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Thomas W. Kensler - One of the best experts on this subject based on the ideXlab platform.
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3h 1 2 dithiole 3 thione targets nuclear factor κb to block expression of inducible nitric oxide synthase prevents hypotension and improves survival in endotoxemic rats
Journal of Pharmacology and Experimental Therapeutics, 2006Co-Authors: Asok R Karuri, Thomas W. Kensler, B D Roebuck, Yong Huang, Sridevi Bodreddigari, Carrie Hayes Sutter, Thomas R. SutterAbstract:Septicemia is a major cause of death associated with noncoronary intensive care. Systemic production of nitric oxide (NO) by inducible nitric-oxide synthase (iNOS) is a major cause of hypotension and poor organ perfusion seen in septic shock. Here, we show that pretreatment of F344 rats with the cancer Chemoprotective Agent 3 H -1,2-dithiole-3-thione (D3T) blocks lipopolysaccharide (LPS)-mediated induction of hepatic iNOS and significantly reduces the associated serum levels of NO metabolites and enzyme markers of toxicity provoked by treatment with LPS. Immunohistochemical analysis shows that this protective effect is largely due to suppression of iNOS expression in hepatocytes. Importantly, pretreatment of animals with D3T blunts LPS-mediated hypotension and dramatically increases their survival. Inasmuch as iNOS expression can be regulated by nuclear factor (NF) κB, mechanistic studies show that D3T blocks NFκB nuclear translocation and DNA binding and that these effects are accompanied by changes in the levels of phospho-inhibitor of NFκB. 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 NFκB and iNOS pathways.
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sensitivity to carcinogenesis is increased and Chemoprotective efficacy of enzyme inducers is lost in nrf2 transcription factor deficient mice
Proceedings of the National Academy of Sciences of the United States of America, 2001Co-Authors: Minerva Ramosgomez, Ken Itoh, Mi Kyoung Kwak, Masayuki Yamamoto, Patrick Dolan, Paul Talalay, Thomas W. KenslerAbstract:Abstract Induction of phase 2 enzymes, which neutralize reactive electrophiles and act as indirect antioxidants, appears to be an effective means for achieving protection against a variety of carcinogens in animals and humans. Transcriptional control of the expression of these enzymes is mediated, at least in part, through the antioxidant response element (ARE) found in the regulatory regions of their genes. The transcription factor Nrf2, which binds to the ARE, appears to be essential for the induction of prototypical phase 2 enzymes such as glutathione S-transferases (GSTs) and NAD(P)H:quinone oxidoreductase (NQO1). Constitutive hepatic and gastric activities of GST and NQO1 were reduced by 50–80% in nrf2-deficient mice compared with wild-type mice. Moreover, the 2- to 5-fold induction of these enzymes in wild-type mice by the Chemoprotective Agent oltipraz, which is currently in clinical trials, was almost completely abrogated in the nrf2-deficient mice. In parallel with the enzymatic changes, nrf2-deficient mice had a significantly higher burden of gastric neoplasia after treatment with benzo[a]pyrene than did wild-type mice. Oltipraz significantly reduced multiplicity of gastric neoplasia in wild-type mice by 55%, but had no effect on tumor burden in nrf2-deficient mice. Thus, Nrf2 plays a central role in the regulation of constitutive and inducible expression of phase 2 enzymes in vivo and dramatically influences susceptibility to carcinogenesis. Moreover, the total loss of anticarcinogenic efficacy of oltipraz in the nrf2-disrupted mice highlights the prime importance of elevated phase 2 gene expression in chemoprotection by this and similar enzyme inducers.
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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
Molecular Medicine, 2001Co-Authors: Mi Kyoung Kwak, Ken Itoh, Thomas R. Sutter, Thomas W. KenslerAbstract:BACKGROUND: The induction of phase 2 enzymes by dithiolethiones such as oltipraz is an effective means for achieving protection against environmental carcinogens in animals and humans. Transcriptional control of the expression of at least some of these protective enzymes is mediated through the antioxidant response element (ARE) found in the upstream regulatory region of many phase 2 genes. The transcription factor Nrf2, which binds to the ARE, appears to be essential for the induction of proto-typical phase 2 enzymes such as glutathione S-transferase (GST) Ya, Yp, and NAD(P)H: quinone reductase (NQO1) in vivo. MATERIALS AND METHODS: In the present study, 3H-1,2-dithiole-3-thione (D3T) was used as a potent model inducer whose effects on gene expression and chemopreventive efficacy have been extensively characterized in the rat. Over a dozen putative D3T-inducible genes were examined in wild-type and nrf2-disrupted mice by Northern blot hybridization and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis to elucidate whether loss of Nrf2 function also affects the induction of a broader representation of phase 2 and antioxidative enzymes. The effects of D3T on hepatic Nrf2 expression and localization were also examined in vivo by Northern blot hybridization, electromobility shift assay, and Western blot analysis. RESULTS: Specific activities of hepatic GST and NQO1 were increased by D3T in wild-type mice and were largely blunted in the nrf2-deficient mice. However, changes in levels of RNA transcripts following D3T treatment of nrf2-disrupted mice were multidirectional, dependent upon the particular gene examined. Although elevation of mRNAs for GST Ya, NQO1, microsomal epoxide hydrolase and gamma-glutamylcysteine synthetase regulatory chain were blocked in the mutant mice, elevation of GST Yp mRNA was largely unimpeded. Increases in levels of mRNA for the heavy and light chains of ferritin were only seen in the nrf2-disrupted mice. Transcript levels of UDP-glucuronyl-transferase 1A6, heme oxygenase-1, maganese superoxide dismutase, which were inducible in the wild-type mice, actually decreased in the mutant mice, whereas levels of mRNA for GST Yc, aflatoxin B1 aldehyde reductase and catalase decreased following D3T treatment in the mutant mice in the absence of any inductive effect by D3T in the wild-type mice. In wild-type mice, treatment with D3T lead to 3-fold increases in hepatic Nrf2 mRNA levels within several hours following dosing as assessed by Northern blot and RT-PCR analyses. Gel shift analyses with oligonucleotide probes for human NQO1 ARE, murine GST Ya ARE, and erythroid transcription factor (NF-E2) binding site showed increased intensity of binding with nuclear extracts prepared from livers of D3T-treated mice compared to vehicle-treated controls. Antibody to Nrf2 supershifted the DNA binding bands of these nuclear extracts. Moreover, immunoblot analysis indicated accumulation of Nrf2 in extracts prepared from hepatic nuclei of D3T-treated mice at the same time points. CONCLUSIONS: Nrf2 plays a central role in the regulation of constitutive and inducible expression of multiple phase 2 and antioxidative enzymes by Chemoprotective dithiolethiones in vivo, although patterns of response vary among different genes. Knowledge of the factors controlling the specificity of actions of enzyme inducers will be exceedingly helpful in the design and isolation of more efficient and selective Chemoprotective Agents.
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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 dithiole 3 thione
Molecular Medicine, 2001Co-Authors: Mi Kyoung Kwak, Ken Itoh, Thomas R. Sutter, Masayuki Yamamoto, Thomas W. KenslerAbstract:The induction of phase 2 enzymes by dithiolethiones such as oltipraz is an effective means for achieving protection against environmental carcinogens in animals and humans. Transcriptional control of the expression of at least some of these protective enzymes is mediated through the antioxidant response element (ARE) found in the upstream regulatory region of many phase 2 genes. The transcription factor Nrf2, which binds to the ARE, appears to be essential for the induction of prototypical phase 2 enzymes such as glutathione S-transferase (GST) Ya, Yp, and NAD(P)H: quinone reductase (NQO1) in vivo. In the present study, 3H-1,2-dithiole-3-thione (D3T) was used as a potent model inducer whose effects on gene expression and chemopreventive efficacy have been extensively characterized in the rat. Over a dozen putative D3T-inducible genes were examined in wild-type and nrf2-disrupted mice by Northern blot hybridization and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis to elucidate whether loss of Nrf2 function also affects the induction of a broader representation of phase 2 and antioxidative enzymes. The effects of D3T on hepatic Nrf2 expression and localization were also examined in vivo by Northern blot hybridization, electromobility shift assay, and Western blot analysis. Specific activities of hepatic GST and NQO1 were increased by D3T in wild-type mice and were largely blunted in the nrf2-deficient mice. However, changes in levels of RNA transcripts following D3T treatment of nrf2-disrupted mice were multidirectional, dependent upon the particular gene examined. Although elevation of mRNAs for GST Ya, NQO1, microsomal epoxide hydrolase and γ-glutamylcysteine synthetase regulatory chain were blocked in the mutant mice, elevation of GST Yp mRNA was largely unimpeded. Increases in levels of mRNA for the heavy and light chains of ferritin were only seen in the nrf2-disrupted mice. Transcript levels of UDP-glucuronyl-transferase 1A6, heme oxygenase-1, maganese superoxide dismutase, which were inducible in the wild-type mice, actually decreased in the mutant mice, whereas levels of mRNA for GST Yc, aflatoxin B1 aldehyde reductase and catalase decreased following D3T treatment in the mutant mice in the absence of any inductive effect by D3T in the wild-type mice. In wild-type mice, treatment with D3T lead to 3-fold increases in hepatic Nrf2 mRNA levels within several hours following dosing as assessed by Northern blot and RT-PCR analyses. Gel shift analyses with oligonucleotide probes for human NQO1 ARE, murine GST Ya ARE, and erythroid transcription factor (NF-E2) binding site showed increased intensity of binding with nuclear extracts prepared from livers of D3T-treated mice compared to vehicle-treated controls. Antibody to Nrf2 supershifted the DNA binding bands of these nuclear extracts. Moreover, immunoblot analysis indicated accumulation of Nrf2 in extracts prepared from hepatic nuclei of D3T-treated mice at the same time points. Nrf2 plays a central role in the regulation of constitutive and inducible expression of multiple phase 2 and antioxidative enzymes by Chemoprotective dithiolethiones in vivo, although patterns of response vary among different genes. Knowledge of the factors controlling the specificity of actions of enzyme inducers will be exceedingly helpful in the design and isolation of more efficient and selective Chemoprotective Agents.
Xingxiang Peng - One of the best experts on this subject based on the ideXlab platform.
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induction of endogenous glutathione by the Chemoprotective Agent 3h 1 2 dithiole 3 thione in human neuroblastoma sh sy5y cells affords protection against peroxynitrite induced cytotoxicity
Biochemical and Biophysical Research Communications, 2004Co-Authors: Zhuoxiao Cao, Seema Hallur, Harry Z Qiu, Xingxiang PengAbstract:Substantial evidence suggests that peroxynitrite generated from the bi-radical reaction of nitric oxide and superoxide is critically involved in the pathogenesis of neurodegenerative disorders, such as Parkinson's disease. Reaction with sulfhydryl (SH)-containing molecules has been proposed to be a major detoxification pathway of peroxynitrite in biological systems. This study was undertaken to determine if chemically elevated intracellular reduced glutathione (GSH), a major SH-containing biomolecule, affords protection against peroxynitrite-mediated toxicity in cultured neuronal cells. Incubation of human neuroblastoma SH-SY5Y cells with the unique chemoprotectant, 3H-1,2-dithiole-3-thione (D3T), led to a significant elevation of cellular GSH in a concentration-dependent fashion. To examine the protective effects of D3T-induced GSH on peroxynitrite-mediated toxicity, SH-SY5Y cells were pretreated with D3T and then exposed to either the peroxynitrite generator, 3-morpholinosydnonimine (SIN-1), or the authentic peroxynitrite. We observed that D3T-pretreated cells showed a markedly increased resistance to SIN-1- or authentic peroxynitrite-induced cytotoxicity, as assessed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium reduction assay. Conversely, depletion of cellular GSH by buthionine sulfoximine (BSO) caused a marked potentiation of SIN-1- or authentic peroxynitrite-mediated cytotoxicity. To further demonstrate the causal role for GSH induction in D3T-mediated cytoprotection, SH-SY5Y cells were co-treated with BSO to abolish D3T-induced GSH elevation. Co-treatment of the cells with BSO was found to significantly reverse the protective effects of D3T on SIN-1- or authentic peroxynitrite-elicited cytotoxicity. Taken together, this study demonstrates for the first time that D3T can induce GSH in cultured SH-SY5Y cells, and that the D3T-augmented cellular GSH defense affords a marked protection against peroxynitrite-induced toxicity in cultured human neuronal cells.
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induction of cellular glutathione linked enzymes and catalase by the unique Chemoprotective Agent 3h 1 2 dithiole 3 thione in rat cardiomyocytes affords protection against oxidative cell injury
Pharmacological Research, 2002Co-Authors: Xingxiang PengAbstract:Abstract Considerable evidence suggests that reactive oxygen species (ROS) are crucially involved in the pathogenesis of cardiovascular diseases, such as myocardial ischemia–reperfusion injury. Consistent with this notion, administration of exogenous antioxidative compounds has been shown to provide protection against oxidative cardiac injury. However, whether induction of endogenous cellular antioxidants by chemicals (drugs) also offers protection against oxidative cardiac injury has not been extensively investigated. In the present study, with rat cardiomyocyte H9C2 cells as an in vitro model, we have investigated the induction of cellular antioxidants by the unique Chemoprotective Agent, 3 H -1,2-dithiole-3-thione (D3T) and the protective effects of the D3T-induced cellular antioxidants against ROS-mediated injury in cardiac cells. Incubation of H9C2 cells with micromolar concentrations of D3T for 24 h resulted in a significant induction of a battery of cellular antioxidants, including reduced glutathione (GSH), GSH peroxidase, GSSG reductase, GSH S-transferase and catalase. To further examine the protective effects of the induced endogenous antioxidants against oxidative cell injury, H9C2 cells were pre-treated with D3T and then incubated with xanthine oxidase (XO) plus xanthine, a system that generates ROS. We observed that D3T pre-treatment of H9C2 cells led to significant protection against XO/xanthine-induced cytotoxicity as determined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) reduction and morphological changes. Taken together, this study demonstrates for the first time that a number of endogenous antioxidants in cardiomyocytes can be induced by exposure to D3T, and that this chemical (drug) induction of cellular antioxidants is accompanied by markedly increased resistance to ROS-mediated cardiac cell injury.
M G Lee - One of the best experts on this subject based on the ideXlab platform.
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effects of acute renal failure induced by uranyl nitrate on the pharmacokinetics of 2 allylthio pyrazine a Chemoprotective Agent in rats the role of cyp3a23 induction
Research Communications in Molecular Pathology and Pharmacology, 2004Co-Authors: Eun Jeong Lee, Yoon Kim, So H Kim, Eun Jin Kim, Hye Chin Chung, Dong Hyun Kim, Sang G Kim, Inchul Lees, M G LeeAbstract:It has been reported that the total body clearance (CL) of 2-(allylthio)pyrazine (2-AP) was significantly faster after intravenous administration of 2-AP to rats pretreated with 3-methylcholanthrene, phenobarbital, and dexamethasone (main inducers of CYP1A1/2, CYP2B1/2, and CYP3A1/2, respectively, in rats) than those in respective control rats. It has also been reported that expression of CYP2E1 and CYP3A1(23) increased 2.3 and 4 times, respectively, in rats with acute renal failure induced by uranyl nitrate (U-ARF) compared with those in control rats. However, CYP1A2 and CYP2B1/2 expression was not changed. Therefore, it could be expected that the pharmacokinetics of 2-AP could be changed in rats with U-ARF due to increase in expression of CYP3A23 in the rats. After intravenous administration of 2-AP at a dose of 50 mg/kg to rats with U-ARF, the area under the plasma concentration-time curve from time zero to time infinity of 2-AP was significantly smaller (1030 versus 1360 microg min/ml) due to significantly faster CL of 2-AP (48.4 versus 36.8 ml/min/kg). This could be due to increased expression of CYP3A23 in rats with U-ARF.
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pharmacokinetic changes of intravenous 2 allylthio pyrazine a Chemoprotective Agent in rats with acute renal failure induced by uranyl nitrate
Research Communications in Molecular Pathology and Pharmacology, 2002Co-Authors: Eun Jeong Lee, Yoon Kim, So H Kim, Eun Jin Kim, Hye Chin Chung, Dong Hyun Kim, Inchul Lee, Sang G Kim, M G LeeAbstract:It was reported that the total body clearance (CL) of 2-(allylthio)pyrazine (2-AP) was significantly faster after intravenous administration of 2-AP to rats pretreated with 3-methylcholanthrene (an inducer of CYP1A1/2 and 2E1 in rats) than that in control rats. It was also found that the CYP2E1 increased 2-4 times in rats with acute renal failure induced by uranyl nitrate (U-ARF) compared with those in control rats. Therefore, it could be expected that the pharmacokinetics of 2-AP could be changed in rats with U-ARF. After intravenous administration of 2-AP, 50 mg/kg, to rats with U-ARF, the area under the plasma concentration-time curve from time zero to time infinity (AUC) of 2-AP was significantly smaller (1030 versus 1360 microg min/ml) and this could be due to significantly faster CL of 2-AP (48.4 versus 36.8 ml/min/kg). This could be due to increased CYP2E1 in rats with U-ARF. More studies are required to find increased metabolite(s) of 2-AP in rats with U-ARF.
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effects of enzyme inducers and inhibitor on the pharmacokinetics of intravenous 2 allylthio pyrazine a new Chemoprotective Agent in rats
Biopharmaceutics & Drug Disposition, 2000Co-Authors: Yoon Kim, So H Kim, M G LeeAbstract:In order to find what types of hepatic cytochrome P450 (CYP) isozymes are involved in the metabolism of 2-(allylthio)pyrazine (2-AP) in rats, enzyme inducers, such as phenobarbital, 3-methylcholanthrene, dexamethasone, or isoniazid, and an enzyme inhibitor, such as SKF 525-A were pretreated. After 1-min intravenous administration of 2-AP, 50 mg/kg, to rats pretreated with SKF 525-A (a non-specific CYP inhibitor in rats), the plasma concentrations were significantly higher, and the area under plasma concentration-time curve from time zero to time infinity (AUC) was significantly greater (1365 compared with 1034 microg min/mL) as a result of significantly slower total body clearance (Cl) (36.6 compared with 48.3 mL/min/kg) than those in control rats, indicating that 2-AP was metabolized by CYP isozymes. After 1-min intravenous administration of 2-AP, 50 mg/kg, to rats pretreated with dexamethasone (an inducer of CYP3A in rats), phenobarbital (an inducer of CYP2B1/2, 2C6, 2C7, and 3A1/2 in rats), and 3-methylcholanthrene (an inducer of CYP1A1/2 and 2A1 in rats), the plasma concentrations were significantly lower, and AUC was significantly smaller (27, 41 and 60% decrease, respectively, compared with respective control rats) owing to faster Cl [37 (p>0.05), 70 (p<0.001), and 150% (p<0.001) increase, respectively, compared with respective control rats].
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pharmacokinetics of a Chemoprotective Agent 2 allylthio pyrazine after intravenous and oral administration to rats hepatic and gastric first pass effects
Drug Metabolism and Disposition, 1999Co-Authors: Kye S Han, M G LeeAbstract:Pharmacokinetic parameters of 2-(allylthio)pyrazine (2-AP) were evaluated after i.v. administration of the drug (10, 20, 50, and 100 mg/kg body weight) and oral administration of the drug (10, 50, and 100 mg/kg body weight) to rats. The hepatic, gastric, and intestinal first-pass effects of 2-AP were also measured after i.v., intraportal, intraduodenal, and oral administration of the drug (10 and 50 mg/kg body weight) to rats. After i.v. administration, the pharmacokinetic parameters of 2-AP were dose-independent at the dose ranges studied. However, after oral administration, the dosenormalized total area under the plasma concentration-time curve from time zero to time infinity values (43.5, 125, and 205 μg min/ml, based on 10 mg/kg body weight) increased significantly with increasing doses. The extent of absolute oral bioavailability (F) values also increased with increasing oral doses; the values were 19.6, 56.7, and 93.6% for 10, 50, and 100 mg/kg body weight, respectively. The oral data above could be due to saturable hepatic, gastric, and/or intestinal first-pass effects. This was proved by saturable gastric first-pass effect (the significant area under the plasma concentration-time curve from time zero to time infinity difference between oral and intraduodenal administration of 2-AP); the values were 62.6 and 26.6% at 2-AP doses of 10 and 50 mg/kg body weight, respectively. Approximately 20% of oral dose was eliminated by liver (hepatic first-pass effect) for both oral doses of 10 and 50 mg/kg body weight. However, the first-pass effects of 2-AP in the intestine, heart, and lung were almost negligible. The low F value after oral administration of 2-AP at low dose (10 mg/kg body weight) was mainly due to gastric first-pass effect in rats.
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stability blood partition and protein binding of a Chemoprotective Agent 2 allylthio pyrazine
Research Communications in Molecular Pathology and Pharmacology, 1998Co-Authors: Kye S Han, S J Woo, Chang Hui Koo, M G LeeAbstract:The stability of 2-AP, a Chemoprotective Agent, in various pH solutions and human gastric juice, the blood partition of 2-AP between plasma and blood cells, and the factors influencing the binding of 2-AP to 4% human serum albumin (HSA) were evaluated. 2-AP was stable in human gastric juice and pH solutions ranging from 1 to 12, however, 2-AP was unstable in pH 13 solution; the disappearance rate constant was 0.00759/h. 2-AP reached equilibrium rapidly between plasma and blood cells of rabbit blood. The equilibrium plasma/blood cells partition ratios were independent of initial rabbit blood concentrations of 2-AP, 1, 5, and 10 micrograms/ml; the values were in the range of 5.99-11.8. Binding of 2-AP to 4% HSA was dependent on HSA concentration, incubation temperature, 'the buffer' pH, and addition of acetylsalicylic acid. The binding of 2-AP was independent of buffers containing various concentrations of chloride ion, heparin, and alpha-1-acid glycoprotein.
Thomas R. Sutter - One of the best experts on this subject based on the ideXlab platform.
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3h 1 2 dithiole 3 thione targets nuclear factor κb to block expression of inducible nitric oxide synthase prevents hypotension and improves survival in endotoxemic rats
Journal of Pharmacology and Experimental Therapeutics, 2006Co-Authors: Asok R Karuri, Thomas W. Kensler, B D Roebuck, Yong Huang, Sridevi Bodreddigari, Carrie Hayes Sutter, Thomas R. SutterAbstract:Septicemia is a major cause of death associated with noncoronary intensive care. Systemic production of nitric oxide (NO) by inducible nitric-oxide synthase (iNOS) is a major cause of hypotension and poor organ perfusion seen in septic shock. Here, we show that pretreatment of F344 rats with the cancer Chemoprotective Agent 3 H -1,2-dithiole-3-thione (D3T) blocks lipopolysaccharide (LPS)-mediated induction of hepatic iNOS and significantly reduces the associated serum levels of NO metabolites and enzyme markers of toxicity provoked by treatment with LPS. Immunohistochemical analysis shows that this protective effect is largely due to suppression of iNOS expression in hepatocytes. Importantly, pretreatment of animals with D3T blunts LPS-mediated hypotension and dramatically increases their survival. Inasmuch as iNOS expression can be regulated by nuclear factor (NF) κB, mechanistic studies show that D3T blocks NFκB nuclear translocation and DNA binding and that these effects are accompanied by changes in the levels of phospho-inhibitor of NFκB. 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 NFκB and iNOS pathways.
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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
Molecular Medicine, 2001Co-Authors: Mi Kyoung Kwak, Ken Itoh, Thomas R. Sutter, Thomas W. KenslerAbstract:BACKGROUND: The induction of phase 2 enzymes by dithiolethiones such as oltipraz is an effective means for achieving protection against environmental carcinogens in animals and humans. Transcriptional control of the expression of at least some of these protective enzymes is mediated through the antioxidant response element (ARE) found in the upstream regulatory region of many phase 2 genes. The transcription factor Nrf2, which binds to the ARE, appears to be essential for the induction of proto-typical phase 2 enzymes such as glutathione S-transferase (GST) Ya, Yp, and NAD(P)H: quinone reductase (NQO1) in vivo. MATERIALS AND METHODS: In the present study, 3H-1,2-dithiole-3-thione (D3T) was used as a potent model inducer whose effects on gene expression and chemopreventive efficacy have been extensively characterized in the rat. Over a dozen putative D3T-inducible genes were examined in wild-type and nrf2-disrupted mice by Northern blot hybridization and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis to elucidate whether loss of Nrf2 function also affects the induction of a broader representation of phase 2 and antioxidative enzymes. The effects of D3T on hepatic Nrf2 expression and localization were also examined in vivo by Northern blot hybridization, electromobility shift assay, and Western blot analysis. RESULTS: Specific activities of hepatic GST and NQO1 were increased by D3T in wild-type mice and were largely blunted in the nrf2-deficient mice. However, changes in levels of RNA transcripts following D3T treatment of nrf2-disrupted mice were multidirectional, dependent upon the particular gene examined. Although elevation of mRNAs for GST Ya, NQO1, microsomal epoxide hydrolase and gamma-glutamylcysteine synthetase regulatory chain were blocked in the mutant mice, elevation of GST Yp mRNA was largely unimpeded. Increases in levels of mRNA for the heavy and light chains of ferritin were only seen in the nrf2-disrupted mice. Transcript levels of UDP-glucuronyl-transferase 1A6, heme oxygenase-1, maganese superoxide dismutase, which were inducible in the wild-type mice, actually decreased in the mutant mice, whereas levels of mRNA for GST Yc, aflatoxin B1 aldehyde reductase and catalase decreased following D3T treatment in the mutant mice in the absence of any inductive effect by D3T in the wild-type mice. In wild-type mice, treatment with D3T lead to 3-fold increases in hepatic Nrf2 mRNA levels within several hours following dosing as assessed by Northern blot and RT-PCR analyses. Gel shift analyses with oligonucleotide probes for human NQO1 ARE, murine GST Ya ARE, and erythroid transcription factor (NF-E2) binding site showed increased intensity of binding with nuclear extracts prepared from livers of D3T-treated mice compared to vehicle-treated controls. Antibody to Nrf2 supershifted the DNA binding bands of these nuclear extracts. Moreover, immunoblot analysis indicated accumulation of Nrf2 in extracts prepared from hepatic nuclei of D3T-treated mice at the same time points. CONCLUSIONS: Nrf2 plays a central role in the regulation of constitutive and inducible expression of multiple phase 2 and antioxidative enzymes by Chemoprotective dithiolethiones in vivo, although patterns of response vary among different genes. Knowledge of the factors controlling the specificity of actions of enzyme inducers will be exceedingly helpful in the design and isolation of more efficient and selective Chemoprotective Agents.
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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 dithiole 3 thione
Molecular Medicine, 2001Co-Authors: Mi Kyoung Kwak, Ken Itoh, Thomas R. Sutter, Masayuki Yamamoto, Thomas W. KenslerAbstract:The induction of phase 2 enzymes by dithiolethiones such as oltipraz is an effective means for achieving protection against environmental carcinogens in animals and humans. Transcriptional control of the expression of at least some of these protective enzymes is mediated through the antioxidant response element (ARE) found in the upstream regulatory region of many phase 2 genes. The transcription factor Nrf2, which binds to the ARE, appears to be essential for the induction of prototypical phase 2 enzymes such as glutathione S-transferase (GST) Ya, Yp, and NAD(P)H: quinone reductase (NQO1) in vivo. In the present study, 3H-1,2-dithiole-3-thione (D3T) was used as a potent model inducer whose effects on gene expression and chemopreventive efficacy have been extensively characterized in the rat. Over a dozen putative D3T-inducible genes were examined in wild-type and nrf2-disrupted mice by Northern blot hybridization and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis to elucidate whether loss of Nrf2 function also affects the induction of a broader representation of phase 2 and antioxidative enzymes. The effects of D3T on hepatic Nrf2 expression and localization were also examined in vivo by Northern blot hybridization, electromobility shift assay, and Western blot analysis. Specific activities of hepatic GST and NQO1 were increased by D3T in wild-type mice and were largely blunted in the nrf2-deficient mice. However, changes in levels of RNA transcripts following D3T treatment of nrf2-disrupted mice were multidirectional, dependent upon the particular gene examined. Although elevation of mRNAs for GST Ya, NQO1, microsomal epoxide hydrolase and γ-glutamylcysteine synthetase regulatory chain were blocked in the mutant mice, elevation of GST Yp mRNA was largely unimpeded. Increases in levels of mRNA for the heavy and light chains of ferritin were only seen in the nrf2-disrupted mice. Transcript levels of UDP-glucuronyl-transferase 1A6, heme oxygenase-1, maganese superoxide dismutase, which were inducible in the wild-type mice, actually decreased in the mutant mice, whereas levels of mRNA for GST Yc, aflatoxin B1 aldehyde reductase and catalase decreased following D3T treatment in the mutant mice in the absence of any inductive effect by D3T in the wild-type mice. In wild-type mice, treatment with D3T lead to 3-fold increases in hepatic Nrf2 mRNA levels within several hours following dosing as assessed by Northern blot and RT-PCR analyses. Gel shift analyses with oligonucleotide probes for human NQO1 ARE, murine GST Ya ARE, and erythroid transcription factor (NF-E2) binding site showed increased intensity of binding with nuclear extracts prepared from livers of D3T-treated mice compared to vehicle-treated controls. Antibody to Nrf2 supershifted the DNA binding bands of these nuclear extracts. Moreover, immunoblot analysis indicated accumulation of Nrf2 in extracts prepared from hepatic nuclei of D3T-treated mice at the same time points. Nrf2 plays a central role in the regulation of constitutive and inducible expression of multiple phase 2 and antioxidative enzymes by Chemoprotective dithiolethiones in vivo, although patterns of response vary among different genes. Knowledge of the factors controlling the specificity of actions of enzyme inducers will be exceedingly helpful in the design and isolation of more efficient and selective Chemoprotective Agents.
Stephen G Chaney - One of the best experts on this subject based on the ideXlab platform.
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hplc and 31p nmr characterization of the reaction between antitumor platinum Agents and the phosphorothioate Chemoprotective Agent s 2 3 aminopropylamino ethylphosphorothioic acid wr 2721
Biochemical Pharmacology, 1995Co-Authors: Charles D Thompson, Steven D Wyrick, David J Holbrook, Stephen G ChaneyAbstract:Abstract In prior studies, we examined the effects of the radioprotective and Chemoprotective Agent WR-2721 [S-2-(3-aminopropylamino)ethylphosphorothioic acid] on the in vivo biotransformation of the cisplatin [cis-diamminedichloroplatinum(II)] analog ormaplatin [(d,l)trans-1-1,2-diaminocyclohexanetetrachloroplatinum(IV), Pt(dach)Cl4, (rmformerly called tetraplatin)]. Those data suggested that a direct interaction between WR-2721 and ormaplatin and/or the corresponding Pt(II) drug, Pt(dach)Cl2, may be occurring in vivo. This would be in contrast to the generally accepted hypothesis that WR-2721 is a prodrug that must first be converted by alkaline phosphatase to a free thiol compound, WR-1065, before any appreciable reactivity would be evident. However, the major biotransformation product observed in the peritoneal fluid, plasma, and all tissues was Pt(dach)(WR-1065). We report here on further investigations into the in vitro reactivity of Pt(dach) compounds with WR-2721 and WR-1065. Separation of reaction products resulting from incubation of Pt(dach)(malonato) with either WR-2721 or WR-1065 under physiological conditions gave profiles that were indistinguishable by reverse phase HPLC and cation exchange HPLC at two different pHs. 31P NMR characterization of the dephosphorylation of WR-2721 revealed essentially no loss of inorganic phosphate for up to 24 hr when incubated in unbuffered water at 30 °. In contrast, when incubated with a 1:1 molar ratio of cisplatin under the same conditions, the WR-2721 signal was decreased markedly in the first 5 min, and had disappeared almost completely by 1 hr. The signal corresponding to inorganic phosphate increased in parallel to the decrease in the WR-2721 signal. No intermediate formation of a complex containing both platinum and phosphate could be detected at any time. These data suggest that the reaction between WR-2721 and platinum complexes results in rapid dephosphorylation of WR-2721, and, consequently, that the reaction products formed with either WR-2721 or WR-1065 and Pt(II) complexes are identical.
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effect of the Chemoprotective Agent wr 2721 on disposition and biotransformations of ormaplatin in the fischer 344 rat bearing a fibrosarcoma
Cancer Research, 1995Co-Authors: D C Thompson, Steven D Wyrick, David J Holbrook, Stephen G ChaneyAbstract:The effects of the phosphorothioate Agent, WR-2721, have been investigated with respect to the biotransformations of ormaplatin in the Fischer 344 rat bearing a transplanted fibrosarcoma. A number of different paradigms of dosing route and schedule for the administration of the two Agents have been investigated. In the first group of experiments, WR-2721 (200 mg/kg, i.p.) was administered 30 min before ormaplatin (12.5 mg/kg, i.p.), and then peritoneal fluid, plasma, and tissues were harvested at 30 min after the ormaplatin administration. Our results suggest that a significant interaction between WR-2721 and ormaplatin is occurring in the peritoneal cavity. The interaction was evident in terms of both effects on distribution and disposition of total platinum and in alterations of the profiles of biotransformation products formed in the various tissues and fluids. Plasma protein binding of ormaplatin was decreased by 50% in the presence of WR-2721. Total platinum in the spleen was decreased by 66% and in the liver by 50%. There were no trends among the findings that would indicate any selectivity between tumor and nontumor tissue with respect to the effects of WR-2721 on the parameters measured. Subsequent investigations examined the effects of dosing the WR-2721 by the i.v. route while continuing with the i.p. administration of the ormaplatin. WR-2721 was administered either 30 or 5 min before the ormaplatin, and the plasma and tissues were harvested at 15, 30, or 60 min after ormaplatin administration. The reverse-phase HPLC peak, which behaved chromatographically as a Pt(dach)(WR-1065) standard, was less prominent after the i.v. administration of WR-2721 than it was after i.p. administration under any of the paradigms tested. There was again no evidence for selectivity between tumor and nontumor tissue in the findings from any of the paradigms. It is concluded that if WR-2721 is capable of selectively protecting nontumor tissue from the toxicities of platinum-based chemotherapy, it is doing so by some mechanism other than its selective uptake into normal tissue and subsequent nonspecific inactivation of any reactive cytosolic platinum species formed. Other possible mechanisms are briefly discussed.