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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The transcription factor NF-E2-related factor 2 (Nrf2) plays an essential role in the mammalian response to chemical and oxidative stress through induction of hepatic phase II detoxification enzymes and regulation of glutathione (GSH). Enhanced liver damage in Nrf2-deficient mice treated with acetaminophen suggests a critical role for Nrf2; however, direct evidence for Nrf2 activation following acetaminophen exposure was previously lacking. We show that acetaminophen can initiate nuclear translocation of Nrf2 in vivo, with maximum levels reached after 1 hour, in a dose dependent manner, at doses below those causing overt liver damage. Furthermore, Nrf2 was shown to be functionally active, as assessed by the induction of epoxide hydrolase, heme oxygenase-1, and glutamate cysteine ligase gene expression. Increased nuclear Nrf2 was found to be associated with depletion of hepatic GSH. Activation of Nrf2 is considered to involve dissociation from a cytoplasmic inhibitor, Kelch-like ECH-associated protein 1 (Keap1), through a redox-sensitive mechanism involving either GSH depletion or direct chemical interaction through Michael addition. To investigate acetaminophen-induced Nrf2 activation we compared the actions of 2 other GSH depleters, diethyl maleate (DEM) and buthionine sulphoximine (BSO), only 1 of which (DEM) can function as a Michael acceptor. For each compound, greater than 60% depletion of GSH was achieved; however, in the case of BSO, this depletion did not cause nuclear translocation of Nrf2. In conclusion, GSH depletion alone is insufficient for Nrf2 activation: a more direct interaction is required, possibly involving chemical modification of Nrf2 or Keap1, which is facilitated by the prior loss of GSH. (HEPATOLOGY 2004;39:1267–1276.)

The cellular defense response to chemical or oxidative stress is characterized by a coordinated induction of phase II drug-metabolizing enzymes and glutathione (GSH) synthesis, which protect the cell through the elimination of electrophiles and reactive oxygen species (ROS).1, 2 Central to this transcriptional response is a common DNA sequence found within the promoter regions of these phase II genes, which is referred to as the antioxidant (or electrophile) responsive element (ARE/EpRE).3, 4

The ARE, first identified in the upstream regulatory region of the rat GSTA2 gene,5 was found to respond to oxidative stress.6 The resemblance of the consensus ARE to the DNA cis element recognized by nuclear factor-erythroid 2 (NF-E2),7 aligned with the discovery of a subset of basic leucine zipper (bZip) transcription factors known as the cap ‘n’ collar proteins that presently comprise the NF-E2-related factors 1, 2, and 3 (Nrf1, Nrf2, and Nrf3), and Bach1 and Bach2, revealed a family of ARE-interacting factors. Studies using forced expression of Nrf2 have demonstrated this bZip protein to be a functionally critical component for ARE activation.8–11 Gene deletion studies have also shed light on the importance of Nrf2 in driving the antioxidant transcriptional response.12–15

Under normal homeostatic conditions, Nrf2 is believed to reside predominantly within the cytoplasm of the cell. Activation of Nrf2, which initially depends on its nuclear translocation, has been postulated to occur through a number of signal transduction pathways (for a review, see Kong et al.16). Intriguingly, it has been shown that Nrf2 is prevented from accessing the nucleus through tethering to an inhibitor protein, Kelch-like ECH-associated protein 1 (Keap1).17, 18 Recent work has shown that the inhibitory mechanism is probably via the ability of Keap1 to direct Nrf2 for proteosome-mediated degradation under conditions of normal cellular homeostasis.19–21 Although the triggering mechanism for the uncoupling event is not known, it has been postulated to depend on 1 or more reactive thiol groups in the Keap1 molecule.17 Since all ARE inducers react with sulfhydryl groups, it has been suggested that Keap1 could be a candidate cellular xenobiotic sensor/trigger.22

Acetaminophen (paracetamol) is a human hepatotoxin at high doses and is still associated with several hundred deaths a year in both the United States23 and the United Kingdom.24 At therapeutic doses, toxicity is an extremely rare event. Despite over 30 years of research into its mechanism of toxicity, the precise biochemical basis remains unknown.25 The role of metabolic activation in acetaminophen hepatotoxicity has been confirmed by studies with cytochrome P450 knockout mice,26, 27 and it has been proposed that an electrophilic species, N-acetyl-p-benzoquinoneimine (NAPQI), underlies the tissue damage observed. NAPQI can react directly with protein and nonprotein thiols, and GSH depletion is a hallmark of acetaminophen poisoning.28 This process occurs remarkably rapidly with protein adducts being detectable in mouse liver within 15 minutes of an intraperitoneal (IP) dose of acetaminophen.29 Furthermore, the formation of NAPQI may be associated with the generation of ROS and oxidative stress, and this has been suggested as a primary cause of the liver damage,30–32 although several other mechanisms have also been implicated. Depressed mitochondrial function aggravated by the formation of peroxynitrite from superoxide and nitric oxide, together with disrupted calcium homeostasis are also believed to be involved in acetaminophen-induced liver injury (for reviews, see Cohen et al.31 and Jaeschke et al.32). Two independent studies with gene knockout mice have shown that acetaminophen hepatotoxicity is exacerbated in the absence of Nrf2,14, 33 suggesting that the antioxidant response is activated by exposure to acetaminophen and affords protection. However, it has not yet been demonstrated whether acetaminophen treatment in vivo actually results in Nrf2 activation. Here we report that administration of acetaminophen to mice does indeed result in increased nuclear levels of Nrf2 in the liver, consistent with a pronounced nuclear translocation of Nrf2 from the cytoplasm. Furthermore, we have investigated the functionality of Nrf2 activation by demonstrating increased expression of several downstream Nrf2 target genes.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

All chemicals were purchased from Sigma (Poole, UK) unless otherwise stated.

Animals were obtained from Charles River (Margate, UK). All experiments were undertaken in accordance with criteria outlined in a license granted under the Animals (Scientific Procedures) Act of 1986 and approved by the Animal Ethics Committee of the University of Liverpool.

Dosing Regime.

Nonfasted animals were dosed as described previously.34 Briefly, male CD-1 mice (25 - 35 g) were administered a single IP dose of acetaminophen (50, 150, 300, 530, 700, and 1000 mg/kg in 0.9% saline), diethyl maleate (DEM; 2.35, 4.7, and 7.05 mmol/kg, administered in corn oil), or buthionine sulfoximine (BSO; 7.2 mmol/kg in 0.9% saline). Untreated animals or animals treated with vehicle alone were used as controls. Various concentrations of acetaminophen in saline (15 mg/mL for the 50 and 150 mg/kg doses and 30 mg/mL for the 300, 530, 700, and 1000 mg/kg doses), DEM in corn oil (0.622 mol for each of the doses) and BSO in saline (1 mol) were prepared. The volumes injected in the acetaminophen studies varied from 100 μL to 1000 μL, and an equal volume of saline was injected into each of the vehicle control groups. For the DEM studies, 100 μL to 300 μL of DEM in corn oil was injected; 100 μL of corn oil was injected into the vehicle control mice. For the BSO study, equal volumes of saline or BSO in saline (approx 200 μL) were injected into the mice. At various time points after dosing, the animals were killed by cervical dislocation and the livers were removed immediately and rinsed in 0.9% saline.

Determination of Serum Alanine Transaminase Levels.

Blood was collected 5 hours (acetaminophen treatment) or 24 hours (DEM treatment) after treatment by cardiac puncture from a satellite group of 2 animals (acetaminophen treatment) or 4 animals (DEM treatment) included with each of the doses investigated. The blood was stored at 4°C and allowed to clot overnight prior to isolation of serum. Serum alanine transaminase (ALT) levels were determined using ThermoTrace Infinity ALT Liquid stable reagent (Alpha Labs, Eastleigh, UK), according to the manufacturer's instructions. Hepatotoxicity was considered to be indicated at levels of ALT greater than 200 IU/L, which in our experience are associated with whole organ manifestations of toxicity.

Determination of Hepatic Reduced GSH Levels.

Hepatic GSH was determined using a microtiter plate assay according to the method of Vandeputte et al.35 The GSH levels were calculated by subtracting the amount of glutathione disulfide from the amount of total GSH in each sample.

Nuclear Extractions.

Mouse hepatic nuclear protein extractions were carried out on fresh, homogenized tissue, using the classical Dignam procedure, as described previously,36, 37 with the exception of the nuclear extracts prepared from the acetaminophen- and BSO-treated mice used for Western analysis. For these samples, the whole nuclei prepared by the conventional centrifugation steps as outlined in the Dignam method were solubilized at 4°C for 10 minutes in a radio-immunoprecipitation assay (RIPA) buffer (Dignam lysis buffer containing 1% sodium deoxycholate and 0.1% sodium dodecyl sulfate (SDS) (wt/vol)) and centrifuged at 14,000g for 10 minutes at 4°C. The supernatants were removed and stored at −80°C prior to analysis. Separate extracts were prepared using the conventional (Dignam) 0.35 mol NaCl nuclear protein extraction for the DEM-treated samples.

Western Analysis.

Hepatic Nrf2 nuclear translocation was determined by Western blot analysis. Briefly, nuclear extracts (25 μg of protein) were separated by denaturing electrophoresis on premade 10% or 12% Nupage Novex Bis-Tris gels (Invitrogen, Paisley, UK) using 3-(N-Morpholino) propane sulfonic acid (MOPS)-SDS running buffer and subsequently transferred to nitrocellulose membranes. After incubation in blocking buffer (10% fat-free milk in Tris-buffered saline (TBS, pH 7) containing 1% Tween 20) for 0.5 hours, membranes were incubated with a rabbit anti-Nrf2 antiserum21 at 1:4000 in TBS-Tween containing 2% milk for 1 hour. To ensure equal loading and transfer in the Western analysis of nuclear extracts, membranes were routinely stained using Ponceau Red. Following multiple washes with TBS-Tween, the secondary antibody was added (peroxidase-conjugated goat anti-rabbit immunoglobulin G; 1:4000 in TBS-Tween containing 2% milk). Visualization of the protein-antibody conjugate was performed using enhanced chemiluminescence, and band volumes were quantified by UVISoft software (UVITech, Cambridge, UK).

Northern Analysis.

Heme oxygenase-1 (HO-1), glutamate cysteine ligase catalytic subunit (GCLC), and microsomal epoxide hydrolase (mEH) messenger RNA (mRNA) levels were determined by conventional Northern blot analysis. The 18S ribosomal RNA band was used as the internal control.

Statistical Analysis.

Results are expressed as mean ± standard deviation. All values to be compared were analyzed for nonnormality using the Shapiro-Wilk test and for equivalence of variance between groups with the F test. Student's unpaired t test was used where parametric analysis was indicated; otherwise, the Mann-Whitney test was used. Results were considered significant when P values were less than .05.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Acetaminophen Administration Triggers Nrf2 Nuclear Translocation In Vivo.

Activation of Nrf2 was determined by Western analysis of nuclear extracts from liver homogenate prepared from CD-1 mice treated with a range of acetaminophen doses. Acetaminophen resulted in a pronounced increase in nuclear Nrf2, consistent with enhanced nuclear translocation. Ideally, this translocation would be monitored as the appearance of the Nrf2 protein in the nuclei concomitant with a decrease in the protein in the cytosol. In fact, we were unable to detect Nrf2 in liver cytosolic fractions from any of the control or treated animals. This is not unexpected given that Nrf2 is known to be constitutively degraded prior to activation, at which point the protein migrates to the nucleus.19–21 Furthermore, we have ruled out the possibility that the chemicals tested in this study might interact with the Nrf2 protein to enhance its immunogenicity in our assay by incubating rNrf2 with NAPQI (the reactive metabolite of acetaminophen) and DEM in vitro: the Nrf2 signal did not change after treatment (data not shown). Fig. 1A shows a representative Western blot of nuclear extracts obtained from animals treated with 700 mg/kg acetaminophen and vehicle controls. The induced Nrf2 protein is identified in the treated nuclei by the inclusion of a co-migrating mouse Nrf2 positive control. Nrf2 positive control was also spiked into several putative Nrf2-induced nuclear extracts to confirm the probable identity of the induced band (data not shown). The nonspecific bands were revealed as abundant liver proteins by the use of Ponceau Red staining. Nrf2 nuclear translocation was observed in each of the 5 treated animals compared to the vehicle-treated controls at and above a dose of 150 mg/kg acetaminophen (Fig. 1B). This occurred at each of the doses within 60 minutes after acetaminophen administration. Only low levels of nuclear Nrf2 were detected in the control animals from any of the treatment groups.

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Figure 1. Acetaminophen induces nuclear translocation of Nrf2 independently of hepatotoxicity. Acetaminophen, or saline vehicle alone, was administered IP to CD-1 mice. After 60 minutes, animals were killed and whole liver nuclei prepared, washed, and extracted. Extracts (25 μg) representing liver nuclear proteins obtained from individual animals were separated by electrophoresis, alongside a mouse Nrf2-transfected 293T cell extract positive control (P) and analyzed by Western blotting. (A) A representative gel showing Nrf2 translocation at 700 mg/kg is shown in full. (B) A close-up image of the gel in the region around the correct migration of Nrf2 is shown at each dose. Each of the analyses was performed at least twice and yielded similar results. The toxicity of each of the doses of acetaminophen is also shown, as assessed by an ALT toxicity assay, carried out on a satellite group of 2 animals per treatment, 5 hours after dosing. APAP, acetaminophen.

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Translocation of Nrf2 was not obviously associated with the toxicity of the acetaminophen dose, at least as assessed by plasma ALT levels (Fig. 1B), where we have assumed hepatotoxicity at ALT values above 200 IU/L in our 5-hour satellite treatment groups. In our experience, ALT levels above 200 IU/L are associated with severe overt toxicity. Nuclear Nrf2 was increased above control levels at both nontoxic and toxic doses, although nuclear Nrf2 levels were highest at the 2 most toxic doses.

To define the relationship between the extent of Nrf2 nuclear translocation and the dose of acetaminophen, we pooled the 5 nuclear extracts obtained at each dose and subjected these to Western analysis (Fig. 2A). The means of data from 3 separate analyses were then plotted against the treatment dose of acetaminophen. The error bars represent the SD obtained within each of the treatment groups in the analyses shown in Fig. 1B and therefore are representative of the interanimal variation in nuclear Nrf2. The amount of Nrf2 present in the nuclear fraction was found to be linearly associated (P < .0001) with the dose of acetaminophen administered, from nontoxic through to toxic doses (Fig. 2B).

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Figure 2. Acetaminophen induces Nrf2 nuclear translocation in vivo in a linear fashion. Acetaminophen, or saline vehicle alone, was administered IP to CD-1 mice. After 60 minutes, animals were killed and whole liver nuclei prepared, washed, and extracted. Extracts (25 μg) were pooled and separated by electrophoresis as described in Fig 1. The analysis was performed 3 times. (A) (top) A representative gel is shown. Membranes probed for Nrf2 by Western blotting were always reversibly stained using Ponceau Red stain prior to blocking and antibody probing. This ensured equal loading of total nuclear protein onto each gel and provided a means for monitoring the integrity of the nuclear extracts from each of the treatment groups. A typical stained membrane is shown and is identical to the membrane probed for Nrf2. The staining shows little difference between the treatment groups with respect to the pattern of abundant nuclear proteins detectable using this technique. The gels were densitometrically scanned, and the amount of nuclear Nrf2 was determined as a percentage of the vehicle-treated animals. (B) The means of these determinations were then plotted against the dose of acetaminophen administered. The errorbars represent the SD of the data (n = 5) for each of the treatment groups as calculated from the densitometry of Nrf2 in each of the nuclear extracts from each group in discrete Western analyses, as shown in Fig. 1B. SD values have been normalized to each of the data points to give an estimate of the interanimal variation within each group.

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Diethyl Maleate Administration Triggers Nrf2 Nuclear Translocation In Vivo.

The direct relationship between dose and nuclear Nrf2 obtained with acetaminophen indicated that hepatotoxicity as assessed by ALT determination was not required to cause Nrf2 activation in vivo. To confirm this observation, we employed the model compound DEM, which is equally as effective as acetaminophen in depleting GSH but lacks its ability to elicit elevated serum transaminases, indicative of liver damage, at the doses used in this study. Treatment of mice with DEM for 60 minutes resulted in hepatic Nrf2 nuclear translocation (Fig. 3A). This translocation was significantly different from corn oil control values at the 4.7 mmol/kg (236 ± 31% of corn oil controls; P < .0005) and 7.05 mmol/kg (652 ± 55% of corn oil controls; P < .0001) doses of DEM. No toxicity was seen at any of the doses of DEM used, as judged by the 24-hour ALT data (ALT data: 2.35 mmol/kg = 28 ± 21 U/L; 4.7 mmol/kg = 26 ± 17 U/L; 7.05 mmol/kg = 17 ± 5.7 U/L).

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Figure 3. Nontoxic doses of diethyl maleate (DEM) induce Nrf2 nuclear translocation in vivo. DEM, with time-matched controls, was administered IP to CD-1 mice (n = 4 for each dose plus controls). After 60 minutes, animals were killed and whole liver nuclei prepared, washed, and extracted. Extracts (25 μg) representing liver nuclear proteins obtained from individual animals were separated by electrophoresis, alongside a mouse Nrf2-transfected 293T cell extract positive control (P), and analyzed by Western blotting. The analyses were performed twice, and representative gels are shown here. The gels were densitometrically scanned, and the amount of nuclear Nrf2 was determined as a percentage of the time-matched control animals.

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Acetaminophen and Diethyl Maleate Treatment Induces Maximal Nuclear Nrf2 Levels 60 Minutes After Treatment.

In order to understand better the nature of the nuclear Nrf2 response to acetaminophen and DEM, we carried out time course studies over a 48-hour period. Both treatments elicited significantly increased levels of nuclear Nrf2 after just 30 minutes (Fig. 4), maximum levels being attained after approximately 1 hour. Thereafter, nuclear Nrf2 levels dropped in both treatments. Nrf2 had returned to control levels between 2 hours and 24 hours after treatment with DEM; this process was slower in the acetaminophen-treated mice, in which baseline levels were reached after approximately 48 hours.

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Figure 4. Acetaminophen and diethyl maleate (DEM) treatment in vivo elicits enhanced hepatic nuclear Nrf2 within 30 minutes. Acetaminophen (530mg/kg in saline) and DEM (4.7 mmol/kg in corn oil) were administered IP to CD-1 mice (n = 3 or 4 for each time point). After the time points indicated, the animals were killed and whole liver nuclei prepared, washed, and extracted. Extracts (25 μg) representing liver nuclear proteins obtained from individual animals were separated by electrophoresis and analyzed by Western blotting. Each analysis was performed twice. Membranes were routinely verified for equal sample loading and equal transfer efficiency by using Ponceau Red staining. In all cases, values are the means ± SD of a representative experiment from duplicate determinations. For all data, values are expressed as a percentage of the zero time-point control value, indicated by the broken line. Statistical significance was assigned relative to untreated control animals as defined in Materials and Methods. *P < .05; **P < .01.

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Nrf2 Nuclear Translocation In Vivo Is Functionally Relevant as Assessed by Northern Blotting of Nrf2-Dependent Genes.

Messenger RNA levels of 3 genes, HO-1, GCLC, and mEH, known to be transcriptionally dependent on Nrf2,9, 10 were analyzed in livers of mice treated with acetaminophen or DEM. At a dose of 530 mg/kg of acetaminophen, at which we observed an approximately 4-fold increase in nuclear Nrf2 (Fig. 2), mRNA levels of mEH, GCLC, and HO-1 were significantly increased 60 minutes after drug administration compared with vehicle-treated controls (Fig. 5A). This confirms that the Nrf2 translocation observed was functionally significant. We also assessed the effect of translocation of Nrf2, at the highest and lowest doses of acetaminophen that affected nuclear accumulation of the bZip protein, on the expression of these genes. Interestingly, at the 150 mg/kg dose, which was the lowest dose that promoted Nrf2 translocation, only the HO-1 mRNA was significantly increased (Fig. 5B). At the 1,000 kg dose, there were no significant differences between the treated and control groups in the mRNA expression of HO-1, GCLC, or mEH (Fig. 5C). We also assessed these genes after administration of 7.05 mmol/kg DEM to check that the effects we observed with acetaminophen were not chemical-specific. In fact, the mRNAs of 2 of these genes, HO-1 and GCLC, were numerically increased upon DEM treatment (Fig. 5D), suggesting that the Nrf2 translocation observed with DEM is also functionally significant.

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Figure 5. Translocation of Nrf2 to the nucleus is functionally relevant as assessed by Northern blotting of Nrf2-dependent genes. Acetaminophen, or saline vehicle alone, and diethyl maleate (DEM), with time-matched controls, were administered IP to CD-1 mice (n = 5). After 60 minutes, animals were killed and the liver was removed and washed. Whole liver RNA was extracted and analyzed using Northern blotting (25 μg of total RNA from each animal), employing gene-specific probes corresponding to mouse mEH, HO-1, and GCLC mRNA sequences. In all cases,values are the means ± SD. For all data, values are standardized against 18S ribosomal RNA. Statistical significance was assigned relative to untreated control animals as defined in Materials and Methods. *P < .05; **P < .01; ns, not significant.

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Translocation of Nrf2 to the Nucleus Is Associated With Levels of Hepatic GSH but Also Requires Chemical Modification of Sensor Protein(s).

Since both acetaminophen and DEM are known to deplete GSH, the primary antioxidant in the liver, the relationship between hepatic GSH and Nrf2 nuclear translocation was investigated. We measured hepatic GSH at each of the doses of acetaminophen and DEM used to investigate translocation, 60 minutes after administration. Fig. 6A shows that nuclear Nrf2 translocation may be associated with GSH for both acetaminophen and DEM treatment; however, the nature of the relationship appears to be nonlinear.

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Figure 6. Nrf2 nuclear translocation is associated with levels of hepatic glutathione (GSH). Acetaminophen, or saline vehicle alone, and diethyl maleate (DEM), with time-matched controls, were administered IP to CD-1 mice. After 60 minutes, animals were killed and the liver was removed and washed. (A) Hepatic GSH levels were determined and plotted against Nrf2 translocation. (B) GSH depletion and Nrf2 translocation were also expressed relative to the dose of acetaminophen. Data are expressed as the mean ± SD, where n = 5.

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The relationship between Nrf2 nuclear translocation and GSH depletion was also expressed relative to the dose of acetaminophen administered. As shown in Fig. 6B, a small increase in Nrf2 translocation is seen when GSH is depleted to 30% of its initial level. This increase is much more dramatic when GSH falls below 30%.

Finally, the association between Nrf2 activation and GSH levels was further investigated using another GSH-depleting agent, BSO. BSO inhibits GCLC, the rate-limiting enzyme in GSH synthesis, leading to a fall in hepatic GSH, but it does not possess the α,β-unsaturated ketone motif characteristic of a functional Michael acceptor, and is thus unable to modify proteins chemically, as is the case for acetaminophen and DEM. Treatment of mice (n = 5) with BSO for 1 and 2 hours resulted in 60% and 56% depletion in hepatic GSH, respectively. When we assayed levels of hepatic nuclear translocation of Nrf2 in these animals, no increase in either of the sets of BSO-treated animals was observed compared to the time-matched control group (data not shown). It therefore appears that, although Nrf2 activation is probably associated with GSH depletion, this alone is insufficient to trigger the event.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The central and critical role played by Nrf2 in coordinating the mammalian cellular defense response to a variety of noxious and potentially harmful stimuli has become increasing well established over the last 10 years. It is now widely accepted that the redox sensitive regulation of this bZip transcription factor represents a convergence point for multiple stress-activated signaling pathways and results in the coordinated up-regulation of a battery of antioxidant proteins involved in cellular defense.2, 38 The involvement of Nrf2 in defense against chemical-induced stress is largely based on in vitro studies in cell lines, in which nuclear translocation has been clearly demonstrated following exposure to a variety of chemicals, including tertiary butyl hydroquinone,39 butylated hydroxy anisole,40 and DEM,41 though not to acetaminophen. In contrast, there is a paucity of data demonstrating chemically induced Nrf2 activation in vivo. To our knowledge, the only documented evidence for Nrf2 translocation in vivo is that describing mouse hepatic nuclear Nrf2 after treatment with 3H-1,2-dithiole-3-thione42 and a related compound, oltipraz.43 Thus, the clear demonstration in the current study that acetaminophen administration to mice elicits a pronounced elevation of nuclear Nrf2 levels represents direct evidence for such activation in either an in vivo or an in vitro model system. Moreover, these data indicate that the nuclear translocation of Nrf2 in vivo is associated with levels of GSH, and thus the redox status, but is also dependent on the presence of chemical species with inherent protein reactivity. This follows from the observation that although acetaminophen, DEM, and BSO all depleted GSH to a similar extent, only DEM (a Michael acceptor) and acetaminophen (which is converted to the Michael acceptor NAPQI) elicited a rise in nuclear Nrf2.

The functional significance of the rise in nuclear Nrf2 was investigated by Northern analysis of 3 unrelated genes that have previously been characterized as downstream targets for Nrf2. Transcription of all 3 genes, HO-1, GCLC, and mEH, was enhanced in line with Nrf2 activation; however, there appeared to be different threshold levels of nuclear Nrf2 required for transcriptional activation in each case. Thus, there was a hierarchical order of induction, with HO-1 being the only gene induced at the nontoxic 150 mg/kg dose, while all 3 genes were induced at the 530 mg/kg dose, which is a threshold dose for overt liver damage. Interestingly, none of the 3 Nrf2-regulated genes was induced at the highest (1000 mg/kg) dose despite the marked Nrf2 nuclear translocation occurring at this dose. Presumably, the level of toxicity associated with this dose results in such widespread cellular malfunctioning that the machinery involved in gene transcription and mRNA synthesis is itself impaired. In fact, cell death caused by acetaminophen may indicate that the toxic insult has overwhelmed repair mechanisms, such as the Nrf2 response, either by causing irreparable damage to it, or by causing other damage too great for the protective responses to deal with effectively. Differential induction of the 3 target genes was also observed with the single, high (7 mmol/kg) but nontoxic, dose of DEM used in this study, at which HO-1 and GCLC were both significantly up-regulated while mEH was unchanged. Thus, it appears that certain genes are more sensitive than others to Nrf2 activation and are induced at lower Nrf2 nuclear levels. Alternatively, since Nrf2 acts as a heterodimer to activate gene transcription, the differential induction of target genes observed may reflect their different preferences with respect to alternative dimerization partners. Precise characterization of the transcription factors recruited by variant forms of the ARE may be required in order to elucidate and define the hierarchical order of Nrf2 target gene transcription.

The linear response in Nrf2 translocation seen at the different doses of acetaminophen contrasts with the nonlinear response in Nrf2 when it is related to GSH. As GSH is depleted to approximately 30% of control levels, a modest increase in Nrf2 translocation is observed up to ≈200% of nuclear control levels (Figs. 6A and B). As GSH drops further, much more pronounced rises are seen in nuclear Nrf2. One could envisage a scenario in which depletion of GSH leads to increasing numbers of the reactive cysteine residues in the cytosolic Nrf2 inhibitor Keap122 becoming available to be oxidized or covalently modified (by the DEM molecule itself, or in the case of acetaminophen, by its reactive metabolite, NAPQI). In fact, the relationship we observe between Nrf2 translocation and GSH depletion is very similar to the relationship between hepatic covalent binding and GSH depletion upon administration of acetaminophen first observed by Mitchell et al.44 This may be supportive of the notion that covalent binding (possibly of Keap1) via Michael addition is the precursor to Nrf2 activation in vivo, although a large number of cellular proteins that are adducted by acetaminophen have been identified45; it is also possible that the modification of 1 or more of these may play a role in triggering Nrf2 activation or other protective responses. Recent evidence suggests that there are 4 highly reactive cysteines within Keap1.46 These are located in the intervening region between the 2 protein-binding domains of Keap1; this increases the likelihood that they may be available to act as sensors. The modification of some or all of these residues would lead to steadily increasing numbers of Nrf2 molecules translocating to the nucleus. At a certain threshold, further oxidation/modification of Keap1 may lead to substantial loss of association with Nrf2, enabling a wide-scale nuclear translocation. Whether Keap1 thiol modification occurs directly, possibly through Michael addition, or indirectly through preceding activation of upstream enzymes, is still unresolved. Recent investigations of the induction of GCLC by indomethacin and related indole compounds point to a role for NADPH (MTHFR; 5,10-methylenetetrahydrofolate reductase) oxidase in Keap1 thiol oxidation, through generation of the superoxide anion.47 Although the universality of this mechanism needs to be clarified, formation of reactive oxygen species, including superoxide, has previously been postulated to be a consequence of acetaminophen metabolism,32 and this may represent a further mechanism for Nrf2 activation. Evidence for a physiologically based mechanism for Nrf2 activation via covalent modification through Michael addition has recently emerged through studies on the prostaglandin J2 family of anti-inflammatory mediators.48 These cyclopentenone compounds are dehydration products of prostaglandin D2, and as such contain the α,β-unsaturated ketone motif characteristic of Michael acceptors. Several of their properties, such as induction of glutathione S-transferases, are consistent with an interaction with the Nrf2 system, and it has recently been shown that they are indeed capable of the covalent modification of the Keap1 protein.49, 50 It is our intention to explore the possibility of direct modification of Keap1 by NAPQI and DEM, in order to clarify the mechanism of induction observed in the present study.

In conclusion, we have detected dynamic changes in the Nrf2 system in vivo, associated with 2 discrete chemical agents. These changes are not dependent on a toxic effect of these agents on the liver but are associated with changes in the hepatic level of reduced GSH and the presence of a chemical species with the potential for covalent modification, with the likely consequent oxidative alterations in cell regulatory and sensor proteins. The induction profile seen with Nrf2-regulated genes following acetaminophen treatment, whereby induction was impaired at very high doses despite the linear increase in nuclear Nrf2, may have therapeutic consequences for the targeting of Nrf2 in chemoprotection. The strategic induction of Nrf2 to boost cellular defense mechanisms may alone provide insufficient protection against some forms of chemical stress without the concomitant maintenance of related cellular functions.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The authors thank Sylvia Newby and Phil Roberts for technical assistance and Dr. Nicola Hanrahan for assistance with the glutathione assay.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  • 1
    Prestera T, Zhang Y, Spencer SR, Wilczak CA, Talalay P. The electrophile counterattack response: protection against neoplasia and toxicity. Adv Enzyme Regul 1993; 33: 281296.
  • 2
    Hayes JD, McMahon M. Molecular basis for the contribution of the antioxidant responsive element to cancer chemoprevention. Cancer Lett 2001; 174: 103113.
  • 3
    Friling RS, Bergelson S, Daniel V. Two adjacent AP-1-like binding sites form the electrophile-responsive element of the murine glutathione S-transferase Ya subunit gene. Proc Natl Acad Sci USA 1992; 89: 668672.
  • 4
    Jaiswal AK. Antioxidant response element. Biochem Pharmacol 1994; 48: 439444.
  • 5
    Rushmore TH, Pickett CB. Transcriptional regulation of the rat glutathione S-transferase Ya subunit gene. Characterization of a xenobiotic-responsive element controlling inducible expression by phenolic antioxidants. J Biol Chem 1990; 265: 1464814653.
  • 6
    Rushmore TH, Morton MR, Pickett CB. The antioxidant responsive element. Activation by oxidative stress and identification of the DNA consensus sequence required for functional activity. J Biol Chem 1991; 266: 1163211639.
  • 7
    Andrews NC, Erdjument-Bromage H, Davidson MB, Tempst P, Orkin SH. Erythroid transcription factor NF-E2 is a haematopoietic-specific basic-leucine zipper protein. Nature 1993; 362: 722728.
  • 8
    Venugopal R, Jaiswal AK. Nrf1 and Nrf2 positively and c-Fos and Fra1 negatively regulate the human antioxidant response element-mediated expression of NAD(P)H:quinone oxidoreductase1 gene. Proc Natl Acad Sci USA 1996; 93: 1496014965.
  • 9
    Wild AC, Moinova HR, Mulcahy RT. Regulation of gamma-glutamylcysteine synthetase subunit gene expression by the transcription factor Nrf2. J Biol Chem 1999; 274: 3362733636.
  • 10
    Alam J, Stewart D, Touchard C, Boinapally S, Choi AM, Cook JL. Nrf2, a Cap'n'Collar transcription factor, regulates induction of the heme oxygenase-1 gene. J Biol Chem 1999; 274: 2607126078.
  • 11
    Jeyapaul J, Jaiswal AK. Nrf2 and c-Jun regulation of antioxidant response element (ARE)-mediated expression and induction of gamma-glutamylcysteine synthetase heavy subunit gene. Biochem Pharmacol 2000; 59: 14331439.
  • 12
    Chanas SA, Jiang Q, McMahon M, McWalter GK, McLellan LI, Elcombe CR, et al. Loss of the Nrf2 transcription factor causes a marked reduction in constitutive and inducible expression of the glutathione S-transferase Gsta1, Gsta2, Gstm1, Gstm2, Gstm3 and Gstm4 genes in the livers of male and female mice. Biochem J 2002; 365: 405416.
  • 13
    Chan K, Kan YW. Nrf2 is essential for protection against acute pulmonary injury in mice. Proc Natl Acad Sci USA 1999; 96: 1273112736.
  • 14
    Enomoto A, Itoh K, Nagayoshi E, Haruta J, Kimura T, O'Connor T, et al. High sensitivity of Nrf2 knockout mice to acetaminophen hepatotoxicity associated with decreased expression of ARE-regulated drug metabolizing enzymes and antioxidant genes. Toxicol Sci 2001; 59: 169177.
  • 15
    McMahon M, Itoh K, Yamamoto M, Chanas SA, Henderson CJ, McLellan LI, et al. The Cap'n'Collar basic leucine zipper transcription factor Nrf2 (NF-E2 p45-related factor 2) controls both constitutive and inducible expression of intestinal detoxification and glutathione biosynthetic enzymes. Cancer Res 2001; 61: 32993307.
  • 16
    Kong AN, Owuor E, Yu R, Hebbar V, Chen C, Hu R, et al. Induction of xenobiotic enzymes by the map kinase pathway and the antioxidant or electrophile response element (ARE/EpRE). Drug Metab Rev 2001; 33: 255271.
  • 17
    Itoh K, Wakabayashi N, Katoh Y, Ishii T, Igarashi K, Engel JD, et al. Keap1 represses nuclear activation of antioxidant responsive elements by Nrf2 through binding to the amino-terminal Neh2 domain. Genes Dev 1999; 13: 7686.
  • 18
    Wakabayashi N, Itoh K, Wakabayashi J, Motohashi H, Noda S, Takahashi S, et al. Keap1-null mutation leads to postnatal lethality due to constitutive Nrf2 activation. Nat Genet 2003; 35: 238245.
  • 19
    Itoh K, Wakabayashi N, Katoh Y, Ishii T, O'Connor T, Yamamoto M. Keap1 regulates both cytoplasmic-nuclear shuttling and degradation of Nrf2 in response to electrophiles. Genes Cells 2003; 8: 379391.
  • 20
    Nguyen T, Sherratt PJ, Huang HC, Yang CS, Pickett CB. Increased protein stability as a mechanism that enhances Nrf2-mediated transcriptional activation of the antioxidant response element. Degradation of Nrf2 by the 26 S proteasome. J Biol Chem 2003; 278: 45364541.
  • 21
    McMahon M, Itoh K, Yamamoto M, Hayes JD. Keap1-dependent proteasomal degradation of transcription factor Nrf2 contributes to the negative regulation of antioxidant response element-driven gene expression. J Biol Chem 2003; 278: 2159221600.
  • 22
    Dinkova-Kostova AT, Massiah MA, Bozak RE, Hicks RJ, Talalay P. Potency of Michael reaction acceptors as inducers of enzymes that protect against carcinogenesis depends on their reactivity with sulfhydryl groups. Proc Natl Acad Sci USA 2001; 98: 34043409.
  • 23
    Ostapowicz G, Fontana RJ, Schiodt FV, Larson A, Davern TJ, Han SH, et al. Results of a prospective study of acute liver failure at 17 tertiary care centers in the United States. Ann Intern Med 2002; 137: 947954.
  • 24
    Fagan E, Wannan G. Reducing paracetamol overdoses. Br Med J 1996; 313: 14171418.
  • 25
    Pierce RH, Franklin CC, Campbell JS, Tonge RP, Chen W, Fausto N, et al. Cell culture model for acetaminophen-induced hepatocyte death in vivo. Biochem Pharmacol 2002; 64: 413424.
  • 26
    Lee SS, Buters JT, Pineau T, Fernandez-Salguero P, Gonzalez FJ. Role of CYP2E1 in the hepatotoxicity of acetaminophen. J Biol Chem 1996; 271: 1206312067.
  • 27
    Zaher H, Buters JT, Ward JM, Bruno MK, Lucas AM, Stern ST, et al. Protection against acetaminophen toxicity in CYP1A2 and CYP2E1 double-null mice. Toxicol Appl Pharmacol 1998; 152: 193199.
  • 28
    Potter WZ, Thorgeirsson SS, Jollow DJ, Mitchell JR. Acetaminophen-induced hepatic necrosis. V. Correlation of hepatic necrosis, covalent binding and glutathione depletion in hamsters. Pharmacology 1974; 12: 129143.
  • 29
    Muldrew KL, James LP, Coop L, McCullough SS, Hendrickson HP, Hinson JA, et al. Determination of acetaminophen-protein adducts in mouse liver and serum and human serum after hepatotoxic doses of acetaminophen using high-performance liquid chromatography with electrochemical detection. Drug Metab Dispos 2002; 30: 446451.
  • 30
    Harman AW. The effectiveness of antioxidants in reducing paracetamol-induced damage subsequent to paracetamol activation. Res Commun Chem Pathol Pharmacol 1985; 49: 215228.
  • 31
    Cohen SD, Hoivik DJ, Khairallah EA. In: PlaaGL, HewittWR, eds. Toxicology of the Liver. Vol. 1. 2nd ed. Washington D.C.: Taylor & Francis; 1998; 159186.
  • 32
    Jaeschke H, Knight TR, Bajt ML. The role of oxidant stress and reactive nitrogen species in acetaminophen hepatotoxicity. Toxicol Lett 2003; 144: 279288.
  • 33
    Chan K, Han XD, Kan YW. An important function of Nrf2 in combating oxidative stress: detoxification of acetaminophen. Proc Natl Acad Sci USA 2001; 98: 46114616.
  • 34
    Kitteringham NR, Powell H, Clement YN, Dodd CC, Tettey JN, Pirmohamed M, et al. Hepatocellular response to chemical stress in CD-1 mice: induction of early genes and gamma-glutamylcysteine synthetase. HEPATOLOGY 2000; 32: 321333.
  • 35
    Vandeputte C, Guizon I, Genestie-Denis I, Vannier B, Lorenzon G. A microtiter plate assay for total glutathione and glutathione disulfide contents in cultured/isolated cells: performance study of a new miniaturized protocol. Cell Biol Toxicol 1994; 10: 415421.
  • 36
    Dignam JD, Lebovitz RM, Roeder RG. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res 1983; 11: 14751489.
  • 37
    Israel N, Gougerot-Pocidalo MA, Aillet F, Virelizier JL. Redox status of cells influences constitutive or induced NF-kappa B translocation and HIV long terminal repeat activity in human T and monocytic cell lines. J Immunol 1992; 149: 33863393.
  • 38
    Nguyen T, Sherratt PJ, Pickett CB. Regulatory mechanisms controlling gene expression mediated by the antioxidant response element. Annu Rev Pharmacol Toxicol 2003; 43: 233260.
  • 39
    Liu RM, Hu H, Robison TW, Forman HJ. Differential enhancement of gamma-glutamyl transpeptidase and gamma-glutamylcysteine synthetase by tert-butylhydroquinone in rat lung epithelial L2 cells. Am J Respir Cell Mol Biol 1996; 14: 186191.
  • 40
    Hayes JD, Chanas SA, Henderson CJ, McMahon M, Sun C, Moffat GJ, et al. The Nrf2 transcription factor contributes both to the basal expression of glutathione S-transferases in mouse liver and to their induction by the chemopreventive synthetic antioxidants, butylated hydroxyanisole and ethoxyquin. Biochem Soc Trans 2000; 28: 3341.
  • 41
    Sekhar KR, Long M, Long J, Xu ZQ, Summar ML, Freeman ML. Alteration of transcriptional and post-transcriptional expression of gamma-glutamylcysteine synthetase by diethyl maleate. Radiat Res 1997; 147: 592597.
  • 42
    Kwak MK, Itoh K, Yamamoto M, Sutter TR, Kensler TW. Role of transcription factor Nrf2 in the induction of hepatic phase 2 and antioxidative enzymes in vivo by the cancer chemoprotective agent, 3H-1, 2-dimethiole-3-thione. Mol Med 2001; 7: 135145.
  • 43
    Ramos-Gomez M, Kwak MK, Dolan PM, Itoh K, Yamamoto M, Talalay P, et al. Sensitivity to carcinogenesis is increased and chemoprotective efficacy of enzyme inducers is lost in nrf2 transcription factor-deficient mice. Proc Natl Acad Sci USA 2001; 98: 34103415.
  • 44
    Mitchell JR, Jollow DJ, Potter WZ, Gillette JR, Brodie BB. Acetaminophen-induced hepatic necrosis. IV. Protective role of glutathione. J Pharmacol Exp Ther 1973; 187: 211217.
  • 45
    Qiu Y, Benet LZ, Burlingame AL. Identification of the hepatic protein targets of reactive metabolites of acetaminophen in vivo in mice using two-dimensional gel electrophoresis and mass spectrometry. J Biol Chem 1998; 273: 1794017953.
  • 46
    Dinkova-Kostova AT, Holtzclaw WD, Cole RN, Itoh K, Wakabayashi N, Katoh Y, et al. Direct evidence that sulfhydryl groups of Keap1 are the sensors regulating induction of phase 2 enzymes that protect against carcinogens and oxidants. Proc Natl Acad Sci USA 2002; 99: 1190811913.
  • 47
    Sekhar KR, Crooks PA, Sonar VN, Friedman DB, Chan JY, Meredith MJ, et al. NADPH Oxidase Activity Is Essential for Keap1/Nrf2-mediated Induction of GCLC in Response to 2-Indol-3-yl-methylenequinuclidin-3-ols. Cancer Res 2003; 63: 56365645.
  • 48
    Shibata T, Kondo M, Osawa T, Shibata N, Kobayashi M, Uchida K. 15-deoxy-delta 12,14-prostaglandin J2. A prostaglandin D2 metabolite generated during inflammatory processes. J Biol Chem 2002; 277: 1045910466.
  • 49
    Shibata T, Yamada T, Ishii T, Kumazawa S, Nakamura H, Masutani H, et al. Thioredoxin as a molecular target of cyclopentenone prostaglandins. J Biol Chem 2003; 278: 2604626054.
  • 50
    Levonen AL, Landar A, Ramachandran A, Ceaser EK, Dickinson DA, Zanoni G, et al. Cellular mechanisms of redox cell signaling: role of cysteine modification in controlling antioxidant defenses in response to electrophilic lipid oxidation products. Biochem J 2004; 378: 373382.