THE ROLE OF NRF2 IN PREVENTING OXIDATIVE/ELECTROPHILIC STRESS-INDUCED LIVER INJURY
Redox maintenance is critical for all biological species. Amplification of mechanisms that reduces oxidative/electrophilic stress promotes health and extends life. Nuclear factor, erythroid derived 2, like 2 (Nrf2) is a master regulator of biochemical mechanisms that respond to oxidative/electrophilic stress. Under basal conditions, Nrf2 is held in an inactive state in the cytoplasm by binding to the cytoskeletal anchoring protein Kelch-like ECH-associated protein 1 (Keap1). Upon generation of oxidative/electrophilic stress, Nrf2 is released from Keap1 and translocates to the nucleus, where it promotes transcription of a battery of cytoprotective genes. The antioxidative role of Nrf2 has been extensively studied for more than ten years, but the global Nrf2 target genes in liver still remain unknown. The present dissertation utilizes a Nrf2 "gene dose-response" model to genetically modulate the levels of Nrf2 in mouse liver, where the transcription profiles in livers of mice with graded Nrf2 activation were correlated with activation of Nrf2-target genes. Thus, the Nrf2-target genes in mouse liver were investigated systematically. Pathway analyses indicate that genes induced by Nrf2 are involved in glutathione synthesis, oxidation/reduction using NADPH as the co-factor, and xenobiotic metabolism. Moreover, mRNA of genes involved in the pentose phosphate pathway and malic enzyme levels had a clear dose-dependent correlation to active levels of Nrf2, indicating that Nrf2 promotes NADPH generation. Among genes suppressed by Nrf2, the majority are involved in lipid synthesis and fatty-acid desaturation. Thus, Nrf2 promotes generation of NADPH, and reduces fatty acid synthesis and desaturation, contributing factors towards protecting liver against environmental oxidative/electrophilic stress. The mRNA abundance of 124 drug processing genes in the Nrf2 "gene dose-response" model was also determined in this dissertation. The results indicate that Nrf2 facilitates electrophile detoxification through inducing non P-450 phase-I enzymes that reduce electrophiles, Gsts that conjugate electrophiles, and efflux transporters that excrete electrophile-GSH conjugates out of cells. In addition to the assessment of the effect of Nrf2 on the global gene transcription profiles in mouse liver, the function of Nrf2 in vivo was examined by testing whether Nrf2 activation protects against chemical-induced oxidative stress and subsequent liver injury in mice. Oxidative stress and lipid accumulation play important roles in ethanol-induced liver injury. Ethanol increased serum ALT and LDH activities in Nrf2-null mice and wild-type mice, but not in Nrf2-enhanced mice, indicating that Nrf2-enhanced mice are resistant to ethanol toxicity. Ethanol increased free fatty acids in livers of Nrf2-null mice, and this increase was blunted in Nrf2-enhanced mice. Mechanistic studies show that Nrf2 prevents ethanol-induced oxidative stress through inducing cytoprotective genes including Nqo1 and Gclc, and prevents accumulation of free fatty acids in liver by suppressing the Srebp1 pathway. Oxidative stress also plays an important role in cadmium-induced liver injury. Nrf2-null mice are susceptible, whereas Nrf2-enhanced mice are resistant to cadmium-induced acute liver injury. Gclc, Gpx2, and Srxn-1, genes that are involved in GSH synthesis and reducing oxidative stress, were only induced in Nrf2-enhanced mice, but not in Nrf2-null mice. Surprisingly, metallothioneins, cysteine-rich proteins that scavenge cadmium to prevent toxicity, were induced markedly in both Nrf2-null and Nrf2-enhanced mice. Thus, there are at least two distinct pathways to protect cadmium-induced toxicity: Nrf2-dependent induction of antioxidative genes and Nrf2-independent induction of metallothioneins. To develop effective and potent Nrf2 activators, a library of synthetic and natural existing compounds was screened to test their efficacy and potency to activate Nrf2. After screening 47,000 compounds, 238 compounds (0.5%) had comparable or better efficacy to activate Nrf2 than tertiary butylhydroquinone (tBHQ), the prototypical Nrf2 activator. Among these 238 compounds, 19 compounds produce more induction of Nrf2 than 2-cyano-3,12-dioxooleana-1,9-dien-28-imidazolide (CDDO-Im), the most effective and potent Nrf2 activator known. However, none of the tested compounds were more potent in activating Nrf2 than CDDO-Im. In addition, chemical structural relationship analysis of these 238 compounds showed enrichment of four chemical scaffolds (diaryl amides and diaryl ureas, oxazoles and thiazoles, pyranones and thiapyranones, and pyridinones and pyridazinones). The top 30 compounds were screened in Hepa1c1c7 cells to increase Nqo1 mRNA, the prototypical Nrf2 target gene, and 17 of the 30 most active hits also increased Nqo1 mRNA in the cell line in a concentration-dependent manner. In addition to synthetic compounds, 54 natural compounds, known antioxidants through activating Nrf2, were tested utilizing the same screening system (AREc32 cells). Andrographolide had the highest efficacy, followed by trans-chalcone, sulforaphane, curcumin, flavone, kahweol, and carnosol, which were all more potent and effective than tBHQ. None of the natural compounds were more potent than CDDO-Im. In conclusion, Nrf2-target genes are involved in NADPH generation, NADPH-facilitated oxidative stress reduction, and detoxification and excretion of electrophiles. Nrf2 activation protects against ethanol-, cadmium-, and diquat-induced liver oxidative stress and liver injury in mice. Lastly, a few synthetic and natural existing compounds were shown to be effective in activating the Keap1-Nrf2-ARE pathway.
Toxicology, High throughput screening, Liver, Microarray, Nrf2, Oxidative stress
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