The response to chemical stress: development of preclinical and translational biomarkers of Nrf2 activity

Nrf2 is a transcription factor that plays a vital role in the cytoprotective response to oxidative stress. Under basal conditions Nrf2 is sequestered in the cytosol by Keap1, a molecule which targets Nrf2 for ubiquitination and subsequent proteasomal degradation. Following oxidative insult, Keap1 is no longer able to facilitate the breakdown of Nrf2. Nrf2 accumulates in the cell and is free to translocate to the nucleus where it binds to the antioxidant response element (ARE) in a range of genes resulting in their expression. Nrf2 regulates genes encoding phase II enzymes, proteins important for glutathione synthesis and antioxidants. Nrf2 knockout (KO) mice have been shown to be more susceptible to the toxicity associated with a range of different compounds, in the liver as well as in other organs. Conversely, pharmacological activation of Nrf2 has been shown to be protective in mouse models of hepatotoxicity. Drug induced liver injury (DILI) is a major concern for the pharmaceutical industry, and there is a clear imperative to improve existing preclinical models of DILI. Oxidative stress is known to result from the administration of a number of model hepatotoxins and has also been associated with cases of idiosyncratic DILI. Biomarkers of Nrf2 activity have potential utility in preclinical models investigating the role of oxidative stress in hepatotoxicity. Furthermore, such biomarkers could also have applications in studies determining the importance and variability of Nrf2 in the human population. Consequently the aim of the work described in this thesis was to characterise the hepatic profiles of mice in which Nrf2 activity had been modulated in order to identify candidate biomarkers of Nrf2 activity. iTRAQ analysis was employed in order to identify the proteins that were differentially expressed in the livers of wild type (WT) and Nrf2 KO mice. Subsequent pathway analysis identified cytoprotection and lipid metabolism as the processes that were most significantly perturbed in the livers of KO animals, with lipid metabolism found to be negatively regulated by Nrf2. The development of an LC-MS/MS assay for the determination of hepatic GSH and GSSG levels in liver homogenates showed that basal GSH levels were reduced by 21.5% in Nrf2 KO mice when compared to their WT counterparts. GC-FID analysis identified a number of fatty acids with levels that differed in the livers of WT and Nrf2 KO animals, constitutively and following carbohydrate restriction. Preliminary lipidomic analysis also identified differences in the wider hepatic lipid profile of the animals. iTRAQ was further employed to investigate the hepatic proteomic profile of mice following the administration of a single 3mg/kg dose of the Nrf2 inducer, CDDO-Me. 5 proteins were found to be regulated at both the basal and inducible level and so have significant potential to be used in the development of biomarkers indicative of Nrf2 activity. The work described in this thesis highlights the importance of the roles that Nrf2 plays in the regulation of hepatic homeostasis in terms of both cytoprotection and lipid metabolism. Furthermore, it has identified proteins and pathways that have potential applications in the development of biomarkers of Nrf2 activity. Such biomarkers would have utility in preclinical assays and in investigations into the importance of the transcription factor in the human population.