This dissertation is presented in two sections. Section one recounts my investigations of modulating the multidrug-resistant protein-three (MRP3) levels, by either activating or silencing the transcription factor Nrf2, in normal lung cells and Keap1 wild-type and mutant non-small cell lung carcinoma cell lines (NSCLC). In section two direct interactions between Nrf2 and the MRP3 promoter were evaluated in NSCLC. Additionally, the relationships between MRP3 mRNA levels and Keap1, Nrf2, and p53 status were also investigated in cell lines and tumor specimens. Combined, these sections explore the complex regulation of the MRP3 gene.

Hepatitis C virus (HCV) is an RNA virus of the Flaviviridae family that is estimated to have infected 170 million people worldwide. HCV infection can lead to serious liver disease such as cirrhosis, steatosis, and hepatocellular carcinoma. In patients, HCV induces an altered redox status and exhibits a high level of genetic variability, existing as quasispecies. Ethanol consumption has been associated with increased HCV pathogenesis, elevated HCV titer, increased sequence heterogeneity of HCV RNA, and decreased efficacy of antiviral treatment, suggesting multiple modes of interaction between ethanol and HCV. The goal of my studies was to elucidate the mechanisms of these multiple modes of interaction between alcohol, oxidative stress, and hepatitis C. By treating Huh7 human hepatoma cells that naturally express CYP2E1 with physiological relevant concentrations of ethanol or its metabolites, we determined that HCV replication was elevated in the context of the complete virus life cycle. This potentiation of HCV replication was dependent on CYP2E1 activity the host mevalonate pathway, fatty acid synthesis, and an increased NADH/NAD+ ratio. In contrast, reactive oxygen species rapidly suppressed HCV replication as previously shown using HCV replicons. These results suggested that lipid metabolism and alteration of the NADH/NAD+ ratio played an important role in the enhancement of HCV replication by ethanol. Finally, we were able to demonstrate that exposing HCV-replicating cells to ethanol and reactive species elevated both the nucleotide and amino acid substitution rates of HCV RNA, which were accompanied by oxidative RNA damage. The overall dN/dS ratio and percentage of sites undergoing positive selection increased with pronounces changes in the serine/threonine/tyrosine sites. In addition, HCV itself induced oxidative RNA damage that was exacerbated by ethanol and BSO. Agents that decreased the RNA damage also reduced the sequence heterogeneity of the HCV RNA. Conclusions/Significance: Therefore, ethanol potentiates HCV replication through the alteration of lipid metabolism and the NADH/NAD+ ratio, which is likely to contribute to the higher viral titer and pronounced pathogenesis in patients who drink alcohol. Ethanol may also synergize with virus-induced oxidative/nitrosative stress to induce RNA damage and likely amplify the error rate of the HCV replicase in the development of quasispecies, iincreasing the probability of immune evasion and antiviral resistance.

Hepatitis C virus (HCV) infection can lead to chronic infection resulting in severe liver diseases, including cirrhosis and hepatocellular carcinoma (HCC). Chronic hepatitis C (CHC) is characterized by ongoing inflammation and generation of reactive oxygen species (ROS) such as superoxide (O2.-) and hydrogen peroxide (H2O2). NADPH oxidase (Nox) enzymes produce ROS as their primary function and have been associated with oxidative stress in CHC. However, the precise role that Nox enzymes play in the pathogenesis of HCC is still unclear. We show that the structural core protein of genotype 1a HCV is sufficient to elevate Nox1 and 4 mRNA, protein expression, and enzyme activity and H2O2 levels in vitro. Human hepatocytes constitutively expressing core exhibited elevated Nox4 in the nucleus and increased DNA damage markers. Furthermore, the novel Nox-specific inhibitor VAS-2870 was capable of decreasing the core-associated increase in Nox enzyme activity. Altogether, these results highlight the importance of developing therapeutic agents targeting Nox enzymes for use as adjunct treatment of CHC to prevent carcinogenesis.

Hepatitis C virus (HCV) is an RNA virus of the Flaviviridae family that is estimated to have infected 170 million people worldwide. HCV can cause serious liver disease in humans, such as cirrhosis, steatosis, and hepatocellular carcinoma. HCV induces a state of oxidative/nitrosative stress in patients through multiple mechanisms, and this redox perturbation has been recognized as a key player in HCV-induced pathogenesis. Studies have shown that alcohol synergizes with HCV in the pathogenesis of liver disease, and part of these effects may be mediated by reactive species that are generated during hepatic metabolism of alcohol. Furthenriore, reactive species and alcohol may influence HCV replication and the outcome of interferon therapy. Alcohol consumption has also been associated with increased sequence heterogeneity of the HCV RNA sequences, suggesting multiple modes of interaction between alcohol and HCV. This review summarizes the current understanding of oxidative and nitrosative stress during HCV infection and possible combined effects of HCV, alcohol, and reactive species in the pathogenesis of liver disease. (c) 2007 Elsevier Inc. All rights reserved.

Reactive species and perturbation of the redox balance have been implicated in the pathogenesis of many viral diseases, including hepatitis C. Previously, we made a surprising discovery that concentrations of H2O2 that are nontoxic to host cells disrupted the hepatitis C virus (HCV) replication complex (RC) in Huh7 human hepatoma cells in a manner that suggested signaling. Here, we show that H2O2 and interferon-gamma have comparable effects on the HCV subgenomic and genomic RNA replication in Huh7 cells. H2O2 induced a gradual rise in the intracellular calcium concentration ([Ca2+](i)). Both rapid and sustained suppression of HCV RNA replication by H2O2 depended on this calcium elevation. The peroxide-induced [Ca2+](i) elevation was independent of extracellular calcium and derived, at least in part, from the endoplasmic reticulum. Likewise, the suppression of the HCV RC by H2O2 was independent of extracellular calcium but required an intracellular calcium source. Other agents that elevated [Ca2+](i) could also suppress the HCV RC, suggesting that calcium elevation might be sufficient to suppress HCV RNA replication. In conclusion, oxidants, may modulate the HCV RC through calcium. Effects on the infectivity and the morphogenesis of HCV remain to be determined. These findings suggest possible regulatory roles for redox and calcium signaling during viral infections. (c) 2006 Elsevier Inc. All rights reserved.