Portal de Periódicos da CAPES

In vivo involvement of cytochrome P450 4A family in the oxidative metabolism of the lipid peroxidation product trans-4-hydroxy-2-nonenal, using PPARα-deficient mice

1999; Elsevier BV; Volume: 40; Issue: 1 Linguagem: Inglês

10.1016/s0022-2275(20)33350-2

1539-7262

Françoise Guéraud, Jacques Alary, Philippe Costet, Laurent Debrauwer, Laurence Dolo, Thierry Pineau, Alain Paris,

Alcohol Consumption and Health Effects

Trans-4-hydroxy-2-nonenal (HNE) is a potent cytotoxic and genotoxic compound originating from the peroxidation of n–6 polyunsaturated fatty acids. Its metabolism has been previously studied in the rat (Alary et al. 1995. Chem. Res. Toxicol., 8: 35–39). In addition to major urinary mercapturic derivatives, some polar urinary metabolites were isolated and could correspond to hydroxylated compounds. 4-Hydroxynonenoic acid (HNA), resulting from the oxidation of the HNE carbonyl group, is a medium chain fatty acid and its ω-hydroxylation might be hypothesized. Therefore, the involvement of the CYP 4A family isoenzymes in the metabolism of [3H]HNE has been investigated in vivo using inducer treatments (fibrates) in wild-type or in peroxisome proliferator-activated receptor α (PPARα)-deficient mice. In wild-type mice, but not in PPARα (−/−) mice, fibrate treatments resulted in an increase of two urinary metabolites characterized, after HPLC purifications and mass spectrometry analyses, as the ω-hydroxylated metabolite of HNA, i.e., 4,9-dihydroxy-2-nonenoic acid, and its oxidized form, 4-hydroxy-2-nonene-1,9-dicarboxylic acid. The formation of the latter is correlated accurately to laurate hydroxylase activity studied concurrently in microsomes prepared from the liver of these animals. Basal levels of these two metabolites were measured in urine of normal and PPARα-deficient mice. These results are in accord with an implication of the P450 4A family in the extended oxidative metabolism of 4-HNE.—Guéraud, F., J. Alary, P. Costet, L. Debrauwer, L. Dolo, T. Pineau, and A. Paris. In vivo involvement of cytochrome P450 4A family in the oxidative metabolism of the lipid peroxidation product trans-4-hydroxy-2-nonenal, using PPARα-deficient mice. J. Lipid Res. 1999. 40: 152–159. Trans-4-hydroxy-2-nonenal (HNE) is a potent cytotoxic and genotoxic compound originating from the peroxidation of n–6 polyunsaturated fatty acids. Its metabolism has been previously studied in the rat (Alary et al. 1995. Chem. Res. Toxicol., 8: 35–39). In addition to major urinary mercapturic derivatives, some polar urinary metabolites were isolated and could correspond to hydroxylated compounds. 4-Hydroxynonenoic acid (HNA), resulting from the oxidation of the HNE carbonyl group, is a medium chain fatty acid and its ω-hydroxylation might be hypothesized. Therefore, the involvement of the CYP 4A family isoenzymes in the metabolism of [3H]HNE has been investigated in vivo using inducer treatments (fibrates) in wild-type or in peroxisome proliferator-activated receptor α (PPARα)-deficient mice. In wild-type mice, but not in PPARα (−/−) mice, fibrate treatments resulted in an increase of two urinary metabolites characterized, after HPLC purifications and mass spectrometry analyses, as the ω-hydroxylated metabolite of HNA, i.e., 4,9-dihydroxy-2-nonenoic acid, and its oxidized form, 4-hydroxy-2-nonene-1,9-dicarboxylic acid. The formation of the latter is correlated accurately to laurate hydroxylase activity studied concurrently in microsomes prepared from the liver of these animals. Basal levels of these two metabolites were measured in urine of normal and PPARα-deficient mice. These results are in accord with an implication of the P450 4A family in the extended oxidative metabolism of 4-HNE. —Guéraud, F., J. Alary, P. Costet, L. Debrauwer, L. Dolo, T. Pineau, and A. Paris. In vivo involvement of cytochrome P450 4A family in the oxidative metabolism of the lipid peroxidation product trans-4-hydroxy-2-nonenal, using PPARα-deficient mice. J. Lipid Res. 1999. 40: 152–159. Trans-4-hydroxy-2-nonenal (HNE), an endogenous α,β-unsaturated aldehydic product resulting from lipid peroxidation (1Esterbauer H. Aldehydic products of lipid peroxidation.in: McBrien D.C.H. Slater T.F. Free Radicals, Lipid Peroxidation and Cancer. Academic Press, London1982: 101-128Google Scholar) is considered to be a potent cytotoxic and genotoxic compound as evidenced by in vitro studies (2Eckl P. Esterbauer H. Genotoxic effects of 4-hydroxynonenal.Adv. Biosci. 1989; 76: 141-157Google Scholar, 3Kruman I. Bruce-Keller A.J. Bredesen D. Waeg G. Mattson M.P. Evidence that 4-hydroxynonenal mediates oxidative stress-induced neuronal apoptosis.J. Neurosci. 1997; 17: 5089-5100Google Scholar). Its important electrophilic nature explains the chemical reactivity towards thiols (4Esterbauer H. Zollner H. Scholz N. Reaction of glutathione with conjugated carbonyls.Z. Naturforsch. 1975; 30: 466-473Google Scholar, 5Esterbauer H. Ertl A. Scholz N. Reaction of cysteine with α,β-unsaturated aldehydes.Tetrahedron. 1976; 32: 285-289Google Scholar), amino groups of amino acids and proteins (lysine, histidine) (6Uchida K. Stadtman E.R. Modification of histidine residues in proteins by reaction with 4-hydroxynonenal.Proc. Natl. Acad. Sci. USA. 1992; 89: 4544-4548Google Scholar, 7Szweda L.I. Uchida K. Tsai L. Stadtman E.R. Inactivation of glucose-6-phosphate dehydrogenase by 4-hydroxynonenal.J. Biol. Chem. 1993; 268: 3342-3347Google Scholar, 8Bolgar M.S. Gaskell S.J. Determination of the sites of 4-hydroxy-2-nonenal adduction to protein by electrospray tandem mass spectrometry.Anal. Chem. 1996; 68: 2325-2330Google Scholar), or DNA bases (deoxyguanosine) (9Winter C.K. Segal H.J. Haddon W.F. Formation of cyclic adducts of deoxyguanosine with the aldehydes trans-4-hydroxy-2-hexenal and trans-4-hydroxy-2-nonenal in vitro.Cancer Res. 1986; 46: 5682-5686Google Scholar, 10Yi P. Zhan D. Samokyszyn V.M. Doerge D.R. Fu P.P. Synthesis and 32P-postlabeling/high-performance liquid chromatography separation of diastereomeric 1,N2-(1,3-propano)-2′-deoxyguanosine 3′-phosphate adducts formed from 4-hydroxy-2-nonenal.Chem. Res. Toxicol. 1997; 10: 1259-1265Google Scholar). In vivo, a substantial level of oxidoreductive metabolism associated with glutathione (GSH) conjugation has been evidenced in the rat (11Alary J. Bravais F. Cravedi J-P. Debrauwer L. Rao D. Bories G. Mercapturic acid conjugates as urinary end metabolites of the lipid peroxidation product 4-hydroxy-2-nonenal in the rat.Chem. Res. Toxicol. 1995; 8: 35-39Google Scholar). A rapid decrease in the HNE pool was observed, resulting in urinary alcohol, acid, and lactone mercapturic derivatives (11Alary J. Bravais F. Cravedi J-P. Debrauwer L. Rao D. Bories G. Mercapturic acid conjugates as urinary end metabolites of the lipid peroxidation product 4-hydroxy-2-nonenal in the rat.Chem. Res. Toxicol. 1995; 8: 35-39Google Scholar). As previously mentioned by Mitchell and Petersen (12Mitchell D.Y. Petersen D.R. The oxidation of α-β unsaturated aldehydic products of lipid peroxidation by rat liver aldehyde dehydrogenase.Toxicol. Appl. Pharmacol. 1987; 87: 403-410Google Scholar) and Esterbauer, Zollner, and Scholz (13Esterbauer H. Zollner H. Scholz N. Metabolism of the lipid peroxidation product 4-hydroxynonenal by isolated hepatocytes and by liver cytosolic fractions.Biochem. J. 1985; 228: 363-373Google Scholar), cytosolic aldehyde and alcohol dehydrogenases seem to be involved in the oxidation and reduction of HNE, respectively, the resulting product being likely processed by a subsequent conjugation to GSH (11Alary J. Bravais F. Cravedi J-P. Debrauwer L. Rao D. Bories G. Mercapturic acid conjugates as urinary end metabolites of the lipid peroxidation product 4-hydroxy-2-nonenal in the rat.Chem. Res. Toxicol. 1995; 8: 35-39Google Scholar). Yet, in this previous in vivo metabolic study of [4-3H]HNE in the rat, almost 40% of the urinary radioactivity was shown to belong to high polarity metabolite classes and remained unidentified to date. 4-Hydroxynonenoic acid (HNA) resulting from the oxidation of HNE belongs to the medium chain fatty acid class. A further oxidative metabolism of HNA mediated by cytochromes P450 4A gene family could be then hypothesized. Noticeably, among other mammalian cytochrome P450s, these isozymes display the specific ability to sustain the ω- and (ω–1)-hydroxylation of medium and long chain fatty acids as well as prostaglandins and leukotrienes (14Aoyama T. Hardwick J.P. Imaoka S. Funae Y. Gelboin H.V. Gonzalez F.J. Clofibrate-inducible rat hepatic P450s IVA1 and IVA3 catalyse the ω- and (ω–1)-hydroxylation of fatty acids and the ω-hydroxylation of prostaglandins E1 and F2α.J. Lipid Res. 1990; 31: 1477-1482Google Scholar). Hepatic levels of cytochrome P450 4A isoenzymes are increased after induction by peroxisome proliferators (15Milton N.N. Elcombe C.R. Gibson G.G. On the mechanism of induction of microsomal cytochrome P450 IVA1 and peroxisome proliferation in rat liver by clofibrate.Biochem. Pharmacol. 1990; 40: 2727-2732Google Scholar, 16Kimura S Hardwick J.P. Kozak C.A. Gonzalez F.J. The rat clofibrate-inducible CYP4A subfamily II. cDNA sequence of IVA3, mapping of the Cyp4a locus to mouse chromosome 4, and coordinate and tissue-specific regulation of the CYP4A genes.DNA. 1989; 8: 517-525Google Scholar, 17Bell D.R. Plant N.J. Rider C.G. Na L. Brown S. Ateitalla I. Acharya S.K. Davies M.H. Elias E. Jenkins N.A. Gilbert D.J. Copeland N.G. Elcombe C.R. Species-specific induction of cytochrome P-450 4A RNAs: PCR cloning of partial guinea pig, human and mouse CYP4A cDNAs.Biochem. J. 1993; 294: 173-180Google Scholar). Such an induction by fibrates is mediated by the peroxisome proliferator-activated receptor α (PPARα), a member of the nuclear receptor family of ligand-activated transcription factors (18Issemann I. Green S. Activation of a member of the steroid hormone receptor superfamily by peroxisome proliferators.Nature. 1990; 347: 645-650Google Scholar, 19Muerhoff A.S. Griffin K.J. Johnson E.F. The peroxisome proliferator-activated receptor mediates the induction of CYP4A6, a cytochrome P450 fatty acid ω-hydroxylase, by clofibric acid.J. Biol. Chem. 1992; 267: 19051-19053Google Scholar, 20Johnson E.F. Palmer C.N.A. 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). In addition, the targeted disruption of the mouse PPARα gene prevents the proliferation of peroxisomes and the subsequent induction of cytochrome P450 4A isoenzymes after exposure of genetically engineered animals to peroxisome proliferators (21Lee S. S-T. 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 α 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). In the present study, biochemical and analytical evidences that ω-hydroxylation of HNA is mediated in vivo by cytochrome P450 4A isoenzymes are given using fibrate induction treatments in wild-type as well as in genetically PPARα-deficient mice. HNE diethyl acetal was synthesized according to Esterbauer and Weger (22Esterbauer H. Weger W. Über die wirkungen von aldehyden auf gesunde und maligne zellen, 3. mitt: Synthese von homologen 4-hydroxy-alkenalen, II (Actions of aldehydes on normal and malignant cells. III. Synthesis of homologous 4-hydroxy-2-alkenals 2).Monatsh.Chem. 1967; 98: 1994-2000Google Scholar). [4-3H]HNE diethyl acetal was synthesized at CEA (Service des Molécules Marquées, CEN, Saclay, France), according to the method developed for the deuterated compound (23Bravais F. Rao D. Alary J. Rao R.C. Debrauwer L. Bories G. Synthesis of 4-hydroxy[4-3H]-2(E)-nonen-1-al-diethylecetal.J. Labelled Comp. Radiopharm. 1994; 36: 471-477Google Scholar). Its radiochemical purity, determined by HPLC, was 95%, and its specific activity was 222 GBq/mmol. HNE was prepared from its ethyl acetal derivative (stored in chloroform) just prior to use by 1 mm HCl hydrolysis for 1 h at room temperature. The hydrolysis was followed by extraction of HNE with CH2Cl2 and evaporation of the solvent. HNE concentration in water was measured spectrophotometrically at 223 nm. HNA was synthesized from HNE as described before (11Alary J. Bravais F. Cravedi J-P. Debrauwer L. Rao D. Bories G. Mercapturic acid conjugates as urinary end metabolites of the lipid peroxidation product 4-hydroxy-2-nonenal in the rat.Chem. Res. Toxicol. 1995; 8: 35-39Google Scholar). [4-3H]HNA was synthesized from [4-3H]HNE in the same way. [1-14C]lauric acid (2.15 GBq/mmol) was purchased from Amersham (Les Ulis, France). Clofibrate was purchased from Sigma (St-Quentin Fallavier, France). Fenofibrate was a generous gift from Fournier (Dijon, France). All solvents and reagents for the preparation of buffers and HPLC eluents were the highest grade commercially available from Sigma, Prolabo (Fontenay-sous-Bois, France), Scharlau (Ferosa, Barcelona, Spain), and Merck (Nogent-sur-Marne, France). Ultrapure water from Milli-Q system (Millipore, St-Quentin-en-Yvelines, France) was used for HPLC eluent preparation. Care of mice was according to institutional guidelines. PPARα (−/−) mice originated from homologous recombinant 129Sv-derived cells as described before (21Lee S. S-T. 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 α 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). Chimeric males were initially backcrossed to C57BL/6 females. Several additional rounds of backcrossing were performed to increase the C57BL/6 genetic background and to generate the animals used in this study. Conventional age-matched C57BL/6 female mice were obtained from Iffa-Credo (L'arbresle, France). During fibrate treatment, mice were fed ad libitum on a standard laboratory diet (UAR, AO3, Epinay-sur-Orge, France) and housed in groups of three in plastic cages at 25°C with a 14-h light/10-h dark cycle. Mice received clofibrate (400 mg/kg body wt per day in ground-nut oil) and fenofibrate (100 mg/kg body wt per day suspended in a 3% aqueous solution of arabic gum) by gavage daily for 3 days. Respective control animals received the appropriate vehicle. Twenty-four hours after the end of this fibrate treatment, animals were individually housed in metabolic cages with fritted glass ground allowing urine/feces separation and injected intraperitoneally with radiolabeled 4-HNE (10 mg/kg body wt in saline; specific activity: 455 MBq/mmol). Animals were killed by cervical dislocation 24 h after 4-HNE injection. The liver was rapidly removed, weighed, and snap frozen in liquid nitrogen before storage at −80°C. A microsomal fraction was prepared for each animal as previously described (24Guéraud F. Masmoudi T. Goudonnet H. Paris A. Differential effect of hypophysectomy and growth hormone treatment on hepatic glucuronosyltransferases in male rats: evidence for an action at a pretranslational level for isoforms glucuronidating bilirubin.Biochem. Pharmacol. 1997; 53: 1637-1647Google Scholar).The amount of microsomal protein was determined by the method of Lowry et al. (25Lowry O.H. Rosebrough N.H. Farr A.G. Randall R.J. Protein measurement with the Folin phenol reagent.J. Biol. Chem. 1951; 193: 265-273Google Scholar) using BSA as a standard. Urine was eluted under vacuum from a fritted glass ground cage with 100 ml distilled water and stored at −20°C. Twenty ml of this eluate was used for subsequent urinary 4-HNE metabolite analysis. Preliminary experiments have shown that most of the intraperitoneally administered radioactivity is excreted in urine (40–60%), less than 2% being excreted in feces. Before HPLC analysis, diluted urine was filtered under vacuum through a 0.45-μm HA Millipore membrane and then concentrated using a Supelclean LC18 SPE 1 g cartridge (Supelco inc., Bellefonte, PA) after acidification to pH 2–3 with 1 m phosphoric acid. The cartridge was washed with 10 mL 0.01 m phosphoric acid solution at pH 2–3, dried under a nitrogen stream, and HNE metabolites were eluted with 5 mL methanol. Part (7–16%) of the urinary radioactivity was unretained on the cartridge. A 0.5-mL aliquot of the methanolic elution was evaporated under nitrogen and analyzed by HPLC for metabolite separation on a Supelco LC18 column (25 × 4.6 mm i.d., 5 μm particle size) (Supelco Inc.) with a Philips model PU4100 apparatus (Argenteuil, France) equipped with a Rheodyne Model 7125 injector (Rheodyne, Cotati, CA), a 500 μL loop and connected to an online radioisotope detector Radiomatic Flo-One/beta model A515 (Packard, Meriden, CT). The HPLC system 1 was used with solvents A [ammonium acetate (20 mm, pH 4.5)–acetonitrile 97.5:2.5 (v/v)], B [ammonium acetate (20 mm, pH 4.5)–acetonitrile 80:20 (v/v)], and C [ammonium acetate (20 mm, pH 4.5)–acetonitrile 30:70 (v/v)] as follows: from 0 to 4 min, 100% A; from 4 to 5 min, linear gradient from 100% A to 85.7% A/14.3% B; from 5 to 25 min, linear gradient from 85.7% A/14.3% B to 80% A/20% B; from 25 to 35 min, 80% A/20% B; from 35 to 40 min, linear gradient from 80% A/20% B to 100% B, which is maintained from 40 to 45 min; from 45 to 50 min, linear gradient from 100% B to 100% C, then maintained from 50 to 60 min, the whole run being achieved at a flow rate of 1 mL/min at 35°C. The HPLC system 2 used the same elution gradient as the HPLC system 1 on a semipreparative column, Shandon Ultrabase C18 (250 × 7.5 mm i.d., 5 μm particle size) (SFCC, Eragny, France), with a 2 mL loop, at a flow rate of 2 mL/min at 35°C. The HPLC system 3 was performed on the same analytical column as for the HPLC system 1 at a flow rate of 1 mL/min at 35°C and lasted 60 min. The elution gradient was used as follows: from 0 to 5 min, 100% A [water–acetonitrile–acetic acid 97.5:2.5/1 (v/v/v), from 5 to 30 min, linear gradient from 100% A to 85.7% A/14.3% B [water–acetonitrile–acetic acid 80:20:1 (v/v/v)], from 30 to 40 min, linear gradient from 85.7% A/14.3% B to 80% A/20% B, which is then maintained for 5 min; from 45 to 46 min, linear gradient from 80% A/20% B to 50% A/50% B, which is finally maintained for 14 min. The two peaks displaying a quantitative increase related to fibrate treatments (peaks A and B in HPLC system 1, eluted at 13.5 and 15.0 min, respectively) were quantitatively purified from the urine of one clofibrate-treated mouse. It was done by using the preparative HPLC system 2 and a Gilson model 202 fraction collector (Gilson Medical Electronics, Viliers-le-Bel, France). Part (5–10%) of the eluted radioactivity was submitted to liquid scintillation counting in a 2200CA Packard spectrophotometer (Packard, Downers Grove, IL) using Ultimagold™ (Packard) as scintillation cocktail. The collected fractions were further purified using HPLC system 3 before being submitted to mass spectrometry analyses. The dry compounds were methylated for 30 min at room temperature by addition of 50 μL etheral diazomethane. Then, solvent was removed under a nitrogen streamand the dry residue was subsequently dissolved in a mixture of N,O-bis(TMS)-trifluoroacetamide–trimethylchlorosilane (Pierce Chemical, Rockford, IL) in a 99:1 ratio and heated at 60°C for 1 h. The solvent was evaporated at 30°C under a nitrogen stream and hexane (20 μL) was added to the dry residue. For GC–MS analyses a Nermag R-10-10-T single quadrupole instrument was coupled to a Delsi DI 200 (Delsi Nermag Instruments) gas chromatograph with a BPX5 (25 m × 0.22 mm × 0.25 μm) capillary column (SGE, Villeneuve-St-Georges, France). The samples (1 μL) were injected in the splitless mode. Helium was used as the carrier gas at a flow rate of 1 mL/min with a back pressure of 0.8 bar. The oven temperature was set as follows: 50°C for 50°s, then from 50°C to 230°C at 25°C/min, and finally from 230 to 270°C at 5°C/min. The injector temperature was 270°C and the interface temperature was 270°C. Electron impact (EI) mass spectra were generated at 70 eV with an emission current of 200 μA at a source temperature of 220°C. Total RNA was isolated from each liver by acid-phenol extraction. Construction of a specific probe for rat P450 4A3 was done as described previously (16Kimura S Hardwick J.P. Kozak C.A. Gonzalez F.J. The rat clofibrate-inducible CYP4A subfamily II. cDNA sequence of IVA3, mapping of the Cyp4a locus to mouse chromosome 4, and coordinate and tissue-specific regulation of the CYP4A genes.DNA. 1989; 8: 517-525Google Scholar, 26Hardwick J.P. Song B.J. Huberman E. Gonzalez F.J. Isolation, complementary DNA sequence, and regulation of rat hepatic lauric acid ω-hydroxylase (cytochrome P-450LAω): identification of a new cytochrome P-450 gene family.J. Biol. Chem. 1987; 262: 801-810Google Scholar). Northern blot analysis of wild-type (+/+) and PPARα homozygous mutant (−/−) mice treated with either clofibrate or fenofibrate were done as described elsewhere (21Lee S. S-T. 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 α 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). The results indicated that PPARα-deficient mice displayed no induction of P450 4A mRNA expression with any of the fibrate treatments used, while wild-type mice presented a great increase in this expression with both treatments (results not shown) as it was pointed out in a previous study (21Lee S. S-T. 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 α 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). Lauric acid ω- and (ω–1)-hydroxylation activities were measured with [1-14C]lauric acid (sp act: 24 MBq/mmol). Reactions mixtures were prepared as described by Orton and Parker (27Orton T.C. Parker G.L. The effect of hypolipidemic agents on the hepatic microsomal drug-metabolizing enzyme system of the rat.Drug. Met. Disp. 1982; 10: 110-115Google Scholar). Incubations lasted 30 min at 37°C with 2 mg microsomal protein. The hydroxylauric acids and unreacted substrate were extracted into diethylether and evaporated to dryness. The formation of 11- and 12-hydroxylauric acids was assayed by HPLC on a Spherisorb C18 column (25 × 4.6 mm i.d., 5 μm particle size) maintained at 35°C with a Philips model PU 4100 apparatus equipped with a 500 μL loop and an on-line radioisotope detector Radiomatic Flo-One/beta model A515. The mobile phase was a mixture of acetonitrile and 0.1 m ammonium acetate buffer (pH 4.6) (35:65, v/v) for 15 min, followed by 100% acetonitrile for another 15 min (28Azerad R. Boucher J.L. Dansette P. Delaforge M. High-performance liquid chromatographic separation of 11-hydroxylauric acid enantiomers. Application to the determination of the stereochemistry of microsomal lauric acid (ω-1) hydroxylation.J. Chromatogr. 1990; 498: 293-302Google Scholar). The flow rate was 1 mL/min. Anti-rat P450 4A1 serum was manufactured by Daiichi Pure Chemicals Co., Ltd (Tokyo, Japan).This serum contains a polyclonal antibody made for studying the inhibition of P450 4A1 catalyzed enzyme activity. Microsome incubations were done according to the supplier's recommendations with 25 μm radiolabeled [4-3H]HNA as substrate (sp act: 680 MBq/mmol). Microsomal proteins (200 μg) were used for preliminary clofibrate-treated rat studies. These studies were achieved to verify the antibody effect with HNA as substrate. Microsomal proteins (500 μg) were used for clofibrate-treated mouse studies. Incubations were stopped by 4 vol methanol. After centrifugation, the liquid phase was submitted to HPLC using the same equipment as for lauric acid hydroxylation studies. The mobile phase was a mixture of acetonitrile and 20 mm ammonium acetate buffer (pH 4.6) (15:85, v/v) for 15 min, then (30:70, v/v) for another 15 min. The flow rate was 1 mL/min. Treatment effects were submitted to variance analyses and comparisons of means were done with the Student-Newman-Keuls procedure. Regression analyses were made using the classical linear model. All statistical analyses were done using SAS software (29SAS SAS/STAT User's Guide (Release 6.09.). SAS Inst., Inc., Cary, NC1989Google Scholar). Urinary metabolites of [4-3H]HNE were quantified in mice by radio-HPLC using the HPLC system 1. Two main metabolites were separated and identified as mercapturic conjugates of 4-hydroxy-2-nonenoic (HNA-MA) and 1,4-dihydroxy-2-nonene (DHN-MA) eluted respectively at 28.7 and 32.4 min (Fig. 1) by comparison with rat urinary metabolites as recently published (11Alary J. Bravais F. Cravedi J-P. Debrauwer L. Rao D. Bories G. Mercapturic acid conjugates as urinary end metabolites of the lipid peroxidation product 4-hydroxy-2-nonenal in the rat.Chem. Res. Toxicol. 1995; 8: 35-39Google Scholar). In control animals, the proportion of polar metabolites eluted between 5.0 and 18.0 min was about 18.0% of the total radioactivity administered. Independent of the peroxisome proliferator administered, the effect of induction was very significant (P < 0.01) on the excretion of polar metabolites of [3H]HNE in urine of treated wild-type mice (Fig. 2A) as compared with respective control animals. We observed 28.8 and 35.9% increases in polar metabolites excretion with clofibrate and fenofibrate treatments, respectively. Noticeable modifications in the ratio of less polar metabolites were not observed at the same time. Polar metabolites were separated into different peaks that contained unresolved complex mixtures of compounds. Two peaks (A: Rt = 13.3 min and B: Rt = 15.0 min, Fig. 1) were specifically increased after clofibrate or fenofibrate treatments (Figs. 2B and 2C). When compared to controls, peak A was increased by 80.3% and 94.7% (P < 0.001) by clofibrate and fenofibrate treatments, respectively. The increase in urinary excretion of metabolites eluted under peak B was also significant: 22.8% (P < 0.02) and 78.8% (P < 0.001) for clofibrate and fenofibrate treatments, respectively. Furthermore,neither peaks A and B nor the total proportion of polar metabolites displayed any increase with fibrate-treatments in control or fibrate treated PPARα-deficient mice (Figs. 2A, 2B, and 2C). Altogether, these results indicate that the relative increase in the amounts of urinary metabolites eluted under peaks A and B after treatment with clofibrate or fenofibrate is linked to a metabolic process related to an enzymatic induction mediated by PPARα.Fig. 2Quantification of urinary polar metabolites of HNE (A), peak A (B), and peak B (C) measured by radio-HPLC using the system 1 as a function of fibrate treatment and animal model.View Large Image Figure ViewerDownload (PPT) The group of polar metabolites eluting at 13.3 min (peak A) in the analytical HPLC system 1 was purified in a larger amount by a semi-preparative method (system 2). These metabolites were further purified on an analytical column using HPLC system 3. A major metabolite (Met-A3), which represents about 36% of the radioactivity belonging to peak A, was separated from numerous minor metabolites (Fig. 3). After methylation and silylation, the metabolite was analyzed by GC-MS using electron impact ionization. A peak was detected at 9.3 min. It shares a fragment ion at m/z 187 with HNA when analyzed in the same conditions. This fragment could correspond to the following ion: [(CH3)3Si–O = CH–CH = CH–COOCH3]+ (Fig. 4A) and could be interpreted as the result from the α-cleavage of the O-TMS group in position 4. Other relevant fragment ions can be explained as follows: m/z 287 [M–CH3]+, m/z 270 [M–CH3OH]+, m/z 239 [M–CH3OH–CH3O]+, m/z 229 [M–CH2COOCH3]+. The absence of any ion at m/z 147 [(CH3)3Si=O–Si(CH3)3]+ rules out the presence of two silylated hydroxy groups for this metabolite, but fragment ions at m/z 75 [(CH3)2Si-OH]+ and m/z 73 [(CH3)3Si]+ correspond to at least one hydroxy group. Besides, the presence of fragment ions at m/z 239 and m/z 229 is indicative of two acidic functions. On the basis of these data, this metabolite may be identified as 4-hydroxy-1,9-dicarboxy-2-nonene.Fig. 4Mass spectra obtained by GC/MS of 4-hydroxy-2-nonene-1,9-dicarboxylic acid (A) and 4,9-dihydroxy-2-nonenoic acid (B).View Large Image Figure ViewerDownload (PPT) As described above, metabolites belonging to peak B were quantitatively purified by semipreparative HPLC. A metabolite eluting at 28.5 min in HPLC system 3 (not shown) was subsequently methylated and silylated before mass spectrometry analysis. When submitted to GC-MS analysis, a metabolite was detected at 9.4 min. As for the methylated and silylated derivative of 4-hydroxy-1,9,dicarboxy-2-nonene, a fragment ion at m/z 187 was detected and could correspond to the same structure: [(CH3)3Si–O =CH–CH=CH–COOCH3]+. This ion clearly representsthe α-cleavage of the 4-O-TMS group (Fig. 4B). The fragment ions at m/z 75 [(CH3)2Si–OH]+ and m/z 73 [(CH3)3Si]+ may correspond to at least one hydroxy group. Moreover, a fragment ion at m/z 147 [(CH3)3Si= O–Si(CH3)3]+ is characteristic of the presence of two silylated hydroxy groups corresponding to two alcohol functions. However, the bis-TMS methylester of an hypothesized dihydroxy-2-nonenoic acid (m/z 346) was not detected. Several fragment ions can bring further evidence for such a structure: m/z 331 [M–CH3]+, m/z 314 [M–CH3OH]+, m/z 299 [M–CH3–CH3OH]+, m/z 241 [M–CH3–(CH3)3Si–OH]+. Last, a minor but significant fragment at m/z 103 [(CH3)3Si–O = CH2]+ would constitute another element in favor of hydroxylation on C-9. These data demonstrate the presence of an ω-hydroxy