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• W1988049205 abstract "Polyunsaturated fatty acids such as arachidonic acid (AA) play an important role in alcohol-induced liver injury. AA promotes toxicity in rat hepatocytes with high levels of cytochrome P4502E1 (CYP2E1) and in HepG2 E47 cells, which express CYP2E1. The possible role of mitogen-activated protein kinase (MAPK) members in this process was evaluated. SB203580, a p38 MAPK inhibitor, and PD98059, an ERK inhibitor, but not wortmannin a phosphatidylinositol 3-kinase (PI3K) inhibitor, prevented AA toxicity in pyrazole hepatocytes and E47 cells. SB203580 prevented the enhancement of AA toxicity by salicylate. SB203580 neither lowered the levels of CYP2E1 nor affected CYP2E1-dependent oxidative stress. The decrease in mitochondrial membrane potential produced by AA was prevented by SB203580. Treating CYP2E1-induced cells with AA activated p38 MAPK but not ERK or AKT. This activation was blocked by antioxidants. AA increased the translocation of NF-κB to the nucleus. Salicylate blocked this translocation, which may contribute to the enhancement of AA toxicity by salicylate. SB203580 restored AA-induced NF-κB translocation, which may contribute to protection against toxicity. In conclusion, AA toxicity was related to lipid peroxidation and oxidative stress, and to the activation of p38 MAPK, as a consequence of CYP2E1-dependent production of reactive oxygen species. Activation of p38 MAPK by AA coupled to AA-induced oxidative stress may synergize to cause cell toxicity by affecting mitochondrial membrane potential and by modulation of NF-κB activation. Polyunsaturated fatty acids such as arachidonic acid (AA) play an important role in alcohol-induced liver injury. AA promotes toxicity in rat hepatocytes with high levels of cytochrome P4502E1 (CYP2E1) and in HepG2 E47 cells, which express CYP2E1. The possible role of mitogen-activated protein kinase (MAPK) members in this process was evaluated. SB203580, a p38 MAPK inhibitor, and PD98059, an ERK inhibitor, but not wortmannin a phosphatidylinositol 3-kinase (PI3K) inhibitor, prevented AA toxicity in pyrazole hepatocytes and E47 cells. SB203580 prevented the enhancement of AA toxicity by salicylate. SB203580 neither lowered the levels of CYP2E1 nor affected CYP2E1-dependent oxidative stress. The decrease in mitochondrial membrane potential produced by AA was prevented by SB203580. Treating CYP2E1-induced cells with AA activated p38 MAPK but not ERK or AKT. This activation was blocked by antioxidants. AA increased the translocation of NF-κB to the nucleus. Salicylate blocked this translocation, which may contribute to the enhancement of AA toxicity by salicylate. SB203580 restored AA-induced NF-κB translocation, which may contribute to protection against toxicity. In conclusion, AA toxicity was related to lipid peroxidation and oxidative stress, and to the activation of p38 MAPK, as a consequence of CYP2E1-dependent production of reactive oxygen species. Activation of p38 MAPK by AA coupled to AA-induced oxidative stress may synergize to cause cell toxicity by affecting mitochondrial membrane potential and by modulation of NF-κB activation. polyunsaturated fatty acid arachidonic acid salicylate mitogen-activated protein kinase extracellular signal-regulated kinase MAPK/ERK kinase stress-activated protein kinase rhodamine 123 phosphatidylinositol 3-kinase mitochondrial membrane potential (±)6-hydroxy-2,5,7,8-teramethylchroman-2-carboxylic acid pyrazole c-Jun NH2-terminal kinase analysis of variance phosphate-buffered saline thiobarbituric acid-reactive products thiobarbituric cytochrome P4502E1 reactive oxygen species Polyunsaturated fatty acids (PUFA)1 such as arachidonic acid (AA) or its metabolites play an important role in a variety of biological processes, such as signal transduction, chemotaxis, and cell proliferation and differentiation (1Chow S.C. Joudal M. J. Biol. Chem. 1990; 265: 902-907Abstract Full Text PDF PubMed Google Scholar, 2Huang J.M. Dian H. Bacaner M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6452-6456Crossref PubMed Scopus (265) Google Scholar, 3Golubic M. Tanaka K. Dobrowolski S. Wood D. Tsai M.H. Marshall M. Tamanoi F. Stacty D.W. EMBO J. 1991; 10: 2897-2903Crossref PubMed Scopus (50) Google Scholar). PUFA also play an important role in alcoholic liver injury (4Tsukamoto H. Gaal K. French S.W. Hepatology. 1990; 12: 599-608Crossref PubMed Scopus (161) Google Scholar, 5French S.W. Crit. Rev. Clin. Lab. Sci. 1992; 29: 83-115Crossref PubMed Scopus (25) Google Scholar, 6Morimoto M. Zern M.A. Hagbjork A.L. Ingelman-Sunberg M. French S.W. Proc. Soc. Exp. Biol. Med. 1994; 207: 197-205Crossref PubMed Scopus (157) Google Scholar). In the intragastric infusion model of ethanol feeding, liver injury occurs when the rats consume diets containing polyunsaturated fatty acid but not saturated fatty acid (7Nanji A.A. Zhao S. Sadzadeh S.M.H. Dannenberg A.J. Tahan S.R. Waxman D.J. Alcohol. Clin. Exp. Res. 1994; 18: 1280-1285Crossref PubMed Scopus (236) Google Scholar, 8Castillo T. Koop D.R. Kamimura S. Triadafilopoulos G. Tsukamoto H. Hepatology. 1992; 16: 992-996Crossref PubMed Scopus (191) Google Scholar). This model is associated with induction of high levels of CYP2E1 and greatly increased lipid peroxidation, which appear to contribute to the liver injury. AA induced toxicity in HepG2 E47 cells, a cell line that expresses CYP2E1 but not control HepG2 cells, which do not express CYP2E1 (9Chen Q. Galleano M. Cederbaum A.I. J. Biol. Chem. 1997; 272: 14532-14541Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). AA also induced toxicity in pyrazole-induced rat hepatocytes with high levels of CYP2E1 but not saline control hepatocytes (10Wu D. Cederbaum A.I. Mol. Cell. Biochem. 2000; 204: 157-167Crossref PubMed Google Scholar). This AA toxicity was prevented by inhibitors of CYP2E1 and by antioxidants (9Chen Q. Galleano M. Cederbaum A.I. J. Biol. Chem. 1997; 272: 14532-14541Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 10Wu D. Cederbaum A.I. Mol. Cell. Biochem. 2000; 204: 157-167Crossref PubMed Google Scholar). AA can activate mitogen-activated protein kinase (MAPK), a ubiquitous group of serine/threonine kinases, which play a crucial role in transmitting transmembrane signals required for cell growth, differentiation, and apoptosis (11Hii C.S.T. Ferrante A. Edwards Y. Huang Z. Hartfield P.J. Rathjen D.A. Poulos A. Murray A.W. J. Biol. Chem. 1995; 270: 4201-4204Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 12Chang L.C. Wang J.P. J. Leukocyte Biol. 2001; 69: 659-665PubMed Google Scholar, 13Rao G.N. Baas A.S. Glasgow W.C. Eling T.E. Runge M.S. Alexander R.W. J. Biol. Chem. 1994; 269: 32586-32591Abstract Full Text PDF PubMed Google Scholar). Members of the kinase family, originally found to be activated by mitogens, have now been found to be activated by a wide variety of mitogenic and non-mitogenic agents via a cascade of kinase/effector molecules which, in mammalian cells, includes protein kinase C, p21, Raf-1, and MEK (MAPK/extracellular signal-regulated protein kinase, ERK) (14Marshall C.J. Curr. Opin. Genet. Dev. 1994; 4: 82-89Crossref PubMed Scopus (902) Google Scholar, 15Minden A. Lin A. McMahon M. Lange-Carter C. Derifark B. Davis R.J. Jonson G.L. Karin M. Science. 1994; 266: 1719-1723Crossref PubMed Scopus (1012) Google Scholar, 16Robinson M.J. Cobb M.H. Curr. Opin. Cell Biol. 1997; 9: 180-186Crossref PubMed Scopus (2286) Google Scholar). AA or its metabolites can activate MAPK members such as ERKs and JNKs/SAPKs, suggesting an important role for AA and its metabolites in mitogenic signaling events (17Paine E. Palmantier R. Akiyama S.K. Olden K. Roberts J.D. J. Biol. Chem. 2000; 275: 11284-11290Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 18Hii C.S.T. Huang Z.H. Bilney A. Costabile M. Murray A.W. Rathjen D.A. Der C.J. Ferrante A. J. Biol. Chem. 1998; 273: 19277-19282Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). We recently reported that salicylate can potentiate the toxicity of AA in CYP2E1-induced hepatocytes (19Wu D. Cederbaum A.I. Mol. Pharmacol. 2001; 59: 795-805Crossref PubMed Scopus (38) Google Scholar). Salicylate was found to increase CYP2E1 levels, preventing its degradation, and this may be one mechanism by which salicylate increases AA-induced toxicity. Sodium salicylate and acetylsalicylic acid are non-steroidal anti-inflammatory agents that prevent activation of nuclear factor κB by inhibition of phosphorylation and subsequent degradation of IκB, or by direct inhibition of IκB kinase (20- 23). Salicylate also interferes with MAPK and other kinase-dependent signaling pathways (24Schwenger P. Bellosta P. Vietor I. Basilico C. Skolnik E.Y. Vilcek J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2869-2873Crossref PubMed Scopus (256) Google Scholar, 25Schwenger P. Skolnik E.Y. Vilcek J. J. Biol. Chem. 1996; 271: 8089-8094Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 26Chen L.C. Kepka-Lenhert D. Wright T.M. Morris S.M. Biochem. J. 1999; 342: 503-507Crossref PubMed Google Scholar) and can affect mitochondrial function (27Biban C. Tassan V. Toninello A. Siliprand D. Siliprandi N. Biochem. Pharmacol. 1995; 50: 497-500Crossref PubMed Scopus (23) Google Scholar, 28Trost C.C. Lemasters J.J. Toxicicol. Appl. Pharmacol. 1997; 147: 431-441Crossref PubMed Scopus (91) Google Scholar). Damage to mitochondria plays an important role in AA plus CYP2E1-dependent toxicity (29Wu D. Cederbaum A.I. Hepatology. 2002; 35: 1420-1430Crossref PubMed Scopus (50) Google Scholar). Whether MAPKs are involved in the AA-induced toxicity, and if so, how MAPKs induce or regulate this toxicity have not been reported, for cytochrome P450-dependent processes in general, and specifically for CYP2E1. In the present study, we characterized the possible role of the MAPKs, p38 and ERK, and PI3K on AA or AA plus salicylate-induced toxicity in pyrazole-induced rat hepatocytes, human hepatocyte cultures, and HepG2 E47 cells. Effects of specific kinase inhibitors on cellular toxicity, CYP2E1 levels, lipid peroxidation, mitochondrial membrane potential, and NF-κB activation and the role of MAPKs in the salicylate enhancement of AA-induced toxicity were evaluated. Results show that AA or AA plus salicylate activated p38 MAPK but not ERK or PI3K, and that p38 MAPK plays a role in AA-induced cytotoxicity in CYP2E1-expressing cells. Rats received humane care, and studies were carried out according to the criteria outline in the Guide for the Care and Use of Laboratory Animals and Institutional Animal Care and Use Committee approval. Male Sprague-Dawley rats, 150–170 g body weight were injected intraperitoneally with pyrazole, 200 mg/kg body weight, once a day for 2 days to induce CYP2E1. After overnight fasting, rat hepatocytes were isolated by a two-step collagenase perfusion method (30Wu D. Clejan L.A. Potter B. Cederbaum A.I. Hepatology. 1990; 12: 1379-1389Crossref PubMed Scopus (61) Google Scholar). Induction of CYP2E1 was validated by Western blot analysis and catalytic activity withp-nitrophenol. Cell viability was generally about 90%. Hepatocytes were seeded onto 100-mm culture dishes, which were coated with the basement membrane Matrigel (BD Biosciences) and cultured in serum-free HeptoZYME-SFM medium (Invitrogen) containing 1% penicillin and streptomycin. One to two hours after seeding, the medium was changed, unattached cells were gently washed out, and the cell culture experiments were initiated. Human hepatocyte cultures plated on rat tail collagen-coated flasks (T-25) in serum-supplemented media were obtained from the Liver Tissue Procurement and Distribution System (University of Minnesota, Minneapolis, MN). E47 cells are HepG2 cells that were transfected with a human CYP2E1 cDNA in the sense orientation and constitutively express CYP2E1. C34 cells are HepG2 cells that were transfected with the pCI vector only; these cells do not express CYP2E1 (9Chen Q. Galleano M. Cederbaum A.I. J. Biol. Chem. 1997; 272: 14532-14541Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). The HepG2 cells were cultured in minimal essential medium supplemented with 10% fetal bovine serum plus 100 units/ml penicillin plus 100 μg/ml streptomycin in 5% CO2 at 37 °C. Hepatocyte or HepG2 cell cultures were treated with arachidonic acid (AA, 60 μm), with or without salicylate (sal, 5 mm) in the presence or absence of 10 μm SB203580 (a specific p38 MAPK inhibitor), 10 μm PD98059 (a specific ERK inhibitor) (Calbiochem Inc.), or 0.1–10 μm wortmannin (a PI3K inhibitor, Sigma Chemical Co.) for 24 h. Inhibitors were dissolved in Me2SO, and controls were incubated with Me2SO (0.6% v/v final concentration). As a non-CYP2E1-dependent control, hepatocytes were treated with 15 ng/ml tumor necrosis factor α plus 40 μmcycloheximide in the presence or absence of MAPK or PI3K inhibitors. Cytotoxicity was determined by assays of either trypan blue exclusion or reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide as described previously (31Wu D. Cederbaum A.I. J. Biol. Chem. 1996; 271: 23914-23919Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). To study whether SB203580 modulates the CYP2E1 level, Pyrazole-induced rat hepatocytes were incubated with either 10 μm SB203580 (or Me2SO solvent control) or 5 mm sal for 1 or 2 days. Microsomes were prepared by differential centrifugation, and SDS-PAGE was carried out using 10 μg of microsomal protein. The blotted membranes were incubated with a monospecific polyclonal CYP2E1 antibody, followed by incubation with goat anti-rabbit antibody conjugated with horseradish peroxidase. Fluorescence was developed using the enhanced chemiluminescence immunoblot-detecting agent (ECL, AmershamBiosciences). The arbitrary unit of density of each band was scanned with a computer software program (UN-SCAN-IT, Automated Digitizing System, version 5.1, Silk Scientific Corp.). Cells were treated with AA or AA plus sal in the presence or absence of 10 μm SB203580, 10 μm PD98059, 10 μm wortmannin, or Me2SO (solvent for the kinase inhibitors) for 24 h. Cells were harvested and sonicated in PBS for 10 s in an ice bath using a W-375 sonicator (50% duty cycle, output at 4 watts). The cellular lysate was collected, and cell extract containing 0.2 mg of protein was incubated with 0.2 ml of trichloroacetic acid-TBA-HCl solution in a 100 °C water bath for 1 h as previously described (19Wu D. Cederbaum A.I. Mol. Pharmacol. 2001; 59: 795-805Crossref PubMed Scopus (38) Google Scholar, 29Wu D. Cederbaum A.I. Hepatology. 2002; 35: 1420-1430Crossref PubMed Scopus (50) Google Scholar). The formation of thiobarbituric acid-reactive products (TBARS) was determined by measuring absorbance at 535 nm and using an extinction coefficient of 1.56 × 105m/cm to calculate malondialdehyde equivalents. As a control, the potential antioxidant activity of SB203580 was evaluated using isolated rat liver microsomes incubated with iron-ADP plus NADPH as previously described (32Carro A.A. Cederbaum A.I. J. Biol. Chem. 2002; 277: 104-113Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Cells were treated with AA or AA plus sal in the presence or absence of 10 μm SB203580, 10 μm PD98059, or 10 μm wortmannin for 10, 20, or 30 min. The cells were harvested and sonicated as described above. The cellular extract (15 μg of protein) was subjected to SDS-PAGE using an 8% gel. The blotted membranes were incubated with either polyclonal p38 or ERK MAPK antibodies or AKT polyclonal antibody to detect the total content of these kinases or incubated with the appropriate phosphorylated monoclonal antibodies, respectively (Santa Cruz), to determine the content of the activated, phosphorylated kinase. The blots were then incubated with either anti-rabbit IgG or anti-mouse IgG conjugated with horseradish peroxidase and fluorescence developed, and results were analyzed as described above. Freshly isolated rat hepatocytes were grown on a glass slide placed in culture dishes. The cells were treated as described above. At the end of treatment, the cells on the glass slide were fixed with 4% paraformaldehyde, washed with PBS, and incubated with NF-κB antibody (Santa Cruz Biotechnology) for 2 h. After rinsing several times in PBS, the slides were incubated with anti-IgG antibody conjugated with fluorescein isothiocyanate. The slides were rinsed several times with PBS and mounted onto a microscopy glass slide with mounting medium for fluorescence (Vector Laboratories, Inc., Burlingame, CA). The localization of NF-κB in the nucleus or the cytosol was observed under a fluorescence microscope. To determine the effect of AA or AA plus sal on NF-κB DNA binding activity, cells were treated with AA or AA plus sal in the presence or absence of 10 μm SB203580, 10 μm PD98059, or 10 μm wortmannin for 30 min or for 24 h. Cells were harvested and lysed with a lysis solution containing 0.32 msucrose, 2 mm CaCl2, 2 mmMgCl2, 0.1 mm EDTA, 0.5% Triton X-100, 1 mm dithiothreitol, 20 mm Hepes, pH 7.5, and 0.1 mm phenylmethylsulfonyl fluoride for 15 min. After centrifugation at 2500 rpm for 10 min, the pellet was collected and resuspended in 1 ml of lysis solution and centrifuged at 6000 rpm for 10 min. The pellet was resuspended in a buffer containing 0.42m NaCl, 20 mm Hepes, pH 7.5, 25% glycerol, 2 mm MgCl2, 0.2 mm EDTA, 1 mm dithiothreitol, 0.1 mm phenylmethylsulfonyl fluoride, 15 μg/ml antipain, 2.5 μg/ml aprotinin, 2.5 μg/ml leupeptin, 2.5 μg/ml pepstatin, and 2.5 μg/ml bestatin, incubated for 10 min and centrifuged at 15,000 rpm for 15 min. The supernatant containing nuclear extract was collected, and 10 μg of nuclear extract was used to carry out the electrophoretic mobility shift assay with a kit (Promega) according the protocol offered by the company. The NF-κB oligonucleotide in the kit was labeled with [32P]ATP, and bands on the gel were visualized by exposure to the Kodak film and then developed. Mitochondrial membrane potential (MMP) was analyzed from the accumulation of rhodamine 123. Hepatocytes were seeded onto six-well dishes for 1–2 h, and unattached hepatocytes were gently removed and replaced with fresh medium. The cells were treated with AA, or AA plus sal with or without 10 μm SB203580, 10 μm PD98059, or 10 μm wortmannin for 20 h. One hour before ending the treatment, rhodamine 123 (5 μg/ml) was added to the medium. Cells were harvested by trypsinization and resuspended in 0.5–0.8 ml of PBS. The intensity of the fluorescence from rhodamine 123 was determined with a Fas-Scan flow cytometer as previously described (29Wu D. Cederbaum A.I. Hepatology. 2002; 35: 1420-1430Crossref PubMed Scopus (50) Google Scholar). One-way ANOVA (ANOVA with subsequent post hoc comparisons by Sheffe) was performed (Version 10.0, SPSS, Chicago, IL).p values of less than 0.05 were considered statistically significant; values reflect means ± S.E., and the number of experiments are given in the figure legends. HepG2 E47 cells were treated with 60 μm AA or 60 μm AA plus 5 mm sal for 12, 24, or 36 h in the presence or absence of 10 μm SB203580, an inhibitor of p38 MAPK, and cell viability (trypan blue exclusion) was determined. As shown in Fig. 1, AA or AA plus sal induced significant toxicity in E47 cells in a time-dependent manner. Salicylate, which was not toxic by itself, increased the toxicity by AA as described previously (19Wu D. Cederbaum A.I. Mol. Pharmacol. 2001; 59: 795-805Crossref PubMed Scopus (38) Google Scholar). SB203580 by itself was not toxic to the E47 cells, however, SB203580 lowered the AA or AA plus sal-induced toxicity e.g. from 60 or 80% at 24 h in the absences of SB203580 to toxicity values of 20 or 30% (p < 0.05) in the presence of SB203580. Similar protection by SB203580 was observed in AA concentration dependence experiments, e.g. in the absence of SB203580, toxicity by 15, 30, or 60 μm AA at 24 h was 24 ± 3, 34 ± 7, and 60 ± 6%, respectively, whereas the toxicity in the presence of 10 μm SB203580 was lowered to values of 18 ± 2, 20 ± 3, and 22 ± 7% (p < 0.05) at 15, 30, or 60 μm AA, respectively (data not shown). C34 cells, which do not express CYP2E1, only exhibited a small response to AA or AA plus sal (TableI) as compared with the E47 cells, which was not altered by SB203580. The effect of SB203580 on another model of toxicity, independent of CYP2E1, was evaluated. In contrast to results with AA or AA plus salicylate in E47 cells, the toxicity by tumor necrosis factor α plus cycloheximide was similar in E47 and C34 cells, and this toxicity was not altered by SB203580 (Table I). Thus, there appears to be some specificity in the protective actions of SB203580.Table IThe inhibition of P38 MAPK prevents AA and AA plus salicylate-induced cytotoxicity in HepG2 E47 cellsTreatmentCytotoxicityE47 cellsC34 cells%None4.8 ± 1.73.8 ± 1.0Salicylate6.5 ± 1.05.3 ± 1.5SB2035806.0 ± 1.86.5 ± 1.0AA31.5 ± 8.71-aNova statistic analysis, p < 0.05 compared with control group.5.3 ± 1.0AA + Sal57.0 ± 7.51-aNova statistic analysis, p < 0.05 compared with control group.13.0 ± 2.9AA + SB20358010.5 ± 2.31-bNova statistic analysis, p < 0.05 compared with AA treatment group.9.5 ± 1.3AA + Sal + SB20358028.0 ± 7.81-cNova statistic analysis, p < 0.05 compared with AA + Sal group.12.0 ± 3.2TNF5.0 ± 3.44.5 ± 1.7CHX5.5 ± 1.76.0 ± 0.8TNF + CHX37.3 ± 6.939.5 ± 3.9TNF + CHX + Sal42.8 ± 6.947.3 ± 7.5TNF + CHX + Sal + SB20358045.8 ± 9.744.0 ± 15.1HepG2 E47 cells, which express CYP2E1, and control HepG2 cells, which do not express CYP2E1 (C34 cells), were incubated with the indicated treatments, 5 mm sodium salicylate, 60 μm AA, AA plus salicylate, 10 μm SB203580 alone or with AA or with AA plus salicylate. Other groups were treated with 20 ng/ml TNFα in the absence or presence of 40 μm cycloheximide (CHX). The cytotoxicity index refers to the percent cells staining with trypan blue. Results are from three experiments.1-a Nova statistic analysis, p < 0.05 compared with control group.1-b Nova statistic analysis, p < 0.05 compared with AA treatment group.1-c Nova statistic analysis, p < 0.05 compared with AA + Sal group. Open table in a new tab HepG2 E47 cells, which express CYP2E1, and control HepG2 cells, which do not express CYP2E1 (C34 cells), were incubated with the indicated treatments, 5 mm sodium salicylate, 60 μm AA, AA plus salicylate, 10 μm SB203580 alone or with AA or with AA plus salicylate. Other groups were treated with 20 ng/ml TNFα in the absence or presence of 40 μm cycloheximide (CHX). The cytotoxicity index refers to the percent cells staining with trypan blue. Results are from three experiments. To extend the results with the HepG2 cells to primary liver cells, rats were treated with pyrazole (PY) to increase hepatic CYP2E1 levels. Hepatocytes from PY-induced rats were treated with AA or AA plus sal, and cell viability was assayed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction. Cell viability was decreased to 44 ± 5 or 31 ± 10% after 24 h of culture with 60 μm AA or 60 μmAA plus 5 mm sal, respectively (data not shown). Adding SB203580 a p38 MAPK inhibitor or PD98059, an ERK MAPK inhibitor, lowered this toxicity. In the presence of SB203580, cell viability increased to 84 ± 4 (AA) and 75 ± 6% (AA plus sal) (p < 0.05), whereas in the presence of PD98059, cell viability increased to 63 ± 6 (AA) and 78 ± 7% (AA plus sal) (p < 0.05), respectively. Wortmannin, a PI3K inhibitor used at concentrations ranging from 0.1 to 10 μm, failed to prevent AA- or AA plus sal-induced toxicity. SB203580, PD98059, or wortmannin had no effect on cell viability; neither did the Me2SO (0.6%) solvent control. Human hepatocyte cultures were treated with AA or AA plus sal in the presence or absence of SB203580, PD98059, or wortmannin for 24 h. AA (60 μm) or AA plus 5 mm sal caused a 50 ± 4 or 84 ± 1% loss of cell viability, respectively (Fig. 2). SB203580 lowered toxicity to 23 ± 7 or 50 ± 2% (p < 0.05), whereas PD98059 lowered toxicity to 36 ± 4 or 63 ± 10% (p < 0.05), respectively. Wortmannin failed to prevent AA- or AA plus sal-induced toxicity in human hepatocyte cultures (p > 0.05, Fig. 2). Generally, with both PY rat hepatocytes and the human hepatocytes, SB203580 was more effective than PD98059 in preventing the CYP2E1 plus AA toxicity. Changes in the level of CYP2E1 could be one mechanism by which SB203580 protects against AA toxicity in the hepatocytes or E47 cells. Pyrazole-induced rat hepatocytes were incubated with 10 μm SB203580 for 1 or 2 days, and levels of CYP2E1 were determined by Western blot analysis. The immunoblots showed the expected decrease in CYP2E1 levels when hepatocytes are placed in culture (30Wu D. Clejan L.A. Potter B. Cederbaum A.I. Hepatology. 1990; 12: 1379-1389Crossref PubMed Scopus (61) Google Scholar) (Fig. 3, lanes 1, 2, and6). SB203580 did not lower CYP2E1 protein levels as compared with control incubations (Fig. 3, lanes 4 and 8); actually, CYP2E1 levels were increased, but this increase was due to the Me2SO solvent used to solubilize the SB203580 (Fig. 3,lanes 5 and 9). Me2SO is a CYP2E1 ligand that protects against CYP2E1 degradation. This suggests that SB203580 prevention of AA- or AA plus sal-induced toxicity in hepatocytes is not mediated via lowering the levels of CYP2E1. As previously shown (19Wu D. Cederbaum A.I. Mol. Pharmacol. 2001; 59: 795-805Crossref PubMed Scopus (38) Google Scholar), salicylate helped to partially maintain CYP2E1 levels at 24 h in the tissue culture (Fig. 3, compare lanes 3 and 7 with lanes 2 and 6), and this maintenance was not altered by SB203580 (not shown). Treating the cells with AA increased lipid peroxidation in hepatocytes about 2-fold, and this was further elevated in the presence of salicylate (Fig. 4 A). Salicylate alone, as previously shown (19Wu D. Cederbaum A.I. Mol. Pharmacol. 2001; 59: 795-805Crossref PubMed Scopus (38) Google Scholar), had no effect on lipid peroxidation. Antioxidants such as Trolox, which prevent the AA-induced lipid peroxidation, were previously shown to protect against the AA toxicity (29Wu D. Cederbaum A.I. Hepatology. 2002; 35: 1420-1430Crossref PubMed Scopus (50) Google Scholar); hence, if SB203580 had any antioxidant action, its ability to prevent lipid peroxidation could explain its protective effects against AA-induced toxicity. However, SB203580, PD98059, or wortmannin (or Me2SO) did not reduce lipid peroxidation induced by AA or AA plus sal (Fig. 4 A). To further exclude a possible antioxidant effect of SB203580, we incubated rat liver microsomes with Me2SO, Me2SO plus SB203580, or, as a positive control, the antioxidant Trolox (100 μm) and determined lipid peroxidation (Fig. 4 B). SB203580 did not affect lipid peroxidation of rat microsomes compared with its Me2SO solvent control. In contrast, Trolox completely inhibited the lipid peroxidation (Fig. 4 B). These results suggest that the prevention by SB203580 of AA- or AA plus sal-induced toxicity is not mediated by an antioxidant action that lowers lipid peroxidation. Mitochondria appear to be a critical target for damage by AA as well as a major organelle for the production of reactive oxygen species. AA was previously shown to decrease MMP, a decrease that was intensified by salicylate; moreover, cyclosporin A, an inhibitor of the mitochondrial permeability transition protected against AA or AA plus salicylate toxicity (29Wu D. Cederbaum A.I. Hepatology. 2002; 35: 1420-1430Crossref PubMed Scopus (50) Google Scholar). Therefore, it was important to study whether the inhibition of p38 MAPK by SB203580 could protect against the AA or AA plus sal reduction of the MMP. Cells were treated with AA or AA plus sal in the presence or absence of kinase inhibitors for 24 h, and the cells were then treated with Rh123 for 1 h. Flow cytometry was carried out to determine the extent of Rh123 fluorescence, an index of the MMP. The percentage of cells with low Rh123 fluorescence (M1 population; reflective of low MMP) was increased from 12 to 31% by AA or to 49% by AA plus sal (Fig. 5). However, in the presence of SB203580, the decline of MMP was prevented as the cells with low MMP decreased to 10% (AA) or 16% (AA plus sal) (p < 0.05, Fig. 5). PD98059 showed only a modest prevention effect (21% or 33% M1 cells), whereas wortmannin had no protective effect against the decline in MMP produced by AA or AA plus salicylate (Fig. 5). The prevention of AA toxicity by SB203580 suggests a role for p38 MAPK in the overall toxicity mechanism of AA. One method to assess activation of p38 MAPK is by determining its extent of phosphorylation. Treatment of rat hepatocytes with AA, sal, or AA plus sal significantly increased the level of phosphorylated p38 MAPK. After 20-min treatment with AA, sal, or AA plus sal, the extent of p38 MAPK phosphorylation increased 2.6-, 2.9-, or 5.2-fold, respectively (Fig. 6 A,lanes 4, 2, and 5, compared withlane 1, respectively), whereas the extent of phosphorylation increased 3.1-, 3.7-, or 3.9-fold, respectively, after 30-min incubation (Fig. 6 A, lanes 4, 2, and 5 compared with lane 1, respectively). AA or AA plus sal did not activate p38 MAPK phosphorylation activity after 30-min treatment in saline hepatocytes with lower levels of CYP2E1 (data not shown). AA, sal, or AA plus sal also induced p38 MAPK phosphorylation in human hepatocyte cultures (Fig. 6 B,top panels, lanes 4, 2, and 5 compared with lane 1, respectively). The ratio of phosphorylated p38 MAPK to total p38 MAPK was increased to values of 2.7, 2.1, and 4.2 after 30-min treatment with salicylate, AA, or AA plus salicylate, respectively, over the control values of 1.0. In contrast to p38 MAPK, AA or AA plus salicylate did not promote the phosphorylation of either ERK or PI3K in PY hepatocytes (data not shown) or human h" @default.
• W1988049205 created "2016-06-24" @default.
• W1988049205 creator A5039170596 @default.
• W1988049205 creator A5046167919 @default.
• W1988049205 date "2003-01-01" @default.
• W1988049205 modified "2023-10-16" @default.
• W1988049205 title "Role of p38 MAPK in CYP2E1-dependent Arachidonic Acid Toxicity" @default.
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