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Toxicol Sci. 2009 October; 111(2): 238–246.
Published online 2009 May 27. doi:  10.1093/toxsci/kfp115
PMCID: PMC2742581
Highlighted Article

Introducing the “TCDD-Inducible AhR-Nrf2 Gene Battery”

Abstract

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) induces genes via the transcription factor aryl hydrocarbon receptor (AhR), including Cyp1a1, NAD(P)H:quinone oxidoreductase 1 (Nqo1), UDP-glucuronosyltransferase 1a6 (Ugt1a6), and glutathione S-transferase a1 (Gsta1). These genes are referred to as the “AhR gene battery.” However, Nqo1 is also considered a prototypical target gene of the transcription factor nuclear factor erythroid 2–related factor 2 (Nrf2). In mice, TCDD induction of Nrf2 and Nrf2 target, Nqo1, is dependent on AhR, and thus TCDD induction of drug-processing genes may be routed through an AhR-Nrf2 sequence. There has been speculation that Nrf2 may be involved in the TCDD induction of drug-processing genes; however, the data are not definitive. Therefore, to address whether TCDD induction of Nqo1, Ugts, and Gsts is dependent on Nrf2, we conducted the definitive experiment by administering TCDD (50 μg/kg, ip) to Nrf2-null and wild-type (WT) mice and collecting livers 24 h later to quantify the mRNA of drug-processing genes. TCDD induction of Cyp1a1 and Ugt1a1 was similar in WT and Nrf2-null mice, whereas TCDD induction of Ugt1a5 and 1a9 was blunted in Nrf2-null mice. TCDD induced Nqo1, Ugt1a6, 2b34, 2b35, 2b36, UDP-glucuronic acid–synthesizing gene UDP-glucose dehydrogenase, and Gsta1, m1, m2, m3, m6, p2, t2, and microsomal Gst1 in WT mice but not in Nrf2-null mice. Therefore, the present study demonstrates the novel finding that Nrf2 is required for TCDD induction of classical AhR battery genes Nqo1, Ugt1a6, and Gsta1, as well as most Ugt and Gst isoforms in livers of mice.

Keywords: Nrf2, AhR, TCDD, Nqo1, Ugts, Gsts

For decades, researchers have sought to characterize the molecular mechanisms of how 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces drug-processing genes in liver (Hankinson, 1995, 2005; Okey and Vella, 1982; Poland and Glover, 1974; Poland and Knutson, 1982; Whitlock, 1990). We now know that TCDD binds to the aryl hydrocarbon receptor (AhR), and the complex translocates to the nucleus and dimerizes with the AhR nuclear translocator (Arnt) (Whitlock, 1990). The AhR/Arnt dimer binds to xenobiotic response elements (XREs) of genes, inducing gene expression of many enzymes responsible for xenobiotic metabolism. The enzyme most thoroughly investigated in regard to the mechanism of TCDD induction is Cyp1a1. However, there are a number of genes included in the mouse AhR gene battery where TCDD induction has not been examined as thoroughly as the Cyps, such as NAD(P)H:quinone oxidoreductase 1 (Nqo1), UDP-glucuronosyltransferase 1a6 (Ugt1a6), glutathione S-transferase a1 (Gsta1), and aldehyde dehydrogenase 3a1 (Aldh3a1) (Nebert et al., 2000). Previous work by this laboratory and others has demonstrated that TCDD induces mRNA expression of most Ugts (Buckley and Klaassen, 2009) and Gsts in livers of mice (Boverhof et al., 2005; Knight et al., 2008; Tijet et al., 2006; Wu et al., 2008).

The mechanism by which TCDD induces Cyp enzymes is well known, but how TCDD induces other members of the AhR battery of drug-processing genes remains unknown. Prochaska and Talalay (1988) proposed that TCDD likely induces Nqo1 and other drug-processing genes by a mechanism in addition to AhR; however, over 20 years later, the complete mechanism of TCDD induction of drug-processing genes in the liver remains unknown.

Nqo1 is considered part of the mouse AhR gene battery, and studies in mice have shown that TCDD induces Nqo1 mRNA expression in liver (Petrick and Klaassen, 2007; Tijet et al., 2006). However, there is another transcription factor, nuclear factor erythroid 2–related factor 2 (Nrf2), which also regulates Nqo1 expression in mice. In fact, Nqo1 is considered a prototypical Nrf2 target gene in mice. Nrf2 binds to its cognate binding sites, known as antioxidant response elements (AREs) and transactivates Nqo1 expression (Kohle and Bock, 2007; Nioi and Hayes, 2004). Upon oxidative insult, Nrf2 avoids kelch-like ECH-associated protein 1 (Keap1)-mediated proteosomal degradation and translocates to the nucleus (Li and Kong, 2009). In the nucleus, Nrf2 heterodimerizes with small Maf proteins (Itoh et al., 1997), binds to AREs, and upregulates a number of Nrf2-dependent drug-processing genes, including Nqo1, as well as Ugts and Gsts.

Zhang et al. (1992) reported that sulforaphane is a potent inducer of Nqo1 in mouse Hepa1c1c7 cells. Thimmulappa et al. (2002) later showed that sulforaphane induces Nrf2-dependent genes, including Nqo1 and Gsta1, in livers of mice. Oltipraz, a prototypical chemical activator of Nrf2, induces Nqo1 (Petrick and Klaassen, 2007), as well as many Ugts (Buckley and Klaassen, 2009) and Gsts (Knight et al., 2008), in livers of mice. Compared to wild-type (WT) mice, Nrf2-null mice have lower basal mRNA expression of Nqo1, Ugt1a6, Ugt1a9, Ugt2b34, Ugt2b35, Ugt2b36, UDP-glucose dehydrogenase (Ugdh), Gsta1, Gstm1, Gstm2, Gstm3, and Gstp2 (Reisman et al., 2009), suggesting that Nrf2 may be important in regulating these genes. Moreover, Keap1 knockdown mice, which have constitutive Nrf2 activation, have increased mRNA expression of Nqo1 as well as many Ugt and Gst isoforms (Reisman et al., 2009). Collectively, previous work by this laboratory and others suggests that Nrf2 is required for both constitutive and inducible mRNA expression of Nqo1, Ugts, and Gsts in livers of mice. Interestingly, Auyeung et al. (2003) reported that olitpraz induction of Ugt1a6 mRNA expression in rat hepatocytes is partially dependent on AhR and XREs, whereas Ramos-Gomez et al. (2001) demonstrated in mice that oltipraz induction of Ugt1a6 is largely Nrf2 dependent. Thus, there appears to be an overlap between the classical AhR battery of drug-processing genes and Nrf2 target genes, which led us to hypothesize that TCDD induction of Nqo1, Ugts, and Gsts is dependent on Nrf2. To test this hypothesis, TCDD induction of drug-processing genes was quantified in livers of WT and Nrf2-null mice.

MATERIALS AND METHODS

Materials.

TCDD was a gift from Dr Karl Rozman (University of Kansas Medical Center, Kansas City, KS). All other materials, unless otherwise specified, were purchased from Sigma-Aldrich (St Louis, MO).

Animals and Husbandry.

Nrf2-null mice have been previously described (Chan et al., 1996), and breeding pairs were obtained from Dr Jefferson Chan (University of California Irvine, Irvine, CA). Nrf2-null mice were backcrossed with C57BL/6 mice (Charles River Laboratories, Inc., Wilmington, MA) and were determined to be > 99% congenic for the C57BL/6 background by Jackson Laboratories (Bar Harbor, ME). Male AhR-null mice (> 99% congenic for C57BL/6J background) from Jackson laboratories (stock # 002831) have been previously described (Schmidt et al., 1996). Compared to C57BL/6 mice, the DBA strain of mice are more resistant to TCDD induction of Cyp1a1 because they are homozygous for the AhR(d)allele, which has a lower affinity for AhR ligands such as TCDD (Poland et al., 1976). DBA mice are resistant to doses of TCDD that induce Cyp1a1 activity in C57BL/6 mice (1.1 μg/kg, po, or 3 μg/kg, ip) (Shen et al., 1991; Weber et al., 1995). The AhR(d) allele has been backcrossed into the C57BL/6 background (Poland et al., 1989), referred to in the manuscript as C57BL/6J mice homozygous for low-affinity AhR<d> allele (AhRd) mice. AhRd mice are a good model of attenuated AhR ligand response in vivo and were purchased from Jackson laboratories (stock # 002921). C57BL/6J WT mice were also purchased from Jackson laboratories. All mice (8–10 weeks old) were fed Teklad Rodent Diet # 8604 (Harlan Laboratories, Madison, WI) ad libitum, given free access to water, and were housed in an AAALAC-accredited animal care facility in temperature-, light-, and humidity-controlled rooms. This study was approved by the University of Kansas Medical Center Institutional Animal Care and Use Committee.

TCDD Administration.

WT C57BL/6 and Nrf2-null mice (n = 4 per group) were administered a single ip injection of either corn oil (5 ml/kg) or TCDD (50 μg/kg). In addition, WT C57BL/6J, AhRd, and AhR-null mice (n = 5 per group) were administered vehicle or TCDD. Livers were collected 24 h after vehicle or TCDD, frozen in liquid nitrogen, and stored at −80°C. Trunk blood was collected and centrifuged to obtain serum.

RNA Isolation.

Total RNA was isolated using RNA-Bee reagent (Tel-Test, Inc., Friendswood, TX) according to the manufacturer's protocol. The concentration of total RNA was quantified spectrophotometrically at 260 nm, and purity was confirmed by 260/280 and 260/230 nm ratios. Total RNA was diluted to 1 μg of total RNA per microliter with diethyl pyrocarbonate–treated deionized water.

Branched DNA Signal Amplification Analysis.

Cyp1a1, Nqo1, Aldh3a1, and Nrf2 mRNAs were quantified using the branched DNA assay (Panomics, Inc., Fremont, CA). Gene sequences were accessed from GenBank, and target sequences were used to design oligonucleotide probe sets (capture, label, and blocker probes) with ProbeDesigner software v 1.0 (Bayer Corp., Emeryville, CA). Sequences for Cyp1a1, Nqo1 (Cheng et al., 2005), Aldh3a1 (Alnouti and Klaassen, 2008), and Nrf2 (Petrick and Klaassen, 2007) probe sets were described previously. In a 96-well plate, 10 μg of total RNA was added to 50 μl of the respective probe set and hybridized overnight at 53°C. Subsequent hybridization steps with label and substrate were carried out according to the manufacturer's protocol, and luminescence was quantified with a Synergy 2 Multi-Detection Microplate Reader interfaced with Gen5 Reader Control and Data Analysis Software (Biotek, Winoosky, VT). Cyp1a1, Nqo1, and Nrf2 mRNAs were presented as relative light units per 10 μg of total liver RNA.

The mRNA expression of Ugts and Gsts was quantified by Quantigene Plex 1.0 technology, Plex 2055 (Panomics, Inc.). In a 96-well plate, 5 μg of total RNA was added to wells containing X-MAP beads coated with capture probes, label extenders, and blockers and hybridized overnight at 54°C. Approximately 16–20 h later, beads and bound target RNA were transferred to filter plates, washed twice, and incubated with amplifier at 46°C for 1 h. Wells were washed twice and then incubated with label (biotin) at 46°C for 1 h. Wells were washed twice and incubated with streptavidin-conjugated R-phycoerythrin that binds biotinylated probes and incubated at room temperature for 30 min. Fluorescence was analyzed using a Bio-Plex 200 system array reader with Luminex 100 X-MAP technology, and data were acquired using Bio-Plex data manager software (Bio-Rad, Hercules, CA). Ugt and Gst mRNAs were normalized to WT controls.

Western Blots.

Nuclear and cytosolic extracts were prepared with the NE-PER nuclear extraction kit according to the manufacturer (Pierce Biotechnology, Rockford, IL). Nrf2 protein was determined in the nuclear fraction, whereas Nqo1 protein was determined in cytosolic extracts. Nrf2 (sc-13032) antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA), whereas Nqo1 (Ab2346) and β-actin (Ab8227) antibodies were purchased from Abcam (Cambridge, MA). PolyADP-ribose polymerase (PARP) (cat. # 556362) antibody was purchased from BD Pharmingen (San Jose, CA).

Protein concentrations were determined in the nuclear and cytosolic fractions by the BCA assay (Pierce Biotechnology). Nuclear and cytosolic proteins (40 micrograms per lane) were electrophoretically resolved using polyacrylamide gels (4% stacking, 10% resolving for Nrf2, or 12% resolving for Nqo1). Gels were transblotted overnight at 4°C onto nitrocellulose (Nrf2) or PVDF (Nqo1). Membranes were blocked for 1 h with 5% nonfat milk in PBS containing 0.05% Tween-20 (PBS-T). Blots were then incubated with primary antibody (1:1000 in 2% nonfat milk/PBS-T) for 3 h. Blots were washed in PBS-T and incubated with species-appropriate secondary antibody conjugated with horseradish peroxidase (1:2000 in 2% nonfat milk/PBS-T) for 1 h at room temperature. Blots were washed again with PBS-T. Protein-antibody complexes were detected using an ECL chemiluminescent kit (Pierce) and exposed to HyBlot CL autoradiography film (Denville Scientific, Inc., Metuchen, NJ). Nrf2, PARP, Nqo1, and β-actin proteins were observed at approximately 110, 116, 25, and 45 kDa, respectively. Intensity of protein bands was quantified by Discovery Series Quantity One 1-D Analysis Software (Bio-Rad Laboratories, Hercules, CA). Equal protein loading was confirmed by loading control (β-actin or PARP), and individual blot densities were normalized to WT control.

Nrf2 Activation (Nuclear Nrf2-ARE Binding).

The amount of Nrf2 available in the nucleus to bind to AREs was determined using the ELISA-based TransAM Nrf2 Kit (Active Motif, Carlsbad, CA). Briefly, nuclear extracts were added to wells containing the immobilized consensus ARE oligonucleotide. A primary antibody against Nrf2 was added to each well. Then a secondary antibody conjugated to horseradish peroxidase that binds to the primary (Nrf2) antibody was added to each well. The signal was detected at 450 nm, and Nrf2-ARE binding was reported as optical density units at 450 nm.

Statistical Analyses.

Differences in mRNA expression were evaluated by ANOVA followed by Duncan's post hoc multiple comparisons. Differences in protein were analyzed by two-tailed t-test (TCDD treated vs. vehicle control) or ANOVA followed by Duncan's post hoc multiple comparisons. SAS v 9.1 (Cary, NC) was used for all statistical analyses, and all differences were considered statistically significant at p ≤0.05.

RESULTS

Effects of TCDD on Nrf2 Activation

To determine whether TCDD activates Nrf2 in vivo, the translocation of Nrf2 from the cytosol to the nucleus and the binding of Nrf2 to AREs were quantified in livers of TCDD-treated WT mice. TCDD increased the amount of Nrf2 protein in the nucleus by 185% and the amount of Nrf2 bound to AREs by 106% (Fig. 1). Therefore, TCDD activated Nrf2 in livers of mice in vivo.

FIG. 1.
(Left) Nuclear Nrf2 protein in livers of WT mice administered vehicle or TCDD. Individual blot densities are normalized to vehicle control ± SEM, n = 3 per group. PARP was used as the loading control. The brackets above the blot identify which ...

Cyp1a1, Nrf2, and Nqo1 mRNA Expression in Livers of TCDD-Treated WT, AhRd, and AhR-null Mice

Cyp1a1 is the prototypical AhR target gene, whereas Nqo1 is considered a prototypical Nrf2 target gene. However, both genes are considered members of the “classical” AhR battery of genes. Therefore, we examined the role of AhR in TCDD induction of Nqo1 by administering TCDD to WT, AhRd, and AhR-null mice. Cyp1a1 was included as a positive control because the mechanism of how TCDD induces its expression is well known. TCDD induction of Cyp1a1 mRNA was marked in livers of both WT and AhRd mice; however, as expected, Cyp1a1 mRNA expression was 28% lower in TCDD-treated AhRd mice than in TCDD-treated WT mice. Cyp1a1 mRNA was almost nondetectable in the TCDD-treated AhR-null mice. Similar to Cyp1a1, TCDD induction of Nrf2 and Nqo1 mRNA was dependent on AhR, that is, TCDD increased mRNA expression of these genes the most in WT mice, intermediate in AhRd, and not at all in AhR-null mice (WT > AhRd > AhR-null) (Fig. 2). Thus, TCDD induction of Nqo1 mRNA was dependent on AhR.

FIG. 2.
Cyp1a1, Nrf2, and Nqo1 mRNA expression in livers of WT, AhRd (low AhR affinity for ligand), and AhR-null mice administered corn oil (TCDD −) or TCDD (TCDD +). mRNA expression is presented as relative light units (RLU) per total liver RNA (RLU ...

Nqo1 Protein in Livers of TCDD-Treated WT, AhRd, and AhR-null Mice

To further examine the role of AhR in mediating TCDD induction of Nqo1 in liver, Nqo1 protein in liver cytosols was quantified by Western blot in the three mouse genotypes. TCDD increased Nqo1 protein in WT livers and to a lesser extent in livers of AhRd mice (Fig. 3). As expected, livers of TCDD-treated AhR-null mice had no induction of Nqo1 protein. Therefore, TCDD induction of Nqo1 protein was dependent on AhR.

FIG. 3.
Cytosolic Nqo1 protein in livers of WT, AhRd (low AhR affinity for ligand), and AhR-null mice administered corn oil (TCDD −) or TCDD (TCDD +). Individual blot densities are normalized to WT control ± SEM, n = 2–3 per group. β-Actin ...

Cyp1a1, Nrf2, and Nqo1 mRNA Expression in Livers of TCDD-Treated WT and Nrf2-null Mice

As noted previously, Cyp1a1 and Nqo1 are both included in the AhR gene battery; however, the role of Nrf2 in TCDD-induced Nqo1 mRNA expression in vivo remains unknown. TCDD markedly increased Cyp1a1 mRNA in livers of both WT and Nrf2-null mice (Fig. 4). Thus, TCDD induction of Cyp1a1 mRNA was not dependent on Nrf2. In livers of WT mice, TCDD increased Nrf2 and Nqo1 mRNA expression by 248 and 619%, respectively (Fig. 4). In contrast, TCDD did not increase Nqo1 mRNA expression in livers of Nrf2-null mice. Therefore, TCDD induction of Nqo1 mRNA expression in vivo was dependent on Nrf2.

FIG. 4.
Cyp1a1, Nrf2, and Nqo1 mRNA expression in livers of WT and Nrf2-null mice administered corn oil (TCDD −) or TCDD (TCDD +). mRNA expression is presented as relative light units (RLU) per total liver RNA (RLU ± SEM, n = 4 per group). *, ...

Nqo1 Protein in Livers of TCDD-Treated WT and Nrf2-null Mice

To further examine the role of AhR in mediating TCDD induction of Nqo1 in liver, Nqo1 protein in liver cytosols was quantified by Western blot in the two mouse genotypes. TCDD administered to WT mice not only increased Nqo1 mRNA by 619% (Fig. 4), but TCDD also increased Nqo1 protein in the liver by 200% (Fig. 5). As expected from the mRNA results, TCDD administered to Nrf2-null mice did not increase Nqo1 protein in their livers.

FIG. 5.
Cytosolic Nqo1 protein in livers of WT and Nrf2-null mice administered corn oil (TCDD −) or TCDD (TCDD +). ND, no protein detected. Individual blot densities are normalized to WT control ± SEM, n = 3 per group. β-Actin was used ...

Nqo1 is considered a prototypical Nrf2 target gene, and the present study demonstrates that Nrf2 is required for TCDD induction of Nqo1 in livers of mice. However, Nrf2 activation also increases the mRNA expression of various Ugts and Gsts in mice. Therefore, it was of interest to determine whether TCDD induction of Ugts and Gsts is dependent on Nrf2.

Ugt mRNA Expression in Livers of TCDD-Treated WT and Nrf2-null Mice

TCDD induces mRNA expression of many Ugt isoforms in livers of mice (Buckley and Klaassen, 2009), and Ugts are expressed at a lower level in livers of Nrf2-null mice (Reisman et al., 2009). Therefore, it was of interest to determine whether TCDD induction of Ugts in mice is also regulated by Nrf2. As observed in previous studies by this laboratory, constitutive mRNA expression of Ugts in livers of Nrf2-null mice was lower than in WT mice (Fig. 6).

FIG. 6.
Ugt mRNA expression in livers of WT and Nrf2-null mice administered vehicle (VC) or TCDD. mRNA expression is normalized to WT control (± SEM, n = 4 per group). For all Ugt mRNAs (except for Ugt1a1), WT TCDD was significantly higher than WT VC ...

TCDD did not induce Ugt2a3, 2b1, 3a1, 3a2, or UDP-glucose pyrophosphorylase in WT mice (data not shown). In WT but not Nrf2-null mice, TCDD induced Ugt1a6, 2b34, 2b35, 2b36, and the UDP-glucuronic acid–synthesizing gene Ugdh (Fig. 6). TCDD induction of Ugt1a1 was similar in WT and Nrf2-null mice, whereas TCDD induction of Ugt1a5 and 1a9 was blunted in Nrf2-null mice. As demonstrated in Figure 6, complete TCDD induction of Ugt1a5, 1a6, 1a9, 2b34, 2b35, 2b36, and Ugdh was dependent on Nrf2, whereas TCDD induction of Ugt1a1 was not dependent on Nrf2.

Gst mRNA Expression in Livers of TCDD-Treated WT and Nrf2-null Mice

TCDD has been shown to increase the mRNA expression of various Gst isoforms in livers of mice (Boverhof et al., 2005; Knight et al., 2008; Tijet et al., 2006; Wu et al., 2008), and many Gst isoforms are more lowly expressed in livers of Nrf2-null mice than WT mice (Reisman et al., 2009). Therefore, it was of interest to determine whether TCDD induction of Gsts in mice is also dependent on Nrf2. As observed in previous studies by this laboratory, constitutive mRNA expression of Gsts in livers of Nrf2-null mice was lower than in WT mice (Fig. 7). TCDD did not induce Gsta4 or p1 in WT mice (data not shown). In WT but not Nrf2-null mice, TCDD increased mRNA expression of Gsta1, m1, m2, m3, m6, p2, t2, and microsomal Gst1 (MGst1). As demonstrated in Figure 7, TCDD induction of all these Gsts was dependent on Nrf2.

FIG. 7.
Gst mRNA expression in livers of WT and Nrf2-null mice administered vehicle (VC) or TCDD. mRNA expression is normalized to WT control (± SEM, n = 4 per group). For all Gst mRNAs, WT TCDD was significantly higher than WT VC and Nrf2-null TCDD ( ...

DISCUSSION

The mouse AhR gene battery consists of genes coordinately induced by TCDD, including Cyp1a1, Cyp1a2, Aldh3a1, Nqo1, Ugt1a6, and Gsta1 (Nebert et al., 1993, 2000). Previous work from this laboratory and others has demonstrated in mice that TCDD induces the mRNA expression of many Ugts (Buckley and Klaassen, 2009) and Gsts (Boverhof et al., 2005; Knight et al., 2008; Tijet et al., 2006; Wu et al., 2008). The present study demonstrates that TCDD activates Nrf2 in livers of WT mice, as demonstrated by increased translocation of Nrf2 into the nucleus and increased amount of Nrf2 in the nucleus that binds to AREs (Fig. 1). Moreover, TCDD induction of Cyp1a1, Nrf2, and Nqo1 mRNAs is AhR dependent, with highest induction in WT mice, intermediate in AhRd mice (low AhR ligand affinity), and no induction in AhR-null mice (Fig. 2). These results agree with previous work by Tijet et al. (2006), who also reported that TCDD induced Nrf2 mRNA in livers of WT mice. They noted that TCDD (1000 μg/kg, po) can induce Nrf2 mRNA expression independent of AhR; however, in their study, TCDD increased Nrf2 mRNA expression by 300% in livers of WT mice, compared to only a 32% increase in livers of AhR-null mice.

In mice, the prototypical AhR target gene is Cyp1a1, whereas a prototypical Nrf2 target gene is Nqo1. In the present study, TCDD induces Cyp1a1 mRNA in livers of both WT and Nrf2-null mice (Fig. 4). This finding is supported by a previous study in which the AhR ligand 3-methylcholanthrene induced Cyp1a1 mRNA in livers of both WT and Nrf2-null mice (Noda et al., 2003). Similar to Cyp1a1, TCDD induction of Aldh3a1 is not dependent on Nrf2 (data not shown). Whereas TCDD induction of Cyp1a1 and Aldh3a1 does not require Nrf2, it was of interest to determine whether Nrf2 is required for TCDD induction of the classical AhR battery of genes (Nqo1, Ugt1a6, and Gsta1), as well as many other Ugt and Gst isoforms in mice that have more recently been shown to be induced by TCDD (Boverhof et al., 2005; Buckley and Klaassen, 2009; Knight et al., 2008; Tijet et al., 2006; Wu et al., 2008). Compared to WT mice, Nrf2-null mice have lower basal mRNA expression of Nqo1, Ugt1a6, Ugt1a9, Ugt2b34, Ugt2b35, Ugt2b36, Ugdh, Gsta1, Gstm1, Gstm2, Gstm3, and Gstp2 (Reisman et al., 2009), suggesting that Nrf2 is important in regulating these drug-processing genes. The present study reports similar findings as Reisman et al. (2009), where constitutive mRNA expression of these same Ugts and Gsts is lower in livers of vehicle-treated Nrf2-null mice compared to WT controls (Figs. 6 and and77).

There has been extensive research on the mechanism of TCDD induction of Cyps; yet, there has been less research on how, other than via AhR, TCDD induces other drug-processing genes. In vitro studies have demonstrated that TCDD induces NQO1 in HepG2 cells (Jaiswal et al., 1988; Venugopal and Jaiswal, 1996), transfected Hepa1 cells (Radjendirane and Jaiswal, 1999), Hepa1c1c7 cells, and mouse embryonic fibroblast cells (Ma et al., 2004; Miao et al., 2005; Zhang et al., 1992). However, in vivo experiments using Nrf2-null mice is the ideal model to determine whether TCDD induction of the AhR battery of genes requires Nrf2.

It has been speculated that Nrf2 may be involved in the TCDD induction of drug-processing genes; however, the data are not definitive, and to our knowledge, no previous studies have reported drug-processing gene expression in livers of TCDD-treated Nrf2-null mice. Therefore, we conducted the definitive experiment by administering TCDD to Nrf2-null and WT mice and quantified drug-processing gene mRNA expression in their livers. TCDD activation of Nrf2 in vivo, by whatever mechanisms involved, supports the present findings that Nrf2 is required for TCDD induction of classical AhR battery genes Nqo1, Ugt1a6, and Gsta1, as well as most Ugt and Gst isoforms in livers of mice (Figs. 4, 6, and 7). Therefore, Nrf2, in addition to AhR, is necessary for TCDD induction of Nqo1 and most Ugts and Gsts in mice. This is the first example of two transcription factors being necessary in succession, yet not sufficient by themselves, for induction of drug-processing genes in vivo.

Theoretically, there are at least two possible mechanisms by which TCDD may induce drug-processing genes via the AhR-Nrf2 pathway presented in the manuscript: (1) TCDD induction of Cyps may increase oxidative stress, which would facilitate the translocation of Nrf2 to the nucleus, and (2) AhR may bind to an XRE in the Nrf2 gene locus and increase the transcription of Nrf2. In livers of mice, it remains unknown whether one or both of the aforementioned mechanisms are involved in the TCDD induction of drug-processing genes (Kohle and Bock, 2007).

One mechanism by which TCDD may induce drug-processing genes via the AhR-Nrf2 pathway is that TCDD induction of Cyps may increase oxidative stress, which would facilitate the translocation of Nrf2 to the nucleus. However, two previous studies have presented data that would be in opposition of this mechanism. Dalton et al. (2000) demonstrated that TCDD (5 μg/kg, ip) increases Nqo1 activity similarly (4-fold) in livers of WT, Cyp1a1-heterozygous, and Cyp1a1-null mice. A recent study by Dragin et al. (2008) examined whether TCDD can induce drug-processing genes in the absence of all three Cyp1 genes. They reported that TCDD (15 μg/kg, ip) induces Nqo1, Ugt1a6, and Gsta1 mRNA in livers of Cyp1a1/1a2/1b1 triple-knockout mice, which have functional AhR. However, it is difficult to compare the extent of TCDD induction between WT and Cyp triple-knockout mice due to altered basal expression of Nqo1, Ugt1a6, and Gsta1 in the Cyp triple-knockout mice. Whereas TCDD induction of Nqo1 may be partially or completely independent of Cyps in livers of C57BL/6 mice, it appears that TCDD induction of human NQO1 may occur via a CYP1A1-mediated oxidative stress pathway because CYP1A1 function corresponds with NQO1 induction (Marchand et al., 2004; Radjendirane and Jaiswal, 1999). Therefore, a potential species difference between rodents and humans may further complicate the understanding of whether Cyp-induced oxidative stress is involved in the TCDD induction of drug-processing genes.

There is debate as to whether TCDD induction of Cyp1 produces oxidative stress in vivo. In mice, TCDD (5 μg/kg, ip) increases Cyp1a1 and 1a2 activities within 24 h; however, the amount of oxidized glutathione is not increased in their livers until much later (Shertzer et al., 1998). Dostalek et al. (2007) reported that Aroclor 1254 and phenobarbital each increased F2-isoprostanes in rat liver and plasma. They proposed that oxidative stress in vivo is probably related to the CYP2B subfamily because Aroclor 1254 and phenobarbital each induced CYP2B, whereas only Arcoclor 1254 induced CYP1A. Other investigators have reported that in mice, TCDD production of mitochondrial reactive oxygen species is dependent on AhR, but not Cyp1a1 or 1a2 (Senft et al., 2002; Shertzer et al., 2006). Taken together, these reports suggest that perhaps AhR is more important than Cyp1 in producing oxidative stress in vivo.

Another mechanism by which TCDD may induce drug-processing genes via the AhR-Nrf2 pathway is that AhR may bind to an XRE in the Nrf2 gene locus and increase the transcription and translation of Nrf2. A previous study by Miao et al. (2005) reported that TCDD (10nM) induces Nrf2 mRNA in Hepa1c1c7 cells and Cyp1a1-deficient cells, but not in AhR-deficient cells. TCDD induction of Nrf2 was attenuated in Nrf2 reporter constructs lacking the XRE-like “GCGTG” element, which is approximately 700 bases upstream in the 5′-flanking region of mouse Nrf2. This XRE-like element is very similar to the XRE core consensus sequence “T(A/T)GCGTG(A/C).” Furthermore, Miao et al. (2005) demonstrated, using chromatin immunoprecipitation in mouse liver cells, that AhR directly binds to the XRE-like sequence in the 5′-flanking region of mouse Nrf2. Collectively, this information suggests that Nrf2 may be a downstream target of AhR, and thus, TCDD induction of Nqo1 and most Ugts and Gsts may be routed through an AhR-Nrf2 sequence (Fig. 8). The present findings could be expanded to study compounds with broader applicability, such as plant polyphenols, that activate XRE and ARE gene expression. Perhaps, such compounds can induce Nqo1, Ugts, and Gsts via an AhR-Nrf2 sequence as well.

FIG. 8.
Proposed pathways of TCDD induction of drug-processing genes in livers of mice: (1) not dependent on Nrf2, (2) partially dependent on Nrf2, and (3) dependent on Nrf2.

Taken together, the results from the present study support our hypothesis that Nrf2, in addition to AhR, is required for TCDD induction of classical AhR battery genes Nqo1, Ugt1a6, and Gsta1, as well as many other Ugt and Gst isoforms in mice that have more recently been shown to be induced by TCDD. TCDD induction of most drug-processing genes is likely dependent on an AhR-Nrf2 sequence, and therefore, the present study introduces the “TCDD-inducible AhR-Nrf2 gene battery.”

FUNDING

National Institutes of Health Grants (ES007079, ES009649, ES009716, ES013714, RR021940).

Acknowledgments

The authors thank the postdoctoral fellows and graduate students of Dr Klaassen's laboratory for critical review of the manuscript. We would also like to thank Dr Jefferson Chan (University of California Irvine, Irvine, CA) for the Nrf2-null mice breeding pairs and Dr Karl Rozman (University of Kansas Medical Center, Kansas City, KS) for the TCDD.

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