Pathomechanisms of Drug-Induced Liver Injury:
Embargoed until 2023-05-06
- Doctoral Thesis
Drug-induced liver injury (DILI) is a rare event, but presents a significant medical issue due to its challenging diagnosis and treatment. DILI can be mild and reversible, or irreversible resulting in acute liver failure requiring liver transplantation. The characterization of acute or long-term toxic effects of drugs, as well as methods of circumventing or treating DILI, are the foundation of this work. This thesis consists of two chapters addressing hepatotoxic effects of two commonly used drugs, nefazodone and valproic acid, and furthermore assessing the possible protective effects of cotreatment with a Farnesoid X Receptor (FXR) agonist. The first part of this thesis is dedicated to valproic acid-induced DILI and the protective effects of the FXR agonist obeticholic acid (OCA). Valproic acid (VPA) has been one of the most widely used antiepileptic drugs over the past 40 years, known to lead to fatty liver disease in more than 40% of patients. Steatosis is a typical clinical manifestation of VPA and is characterized by an abnormal accumulation of lipids in the cell. Oxidative stress plays a crucial part in the progression of non-alcoholic fatty liver disease (NAFLD). This study aimed at investigating whether activation of FXR by OCA, which is an effective treatment for NAFLD, can also prevent VPA-induced steatosis in an in vivo mouse model. C57BL/6 mice were treated for four weeks with OCA and then divided into two groups: co-treatment with OCA and VPA for an additional four weeks or treatment with VPA alone to induce fatty liver disease. Biochemical parameters, body mass, liver lab tests, and histology were examined. Gene expression in the liver was tested by RT-PCR, comparing VPA-treated mice with or without OCA co-treatment. In addition, next-generation RNA sequencing was employed on liver samples from control, VPA and VPA+OCA-treated mice to explore gene-expression profiles. Liver sections, stained with hematoxylin-eosin, BODIPY 493/503 and Oil Red O confirmed hepatocellular lipid accumulation in mice treated with VPA, with a marked reduction in the accumulation of lipids in mice cotreated with OCA. FXR activation was associated with the inhibition of VPA-induced oxidative stress, confirmed by RNA-sequencing analysis. Specific genes necessary for the most abundant defence against oxidative stress, glutathione, were confirmed to be upregulated by OCA with RT-PCR. The data suggest that OCA reduced the accumulation of VPA-induced lipids in the liver of mice as well as up-regulated glutathione antioxidant protective pathways. Our findings provide evidence for the protective role of OCA against VPA-induced fatty liver. The second part of this work is dedicated to nefazodone-induced hepatotoxicity. Acute toxic effects of nefazodone were characterized by assessing intracellular ATP levels, oxygen consumption rate (OCR), and the extracellular acidification rate (ECAR) using the hepatocarcinoma Huh7 cell line and primary cultured hepatocytes. Additionally, enzymatic assays were performed on purified enzymes or cell lysates and a metabolomic profile of Huh7 cells was generated by liquid chromatography and mass spectrometry. Nefazodone rapidly depleted Huh7 intracellular ATP content (IC50 44 µM, 95% CI 39-50), which was aggravated in galactose-containing medium (IC50 9.8 µM, 95% CI 9.0-11). Similarly, low concentrations of nefazodone inhibited only the OCR (mitochondrial activity), whereas at higher concentrations both OCR and the ECAR (anaerobic glycolysis) were inhibited simultaneously. Short-term exposure to nefazodone altered the metabolite pattern associated with the ATP biosynthetic pathways (e.g. tricarboxylic acid, glycolysis). Furthermore, enzymatic assays confirmed that nefazodone inhibits the activity of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). At low concentrations, nefazodone inhibited mitochondrial ATP production in Huh7 cells, while acting as an inhibitor of anaerobic glycolysis at higher concentrations. Besides, we detected the protective effects of FXR activation against nefazodone induced ATP depletion. In summary, the Huh7 cell model used in this study, representing hepatocarcinoma cells that are relying heavily on glycolysis, allowed us to identify glycolysis as a novel target of nefazodone. Programmed cell death or apoptosis requires ATP, unlike necrosis, and worsens a hepatic injury. ATP availability may be a key factor determining the outcome of DILI induced by nefazodone. Studies described in this thesis evaluated hepatotoxicity of nefazodone and valproic acid with the aim, on the one hand, to better understand the risk as well as the severity of DILI, and, on the other hand, to determine the potential protective effects of treatment with OCA in combination with DILI causing drugs. In the case of nefazodone, it protects energy production at the ATP level in vitro and in the case of VPA, it protects the liver against oxidative stress in vivo. Such studies can open avenues for the potential alleviation of long-term liver injury, generally reducing the risk of developing non-alcoholic fatty liver disease, acute liver failure and, potentially requiring liver transplantation. Show more
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ContributorsExaminer: Zeilhofer, Hanns Ulrich
Examiner: Kullak-Ublick, Gerd A.
Examiner: Stoffel, Markus
Organisational unit03742 - Zeilhofer, Hanns U. / Zeilhofer, Hanns U.
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