The overall goals of the proposed studies are to assess the functional significance of phase 2 enzymes in chemoprotection. An extraordinary variety of chemical agents protect animals against the neoplastic effects of many different types of carcinogens. Many of these chemoprotectors exert their anticarcinogenic effects by selectively inducing (by enhanced transcription) phase 2 and antioxidative genes that serve to detoxify the DNA-damaging forms of electrophilic ultimate carcinogens and free radicals. Most of these protective genes appear to be induced through a common enhancer, the Antioxidant Response Element (ARE). Transcription factors which in turn bind to the ARE are being identified and include Nrf2 and several small Maf proteins. In this project we seek to use molecular genetic approaches to test the hypothesis that the induction of genes important for the chemoprotective efficacy of clinically relevant phase 2 enzyme inducers (isothiocyanates and dithiolethiones) is mediated, at least in part, through activation of Nrf2.
Aim 1 is designed to investigate the role of the Nrf2 chaperone, Keapi, as the sensor for phase 2 enzyme induction, and establish the role of this target in signaling phase 2 gene expression.
Aim2 seeks to use microarray technology to identify which genes are induced through the Nrf2 pathway by comparing patterns of expression in wild-type and nrJ2-mutant mice.
Aim 3 will assess the importance of phase 2 and antioxidative enzyme induction in chemoprotection. Using gene-disrupted mice, the influence of nrJ2, keapi and maf null genotypes on enzyme induction in vivo by our lead protectors (e.g., sulforaphane and oltipraz) and their chemoprotective efficacy as inhibitors of experimental carcinogenesis will be evaluated. Collectively, the approaches encompass a broad range of experimental systems from molecular/genetic systems to susceptibility to carcinogenesis in gene knockout mice. These studies will firmly establish the role of induction of phase 2 and antioxidative enzymes/proteins in chemoprotection. Knowledge of the mechanisms by which chemoprotectors interact with sensor protein(s) to communicate to the ARE to signal enzyme induction will facilitate the identification and design of more potent and specific enzyme inducers and enhance their effective use in humans.
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