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• W1966322286 abstract "Phenolic antioxidant butylated hydroxyanisole (BHA) is a commonly used food preservative with broad biological activities, including protection against acute toxicity of chemicals, modulation of macromolecule synthesis and immune response, induction of phase II detoxifying enzymes, and especially its potential tumor-promoting activities. Understanding the molecular basis underlying these diverse biological actions of BHA is thus of great importance. Here we demonstrate that BHA is capable of activating distinct mitogen-activated protein kinases (MAPKs), extracellular signal-regulated protein kinase 2 (ERK2), and c-Jun N-terminal kinase 1 (JNK1). Activation of ERK2 by BHA was rapid and transient, whereas the JNK1 activation was relatively delayed and persistent. A major metabolite of BHA, tert-butylhydroquinone (tBHQ), also activated ERK2 but weakly stimulated JNK1 activity. Furthermore, tBHQ activation of ERK2 was late and prolonged, showing a kinetics different from that induced by BHA. ERK2 activation by both compounds required the involvement of an upstream signaling kinase MAPK/ERK kinase (MEK), as evidenced by the inhibitory effect of a MEK inhibitor, PD98059. Pretreatment with N-acetyl-l-cysteine, glutathione, or vitamin E attenuated ERK2 but not JNK1 activation by BHA and tBHQ. Modulation of intracellular H2O2levels by direct addition of catalase or pretreatment with a catalase inhibitor, aminotriazole, also affected BHA- and tBHQ-stimulated ERK2 activity but not JNK1, indicating the involvement of oxidative stress in the ERK2 activation by these two compounds. However, we did not observe any generation of H2O2 after exposure of cells to BHA or tBHQ using a H2O2-sensitive fluorescent probe, 2′,7′-dichlorofluorescein diacetate. Instead, BHA and tBHQ substantially reduced the amount of intracellular H2O2. Furthermore, BHA and tBHQ activation of ERK2 was strongly inhibited by ascorbic acid and a peroxidase inhibitor, sodium azide, suggesting the potential role of phenoxyl radicals and/or their derivatives. Taken together, our results indicate that (i) BHA and its metabolite tBHQ differentially regulate MAPK pathways, and (ii) oxidative stress due to the generation of reactive intermediates, possibly phenoxyl radicals but not H2O2, is responsible for the ERK2 activation by BHA and tBHQ, whereas the JNK1 activation may require a distinct yet unknown mechanism. Phenolic antioxidant butylated hydroxyanisole (BHA) is a commonly used food preservative with broad biological activities, including protection against acute toxicity of chemicals, modulation of macromolecule synthesis and immune response, induction of phase II detoxifying enzymes, and especially its potential tumor-promoting activities. Understanding the molecular basis underlying these diverse biological actions of BHA is thus of great importance. Here we demonstrate that BHA is capable of activating distinct mitogen-activated protein kinases (MAPKs), extracellular signal-regulated protein kinase 2 (ERK2), and c-Jun N-terminal kinase 1 (JNK1). Activation of ERK2 by BHA was rapid and transient, whereas the JNK1 activation was relatively delayed and persistent. A major metabolite of BHA, tert-butylhydroquinone (tBHQ), also activated ERK2 but weakly stimulated JNK1 activity. Furthermore, tBHQ activation of ERK2 was late and prolonged, showing a kinetics different from that induced by BHA. ERK2 activation by both compounds required the involvement of an upstream signaling kinase MAPK/ERK kinase (MEK), as evidenced by the inhibitory effect of a MEK inhibitor, PD98059. Pretreatment with N-acetyl-l-cysteine, glutathione, or vitamin E attenuated ERK2 but not JNK1 activation by BHA and tBHQ. Modulation of intracellular H2O2levels by direct addition of catalase or pretreatment with a catalase inhibitor, aminotriazole, also affected BHA- and tBHQ-stimulated ERK2 activity but not JNK1, indicating the involvement of oxidative stress in the ERK2 activation by these two compounds. However, we did not observe any generation of H2O2 after exposure of cells to BHA or tBHQ using a H2O2-sensitive fluorescent probe, 2′,7′-dichlorofluorescein diacetate. Instead, BHA and tBHQ substantially reduced the amount of intracellular H2O2. Furthermore, BHA and tBHQ activation of ERK2 was strongly inhibited by ascorbic acid and a peroxidase inhibitor, sodium azide, suggesting the potential role of phenoxyl radicals and/or their derivatives. Taken together, our results indicate that (i) BHA and its metabolite tBHQ differentially regulate MAPK pathways, and (ii) oxidative stress due to the generation of reactive intermediates, possibly phenoxyl radicals but not H2O2, is responsible for the ERK2 activation by BHA and tBHQ, whereas the JNK1 activation may require a distinct yet unknown mechanism. Butylated hydroxyanisole (BHA), 1The abbreviations used are: BHA, butylated hydroxyanisole; tBHQ, tert-butylhydroquinone; MAPK, mitogen-activated protein kinase; ERK2, extracellular signal-regulated protein kinase 2; JNK1, c-Jun N-terminal kinase 1; NAC,N-acetyl-l-cysteine; V-E, vitamin E; ROS, reactive oxygen species; DCF-DA, 2′,7′-dichlorofluorescein-diacetate; MEK, MAP kinase kinase; GST, glutathione S-transferase; PBS, phosphate-buffered saline; MBP, myelin basic protein. a synthetic phenolic antioxidant due to its chain-breaking action during the autooxidation of lipid, is widely used as a food preservative, probably ubiquitous in almost all food additives (1World Health Organization WHO Food Additive Ser. 1987; 21: 25-46Google Scholar, 2Rehwoldt R. Food Chem. Toxicol. 1986; 24: 1039-1041Crossref PubMed Scopus (24) Google Scholar). In addition to its action as an inhibitor of lipid peroxidation, this compound exhibits a wide range of biological activities. BHA protects animals against radiation and the acute toxicity of various xenobiotics and mutagens (3Kahl R. Toxicology. 1984; 33: 185-228Crossref PubMed Scopus (210) Google Scholar, 4Wattenberg L.W. Lam L.K. Nygaard O.F. Simig M.A. Radioprotectors and Anti-carcinogens. Academic Press, Inc., NY1983: 461-469Google Scholar). Dietary administration of BHA also leads to the protection against various carcinogens, presumably through the induction of many phase II detoxifying enzymes such as epoxide hydrolases (5Monroe D. Eaton D. Toxicol. Appl. Pharmacol. 1987; 90: 401-409Crossref PubMed Scopus (109) Google Scholar, 6Cha Y.-N. Martz F. Beuding E. Cancer Res. 1978; 38: 4496-4498PubMed Google Scholar), glutathioneS-transferases (7Benson A.M. Batzinger R.P. Ou S.-Y.L. Bueding E. Cha Y.-N. Talalay P. Cancer Res. 1978; 38: 4486-4495PubMed Google Scholar), and glucuronosyltransferases (8Moldeus P. Dock L. Cha Y.-N. Berggren M. Jernstrom B. Biochem. Pharmacol. 1982; 31: 1907-1910Crossref PubMed Scopus (14) Google Scholar), as well as through the inhibition of cytochrome P-450 monooxygenase (9Cummings S.W. Prough R.A. J. Biol. Chem. 1983; 258: 12315-12319Abstract Full Text PDF PubMed Google Scholar). On the other hand, a growing body of evidence indicates that BHA may also be a tumor initiator or a tumor promoter in some tissues of animals. For example, BHA induced papilloma and carcinoma formation in the forestomachs of rats, mice, and hamsters when fed continuously at high concentrations (10Ito N. Fukushima S. Hagiwara A. Shibata M. Ogiso T. J. Natl. Cancer Inst. 1983; 70: 343-352PubMed Google Scholar, 11Clayson D.B. Iverson F. Nera E.A. Lok E. Annu. Rev. Pharmacol. Toxicol. 1990; 30: 441-463Crossref PubMed Scopus (55) Google Scholar). Chronic dietary administration of BHA also enhances the development of preneoplastic and neoplastic lesions in the rat kidney and urinary bladder (12Nera E.A. Iverson F. Lok E. Armstrong C.L. Karpinski K. Clayson D.B. Toxicology. 1988; 53: 251-268Crossref PubMed Scopus (64) Google Scholar, 13Peters M.M.C. Rivera M.I. Jones T.W. Monks T.J. Lau S.S. Cancer Res. 1996; 56: 1006-1011PubMed Google Scholar). Furthermore, BHA appears to have initiating activity in two-stage mouse skin carcinogenesis assay and in two-stage transformation of BALB/3T3 cells (14Sakai A. Miyata N. Takahashi A. Carcinogenesis. 1990; 11: 1985-1988Crossref PubMed Scopus (17) Google Scholar). Most notably, BHA induced proliferative effects not only in rodent forestomachs but also in the esophagus of pigs and primates (15Wurtzen G. Olsen P. Food Chem. Toxicol. 1986; 24: 1229-1233Crossref PubMed Scopus (35) Google Scholar,16Iverson F. Truelove J. Nera E. Wong J. Clayson D.B. Cancer Lett. 1985; 26: 43-50Crossref PubMed Scopus (42) Google Scholar). Thus, this well known antioxidant exerts opposing biological effects. Although both anti-carcinogenic and carcinogenic effects of BHA are well described, the precise mechanisms of how these effects are achieved remain obscure but probably are dose- and/or tissue-dependent. Studies on the metabolism of BHA revealed that several metabolic pathways may exist, including dimerization, conjugation, andO-demethylation (17Verhagen H. Shilderman P.A. Kleinjans J.C. Chem. Biol. Interact. 1991; 80: 109-134Crossref PubMed Scopus (81) Google Scholar). One of the major metabolites of BHA, as shown in dogs (18Astill B.D. Mills J. Rassett R.L. Roundbash R.L. Terhaar C.J. Agric. Food Chem. 1962; 10: 315-318Crossref Scopus (56) Google Scholar), rats, and man (19Verhagen H. Furnee C. Schutte B. Hermans R.J.J. Bosman F.T. Blijhan G.H. Hoor F.Th. Henderson P. Kleinjans J.C.S. Carcinogenesis. 1989; 10: 1947-1951Crossref PubMed Scopus (13) Google Scholar), and in rat liver microsomes (20Rahimthula A. Chem. Biol. Interact. 1983; 45: 125-135Crossref PubMed Scopus (50) Google Scholar), is the demethylated product, tert-butylhydroquinone (tBHQ), which also exhibits anti-carcinogenic properties in some animal models of cancer in a manner similar to that described for BHA. This includes modulation of the enzyme systems responsible for metabolic activation or deactivation of chemical carcinogens (21Wattenberg L.W. Coccia J.B. Lam L.K.T. Cancer Res. 1980; 40: 2820-2823PubMed Google Scholar). Thus, metabolic formation of tBHQ is thought to contribute to the anti-carcinogenic activities of BHA. However, tBHQ is also shown to be a carcinogen in many animal tissues. Although the reason why tBHQ is carcinogenic remains unknown, the oxidation of tHBQ to its corresponding quinone, tert-butylquinone, accompanied by the generation of reactive oxygen species (ROS), presumes to play an important role (22Rossing D. Kahl R. Hildebrandt A.G. Toxicology. 1985; 34: 67-77Crossref PubMed Scopus (40) Google Scholar, 23Kahl R. Weinke S. Kappus H. Toxicology. 1989; 59: 179-194Crossref PubMed Scopus (53) Google Scholar, 24Schilderman P.A.E.L. Maanen J.M.S. Smeets E.J. Hoor F. Kleinjans J.C.S. Carcinogenesis. 1993; 14: 347-353Crossref PubMed Scopus (36) Google Scholar). Since BHA did not show genotoxic activity in most tests for mutagenicity (25Rogers C.G. Boyes B.G. Matula T.I. Stapley R. Mutat. Res. 1992; 280: 17-27Crossref PubMed Scopus (17) Google Scholar, 26Schilderman P.A.E.L. Rhijinsburger E. Zwingmann I. Kleinjans J.C.S. Carcinogenesis. 1995; 16: 507-512Crossref PubMed Scopus (23) Google Scholar), tBHQ-mediated generations oftert-butylquinone and ROS, which are known to cause DNA damage, is believed to be responsible for BHA-induced carcinogenesis. However, several studies suggested that such carcinogenic effects of BHA are tBHQ- or tert-butylquinone-independent and are epigenetic (19Verhagen H. Furnee C. Schutte B. Hermans R.J.J. Bosman F.T. Blijhan G.H. Hoor F.Th. Henderson P. Kleinjans J.C.S. Carcinogenesis. 1989; 10: 1947-1951Crossref PubMed Scopus (13) Google Scholar, 23Kahl R. Weinke S. Kappus H. Toxicology. 1989; 59: 179-194Crossref PubMed Scopus (53) Google Scholar, 27Hirose M. Inoue T. Masuda H. Tsuda H. Ito N. Carcinogenesis. 1987; 8: 1555-1558Crossref PubMed Scopus (31) Google Scholar). While the relevance of metabolism of BHA for its biological action has been the focus of many studies over the years, the signal transduction pathways that control the cellular responses to BHA and its metabolites have not been elucidated. Mitogen-activated protein kinases (MAPKs), characterized as proline-directed serine/threonine kinases (28Cobb M.H. Goldsmith E.J. J. Biol. Chem. 1995; 270: 14843-14846Abstract Full Text Full Text PDF PubMed Scopus (1663) Google Scholar), are important cellular signaling components that convert various extracellular signals into intracellular responses through serial phosphorylation cascades (29Marshall C.J. Curr. Opin. Gen. & Dev. 1994; 4: 82-89Crossref PubMed Scopus (902) Google Scholar). At the present time, three distinct but parallel MAPK cascades have been identified in mammalian cells (30Cano E. Mahadevan L.C. Trends Biochem. Sci. 1995; 20: 117-122Abstract Full Text PDF PubMed Scopus (1001) Google Scholar). Each consists of a module of three kinases as follows: a MAPK kinase kinase, which phosphorylates and activates a MAPK kinase, which, in turn, phosphorylates and activates a MAPK. The best characterized MAPK pathway is a Ras-dependent activation of extracellular signal-regulated protein kinases (ERKs) in response to growth factors. In this pathway, tyrosine-phosphorylated transmembrane receptors associate with the SH2 domain of the adapter protein Grb2 (31Skolnik E.Y. Lee C.H. Batzer A. Vincentini L.M. Zhou M. Daly R. Myers Jr., M.G. Backer G.M. Ullrich A. White M.F. Schlessinger J. EMBO J. 1993; 12: 1929-1936Crossref PubMed Scopus (607) Google Scholar) and target nucleotide exchange factor SOS to the membrane-bound small G-protein Ras (32Egan S.E. Giddings B.W. Brooks M.W. Buday L. Sizeland A.M. Weinberg R.A. Nature. 1993; 363: 45-51Crossref PubMed Scopus (1011) Google Scholar). Activated Ras recruits Raf-1 (a MAPK kinase kinase) to the membrane, leading the activation of Raf-1 (33Stokeo D.M. Caddwallader K. Symons M. Hancock J.F. Science. 1994; 264: 1463-1467Crossref PubMed Scopus (847) Google Scholar). Once activated, Raf-1 can phosphorylate and activate a dual specificity kinase MEK (a MAPK kinase), which, in turn, activates ERK (a MAPK). In addition to tyrosine kinase receptors, certain G-protein-coupled receptors and protein kinase C are also capable of activating ERK cascade (34Burgering B.M.T. Bos J.L. Trends Biochem. Sci. 1995; 20: 18-22Abstract Full Text PDF PubMed Scopus (292) Google Scholar). Another emerging MAPK group is c-Jun N-terminal kinase (JNK), which is operated by a parallel signaling module, consisting of MEKK1/MKK4 (or SEK1, JNKK)/JNK (35Hibi M. Lin A. Smeal T. Minden A. Karin M. Genes Dev. 1993; 7: 2135-2148Crossref PubMed Scopus (1710) Google Scholar,36Kyriakis J.M. Banerjee P. Nikolakaki E. Dai T. Rubie E.A. Ahmad M.F. Avruch J. Woodgett J.R. Nature. 1994; 369: 156-160Crossref PubMed Scopus (2415) Google Scholar). However, JNK cascade, unlike ERK pathway, is only modestly activated by growth factors and phorbol esters and is instead strongly activated by stress signals such as UV light (37Chen Y.-R. Wang X. Templeton D. Davis R.J. Tan T.-H. J. Biol. Chem. 1997; 271: 31929-31936Abstract Full Text Full Text PDF Scopus (856) Google Scholar), γ-radiation (38Chen Y.-R. Meyer C.F. Tan T.-H. J. Biol. Chem. 1996; 271: 631-634Abstract Full Text Full Text PDF PubMed Scopus (461) Google Scholar), protein synthesis inhibitors (39Cano E. Hazzalin C.A. Mahadevan L.C. Mol. Cell. Biol. 1994; 14: 7352-7362Crossref PubMed Scopus (271) Google Scholar), ceramide (40Verheij M. Bose R. Lin X.H. Yao B. Jarvis W.D. Grant S. Birrer M. Szabo E. Zon L.I. Kyriakis J.M. Haimovitz-Friendman A. Fuks Z. Kolesnick R. Nature. 1996; 380: 75-79Crossref PubMed Scopus (1718) Google Scholar), DNA damaging agents (41Yu R. Shtil A.A. Tan T.-H. Roninson I.B. Kong A.-N.T. Cancer Lett. 1996; 107: 73-81Crossref PubMed Scopus (104) Google Scholar), inflammatory cytokines (42Raingeaud J. Gupta S. Rogers J.S. Dickens M. Han J. Ulevitch R.J. Davis R.J. J. Biol. Chem. 1995; 270: 7420-7426Abstract Full Text Full Text PDF PubMed Scopus (2046) Google Scholar), and chemopreventive agents (43Yu R. Jiao J.J. Duh J.L. Tan T.-H. Kong A-N.T. Cancer Res. 1996; 56: 2954-2959PubMed Google Scholar). Therefore, JNK is also termed as stress-activated protein kinase. Both ERK and JNK activation culminate in the phosphorylation of downstream cytosolic and nuclear substrates, including transcription factors, c-Myc, p62TCF/Elk-1, c-Jun, and ATF2, ultimately leading to the changes in gene expression (44Karin M. J. Biol. Chem. 1995; 270: 16483-16486Abstract Full Text Full Text PDF PubMed Scopus (2258) Google Scholar). Given the fact that MAPKs are activated by such a wide range of factors, these signaling cascades may serve as a common mechanism and integrate with other signaling pathways to control cellular responses to various extracellular stimuli. In this study, we examined ERK and JNK activation by BHA and its principal metabolite tBHQ in two cell lines, HeLa cells and human hepatoma Hep G2 cells, with particular interest in the relationship between these two chemicals and the possible mediators in modulating ERK and JNK cascades. We demonstrated that BHA strongly activated ERK2 and JNK1 in both cell lines, whereas tBHQ stimulated ERK2 only in Hep G2 cells and weakly activated JNK1 in both cell lines. ERK2 activation by both agents requires involvement of upstream signaling components as demonstrated by the inhibitory effects of a MEK-specific inhibitor PD98059. Further studies using different free radical scavengers and flow cytometry revealed that formation of non-oxygen free radicals, possibly phenoxyl free radicals, from parent molecules of BHA and tBHQ, represents a major mechanism for ERK2 activation but not for JNK1. Human HeLa cells and Hep G2 cells, obtained from American Type culture Collection (Rockville, MD) were maintained as monolayer cultures in minimum essential medium supplemented with 10% fetal bovine serum, 2.2 g/liter sodium bicarbonate, 100 units/ml penicillin, and 100 μg/ml streptomycin. Cells were normally starved overnight in serum-free medium without phenol red before treatment. Polyclonal antibody to ERK2 was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Rabbit anti-JNK1 antiserum (Ab101) was described previously (38Chen Y.-R. Meyer C.F. Tan T.-H. J. Biol. Chem. 1996; 271: 631-634Abstract Full Text Full Text PDF PubMed Scopus (461) Google Scholar, 43Yu R. Jiao J.J. Duh J.L. Tan T.-H. Kong A-N.T. Cancer Res. 1996; 56: 2954-2959PubMed Google Scholar). GST-c-Jun-(1–79) expressing plasmid was kindly provided by Dr. M. Karin (University of California, San Diego, CA), and the GST-c-Jun fusion protein was purified from Escherichia coli lysates with the aid of glutathione-Sepharose beads (Pharmacia Biotech Inc.). PD95089, a specific inhibitor of MEK, was obtained from New England Biolabs Inc. Fluorescence dyes 2′,7′-dichlorofluorescein diacetate (DCF-DA) were purchased from Molecular Probes, Inc. (Eugene, OR). Myelin basic protein, catalase, dismutase, NAC, reduced glutathione (GSH), α-tocopherol (vitamin E; V-E), ascorbic acid (vitamin C), 3-aminotriazole, hydrogen peroxide (H2O2), and BHA were obtained from Sigma. tBHQ was purchase from Aldrich. [γ-32P]ATP (3,000 Ci/mmol) was obtained from NEN Life Science Products. After treatment with BHA or tBHQ (both agents were dissolved in ethanol, the final concentration of ethanol in the culture medium was controlled to less than 0.1%), monolayer cells, grown in a 10-cm diameter plate, were washed with ice-cold phosphate-buffered saline (PBS) and harvested in 700 μl of lysis buffer containing 10 mm Tris-HCl (pH 7.1), 50 mm sodium chloride, 30 mm inorganic sodium pyrophosphate, 50 mm sodium fluoride, 100 μm sodium orthovanadate, 2 mm iodoacetic acid, 5 μm zinc chloride, 1 mmphenylmethylsulfonyl fluoride, and 0.5% Triton X-100. The lysate was homogenized by passing through a 23-gauge needle three times. After 15 min on ice, the homogenate was centrifuged at high speed for 10 min at 4 °C, and the supernatant was transferred into a new tube. ERK activity was determined by an immunocomplex kinase assay as described previously (43Yu R. Jiao J.J. Duh J.L. Tan T.-H. Kong A-N.T. Cancer Res. 1996; 56: 2954-2959PubMed Google Scholar). Briefly, equal amounts of protein, as measured by Bio-Rad protein Assay (Bio-Rad, CA), were incubated with rabbit anti-ERK2 polyclonal antibody and protein A-Sepharose beads for 2 h at 4 °C. The immunocomplex was spun down at high speed for 1 min and washed twice with lysis buffer and twice with kinase buffer (20 mm HEPES (pH 7.9), 10 mm magnesium chloride, 2 mm manganese chloride, 0.1 mmsodium orthovanadate, 50 mm β-glycerophosphate, 10 mm ρ-nitrophenyl phosphate). Kinase reaction was initiated by resuspending the immunoprecipitate in 30 μl of kinase assay buffer containing 10 μg of myelin basic protein, 2 μCi of [γ-32P]ATP, and 20 μm ATP. After incubation for 15 min at 30 °C, the reaction was terminated by adding 10 μl of 4 × Laemmli's buffer and heating to 95 °C for 5 min; samples were resolved in 13.5% SDS-polyacrylamide gel electrophoresis. Gel was stained with Coomassie Blue and then washed overnight and dried. The phosphorylation of myelin basic protein was visualized by autoradiography and quantified with a PhosphorImager (AMBIS, Inc., San Diego, CA). JNK1 activity was assayed according to the procedures described above for ERK2 assay, with the following changes. After immunoprecipitation with rabbit anti-JNK1 antiserum, JNK1 activity was detected using 10 μg of GST-c-Jun-(1–79) fusion protein as substrate. Kinase reaction was performed at 30 °C for 30 min. The phosphorylated product was resolved in 10% SDS-polyacrylamide gel electrophoresis and analyzed by autoradiography and phosphorimage. Confluent, serum-starved HeLa cells and Hep G2 cells were treated with different agents and then incubated with 5 μm DCF-DA (dissolved in Me2SO) for an additional 30 min at 37 °C. After chilling on ice, cells were washed with ice-cold PBS, scraped from the plate, and resuspended at 5 × 105 cells/ml in PBS containing 2% fetal bovine serum and 5 μm DCF-DA. The fluorescence intensities of DCF-DA of more than 10,000 viable cells from each sample were analyzed by flow cytometry using a Becton Dickinson FACScan flow cytometer with excitation and emission settings of 488 and 525 nm, respectively. Prior to data collection, propidium iodide was added to the sample for gating out dead cells. Cells were plated at a density of 5 × 104 cells/well into 96-well plates, with each well containing 100 μl of medium. After overnight recovery, cells were treated with a series of concentrations of BHA or tBHQ. Drugs were removed after 4 h of treatment, and cells were cultured in the fresh medium for an additional 24 h and then assayed for viability using CellTiter 96 Non-Radioactive Cell Proliferation Assay Kit (Promega, Madison, WI). Briefly, 20 μl of combined solution of a tetrazolium compound 3-(4,5dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt, and an electron coupling reagent phenazine methosulfate was added to each well. After incubation for 1 h at 37 °C in a humidified 5% CO2 atmosphere, the absorbance at 490 nm was recorded using an enzyme-linked immunosorbent assay plate reader. HeLa cells, which are derived from a human cervical squamous cell carcinoma, are used as a model system for the studies of MAPK in response to various factors. Hep G2, a human hepatoma cell line, has been widely used to study the role of metabolism of different compounds, because of the extensive metabolic capacity of this cell line. In the present experiment, we have examined the effects of BHA on ERK2 and JNK1 activities in these cell lines. As illustrated in Fig.1 A, BHA strongly stimulated ERK2 activity in a dose-dependent fashion in both cell lines after 25 min of treatment. In HeLa cells, ERK2 activation occurred when the concentration of BHA reached 250 μm. In Hep G2 cells, the stimulated ERK2 activity, however, appeared at a much lower concentration, around 50 μm. The maximal induction of ERK2 activity was observed at 500 μm in HeLa cells and 250 μm in Hep G2 cells. Further increasing concentrations of BHA to 750 μm dramatically reduced ERK2 activity to control level in Hep G2 cells or to the level slightly higher than control in HeLa cells. Similar to ERK2, JNK1 was also strongly activated in both cell lines after 90 min of treatment with BHA (Fig.1 B). Interestingly, activation of both kinases followed a similar dose-response pattern. Based on the dose-response data, we next studied the kinetics of ERK2 and JNK1 activation by BHA. In HeLa cells, 500 μm BHA activated ERK2 in a time-dependent fashion (Fig. 2 A). The induced ERK2 activity reached maximum within 15 min and disappeared after 60 min of treatment with BHA. Similar to that observed in HeLa cells, activation of ERK2 in Hep G2 cells by 250 μm BHA peaked at 15 min. Afterward, a rapid decrease in ERK2 activity ensued. Unlike ERK2 activation, JNK1 activation was delayed and seen at 15 min in HeLa cells or at 30 min in Hep G2 cells, instead of 5 min in ERK2 activation (Fig. 2 B). Furthermore, after peaking at 2 h, JNK1 activity gradually declined to control levels in both cell lines, showing a sustained activation pattern. Different kinetics of ERK2 and JNK1 activation suggest that these two kinases are differentially regulated in response to BHA. When Western blotting analysis was performed with anti-ERK2 or anti-JNK1 antibodies, no changes in the protein levels of ERK2 or JNK1 occurred throughout the kinetic studies, indicating that the activation of ERK2 or JNK1 resulted from the phosphorylation of pre-existing kinase molecules instead of thede novo protein synthesis (data not shown). tBHQ is a major metabolite of BHA, as demonstrated in many animal species as well as in human (18Astill B.D. Mills J. Rassett R.L. Roundbash R.L. Terhaar C.J. Agric. Food Chem. 1962; 10: 315-318Crossref Scopus (56) Google Scholar, 19Verhagen H. Furnee C. Schutte B. Hermans R.J.J. Bosman F.T. Blijhan G.H. Hoor F.Th. Henderson P. Kleinjans J.C.S. Carcinogenesis. 1989; 10: 1947-1951Crossref PubMed Scopus (13) Google Scholar). A number of previous studies implicated that this compound may mediate many biological activities of BHA, including anti-carcinogenic and carcinogenic effects. Thus, it would be of particular interest to see whether tBHQ could mimic the effects of BHA with respect to the activation of MAPK. In Hep G2 cells, tBHQ stimulated ERK2 activation, which appeared at 50 μm and reached a maximum at 250–500 μm (Fig.3 A). Further increasing tBHQ concentration to 750 μm dramatically reduced the ERK2 activity. Thus, ERK2 activation by tBHQ exhibits a dose-dependent manner similar to that observed with BHA. However, time course analyses showed that tBHQ, unlike BHA, induced delayed and sustained ERK2 activation. The stimulated ERK2 activity appeared at 15 min, reached maximum at 30 min, and was persistent for more than 2 h. Furthermore, tBHQ seemed to be less potent than BHA in activation of ERK2. For example, a maximum of more than 10-fold induction was observed in the treatment with BHA (250 μm, 15 min), whereas the maximal ERK2 activation by tBHQ (500 μm, 30 min) was around 7-fold. The differences in kinetics and extent of ERK2 activation suggest that BHA and tBHQ differentially regulate this kinase cascade. This observation was further supported by the experiment in HeLa cells. In this experiment, HeLa cells were treated with either 500 μm tBHQ for different time periods or with different concentrations of tBHQ for 30 min, as shown in Fig. 3 B. No significant induction of ERK2 activity was observed, regardless of the dose and time of the treatments. However, under similar conditions, BHA strongly activated ERK2, with a maximum of more than 18-fold induction, as seen in Fig. 2 A. Differential regulation of ERK2 signaling pathway by BHA and tBHQ prompted us to examine whether these two compounds also differentially regulate another member of the MAPK family, JNK. To do so, we first studied the time course of JNK1 activation by tBHQ. Both HeLa cells and Hep G2 cells were treated with 500 μm tBHQ for different periods. As shown in Fig. 3 C, JNK1 activity was weakly stimulated by tBHQ, with a maximum of 2.2-fold induction in HeLa cells and 2.7-fold induction in Hep G2 cells. To exclude the possibility that JNK1 activity was measured at an unfavorable concentration, we then investigated the dose response of JNK1 activation by tBHQ. Similar to the results obtained in the kinetic studies (Fig. 3 B), no substantial JNK1 activation was observed (data not shown). Therefore, unlike BHA, tBHQ was a weak inducer of JNK1 activity. Activation of ERK2 is a consequence of serial phosphorylation of upstream kinases (28Cobb M.H. Goldsmith E.J. J. Biol. Chem. 1995; 270: 14843-14846Abstract Full Text Full Text PDF PubMed Scopus (1663) Google Scholar). Among these kinases is MEK (a MAPK kinase), which activates ERK2 in response to various stimuli, such as growth factors (45Lange-Carter C.A. Pleiman C.M. Gardner A.M. Blumer K.J. Johnson G.L. Carol A. Science. 1993; 260: 315-319Crossref PubMed Scopus (875) Google Scholar) and oxidative stress (46Chen Q. Olashaw N. Wu J. J. Biol. Chem. 1995; 270: 28499-28502Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar), by phosphorylation of ERK2 on threonine and tyrosine residues in a consensus TEY motif. To determine whether BHA and tBHQ utilize a MEK-dependent pathway, we took advantage of the recently identified MEK inhibitor, PD98059 (47Dudley D.T. Pang L. Decker S.J. Bridges A. Saltiel A.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7686-7689Crossref PubMed Scopus (2595) Google Scholar, 48Servant M.J. Giasson E. Meloche S. J. Biol. Chem. 1996; 271: 16047-16052Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). Since both BHA and tBHQ activated ERK2" @default.
• W1966322286 created "2016-06-24" @default.
• W1966322286 creator A5005384948 @default.
• W1966322286 creator A5038293206 @default.
• W1966322286 creator A5083630398 @default.
• W1966322286 date "1997-11-01" @default.
• W1966322286 modified "2023-10-10" @default.
• W1966322286 title "Butylated Hydroxyanisole and Its Metabolitetert-Butylhydroquinone Differentially Regulate Mitogen-activated Protein Kinases" @default.
• W1966322286 cites W1510592624 @default.
• W1966322286 cites W1559986791 @default.
• W1966322286 cites W1577965127 @default.
• W1966322286 cites W1591823948 @default.
• W1966322286 cites W1964045671 @default.
• W1966322286 cites W1969158591 @default.
• W1966322286 cites W1970590879 @default.
• W1966322286 cites W1971475788 @default.
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• W1966322286 cites W1976630097 @default.
• W1966322286 cites W1977687557 @default.
• W1966322286 cites W1978798502 @default.
• W1966322286 cites W1989480480 @default.
• W1966322286 cites W2001521752 @default.
• W1966322286 cites W2002075356 @default.
• W1966322286 cites W2007986629 @default.
• W1966322286 cites W2008577285 @default.
• W1966322286 cites W2008682894 @default.
• W1966322286 cites W2009521921 @default.
• W1966322286 cites W2015471917 @default.
• W1966322286 cites W2022500726 @default.
• W1966322286 cites W2024270933 @default.
• W1966322286 cites W2025850775 @default.
• W1966322286 cites W2026228228 @default.
• W1966322286 cites W2026951979 @default.
• W1966322286 cites W2029165306 @default.
• W1966322286 cites W2030570585 @default.
• W1966322286 cites W2036118421 @default.
• W1966322286 cites W2047141987 @default.
• W1966322286 cites W2047182549 @default.
• W1966322286 cites W2048569323 @default.
• W1966322286 cites W2048983596 @default.
• W1966322286 cites W2049523243 @default.
• W1966322286 cites W2049656784 @default.
• W1966322286 cites W2051023424 @default.
• W1966322286 cites W2051333534 @default.
• W1966322286 cites W2051538397 @default.
• W1966322286 cites W2057580074 @default.
• W1966322286 cites W2062375503 @default.
• W1966322286 cites W2065661936 @default.
• W1966322286 cites W2065863516 @default.
• W1966322286 cites W2068502127 @default.
• W1966322286 cites W2071662241 @default.
• W1966322286 cites W2073541193 @default.
• W1966322286 cites W2074169536 @default.
• W1966322286 cites W2074815894 @default.
• W1966322286 cites W2077880395 @default.
• W1966322286 cites W2078031827 @default.
• W1966322286 cites W2078757969 @default.
• W1966322286 cites W2084720534 @default.
• W1966322286 cites W2107612680 @default.
• W1966322286 cites W2117669008 @default.
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• W1966322286 cites W2140355533 @default.
• W1966322286 cites W2412099857 @default.
• W1966322286 cites W912507531 @default.
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