Drug and alcohol induced hepatotoxicity

Sammanfattning: Drug induced hepatotoxicity is the most common reason cited for withdrawal of already approved drugs from the market and accounts for more than 50 percent of cases of acute liver failure in the United States. Ethanol (EtOH) causes a further substantial amount of liver insufficiencies world wide. The current thesis was focused on the mechanisms behind hepatotoxicity caused by these agents. Using a rat in vivo model for alcoholic liver disease (ALD) it was found that cytokine and chemokine levels in blood accompanied the fluctuating levels of blood EtOH, indicating that they are directly influenced by absolute EtOH concentration. During the early phases of ALD in this model, a strong initial Th1 response was observed as revealed by increased levels of cytokine as well as transcription factor mRNAs, followed by a downregulation, whereas Th2 response was decreased by EtOH over the entire treatment period of four weeks. We found that supplementation with the antioxidant NAC to ethanol treated animals decreases severity of liver damage and somewhat decreases initial inflammatory response mediated by TNFalpha. NAC also diminished the ethanol-induced formation of protein adducts of lipid peroxidation products like MDA and HNE. Also, the formation of antibodies against neo-antigens formed by MDA, HNE and HER protein adducts was lowered. In order to further study the influence of oxidative stress in ALD we utilized a transgenic mouse model overexpressing the human form of CYP2E1. Pathological changes were significantly increased after EtOH treatment, and principal component analysis showed that among parameters influencing total pathology score, para-nitrophenol activity mirroring CYP2E1 activity, had the highest impact. Analysis of 39,000 gene transcripts revealed that the expression of several genes previously known to be associated with ALD as well as several TNFalpha induced transcripts increased in the transgenic EtOH treated mice. We also show that cytokeratins 8 and 18, known to be of importance for formation of Mallory bodies, correlated highly to total pathology score. The results strongly support the view of an important role of oxidative stress and CYP2E1 in ethanol mediated hepatotoxicity and suggest that cytokeratin 8 can be used as an in vivo marker for ALD. There are many different models available to study liver toxicity in vitro, with the major drawback of low predictability. We showed that the human hepatoma cell line B16A2 differentiates spontaneously after long term confluent growth, into a more mature hepatocyte like phenotype. We developed a co-culture system using human hepatoma cells and monocytes and found that, compared to single cell cultures, co-cultures showed increased cytotoxicity and elevated mitochondrial oxidative stress in response to troglitazone whereas rosiglitazone was without effect. The data underline the importance of using in vitro models harboring different cell types for studies of drug induced hepatotoxicity.