**7.6 With or without 'reactive oxygen species (ROS) and Antioxidant dynamics'**

A 'two-hit' theory has been postulated to help explain the mechanisms underlying the development of advanced NAFLD. The 'second hit' has yet to be completely described; extensive research has identified several possible mechanisms, including oxidative stress (OS)-induced inflammation with lipid peroxidation, cytokine activation and excess production of reactive oxygen and nitrogen species (ROS/RNS) [124].

The main source of radicals in biological systems is molecular oxygen, which readily accepts electrons, the most important of which being the hydroxyl radical (•OH), the superoxide anion (O2 •−) and nitric oxide radical (NO•). These unstable and reactive radicals are natural by-products of the intracellular metabolism and from exogenous substances, which have the ability to react with biological compounds including proteins, FFA and DNA [136, 137]. On the other hand, the main endogenous intracellular sources of ROS are mitochondria, the endoplasmic reticulum (ER) and peroxisomes, superoxide anion radicals (O2 •−) are produced because of enzymatic activity, such as with xanthine oxidase (XO) and cytochrome P450 metabolism [107, 138].

In a normal situation, a fine balance exists between prooxidant and antioxidant mechanisms, and OS, which has been long recognised as a key mechanism responsible for liver damage and disease progression in NAFLD, is believed to occur due to an imbalance in favour of prooxidation [139]. Numerous pieces of evidence accumulated over the past decade suggest that mitochondrial dysfunction plays a significant role in steatosis and steatohepatitis. ROS overproduction is induced by mitochondrial dysfunction, and the ensuing increase in the lipid peroxidation and protein oxidation has a detrimental effect on fat homeostasis in the liver. Mitochondria remain the main source of ROS in hepatocytes, although other subcellular organelles have also been shown to participate in the process [140, 141].

As a matter of fact, peroxisomes can oxidise long-chain FFA more rapidly than mitochondria, thereby increasing the cell's capacity to metabolise FFA. However, H2O2, which is an end-product of peroxisomal β-oxidation, is converted into the highly reactive OH radical with ease. By promoting toxic accumulation of ROS, which *Regulation of Iron Metabolism in NAFLD/NASH DOI: http://dx.doi.org/10.5772/intechopen.107221*

triggers other signalling pathways within the cell, chronic ER stress may also contribute to OS. The relationship between ER stress and OS works both ways because ROS generated through inflammation or damage to organelles (e.g., mitochondria) may also accelerate ER dysfunction [140, 141].

Because of either excessive production of ROS within the hepatocyte or reduced antioxidant defences, oxidative stress occurs and accumulates within the hepatocytes. Most antioxidant enzymes, copper/zinc superoxide dismutase (Cu/Zn SOD) and manganese-superoxide dismutase (MnSOD), which are mainly present in the cytoplasm and mitochondria, promote the reduction of O2 •− to H2O2. Another antioxidant enzyme, glutathione peroxidase (GPx), facilitates the subsequent conversion of H2O2 to H2O [137]. In correlation with disease severity, a breakdown in the antioxidant defences plays a significant role in OS associated with NASH, as evidenced by decreased hepatic glutathione (GSH) and diminished SOD, GPx, catalase and glutathione transferase activities [107].

Lipid peroxidation to release more reactive aldehydes is augmented by the resultant increase in mitochondrial ROS, which further damages the mitochondrial DNA (mtDNA) and respiratory chain polypeptides [142].

In summary, mitochondrial dysfunction not only impairs fat homeostasis in the liver but also leads to an overproduction of ROS, which is deliberated to be an important factor in producing lethal hepatocyte injury associated with NAFLD [107].
