Abstract
Oxidative stress and mitochondrial dysfunction play an important role in the pathogenesis of nonalcoholic fatty liver disease and toxic liver injury. The present study was designed to evaluate the effect of exogenous inducer of oxidative stress (tert-butyl hydroperoxide, tBHP) on nonfatty and steatotic hepatocytes isolated from the liver of rats fed by standard and high-fat diet, respectively. In control steatotic hepatocytes, we found higher generation of ROS, increased lipoperoxidation, an altered redox state of glutathione, and decreased ADP-stimulated respiration using NADH-linked substrates, as compared to intact lean hepatocytes. Fatty hepatocytes exposed to tBHP exert more severe damage, lower reduced glutathione to total glutathione ratio, and higher formation of ROS and production of malondialdehyde and are more susceptible to tBHP-induced decrease in mitochondrial membrane potential. Respiratory control ratio of complex I was significantly reduced by tBHP in both lean and steatotic hepatocytes, but reduction in NADH-dependent state 3 respiration was more severe in fatty cells. In summary, our results collectively indicate that steatotic rat hepatocytes occur under conditions of enhanced oxidative stress and are more sensitive to the exogenous source of oxidative injury. This confirms the hypothesis of steatosis being the first hit sensitizing hepatocytes to further damage.
Highlights
Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in the USA and the Western world, and the prevalence of NAFLD has recently dramatically increased and it seems that this trend will continue [1, 2]
There are no differences between control lean and steatotic hepatocytes in albumin production (Figure 2(c)), percentage of hepatocytes with energized mitochondria (Figures 4(a), 4(b), and 5), and oxygen consumption at state 4 respiration whereas oxygen consumption at state 3 respiration was significantly reduced in steatotic cells (Table 2)
WST-1 test showed (Figure 1(a)) that lean hepatocytes exposed to tBHP for a period of 60 min are significantly affected from the concentration of 0.25 mmol/L, whereas in fatty hepatocytes, WST-1 test was already decreased at tBHP concentration of 0.1 mmol/L
Summary
Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in the USA and the Western world, and the prevalence of NAFLD has recently dramatically increased and it seems that this trend will continue [1, 2]. There is accumulating evidence that hepatic mitochondrial dysfunction and oxidative stress play an important role in the pathogenesis of NAFLD [3]. Oxidative stress and resulting altered redox balance seem to be crucial in the pathogenesis of steatosis, steatohepatitis, and fibrosis [7]. Altered hepatic redox status was proofed in the advanced forms of NAFLD, such as nonalcoholic steatohepatitis (NASH), and in simple steatosis [10]. Enhanced oxidative stress is thought to belong to second hits that participate in the progression of simple steatosis to steatohepatitis [11]. Increased generation of free radicals and other highly reactive substances results from fat accumulation in the liver, due to direct toxicity of fatty acids, increased peroxisomal and microsomal oxidation of fatty acids, and mitochondrial dysfunction [12, 13].
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