**2. Impact of oxidative DNA damage during hepatocarcinogenesis**

Many previous studies have indicated a close relationship between metal overload and oxidative DNA damage (Imlay et al., 1988). For example, when DNA is exposed to hydrogen peroxide with iron, the Fenton reaction, in which hydrogen peroxide (H2O2) is catalysed to hydroxyl radicals (OH•) by iron (II) (Lloyd et al., 1998), causes the production of carcinogenic malondialdehyde, 4-hydroxynonenal (4-HNE) and other exocyclic DNA

Molecular Mechanism of DNA Damage Response Pathway During Hepatic Carcinogenesis 315

related toxic products (Niu et al., 2009). In turn, ROS stimulate HBx expression (Ha et al., 2010), suggesting that ROS may be an autocrine transducer of HBx-mediated carcinogenesis. Very recently, HBX was shown to activate the transcriptional activity of Forkhead box class O 4 (Foxo4) via JNK, leading to enhancement of the resistance to oxidative stress-induced

HCV infection is a leading cause of HCC development throughout the world. Several lines of evidence have suggested that HCV plays a critical role in the state of oxidative stress in the liver. HCV is constructed of core, E1, E2 and nonstructural (NS2, NS3, NS4A, NS4B, NS5, NS5A, NS5B) proteins, and each of these encoded proteins has been shown to be essential for the pathogenesis of HCV. HCV core protein plays a role in cell proliferation, while NS5A interacts with the double-stranded RNA-dependent PKR to promote viral replication. Many studies have revealed that chronic HCV infection leads to doublestranded DNA breaks and enhances the mutation frequency of whole cellular genes (Machida et al., 2004). Okuda et al. (2002) reported that HCV core protein localizes to mitochondria, leading to redistribution of cytochrome c from the mitochondria to the cytoplasm. Their data clearly indicate that HCV can be a direct source of ROS production. Moreover, HCV core protein has been shown to be strongly associated with the outer membrane of mitochondria to increase Ca2+ uptake, leading to oxidation of the glutathione pool and a decrease in the NADPH content in vivo (Korenaga et al., 2005). Recently, the mechanism by which hepatic iron overload develops in patients with HCV-associated chronic liver disease has been elucidated. Miura et al. (2008) reported that hepcidin, which plays a pivotal role as a negative regulator of iron absorption, was significantly decreased in HCV replicon cells and HCV core-expressing cells. Their findings should be important, because the decreased level of hepcidin may lead to increased duodenal iron transport as well as macrophage iron release, thereby causing hepatic iron accumulation. Since increased activity of histone deacetylase (HDAC) was found to be the main reason for the decreased levels of hepcidin (Miura et al., 2008), HDAC may play a critical role in HCV-related

It is widely known that ethanol is a strong inducer of DNA damage. The ethanol derivative acetaldehyde causes DNA damage by directly binding to DNA and inhibiting DNA repair systems (Seitz et al., 2006). Furthermore, ethanol treatment increases the production of intracellular ROS and lowers the levels of antioxidants, leading to enhancement of oxidative stress (Wu et al., 2009). Ethanol-induced oxidative stress plays a critical role in the pathogenesis of DNA damage as well as liver injury, and mitochondrial dysfunction is also induced by oxidation of various mitochondrial proteins (Suh et al., 2004). One of the most well-known mediators of alcohol-induced ROS is cytochrome P450 2E1 (CYP2E1) (Lu et al., 2008; Wu et al., 2009; Beier et al., 2010). CYP2E1 is an important enzyme for the conversion of ethanol to acetaldehyde and acetate, and is involved in the metabolism of xenobiotics. Ethanol intoxication increases CYP2E1 not only in the endoplasmic reticulum but also in mitochondria, leading to oxidative stress in these compartments (Robin et al., 2005). Intriguingly, Wang et al. (2009) reported that both protein-bound 4-HNE and etheno-DNA

cell death (Srisuttee et al., 2011).

**2.1.2 HCV and ROS** 

hepatocarcinogenesis.

**2.1.3 Alcohol consumption and ROS** 

adducts including 8-hydroxy-2'-deoxyguanosine (8-OHdG) (Jomova et al., 2011). Among these products, 8-OHdG is considered to be an oxidative DNA marker produced by reactive oxygen species (ROS), because the numbers of 8-OHdG-positive hepatocytes are significantly increased with progression of the severity of chronic hepatitis activity (Kitada et al., 2001; Ichiba et al., 2003) with iron content (Tanaka et al., 2008). Kato et al. (2001) investigated whether therapeutic iron reduction by phlebotomy with a low-iron diet could decrease the risk of HCC development in patients with chronic HCV. They found that patients treated with phlebotomy for 6 years showed significantly decreased levels of 8- OHdG in the liver, with improved severity of chronic hepatitis. Interestingly, all the patients who received phlebotomy did not develop HCC, suggesting a strong correlation between oxidative DNA damage and hepatocarcinogenesis. Subsequent studies reported that the level of hepatic 8-OHdG can be a predictive factor for the risk of recurrence in HCC patients after surgery (Matsumoto et al., 2003; Tanaka et al., 2008) and for individuals with naive chronic HCV infection (Chuma et al., 2008).

Intracellular ROS not only induce DNA damage but also regulate various types of intracellular signaling. In hepatoma cells, ROS potentiate cell growth by activating the signaling pathways of the stress kinases Akt, extracellular signal-regulated kinase (ERK) and Jun N-terminal kinase (JNK) (Liu et al., 2002). On the other hand, ROS accelerate tumor invasiveness in many types of cancers. In the case of hepatoma cells, Lim et al. (2008) reported tight correlations among ROS induction, E-cadherin downregulation, Snail upregulation and E-cadherin promoter methylation (Lim et al., 2008). Since E-cadherin is a master regulator of the epithelial-to-mesenchymal transition in HCC cells, it is plausible that tumor invasiveness is significantly accelerated by ROS. Importantly, recent studies have revealed that hepatitis viruses might have the property of producing ROS, supporting the idea that oxidative DNA damage might be directly induced in viral hepatitis.
