1. Introduction The cellular defence response to chemical or oxidative stress is characterised by a co-ordinated induction of phase II detoxifying enzymes (including UDP-glucuronosyltransferase and glutathione- S-transferases) and antioxidant stress proteins (such as  - glutamylcysteine synthesase 1 and haem oxygenase 1 2 ). These enzymes are responsible for the elimination of electrophiles, free radicals and reactive oxygen species (ROS), the accumulation of these would otherwise provoke cellular damage and cause cell death. This concerted up-regulation of drug metabolising and antioxidant enzymes is pivotal in providing cellular protection after xenobiotic exposure. These metabolising and antioxidant proteins are transcriptionally regulated by a common element, known as the antioxidant response element (ARE) in their gene regulatory regions and nrf2 (a Cap’n’Collar leucine zipper transcription factor) has been shown to bind to this sequence and hence regulate gene expression 3. In recent years the Keap1/nrf2 system has been shown to be a key cellular sensor responsible for the initial response to oxidative stress. Under normal conditions Nrf2 is tethered to the cytoskeletal negative regulator Keap1 4. On exposure to xenobiotics there is spontaneous dissociation with immediate nuclear translocation of Nrf2. Once in the nucleus, Nrf2 forms heterodimers with small Maf proteins to facilitate induction of metabolising and antioxidant genes. We have attempted here for the first time to set up an animal model to investigate Nrf2 over a 24 hour time period after hepatotoxic doses of paracetamol, carbon tetrachloride (CCL 4 ), diethyl maleate (DEM) and buthionine sulphoximine (BSO). Assays measuring nuclear Nrf2 translocation to antioxidant gene induction were performed, as previous studies were done either in vitro 1-2,4-5 or in Nrf2 knockout mice 6 to determine the role of this transcription factor in the response to chemical stress. Figure 1. Schematic representation of Nrf2 nuclear translocation, ARE- binding and gene regulation after chemical stress. Conclusions Our data indicate that depletion of GSH per se may provide a sufficient signal for activation of the Nrf2/Keap1 sensor system. This may be due to spontaneous activation resulting in the release of active Nrf-2 from its cytosolic-tethering partner, Keap 1, or an activation of the Nrf2 protein itself, or both. There is an immediate translocation of Nrf2 after paracetamol, CCl 4 and DEM treatment and this trend follows in a similar manner the glutathione depletion caused by either conjugation with reactive metabolites (paracetamol and CCl 4 ) or direct chemical reaction (DEM). BSO depletes glutathione by binding to  -glutamyl-cysteine synthetase (the rate-limiting enzyme in glutathione synthesis) and inhibiting its activity. Consequently, glutathione depletion is delayed with BSO and this is mirrored in the delay in Nrf2 translocation. The trend of increasing nuclear Nrf2 is reflected in the increased binding to the ARE sequence by mobility shift assay after chemical insult, suggesting a functional role in Nrf2 re- compartmentalisation and its function in oxidative stress. We have no evidence for the enhanced transcription of Nrf2 upon exposure to chemical insult. In fact, DEM, paracetamol, and CCl 4 cause a large depletion in the steady state level of hepatic Nrf2 mRNA, as indicated by Northern assays. The possibility that a negative feedback loop exists to restrict Nrf2 activation is currently being investigated. An interesting aspect of the study was to determine whether Nrf2 movement from cytoplasm to nucleus had any functional role after chemical insult. Early induction of haem oxygenase mRNA (a known Nrf2-regulated gene) 2 hrs following paracetamol, CCl 4 and DEM treatment suggests probable involvement of Nrf2 in antioxidant stress protein regulation. With BSO antioxidant induction occurred only after 24hrs which follows the time course of Nrf2 nuclear translocation. References: 1 1 Wild AC, Moinova H, Mulcahy RT (1999) Regulation of  - glutamylcysteine synthetase subunit gene expression by the transcription factor Nrf2. J Biol Chem 274(47): 33627-36. 2 2Alam J, Stewart D, Touchard C, Boinapally S, Choi AK, Cook JL (1999) Nrf2, a Cap’n’Collar transcription factor, regulates induction of the heme oxygnease-1 gene. J Biol Chem 274(37):26071-8. 3 3Wolf CR. (2001) Chemoprevention:Increased potential to bear fruit. Proc Natl Acad Sci U.S.A. 98(6): 2941-43. 4 4Itoh K, Wakabayashi N, Katoh Y, Ishii T, Igarashi K, Engel JD, Yamamoto M. (1999) Keap 1 represses nuclear translocation of antioxidant responsive elements by Nrf2 through binding to the amino- terminal Neh2 domain. Gene and Development. 13:76-86. 5 5Enomoto A, Itoh K, Nagayoshi E, Haruta J, Kimura T, O’Connor T, Harada T, Yamamoto M (2001) High sensitivity of Nrf2 knockout mice to acetaminophen hepatotoxicity associated with decreased expression of ARE-regulated drug metabolising enzymes and antioxidant genes. Toxicol Sci 59:169-177. 6 6Lee JM, Moehlenkamp JD, Hanson JM, Johnson JA (2001) Nrf2- dependent activation of the antioxidant responsive element by tert- butylhydroquinone is independent of oxidative stress in IMR-32 human neuroblastoma cells. Biochem Biophys Res Commun 280: 286- 92. This work was supported by The Wellcome Trust. YC is a Wellcome Travelling Fellow. BKP is a Wellcome Principal Research Fellow. Results Nuclear translocation (Western blotting): Nrf2 was rapidly translocated to the nucleus after chemical insult with paracetamol, CCL 4 and DEM. This event occurred within 30 min with maximal effect (between 2.5 to 7-fold increases, p<0.05) after 1 hr, there was a return to basal levels after 24hrs (Figure 3-5). A delayed 5- fold maximal increase of nuclear Nrf2 was observed with BSO 2 hr after administration with elevated levels persisting up to 24hrs (Figure 6). Nrf2-Antioxidant responsive element binding (EMSA): Figure 7. Control experiments Nrf2:Are-binding pattern was similar to protein translocation. Nrf2 regulation (Northern blotting): After administration of paracetamol and DEM there was an immediate significant decrease in mRNA levels up, with maximal decrease at 2hr with decline to 25% basal levels, p<0.05 (Figures 10 and 11) with a marginal non-significant increase after 24 hrs. After CCl 4 there was a maximal decline in Nrf2 mRNA to about 50% basal levels after 1h (Figure 12). With BSO there were no significant changes in Nrf2 mRNA levels (Figure 13). Heme oxygenase induction after CCl 4 and BSO There was an early induction of HO-1 mRNA after CCl 4, DEM and paracetamol (data not shown). With BSO there is a delayed induction at 24hrs. Regulation of Hepatic NRF2 in Mice Exposed to Chemical Stress Yuri Clement, Chris Goldring, Neil R Kitteringham, B Kevin Park Department of Pharmacology and Therapeutics, The University of Liverpool, L69 3GE, UK. Materials and methods CD-1 mice were treated with paracetamol (3.5mmol/kg in saline), CCL 4 (1.0mmol/kg in corn oil), DEM (4.2mmol/kg) and BSO (7.2 mmol/kg in saline) and killed at 30min, 1hr, 2hr, and 24hr and livers were removed. Nuclear extracts were made from fresh tissue at each time point and used to determine Nrf2 nuclear translocation (by Western blotting) and antioxidant responsive element (ARE) binding by electrophoretic mobility shift assay. The tissue samples were also used to prepare total RNAs which were used to determine (by Northern blotting) the regulation of Nrf2. The regulation of heam oxygenase (an antioxidant stress protein) was also investigated after drug treatment by Northern blotting. Abstract Cells exposed to chemical stress develop a complex, multilevel response that involves activation of cellular defence mechanisms. The initial signal by which the cell detects such stress is not known, although redox-sensitive protein interactions between transcription factors and cytosolic inhibitors (e.g. Nrf- 2/keap1) have recently been discovered to be a possible redox sensor. Previously, we have examined the AP-1 response using specific chemical agents (paracetamol – 3.5mmol/kg, CCl 4 – 1.0mmol/kg, DEM – 4.2 mmol/kg and BSO – 7.2 mmol/kg) which deplete the critical cell defence tripeptide glutathione (GSH) by different mechanisms. The dose chosen for each compound depleted GSH to a similar extent. This approach is informative of the relationship between GSH and early cellular responses as well as addressing the question, do particular forms of chemical stress have a characteristic cellular defence response? We have now investigated the nuclear translocation and activation of the nrf-2 factor in the liver of mice exposed to these specific chemical agents. The protein is rapidly translocated to the nucleus upon each chemical insult (paracetamol, CCl 4, DEM within 30 min; BSO within 2h). Using gel shift assays, nrf-2-specific binding to an antioxidant responsive element derived from the heavy chain of the human  - glutamylcysteine synthetase gene enhancer sequence is similarly enhanced. Thus, our data indicate that depletion of GSH per se may provide sufficient signal for activation of the nrf-2 sensor. This swift activation is probably due to either the release of active nrf-2 from its cytosolic-tethering partner, keap1, or an activation of the nrf-2 protein itself, or a function of both. Certainly, we have no evidence for the enhanced transcription of nrf-2 upon exposure to chemical insult. In fact, both DEM and APAP cause a large depletion in the steady-state level of hepatic nrf-2 mRNA, as measured using Northern assays. The possibility that a negative feedback loop exists to restrict nrf-2 activation is therefore currently being investigated. Non- competitor Competitor control antibody anti- nrf2 antibody 0’ 32 P-mut oligo 60’ 32 P-mut oligo Putative nrf2- containing complex(es ) Cytoplasm Nucleus Chemical Stress Chemical Insult Keap 1 Nrf2 Nrf2 Maf Cytoprotective genes ARE Phase II enzymes,  -GCS, HO-1, etc Controls 30mins 60mins 120mins 24h Representative Northern showing Nrf2 mRNA extracted from individual animals treated with DEM Controls 30mins 60mins 120mins 24h Figure 2. Representative Western showing nrf2 protein obtained from nuclear extracts from individual animals treated with DEM Northern showing heme oxygenase-1 mRNA extracted from individual animals treated with BSO Controls 30mins 60mins 120mins 24h Northern showing heme oxygenase-1 mRNA extracted from individual animals treated with CCl 4 Controls 30mins 60mins 120mins 24h