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GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL
B to Block Expression of Inducible Nitric-Oxide Synthase, Prevents Hypotension, and Improves Survival in Endotoxemic Rats
W. Harry Feinstone Center for Genomic Research, University of Memphis, Memphis, Tennessee (A.R.K., Y.H., S.B., C.H.S., T.R.S.); Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, New Hampshire (B.D.R.); and Department of Environmental Health Sciences, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland (T.W.K.)
Received October 3, 2005; accepted December 20, 2005.
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Abstract |
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B, mechanistic studies show that D3T blocks NFB nuclear translocation and DNA binding and that these effects are accompanied by changes in the levels of phospho-inhibitor of NFB. In conclusion, this study identifies new drug classes and targets that may improve the prevention and treatment of septic shock, as well as chronic diseases associated with the NFB and iNOS pathways.
; Thiemermann and Vane, 1990). Studies of iNOS null mutant mice have shown such mice to be resistant to the hypotension and death caused by lipopolysaccharide (LPS) (MacMicking et al., 1995; Wei et al., 1995). Inhibition of nitric-oxide synthase activity with selective inhibitors of iNOS have been shown to have beneficial effects in rodent models of septic shock, whereas inhibition of endothelial nitric-oxide synthase (eNOS) may lead to adverse effects including enhanced organ damage and excessive vasoconstriction (Thiemermann, 1997; Vos et al., 1997). Treatments such as dexamethasone that act to block the expression of iNOS protein decrease circulatory and organ failure, although such agents must be administered prior to endotoxin to achieve these beneficial outcomes (for review, see Thiemermann, 1997).
Dithiolethiones such as 3H-1,2-dithiole-3-thione (D3T) are potent chemopreventive agents that induce phase 2 and antioxidative enzymes to enhance the detoxication of chemical carcinogens. Moreover, dithiolethiones have been shown in animals to protect against neoplasia in multiple target organs (Kensler et al., 1999). Use of these agents for the prevention of cancer in humans is currently under clinical investigation (Wang et al., 1999; Lam et al., 2002). Both the cancer chemopreventive and gene expression effects of D3T have been shown to be mediated by the Keap1-Nrf2 pathway (Kwak et al., 2001, 2003). Keap1 is a sulfhydryl-rich protein that sequesters Nrf2 in the cytoplasm. Administration of sulfhydryl reactive compounds such as sulforaphane (SFN) or D3T abolishes the Keap1-Nrf2 interaction, facilitating nuclear translocation of Nrf2 (Venugopal and Jaiswal, 1996; Itoh et al., 1997; Dinkova-Kostova et al., 2002; Sekhar et al., 2002; Zipper and Mulcahy, 2002). Nrf2, a "cap n collar" basic region leucine zipper transcription factor, forms heterodimers with small Maf proteins to regulate genes containing antioxidant response elements in their upstream regulatory regions (Venugopal and Jaiswal, 1996; Itoh et al., 1997). Of particular relevance to this study, Vos et al. (1999) reported that depletion of glutathione by pretreatment of rats with diethylmaleate prevented the induction of hepatic iNOS RNA and protein 6 h post-treatment with LPS. Although these authors related this effect only to intracellular glutathione status, earlier studies on the regulation of phase 2 enzyme induction showed that agents that lowered glutathione levels were effective activators of reporter gene transcription driven by an antioxidant response element (Bergelson et al., 1994). In 2001, Heiss et al. (2001) showed that cotreatment of RAW cells with SFN blocked the LPS-mediated induction of iNOS mRNA and protein. SFN did not directly interact with NO or inhibit iNOS activity. Rather, SFN was shown to inhibit DNA binding of NFB, without affecting the degradation of the inhibitor of NFB, IB. Earlier this year, the link between the Keap1-Nrf2 phase 2 regulatory pathway and protection against oxidant and inflammatory stress was established in vitro (Dinkova-Kostova et al., 2005). Potent anti-inflammatory triterpenoids (Honda et al., 2000) were shown to block the induction of iNOS and cyclooxygenase-2 by 
-interferon (IFN-) in RAW cells. A strong correlation of the potency of these triterpenoids for this anti-inflammatory effect and induction of a phase 2 response was demonstrated over a range of several log concentrations. Using mouse embryo fibroblast from wild-type and Nrf 2-deficient mice, these authors established that the effect of triterpenoids to block NO production in response to treatment of these cells with IFN- and tumor necrosis factor-
was Nrf2-dependent (Dinkova-Kostova et al., 2005).
Collectively, these reports support the hypothesis that agents that activate Nrf2 signaling could effectively suppress iNOS elevation, NO production, and lessen the severity of endotoxin-induced toxicity in vitro. Herein, we report the results of a study of an in vivo model of LPS-induced toxicity. Pretreatment with a potent activator of Nrf2 blocked induction of iNOS, lessened hypotension and other indices of systemic toxicity, and dramatically improved survival of LPS-treated rats.
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). Twenty-four hours after the last treatment with either vehicle or D3T, animals in groups 1 (V) and 3 (D) were injected i.p. with PBS (pH 7.4); groups 2 (VL) and 4 (DL) were injected i.p. with LPS in PBS, pH 7.4, at a dose of 5 mg/kg b.wt., per Vos et al. (1999). For biochemical endpoints, four animals per group were treated for 6 h with PBS or LPS. The animals were euthanized with pentobarbital (60 mg/kg i.p.), and the livers were removed, washed in PBS, cut into small pieces, and either snap frozen in liquid nitrogen or used directly for nuclear protein isolation. Heparinized blood samples were drawn by cardiac puncture for determination of serum aspartate transaminase (AST), alanine transaminase (ALT), and the NO metabolites, nitrite plus nitrate. To determine the impact of D3T pretreatment on the LPS-induced hypotension, blood pressure (BP) was measured on the ventral artery of tails by simultaneously detecting the pulse of the artery and the pressure of an inflatable tail cuff (Krege et al., 1995; Johns et al., 1996). The rats were held in a restrainer of appropriate size to immobilize the tail and trained 15 min per session for 3 days to familiarize the animals with the restraint and to reduce the variation of the BP measurement. The cuff was fitted around the tail and inflated/deflated using a manual rubber bulb. The pulse sensor SS4LA and pressure sensor SS19L were connected to the monitor of a Biopac MP35 system with multiple channels (Biopac Systems, Inc., Goleta, CA). The environment temperature around the rat tail was controlled at 32°C throughout the BP measurement. After catching the pulse, the cuff was inflated to block the blood flow on the tail artery. The cuff was then slowly deflated until recovery of the pulse. The pressure change and pulse were recorded by computer to determine the systolic pressure, which was used to represent BP in this study. For survival analysis, animals were individually housed and monitored for a period of 18 h, following the last measurement of BP. Surviving animals were euthanized by asphyxiation with CO2.
Enzymes, NO Metabolites, and GSH Assays. The activities of ALT and AST in plasma were measured using kits from Biotron Diagnostics Inc. (Hemet, CA) (Vos et al., 1999). To measure total nitrite plus nitrate, plasma samples were thawed and deproteinized by incubating them with 95% ethanol at 4°C for 30 min, followed by centrifugation for 10 min at 13,000g. NO metabolites were measured according to the manufacturer's protocol (Roche Molecular Biochemicals, Mannheim Germany). Levels of intracellular GSH equivalents were determined using a colorimetric detection assay (BioVision Research Products, Mountain View, CA) based on the method of Akerboom and Sies (1981). Tissue samples were homogenized in buffer, mixed with 5% sulfosalicylic acid, and centrifuged at 8000g for 10 min. Glutathione content in the supernatant was determined as the conversion of 5.5'-dithiobis-(2-nitrobenzoic acid) to colored 2-nitro-5-thiobenzoic acid by measuring the spectrophotometric absorbance at 415 nm in a Benchmark Microplate Reader (Bio-Rad, Hercules, CA). A standard curve was made using GSH.
Protein Isolation and Western Blot Analysis. Total protein was isolated from the frozen tissue and used for iNOS analysis by Western blot (Vos et al., 1999). Sixty micrograms of each sample was separated by SDS-PAGE under reducing conditions and transferred onto 0.2-µm nitrocellulose membranes (Schleicher and Schuell, Keene, NH). iNOS was identified using an affinity-purified rabbit polyclonal antibody (50 µg/ml) (BD Biosciences, San Jose, CA). Nuclear and cytosolic proteins were extracted with nuclear and cytoplasmic extraction reagents (Pierce Laboratories, Rockford, IL) following the company's protocol and stored at –80°C in aliquots. Nuclear (2 µg) and cytosolic (50 µg) proteins of each sample were used for the assay of p65 by Western blot, using rabbit polyclonal anti-p65 antibody (Santa Cruz Biotechnology, Santa Cruz, CA). IkB- (Santa Cruz Biotechnology), phospho-IkB- (Cell Signaling, Beverly, MA), and IkB-
(Santa Cruz Biotechnology) were used for other Western blot analyses. For detection of eNOS, protein-containing particulate fractions were prepared from frozen liver samples according to Pollock et al. (1991), with some modifications. Tissues were suspended in 10 volumes of ice-cold buffer A (50 mM Tris-HCl, pH 7.4, 0.1 mM EDTA, and 0.1% 2-mercaptoethanol), containing 1 mM phenylmethylsulfonyl fluoride and 2 µM protease inhibitor cocktail (P8340; Sigma), and homogenized in a Potter-Elvehjem tissue grinder. The homogenate was centrifuged at 600g for 10 min, and the supernatant was again centrifuged at 100,000g for 60 min. The pellet was resuspended in buffer A with 10% (v/v) glycerol. Electrophoresis was carried out in the Bio-Rad minigel apparatus using 7.5% SDS-PAGE gels under reducing conditions. Levels of eNOS protein were detected using specific antibodies against eNOS (catalog no. 610296; BD Transduction, San Jose, CA) at a 1:1000 dilution. The immunocomplexes were detected by enhanced chemiluminescence (SuperSignal System; Pierce Laboratories). The density of bands was quantified by using a scanner (Expression 836XL; EPSON, Long Beach, CA) and measurement software (Image J, W. S. Rasband; U.S. National Institutes of Health, Bethesda, MD; http://rsb.info.nih.gov/ij/, 1997–2005).
Immunohistochemistry. Immunohistochemistry was carried out as previously described (Vos et al., 1999). Briefly, samples were prepared by mounting 4-µm slices of formalin-fixed/paraffin-embedded liver sections. Slides were deparaffinized with xylyne and stained with hematoxylin and eosin for routine histology. For immunohistochemistry, polyclonal iNOS antibody (2 µg/ml) (Santa Cruz Biotechnology) or normal rabbit serum, as a negative control, were used. After incubation with the secondary antibody, the slides were washed with PBS and labeled with a biotin-horseradish peroxidase conjugate and developed by incubation with 3,3'-diaminobenzidine and hydrogen peroxide (ABC kit; Vector Laboratories, Burlingame, CA). The slides were dehydrated and counterstained with hematoxylin.
Electrophoretic Mobility Shift Assay. DNA binding conditions for NFB and electrophoretic mobility shift assay (EMSA) were as described (Lenardo and Baltimore, 1989). Oligonucleotides from the consensus binding site for NFB were used: 5'-AGT TGA GGG GAC TTT CCC AGG C-3' and reverse complement (Integrated DNA Technologies, Coralville, IA). Each NFB binding oligonucleotide was end-labeled with biotin using a biotin 3'-end labeling kit (Pierce Laboratories) and annealed by incubating equal concentrations and heating to 75°C, followed by gradual cooling to room temperature. Binding reactions were performed at room temperature for 25 min using 1 µg of nuclear protein and 20 fmol biotin-labeled NFB. DNA-protein complexes were separated on native 6% polyacrylamide gels and electroblotted onto positively charged Biodyne B nylon membranes. Detection of biotin-labeled DNA by chemiluminescence was performed according to protocol provided in the Lightshift Chemiluminescent EMSA kit (Pierce Laboratories). Cold competition was performed using consensus or mutant NFB oligonucleotides. Supershift experiments were carried out by adding anti-p65 or -p50 antibodies (Santa Cruz Biotechnology) to the binding mixture prior to PAGE.
Statistical Analysis. All data were expressed as mean ± S.D. Pair-wise comparisons were performed by Student's t test. Survival following LPS challenge was assessed by the 
2 test. Differences were considered statistically significant at values of p 
0.05.
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Histology and iNOS Immunohistochemistry. Hepatic tissue of vehicle- or D3T-treated animals appeared normal in structure, with no or few inflammatory cells in the portal tracts or parenchymas. There were no major differences in the histology of D3T plus LPS-treated animals and LPS-treated animals, 6 h post-LPS treatment. Both sets of samples showed occasional areas of perivascular lymphocytes and scattered areas of cell necrosis. In the D3T plus LPS-treated animals, one animal showed two large areas of necrosis, whereas the other animals showed only occasional foci of necrosis and occasional neutrophils. In the LPS-treated animals, two animals showed more than 10 regions of necrosis involving 10 to 30 cells and frequent neutrophil infiltration, whereas the other two LPS-treated animals showed focal necrosis and occasional neutrophils (data not shown). At 6 h after treatment with LPS, iNOS staining was pronounced in hepatocytes (Fig. 2C). A marked decrease of iNOS observed in the hepatic tissue of D3T plus LPS-treated animals was a result of diminished iNOS expression in hepatocytes (Fig. 2D).
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), we determined the effect of the treatments on the expression of eNOS protein. The level of expression of hepatic eNOS expression was not altered by any of the treatments as determined by Western blot analysis (Fig. 3).
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Effects of Treatment on GSH Levels. Previously, it had been reported (Vos et al., 1999) that depletion of glutathione prevented the induction of hepatic iNOS protein 6 h post-treatment with LPS. To rule out similar effects of D3T, we determined hepatic levels of GSH (Fig. 4). As expected, treatment of animals with D3T resulted in approximately a 3-fold increase in hepatic GSH levels (Fig. 4, V and D). Although LPS treatment slightly decreased GSH content, this effect was not statistically significant. However, in animals pretreated with D3T and then challenged with LPS, the levels of GSH were lower than those observed for animals treated only with D3T. Moreover, animals pretreated with D3T and then challenged with LPS maintained significantly higher levels of GSH (Fig. 4, VL and DL).
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D3T Targets NFB Regulation of iNOS. Based on the published results of in vitro studies of SFN in RAW cells (Heiss et al., 2001) and diethylmaleate in primary cultures of rat hepatocytes (Vos et al., 1999), we focused our initial investigation of the mechanism of the D3T effect to block LPS-mediated induction of iNOS on NFB, an established regulator of iNOS expression. By EMSA, no NFB DNA binding was observed in nuclear extracts prepared from liver tissue of either vehicle- or D3T-treated rats (Fig. 5A, lanes 1 and 2). NFB DNA binding was observed in samples prepared from LPS-treated animals (Fig. 5A, lane 3). This binding complex was shown by supershift assay to contain both p65 and p50 proteins (Fig. 5A, lanes 6 and 7). Pretreatment of animals with D3T blocked the LPS-stimulated increase in NFB DNA binding (Fig. 5A, lane 4). Consistent with these results, Western blot analysis detected p65 protein only in nuclear extracts prepared from liver tissue of animals treated with LPS alone (Fig. 5B, top, lanes 5 and 6). Levels of p65 protein in cytosolic fractions did not vary greatly between the treatment groups (Fig. 5B, bottom), consistent with the knowledge that nuclear localization of NFB is regulated through the degradation of its cytosolic protein partner/inhibitor, IB (Baeuerle and Baltimore, 1988; Zandi et al., 1997). Canonical cytokine-mediated activation of the NFB pathway involves phosphorylation of IB by IB kinase, followed by IB ubiquitination and proteosome degradation (Courtois et al., 2001; Ghosh and Karin, 2002). Western blot analysis of cytosolic proteins showed no treatment-related differences in the levels of IB or IB proteins (Fig. 5C). However, levels of phospho-IB were shown to be lower in samples from animals treated for 6 h with LPS than in other treatment groups (Fig. 5C, P-IB). Relative densitometric data for this observation are shown in the bottom panel of Fig. 5C, expressed as the ratio of P-IB to IB.
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Effect of D3T on Physiologic Endpoints of Endotoxic Shock. Because induction of iNOS in many organs contributes to hypotension, vascular hyporeactivity to vasoconstrictors and organ injury (Millar and Thiemermann, 1997; Nathan, 1997; Rosselet et al., 1998), we expanded our study to include endpoints of systemic effects of LPS. We chose a noninvasive tail cuff procedure to determine atrial blood pressure. Doing so eliminated the need for survival surgery and allowed us to determine BP measurements at baseline, after pretreatment regimens and following LPS challenges, all on the same animals. The decision to measure BP 6 h post-treatment with LPS was based on other studies showing that significant differences in BP occur at this time with LPS (Millar and Thiemermann, 1997; Rosselet et al., 1998). The baseline atrial BP of animals treated with either vehicle or D3T was 140.3 ± 6.1 and 135.5 ± 9.5 mm Hg, respectively (Fig. 6A). There were no significant differences in these values. As expected, the BP was decreased in vehicle plus LPS group. An average reduction of 30.8 mm Hg (21.9%) was observed in theses animals 2 h after LPS injection, and this value was significantly different (p = 0.003) from its baseline value. The BP was further decreased 6 h after LPS injection, with an average reduction of 57.5 mm Hg (40.1%) from baseline. Again, this value was statistically different (p = 0.001) from its baseline value. Interestingly, the BP value was not reduced after LPS injection in D3T-pretreated animals. Actually, there was a slight increase of 9 mm Hg (12.5%) from the baseline value 2 h after injection of LPS (p = 0.049). The reason for this increase is not known but may be related to the procedure. It is important to note that this effect is transient, whereby the BP returns to baseline by 6 h after injection of LPS in the D3T-pretreated animals. Survival during the period of 24 h post-LPS injection was dramatically improved in animals pretreated with D3T. One hundred percent of the animals pretreated with D3T survived, whereas only 37.5% (three of eight) of the animals given vehicle plus LPS survived (Fig. 6B).
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Discussion |
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As others have reported that depletion of glutathione by pretreatment of rats with diethylmaleate prevented the induction of hepatic iNOS protein (Vos et al., 1997), we studied these effects of D3T. As expected from the pioneering work of Bueding on dithiolethiones (De Long et al., 1986), hepatic levels of GSH were increased 3-fold by treatment with D3T. Of note, challenge with LPS of animals pretreated with D3T decreased by 50% the levels of GSH relative to the D3T-treated-only group. Thus, it appears that one action of D3T is to protect against the oxidative stress component of LPS-mediated toxicity.
A fundamental aspect of the LPS-mediated induction of iNOS is the activation of the transcription factor NFB. EMSA and Western blot analysis of nuclear protein extracts established that pretreatment of animals with D3T blocked LPS-mediated nuclear translocation and DNA binding of NFB. This action of D3T was distinct from what has been reported for SFN in RAW cells. Although SFN also inhibited the DNA binding of NFB, it did not prevent nuclear translocation of p65. Thus, unlike the in vitro effect of SFN (Heiss et al., 2001) and other vicinal dithiol-binding agents such as phenylarsine oxide that modify the structure of NFB and inhibit its binding to DNA (Oda et al., 2000), the in vivo effect of D3T appears to act upstream of nuclear translocation of NFB. Analysis of the IB proteins showed that pretreatment with D3T resulted in higher levels of phospho-IB relative to samples from untreated animals. Furthermore, D3T pretreatment appeared to alter the LPS-directed decrease in phospho-IB protein levels. The observed change in phospho-IB and not total IB in rat liver is consistent with current knowledge that most of the cellular IB protein exists in the nonphosphorylated form (Menon et al., 1995) and that phosphorylation of this protein results in its degradation and activation of NFB (Courtois et al., 2001; Ghosh and Karin, 2002). Since our analysis was performed at only a single time point and the kinetics of IB synthesis, phosphorylation, and turnover are complex, we are cautious not to overinterpret these data. However, our results are consistent with several other studies of structurally diverse antioxidants that have been shown to alter IB degradation (for review, see Surh et al., 2001). In the case of (–)-epigallocatechin gallate, phosphorylation of IB was shown to be the target (Nomura et al., 2000), and further studies of IB and its associated IK kinase are warranted for D3T.
What then is the mechanism of action of D3T? Our current study design employed an intermittent dose schedule of D3T, followed 24 h after the last dose with LPS challenge. This model is the same design we have used in cancer chemoprevention (Kensler et al., 1999; Roebuck et al., 2003), where D3T has been shown to induce gene expression through Nrf2 (Kwak et al., 2001, 2003; Dinkova-Kostova et al., 2005). A recent report from the laboratories of Sporn and Talalay showed a strong correlation of the potencies of triterpenoids for anti-inflammatory effects, including blocking iNOS expression, and phase 2 enzyme responses. Using mouse embryo fibroblasts from wild-type and Nrf2-disrupted mice, these authors established that the effect of triterpenoids to block NO production in response to treatment of these cells with IFN- and tumor necrosis factor- was Nrf2-dependent (Dinkova-Kostova et al., 2005). Therefore, we believe that it is likely that D3T acts through Nrf2 to inhibit iNOS expression. Ongoing studies in Nrf 2 null mice should clarify this point.
Although our studies focused on expression of hepatic iNOS, it is known that several organs are sites of LPS-mediated toxicity, including lung, intestine, kidney, and liver. Two important physiologic endpoints of the systemic effect of LPS include hypotension and death (Mailman et al., 2001). That iNOS is an important mediator of these endpoints has been established in studies of iNOS null mice (Julou-Schaeffer et al., 1990; Thiemermann and Vane, 1990). Furthermore, iNOS is one of three nitric-oxide synthase enzymes. The other two forms are constitutively expressed in either neuronal or eNOS cells. Studies using NOS inhibitors either specific to iNOS (S-methyl-isothiourea) or those that are better inhibitors of eNOS (N6-nitro-L-arginine methyl ester) have shown that inhibitors of iNOS afforded protection against endotoxemia, whereas the eNOS inhibitors augmented toxicity (Vos et al., 1997). Thus, iNOS induction has been causally linked with the systemic toxicity of LPS, yet the relative contribution of different organ systems is still under investigation. By measuring the systemic endpoints of hypotension and survival during endotoxic shock, we demonstrated that the protective effect of D3T pretreatment extends beyond effects on hepatic function. One hundred percent of animals pretreated with D3T and then challenged with LPS survived and maintained normal blood pressure throughout the measured period. Only 37.5% of the animals treated with LPS alone survived the 24-h follow-up period. All animals in the vehicle-pretreated group exhibited marked hypotension.
To our knowledge, this is the first report showing that D3T or other inducers of phase 2 enzymes protect against endotoxic shock in vivo. Although the current model of acute endotoxic shock may not adequately simulate human septic shock (Rosselet et al., 1998), the results of this study strongly support the need for further studies on this class of compounds for the prevention and treatment of sepsis and septic shock, as well as other chronic diseases associated with the NFB and iNOS pathways.
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Acknowledgements |
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Footnotes |
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A.R.K. and Y.H. contributed equally to this work.
ABBREVIATIONS: NO, nitric oxide; iNOS, inducible nitric-oxide synthase; LPS, lipopolysaccharide; eNOS, endothelial nitric-oxide synthase; D3T, 3H-1,2-dithiole-3-thione; SFN, sulforaphane; NF, nuclear factor; IB, inhibitor of NFB; IFN, interferon; PBS, phosphate-buffered saline; AST, aspartate transaminase; ALT, alanine transaminase; BP, blood pressure; GSH, glutathione; PAGE, polyacrylamide gel electrophoresis; EMSA, electrophoretic mobility shift assay.
Address correspondence to: Thomas R. Sutter, W. Harry Feinstone Center for Genomic Research, 201 Life Science Building, University of Memphis, Memphis, TN 38152. E-mail: tsutter{at}memphis.edu
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References |
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|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Akerboom TP and Sies H (1981) Assay of glutathione, glutathione disulfide and glutathione mixed disulfides in biological samples. Methods Enzymol 77: 373–382.[Medline]
Baeuerle PA and Baltimore D (1988) I kappa B: a specific inhibitor of the NF-kappa B transcription factor. Science (Wash DC) 242: 540–546.
Bergelson S, Pinkus R, and Daniel V (1994) Intracellular glutathione levels regulate Fos/Jun induction and activation of glutathione S-transferase gene expression. Cancer Res 54: 36–40.
Courtois G, Smahi A, and Israel A (2001) NEMO/IKK gamma: linking NF-kappa B to human disease. Trends Mol Med 7: 427–430.[CrossRef][Medline]
De Long MJ, Dolan P, Santamaria AB, and Bueding E (1986) 1,2-Dithiol-3-thione analogs: effects on NAD(P)H:quinone reductase and glutathione levels in murine hepatoma cells. Carcinogenesis 7: 977–980.
Dinkova-Kostova AT, Holtzclaw WD, Cole RN, Itoh K, Wakabayashi N, Katoh Y, Yamamoto M, and Talalay P (2002) 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 99: 11908–11913.
Dinkova-Kostova AT, Liby KT, Stephenson KK, Holtzclaw WD, Gao X, Suh N, Williams C, Risingsong R, Honda T, Gribble GW, et al. (2005) Extremely potent triterpenoid inducers of the phase 2 response: correlations of protection against oxidant and inflammatory stress. Proc Natl Acad Sci USA 102: 4584–4589.
Ghosh S and Karin M (2002) Missing pieces in the NF-kappaB puzzle. Cell 109 (Suppl): S81–S96.[CrossRef][Medline]
Heiss E, Herhaus C, Klimo K, Bartsch H, and Gerhauser C (2001) Nuclear factor kappa B is a molecular target for sulforaphane-mediated anti-inflammatory mechanisms. J Biol Chem 276: 32008–32015.
Honda T, Gribble GW, Suh N, Finlay HJ, Rounds BV, Bore L, Favaloro FG Jr, Wang Y, and Sporn MB (2000) Novel synthetic oleanane and ursane triterpenoids with various enone functionalities in ring A as inhibitors of nitric oxide production in mouse macrophages. J Med Chem 43: 1866–1877.[CrossRef][Medline]
Itoh K, Chiba T, Takahashi S, Ishii T, Igarashi K, Katoh Y, Oyake T, Hayashi N, Satoh K, Hatayama I, et al. (1997) An Nrf2/small Maf heterodimer mediates the induction of phase II detoxifying enzyme genes through antioxidant response elements. Biochem Biophys Res Commun 236: 313–322.[CrossRef][Medline]
Johns C, Gavras I, Handy DE, Salomao A, and Gavras H (1996) Models of experimental hypertension in mice. Hypertension 28: 1064–1069.
Julou-Schaeffer G, Gray GA, Fleming I, Schott C, Parratt JR, and Stoclet JC (1990) Loss of vascular responsiveness induced by endotoxin involves L-arginine pathway. Am J Physiol 259: H1038–H1043.[Medline]
Kensler TW, Groopman JD, Sutter TR, Curphey TJ, and Roebuck BD (1999) Development of cancer chemopreventive agents: oltipraz as a paradigm. Chem Res Toxicol 12: 113–126.[CrossRef][Medline]
Krege JH, Hodgin JB, Hagaman JR, and Smithies O (1995) A noninvasive computerized tail-cuff system for measuring blood pressure in mice. Hypertension 25: 1111–1115.
Kwak MK, Itoh K, Yamamoto M, Sutter TR, and Kensler TW (2001) 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 7: 135–145.[Medline]
Kwak MK, Wakabayashi N, Itoh K, Motohashi H, Yamamoto M, and Kensler TW (2003) Modulation of gene expression by cancer chemopreventive dithiolethiones through the Keap1-Nrf2 pathway: identification of novel gene clusters for cell survival. J Biol Chem 278: 8135–8145.
Lam S, MacAulay C, Le Riche JC, Dyachkova Y, Coldman A, Guillaud M, Hawk E, Christen MO, and Gazdar AF (2002) A randomized phase IIb trial of anethole dithiolethione in smokers with bronchial dysplasia. J Natl Cancer Inst 94: 1001–1009.
Lenardo MJ and Baltimore D (1989) NF-kappa B: a pleiotropic mediator of inducible and tissue-specific gene control. Cell 58: 227–229.[CrossRef][Medline]
MacMicking JD, Nathan C, Hom G, Chartrain N, Fletcher DS, Trumbauer M, Stevens K, Xie QW, Sokol K, Hutchinson N, et al. (1995) Altered responses to bacterial infection and endotoxic shock in mice lacking inducible nitric oxide synthase. Cell 81: 641–650.[CrossRef][Medline]
Mailman D, Guntuku S, Bhuiyan MB, and Murad F (2001) Organ sites of lipopolysaccharide-induced nitric oxide production in the anesthetized rat. Nitric Oxide 5: 243–251.[CrossRef][Medline]
Menon SD, Guy GR, and Tan YH (1995) Involvement of a putative protein-tyrosine phosphatase and I kappa B-alpha serine phosphorylation in nuclear factor kappa B activation by tumor necrosis factor. J Biol Chem 270: 18881–18887.
Millar CG and Thiemermann C (1997) Intrarenal haemodynamics and renal dysfunction in endotoxaemia: effects of nitric oxide synthase inhibition. Br J Pharmacol 121: 1824–1830.[CrossRef][Medline]
Nathan C (1997) Inducible nitric oxide synthase: what difference does it make? J Clin Investig 100: 2417–2423.[Medline]
Nomura M, Ma W, Chen N, Bode AM, and Dong Z (2000) Inhibition of 12-O-tetradecanoylphorbol-13-acetate-induced NF-kappaB activation by tea polyphenols, (–)-epigallocatechin gallate and theaflavins. Carcinogenesis 21: 1885–1890.
Oda M, Sakitani K, Kaibori M, Inoue T, Kamiyama Y, and Okumura T (2000) Vicinal dithiol-binding agent, phenylarsine oxide, inhibits inducible nitric-oxide synthase gene expression at a step of nuclear factor-kappaB DNA binding in hepatocytes. J Biol Chem 275: 4369–4373.
Pollock JS, Forstermann U, Mitchell JA, Warner TD, Schmidt HH, Nakane M, and Murad F (1991) Purification and characterization of particulate endothelium-derived relaxing factor synthase from cultured and native bovine aortic endothelial cells. Proc Natl Acad Sci USA 88: 10480–10484.
Roebuck BD, Curphey TJ, Li Y, Baumgartner KJ, Bodreddigari S, Yan J, Gange SJ, Kensler TW, and Sutter TR (2003) Evaluation of the cancer chemopreventive potency of dithiolethione analogs of oltipraz. Carcinogenesis 24: 1919–1928.
Rosselet A, Feihl F, Markert M, Gnaegi A, Perret C, and Liaudet L (1998) Selective iNOS inhibition is superior to norepinephrine in the treatment of rat endotoxic shock. Am J Respir Crit Care Med 157: 162–170.[Medline]
Sekhar KR, Spitz DR, Harris S, Nguyen TT, Meredith MJ, Holt JT, Gius D, Marnett LJ, Summar ML, and Freeman ML (2002) Redox-sensitive interaction between KIAA0132 and Nrf2 mediates indomethacin-induced expression of gamma-glutamylcysteine synthetase. Free Radic Biol Med 32: 650–662.[CrossRef][Medline]
Surh YJ, Chun KS, Cha HH, Han SS, Keum YS, Park KK, and Lee SS (2001) Molecular mechanisms underlying chemopreventive activities of anti-inflammatory phytochemicals: down-regulation of COX-2 and iNOS through suppression of NF-kappa B activation. Mutat Res 480–481: 243–268.
Thiemermann C (1997) Nitric oxide and septic shock. Gen Pharmacol 29: 159–166.[Medline]
Thiemermann C and Vane J (1990) Inhibition of nitric oxide synthesis reduces the hypotension induced by bacterial lipopolysaccharides in the rat in vivo. Eur J Pharmacol 182: 591–595.[CrossRef][Medline]
Venugopal R and Jaiswal AK (1996) 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 93: 14960–14965.
Vos TA, Gouw AS, Klok PA, Havinga R, van Goor H, Huitema S, Roelofsen H, Kuipers F, Jansen PL, and Moshage H (1997) Differential effects of nitric oxide synthase inhibitors on endotoxin-induced liver damage in rats. Gastroenterology 113: 1323–1333.[CrossRef][Medline]
Vos TA, Van Goor H, Tuyt L, De Jager-Krikken A, Leuvenink R, Kuipers F, Jansen PL, and Moshage H (1999) Expression of inducible nitric oxide synthase in endotoxemic rat hepatocytes is dependent on the cellular glutathione status. Hepatology 29: 421–426.[CrossRef][Medline]
Wang JS, Shen X, He X, Zhu YR, Zhang BC, Wang JB, Qian GS, Kuang SY, Zarba A, Egner PA, et al. (1999) Protective alterations in phase 1 and 2 metabolism of aflatoxin B1 by oltipraz in residents of Qidong, People's Republic of China. J Natl Cancer Inst 91: 347–354.
Wei XQ, Charles IG, Smith A, Ure J, Feng GJ, Huang FP, Xu D, Muller W, Moncada S, and Liew FY (1995) Altered immune responses in mice lacking inducible nitric oxide synthase. Nature (Lond) 375: 408–411.[CrossRef][Medline]
Zandi E, Rothwarf DM, Delhase M, Hayakawa M, and Karin M (1997) The IkappaB kinase complex (IKK) contains two kinase subunits, IKKalpha and IKKbeta, necessary for IkappaB phosphorylation and NF-kappaB activation. Cell 91: 243–252.[CrossRef][Medline]
Zipper LM and Mulcahy RT (2002) The Keap1 BTB/POZ dimerization function is required to sequester Nrf2 in cytoplasm. J Biol Chem 277: 36544–36552.
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