PHARMACOLOGIC AND TRANSGENIC ACTIVATION OF NUCLEAR FACTOR-ERYTHROID 2-RELATED FACTOR 2 (NRF2) ALTERS KINETICS AND TOXICODYNAMICS OF XENOBIOTICS

Abstract

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor, which, upon translocation into the nucleus, is capable of inducing a variety of cytoprotective genes, such as NAD(P)H:quinone oxidoreductase 1 (Nqo1), glutamate-cysteine ligase, catalytic subunit (Gclc), glutathione-S-transferases (Gsts), and multidrug-associated resistance proteins (Mrps). Because Nrf2 can induce many cytoprotective enzymes and transporters, it is a potential target for the prevention of liver injury, for which there are limited treatments. Therefore, it was hypothesized that pharmacologic activation of Nrf2 would protect against acetaminophen (AA) hepatotoxicity, and that genetic ablation and enhancement of Nrf2 activation would alter pharmacokinetics of AA and sulfobromopthalein (BSP). Pharmacokinetic analysis will provide insight into a potentially novel hepatoprotective role for Nrf2. It was determined whether the natural triterpenoid oleanolic acid and a synthetic derivative 2-cyano-3,12-dioxooleana-1,9-diene-28-imidazolide (CDDO-Im) could protect liver from AA toxicity through activation of Nrf2. Oleanolic acid increased mRNA expression of the Nrf2 target genes Nqo1, Gclc, and heme oxygenase-1 (Ho-1) in wild-type but not in Nrf2-null mice, and protected against acetaminophen hepatotoxicity in wild-type mice, but to a lesser extent in Nrf2-null mice. The synthetic triterpenoid CDDO-Im also protected the liver from AA-induced injury and induced the Nrf2 target genes Nqo1, Gclc, and Ho-1 in a dose- and time-dependent manner. In contrast, this protection and mRNA induction was ablated in Nrf2-null mice. These studies demonstrate that oleanolic acid and CDDO-Im protect the liver from AA-induced injury by activating the antioxidant transcription factor Nrf2. A recently engineered mouse with knockdown of Keap1 (Keap1-kd mice), the cytosolic repressor of Nrf2, has a 55% decrease in Keap1 mRNA and a 200% increase in Nrf2 protein in liver. Several experiments with Nrf2-null mice have demonstrated the effects of a lack of Nrf2. However, little is known about the biological effects of more Nrf2 activation. Accordingly, the phenotype of Keap1-kd mice, as well as mRNA expression of detoxifying and antioxidant genes, were compared with Nrf2-null and wild-type mice. The present study revealed three patterns of gene expression, which collectively suggest that hepatic Nrf2 is more important for the detoxification and elimination of electrophiles rather than reactive oxygen species. Numerous studies have shown that Nrf2 protects against toxicity, which is frequently attributed to decreased toxicodynamic effects of chemical insults, as exemplified by increased expression of cytoprotective genes. However, the effects of Nrf2 on the kinetics of xenobiotics have not been examined. It was found that Nrf2 increases biliary excretion of BSP by increasing glutathione (GSH) conjugation and biliary excretion of the BSP conjugate. In addition, lack of Nrf2 decreases AA glucuronidation, leading to increased NAPQI formation and hepatotoxicity, whereas activation of Nrf2 enhances detoxification of NAPQI by Nqo1 and elimination of AA glucuronide conjugate via Mrp3. Therefore, the effects of Nrf2 on the kinetics of xenobiotics should also be considered as a means of protection. Collectively, these studies have shown that liver can be protected by activation of Nrf2, either pharmacologically or genetically, as in the case of genetically-engineered Keap1-kd mice. Nrf2 can also dramatically affect the kinetics of xenobiotics because of its ability to transcriptionally control genes important in the biotransformation and excretion of chemicals.