Characterization of the Oxidative Responsive Element (ORE) in the human thioredoxin promoter
H. Masutani, M. Ueno, Y. Yamaguchi, T. Sasada, and J. Yodoi
Institute for Virus Research, Kyoto University 53 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-01, Japan
Adult T cell leukemia derived factor (ADF)/human thioredoxin (TRX), which has thiol reducing and radical scavenging activities, plays an essential role on cellular protection against oxidative stress and cell death. TRX as well as Ref-1 potentiated the sequence specific binding of p53 to the consensus binding site of p53, suggesting that TRX protects cells against oxidative stress not only as a radical scavenger but via p53 function. TRX itself is induced by various oxidative stress including hydrogen peroxide, thiol oxidizing agent (diamide), menadione, UV or X-irradiation, and hemin, but not by heat shock. To investigate the mechanism of this induction, the promoter region of the TRX gene was analyzed. Oxidative agents activated the TRX promoter through a newly identified oxidative responsive element (ORE). Complexes CI, CII, CIII were detected in an electrophoretic mobility shift assay using the ORE as a probe. Binding proteins to the ORE were purified to homogeneity from nuclear extracts of Jurkat T cells. The binding was not competed by oligonucleotide encoding the heat shock element, suggesting that TRX is induced by oxidative stress through novel Oxidative Response Factors (ORFs) distinct from Heat shock factors.
Direct evidence for catalase as the predominant H2O2removing enzyme in human erythrocytes
Sebastian Mueller *, Hans-Dieter Riedel, and Wolfgang Stremmel
*Department of Molecular Pharmacology and Toxicology, University of Southern California, Los Angeles, CA, USA, and Department of Internal Medicine IV, University of Heidelberg, Heidelberg, Germany
The erythrocyte is a traditional target for studying the metabolism of H2O2, which is considered an oxygen key metabolite with importance in cell toxicity and, more recently, in cell signaling cascades. Since the first description of glutathione peroxidase (GPO), a debate exists on whether catalase or GPO is the primary enzyme in the removal of H2O2 in erythrocytes. Most drawbacks in studies of H2O2 decomposition stem from the lack of sensitive, direct and nontoxic H2O2 assays. Here, we have studied the decomposition of H2O2 at physiological levels in human erythrocytes by means of a recently developed sensitive H2O2 assay (luminol-hypochlorite assay). The exponential decay of H2O2 in the presence of purified erythrocyte catalase was followed down to 109 M H2O2 at pH 7.4. H2O2 decomposition by purified erythrocyte GPO could be directly observed down to 10-7 M H2O2. No enzyme inhibition was observed at these low H2O2 concentrations. Catalase and GPO activities can be determined separately in a titrated mixture of purified enzymes, which simulates the conditions of H2O2 removal by the erythrocyte. Experiments with fresh human hemolysate allowed us to determine H2O2 decomposition by catalase and GPO using these enzymes in their original quantitative ratio. The different kinetics of these enzymes are demonstrated: H2O2 decomposition by catalase depends linearly on H2O2 concentration, whereas that by GPO becomes saturated at concentrations above 10-6 M H2O2. At submicromolar H2O2 concentrations, the rate of H2O2 removal is 8% of that by catalase. These data indicate an almost exclusive role for catalase in the removal of H2O2 in normal human erythrocytes.
Mueller S, Arnhold J (1995) J. Biolumin. Chemilumin. 10(4):229-37
Mueller S, Riedel DH, Stremmel W (1997 Anal. Biochem. 245, 55-60
Mueller S, Riedel HD, Stremmel W (1997) Blood 90, 4973-497
Role of p53 in the induction of p21 by redox-cycling anticancer quinones
Sebastian Mueller, Ray-Chang Wu*, Axel H. Schnthal*, and Enrique Cadenas
Department of Molecular Pharmacology and Toxicology, School of Pharmacy, *Department of Molecular Microbiology and Immunology, School of Medicine, University of Southern California, Los Angeles, CA 90033
Quinones are the second largest family of anticancer drugs clinically used in the United States. Their exact mode of action is not completely understood. We have earlier shown that DZQ leads to the increased expression of p21 protein, an inhibitor of cyclin-dependent kinases which has been established as an important negative regulator of the cell cycle. Here, we show that in HL-60 myeloid leukemia cells that lack the tumor supressor p53, p21 protein is below the detection level. However, restoration of p53 status in HL60 cells (HL60-p53) significantly increases basal levels of p21 and p21 induction by DZQ. The importance of functional p53 for the p21 induction is further demonstrated in an immortalized p53 knockout cell line (MEF p53-/-) and in a cell line with mutated p53 (HT-29): no induction of p21 by DZQ is observed in these cell lines.
Transient tranfection with p21-promotor-luciferase contructs indicate that the p21 induction by DZQ is mainly regulated at the transcriptional level. In cytotoxicity studies with several anticancer drugs and quinones the restoration of p53 increases the sensitivity to redox-cycling drugs (DZQ, daunorubicin), but not to alkylating agents (juglone, chlorambucil). The increased sensitivity of HL60-p53 cells to DZQ is accompanied by an increased generation of reactive oxygen species as measured by the lucigenin assay. The antioxidant N-acetylcysteine prevents the generation of reactive oxygen species, p53 and p21 induction, and cell cytotxicity by DZQ. Our results indicate, that the p53 p21 pathway and possibly reactive oxygen species may play a central role in mediating the cytotoxic effects of aziridinylbenzoquinones and other redox cycling anticancer drugs.
Qiu X, Forman HJ, Schnthal AH, Cadenas E (1996) J. Biol. Chem. 271, 31915- 31921
Qiu X, Cadenas E (1997) Arch. Biochem. Biophys. 346, 241-251
Qiu X, Schnthal AH, Cadenas E (1998): Free Rad. Biol. Med. (in press)
Ziad A. Muna, Lise Madsen, and Rolf K. Berge
Department of Clinical Biology, Division of Biochemistry, University of Bergen, Haukeland University Hospital, N-5021 Bergen, Norway
Tetradecylthioacetic acid (TTA) which can not be -oxidized, lowers plasma VLDL-triacylglycerol (TG) and LDL-cholesterol (Chol). Increased mitochondrial -oxidation with a concomitant decrease in TG synthesis and secretion, seems to be the primary mechanism underlying the hypotriglyceridemic effect not only of TTA but also of w-3 fatty acids as well as fibrates in rats, rabbits, dogs and possibly also in humans. TTA is an inhibitor of HMG-CoA reductase. We have generated results both in vivo and in vitro that present evidence that TTA besides being a lipid lowering agent, also possesses antioxidant properties. First, TTA inhibits the oxidative modification of LDL which is considered as the key step in the formation of foam cells and in initiation and progression of atherosclerotic plaque. Also TTA changes the antioxidant defense system in a beneficial way i.e. glutathion (GSH) is increased, the total antioxidant status is elevated and TBARS are decreased. Second, TTA has an olive oil effect since the plasma was enriched with oleic acid (18:1 n-9) and a 9-desaturated metabolite of TTA. This was due to upregulation of the hepatic enzyme 9-desaturase gene expression. Third, TTA lowers the plasma homocysteine level and inhibits restenosis. Fourth, TTA reduces the proliferation of smooth muscle cells. In conclusion, TTA is a hypolipidemic drug but also a new antioxidant. This novel bioactive compound is promising as a new therapeutic drug against atherosclerosis as it changes the plasma profile from atherogenic to cardioprotective.
M.P.N. Nair, *C. Kandaswam, A. Sweet, and S.A. Schwartz
Department of Medicine, State University of NY at Buffalo, Buffalo, NY 14203 and Canandaigua West Tech.Ctr, Madera,CA 93639*
Discovery of effective and less toxic antiviral agents is a high priority in the search for more effective therapies against HIV. Flavonoids and several other antioxidants possess antiviral, antiin flammatory and antitumor activities with no apparent toxic side effects on cells. We recently demonstrated that quercetin, a polyhydroxylated flavonoid, significantly inhibited the expression of the gene for the proinflammatory cytokine, TNFa and induced the gene expression of the anti-inflammatory cytokine, IL-13, by normal PBMC in a dose-dependent manner. Recent studies show that the chemokine receptors, CCR3 and CCR5, also serve as entry coreceptors for HIV-1. Although flavonoids possess antiviral effects, the molecular mechanisms underlying these activities remain to be elucidated. In the present studies we investigated the effect of quercetin on the constitutive expression of the CCR5 gene by normal PBMC. Cultures of PBMC received either media alone, or different concentrations of quercetin (0.1 to 100 M). After 24 hr of incubation, total RNA was extracted and reverse transcribed. The newly synthesized cDNA products were mixed with the housekeeping gene, G3PDH, plus CCR5 specific primers in a PCR assay. The PCR products were separated by 1.2% agarose gel electrophoresis containing ethidium bromide. Our results show that quercetin significantly suppressed the expression of the HIV-1 entry coreceptor, CCR5, by normal PBMC. Further investigation of the mechanisms underlying the anti-HIV effects of quercetin may help to identify promising natural products useful in the treatment of patients with HIV infections.
Effect of caffeic acid on t-BOOH induced oxidative stress in U937
M. Nardini, E. Piccolella, P. Pisu, and C. Scaccini
National Institute of Nutrition, Rome; Department of Cellular and Development Biology, University La Sapienza, Rome, Italy
The impairment of the antioxidant defense system is considered to be critically involved in a number of pathological conditions, such as atherosclerosis, cancer, inflammation, cataract. Diet can contribute to the overall redox balance being a source of natural antioxidants. Phenolic compounds are bioactive substances occurring widely in food plants and therefore potentially present in human plasma in a diet-dependent concentration. Among these, caffeic acid (CA) is found naturally in various agricultural products such fruits, vegetables, wine, olive oil, coffee beans. In previous studies we demonstrate the physiological relevance of caffeic acid and its antioxidant action both in vitro and in vivo. In this study we examined the effect of caffeic acid supplementation (50M) on cultured human monocyte (U937) cells and their resistance to t-butyl hydroperoxide (t-BOOH) induced toxicity. Preliminary results indicate that CA treatment does not induce significant differences in GSH and alpha-tocopherol content between treated and control cells. Moreover, CA was incorporated into cells at concentration ranging from 1 to 10 ng/mg protein. Cells treated with CA were somewhat more resistant against t-BOOH induced cytotoxicity than control cells, as demonstrated by the time course of cell viability, determined by Trypan blue exclusion. The intracellular glutathione level in control cells significantly dropped to about 50% of the initial value after 2-h treatment with t-BOOH, while in CA-treated cells only a slight decrease was observed (CA-treated cells: from 22.7 4.0 to 16.0 3.5 nmol/mg protein, ns; control cells: from 21.5 4.3 to 11.1 1.2 nmol/mg, P= 0.0174). Moreover a markedly longer lag phase was observed in the kinetic of TBA-RS formation in CA-treated cells than in control cells, resulting in TBA-RS levels after 2 h incubation about one third of those found in control cells. No statistically significant differences were observed in alpha-tocopherol concentration after 2h incubation (CA-treated cells: 84.5 17.1 % residual; control cells 74.6 10.5 % , P=0.343) with t-BOOH. CA did not affect proliferation rate in U937. Nevertheless treatment with t-BOOH (1 h) resulted in 50% inhibition of cell growth in control cells in respect to CA-treated cells. In conclusion, the overall preliminary data support the view that, under conditions of t-BOOH challenge, caffeic acid is able to effectively protect cells from oxidative injury.
The respiratory burst in activated rat neutrophils: Effects of lipoic acid and magnetic fields
Y. Noda, R.P. Liburdy*, A. Mori, and L. Packer
University of California, Berkeley, CA, USA and the *Lawrence Berkeley National Laboratory, Berkeley, CA, USA.
The role of lipoic acid on free radical generation in PMA-activat ed rat neutrophils is being investigated in these studies. We have observed that pretreatment of rat neutrophils with lipoic acid significantly enhances free radical production during the respiratory burst in a dose-dependent manner (0-500 mM). In these studies we have loaded cells with the fluorescent probe DCFH-DA to follow production of reactive oxygen species in real time during the respiratory burst. We are using reagents such as oxypurinol, an inhibitor of xanthine oxidase, and structural analogs of lipoic acid to investigate the influence on lipoic acid on the respiratory burst. Additional studies have employed the reduced form of lipoic acid to pretreat cells; preliminary observations suggest that this form of lipoic acid appears less effective in enhancing the respiratory burst. We have conducted parallel studies to investigate the potential effects of magnetic fields on activated neutrophils that have been preteated with lipoic acid, or not. In these studies we have been employed a cuvette-based, magnetic field exposure system that permits continuous field exposure during fluorescence measurements. We have previously observed that a 1 Gauss (60Hz) magnetic field alone can significantly increase the respiratory burst [FEBS Letters (1995) 376: 164-166]. Experiments are underway in which we are examining the combined effect of lipoic acid plus magnetic fields on the respiratory burst. In these studies we are varying field strength from 0-20 Gauss (rms), 60Hz.
Nitric oxide scavenging activity of melatonin and its presursors
Y. Noda, A. Mori, and L. Packer
251 LSA, Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, CA 94720-3200, U.S.A.
Melatonin, which exists in pineal body, protects against oxidative stress-induced DNA damage, lipid peroxidation, and apoptosis. This compound has functions such as (i) hydroxyl and peroxy radical scavenging activities, (ii) stimulation of gluthathione peroxidase, and (iii) inhibition of nitric oxide synthase. However, direct evidence of nitric oxide scavenging activity is not yet known. We hypothesized that this indol compound could also directly scavenge nitric oxide.
In this in vitro study, the possible nitric oxide scavenging activities of melatonin including its precursors were examined. Melatonin, N-acetyl-5-hydroxytryptamin (N-acetylserotonin), serotonin, 5-hy- droxytryptophan and L-tryptophanwere examined using flow injection analysis. 1-Hydroxy-2-oxo-3-(N-methyl-3-aminopropyl)-3-me- thyl-1-triazene (NOC-7) was used as a nitric oxide generator. These compounds, except L-tryptophan, scavenged nitric oxide in a dose- dependent manner during the incubation with NOC-7. Melatonin, which has the methoxy group and the acetyl group, showed most potent scavenging activity. Serotonin, N-acetylserotonin and 5-hy droxy-tryptophan scavenged nitric oxide moderately. These results imply that melatonin may also have a neural regulation by the direct scavenging activity of nitric oxide. Physiological relevance is being investigated.
I.G. Obrosova, M.J. Stevens, and D.A. Greene
Department of Internal Medicine, University of Michigan, Ann Arbor, MI, USA
VEGF production, neovascularization, and vascular proliferation in diabetic retinopathy (DR) have been attributed either to decreased retinal blood flow (RBF) and hypoxia, or to increased RBF and "pseudohypoxia" (decrease in free cytosolic NAD+/NADH ratio putatively associated with increased conversion of sorbitol to fructose by sorbitol dehydrogenase). In consideration of importance of the current debate for understanding the pathogenetic mechanisms of DR and development of therapeutic strategies, we compared the effects of two potential retinal vasodilators: prazosin (P), a vasoactive a1-adreno ceptor antagonist that does not alter sorbitol pathway activity, and DL a-lipoic acid (LA), an antioxidant that also increases sorbitol pathway metabolites, on mitochondrial and cytosolic redox state of NAD(P) couples in diabetic retina. Control (C) and 3-wk STZ-diabetic (D) rats were treated with/without P (5 mg/kg/d, in the drinking water), as well as C and 6-wk STZ-D rats were treated with/without LA (100 mg/kg/d, i.p.). Glucose, sorbitol, fructose, lactate, pyruvate, malate, glutamate, a ketoglutarate, and ammonia were assayed spectrofluorometrically in individual retinas by enzymatic procedures. Free mitochondrial NAD+/NADH, free cytosolic NAD+/NADH and NADP+/NADPH ratios were calculated from the glutamate dehydrogenase, lactate dehydrogenase and malic enzyme systems. The data are presented as mean SD. Free mitochondrial redox state of NAD-couples (an indicator of tissue oxygenation) was decreased in both 3-(11.73.9) and 6-wk D (16.39.0) vs C (48.912.3 and 48.017.8, p<0.01 for both), and this reduction was ameliorated by both P and LA (23.54.3 and 38.015.5, p <0.01 and <0.05 vs D). Free cytosolic NAD+/NADH ratios were decreased to 22936 and 28650 in 3- and 6-wk D (vs 349 58 and 437118 in C, p<0.01 for both) and were ameliorated by both P and LA (31541 and 462112, p<0.01 vs D for both) in spite of the fact that P did not affect and LA even further increased the sorbitol pathway metabolites (levels of glucose, sorbitol, and fructose were 2-, 1.8-, and 1.6-fold higher in D+LA vs D). Free cytosolic NADP+/ NADPH ratios were decreased in both 3- and 6-wk D vs C, and this decrease was prevented by P but not LA. The findings support a role for hypoxia rather than "pseudohypoxia" in diabetes-induced changes of retinal NAD(P)-redox status, and evoke a possibility of a1-adreno ceptor involvement in the regulation of RBF in diabetes. They provide metabolic indication for amelioration of BF in the diabetic retina by both a1-adrenoceptor antagonist and DL-a-lipoic acid therapy. Both compounds are recommended for further studies in experimental DR, to assess their efficacy in preventing VEGF production and neo-vascularization.
Chloramines modulate protein kinase C activity
Tetsuya Ogino, Hirotsugu Kobuchi, Chandan K. Sen, Sashwati Roy, Lester Packer, and John J. Maguire
Department of Molecular and Cell Biology and Environmental Energy Technologies Division Lawrence Berkeley National Laboratory, University of California, Berkeley, CA 94720
Monochloramine derivatives are long-lived physiological oxidants produced by neutrophils during the respiratory burst. The effects of chemically prepared monochloramine (NH2Cl) on protein kinase C (PKC) and PKC-mediated cellular responses were studied in elicited rat peritoneal neutrophils and human Jurkat T cells. Neutrophils pretreated with NH2Cl (30-50 M) showed a marked decrease in the respiratory burst activity induced by phorbol 12-myristate 13-acetate (PMA), which is a potent PKC activator. These cells, however, were viable and showed a complete respiratory burst upon arachidonic acid stimulation, which induces the respiratory burst by a PKC-indepen dent mechanism. The NH2Cl-treated neutrophils showed a decrease in both PKC activity and PMA-induced phosphorylation of a 47 kDa protein, which corresponds to the cytosolic factor of NADPH oxidase, p47phox. Jurkat T cells pretreated with NH2Cl (20-70 M) showed a decrease in the expression of the interleukin-2 receptor chain following PMA stimulation. This was also accompanied by a decrease in both PKC activity and nuclear transcription factor-B activation, also without loss of cell viability. These results show that NH2Cl inhibits PKC-mediated cellular responses through inhibition of the inducible PKC activity. J. Biol. Chem. (1997) 272: 26247-26252