Nrf2 is a transcription factor that upon activation by oxidative/electrophilic insult induces a battery of cytoprotective genes. Nrf2-null mice are highly susceptible to a variety of oxidative/electrophilic stress-induced pathologies because of reduced basal and inducible expression of detoxification enzymes (Aleksunes and Manautou, 2007
). In contrast, chemicals that activate Nrf2 protect rodents from pathologies linked to or caused by oxidative/electrophilic stress, leading to the hypothesis that Nrf2 is a target for chemoprevention (Lee and Surh, 2005
). However, because chemical compounds typically have many off-target effects, it would be more informative to study activation of Nrf2 in a mouse model of increased Nrf2 activation. For example, the Nrf2 activator oltipraz, which has been used in the identification of possible Nrf2-target genes, also activates the CAR, inducing its target gene Cyp2b10 (Merrell et al., 2008
). Therefore, the present study utilized genetic models of Nrf2 under- and overactivation to determine which genes have decreased mRNA expression with lack of Nrf2, and which genes have more mRNA expression when there is increased activation of Nrf2.
Upon activation, Nrf2 translocates into the nucleus, as reflected by the amount of Nrf2 protein in the nucleus in wild-type, Nrf2-null, and Keap1-kd mice. As shown in , wild-type mice have very little Nrf2 protein in the nucleus, similar to previously published results (Kwak et al., 2001
; Maher et al., 2007
). Low Nrf2 protein in the nucleus in wild-type mice is most likely due to the rapid turnover of Nrf2 under basal or unchallenged conditions (Kobayashi et al., 2004
). As expected, no hepatic Nrf2 mRNA or protein was detected in Nrf2-null mice, similar to previous results (Tanaka et al., 2008a
). Nrf2-null mice also have lower Keap1 mRNA expression, most likely due to loss of Nrf2 binding to a functional ARE in the promoter region of Keap1 in a proposed negative feedback mechanism (Lee et al., 2007
). In contrast, Keap1-kd mice have 55% lower Keap1 mRNA expression, which is likely the mechanism for tripling the amount of Nrf2 protein in the nucleus of Keap1-kd mice. Keap1-kd mice also have a modest decrease in Nrf2 mRNA expression, which suggests a possible negative feedback pathway for Nrf2 mRNA expression when Nrf2 is overactivated.
Most basal hepatic physiological parameters in serum, liver, and bile were not different among wild-type, Nrf2-null, and Keap1-kd mice (). However, bile flow tended to be lower in Nrf2-null mice and was significantly higher in Keap1-kd mice. Increased bile flow in Keap1-kd mice is due to increased biliary excretion of GSH, as GSH excretion regulates bile-acid–independent bile flow (Ballatori and Truong, 1989
). Increased expression of GSH synthetic enzymes (Gclc and Gclm), as discussed in detail below, likely contributes to increased GSH concentrations in liver and biliary excretion of GSH in Keap1-kd mice. There were no differences in total biliary bile acids among genotypes (data not shown). Biliary excretion, possibly as a result of increase bile flow, but not biliary concentrations of phospholipids and cholesterol were increased in Keap1-kd mice.
Hepatic concentrations of GSH in the three mouse genotypes were proportional to the amount of activated Nrf2. GSH is a prominent cellular antioxidant that protects against oxidative/electrophilic stress by directly scavenging reactive oxygen species or acting as a cosubstrate for Gst-mediated detoxification of reactive electrophiles (Meister and Anderson, 1983
). The rate-limiting enzymatic process in GSH synthesis is catalyzed by glutamate-cysteine ligase, which is made up of the catalytic and modifier subunits, Gclc and Gclm, respectively. Nrf2-null mice have lower, whereas Keap1-kd mice have higher hepatic mRNA expression of Gclc and Gclm, genes positively regulated by Nrf2 (Lee et al., 2007
; Moinova and Mulcahy, 1999
; Wild et al., 1999
) (). Furthermore, mRNA expression of Gsr, the enzyme responsible for the reduction of oxidized glutathione (GSSG) to reduced GSH, is lower in Nrf2-null mice and higher in Keap1-kd mice. Because Gclc, Gclm, and Gsr mRNA expression is lower in Nrf2-null mice and higher in Keap1-kd mice, the amount of GSH in the liver was determined. Nrf2-null mice have less than half, whereas Keap1-kd mice have almost double the hepatic reduced GSH as wild-type mice (). Higher GSH concentrations in the Keap1-kd mouse should increase resistance to a variety of oxidative/electrophilic stress-induced pathologies, whereas lower reduced GSH concentrations render the Nrf2-null mice more susceptible to such pathologies.
With few exceptions, typical Nrf2-target genes are lower in Nrf2-null mice and higher in Keap1-kd mice. Nqo1 is a Nrf2-dependent flavoprotein that catalyzes the two-electron reduction and detoxification of electrophilic quinones and its derivatives (Jaiswal, 2000
). Nqo1 is a prototypical Nrf2 target gene and is expressed lower in Nrf2-null mice and higher in Keap1-kd mice (). Eh-1 is induced by sulforophane and 3H
-1,2-dithiole-3-thione (D3T) in wild-type but not Nrf2-null mice (Kwak et al., 2001
; Thimmulappa et al., 2002
). However, Eh-1 mRNA is not increased in Keap1-kd mice, suggesting that other mechanisms aside from Nrf2 nuclear translocation are important for Eh-1 mRNA induction in the liver. Ho-1 catalyzes the breakdown of heme into iron, carbon monoxide, and biliverdin. Biliverdin is then reduced to bilirubin, an antioxidant, via biliverdin reductase. Ho-1 is induced, in mice, by many Nrf2 activators or stressors, such as CDDO-Im (Liby et al., 2005
), butylated hyroxyanisole (Keum et al., 2006
), hyperoxia (Cho et al., 2002
), and arsenite (Gong et al., 2002
). However, no difference in Ho-1 expression was observed among wild-type, Nrf2-null, and Keap1-kd mice, a result that is consistent with previous reports (Okawa et al., 2006
). The lack of induction of Ho-1 in Keap1-kd mice is attributed to the presence of Bach1, a protein that suppresses the activity of Maf proteins, which are important heterodimers for Nrf2-ARE binding and subsequent Ho-1 gene transcription (Keum et al., 2006
The superoxide and hydrogen peroxide detoxifying enzymes Sod1, Sod2, Cat, and Gpx are inducible in response to Nrf2 activators, such as D3T (Zhu et al., 2005
), phenolic acids (Yeh and Yen, 2006
), and shear stress (Jones et al., 2007
). In the present study, a minor decrease in hepatic mRNA expression of Sod1, Sod2, and Cat was observed in Nrf2-null mice (10-30%), whereas there was no increase in Keap1-kd mice. Gpx mRNA expression was not different among the three genotypes (). There were also no differences among the three genotypes in Sod, Cat, or Gpx enzyme activities. The lack of induction of Sods, Cat, and Gpx in Keap1-kd mice suggests that nuclear translocation of Nrf2 does not induce enzymes capable of detoxifying superoxide and related reactive oxygen species in liver.
Redoxins are important for protein repair, as they are responsible for the reduction of protein disulfide bonds that could disrupt proper protein folding and function. Peroxiredoxins reduce hydrogen peroxide to water, protecting against hydroxyl radical formation. Glrx1, Prx1, Txn1, and Txnrd1 have lower hepatic mRNA expression in Nrf2-null mice, whereas only Txn1 and Txnrd1 are induced in Keap1-kd mice (). NADPH is a cofactor for many oxidoreductase reactions that are important in detoxifying reactive oxygen species. Me1 and H6pdh are enzymes that regenerate NADPH after it has been oxidized. Both Me1 and H6pdh are lower in Nrf2-null mice but not increased in Keap1-kd mice ().
Chemicals, such as acetaminophen, benzo[a]pyrene, carbon tetrachloride, and benzene, are activated to reactive intermediates by Cyps, which can result in toxicity. Knowledge of Cyp activity in toxicity studies utilizing Nrf2-null and Keap1-kd mice is critical, because changes in Cyp activity could alter the amount of toxic metabolite generated, leading to an incorrect interpretation of results, that is, less injury caused by decreased toxic metabolite formation and not increased detoxification of the metabolite. Even though there were differences among genotypes in mRNA expression of Cyp1a1, 2b10, and 4a14, minor differences, if any, were detected in the enzyme activity of major Cyps. Thus, Cyps most likely will not play a role in the interpretation of results from toxicity studies involving Nrf2-null and Keap1-kd mice.
Aldhs catalyze the oxidation of a wide variety of electrophilic aliphatic and aromatic aldehydes to carboxylic acids (Parkinson and Ogilvie, 2008
). Nrf2-null mice have lower expression of Aldh1a1, 1a7, 3a2, 6a1, and 7a1, whereas Keap1-kd mice were not different from wild-type mice (). The lack of induction of Aldhs in Keap1-kd mice suggests that Aldhs are not induced by Nrf2 in this mouse model.
Carboxylesterases (Cess) catalyze the hydrolysis of ester- and amide-containing chemicals and are important in the activation of prodrugs (Parkinson and Ogilvie, 2008
). Produrg strategies allow for improvement of oral bioavailability of poorly absorbed drugs. Expression of Ces1b4, 1d1, 1e1, 1h1, and 2a6 were lower in Nrf2-null mice, whereas only Ces1e1 and 2a6 were higher in Keap1-kd mice (). An increase in expression of Cess in Keap1-kd mice suggests that activation of Nrf2 increases expression of Cess and metabolism of xenobiotics by Cess. An increase in Ces1e1 also might increase the amount of prodrug (i.e., the chemotherapeutics floxuridine and gemcitabine) converted to the active form, providing a more efficacious result (Landowski et al., 2006
; Marsh et al., 2004
; Taketani et al., 2007
Hepatic mRNA expression of phase-II enzymes is also altered in Nrf2-null and Keap1-kd mice. Ugts catalyze the conjugation of a glucuronosyl group from UDP-GA to a variety of substrate molecules, making them more water soluble and readily excreted. Increased excretion of xenobiotics decreases the amount of compound available for biotransformation to a toxic electrophilic metabolite. In addition, Ugt-catalyzed reactions are responsible for approximately 35% of all drugs metabolized by phase-II enzymes (Evans and Relling, 1999
). In general, Ugt mRNA expression is lower in Nrf2-null mice, whereas Keap1-kd mice are not different from wild-type mice, with the exception of a minor increase in mRNA expression of Ugt1a6 and Ugt2b35 (). The availability of the cosubstrate UDP-GA is required for the enzyme activity of Ugts. Both Ugdh and Ugp2 mRNA expression were lower in Nrf2-null mice, whereas Ugdh was significantly higher and Ugp2 tended to be higher in Keap1-kd mice.
Sults catalyze approximately 20-25% of phase-II reactions by transferring a sulfonic acid group from the cosubstrate PAPS (Evans and Relling, 1999
). There are few differences in the amount of the various Sult mRNAs among wild-type, Nrf2-null, and Keap1-kd mice, suggesting that Nrf2 does not play a major transcriptional role in the expression of Sults.
Gsts catalyze the conjugation of nucleophilic GSH with reactive and potentially damaging electrophiles (Parkinson and Ogilvie, 2008
). Substrates for Gsts include hydroperoxides of fatty acids, phospholipids, cholesterol, and quinone-containing compounds (Hayes et al., 2005
). The expression of many Gsts is dependent on Nrf2, with most Gsts having 41-85% lower expression in Nrf2-null mice and 45-585% higher expression in Keap1-kd mice (). A decrease in hepatic GSH concentration and Gst mRNA expression contributes to Nrf2-null mice being highly susceptible to electrophilic stress. In contrast, Keap1-kd mice have increased hepatic GSH concentrations and Gst mRNA expression and most likely have an increased resistance against damaging electrophiles that can be neutralized via GSH conjugation. The ability to produce and use more GSH appears to be one of the most important benefits of increased hepatic activated Nrf2, as observed in Keap1-kd mice.
Uptake transporters are important for the hepatic uptake and clearance of xenobiotics, an important process in the first-pass effect (Klaassen and Lu, 2008
). In general, mRNA expression of hepatic uptake transporters was similar among genotypes, with the exception of Oatp1a1, 1b2, and Oct1, which exhibit relatively minor differences (< 25%) in expression (). Of note, Oatp1a1 expression is lower in Keap1-kd mice, which is consistent with what has been observed upon administration of Nrf2 activators (Cheng et al., 2005
). Furthermore, Oatp and Oct1 mRNA are generally not altered by Nrf2 activators or other microsomal enzyme inducers (Cheng et al., 2005
). Thus, this data suggests that uptake transporters in the liver are not activated by Nrf2.
Hepatic efflux transporters are important for the elimination and overall clearance of xenobiotics from the liver. Mrps are a group of ATP-dependent transporters that are important in cytoprotection, because Mrps can remove potentially toxic xenobiotics, metabolites, and endogenous substrates from cells (Maher et al., 2007
). Mrp2 and Mrp3 mRNA expression are lower in Nrf2-null mice, but higher in Keap1-kd mice. Mrp4 mRNA was lowly expressed and not different between Nrf2-null and wild-type mice, but was increased 55% over wild-type in Keap1-kd mice (). Bcrp is an efflux transporter for substrates, such as mitoxantrone, anthracyclines, camptothecins, topotecan, and SN-38, the active metabolite of irinotecan (Brangi et al., 1999
; Doyle et al., 1998
; Litman et al., 2000
; Miyake et al., 1999
; Ross et al., 1999
). Bcrp mRNA expression is 28% lower in Nrf2-null mice and 15% higher in Keap1-kd mice. Mate1 effluxes organic cations, such as metformin and tetraethylammonium, from hepatocytes into bile (Hiasa et al., 2006
; Terada et al., 2006
). Mate1 mRNA is expressed at a 27% lower level in Nrf2-null mice. There was no difference in Mate1 mRNA expression between Keap1-kd and wild-type mice, similar to a previous report in which Nrf2 activators did not induce Mate1 mRNA expression (Lickteig et al., 2008
). Abcg5 and Abcg8, transporters involved in the efflux of cholesterol and potentially toxic plant sterols from the liver into bile, were lower in Nrf2-null mice and higher in Keap1-kd mice. The increase of mRNA expression of efflux transporters may provide Keap1-kd mice the ability to increase the clearance of potentially toxic xenobiotics, thereby decreasing time of exposure and toxicity.
categorizes mRNA expression of detoxifying and transporter genes among wild-type, Nrf2-null, and Keap1-kd mice into three different patterns. The first pattern encompasses genes that have decreased mRNA expression in Nrf2-null mice and increased expression in Keap1-kd mice compared with wild-type mice. Pattern 1 genes include Nqo1, Gsts, and Mrps, which detoxify and eliminate electrophiles. The second pattern consists of genes that have decreased mRNA expression in Nrf2-null mice but no difference between Keap1-kd and wild-type mice. Prominent genes in pattern 2 are the superoxide detoxifying enzymes Sod1, Sod2, Cat, and Prx1, as well as some Ugts and Aldhs. The third pattern includes genes that were not different among wild-type, Nrf2-null, and Keap1-kd mice and includes genes such as some Ugts, Sults, some Aldhs, Gpx, Ho-1, and uptake transporters. It should be noted that the genes in pattern 3, with the exception of Ho-1, are not known Nrf2-target genes.
Categorization of Antioxidant, Phase-I, Phase-II, and Transporter mRNA Expression
Keap1 has been shown in vitro
to bind and regulate the ubiquitination and subsequent proteasomal degradation of one other protein, phosphoglycerate mutase family member 5 (PGAM5) (Lo and Hannink, 2006
). Phosphoglycerate mutases catalyze the conversion of 3-phosphoglycerate to 2-phosphoglycerate, which is an important substrate in glycolysis. Therefore, because Keap1 is capable of binding other proteins, at least in vitro
, it is possible that some of the genes that are upregulated in Keap1-kd mice could be independent of Nrf2. However, a follow-up study by Lo et al. demonstrated in vitro
that PGAM5 tethers a ternary complex containing both Keap1 and Nrf2 in mitochondria (Lo and Hannink, 2008
). Lo and Hannick (2008) also hypothesized that Keap1 may use other proteins, such as PGAM5, to regulate Nrf2 at other subcellular localizations, such as the mitochondria. In addition, PGAM5 has not been shown to transcriptionally regulate any gene. Therefore, it seems unlikely that PGAM5 would be responsible for the upregulation of genes in Keap1-kd mice.
In conclusion, this study has shown that whereas Nrf2-null and Keap1-kd mice have normal livers under standard institutional animal care conditions, baseline defenses against electrophilic stress are lower in Nrf2-null mice and higher in Keap1-kd mice. In addition, classical reactive oxygen species reducing enzymes, such as Cat, Gpx, and Sods, were not induced in livers of Keap1-kd mice, whereas genes, such as Gsts, Nqo1, and Mrps, important in detoxifying and eliminating electrophiles are markedly increased in Keap1-kd mice. The major advantage Keap1-kd mice have against reactive oxygen and nitrogen species in the liver appears to be an increase in hepatic GSH concentrations. Collectively, these results suggest that activation of Nrf2 in liver results in induction of genes, whose protein products, are more important in the direct detoxification of highly reactive electrophilies formed from xenobiotic exposure than for detoxification of reactive oxygen species.