form of 'motifs' or combinations; we showed that these multiple motifs are associated with a common functional effect.

Lobar selective internal radiation therapy (SIRT) is widely used to treat liver tumors inducing atrophy of the treated lobe and contralateral hypertrophy. The lack of animal model has precluded further investigations to improve this treatment. We developed an animal model of liver damage and atrophy–hypertrophy complex after SIRT. Three groups of 5–8 rabbits received transportal SIRT with Yttrium 90 resin microspheres of the cranial lobes with diferent activities (0.3, 0.6 and 1.2GBq), corresponding to predicted absorbed radiation dose of 200, 400 and 800 Gy, respectively. Another group received non-loaded microspheres (sham group). Cranial and caudal lobes volumes were assessed using CT volumetry before, 15 and 30 days after SIRT. Liver biochemistry, histopathology and gene expression were evaluated. Four untreated rabbits were used as controls for gene expression studies. All animals receiving 1.2GBq were euthanized due to clinical deterioration. Cranial SIRT with 0.6GBq induced caudal lobe hypertrophy after 15 days (median increase 34% -ns-) but produced signifcant toxicity. Cranial SIRT with 0.3GBq induced caudal lobe hypertrophy after 30 days (median increase 82%, p = 0.04). No volumetric changes were detected in sham group. Transient increase in serum transaminases was detected in all treated groups returning to normal values at 15 days. There was dose-dependent liver dysfunction with bilirubin elevation and albumin decrease. Histologically, 1.2GBq group developed permanent severe liver damage with massive necrosis, 0.6 and 0.3GBq groups developed moderate damage with infammation and portal fbrosis at 15 days, partially recovering at 30 days. There was no diference in the expression of hepatocyte function and diferentiation genes between 0.3GBq and control groups. Cranial SIRT with 0.3GBq of 90Y resin microspheres in rabbits is a reliable animal model to analyse the atrophy–hypertrophy complex and liver damage without toxicity.

Hepatitis delta virus (HDV) is a defective RNA virus that has an absolute requirement for a virus belonging to the hepadnaviridae family like hepatitis B virus (HBV) for its replication and formation of new virions. HDV infection is usually associated with a worsening of HBV-induced liver pathogenesis, which leads to more frequent cirrhosis, increased risk of hepatocellular carcinoma (HCC), and fulminant hepatitis. Importantly, no selective therapies are available for HDV infection. The mainstay of treatment for HDV infection is pegylated interferon alpha; however, response rates to this therapy are poor. A better knowledge of HDV–host cell interaction will help with the identification of novel therapeutic targets, which are urgently needed. Animal models like hepadnavirus-infected chimpanzees or the eastern woodchuck have been of great value for the characterization of HDV chronic infection. Recently, more practical animal models in which to perform a deeper study of host virus interactions and to evaluate new therapeutic strategies have been developed. Therefore, the main focus of this review is to discuss the current knowledge about HDV host interactions obtained from cell culture and animal models.

Tissue samples from spotted gar (Lepisosteus oculatus) and largemouth bass (Micropterus salmoides) were collected from Caddo Lake. Gar and bass livers were subjected to histological investigation and color analysis. Liver color (as abs at 400 nm) was significantly correlated with total mercury in the liver (r2 = 0.57, p = 0.02) and muscle (r2 = 0.58, p = 0.01) of gar. Evidence of liver damage as lipofuscin and discoloration was found in both species but only correlated with liver mercury concentration in spotted gar. Inorganic mercury was the predominant form in gar livers. In order to determine the role of mercury speciation in fish liver damage, a laboratory feeding study was employed. Zebrafish (Danio rerio) were fed either a control (0.12 ± 0.002 µg Hg.g-1 dry wt), inorganic mercury (5.03 ± 0.309 µg Hg.g-1 dry wt), or methylmercury (4.11 ± 0.146 µg Hg.g-1 dry wt) diet. After 78 days of feeding, total mercury was highest in the carcass of zebrafish fed methylmercury (12.49 ± 0.369 µg Hg.g-1 dry wt), intermediate in those fed inorganic mercury (1.09 ± 0.117 µg Hg.g-1 dry wt), and lowest in fish fed the control diet (0.48 ± 0.038 µg Hg.g-1 dry wt). Total mercury was highest in the viscera of methylmercury fed zebrafish (11.6 ± 1.86 µg Hg.g-1 dry wt), intermediate in those fed inorganic diets (4.3 ± 1.08 µg Hg.g-1 dry wt), and lowest in the control fish (below limit of detection). Total mercury was negatively associated with fish length and weight in methylmercury fed fish. Condition factor was not associated with total mercury and might not be the best measure of fitness for these fish. No liver pathologies were observed in zebrafish from any treatment.

Dietary methionine is mainly metabolized in the liver where it is converted into S-adenosylmethionine (AdoMet), the main biologic methyl donor. This reaction is catalyzed by methionine adenosyltransferase I/III (MAT I/III), the product of MAT1A gene, which is exclusively expressed in this organ. It was first observed that serum methionine levels were elevated in experimental models of liver damage and in liver cirrhosis in human beings. Results of further studies showed that this pathological alteration was due to reduced MAT1A gene expression and MAT I/III enzyme inactivation associated with liver injury. Synthesis of AdoMet is essential to all cells in the organism, but it is in the liver where most of the methylation reactions take place. The central role played by AdoMet in cellular function, together with the observation that AdoMet administration reduces liver damage caused by different agents and improves survival of alcohol-dependent patients with cirrhosis, led us to propose that alterations in methionine metabolism could play a role in the onset of liver disease and not just be a consequence of it. In the present work, we review the recent findings that support this hypothesis and highlight the mechanisms behind the hepatoprotective role of AdoMet.

Methionine adenosyltransferase (MAT) is an essential enzyme that catalyzes the synthesis of S-adenosylmethionine (AdoMet), the most important biological methyl donor. Liver MAT I/III is the product of the MAT1A gene. Hepatic MAT I/III activity and MAT1A expression are compromised under pathological conditions such as alcoholic liver disease and hepatic cirrhosis, and this gene is silenced upon neoplastic transformation of the liver. In the present work, we evaluated whether MAT1A expression could be targeted by the polycyclic arylhydrocarbon (PAH) 3-methylcholanthrene (3-MC) in rat liver and cultured hepatocytes. MAT1A mRNA levels were reduced by 50% following in vivo administration of 3-MC to adult male rats (100 mg/kg, p.o., 4 days' treatment). This effect was reproduced in a time- and dose-dependent fashion in cultured rat hepatocytes, and was accompanied by the induction of cytochrome P450 1A1 gene expression. This action of 3-MC was mimicked by other PAHs such as benzo[a]pyrene and benzo[e]pyrene, but not by the model arylhydrocarbon receptor (AhR) activator 2,3,7,8-tetrachlorodibenzo-p-dioxin. 3-MC inhibited transcription driven by a MAT1A promoter-reporter construct transfected into rat hepatocytes, but MAT1A mRNA stability was not affected. We recently showed that liver MAT1A expression is induced by AdoMet in cultured hepatocytes. Here, we observed that exogenously added AdoMet prevented the negative effects of 3-MC on MAT1A expression. Taken together, our data demonstrate that liver MAT1A gene expression is targeted by PAHs, independently of AhR activation. The effect of AdoMet may be part of the protective action of this molecule in liver damage.

The measurement of urinary taurine and the liver specific enzymes AST and GGT in sheep with hepatic damage was undertaken to determine if urinary taurine could be used as a biomarker of hepatic damage. Two treatment groups (n = 5) were treated with O.125ml/Kg BW of carbon tetrachloride (CCl₄) (Group A), and O.125mg/Kg BW/day of sporidesmin (SPDM) (Group B) respectively. Two other treatment groups (n = 4) were used as control groups. One group was placed on a restricted diet and treated with O.625ml/Kg BW of paraffin (Group C), the other group was not dosed (Group D). Following dosing blood and urine samples were taken every 12 h for five days, before weekly sampling commenced. On day 27 animals in group A were redosed with CCl₄ , due to insignificant liver damage in this group. Blood and urine samples were again taken every 12h for 5 days, then sampling occurred weekly until the end of the trial, when all animals, except group D animals, were sacrificed. Tissue samples were then from liver, kidney, and pancreatic tissue, for metal analysis, and the livers were scored for liver damage. Liver specific enzyme activity increased in group A animals after second dosing. Enzyme activity did not increase in group B animals, even though animals' livers demonstrated fibrosis. Urinary taurine output was patchy and inconsistent with no conclusions able to be reached about this data. Results are inconclusive, with there being no indication of the effectiveness of urinary taurine as a biomarker of liver damage.