P450 CYP2C epoxygenase and CYP4A ω-hydroxylase mediate ciprofibrate-induced PPARα-dependent peroxisomal proliferation
2007; Elsevier BV; Volume: 48; Issue: 4 Linguagem: Inglês
10.1194/jlr.m700002-jlr200
ISSN1539-7262
AutoresArnaldo Gatica, Mauricio C. Aguilera, David Contador, Gloria Loyola, Claudio Pinto, Ludwig Amigo, Juan E. Tichauer, Silvana Zanlungo, Miguel Bronfman,
Tópico(s)Pharmacogenetics and Drug Metabolism
ResumoPeroxisomal proliferators, such as ciprofibrate, are used extensively as effective hypolipidemic drugs. The effects of these compounds on lipid metabolism require ligand binding activation of the peroxisome proliferator-activated receptor (PPAR) α subtype of nuclear receptors and involve transcriptional activation of the metabolic pathways involved in lipid oxidative metabolism, transport, and disposition. ω-Hydroxylated-eicosatrienoic acids (HEETs), products of the sequential metabolism of arachidonic acid (AA) by the cytochrome P450 CYP2C epoxygenase and CYP4A ω-hydroxylase gene subfamilies, have been identified as potent and high-affinity ligands of PPARα in vitro and as PPARα activators in transient transfection assays. Using isolated rat hepatocytes in culture, we demonstrate that specific inhibition of either the CYP2C epoxygenase or the CYP4A ω-hydroxylase abrogates ciprofibrate-induced peroxisomal proliferation, whereas inhibition of other eicosanoid-synthesizing pathways had no effect. Conversely, overexpression of the rat liver CYP2C11 epoxygenase leads to spontaneous peroxisomal proliferation, an effect that is reversed by a CYP inhibitor. Based on these results, we propose that HEETs may serve as endogenous PPARα ligands and that the P450 AA monooxygenases participate in ciprofibrate-induced peroxisomal proliferation and the activation of PPARα downstream targets. Peroxisomal proliferators, such as ciprofibrate, are used extensively as effective hypolipidemic drugs. The effects of these compounds on lipid metabolism require ligand binding activation of the peroxisome proliferator-activated receptor (PPAR) α subtype of nuclear receptors and involve transcriptional activation of the metabolic pathways involved in lipid oxidative metabolism, transport, and disposition. ω-Hydroxylated-eicosatrienoic acids (HEETs), products of the sequential metabolism of arachidonic acid (AA) by the cytochrome P450 CYP2C epoxygenase and CYP4A ω-hydroxylase gene subfamilies, have been identified as potent and high-affinity ligands of PPARα in vitro and as PPARα activators in transient transfection assays. Using isolated rat hepatocytes in culture, we demonstrate that specific inhibition of either the CYP2C epoxygenase or the CYP4A ω-hydroxylase abrogates ciprofibrate-induced peroxisomal proliferation, whereas inhibition of other eicosanoid-synthesizing pathways had no effect. Conversely, overexpression of the rat liver CYP2C11 epoxygenase leads to spontaneous peroxisomal proliferation, an effect that is reversed by a CYP inhibitor. Based on these results, we propose that HEETs may serve as endogenous PPARα ligands and that the P450 AA monooxygenases participate in ciprofibrate-induced peroxisomal proliferation and the activation of PPARα downstream targets. Several structurally unrelated xenobiotics with hypolipidemic properties induce peroxisome proliferation in the livers of rodents. These agents, termed hypolipidemic peroxisome proliferators (PPs), include an extensive number of synthetic compounds, such as ciprofibrate and related drugs used in the control of hyperlipemia, as well as environmental contaminants, such as plasticizers and herbicides (reviewed in Refs. 1Desvergne B. Wahli W. Peroxisome proliferator-activated receptors: nuclear control of metabolism. Endocr. Rev. 1999; 20: 649-688Google Scholar, 2Reddy J.K. Hashimoto T. Peroxisomal beta-oxidation and peroxisome proliferator-activated receptor alpha: an adaptive metabolic system. Annu. Rev. Nutr. 2001; 21: 193-230Google Scholar). In spite of their slight structural similarity, PPs as a group induce in rat and mouse a qualitatively similar pleiotropic response, consisting of hypolipidemia, hepatomegaly, proliferation of peroxisomes, and the induction of several hepatic enzymes involved in lipid metabolism (2Reddy J.K. Hashimoto T. Peroxisomal beta-oxidation and peroxisome proliferator-activated receptor alpha: an adaptive metabolic system. Annu. Rev. Nutr. 2001; 21: 193-230Google Scholar, 3Lazarow P.B. De Duve C. A fatty acyl-CoA oxidizing system in rat liver peroxisomes: enhancement by clofibrate, a hypolipidemic drug. Proc. Natl. Acad. Sci. USA. 1976; 73: 2043-2046Google Scholar, 4Reddy J.K. Goel S.K. Nemali M.R. Carrino J.J. Laffler T.G. Reddy M.K. Sperbeck S.J. Osumi T. Hashimoto T. Lalwani N.D. Transcription regulation of peroxisomal fatty acyl-CoA oxidase and enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase in rat liver by peroxisome proliferators. Proc. Natl. Acad. Sci. USA. 1986; 83: 1747-1751Google Scholar). In addition, certain PPs are rapidly and abundantly transformed into nonmetabolizable acyl-CoA thioesters in vitro and in vivo (5Bronfman M. Morales M.N. Amigo L. Orellana A. Nunez L. Cardenas L. Hidalgo P.C. Hypolipidaemic drugs are activated to acyl-CoA esters in isolated rat hepatocytes. Detection of drug activation by human liver homogenates and by human platelets. Biochem. J. 1992; 284: 289-295Google Scholar, 6Hertz R. Sheena V. Kalderon B. Berman I. Bar-Tana J. Suppression of hepatocyte nuclear factor-4alpha by acyl-CoA thioesters of hypolipidemic peroxisome proliferators. Biochem. Pharmacol. 2001; 61: 1057-1062Google Scholar), and significant changes are also produced in the levels of Coenzyme A (CoASH) and their fatty acyl-CoA thioesters (7Voltti H. Savolainen M.J. Jauhonen V.P. Hassinen I.E. Clofibrate-induced increase in coenzyme A concentration in rat tissues. Biochem. J. 1979; 182: 95-102Google Scholar, 8Halvorsen O. Effects of hypolipidemic drugs on hepatic CoA. Biochem. Pharmacol. 1983; 32: 1126-1128Crossref PubMed Scopus (19) Google Scholar). Long-term exposure to PPs leads to the development of hepatocellular carcinoma in rats and mice (9Reddy J.K. Azarnoff D.L. Hignite C.E. Hypolipidaemic hepatic peroxisome proliferators form a novel class of chemical carcinogens. Nature. 1980; 283: 397-398Google Scholar). PP effects are mediated by peroxisome proliferator-activated receptor (PPAR) α, as these effects are abrogated in PPARα null mice (10Lee S.S. Pineau T. Drago J. Lee E.J. Owens J.W. Kroetz D.L. Fernandez-Salguero P.M. Westphal H. Gonzalez F.J. Targeted disruption of the alpha isoform of the peroxisome proliferator-activated receptor gene in mice results in abolishment of the pleiotropic effects of peroxisome proliferators. Mol. Cell. Biol. 1995; 15: 3012-3022Google Scholar). Fatty acids, and particularly their eicosanoid products, resulting from the lipoxygenase, cyclooxygenase, and P450 pathways, show high affinity for PPARs, and some of them have been suggested as endogenous PPARα ligands (11Forman B.M. Chen J. Evans R.M. Hypolipidemic drugs, polyunsaturated fatty acids, and eicosanoids are ligands for peroxisome proliferator-activated receptors alpha and delta. Proc. Natl. Acad. Sci. USA. 1997; 94: 4312-4317Google Scholar, 12Lin Q. Ruuska S.E. Shaw N.S. Dong D. Noy N. Ligand selectivity of the peroxisome proliferator-activated receptor alpha. Biochemistry. 1999; 38: 185-190Google Scholar, 13Murakami K. Ide T. Suzuki M. Mochizuki T. Kadowaki T. Evidence for direct binding of fatty acids and eicosanoids to human peroxisome proliferator-activated receptor alpha. Biochem. Biophys. Res. Commun. 1999; 260: 609-613Google Scholar, 14Cowart L.A. Wei S. Hsu M.H. Johnson E.F. Krishna M.U. Falck J.R. Capdevila J.H. The CYP4A isoforms hydroxylate epoxyeicosatrienoic acids to form high affinity peroxisome proliferator-activated receptor ligands. J. Biol. Chem. 2002; 277: 35105-35112Google Scholar). ω-Hydroxylated-eicosatrienoic acids (HEETs), products of the sequential metabolism of arachidonic acid (AA) by the cytochrome P450 CYP2C epoxygenase and CYP4A ω-hydroxylase gene subfamilies, have been identified as the ligands with the highest affinity for PPARα (14Cowart L.A. Wei S. Hsu M.H. Johnson E.F. Krishna M.U. Falck J.R. Capdevila J.H. The CYP4A isoforms hydroxylate epoxyeicosatrienoic acids to form high affinity peroxisome proliferator-activated receptor ligands. J. Biol. Chem. 2002; 277: 35105-35112Google Scholar). These P450 isoforms play important physiological roles in the control of tissue and body homeostasis (15Capdevila J.H. Harris R.C. Falck J.R. Microsomal cytochrome P450 and eicosanoid metabolism. Cell. Mol. Life Sci. 2002; 59: 780-789Crossref PubMed Scopus (65) Google Scholar). CYP2C P450s catalyze the regioselective and enantioselective metabolism of AA to epoxyeicosatrienoic acids (EETs) and account for most of the epoxygenase activity in rat and human liver and kidney (16Capdevila J.H. Falck J.R. The CYP P450 arachidonic acid monooxygenases: from cell signaling to blood pressure regulation. Biochem. Biophys. Res. Commun. 2001; 285: 571-576Crossref PubMed Scopus (116) Google Scholar). EETs have vasoactive properties, modulate ion channels (17Maier K.G. Roman R.J. Cytochrome P450 metabolites of arachidonic acid in the control of renal function. Curr. Opin. Nephrol. Hypertens. 2001; 10: 81-87Crossref PubMed Scopus (86) Google Scholar), and function as second messengers of pathways, such as epidermal growth factor-dependent signaling (18Chen J.K. Wang D.W. Falck J.R. Capdevila J. Harris R.C. Transfection of an active cytochrome P450 arachidonic acid epoxygenase indicates that 14,15-epoxyeicosatrienoic acid functions as an intracellular second messenger in response to epidermal growth factor. J. Biol. Chem. 1999; 274: 4764-4769Google Scholar). EETs are excellent substrates for CYP4A isoforms (14Cowart L.A. Wei S. Hsu M.H. Johnson E.F. Krishna M.U. Falck J.R. Capdevila J.H. The CYP4A isoforms hydroxylate epoxyeicosatrienoic acids to form high affinity peroxisome proliferator-activated receptor ligands. J. Biol. Chem. 2002; 277: 35105-35112Google Scholar), which selectively hydroxylate saturated and unsaturated fatty acids in the ω/ω-1 position (19Hardwick J.P. Song B.J. Huberman E. Gonzalez F.J. Isolation, complementary DNA sequence, and regulation of rat hepatic lauric acid omega-hydroxylase (cytochrome P-450LA omega). Identification of a new cytochrome P-450 gene family. J. Biol. Chem. 1987; 262: 801-810Google Scholar, 20Helvig C. Dishman E. Capdevila J.H. Molecular, enzymatic, and regulatory characterization of rat kidney cytochromes P450 4A2 and 4A3. Biochemistry. 1998; 37: 12546-12558Google Scholar, 21Hoch U. Zhang Z. Kroetz D.L. Ortiz de Montellano P.R. Structural determination of the substrate specificities and regioselectivities of the rat and human fatty acid omega-hydroxylases. Arch. Biochem. Biophys. 2000; 373: 63-71Google Scholar). Some of the CYP4A isoforms are under the control of PPARα, which regulates an adaptive CYP4A induction in response to starvation and diabetes (22Kroetz D.L. Yook P. Costet P. Bianchi P. Pineau T. Peroxisome proliferator-activated receptor alpha controls the hepatic CYP4A induction adaptive response to starvation and diabetes. J. Biol. Chem. 1998; 273: 31581-31589Google Scholar). Cyp4A is also under the control of other physiological stimuli, including fatty acids and hormones (23Johnson E.F. Palmer C.N. Griffin K.J. Hsu M.H. Role of the peroxisome proliferator-activated receptor in cytochrome P450 4A gene regulation. FASEB J. 1996; 10: 1241-1248Google Scholar). We report here that inhibition of the CYP2C epoxygenase or the CYP4A ω-hydroxylase abrogates ciprofibrate-induced peroxisomal proliferation in isolated rat hepatocytes. Conversely, overexpression of the rat liver CYP2C11 epoxygenase leads to spontaneous peroxisomal proliferation. Our data suggest that HEETs are endogenous PPARα ligands and that the P450 AA monooxygenases participate in ciprofibrate-induced peroxisomal proliferation. Ciprofibrate was provided by the Sterling-Winthrop Institute. Williams E medium and FBS were obtained from GIBCO (Grand Island, NY). Indomethacin, lipoxygenase-specific inhibitors, and EET standards were from Cayman Chemical (Ann Arbor, MI). All other drugs and chemicals were obtained from Sigma Chemical Co. (St. Louis, MO). PPARα-specific antibodies were purchased from Affinity BioReagents (Golden, CO). Antibodies against the integral peroxisomal membrane protein (PMP70) and catalase were a kind gift from Dr. M. Santos (Department of Cellular and Molecular Biology, Faculty of Biological Sciences, P. Universidad Catolica de Chile). Antibodies against alkyl-dihydroxyacetone phosphate synthase (ADAPS) were a generous gift from Dr. W. Just (Biochemie-Zentrum, Ruprecht-Karls-Universität, Heidelberg, Germany). Rat hepatocytes were isolated as described (24Berry M.N. Friend D.S. High-yield preparation of isolated rat liver parenchymal cells: a biochemical and fine structural study. J. Cell Biol. 1969; 43: 506-520Google Scholar). Cell viability was 80–90%, as determined by trypan blue exclusion. Isolated hepatocytes were seeded at 7.5 × 105 cells in 3.5 cm plastic dishes coated with collagen and cultured in Williams E medium supplemented with 5% FBS, as described previously (5Bronfman M. Morales M.N. Amigo L. Orellana A. Nunez L. Cardenas L. Hidalgo P.C. Hypolipidaemic drugs are activated to acyl-CoA esters in isolated rat hepatocytes. Detection of drug activation by human liver homogenates and by human platelets. Biochem. J. 1992; 284: 289-295Google Scholar). At 8 h, the medium was replaced by drugs containing medium. Inhibitor drugs were dissolved in DMSO, yielding a final DMSO concentration of 0.01%. For [1-14C]AA metabolic experiments, cells were incubated for various times in medium containing 4 μM AA and 0.25 μCi/dish [1-14C]AA (53 mCi/mmol; Perkin-Elmer Life and Analytical Science, Boston, MA). Cells were fixed with 1 ml of methanol containing 0.1% acetic acid and triphenylphosphine (0.1 mM) and extracted twice with 2 ml of chloroform, according to published methods (25Capdevila J.H. Dishman E. Karara A. Falck J.R. Cytochrome P450 arachidonic acid epoxygenase: stereochemical characterization of epoxyeicosatrienoic acids. Methods Enzymol. 1991; 206: 441-453Google Scholar). The combined organic phases were evaporated under argon and dissolved in 300 μl of 50% acetonitrile containing 0.1% acetic acid. Separation of [1-14C]AA from its radioactive metabolites was performed by reverse-phase HPLC with a 5 μ C18 symmetric 300 column (4 × 250 mm; Waters Corp., Milford, MA) and a linear solvent gradient from initially acetonitrile-water-acetic acid (49.9:49.9:0.1, v/v/v) to acetonitrile-acetic acid (99.9:0.1, v/v) over 40 min at 1 ml/min (26Capdevila J. Falck J. Dishman E. Karara A. Cytochrome P-450 arachidonate oxygenase. Methods Enzymol. 1989; 187: 385-394Google Scholar). Fractions of 0.8 ml were collected, and their radioactivity was determined by liquid scintillation counting. Chemical synthesis of [1-14C]14,15-EET was performed according to published methods and purified by reverse-phase HPLC (25Capdevila J.H. Dishman E. Karara A. Falck J.R. Cytochrome P450 arachidonic acid epoxygenase: stereochemical characterization of epoxyeicosatrienoic acids. Methods Enzymol. 1991; 206: 441-453Google Scholar, 27Falck J.R. Yadagiri P. Capdevila J. Synthesis of epoxyeicosatrienoic acids and heteroatom analogs. Methods Enzymol. 1990; 187: 357-364Crossref PubMed Scopus (54) Google Scholar). Incubation of hepatocytes with [1-14C]14,15-EET and separation from its metabolites were performed as described above for [1-14C]AA. Cells were incubated with 4 μM 14,15-EET and 0.1 μCi/dish [1-14C]14,15-EET. Nucleic acid hybridizations were performed using total rat hepatocyte RNA, which was isolated using the guanidinium-phenol method described previously (28Chomczynski P. Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate- phenol-chloroform extraction. Anal. Biochem. 1987; 162: 156-159Google Scholar) and quantified by standard spectrophotometric methods. Total hepatocyte RNA samples were fractionated by denaturing agarose electrophoresis and transferred to nylon membranes (Gene Screen Plus; NEN-PerkinElmer, Waltham, MA). Membranes were then cross-linked by UV irradiation and prehybridized in ULTRAHyb (Ambion, Austin, TX) for 2 h at 42°C. Hybridizations were done using specific cDNA probes for acyl-coenzyme A oxidase (AOX), peroxisomal membrane marker PMP70, and PPARα. Probes were radiolabeled using Klenow amplification in the presence of [32P]deoxythymidine. Hybridization was done at 48°C for 14 h. The probes were synthesized by PCR amplification of cDNAs. The following primers were used: for AOX, forward (5′-TGA-CAC-CAT-ACC-ACC-CAC-CAA-C-3′) and reverse (5′-ACG-CAC-ATC-TTGGAT-GGC-AG-3′); for PMP70, forward (5′-TGT-GCG-GCT-CAC-TAG-ATA-CC-3′) and reverse (5′-TAC-GAG-GAG-GAT-TGT-GGA-GG-3′); and for PPARα, forward (5′-GTG-CCT-GTC- CGT-CGG-GAT-GT-3′) and reverse (5′-GTG-AGC-TCG-GTG-ACG-GTC-TC-3′). A reporter plasmid containing three tandem repeats of the peroxisome proliferator response element (PPRE) from the AOX gene, fused to the herpes virus thymidine kinase promoter upstream of the coding sequence for luciferase, and an expression vector containing murine PPARα were a kind gift of Dr. R. M. Evans (Howard Hughes Medical Institute, Gene Expression Laboratory, Salk Institute for Biological Studies, La Jolla, CA). The expression vector containing only the coding sequence for luciferase under the control of the herpes virus thymidine kinase promoter (Clontech, Palo Alto, CA) was used as a control. Transient transfections of CHO cells were carried out according to the manufacturer's instructions, using LipofectAmine 2000 (Life Technologies, Carlsbad, CA). CHO cells were grown in DMEM, supplemented with 4% FBS until 70–80% confluent, and transfected with 0.3 μg of reporter plasmid or cotransfected with the reporter plasmid, 0.2 μg of the PPARα plasmid and 40 ng of a CMV-β-Gal vector (Clontech, Palo Alto, CA), for normalization. The recombinant adenovirus containing the predominant rat liver arachidonate epoxygenase (CYP2C11), fused to rat P450 oxidoreductase (Ad-EPOX) (29Helvig C. Capdevila J.H. Biochemical characterization of rat P450 2C11 fused to rat or bacterial NADPH-P450 reductase domains. Biochemistry. 2000; 39: 5196-5205Google Scholar) and the bacterial β-galactosidase (Ad-βgal) transgenes, was under the control of the cytomegalovirus promoter. Antibodies against CP2C11 and β-galactosidase, as well as CYP2C and CYP4A P450 inhibitors, were kindly donated by Dr. Jorge Capdevila (Department of Medicine, Vanderbilt University Medical School, Nashville, TN). Large-scale production of recombinant adenoviruses was performed from HEK 293 infected cells, as described (30Kozarsky K.F. Jooss K. Donahee M. Strauss J.F. Wilson J.M. Effective treatment of familial hypercholesterolaemia in the mouse model using adenovirus-mediated transfer of the VLDL receptor gene. Nat. Genet. 1996; 13: 54-62Google Scholar). For adenoviral infections, primary rat hepatocyte cultures were plated on 3.5 cm tissue culture dishes (200,000 total). Twenty-four hours after plating, cells were infected with 7 × 107 viral particles/cm2. After 2 h of infection, unbound virus was removed and replaced with fresh medium. The cells were incubated at 37°C in 5% CO2 for 48 h. Cells were then harvested and processed for blotting or immunocytochemistry. As stated previously, eicosanoid products of the cyclooxygenase, lipoxygenase, and cytochrome P450 pathways have been proposed as endogenous PPARα activators (11Forman B.M. Chen J. Evans R.M. Hypolipidemic drugs, polyunsaturated fatty acids, and eicosanoids are ligands for peroxisome proliferator-activated receptors alpha and delta. Proc. Natl. Acad. Sci. USA. 1997; 94: 4312-4317Google Scholar, 31Devchand P.R. Keller H. Peters J.M. Vazquez M. Gonzalez F.J. Wahli W. The PPARalpha-leukotriene B4 pathway to inflammation control. Nature. 1996; 384: 39-43Google Scholar, 32Keller H. Dreyer C. Medin J. Mahfoudi A. Ozato K. Wahli W. Fatty acids and retinoids control lipid metabolism through activation of peroxisome proliferator-activated receptor-retinoid X receptor heterodimers. Proc. Natl. Acad. Sci. USA. 1993; 90: 2160-2164Google Scholar, 33Yu K. Bayona W. Kallen C.B. Harding H.P. Ravera C.P. McMahon G. Brown M. Lazar M.A. Differential activation of peroxisome proliferator-activated receptors by eicosanoids. J. Biol. Chem. 1995; 270: 23975-23983Abstract Full Text Full Text PDF PubMed Scopus (635) Google Scholar, 34Kliewer S.A. Sundseth S.S. Jones S.A. Brown P.J. Wisely G.B. Koble C.S. Devchand P. Wahli W. Willson T.M. Lenhard J.M. Fatty acids and eicosanoids regulate gene expression through direct interactions with peroxisome proliferator-activated receptors alpha and gamma. Proc. Natl. Acad. Sci. USA. 1997; 94: 4318-4323Google Scholar). On this basis, we first determined whether inhibitors of these pathways had any effect on ciprofibrate-induced peroxisomal proliferation in isolated rat hepatocytes. We found that the CYP P450 general inhibitors, clotrimazole and ketoconazole (35Capdevila J. Gil L. Orellana M. Marnett L.J. Mason J.I. Yadagiri P. Falck J.R. Inhibitors of cytochrome P-450-dependent arachidonic acid metabolism. Arch. Biochem. Biophys. 1988; 261: 257-263Crossref PubMed Scopus (168) Google Scholar), strongly decrease ciprofibrate-induced mRNA expression of the peroxisomal proliferation markers AOX and the peroxisomal membrane protein PMP70 (36Santos M.J. Imanaka T. Shio H. Lazarow P.B. Peroxisomal integral membrane proteins in control and Zellweger fibroblasts. J. Biol. Chem. 1988; 263: 10502-10509Abstract Full Text PDF PubMed Google Scholar) in a concentration-dependent manner, using GAPDH as a control (Fig. 1 ). Although GAPDH increases its expression upon long-term treatment of rats with some PPs (37Garcia-Allan C. Loughlin J. Orton T. Lord P. Changes in protein and mRNA levels of growth factor/growth factor receptors in rat livers after administration of phenobarbitone or methylclofenapate. Arch. Toxicol. 1997; 71: 409-415Google Scholar), we did not observe significant changes in its expression in our short-term (12 h) experiments. Under the same experimental conditions, neither the specific lipoxygenase inhibitors, caffeic acid [a 5- and a 12-lipoxygenase inhibitor (38Koshihara Y. Neichi T. Murota S. Lao A. Fujimoto Y. Tatsuno T. Caffeic acid is a selective inhibitor for leukotriene biosynthesis. Biochim. Biophys. Acta. 1984; 792: 92-97Crossref PubMed Scopus (248) Google Scholar)], baicalein [a 12-lipoxygenase inhibitor (39Sekiya K. Okuda H. Selective inhibition of platelet lipoxygenase by baicalein. Biochem. Biophys. Res. Commun. 1982; 105: 1090-1095Crossref PubMed Scopus (247) Google Scholar)], or sculetin [a 15-lipoxygenase inhibitor (40Gleason M.M. Rojas C.J. Learn K.S. Perrone M.H. Bilder G.E. Characterization and inhibition of 15-lipoxygenase in human monocytes: comparison with soybean 15-lipoxygenase. Am. J. Physiol. 1995; 268: C1301-C1307Google Scholar)], nor the selective cyclooxygenase inhibitor indomethacin (41Jaradat M.S. Wongsud B. Phornchirasilp S. Rangwala S.M. Shams G. Sutton M. Romstedt K.J. Noonan D.J. Feller D.R. Activation of peroxisome proliferator-activated receptor isoforms and inhibition of prostaglandin H(2) synthases by ibuprofen, naproxen, and indomethacin. Biochem. Pharmacol. 2001; 62: 1587-1595Google Scholar), all in the 10–100 μM concentration range, had any effect on the ciprofibrate-induced increase of peroxisomal proliferation markers (data not shown). Next, we used 6-(2-propargyloxyphenyl) hexanoic acid (PPOH) and N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS), specific inhibitors of CYP4A-dependent ω-hydroxylation and CYP2C-dependent epoxidation, respectively (42Wang M.H. Brand-Schieber E. Zand B.A. Nguyen X. Falck J.R. Balu N. Schwartzman M.L. Cytochrome P450-derived arachidonic acid metabolism in the rat kidney: characterization of selective inhibitors. J. Pharmacol. Exp. Ther. 1998; 284: 966-973Google Scholar), to dissect the P450 pathways involved in the inhibition of peroxisomal proliferation marker induction by ciprofibrate. Both inhibitors abrogate the ciprofibrate-induced expression of AOX and PMP70 in a concentration-dependent manner (Fig. 2A ,B). To evaluate whether these inhibitors could act as direct antagonists of PPARα, we transiently cotransfected CHO cells with a PPRE-luciferase reporter plasmid and a mouse PPARα expression plasmid (see Materials and Methods), and the transfected cells were treated for 24 h with 100 μM ciprofibrate in the presence or absence of increasing concentrations of DDMS or PPOH. No effect on the relative ciprofibrate-induced luciferase activity was observed for either drug (Fig. 2C), showing that they do not affect PPARα transcriptional activation. We used CHO cells because of the low efficiency of cultured hepatocytes in transfection experiments with the reporter plasmid. Similarly, no effect of the drugs was found on PPARα mRNA expression in isolated hepatocytes at the maximal concentration used (Fig. 2D). Because both CYP4A and CYP2C are also regulated by PPARα activators in opposite directions (43Corton J.C. Fan L.Q. Brown S. Anderson S.P. Bocos C. Cattley R.C. Mode A. Gustafsson J.A. Down-regulation of cytochrome P450 2C family members and positive acute-phase response gene expression by peroxisome proliferator chemicals. Mol. Pharmacol. 1998; 54: 463-473Google Scholar), we determined the levels of these proteins in control and ciprofibrate-treated cells in the absence and presence of CYP inhibitors (Fig. 2E; quantified in Fig. 2F). No effect of inhibitors was found in control cells for both proteins; however, in ciprofibrate-treated cells, CYP4A increased by 1.5- to 1.8-fold. Consistent with previous data, ciprofibrate-induced CYP4A upregulation was reversed by CYP inhibitors. Ciprofibrate and the inhibitors were without effect on CYP2C expression, either in the absence or presence of ciprofibrate. The lack of ciprofibrate effect on CYP2C11 level is in agreement with published data showing that downregulation of CYP2C occurs only after 48 h of exposure of hepatocytes to the strong PP WY-14,643 (43Corton J.C. Fan L.Q. Brown S. Anderson S.P. Bocos C. Cattley R.C. Mode A. Gustafsson J.A. Down-regulation of cytochrome P450 2C family members and positive acute-phase response gene expression by peroxisome proliferator chemicals. Mol. Pharmacol. 1998; 54: 463-473Google Scholar). A short (12 h) exposure to ciprofibrate was used in our experiments, which also explains the low, although significant, effect of ciprofibrate on CYP4A expression. These results suggest that CYP2C and CYP4A activities are important for ciprofibrate-induced peroxisomal proliferation and that HEETs, the products of the sequential action of CYP2C and CYP4A on unsaturated fatty acids, may be the physiological activators of PPARα in rat liver. To assess whether ciprofibrate induces measurable changes in AA oxidized metabolites, hepatocytes were incubated with [1-14C]AA to label only oxidized AA metabolites and not chain-shortened fatty acids, which are formed from AA as well as from EETs (44Fang X. Kaduce T.L. VanRollins M. Weintraub N.L. Spector A.A. Conversion of epoxyeicosatrienoic acids (EETs) to chain-shortened epoxy fatty acids by human skin fibroblasts. J. Lipid Res. 2000; 41: 66-74Google Scholar). Hepatocytes were incubated with [1-14C]AA for 1 and 6 h in the presence or absence of 100 μM ciprofibrate. The labeled oxidized metabolites and the remaining [1-14C]AA were extracted with chloroform and resolved by reverse-phase HPLC, according to published methods (25Capdevila J.H. Dishman E. Karara A. Falck J.R. Cytochrome P450 arachidonic acid epoxygenase: stereochemical characterization of epoxyeicosatrienoic acids. Methods Enzymol. 1991; 206: 441-453Google Scholar, 26Capdevila J. Falck J. Dishman E. Karara A. Cytochrome P-450 arachidonate oxygenase. Methods Enzymol. 1989; 187: 385-394Google Scholar) (see Materials and Methods). AA is rapidly metabolized by hepatocytes, and only 30–35% of the radioactivity remained in the organic phase as [1-14C]AA after 1 h of incubation; however, after 6 h, only ∼8% of radioactivity was still present as AA, as assessed by reverse-phase HPLC of the organic extracts (data not shown). No radioactivity was detected in the retention time corresponding to EETs, either in the presence or absence of ciprofibrate (Fig. 3 ), suggesting that EETs are very rapidly metabolized. For both incubation times, radioactivity was recovered mainly as [1-14C]AA and also in metabolites eluting earlier than EETs (peak X in Fig. 3A–C). Radioactivity in peak X, which presents a retention time corresponding roughly to that of ω/ω-1 hydroxy acids, such as HEETs (26Capdevila J. Falck J. Dishman E. Karara A. Cytochrome P-450 arachidonate oxygenase. Methods Enzymol. 1989; 187: 385-394Google Scholar), increased 10–15% in ciprofibrate-treated cells. However, ω/ω-1 hydroxylation is one of the known routes for EET metabolism (45Spector A.A. Fang X. Snyder G.D. Weintraub N.L. Epoxyeicosatrienoic acids (EETs): metabolism and biochemical function. Prog. Lipid Res. 2004; 43: 55-90Crossref PubMed Scopus (490) Google Scholar), and peak X probably contains other oxidized metabolites of AA. To indirectly assess whether this fraction may contain HEETs, hepatocytes were incubated for 1, 2, and 6 h in the presence of 14,15-[1-14C]EET in the absence or presence of 20 μM of the CYP4A inhibitor PPOH. Labeled EET disappeared from the incubation medium even faster than AA in both conditions, and only 20% of the initial radioactivity eluted in the retention time of 14,15-EET after 1 h of incubation; after 6 h, it was <5%, as assessed by reverse-phase HPLC of the organic extracts (data not shown). After 1 h of incubation, no differences were induced by PPOH in the amount of radioactivity eluting with the EET peak. However, the radioactivity eluting in fractions around the retention time of peak X was reduced by almost 50% in the presence of PPOH (Fig. 3C). Quantification of the time course of radioactivity eluting in peak X in hepatocytes metabolizing 14,1
Referência(s)