Emerging Role of Nrf2 in Protecting Against Hepatic and Gastrointestinal Disease

Abstract

Transcription factor NF-E2-related factor 2 (Nrf2) belongs to the basic region-leucine zipper family and is activated in response to electrophiles and reactive oxygen species. Nrf2 coordinately regulates the constitutive and inducible transcription of a wide array of genes involved in drug metabolism, detoxification, and antioxidant defenses. During periods of oxidative stress, Nrf2 is released from sequestration in the cytoplasm and translocates to the nucleus. Nrf2 binds antioxidant response elements (AREs) in the regulatory regions of target genes and activates transcription. Genetically modified mice lacking Nrf2 serve as a useful tool for identifying new ARE-regulated genes and assessing the ability of Nrf2 to confer protection against a variety of pathologies in numerous organs including the liver, intestine, lung, skin, and nervous system. With regards to the liver and gastrointestinal tract, Nrf2 knockout mice are more susceptible to acetaminophen-induced hepatocellular injury, benzo[a]pyrene-induced tumor formation and Fas-and TNFα-mediated hepatocellular apoptosis. The higher sensitivity of Nrf2 knockout mice to chemical toxicity is due in part to reduced basal and inducible expression of detoxification enzymes. Nrf2 may also be important in protecting against liver fibrosis, gallstone development, and formation of aberrant crypt foci. Research of Nrf2 has opened up new opportunities in understanding how antioxidant defense pathways are regulated, how oxidative stress contributes to disease progression and may serve as a novel target for designing therapies to prevent and treat diseases in which oxidative stress is implicated.

Introduction

As sites of entry for xenobiotics, the liver and gastrointestinal tract are continuously exposed to diverse chemicals that are subsequently distributed into the systemic circulation. Metabolic enzymes in both organs often convert these xenobiotics into less toxic and more water-soluble forms. In some cases, however, metabolism of chemicals generates more toxic species making the liver and gastrointestinal tract particularly susceptible to oxidative-type diseases such as chemical toxicity and carcinogenesis. Both organs are equipped with defense mechanisms to detoxify reactive intermediates and minimize oxidative stress. Recent work suggests that the Nuclear factor E2-related factor 2 (Nrf2) transcription factor is critical for protecting the liver and gastrointestinal tract against disease by regulating a multifaceted cellular antioxidant defense.

In this review, the current knowledge on the regulation of a coordinated battery of Nrf2-responsive genes will be examined with particular emphasis on how perturbations in this signaling pathway alter the development and/or progression of hepatic and gastrointestinal diseases. The molecular signaling pathways responsible for Nrf2 transactivation will be explained in the first portion of this review. This will be followed by detailed descriptions of ten Nrf2-responsive genes and their role(s) in cellular antioxidant defense. The final section of this review highlights phenotypic changes in mice deficient in Nrf2-related signaling pathways and the use of these mice to study the influence of Nrf2 on diseases of the liver and gastrointestinal tract. These diseases often have an oxidative stress component and include acetaminophen-induced hepatocellular necrosis, liver fibrosis, chemical carcinogenesis, gallstone development, heavy metal toxicity, and immune-mediated hepatocellular apoptosis. Understanding how Nrf2 signaling protects against these diseases may enable the development of chemopreventive agents that augment the innate Nrf2 antioxidant defense system.

Transcriptional Responses to Oxidative Stress

Reactive oxygen species (ROS) such as hydrogen peroxide, superoxide, peroxynitrite, and the hydroxyl radical are transient species formed during the normal course of cellular metabolism. Elevated levels of highly reactive ROS damage cellular macromolecules including proteins, lipids, and mitochondrial and nuclear DNA. These biological perturbations often result in the propagation of more ROS molecules and lipid peroxides. The end result of this cascade is cellular dysfunction and/or death. Similarly, electrophilic compounds can interfere with normal cell function directly by binding to cell structures or indirectly by producing ROS. Cells possess defense systems that provide protection against oxidative stress and ameliorate oxidative injury. Endogenous antioxidants such as glutathione (GSH) can scavenge and inactivate ROS, thereby restoring cellular homeostasis. In addition, there is a select set of genes encoding detoxifying enzymes, antioxidant proteins, and stress proteins that can remove compounds and/or intermediates capable of generating ROS. Induction of these genes is an adaptive defense to counteract oxidative stress. The transcription factor Nrf2 is of particular importance to the regulation of detoxifying and antioxidant genes. This is one of multiple cellular redox status sensors. Nrf2 binds the promoters of these genes through specific responsive elements, regulating both their constitutive and inducible expression (Friling et al., 1990; Rushmore et al., 1991; Wasserman and Fahl, 1997).

Nrf2: Key Regulator of Oxidative Stress Response

A plethora of evidence from the past 10 years points to Nrf2 as a key regulator of the cellular response to oxidative stress in multiple tissue and cell types. Nrf2 was originally identified during a screen for proteins that bind to the control region of the β-globin gene (Moi et al., 1994). Nrf2 was subsequently cloned from human, chicken, and mouse erythroid cDNA libraries (Moi et al., 1994; Chui et al., 1995; Itoh et al., 1995). High sequence homology between Nrf2 and the p45 subunit of the previously identified transcription factor nuclear factor erythroid 2 (p45-NF-E2) was observed. Comparison of the two transcription factors demonstrated that p45-NF-E2 is expressed solely in megakaryocytes, erythroid cells, and mast cells while Nrf2 is present in a wide number of tissues including liver, lung, intestine, and kidney (Andrews et al., 1993; Moi et al., 1994; Chui et al., 1995; Itoh et al., 1995; Chan et al., 1996; McMahon et al., 2001). Nrf2 belongs to the cap ‘n’ collar family of transcription factors that share a conserved basic region-leucine zipper (bZIP) structure (Moi et al., 1994). Nrf1, Nrf3, Bach1, and Bach2 are also members of this family of transcription factors.

Negative Regulation of Nrf2 by Keap1

Sequestration

Under basal conditions, Nrf2 is largely bound to the cytoskeletal anchoring protein Kelch-like ECH-associated protein 1 (Keap1) in the cytoplasm (Itoh et al., 1999; Kang et al., 2004) (Figure 1). Keap1 is a homologue of the Drosophila actin-binding protein Kelch (Xue and Cooley, 1993; Itoh et al., 1999). The double glycine repeat (DGR) region of Keap1 binds to the Nrf2-ECH homology 2 (Neh2) domain on the N-terminal portion of Nrf2 causing sequestration and repression of Nrf2 activity (Itoh et al., 1999). The Keap1:Nrf2 complex is conserved in rodents and humans as well as zebrafish illustrating an indispensable function for this interaction in regulating the antioxidant defense (Kobayashi et al., 2002).

Dissociation

During periods of oxidative stress or following exposure to electrophiles, Keap1 releases Nrf2 from sequestration (Itoh et al., 1999) (Figure 1). After dissociation, Nrf2 translocates to the nucleus enabling gene transcription. Multiple steps appear to be important in triggering the release of Nrf2 from Keap1 during oxidative stress. Two of these events include oxidation of critical cysteine residues within Keap1 and phosphorylation of Nrf2. Murine Keap1 contains 25 cysteine residues that are also conserved in the rat and human proteins (Dhakshinamoorthy and Jaiswal, 2001; Dinkova-Kostova et al., 2002). Of these residues, 4 cysteines (including Cys273 and Cys 288) were identified as critical for the release of Nrf2 from the Keap1:Nrf2 complex (Dinkova-Kostova et al., 2002; Zhang and Hannink, 2003; Levonen et al., 2004; Wakabayashi et al., 2004). In addition, the chemopreventive actions of Nrf2-activating chemicals such as sulforaphane may occur through modulation of cysteine residues in Keap1 (Hong et al., 2005). Exactly how these cysteine residues act as “redox sensors” to modulate Nrf2 turnover and stabilization is currently under investigation (Eggler et al., 2005; Kobayashi et al., 2006).

Although somewhat controversial, accumulating evidence suggests phosphorylation of Nrf2 is a requirement for dissociation of Nrf2 from Keap1 (Figure 1). Multiple kinases (including protein kinase C, extracellular signal-regulated kinase, phosphatidylinositol 3-kinase, and p38 MAP kinase) can phosphorylate Nrf2 and alter transcription of Nrf2 target genes (Yu et al., 1999; Huang et al., 2000; Yu et al., 2000; Zipper and Mulcahy, 2000; Kang et al., 2001; Lee et al., 2001; Reichard and Petersen, 2006). In one example, however, phosphorylation of Nrf2 by protein kinase C resulted in dissociation of Nrf2 from Keap1 without nuclear translocation of Nrf2 or activation of gene expression (Bloom and Jaiswal, 2003). Consequently, the precise role of Nrf2 phosphorylation in each step of Nrf2-mediated gene transactivation (translocation, binding to critical response elements and/or recruitment of transcriptional machinery) remains a source of controversy within the field (Huang et al., 2000; Bloom and Jaiswal, 2003).

Ubiquitination

Nrf2 protein has a short half-life (approximately 13–20 minutes) (Itoh et al., 2003; Stewart et al., 2003). Instability of Nrf2 protein under basal conditions has been attributed to constitutive ubiquitin-proteasomal degradation (McMahon et al., 2003; Nguyen et al., 2003a; Zhang and Hannink, 2003) (Figure 1). Blockade of the proteasomal system with specific inhibitors increases levels of Nrf2 (Itoh et al., 2003) and allows for detection of ubiquitinated Nrf2 protein (Stewart et al., 2003).

Keap1 has recently been shown to function as an adaptor protein in a ubiquitin-proteasome complex named Cul3-based E3 ligase complex (Cullinan et al., 2004; Kobayashi et al., 2004; Zhang et al., 2004; Furukawa and Xiong, 2005). E3 ligases catalyze the binding of ubiquitin to substrate protein. Cul-based ligase enzymes are particularly important for recycling of transcription factors. Keap1 interacts with the Cul3-based E3 ligase and promotes ubiquitination of Nrf2 suggesting that Keap1 is responsible not only for sequestering Nrf2, but also for its normal proteasomal targeting and degradation.

Localization Sequences

In addition to posttranslational modifications of Keap1 and Nrf2, recently identified localization signals in Nrf2 may dictate its subcellular distribution (Figure 1). Nrf2 contains a nuclear localization signal in its basic region that stimulates translocation into the nucleus. There are also two nuclear export signals in the leucine zipper and transactivation domains of Nrf2 which are responsible for cytoplasmic localization (Jain et al., 2005; Li et al., 2005, 2006). One of the nuclear export signals is redox sensitive and can be disabled in the presence of ROS allowing Nrf2 to translocate into the nucleus (Li et al., 2006).

The aforementioned Keap1:Nrf2 regulatory modifications are generally thought to dictate Nrf2 subcellular localization and activity. Nonetheless, questions regarding how Nrf2 maintains constitutive transcription of drug metabolizing enzymes in the current model of Keap1:Nrf2 sequestration have recently prompted researchers to put forth alternative models of Nrf2 signaling (Nguyen et al., 2005; Velichkova and Hasson, 2005). One hypothesis includes the constitutive targeting of de novo Nrf2 protein to the nucleus (Nguyen et al., 2005). In this model, excessive ARE gene transcription is prevented by continuously shuttling Keap1 to the nucleus for removal of Nrf2 to the cytoplasm and proteasomal degradation. This shuttling would be disrupted during periods of oxidative stress, thus allowing Nrf2 to accumulate in the nucleus and increase gene transcription. There is limited mechanistic explanation to understand such disruption in Keap1 and Nrf2 interaction. However, it can be speculated that events similar to the current model describing Nrf2:Keap1 dissociation may be involved.

Molecular Mechanisms Underlying Nrf2-Mediated Transcription

Once in the nucleus, Nrf2 can heterodimerize with a variety of transcriptional regulatory proteins. These protein complexes then bind to motifs known as antioxidant or electro phile response elements (ARE/EpRE) located in the promoters or upstream promoter regions of detoxification genes (Friling et al., 1990; Rushmore et al., 1991) (Figure 1). Because ARE and EpRE motifs share homology in their core sequences, the two terms are used interchangeably. The functional ARE sequence contains a common core 5′-GTGACnnnGC-3′ motif with ‘n’ representing any nucleotide (Rushmore et al., 1991). Variations and extensions of this consensus sequence have also been reported (Wasserman and Fahl, 1997; Erickson et al., 2002; Nioi et al., 2003). Nrf2 cannot homodimerize (Moi et al., 1994). Obligatory partners of Nrf2 are typically members of the activator protein-1 (AP-1) family (such as Jun and Fos) (Venugopal and Jaiswal 1998; Alam et al., 1999; Wild et al., 1999; Jeyapaul and Jaiswal, 2000) or the small Maf family of transcription factors (Itoh et al., 1997; Wild et al., 1999; Nguyen et al., 2000; Zhu and Fahl, 2001).

Jun and Fos are protein products of the oncogenes c-jun and c-fos (reviewed in Curran and Franza, 1988) (Figure 1). Jun and Fos bind ARE sequences (Friling et al., 1992; Tsuji, 2005) and act as positive and negative regulators of ARE gene transcription, respectively (Venugopal and Jaiswal 1996, 1998; Jeyapaul and Jaiswal, 2000). Jun has been shown to interact with Nrf2 and overexpression of both Jun and Nrf2 proteins in hepatoma cells activates ARE-mediated transcription (Venugopal and Jaiswal, 1998). In a similarly designed experiment, co-expression of Fos and Nrf2 repressed ARE reporter gene activity (Venugopal and Jaiswal, 1996). These data suggest that members of the AP-1 family of transcription factors differentially regulate Nrf2 transcriptional activity.

Small Maf proteins are a family of transcription factors with homology to the avian transforming retroviral oncogene, v-maf (musculo-aponeurotic fibrosarcoma) (Fujiwara et al., 1993). The precise role of small Maf proteins in the transactivation of ARE-containing genes remains controversial. Early in vitro data in Nrf2-overexpressing cells suggested that small Maf proteins repress Nrf2 activity (Wild et al., 1999; Dhakshinamoorthy and Jaiswal, 2000; Nguyen et al., 2000, 2003b). However, more recent in vivo rodent data demonstrate that small Maf proteins MafF and MafG function cooperatively with Nrf2 to activate gene transcription (Motohashi et al., 2004).

There is some evidence that Nrf2 can regulate its own expression. Two ARE-like elements have been identified in the 5′ flanking region of the mouse Nrf2 promoter and can be transcriptionally activated following chemoprotectant exposure (Kwak et al., 2002). These data suggest that Nrf2 may auto-regulate its own expression through direct binding to its promoter and in turn, further stimulate expression of downstream target genes (Kwak et al., 2002). However, some Nrf2-activating chemicals do not significantly enhance transcription of Nrf2 mRNA (Dhakshinamoorthy and Jaiswa, 2001; Itoh et al., 2003; McMahon et al., 2003; Nguyen et al., 2003a). These chemicals tend to increase Nrf2 levels by interfering with degradation of existing Nrf2 protein. This does not rule out the possibility that auto-induction of Nrf2 occurs under a limited number of experimental conditions, such as the case of chemoprotectant exposure reported by Kwak et al. (2002).

In summary, the Keap1:Nrf2 complex is an important mechanism for negatively regulating Nrf2 activity. This occurs primarily through localization of Nrf2 to the cytoplasm and Keap1-directed degradation. Multiple signals are likely required for dissociation of this complex, which then permits Nrf2 to translocate to the nucleus. In the nucleus, Nrf2-driven transcription is further influenced by the identity of the heterodimer partner. Additional reviews have been published which describe the molecular mechanisms underlying Nrf2 activation and transcription in greater detail (Itoh et al., 2004; Jaiswal, 2004; Motohashi and Yamamoto, 2004; Nguyen et al., 2004; Kang et al., 2005).

Coordinate Battery of Nrf2-Responsive Genes

ARE-containing genes identified as downstream targets of Nrf2 are involved in a variety of cellular functions including drug metabolism, ROS scavenging, GSH homeostasis, stress proteins, and efflux transport pathways (reviewed in Nguyen et al., 2003b) (Table 1). Impaired expression of these genes often increases sensitivity of cells to oxidative-type damage. In particular, polymorphisms in some ARE genes are associated with an increased risk of numerous cancers as well as enhanced susceptibility to chemical toxicity (reviewed in Wormhoudt et al., 1999; Nebert et al., 2002; Ross, 2004; Hayes et al., 2005; McIlwain et al., 2006; Nagar and Remmel, 2006). On the other hand, chronic exposure to electrophiles, such as carcinogens, activates the Nrf2 pathway leading to overexpression of ARE-containing genes. High levels of these proteins in tumor cells are advantageous and have been linked to chemotherapeutic drug resistance. Therefore, a fine balance in the regulation of Nrf2 target genes dictates a myriad of responses from cytotoxicity to cytoprotection.

Nrf2-activating chemicals that induce ARE genes have been categorized as cytoprotective agents. These include phenolic antioxidants (β-naphthoflavone, β-NF, butylated hydroxyanisole, BHA, and tert-butyl-hydroquinone, tBHQ), synthetic antioxidants (ethoxyquin), derivatives of 1,2-dithiole-3-thiones (oltipraz, 3H-1,2-dithiol-3-thione, D3T), phorbol esters (phorbol 12-myristate 13-acetate, PMA), triterpenoid analogues (oleanolic acid derivatives including 1-[2-cyano-3-,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole, CDDO-Im), and isothiocyanates (sulforaphane from broccoli, phenethyl isothiocyanate) (reviewed in Nguyen et al., 2003b). Researchers routinely use these compounds to identify Nrf2-inducible genes.

NAD(P)H Quinone Oxidoreductase 1

NAD(P)H quinone oxidoreductase 1 (Nqo1) is a cytosolic flavoprotein that is constitutively expressed in a wide number of tissues. Nqo1 catalyzes the two electron reductive metabolism and detoxification of endogenous and exogenous chemicals (reviewed in Ross, 2004). Apart from its role in drug metabolism, Nqo1 also defends against intracellular oxidative stress by scavenging superoxide (Siegel et al., 2004) and maintaining the reduced form of endogenous antioxidants including alpha-tocopherol-hydroquinone and coenzyme Q (Beyer et al., 1996; Landi et al., 1997; Siegel et al., 1997).

Early research with the Nqo1 promoter focused on the potential role of the aryl hydrocarbon receptor for regulation. However, it was shown that response elements distinct from those required for activation by the aryl hydrocarbon receptor were necessary for the basal and inducible expression of Nqo1. Mutations and deletions in the mouse and rat Nqo1 promoter aided in identifying the core ARE sequence (Rushmore et al., 1991; Favreau and Pickett, 1995; Nioi et al., 2003). Knockout mice lacking Nrf2 have reduced constitutive expression and activity of Nqo1 in liver, forestomach, and small intestine (Li and Jaiswal, 1992; Itoh et al., 1997; McMahon et al., 2001; Ramos-Gomez et al., 2001). In addition, chemical treatment with the Nrf2 activator BHA increases hepatic and intestinal Nqo1 levels in wild-type, but not Nrf2 knockout mice (Ishii et al., 2002). Collectively, these data confirm a role for Nrf2 in both the basal and inducible expression of Nqo1 in numerous tissues.

Glutamate-Cysteine Ligase and Glutathione Synthetase

The nonprotein thiol GSH (L-γ-glutamyl-cysteinyl-glycine) maintains intracellular redox balance and protects against oxidative insult. Additionally, GSH can detoxify chemicals through direct binding or enzymatic conjugation by glutathione- S- transferases (Gst). GSH also plays an important role in free radical scavenging. Glutamate- cysteine ligase (Gcl), also known as γ-glutamyl cysteine synthetase, performs the rate-limiting step in GSH synthesis by catalyzing the formation of γ-glutamylcysteine from glutamine and cysteine in the presence of ATP. Glutathione synthetase (GS) subsequently adds glycine to form the GSH tripeptide. Decreased levels of GSH are observed in hepatocytes and fibroblasts from Nrf2 knockout mice (Chan and Kwong, 2000). Reduced GSH biosynthesis results from lower amounts of synthetic enzymes such as Gcl. Gcl is a holoenzyme comprised of two subunits, a light chain or modifier subunit (Gclm, Mr = 27,700) and a heavy chain or catalytic subunit (Gclc, Mr = 73,000), both of which contain ARE sequences in their promoters (Seelig et al., 1984; Moinova and Mulcahy 1998; Mulcahy and Gipp, 1995; Mulcahy et al., 1997). Similarly, 2 potential binding sites for Nrf2 were identified during cloning of the human GS promoter. Livers from Nrf2 knockout mice exhibit 4- to 5-fold lower expression of both Gclm and Gclc genes corresponding with diminished GSH stores (Chan and Kwong, 2000; Chanas et al., 2002). Nrf2 binds in vitro to ARE sequences present in the human Gclm (Chan and Kwong, 2000) and Gclc promoters (Wild et al., 1999). Nrf2 also indirectly controls induction of rat Gclc mRNA by increasing expression of key members of the AP-1 and nuclear factor kappa B (NF-κB) families that participate in Gclc transcription (Yang et al., 2005).

Regulation of both Gcl and GS enzymes by Nrf2 points to a critical role for this transcription factor in maintaining cellular GSH homeostasis. Lower levels of hepatic GSH are observed in older animals. One explanation is that diminished GSH results from reduced transcriptional activity of Nrf2 with increasing age (Suh et al., 2004). Treatment of older rats with lipoic acid enhances Nrf2-mediated transcription and restores Gcl expression and activity to normal levels (Suh et al., 2004). In this context, direct activation of Nrf2 and restoration of GSH levels is one means for counteracting age-related hepatic oxidative stress.

Glutathione-S-Transferase

Gst enzymes catalyze the conjugation of chemicals and electrophilic species with GSH. These conjugates are subsequently degraded to mercapturates and excreted from the body (reviewed in Hayes et al., 2005). Numerous Gst isoforms are regulated at least in part by Nrf2 (Itoh et al., 1997). For example, constitutive gene expression of Gst isoforms (a1, a2, a3, a4, m1, m3, m4) is markedly less in livers from Nrf2 knockout mice (Chanas et al., 2002). Basal levels of Gsta1/2, Gsta3, Gstm1, but not Gstp1/2, are similarly decreased in the small intestine of these mutant mice (McMahon et al., 2001). Liver, forestomach, and intestine from Nrf2 knockout mice exhibit reduced total Gst activity corresponding with lower mRNA levels (McMahon et al., 2001; Ramos-Gomez et al., 2001). In line with these observations, induction of hepatic and intestinal Gst isoforms by BHA and ethoxyquin is also impaired in the absence of Nrf2 (Itoh et al., 1997; Hayes et al., 2000; Ishii et al., 2002). Of the Gst isoforms which are inducible by Nrf2 activating chemicals, ARE sequences have been identified in the promoter regions of their genes (Friling et al., 1990, 1992; Reinhart and Pearson, 1993; Ahlgren-Beckendorf et al., 1999; Ikeda et al., 2002).

Glutathione Peroxidase

The gastrointestinal isoform of glutathione peroxidase (GI-GPx) scavenges hydrogen peroxide and alkyl hydroperoxides and protects against intestinal inflammation and malignancies (Brigelius-Flohe, 1999). The cytoprotective actions of GI-GPx are limited not just to the gastrointestinal tract. Hyperoxia induces expression of GI-GPx in the lungs of wild-type, but not Nrf2 knockout mice (Cho et al., 2002). Both the mouse and human GI-GPx promoters contain ARE sequences that bind Nrf2 and are transcriptionally active (Banning et al., 2005). In addition, tBHQ and sulforaphane activate the GI-GPx promoter in transfected HepG2 cells and induce GI-GPx mRNA and protein expression in CaCo-2 cells (Banning et al., 2005). Further research is necessary to determine if Nrf2 similarly regulates expression of additional isoforms of GPx, particularly in the liver.

UDP-Glucuronosyltransferase

UDP- glucuronosyl transferase (Ugt) enzymes catalyze the conjugation of exogenous and endogenous chemicals with glucuronic acid (reviewed in Bock and Kohle, 2005). Although multiple isofoms of Ugt enzymes exist, one isoform Ugt1a6 has received particular attention as an Nrf2 target gene. In Nrf2-deficient mice, the basal expression of Ugt1a6 in liver and lung is depressed to about 56% of wild-type (Chan and Kan, 1999; Enomoto et al., 2001). Similarly, induction of hepatic Ugt1a6 by oltipraz is abolished in Nrf2 knockout mice (Ramos-Gomez et al., 2001). More recently, ARE-like elements were identified in the human Ugt1a6 promoter suggesting that the regulation of both the human and mouse Ugt1a6 gene is dependent upon Nrf2 (Munzel et al., 2003).

A recent study demonstrates induction of multiple Ugt isoforms in the liver and intestinal tract in male rats given oltipraz (Shelby and Klaassen, 2006). Higher levels of hepatic Ugt1a3, 1a6, and 1a7 mRNA and intestinal Ugt1a2, 1a3, 2b1, 2b3, 2b8, and 2b12 mRNA were seen after oltipraz treatment. Additional analysis is needed to determine Nrf2 involvement in the up-regulation of multiple Ugt isoforms.

Heme oxygenase-1

Heme oxygenase-1 (Ho-1) catalyzes the first and rate-limiting step in the catabolism of the pro-oxidant heme to carbon monoxide, biliverdin, and free iron. Ho-1 is also known as heat shock protein 32. This name is not surprising since mRNA and protein expression of Ho-1 is commonly up-regulated following oxidative stress and cellular injury (reviewed in Guo et al., 2001). Nrf2 directly regulates Ho-1 promoter activity (Alam et al., 1999). Analysis of the Ho-1 promoter identified binding sites resembling the ARE (Prestera et al., 1995; Inamdar et al., 1996). Similarly, in vitro exposure to diverse toxicants including diethyl maleate, paraquat, and cadmium chloride induced Ho-1 gene and protein expression in wild-type, but not Nrf2 knockout peritoneal macrophages (Ishii et al., 2000). Unlike Gst, Nqo1, and Ugt, which have been classically termed drug metabolizing enzymes, Ho-1 is more often thought of as a stress-related Nrf2 target gene.

Ferritin

As mentioned before, one of the breakdown products of heme metabolism is iron. Release of free iron during oxidative stress turns on gene transcription for the iron sequestrant, ferritin (Anderson and Frazer, 2005). Ferritin binds free iron and prevents it from participating in iron-mediated free radical reactions and subsequent cellular oxidative stress. High levels of ferritin are known to protect endothelial cells from oxidant-mediated insult (Juckett et al., 1996). Transcriptional control of ferritin mRNA expression by Nrf2 is mediated throughARE elements in the promoters of the heavy and light chains of both the mouse and human genes (Wasserman and Fahl, 1997; Tsuji et al., 2000; Chen et al., 2003; Pietsch et al., 2003; Hintze and Theil, 2005; Tsuji, 2005).

Microsomal Epoxide Hydrolase

Microsomal epoxide hydrolase (mEH) hydrolyzes and inactivates epoxides within the cell. Some information implicates Nrf2 in the regulation of mEH expression. This evidence includes reduced basal mRNA expression of mEH in multiple tissues of Nrf2 knockout mice (Ramos-Gomez et al., 2001; Hu et al., 2006a; Ma et al., 2006). Similarly, treatment of these mutants with prototypical Nrf2 inducers fails to induce mEH pointing to a role for Nrf2-mediated control.

Transporters

Cellular injury can be minimized not only by detoxification of chemicals but also through enhanced elimination via plasma membrane efflux transport proteins. Recent work suggests that ATP-dependent efflux transporters such as the multidrug resistance-associated proteins (Mrp) and multidrug resistance proteins (Mdr) may be part of the antioxidant response (reviewed in Shih et al., 2003; Klaassen and Slitt, 2005; Yeh and Yen, 2006). Overexpression of Keap1 in human hepatoma cells reduces Mrp efflux activity suggesting that Nrf2 positively regulates Mrp proteins (Sekhar et al., 2003). Using knockout mice, Nrf2 was shown to be required for both the constitutive and chemical inducible expression of Mrp1 in fibroblasts (Hayashi et al., 2003). ARE sequences exist in the mouse Mrp1 (Hayashi et al., 2003) and Mrp2 (Vollrath et al., 2006) promoters. Treatment of mice with Nrf2 activating chemicals coordinately induces hepatic Nqo1 and Mrp isoforms 2 through 6 mRNA (Maher et al., 2005). Similarly, expression of Nqo1, Mrp2, Mrp3, and Mdr1 in primary human hepatocytes is increased following oltipraz exposure (Jigorel et al., 2006). Up-regulation of liver Mdr genes (isoforms 1a and 1b) is also seen in mice administered the cancer chemopreventive agent, sulforaphane. This induction was absent in treated Nrf2 knockout mice (Hu et al., 2006a). Due to the documented role of efflux transporters in drug resistance, they are being evaluated as potential members of the Nrf2 defense response.

Additional Genes

In addition to the aforementioned genes, other genes have been proposed as Nrf2 targets. This includes the cystine/glutamate exchange transporter ${\text{X}}_c^{-}$ that contains an ARE sequence and can be induced by electrophile and oxidant exposure in an Nrf2-dependent manner (Ishii et al., 2000; Sasaki et al., 2002; Shih et al., 2003). More recently, gene array studies evaluated hepatic and intestinal gene expression of wild-type and Nrf2 knockout mice given a prototypical Nrf2 activator compound (Thimmulappa et al., 2002; Kwak et al., 2003; Hu et al., 2006a, 2006b; Shen et al., 2006). These studies identified additional Nrf2 targets including malic enzyme, peroxiredoxin 1, thioredoxin, thioredoxin reductase 1 and 3 as well as genes related to the ubiquitin-proteasome system, stress response, protein synthesis and trafficking, immunity, cell cycle, fatty acid metabolism, and phosphorylation/dephosphorylation cascades (Kim et al., 2001; Ishii et al., 2002; Thimmulappa et al., 2002; Kim et al., 2003; Kwak et al., 2003; Hu et al., 2006a, 2006b; Nair et al., 2006; Shen et al., 2006). Additional analysis is necessary to identify functional ARE sequences in the upstream regions of these novel Nrf2 genes.

Mice Deficient in Nrf2 and Keap1 Signaling Pathways

Gene-disrupted mouse models with perturbations in the Keap1:Nrf2 pathway have been established. These include embryos and mice lacking Nrf2, Keap1, both Nrf2/Nrf1, liver-specific Nrf1, and liver-specific Keap1 (Chan et al., 1996, 1998; Itoh et al., 1997; Kuroha et al., 1998; Leung et al., 2003; Wakabayashi et al., 2003; Okawa et al., 2006). In some cases, deletion of these genes leads to in utero lethality. Development of these gene knockout systems in the intact animal validates the importance of the Nrf2 signaling pathway in regulating antioxidant and drug metabolism enzymes and its role in protecting against toxicity.

Nrf2 Knockout Mice

Since Nrf2 was first identified in a screen for proteins involved in the developmental regulation of the β-globin gene, it was anticipated that mice lacking the Nrf2 gene would have impaired erythropoesis. Nrf2 knockout mice have been generated on multiple mouse backgrounds and none of the lines exhibit changes in erythropoesis during ontogeny (Chan et al., 1996; Itoh et al., 1997; Kuroha et al., 1998). Phenotypic changes in hematopoesis and lupus-like changes in Nrf2 knockout mice are typically associated with aging. For example, male and female Nrf2 knockout mice greater than 24 weeks old demonstrate anemia with secondary splenomegaly (Lee et al., 2004).

Peripheral blood smears show morphological abnormalities of red blood cells including Howell-Jolly bodies, schistocytes, and acantocytes indicating that the anemia results from hemolysis of damaged erythrocytes (Lee et al., 2004). Similarly, female Nrf2 knockout mice between 48 and 60 weeks develop lupus-like lesions including membranoproliferative glomerulonephritis with segmental hyalinization and sclerosis and hepatitis with severe lymphocytic infiltration and vasculitis surrounding the central vein and portal tracts (Yoh et al., 2001; Li et al., 2004; Ma et al., 2006). Both anemia and lupus-like symptoms in aged Nrf2-null mice have been attributed in part to long-standing deficiencies in antioxidant defenses and corresponding changes in immune system function.

Since Nrf2 is critical in mitigating cellular oxidative stress, researchers have been interested in determining the impact of knocking out Nrf2 on the ability of the liver and kidneys to eliminate free radicals. Electron paramagnetic resonance testing measures levels of the free radical spin probe 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (Carbamoyl-PROXYL) three dimensionally in whole animals (reviewed in Krishna et al., 2001). Nrf2 knockout mice show decreased liver and kidney Carbamoyl-PROXYL reducing activity compared to their wild-type counterparts (Hirayama et al., 2003). This difference in in vivo redox status is even more pronounced in older (50 weeks old) rather than younger (10 weeks old) Nrf2-deficient mice (Hirayama et al., 2003). Lower free radical reducing activity and GSH synthesis in older mutant mice may play a role in their age-related conditions (such as glomerulonephritis and anemia).

Nrf1 Knockout Mice

In contrast to Nrf2 knockout mice, which are considered to have essentially normal development, embryos with a targeted deletion of the Nrf1 gene die during mid-gestation (days 15 to 18 of gestation) as a result of fetal liver abnormalities and subsequent anemia (Chan et al., 1998). The combined deletion of both Nrf1 and Nrf2 genes results in embryonic lethality by days 9 and 10 of gestation due to intracellular accumulation of ROS and extensive apoptosis (Leung et al., 2003). Earlier lethality in the combined mutant embryos indicates cooperation between Nrf1 and Nrf2 in attenuating oxidative stress during fetal development.

Nonlethal, hepatocyte-specific Nrf1 knockout mice were recently developed (Xu et al., 2005). By fours weeks, serum alanine aminotransferase and triglyceride levels in these animals are elevated. Histologic examination of livers from these mice demonstrates steatosis, apoptotic and necrotic cells, and infiltration of inflammatory cells (Xu et al., 2005). As early as four months, Nrf1 knockout mice begin to develop foci of neoplastic growth (both hepatocellular adenomas and carcinomas). In addition, Masson’s trichrome staining shows fibrosis in livers from 6- to 12-month-old hepatocyte-specific Nrf1 knockout mice. Molecular analysis of hepatic mRNA expression revealed reduced expression of some ARE-containing genes (multiple Gst isoforms) in the mutant mice. In addition, Nrf1 knockout mice had markedly increased levels of cytochrome P450 4a genes, which mediate microsomal fatty acid oxidation (Xu et al., 2005). Differential changes in antioxidant and oxidant gene expression likely predisposes Nrf1 knockout mice to oxidative stress and development of steatohepatitis and liver cancer.

Keap1 Knockout Mice

Homozygous mice with a targeted deletion of the Keap1 gene die within 3 weeks after birth (Wakabayashi et al., 2003). Lethality was attributed to esophageal constriction, obstruction of the upper gastrointestinal tract and progressive asthenia in these mice. Postmortem examination of the homozygous Keap1 knockouts reveals severe hyperkeratosis of the esophagus and forestomach. Keap1 knockout mice demonstrated pronounced nuclear accumulation of Nrf2 and overexpression of Nrf2 target genes, Nqo1 and Gst (Wakabayashi et al., 2003). Interestingly, simultaneous deletion of both Nrf2 and Keap1 reverses this phenotype with double mutants exhibiting normal development and survival similar to wild-type mice. Further analysis identified functional AREsequences in genes regulating squamous cell differentiation including loricrin and keratin K6a genes. Therefore, Keap1 deficiency likely causes nuclear accumulation of Nrf2 and activation of loricrin and keratin K6a genes, resulting in severe hyperkeratosis of the esophagus and forestomach of mice.

More recently, mice with a hepatocyte-specific conditional deletion of the Keap1 gene were generated (Okawa et al., 2006). Selective loss of hepatic Keap1 does not appear to interfere with development of these mice. Histological examination of their livers demonstrates normal tissue morphology. Similar to the Keap1 knockout mice, Nrf2 accumulates in the nuclei of hepatocytes from the conditional knockouts, resulting in constitutive overexpression of numerous Nrf2 target genes (including Nqo1 and Gst isoforms). This mouse line should prove to be highly advantageous in studying the constitutive activation of Nrf2 defense genes in an intact animal.

Influence of Nrf2 on Diseases of the Liver and Gastrointestinal Tract

Recently, a number of review papers have highlighted the involvement of Nrf2 in a number of diseases of the lung, nervous system, and immune system (Chen and Kunsch, 2004; Lee and Johnson, 2004; Lee et al., 2005; van Muiswinkel and Kuiperij, 2005; Cho et al., 2006). Similarly, it is the intention of the authors to provide a comprehensive overview of current investigations into the role of Nrf2 in mitigating different liver and gastrointestinal diseases. These two organs express Nrf2 and are sites of continuous xenobiotic exposure, metabolism and detoxification. As such, an Nrf2-mediated defense is critical to their protection.

Drug-Induced Hepatotoxicity

Acetaminophen (APAP) has classically been used as a model hepatotoxicant in mechanistic studies evaluating drug-induced liver injury (Jaeschke and Bajt, 2006). APAP-induced hepatotoxicity involves generation of a highly reactive electrophile, depletion of GSH stores, protein covalent adduct formation, and oxidative stress. A role for oxidative stress in the pathogenesis of APAP hepatotoxicity is supported by the efficacy of antioxidant-type therapies such as N-acetyl-cysteine. This is a GSH replenishing agent used as antidote for APAP poisoning (Oz et al., 2004; Rowden et al., 2005). Therefore, it was hypothesized that Nrf2 could be involved in an innate cellular defense system against APAP toxicity and other xenobiotics with a similar mode of toxicity.

APAP causes nuclear accumulation of Nrf2 in mouse liver as early as 60 minutes after treatment (Goldring et al., 2004). Translocation of Nrf2 corresponds with increased expression of downstream Nrf2 target genes including mEH, Ho-1, and Gclc. In the study by Goldring et al., induction of these genes was only observed at a dose of APAP, which caused mild hepatotoxicity. Higher doses of APAP caused Nrf2 translocation but did not induce mRNA of mEH, Ho-1, and Gclc suggesting that severe degeneration and necrosis impaired mRNA synthesis. Similar studies have confirmed the hepatic induction of mouse Nrf2 target genes Nqo1 and Ho-1 after APAP (Roberts et al., 1998; Aleksunes et al., 2005, 2006b). Up-regulation of Nqo1 protein and activity is also seen in human liver specimens obtained during transplantation following APAP overdose (Aleksunes et al., 2006a).

Treatment of Nrf2 knockout mice with APAP results in enhanced liver injury and mortality compared to wild-type and heterozygote counterparts (Chan et al., 2001; Enomoto et al., 2001). N-acetyl-cysteine rescue therapy was effective in preventing mortality in APAP-treated wild-type mice, however, lower efficacy was observed in the Nrf2 knockouts (Chan et al., 2001). Enhanced toxicity in Nrf2 knockout mice was attributed to altered Phase 1 and 2 metabolism of APAP. These mice exhibit lower mRNA expression and activity of Ugt1a6 which is considered a detoxification pathway for APAP (Enomoto et al., 2001). Reduced APAP-glucuronide formation was hypothesized to increase availability of APAP for bioactivation to its reactive metabolite by cytochrome P450 enzymes. This is supported by enhanced immunohisto-chemical staining of APAP-adducted proteins in Nrf2 knockout liver sections (Enomoto et al., 2001). Increased staining may be also due to lower availability and synthesis of GSH (Chan et al., 2001; Enomoto et al., 2001). Differences in basal and inducible expression of GSH-related genes, including Gclc, Gclm, and Gst in Nrf2 knockout mice likely contribute to enhanced APAP toxicity. From these data, Nrf2 not only regulates the expression of enzymes that metabolize APAP, but also influences genes known to counteract the deleterious effects set in motion by the reactive intermediate of APAP. Further work is necessary to determine if Nrf2 signaling also participates in cellular repair mechanisms following APAP toxicity.

As would be expected, mice bearing the hepatocyte-specific disruption of the Keap1 gene exhibit dramatic resistance to APAP hepatotoxicity (Okawa et al., 2006). Higher nuclear accumulation of Nrf2 in these mice markedly enhances the constitutive expression of ARE-containing genes such as Nqo1, Gst (numerous isoforms), and Gclc. Decreased formation of APAP adducts is attributed to a greater detoxification capacity in Keap1-disrupted livers. However, differences in APAP bioactivation to its reactive metabolite have yet to be ruled out (Okawa et al., 2006). Taken together, data generated from treatment of both Nrf2 and Keap1 knockout mice with APAP point to a strong role for Nrf2 in attenuating drug-induced hepatotoxicity.

Cytochrome P450 2E1-Associated Oxidative Stress

Overexpression of cytochrome P450 2E1 (Cyp2E1) in mouse liver and cultured hepatoma cells results in cellular oxidative stress without exposure to any toxicant (Gong et al., 2003; Bai and Cederbaum, 2006). This likely occurs because ROS including superoxide, hydroxyl radical, and hydrogen peroxide are generated during the normal catalytic cycle of Cyp2E1. This Cyp450 isoform is notorious for higher oxidase activity and generating greater amounts of ROS compared to other Cyp450s (Gorsky et al., 1984). Production of ROS is significantly amplified with conditions resulting in Cyp2E1 induction (Kessova and Cederbaum, 2003)

Cyp2E1-induced oxidative stress is thought to be a central mechanism underlying ethanol-mediated hepatotoxicity (reviewed in Dey and Cederbaum, 2006). Exposure of mice to ethanol or chemicals such as pyrazole increases hepatic expression of Cyp2E1 (Castillo et al., 1992). This leads to a compensatory up-regulation in Nrf2 mRNA and protein (Castillo et al., 1992; Gong and Cederbaum 2006). In vitro overexpression of Cyp2E1 in cultured hepatoma cells similarly enhances mRNA and protein levels of Nrf2 and its targets (including Ho-1 and Gclc) due to elevated ROS generation. Knocking down Nrf2 levels in Cyp2E1-overexpressing cells using siRNA prevents induction of Gclc and Ho-1 protein (Gong and Cederbaum, 2006). These changes are accompanied by marked increases in ROS and lipid peroxidation and reduced cell viability. Consequently, activation of Nrf2 signaling likely represents an early adaptive mechanism to thwart oxidative stress associated with Cyp2E1 induction following ethanol or chemical exposure (reviewed in Dey and Cederbaum, 2006).

Liver Fibrosis

One consequence of inflammation or direct toxic insult to the liver is fibrosis. During fibrosis, there is excessive deposition of extracellular matrix proteins (namely, collagen) by stellate cells. Under normal conditions, stellate cells are quiescent. In response to accumulating mediators of oxidative stress and lipid peroxidation, stellate cells become “activated” and begin to proliferate and increase production of collagen. These changes are accompanied by acquired resistance of activated stellate cells in culture to injury mediated by electrophiles, such as menadione (Vasiliou et al., 2003). Resistance of activated stellate cells to electrophile cytotoxicity was hypothesized to be due to direct detoxification of menadione and/or increased scavenging of ROS (Vasiliou et al., 2003). Stellate cells isolated from a cirrhotic rat liver demonstrate marked induction of Nqo1 protein and activity. This observation provided a potential clue for the resiliency of these cells, since Nqo1 can scavenge superoxide as well as detoxify menadione intermediates (Vasiliou et al., 2003). Similar to the rat liver fibrosis model, normal stellate cells can be activated in culture using a known inducer of antioxidant responsive genes, tBHQ (Reichard and Petersen, 2006). Using nuclear protein extracts from normal and activated stellate cells, Nrf1 and Nrf2 proteins were identified as part of the Nqo1 ARE DNA/protein complex. Concomitant activation of the ARE defense and enhanced cell proliferation may confer protection against electrophile-mediated stellate cell toxicity during liver fibrosis.

Chemical Carcinogenesis

Although chemical carcino-genesis is multifactorial, oxidative stress is a key component for cellular transformation (Klaunig and Kamendulis, 2004). Polymorphisms in some Nrf2-regulated antioxidant and detoxification enzymes are risk factors for the development of cancer (reviewed in Wormhoudt et al., 1999; Ross, 2004; Hayes et al., 2005; McIlwain et al., 2006; Nagar and Remmel, 2006; Nebert et al., 2002). These risk factors hint at a role for Nrf2 antioxidant defenses in preventing chemical-induced carcinogenesis. To investigate this hypothesis, benzo[a]pyrene-induced carcinogenesis was evaluated in Nrf2 knockout mice. Benzo[a]pyrene-induced neoplasia in the forestomach was observed in both Nrf2 wild-type and knockout mice (Ramos-Gomez et al., 2001). Histopathological analysis demonstrated enlarged forestomachs with narrowed lumens due to papillomas of varying sizes. Notably, a greater number of tumors were seen in the Nrf2 knockout mice compared to wild-types. Other sets of mice received doses of the Nrf2 activator, oltipraz, in conjunction with benzo[a]pyrene. Oltipraz treatment reduced the incidence of benzo[a]pyrene-induced tumors by 50% in wild-type mice. Disruption of the Nrf2 gene in knockout mice completely abrogated the chemopreventive efficacy of oltipraz. A higher incidence of tumors was attributed to negligible induction of multiple Nrf2 enzymes (including Gst, Nqo1, Ugt1a6, and mEH) in liver and forestomach of oltipraz-treated Nrf2-deficient mice. Subsequent work confirmed these findings and demonstrated that the protective effects of oltipraz were not due to inhibition of Cyp450-mediated benzo[a]pyrene metabolism (Ramos-Gomez et al., 2003).

Similar chemopreventive efficacy against benzo[a]pyrene-induced neoplasia is documented in mice treated with sulforaphane, an Nrf2 activating compound that is a constituent of cruciferous vegetables such as broccoli (Fahey et al., 2002). Again, benzo[a]pyrene-treated Nrf2-null mice had more tumors in the forestomach compared to wild-type mice which were not attenuated by dietary sulforaphane administration (Fahey et al., 2002). Early findings suggest that other Nrf2 activating compounds such as CDDO-Im similarly protect against formation of aflatoxin-induced preneoplastic lesions (identified as Gstp-positive foci) in rat liver (Roebuck et al., 2003; Yates et al., 2006).

In addition to prevention of gastric and hepatocellular tumors, Nrf2 activators may similarly protect against azoxymethane (AOM)- and N-methyl-nitrosourea-induced colonic aberrant crypt foci (ACF) in rats (Wargovich et al., 1996, 2000; Chung et al., 2000; Begleiter et al., 2003). ACF are preneoplastic lesions observed in carcinogen-treated rodents. Their appearance is considered an early indicator of colon carcinogenesis. In one study, treatment with sulforaphane or phenethyl isothiocyanate reduced the formation of total ACF and multicrypt foci (more than 4 crypts/focus) in rats exposed to AOM (Chung et al., 2000). Similar findings were observed in oltipraz-treated rats exposed to AOM. This was mechanistically linked to increased hepatic and intestinal Nqo1 activity (Begleiter et al., 2003). Although these results further support a role for Nrf2 in chemoprevention, it is unknown whether the protection against ACF formation afforded by oltipraz is occurring via Nrf2-dependent pathways. Reports of reduced tumor burden following treatment of rodents with Nrf2-activating compounds have prompted clinical trials of these agents for cancer prevention in humans (Zhang and Gordon, 2004; Shen et al., 2006).

Gallstones

Susceptibility to gallstone formation in rodents is thought to be dictated by genetics. Microarray analysis has been used to identify genes differentially expressed in mouse strains that are either resistant or susceptible to gallstones following a lithogenic diet. Differential gene expression profiles between gallstone-susceptible C57L/J mice and gallstone-resistant AKR/J mice fed a normal diet demonstrated greater basal expression of Nrf2 target genes including Gst and mEH in AKR/J mice (Dyck et al., 2003). Similarly, higher basal expression of Nrf2 mRNA and protein was observed in AKR/J mice. Genetic analysis previously identified Lith 1 as a major gallstone gene locus (Khanuja et al., 1995; Paigen et al., 2000). Interestingly, Nrf2 maps to the Lith 1 locus, suggesting that it may be a candidate gene for determining gallstone susceptibility in mice.

Environmental Toxicants

Chronic exposure to the environmental pollutant, penta chloro phenol (PCP), causes hepatocellular proliferation, necrosis and acts as a tumor promoter in mice (Umemura et al., 1999). Mice fed PCP in their diet for 4 weeks showed marked induction of hepatic Nqo1 protein and Ugt activity (Umemura et al., 2006). As expected, up-regulation of these two pathways is absent in Nrf2 knockout mice that exhibit slightly greater hepatic oxidative stress and toxicity as indicated by malondialdehyde and alkaline phosphatase levels, respectively. Notably, there is no difference in plasma ALT and AST activity in Nrf2 wild-type and knockout mice treated with PCP. PCP toxicity is an example in which lack of Nf2 signaling does not always translate into enhanced susceptibility to chemical toxicity.

Hepatocellular Apoptosis

Though research has linked Nrf2 to the prevention of apoptosis in the lung (Rangasamy et al., 2004), ovarian follicles (Hu et al., 2006c), and nervous system (Lee et al., 2003; Vargas et al., 2006), there is limited information regarding Nrf2 and liver apoptosis (Li et al., 2004). In order to evaluate the contribution of Nrf2 to apoptosis, two rodent models of hepatocyte apoptosis were utilized by Morito et al. (2003) In the first model, apoptosis was induced by administering antibodies against the cytokine receptor Fas (APO-1/CD95). In the second model, mice were first primed with D-galactosamine and then administered tumor necrosis factor-alpha (TNFα) (reviewed in Lehmann et al., 1987; Schulze-Osthoff et al., 1998).

Activation of both the Fas and TNFα death receptors results in apoptosis events including translocation of phosphatidylserine to the outer leaflet of the cell membrane, activation of endonucleases which cleave genomic DNA, and stimulation of caspase signaling cascades (Ogasawara et al., 1993; Leist et al., 1996; Medema et al., 1997; Schulze-Osthoff et al., 1998). Administration of either anti-Fas antibodies or TNFα/D-galactosamine to Nrf2 knockout mice resulted in greater hepatotoxicity (plasma ALT leakage) as compared to wild-type counterparts (Morito et al., 2003). Histopathological examination confirmed increased numbers of apoptotic hepatocytes and diffusely scattered hemorrhagic foci in treated Nrf2 knockout mice. Enhanced hepatocyte apoptosis in Nrf2 knockout mice was associated with lower intracellular GSH content. Administration of the ethyl monoester of GSH to Nrf2 knockout mice decreased the extent of hepatocyte apoptosis after anti-Fas antibody or TNFα treatment to levels seen in wild-type mice (Morito et al., 2003). Although the current information available links Nrf2 and apoptosis, additional work is necessary to determine if this relationship is direct (transcriptional activation of apoptosis-related genes by Nrf2) or indirect (secondary pathways such as GSH homeostasis).

Additional in vitro evidence connects Nrf2 to apoptosis. Using random gene knockdowns to screen for inhibitors of apoptosis, Nrf2 was found to inhibit the Fas-mediated apoptosis pathway in HeLa cells (Kotlo et al., 2003). Forced over-expression of Nrf2 in transfected cells protects against anti-Fas antibody cell death (Kotlo et al., 2003). Furthermore, Nrf2 can be cleaved by caspase proteases in vitro and in vivo (Ohtsubo et al., 1999). Degradation of Nrf2 by caspases may be a mechanism to down-regulate opposing cell survival pathways.

Heavy Metal Toxicity

In vitro exposure of mouse hepatoma cells to heavy metals including inorganic mercury and lead results in the transcriptional activation of Nqo1 and Gst genes (Korashy and El-Kadi, 2006). Similarly, arsenic induces Nqo1 in hepatoma cells by stabilizing Nrf2 protein (He et al., 2006). Arsenic-induced cytotoxicity in primary mouse hepatocytes can be suppressed by prior incubation with sulforaphane and this is likely due to enhanced Ho-1, Gclc, Gclm, and Gst protein expression (Shinkai et al., 2006). Animal experiments examining Nrf2 signaling in response to heavy metal exposure are needed to determine if a similar induction of xenobiotic metabolizing enzymes occurs in vivo.

Conclusions

Nrf2 is critical for cytoprotection by coordinately activating detoxification genes and preventing the pathogenesis of liver and gastrointestinal diseases. From a toxicologic standpoint, Nrf2 is likely a key mediator dictating susceptibility to oxidative and chemical-induced injury. Since the generation of Nrf2 knockout mice, researchers have identified potential mechanisms through which Nrf2 mitigates oxidative stress in numerous disease models of the liver, gastrointestinal tract, lung, skin, and central nervous system. Enhanced susceptibility of these mutant mice commonly arises from impaired expression of ARE-containing genes or an inability to adapt to electrophilic toxicity and/or oxidative stress. In some cases, it is likely that both scenarios are occurring. Nrf2 knockout mice are also a useful model for investigating Nrf2 signaling pathways and identifying novel Nrf2 target genes.

Given the critical role of Nrf2 in modulating numerous cellular processes including GSH homeostasis, drug metabolism, antioxidant defense, and cell cycle progression, it is not surprising that multiple regulatory mechanisms for this transcription factor have developed. Regulation of Nrf2 signaling occurs in the cytoplasm (Keap1 sequestration, modifications of Nrf2 and Keap1 proteins) and the nucleus (identity and availability of heterodimeric partners, multiple DNA response elements). There are likely additional regulatory mechanisms for Nrf2 signaling that have yet to be defined.

Loss of Nrf2 does not always result in enhanced disease, as in the case of PCP-induced hepatotoxicity. In this model of injury, adaptive mechanisms as a result of the Nrf2 deletion could be impacting the response of these animals to insult. Therefore, alternative antioxidant and cell survival pathways involved in chemoprevention need to be thoroughly characterized in Nrf2 knockout mice since compensation in their signaling could influence susceptibility to liver and gastrointestinal diseases. Mutations in Nrf2 target genes alter disease susceptibility, particularly to the development of malignancies. More recently, polymorphisms were identified in the promoter region of the human Nrf2 gene (Yamamoto et al., 2004). Functional characterization of these polymorphisms may provide insight into the susceptibility of individuals to oxidative-type diseases as well as chemical-induced toxicities.