Laura Faye Wetherill

Non-alcoholic fatty liver disease (NAFLD) is a growing clinical problem, which manifests itself particularly in obese subjects who may have the metabolic syndrome. A two-hit hypothesis for the pathogenesis of the disease has been proposed. The first hit is the development of insulin resistance leading to fat accumulation specifically in the liver. The second hit involves oxidative damage to the liver when intracellular triglyceride is metabolized by beta-oxidation in the mitochondria to produce harmful reactive oxygen species (ROS) and their hydroperoxide by-products. An in vitro model for NAFLD along with a method to detect the levels of oxidative stress would be useful for testing this hypothesis. Such a model would also allow investigation of the ability of antioxidants such as selenium to prevent oxidative damage. This study aimed to develop a method for assessing the levels of oxidative stress in cultured fat-loaded human hepatocytes (C3A cells) and hepatic stellate cells (LX-2 cells) using electron spin resonance with the spin trap 1-hydroxy-2,2,6,6-tetramethyl-4-oxopiperidine (TEMPONE-H). Cells were fat-loaded with either LPON (lactate, pyruvate, octanoate and NH4+) or oleate. Initial experiments showed that the culture media alone generated free radicals but this was minimal when Dulbecco's phosphate-buffered saline was used as the TEMPONE-H carrier. It proved difficult to detect the free radical production by cells cultured in the basal state; however, when marked oxidative stress was induced in the cells by adding tertiary butyl hydroperoxide (t-BuOOH), free radical production by cells could be identified. Pre-treating cells with selenium, to induce the synthesis of selenoenzymes with antioxidant action, protected cells from the harmful effects of t-BuOOH. This supported selenium's role as an antioxidant, which may have the potential to prevent the onset of non-alcoholic steato-hepatitis. The human vascular endothelial cell line EAhy926 also accumulates lipid as triglyceride when pre-treated with oleate but not with LPON. This suggests that the use of LPON rather than oleate may be a more appropriate model of NAFLD.