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• W2005891669 abstract "Phenolic antioxidants exhibit anti-inflammatory activity in protection against chemical toxicity and cancer. To investigate the molecular mechanism of anti-inflammation, we analyzed the regulation of tumor necrosis factor α (TNF-α) expression in macrophages, a key step in inflammation, by the antioxidants. Whereas lipopolysaccharide (LPS), an inflammatory inducer, stimulates rapid synthesis of TNF-α protein, phenolic antioxidants, exemplified bytert-butyl hydroquinone and 1,4-dihydroquinone, block LPS-induced production of TNF-α protein in a time- and dose-dependent manner. Inhibition of TNF-α induction correlates with the capacity of the antioxidants to undergo oxidation-reduction cycling, implicating oxidative signaling in the inhibition. The antioxidants blocked LPS-induced increase of the steady-state mRNA of TNF-α but did not affect the half-life of the mRNA. Electrophoretic mobility shift assay reveals a total inhibition of LPS-induced formation of nuclear factor κB·DNA binding complexes by phenolic antioxidants. Finally, 1,4-dihydroquinone blocks the induction of TNF-α target genes interleukin 1β and interleukin 6 at both mRNA and protein levels. Our findings demonstrate that phenolic antioxidants potently inhibit signal-induced TNF-α transcription and suggest a mechanism of anti-inflammation by the antioxidants through control of cytokine induction during inflammation. Phenolic antioxidants exhibit anti-inflammatory activity in protection against chemical toxicity and cancer. To investigate the molecular mechanism of anti-inflammation, we analyzed the regulation of tumor necrosis factor α (TNF-α) expression in macrophages, a key step in inflammation, by the antioxidants. Whereas lipopolysaccharide (LPS), an inflammatory inducer, stimulates rapid synthesis of TNF-α protein, phenolic antioxidants, exemplified bytert-butyl hydroquinone and 1,4-dihydroquinone, block LPS-induced production of TNF-α protein in a time- and dose-dependent manner. Inhibition of TNF-α induction correlates with the capacity of the antioxidants to undergo oxidation-reduction cycling, implicating oxidative signaling in the inhibition. The antioxidants blocked LPS-induced increase of the steady-state mRNA of TNF-α but did not affect the half-life of the mRNA. Electrophoretic mobility shift assay reveals a total inhibition of LPS-induced formation of nuclear factor κB·DNA binding complexes by phenolic antioxidants. Finally, 1,4-dihydroquinone blocks the induction of TNF-α target genes interleukin 1β and interleukin 6 at both mRNA and protein levels. Our findings demonstrate that phenolic antioxidants potently inhibit signal-induced TNF-α transcription and suggest a mechanism of anti-inflammation by the antioxidants through control of cytokine induction during inflammation. tert-butylhydroquinone 1,4-dihydroquinone lipopolysaccharide N-acetylcysteine tumor necrosis factor α interleukin heme oxygenase-1 NfE2-related factor enzyme-linked immunosorbent assay nuclear factor κB activator protein reactive oxygen species antioxidant response element nuclear factor of activated T cells TGF-β-activating kinase MAPK kinase kinase A variety of chemicals can protect animals against the toxic and carcinogenic effects of chemicals and microbes (1Wattenberg L.W. Cancer Res. 1985; 45: 1-8Crossref PubMed Scopus (166) Google Scholar, 2Yang C.S. Landau J.M. Huang M.T. Newmark H.L. Annu. Rev. Nutr. 2001; 21: 381-406Crossref PubMed Scopus (1113) Google Scholar), a phenomenon termed chemoprotection. Many protective chemicals manifest antioxidant activities (3Garewal H.S. Antioxidants and Disease Prevention (Modern Nutrition Series). CRC Press, New York1997: 1-186Google Scholar, 4Ahmad S. Oxidative Stress and Antioxidant Defenses in Biology. Chapman & Hall, New York1995: 1-457Crossref Google Scholar). Synthetic phenolic antioxidants, such as tBHQ,1 are commonly used as food preservatives due to their potent anti-lipid peroxidation activity (5Verhugen H. Schilderman P.A.E.L. Kleinjans J.C.S. Chem. Biol. Interact. 1991; 80: 109-134Crossref PubMed Scopus (80) Google Scholar, 6Nunn C.J. Verhagen H. Kleinjans J.C.S. Food Chem. Toxicol. 1991; 29: 73-75Crossref PubMed Scopus (10) Google Scholar), whereas natural phenolic antioxidants exist in a wide range of edible plants and animal tissues (7Bishop A. Gallop P.M. Karnovsky M.L. Nutr. Rev. 1998; 56: 287-293Crossref PubMed Scopus (37) Google Scholar). As such, human beings consume appreciable amounts of phenolic antioxidants from dietary sources. Phenolic antioxidants exhibit anticarcinogenic, anti-inflammatory, antiatherosclerotic, and antidiabetic functions in animals (8Wattenberg L.W. J. Natl. Cancer Inst. 1972; 48: 1425-1430PubMed Google Scholar, 9Ulland B.M. Weisburger J.H. Yammanoto R.S. Weisburger E.K. Food Cosmet. Toxicol. 1973; 11: 199-207Crossref PubMed Scopus (149) Google Scholar, 10Hirose M. Ito T. Takahashi S. Ozaki M. Ogiso T. Nihro Y. Miki T. Shirai T. Eur. J. Cancer Prev. 1998; 7: 233-241Crossref PubMed Scopus (25) Google Scholar, 11Nagakawa J. Hirota K. Hishinuma I. Miyamoto K. Sonoda J. Yamanaka T. Katayama K. Yamatsu I. J. Pharmacol. Exp. Ther. 1993; 264: 496-500PubMed Google Scholar, 12Nagakawa J. Hishimuma I. Miyamoto K. Hirota K. Abe S. Yamanaka T. Katayama K. Yamatsu I. J. Pharmacol. Exp. Ther. 1992; 262: 145-150PubMed Google Scholar, 13Bjorkhem I. Henriksson-Freyschuss A. Breuer O. Diczfalusy U. Berglund L. Henriksson P. Arterioscler. Thromb. 1991; 11: 15-22Crossref PubMed Google Scholar, 14Nishizono S. Hayami T. Ikeda I. Imaizumi K. Biosci. Biotechnol. Biochem. 2000; 64: 1153-1158Crossref PubMed Scopus (39) Google Scholar). The broad spectrum of the biological functions of the antioxidants suggests the existence of multiple molecular targets that mediate diverse responses to the chemicals in cells and whole animals. Identifying the target molecules can facilitate the design of better preventive and/or therapeutic agents for protection against pathological responses to various occupational, environmental, or therapeutic chemicals as well as against certain conditions associated with aging, such as cancer and chronic inflammatory and cardiovascular diseases. Current understanding of the mechanism of action by phenolic antioxidants comes mostly from studies on the induction of phase II detoxification enzymes by the antioxidants. Phase II enzymes, such as NAD(P)H:quinone oxidoreductase (15Ernster L. Estabrook R.W. Hochstein P. Orrenius S. DT-diaphorase: A Quinone Reductase with Special Functions in Cell Metabolism and Detoxication. Vol. 27A. Cambridge University Press, New York1987: 1-207Google Scholar, 16Ma Q. Cui K. Xiao F. Lu A.Y.H. Yang C.S. J. Biol. Chem. 1992; 267: 22298-22304Abstract Full Text PDF PubMed Google Scholar) and glutathioneS-transferase (17Pickett C.B. Lu A.Y.H. Annu. Rev. Biochem. 1989; 58: 743-764Crossref PubMed Scopus (547) Google Scholar), metabolize chemicals to water-soluble products through reduction and conjugation reactions. Induction of phase II enzymes alters the metabolic fate of chemicals by enhancing their metabolism and excretion. Induction of glutathioneS-transferase A1 and NAD(P)H:quinone oxidoreductase by phenolic antioxidants requires an ARE located in the enhancers of the genes (18Favreau L. Pickett C. J. Biol. Chem. 1991; 266: 4556-4561Abstract Full Text PDF PubMed Google Scholar, 19Rushmore T.H. King R.G. Paulson K.E. Pickett C.B. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3826-3830Crossref PubMed Scopus (389) Google Scholar) and is mediated through an Nrf2-dependent signal transduction (20Itoh K. Chiba T. Takahashi S. Ishii T. Igarashi K. Katoh Y. Oyake T. Hayashi N. Satoh K. Htayama I. Yamamoto M. Nabeshima Y. Biochem. Biophys. Res. Commun. 1997; 236: 313-322Crossref PubMed Scopus (3145) Google Scholar). Nrf2 is a member of the Cap'n Colar bZip family of transcription factors (21Chan K. Kan Y.W. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12731-12736Crossref PubMed Scopus (522) Google Scholar) that forms a cytoplasmic complex with Keap1 (22Itoh K. Wakabayashi N. Katoh Y. Ishii T. Igarashi K. Engel J.D. Yamamoto M. Genes Dev. 1999; 13: 76-78Crossref PubMed Scopus (2734) Google Scholar). Upon exposure to phenolic chemicals, Nrf2 dissociates from Keap1, translocates into the nucleus, dimerizes with an as-yet-unidentified partner transcription factor, and mediates the transcription of target genes through ARE-dependent transcription. Studies on Nrf2-null mice revealed that loss of expression of Nrf2 markedly enhances the susceptibility of the mice to toxicity by acetaminophen (23Chan K. Han X.-D. Kan Y.W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4611-4616Crossref PubMed Scopus (643) Google Scholar) or cancer by benzo(a)pyrene (24Ramos-Gomez M. Kwak M.-K. Dolan P.M. Itoh K. Yamamoto M. Talalay P. Kensler T.W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3410-3415Crossref PubMed Scopus (979) Google Scholar), which is attributable to diminished detoxification of the chemicals in the Nrf2-null mice. Thus, induction of phase II enzymes through Nrf2 can account for chemoprotection by phenolic antioxidants against certain carcinogens and toxins by altering the pharmacokinetic fate of the chemicals. However, such mechanisms do not readily explain the anti-inflammatory function of the antioxidants, which is largely unaddressed at present. The importance of inflammation in the pathological responses to chemicals of endogenous or foreign origin is well recognized because more diseases, ranging from silicosis, asthma, and chronic hepatitis to idiopathic pulmonary fibrosis, Alzheimer's disease, and certain forms of neoplasia, manifest an inflammatory component that either causes or increases the severity of the diseases (25Luster M.I. Environ. Health Perspect. 1998; 106: A418-A419Crossref PubMed Google Scholar, 26Wills-Karp M. Annu. Rev. Immunol. 1999; 17: 255-281Crossref PubMed Scopus (955) Google Scholar, 27Pohl L.R. Pumford N.R. Martin J.L. Eur. J. Haematol. 1996; 60: 98-104Google Scholar, 28Laskin D.L. Pendino K.J. Annu. Rev. Pharmacol. Toxicol. 1995; 35: 655-677Crossref PubMed Scopus (579) Google Scholar, 29Locksley R.M. Killeen N. Lenardo M.J. Cell. 2001; 104: 487-501Abstract Full Text Full Text PDF PubMed Scopus (2984) Google Scholar). Inflammation, in general, is a directed tissue response to tissue damage caused by noxious and injurious stimuli. Inflammation serves to isolate the injured tissue, inactivate the toxic stimuli, and repair the tissue damage. In many cases, however, inflammation can lead to further tissue damage and the development of inflammatory diseases. Thus, inhibition of inflammation constitutes an effective mechanism of chemoprotection against chemical-induced inflammatory lesions or disease conditions. Inflammatory stimuli induce cytokines, which mediate tissue responses in different phases of inflammation in a sequential and concerted manner (28Laskin D.L. Pendino K.J. Annu. Rev. Pharmacol. Toxicol. 1995; 35: 655-677Crossref PubMed Scopus (579) Google Scholar). Regulation of cytokine induction serves as a key mechanism of inflammation control by endogenous or exogenous chemicals. TNF-α is produced in the early phase of inflammation in cells of reticuloendothelial origin, such as macrophages. TNF-α mediates early-stage responses of inflammation by regulating the production of other cytokines, including IL-1β and IL-6. Cumulative evidence indicates that abnormalities in the production or function of TNF-α play essential roles in many inflammatory lesions (25Luster M.I. Environ. Health Perspect. 1998; 106: A418-A419Crossref PubMed Google Scholar, 29Locksley R.M. Killeen N. Lenardo M.J. Cell. 2001; 104: 487-501Abstract Full Text Full Text PDF PubMed Scopus (2984) Google Scholar, 30Feldmann M. Maini R.N. Annu. Rev. Immunol. 2001; 19: 163-196Crossref PubMed Scopus (1145) Google Scholar). For instance, exposure to the bacterial endotoxin LPS can cause inflammatory liver damage and septic shock. Induction of TNF-α by LPS is a key step in the response; administration of TNF-α mimics the response, whereas blocking the function of TNF-α by using neutralizing agents of TNF-α protects animals from the LPS-induced lesions. The pivotal role of TNF-α in inflammation and the potent anti-inflammatory activity of phenolic antioxidants raise the question of whether the induction of TNF-α during inflammation serves as a target of anti-inflammation by phenolic antioxidants. In this study, we tested this hypothesis by examining the effect of phenolic antioxidants on the induction of TNF-α by LPS in macrophage cells. Our data reveal that phenolic antioxidants block LPS-induced expression of TNF-α both time- and dose-dependently; the inhibition occurs at a transcriptional level and involves inhibition of NF-κB activation, the major regulator of TNF-α transcription in macrophage cells. To our knowledge, this study is the first report of inhibition of signal-induced TNF-α production by phenolic chemicals. Our findings provide new insights into the mechanism of chemoprotection against inflammatory diseases by phenolic antioxidants. Restriction endonucleases and other general molecular biology reagents were purchased from New England Biolabs (Beverly, MA), Roche Molecular Biochemicals, and Promega (Madison, WI). Radioactive compounds were from Amersham Biosciences, Inc. LPS, tBHQ, HQ, catechol, resorcinol, para-benzoquinone, α-naphthoflavone, and NAC were purchased from Sigma. Cell culture materials were from Invitrogen. Reagents for Northern blotting and ELISA are as described below. The mouse monocyte-macrophage RAW 264.7 cell line was purchased from American Type Culture Collection (Manassas, VA). The macrophage cells were grown as a monolayer in Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum at 5% CO2 and 37 °C. The cells were treated with LPS, phenolic antioxidants, or other reagents as described in the figure legends; Me2SO or water was used as the solvent control for the antioxidants and LPS, respectively. cDNA fragments for mouse TNF-α, IL-1β, IL-6, and HO-1 were synthesized by PCR amplification of the corresponding cDNA templates fromCLONTECH using the primer sets specific for each mouse gene from the same company. The cDNAs were subcloned into the pCRII TA cloning vector (Invitrogen), verified by sequencing, and used to generate riboprobes for the corresponding mRNA species. A mouse actin cDNA fragment (∼500 bp) was used to generate a riboprobe for actin. The riboprobes were synthesized in the presence of digoxygenin-UTP using a digoxygenin labeling kit (Roche Molecular Biochemicals). Total RNA was isolated from cells using the RNeasy kit (Qiagen, Valencia, CA). Total RNA (5 μg each lane) was fractionated on a 1% agarose-formaldehyde gel and transferred to a Nytran membrane by capillary action. After UV cross-linking, the membrane was hybridized overnight with a digoxygenin-labeled riboprobe at 68 °C. Signals were visualized by chemiluminescence using a digoxygenin RNA detection kit with CDP star as a substrate (Roche Molecular Biochemicals). Quantitation of the blotting result was performed by using the ImageQuaNT program (Molecular Dynamics, San Jose, CA). All data were corrected for loading variations by comparing the amount of actin of each sample analyzed. The macrophage cells were plated in a 48-well cell culture dish at a density of 2 × 105 cells/well in 500 μl of medium. The cells were grown at 37 °C for 24 h before treatment. In a typical experiment, the cells were treated with a phenolic antioxidant or Me2SO for 1 h, followed by stimulation with LPS for 5 h. The medium was collected and assayed for TNF-α, IL-1β, or IL-6 by using ELISA kits specific for each of the murine cytokines from R&D Systems (Minneapolis, MN). For TNF-α and IL-6, the medium was diluted with water at a 1:20 or 1:10 ratio, respectively; 50 μl of each diluted sample was used for ELISA. Quantitation of the ELISA results was performed using a Microplate Spectrophotometer (Molecular Devices, Sunnyvale, CA) set to 450 nm and corrected for absorbance at 570 nm according to the manufacturer's instructions. The nuclear extracts were prepared with a three-step procedure as described elsewhere (31Ye J. Shi X. Jones W. Rojanasakul Y. Cheng N. Schwegler-Berry D. Baron P. Deye G.J. Li C. Castronova V. Am. J. Physiol. 1999; 276: L426-L434Crossref PubMed Google Scholar). Briefly, the cells were grown in a 100-mm dish to near confluence. After treatment, the cells were collected with a rubber policeman, washed with 1× phosphate-buffered saline, and lysed in 500 μl of a lysis buffer on ice for 4 min. The lysis buffer contains 50 mm KCl, 0.5% Nonidet P-40, 25 mm HEPES, pH 7.8, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 20 μg/ml aprotinin, and 100 μm1,4-dithiothreitol. The cell lysate was centrifuged at 14,000 rpm for 1 min in a microcentrifuge. In the second step, the pellet (the nuclei fraction) was washed once in washing buffer (lysis buffer without Nonidet P-40). In the final step, the nuclei were treated with an extraction buffer, which contains 500 mm KCl, 10% glycerol, and several other reagents as in the lysis buffer. The nuclei/extraction buffer mixture was frozen at −80 °C and then thawed on ice and centrifuged at 14,000 rpm for 5 min. The supernatant was collected as the nuclear extract and stored at −80 °C for further use. A NF-κB DNA-binding sequence (5′-GATTTTCCCATGAGTCT-3′) was used to synthesize oligonucleotides as the NF-κB binding probe (31Ye J. Shi X. Jones W. Rojanasakul Y. Cheng N. Schwegler-Berry D. Baron P. Deye G.J. Li C. Castronova V. Am. J. Physiol. 1999; 276: L426-L434Crossref PubMed Google Scholar). The complementary single-strand oligomers were denatured at 80 °C for 5 min and annealed at room temperature. An activator protein (AP)-1 DNA-binding sequence derived from the AP-1 binding site in the collagenase enhancer was used as a nonspecific competitor probe (31Ye J. Shi X. Jones W. Rojanasakul Y. Cheng N. Schwegler-Berry D. Baron P. Deye G.J. Li C. Castronova V. Am. J. Physiol. 1999; 276: L426-L434Crossref PubMed Google Scholar). Double-stranded oligonucleotide probes were labeled with [32P]ATP using T4 kinase (New England Biolabs). The DNA-protein binding reaction was conducted in a 24-μl reaction mixture containing 1 μg of poly(dI-dC) (Sigma), 3 μg of nuclear extract, 3 μg of bovine serum albumin, and 12 μl of a reaction buffer (12% glycerol, 24 mm HEPES, pH 7.9, 8 mm Tris-HCl, 2 mm EDTA, and 1 mm 1,4-dithiothreitol). The mixture was incubated on ice for 10–20 min, followed by the addition of 4 × 104 counts/min of a 32P-labeled oligonucleotide probe; the incubation was continued at room temperature for 20 min. For competition experiments, 100 ng of cold (unlabeled) double-stranded NF-κB or AP-1 probe was added to the reaction mixture for competition with labeled NF-κB probe. For supershift assays, antibodies specific for the p50 or p65 subunit of NF-κB, c-Jun, NF-AT, or c-Fos were added to the reaction mixture respectively (2 μg/reaction). The antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The DNA·protein complexes were resolved in a 5% acrylamide gel (pre-run at 170 V for 30 min with 0.5× Tris-boric acid-EDTA buffer) at 200 V for 90 min and visualized by exposure to films. Macrophage cells were plated in a 6-well dish at 5 × 105 cells/well 16 h before treatment. 2′,7′-Dichlorofluorescin diacetate (5 μm; Sigma) was used to stain cells for H2O2 for 45 min before the treatment was completed. The cells were washed in phosphate-buffered saline and harvested in 1 ml of phosphate-buffered saline using a rubber policeman for quantitation of H2O2 by fluorescence-activated cell-sorting analysis (32Ye J. Wang S. Leonard S.S. Sun Y. Butterworth L. Antonini J. Ding M. Rojanasakul Y. Vallyathan V. Castronova V. Shi X. J. Biol. Chem. 1999; 274: 34974-34980Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). 2′,7′-Dichlorofluorescin diacetate was excited at 488 nm and detected at 525 nm. Lactate dehydrogenase activity in cell culture medium was measured by using a lactate dehydrogenase kit from Roche Diagnostics Corp. (Indianapolis, IN) according to the manufacturer's instructions. The lactate dehydrogenase activity was measured spectrophotometrically by using the COBAS chemistry system FARA (Roche Diagnostics Corp.) and expressed in units/liter. Protein concentration was determined by using the Bradford method (33Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (214351) Google Scholar) with reagents from Bio-Rad. To analyze the mechanism of anti-inflammation by phenolic antioxidants, we examined the effect of the chemicals on signal-induced production of TNF-α in macrophage cells. tBHQ and HQ, two prototypical phenolic antioxidants, were chosen as testing agents in the study. Little TNF-α protein was detected by ELISA specific for mouse TNF-α in controls (Fig. 1A,DMSO). tBHQ or HQ alone does not affect TNF-α production (Fig. 1A, tBHQ and HQ). Upon exposure to LPS, large quantities of TNF-α were produced, and the induction was inhibited by tBHQ or HQ (Fig. 1A, compare LPSwith tBHQ+LPS and HQ+LPS). Thus, tBHQ or HQ totally blocks LPS-induced production of TNF-α protein in macrophages. Next, we analyzed the time- and concentration-response curves. As shown in Fig. 1B, LPS induces a 5-fold increase in TNF-α production within 1 h after treatment, reaching a maximum of ∼300-fold at 5 h. The maximum induction is maintained for over 24 h. HQ blocks the induction of TNF-α by LPS throughout the testing time course. These results suggest that inhibition of TNF-α production by HQ occurs early and is therefore likely to be a primary response; furthermore, the inhibition is complete and long-lasting. HQ inhibits LPS-induced TNF-α production dose-dependently (Fig. 1C). The IC50 value of the inhibition by HQ is ∼15 μm, which is similar to the potency of HQ for other biological responses, such as the induction of phase II drug-metabolizing enzymes (34Prestera T. Holtzclaw W.D. Zhang Y. Talalay P. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2965-2969Crossref PubMed Scopus (404) Google Scholar). Therefore, the inhibition by HQ is potent and may be mediated through a mechanism analogous to the induction of phase II enzymes by phenolic antioxidants. tBHQ inhibits LPS-induced TNF-α production both time- and dose-dependently in a manner similar to that of HQ (data not shown). Certain phenolic chemicals undergo oxidation-reduction cycling in cells. We tested whether the inhibition of TNF-α production by phenolic chemicals involves redox cycling of the chemicals. HQ, catechol, and resorcinol are structural analogs of diphenols, which differ with regard to the positions of hydroxyl groups on the aromatic ring. HQ and catechol undergo facile reversible oxidations to the corresponding quinones, whereas resorcinol can not. HQ and catechol strongly inhibit LPS-induced TNF-α production; in contrast, resorcinol is inactive for the inhibition (Fig. 2A). To exclude cell toxicity as a possible cause of the inhibition, we measured lactate dehydrogenase activity in the medium, which reflects cellular damage. As shown in Fig. 2B, LPS, HQ, catechol, and resorcinol did not cause a significant increase in lactate dehydrogenase activity, given either alone or in combination. These data demonstrate that the antioxidants do not cause marked damage to the cells at the concentrations tested. Several related chemicals were similarly tested for inhibition of TNF-α induction (Fig. 2A). Phenol, which has a single hydroxyl group, does not inhibit TNF-α production at 100 μm. para-Benzoquinone, which is reduced to hydroquinone through a number of reductive pathways (15Ernster L. Estabrook R.W. Hochstein P. Orrenius S. DT-diaphorase: A Quinone Reductase with Special Functions in Cell Metabolism and Detoxication. Vol. 27A. Cambridge University Press, New York1987: 1-207Google Scholar), exhibits dose-dependent inhibition with a potency similar to that of HQ (IC50 = ∼15 μm). α-Naphthoflavone (ANF), a polycyclic aromatic hydrocarbon that is metabolized to oxidizable products in cells (34Prestera T. Holtzclaw W.D. Zhang Y. Talalay P. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2965-2969Crossref PubMed Scopus (404) Google Scholar), inhibits TNF-α production, whereas 2,3,7,8-tetrachlorodiben-zo-p-dioxin (TCDD), which is metabolically stable, does not. These data demonstrate that inhibition of LPS-induced TNF-α production by phenolic antioxidants correlates with the oxidation-reduction capability of the chemicals. LPS stimulates the generation of reactive oxygen species (ROS), such as H2O2, in macrophages (35Sanlioglu S. Williams C.M. Samavati L. Butler N.S. Wang G. McCray Jr., P.B. Ritchie T.C. Hunninghake G.W. Zandi E. Engelhardt J.F. J. Biol. Chem. 2001; 276: 30188-30198Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar). We tested whether phenolic antioxidants affect LPS-induced ROS production. As shown in Fig. 3A, LPS induces a marked increase in H2O2; tBHQ or HQ blocks LPS-induced H2O2 production. Resorcinol does not affect H2O2 induction. This observation can be explained in two ways: first, the LPS-induced ROS production and its inhibition by the antioxidants are unrelated to TNF-α induction and inhibition; alternatively, they are integral to the LPS response and the antioxidant action. To test the possibilities, we examined whether ROS inhibitors block TNF-α induction. NAC, which inhibits LPS-induced H2O2 production in macrophages (35Sanlioglu S. Williams C.M. Samavati L. Butler N.S. Wang G. McCray Jr., P.B. Ritchie T.C. Hunninghake G.W. Zandi E. Engelhardt J.F. J. Biol. Chem. 2001; 276: 30188-30198Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar), inhibits TNF-α induction in a manner similar to that of HQ (Fig.3B). Together, these findings support the notion that oxidative signaling contributes to the inhibition of LPS responses by phenolic antioxidants. To analyze the mechanism of the inhibition, we measured the mRNA expression of TNF-α. The macrophage cells express a barely detectable level of TNF-α mRNA in the absence of LPS stimulation (Fig. 4, A and B,lane 1). LPS induces a ∼50-fold increase in the expression of TNF-α mRNA (Fig. 4, A and B, lane 2). hq (100 μm) alone does not affect TNF-α mRNA expression (Fig. 4, A and B,lane 3), but it blocks LPS-induced expression of TNF-α mRNA dose-dependently, with an IC50 of ∼15–20 μm (Fig. 4, A and B,lanes 4–7), which is similar to the IC50measured by ELISA (Fig. 1C). On the contrary, resorcinol, given either alone or with LPS (Fig. 4, A and B,lanes 8 and 9), does not affect the expression of TNF-α mRNA. Thus, inhibition of TNF-α mRNA expression by phenolic antioxidants correlates with the inhibition of TNF-α protein production. To exclude the possibility that HQ inhibits gene transcription in general, we tested whether HQ induces HO-1, a known inducible gene by phenolic antioxidants (36Ishii T. Itoh K. Takahashi S. Sato H. Yanagawa T. Katoh Y. Bannai S. Yamamoto M. J. Biol. Chem. 2000; 275: 16023-16029Abstract Full Text Full Text PDF PubMed Scopus (1225) Google Scholar), under the same condition used for TNF-α analysis. HO-1 is expressed at a low level in macrophage cells (Fig. 4, A and C, lane 1). HQ induces HO-1 to a level ∼6-fold higher than control at 1 μm (Fig. 4, A and C, lane 4) and to a level >10-fold higher than control at 25, 50, and 100 μm (Fig. 4, A and C, lanes 3 and 5–7). LPS does not induce HO-1 or affect HO-1 induction by HQ. Resorcinol induces HO-1 to a minor extent (Fig. 4,A and C, lanes 8 and 9). Thus, HQ maintains its capacity to induce HO-1 under the same condition it inhibits TNF-α induction by LPS. Inhibition of LPS-induced increase in TNF-α mRNA can be due to a decrease in the synthesis of TNF-α mRNA, an increase in the degradation of TNF-α mRNA, or both. In Fig.5, we measured the half-life (t1/2) of LPS-induced TNF-α mRNA in the absence or presence of HQ. The data indicate that HQ does not affect the stability of LPS-induced TNF-α mRNA in either the absence or presence of actinomycin D, an inhibitor of RNA synthesis (Fig.5A). The t1/2 of LPS-induced TNF-α mRNA is ∼1.2 h, whereas the t1/2 in the presence of HQ is ∼1.4 h (Fig. 5B). These data indicate that HQ inhibits LPS-induced expression of TNF-α through inhibition of the transcription of the gene. Previous studies have established that NF-κB is the major transcription factor that mediates the induction of TNF-α by LPS in macrophage cells (37Ghosh S. May M.J. Kopp E.B. Annu. Rev. Biochem. 1998; 16: 225-260Google Scholar). Therefore, we tested whether phenolic antioxidants inhibit the LPS-induced activation of NF-κB by using an electrophoretic mobility shift assay, which measures the formation of nuclear NF-κB dimers that bind to the NF-κB DNA response element, as a mechanism of inhibition of TNF-α transcription. As shown in Fig.6, LPS induces the formation of two NF-κB·DNA complexes (compare lanes 1 and 2). The LPS-induced NF-κB bands are specific for NF-κB binding element because unlabeled NF-κB DNA probe can compete off the binding of NF-κB to labeled NF-κB DNA probe (compare lanes 7 and8), whereas the AP-1 DNA probe, which is the DNA binding element for AP-1 proteins, fails to compete with the labeled NF-κB probe for binding (lane 9). The two NF-κB/DNA bands represent the p65/p50 heterodimer and the p50 homodimer of NF-κB, respectively, because antibodies against p50 supershift both bands (lane 10), whereas antibodies against p65 disrupt the upper band (p65/p50; lane 11), but not the p50 homodimer band. However, antibodies against c-Jun, NF-AT, or c-Fos have no effect on NF-κB band formation (lanes 12–14). Treatment with HQ alone does not induce NF-κB activation (lane 3), but it completely blocks LPS-induced formation of NF-κB dimer·DNA compl" @default.
• W2005891669 created "2016-06-24" @default.
• W2005891669 creator A5018053837 @default.
• W2005891669 creator A5076745184 @default.
• W2005891669 date "2002-01-01" @default.
• W2005891669 modified "2023-10-18" @default.
• W2005891669 title "Chemoprotection by Phenolic Antioxidants" @default.
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