Abstract
Non-alcoholic fatty liver disease (NAFLD) is characterised by the accumulation of lipid in liver. Liver is the principal organ involved in drug biotransformation. The central hypothesis of this project is that alterations in the liver environment due to lipid accumulation aggravate sensitivity of hepatocytes to toxicity of some commonly prescribed drugs (paracetamol, alcohol, phenobarbital, cisplatin or doxorubicin). The aim of this study was to investigate the effect of lipid overloading (steatosis) on drug cytotoxicity in the liver model Huh7 cell line. Hepatic steatosis was induced in Huh7 cells by exposing cells to 300 μM FFA mixture or 1 mM oleic acid. Impact of steatosis on drug toxicity was examined by co-treating the cells with FFA and paracetamol, alcohol, phenobarbital, cisplatin or doxorubicin. Cell viability MTT assay showed that neither 300 μM FFA mixture nor 1 mM OA caused significant reduction in cell viability after 24 h incubation. However, a significant reduction seen after incubation for 48 h, therefore for the subsequent experiments the time frame chosen was 24 h. The results showed that 300 μM FFA mixture did not induce a significant amount of intracellular lipid, whereas 1 mM OA induced significant amount of intracellular lipid. The subsequent experiments were carried out to test the hypothesis that lipid-loaded Huh7 cells are more sensitive to drug toxicity. Co-treatment with FFA and either paracetamol, ethanol or doxorubicin resulted in further reduction in cell viability. Phenobarbital resulted in enhanced cell viability, and no significant changes in cell viability observed after co-treatment with cisplatin. To investigate mode of cell death, caspase3/7 activity was measured as mediator of apoptosis. Generally, an advance apoptosis was observed in steatotic cells treated with the different drugs. Reactive oxygen species were measured as a possible trigger of apoptosis. A significant amount of ROS were generated by FFAs, and generally, drug treatment induced a higher significant amount of ROS in the steatotic Huh7 cells. To elucidate the specific changes observed upon treatment of steatotic cells with hepatotoxic drugs, a single FFA dose (300 μM FFA mixture) and a single drug (doxorubicin) were selected to identify major alterations in gene expression. The microarray data confirmed alterations in oxidative stress-related genes. Such changes were functionally relevant as confirmed by cellular assay and In-Cell Western blot. In conclusion, the hypothesised effect of steatosis on drug toxicity has been confirmed and steatosis may enhance drug toxicity through changing cellular oxidative state and activating the apoptotic pathway.