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• W2103708921 abstract "Lipoxygenases (LOs) convert polyunsaturated fatty acids into lipid hydroperoxides. Homolytic decomposition of lipid hydroperoxides gives rise to endogenous genotoxins such as 4-oxo-2(E)-nonenal, which cause the formation of mutagenic DNA adducts. Chiral lipidomics analysis was employed to show that a 5-LO-derived lipid hydroperoxide was responsible for endogenous DNA-adduct formation. The study employed human lymphoblastoid CESS cells, which expressed both 5-LO and the required 5-LO-activating protein (FLAP). The major lipid peroxidation product was 5(S)-hydroperoxy-6,8,11,14-(E,Z,Z,Z)-eicosatetraenoic acid, which was analyzed as its reduction product, 5(S)-hydroxy-6,8,11,14-(E,Z,Z,Z)-eicosatetraenoic acid (5(S)-HETE)). Concentrations of 5(S)-HETE increased from 0.07 ± 0.01 to 45.50 ± 4.05 pmol/107 cells upon stimulation of the CESS cells with calcium ionophore A23187. There was a concomitant increase in the 4-oxo-2(E)-nonenal-derived DNA-adduct, heptanone-etheno-2′-deoxyguanosine (HϵdGuo) from 2.41 ± 0.35 to 6.31 ± 0.73 adducts/107 normal bases. Biosynthesis of prostaglandins, 11(R)-hydroxy-5,8,12,14-(Z,Z,E,Z)-eicosatetraenoic acid, and 15(R,S)-hydroxy-5,8,11,13-(Z,Z,Z,E)-eicosatetraenoic acid revealed that there was cyclooxygenase (COX) activity in the CESS cells. Western blot analysis revealed that COX-1 was expressed by the cells, but there was no COX-2 or 15-LO-1. FLAP inhibitor reduced HϵdGuo-adducts and 5(S)-HETE to basal levels. In contrast, aspirin, which had no effect on 5(S)-HETE, blocked the formation of prostaglandins, 15-HETE, and 11-HETE but did not inhibit HϵdGuo-adduct formation. These data showed that 5-LO was the enzyme responsible for the generation of the HϵdGuo DNA-adduct in CESS cells. Lipoxygenases (LOs) convert polyunsaturated fatty acids into lipid hydroperoxides. Homolytic decomposition of lipid hydroperoxides gives rise to endogenous genotoxins such as 4-oxo-2(E)-nonenal, which cause the formation of mutagenic DNA adducts. Chiral lipidomics analysis was employed to show that a 5-LO-derived lipid hydroperoxide was responsible for endogenous DNA-adduct formation. The study employed human lymphoblastoid CESS cells, which expressed both 5-LO and the required 5-LO-activating protein (FLAP). The major lipid peroxidation product was 5(S)-hydroperoxy-6,8,11,14-(E,Z,Z,Z)-eicosatetraenoic acid, which was analyzed as its reduction product, 5(S)-hydroxy-6,8,11,14-(E,Z,Z,Z)-eicosatetraenoic acid (5(S)-HETE)). Concentrations of 5(S)-HETE increased from 0.07 ± 0.01 to 45.50 ± 4.05 pmol/107 cells upon stimulation of the CESS cells with calcium ionophore A23187. There was a concomitant increase in the 4-oxo-2(E)-nonenal-derived DNA-adduct, heptanone-etheno-2′-deoxyguanosine (HϵdGuo) from 2.41 ± 0.35 to 6.31 ± 0.73 adducts/107 normal bases. Biosynthesis of prostaglandins, 11(R)-hydroxy-5,8,12,14-(Z,Z,E,Z)-eicosatetraenoic acid, and 15(R,S)-hydroxy-5,8,11,13-(Z,Z,Z,E)-eicosatetraenoic acid revealed that there was cyclooxygenase (COX) activity in the CESS cells. Western blot analysis revealed that COX-1 was expressed by the cells, but there was no COX-2 or 15-LO-1. FLAP inhibitor reduced HϵdGuo-adducts and 5(S)-HETE to basal levels. In contrast, aspirin, which had no effect on 5(S)-HETE, blocked the formation of prostaglandins, 15-HETE, and 11-HETE but did not inhibit HϵdGuo-adduct formation. These data showed that 5-LO was the enzyme responsible for the generation of the HϵdGuo DNA-adduct in CESS cells. PUFAs 2The abbreviations used are: PUFApolyunsaturated fatty acidAPCIatmospheric pressure chemical ionizationECAPCIelectron capture APCICOXcyclooxygenasedGuo2′-deoxyguanosineϵdGuoetheno-dGuoHϵdGuohepatanone-etheno-dGuoCPϵdGuocarboxypentanone-2′-deoxyguanosineFLAP5-lipoxygenase-activating protein5-HETE5-hydroxy-6,8,11,14-(E,Z,Z,Z)-eicosatetraenoic acid8-HETE8-hydroxy-5,9,11,14-(Z,E,Z,Z)-eicosatetraenoic acid11-HETE11-hydroxy-5,8,12,14-(Z,Z,E,Z)-eicosatetraenoic acid12-HETE12-hydroxy-5,8,10,14-(Z,Z,E,Z)-eicosatetraenoic acid15-HETE15-hydroxy-5,8,11,13-(Z,Z,Z,E)-eicosatetraenoic acid13-HODE13-hydroxy-9,11-(Z,E)-octadecadienoic acidHpETEhydroperoxyeicosatetraenoic acidLOlipoxygenaseLTB4leukotriene B4MOPS3-morpholinopropanesulfonic acidMRMmultiple reaction monitoringMSmass spectrometryONE4-oxo-2(E)-nonenalPFB-Brpentafluorobenzyl bromidePGprostaglandinROSreactive oxygen speciesHPLChigh performance liquid chromatography (LC)Bis-Tris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol. 2The abbreviations used are: PUFApolyunsaturated fatty acidAPCIatmospheric pressure chemical ionizationECAPCIelectron capture APCICOXcyclooxygenasedGuo2′-deoxyguanosineϵdGuoetheno-dGuoHϵdGuohepatanone-etheno-dGuoCPϵdGuocarboxypentanone-2′-deoxyguanosineFLAP5-lipoxygenase-activating protein5-HETE5-hydroxy-6,8,11,14-(E,Z,Z,Z)-eicosatetraenoic acid8-HETE8-hydroxy-5,9,11,14-(Z,E,Z,Z)-eicosatetraenoic acid11-HETE11-hydroxy-5,8,12,14-(Z,Z,E,Z)-eicosatetraenoic acid12-HETE12-hydroxy-5,8,10,14-(Z,Z,E,Z)-eicosatetraenoic acid15-HETE15-hydroxy-5,8,11,13-(Z,Z,Z,E)-eicosatetraenoic acid13-HODE13-hydroxy-9,11-(Z,E)-octadecadienoic acidHpETEhydroperoxyeicosatetraenoic acidLOlipoxygenaseLTB4leukotriene B4MOPS3-morpholinopropanesulfonic acidMRMmultiple reaction monitoringMSmass spectrometryONE4-oxo-2(E)-nonenalPFB-Brpentafluorobenzyl bromidePGprostaglandinROSreactive oxygen speciesHPLChigh performance liquid chromatography (LC)Bis-Tris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol. can be converted into lipid hydroperoxides enzymatically by the action of LOs (1Zhu P. Oe T. Blair I.A. Rapid Commun. Mass Spectrom. 2008; 22: 432-440Crossref PubMed Scopus (62) Google Scholar) and COXs (2Lee S.H. Rangiah K. Williams M.V. Wehr A.Y. DuBois R.N. Blair I.A. Chem. Res. Toxicol. 2007; 20: 1665-1675Crossref PubMed Scopus (33) Google Scholar) or nonenzymatically by reactive oxygen species (ROS) (3Porter N.A. Caldwell S.E. Mills K.A. Lipids. 1995; 30: 277-290Crossref PubMed Scopus (995) Google Scholar). Arachidonic acid, one of the essential PUFAs present in cell membranes, is released from phospholipids by different phospholipase A2 isoforms upon diverse physical, chemical, inflammatory, and mitogenic stimuli (4Schaloske R.H. Dennis E.A. Biochim. Biophys. Acta. 2006; 1761: 1246-1259Crossref PubMed Scopus (720) Google Scholar). The free arachidonic acid then serves as a substrate for LOs, COXs, or ROS to produce a variety of lipid hydroperoxides (5Blair I.A. Exp. Gerontol. 2001; 36: 1473-1481Crossref PubMed Scopus (169) Google Scholar, 6Blair I.A. J. Biol. Chem. 2008; 283: 15545-15549Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). ROS, 12-LO, and 15-LO can also act directly upon arachidonic acid esterified in phospholipids to produce lipid hydroperoxides (7Kuhn H. Walther M. Kuban R.J. Prostaglandins Other Lipid Mediat. 2002; 68–69: 263-290Crossref PubMed Scopus (193) Google Scholar), which are reduced (8Kühn H. Borchert A. Free Radic. Biol. Med. 2002; 33: 154-172Crossref PubMed Scopus (213) Google Scholar), hydrolyzed by phospholipase A2 (4Schaloske R.H. Dennis E.A. Biochim. Biophys. Acta. 2006; 1761: 1246-1259Crossref PubMed Scopus (720) Google Scholar), and then secreted as the corresponding free HETEs (9Chaitidis P. Schewe T. Sutherland M. Kühn H. Nigam S. FEBS Lett. 1998; 434: 437-441Crossref PubMed Scopus (32) Google Scholar). COX-2-mediated (10Hamberg M. Arch. Biochem. Biophys. 1998; 349: 376-380Crossref PubMed Scopus (41) Google Scholar) and 15-LO-1-mediated (11Kühn H. Thiele B.J. Ostareck-Lederer A. Stender H. Suzuki H. Yoshimoto T. Yamamoto S. Biochim. Biophys. Acta. 1993; 1168: 73-78Crossref PubMed Scopus (29) Google Scholar) metabolism of linoleic acid results in the formation of 13(S)-hydroperoxy-9,11-(Z,E)-octadecadienoic acid, which is rapidly reduced to 13(S)-hydroxy-9,11-(Z,E)-octadecadienoic acid (HODE) and secreted from cells. Arachidonic acid is specifically metabolized by 5-LO into 5(S)-HpETE, which is either reduced to 5(S)-HETE or serves as precursor to the formation of leukotrienes (LTs) (Scheme 1) (12Lötzer K. Funk C.D. Habenicht A.J. Biochim. Biophys. Acta. 2005; 1736: 30-37PubMed Google Scholar). In contrast, ROS-mediated reactions produce racemic mixtures of all possible regioisomers of HpETEs and 13(S)-hydroperoxy-9,11-(Z,E)-octadecadienoic acids (3Porter N.A. Caldwell S.E. Mills K.A. Lipids. 1995; 30: 277-290Crossref PubMed Scopus (995) Google Scholar) that are subsequently secreted from cells as complex mixtures of racemic HETEs and HODEs. Therefore, the ability to analyze different HETE and HODE enantiomers and regioisomers is important for elucidating specific cellular lipid peroxidation pathways (13Lee S.H. Williams M.V. DuBois R.N. Blair I.A. Rapid Commun. Mass Spectrom. 2003; 17: 2168-2176Crossref PubMed Scopus (156) Google Scholar). polyunsaturated fatty acid atmospheric pressure chemical ionization electron capture APCI cyclooxygenase 2′-deoxyguanosine etheno-dGuo hepatanone-etheno-dGuo carboxypentanone-2′-deoxyguanosine 5-lipoxygenase-activating protein 5-hydroxy-6,8,11,14-(E,Z,Z,Z)-eicosatetraenoic acid 8-hydroxy-5,9,11,14-(Z,E,Z,Z)-eicosatetraenoic acid 11-hydroxy-5,8,12,14-(Z,Z,E,Z)-eicosatetraenoic acid 12-hydroxy-5,8,10,14-(Z,Z,E,Z)-eicosatetraenoic acid 15-hydroxy-5,8,11,13-(Z,Z,Z,E)-eicosatetraenoic acid 13-hydroxy-9,11-(Z,E)-octadecadienoic acid hydroperoxyeicosatetraenoic acid lipoxygenase leukotriene B4 3-morpholinopropanesulfonic acid multiple reaction monitoring mass spectrometry 4-oxo-2(E)-nonenal pentafluorobenzyl bromide prostaglandin reactive oxygen species high performance liquid chromatography (LC) 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol. polyunsaturated fatty acid atmospheric pressure chemical ionization electron capture APCI cyclooxygenase 2′-deoxyguanosine etheno-dGuo hepatanone-etheno-dGuo carboxypentanone-2′-deoxyguanosine 5-lipoxygenase-activating protein 5-hydroxy-6,8,11,14-(E,Z,Z,Z)-eicosatetraenoic acid 8-hydroxy-5,9,11,14-(Z,E,Z,Z)-eicosatetraenoic acid 11-hydroxy-5,8,12,14-(Z,Z,E,Z)-eicosatetraenoic acid 12-hydroxy-5,8,10,14-(Z,Z,E,Z)-eicosatetraenoic acid 15-hydroxy-5,8,11,13-(Z,Z,Z,E)-eicosatetraenoic acid 13-hydroxy-9,11-(Z,E)-octadecadienoic acid hydroperoxyeicosatetraenoic acid lipoxygenase leukotriene B4 3-morpholinopropanesulfonic acid multiple reaction monitoring mass spectrometry 4-oxo-2(E)-nonenal pentafluorobenzyl bromide prostaglandin reactive oxygen species high performance liquid chromatography (LC) 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol. The conversion of arachidonic acid to 5(S)-HpETE by 5-LO is critically dependent upon the presence of FLAP (14Woods J.W. Evans J.F. Ethier D. Scott S. Vickers P.J. Hearn L. Heibein J.A. Charleson S. Singer I.I. J. Exp. Med. 1993; 178: 1935-1946Crossref PubMed Scopus (359) Google Scholar). 5-LO and FLAP are expressed primarily in inflammatory cells such as polymorphonuclear leukocytes, monocytes, macrophages, and mast cells (12Lötzer K. Funk C.D. Habenicht A.J. Biochim. Biophys. Acta. 2005; 1736: 30-37PubMed Google Scholar, 15Peters-Golden M. Henderson Jr., W.R. Ann. Allergy Asthma Immunol. 2005; 94: 609-618Abstract Full Text PDF PubMed Google Scholar, 16Murphy R.C. Gijón M.A. Biochem. J. 2007; 405: 379-395Crossref PubMed Scopus (255) Google Scholar, 17Werz O. Curr. Drug Targets Inflamm. Allergy. 2002; 1: 23-44Crossref PubMed Scopus (171) Google Scholar). Therefore, 5-LO-mediated LT formation is thought to play a critical role in inflammation and allergic disorders (18Sharma J.N. Mohammed L.A. Inflammopharmacology. 2006; 14: 10-16Crossref PubMed Scopus (93) Google Scholar, 19Hicks A. Monkarsh S.P. Hoffman A.F. Goodnow Jr., R. Expert Opin. Investig. Drugs. 2007; 16: 1909-1920Crossref PubMed Scopus (77) Google Scholar, 20Wymann M.P. Schneiter R. Nat. Rev. Mol. Cell Biol. 2008; 9: 162-176Crossref PubMed Scopus (940) Google Scholar, 21Peters-Golden M. Curr. Allergy Asthma Rep. 2008; 8: 367-373Crossref PubMed Scopus (48) Google Scholar). In addition, a number of studies have implicated 5-LO-derived arachidonic acid metabolites as mediators of atherogenesis and heart disease (12Lötzer K. Funk C.D. Habenicht A.J. Biochim. Biophys. Acta. 2005; 1736: 30-37PubMed Google Scholar, 22Zhao L. Funk C.D. Trends Cardiovasc. Med. 2004; 14: 191-195Crossref PubMed Scopus (177) Google Scholar, 23Fairweather D. Frisancho-Kiss S. Cardiovasc. Hematol. Disord. Drug Targets. 2008; 8: 80-90Crossref PubMed Scopus (32) Google Scholar). The 5-LO pathway of arachidonic acid metabolism has also been proposed to play a role in prostate and pancreatic cancer (24Gupta S. Srivastava M. Ahmad N. Sakamoto K. Bostwick D.G. Mukhtar H. Cancer. 2001; 91: 737-743Crossref PubMed Scopus (197) Google Scholar, 25Hennig R. Ding X.Z. Tong W.G. Schneider M.B. Standop J. Friess H. Büchler M.W. Pour P.M. Adrian T.E. Am. J. Pathol. 2002; 161: 421-428Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 26Chen X. Sood S. Yang C.S. Li N. Sun Z. Curr. Cancer Drug Targets. 2006; 6: 613-622Crossref PubMed Scopus (56) Google Scholar). Lipid hydroperoxides undergo homolytic decomposition into bifunctional electrophiles such as 4-hydroxy-2(E)-nonenal, ONE, 4,5-epoxy-2(E)-decenal, and 4-hydroperoxy-2(E)-nonenal (27Lee S.H. Oe T. Blair I.A. Science. 2001; 292: 2083-2086Crossref PubMed Scopus (409) Google Scholar). These bifunctional electrophiles are highly reactive and can readily modify intracellular molecules including glutathione (GSH) (28Jian W. Arora J.S. Oe T. Shuvaev V.V. Blair I.A. Free Radic. Biol. Med. 2005; 39: 1162-1176Crossref PubMed Scopus (46) Google Scholar, 29Jian W. Lee S.H. Mesaros C. Oe T. Elipe M.V. Blair I.A. Chem. Res. Toxicol. 2007; 20: 1008-1018Crossref PubMed Scopus (44) Google Scholar), DNA, (5Blair I.A. Exp. Gerontol. 2001; 36: 1473-1481Crossref PubMed Scopus (169) Google Scholar, 6Blair I.A. J. Biol. Chem. 2008; 283: 15545-15549Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar), and proteins (30Oe T. Arora J.S. Lee S.H. Blair I.A. J. Biol. Chem. 2003; 278: 42098-42105Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 31Sayre L.M. Lin D. Yuan Q. Zhu X. Tang X. Drug Metab. Rev. 2006; 38: 651-675Crossref PubMed Scopus (289) Google Scholar). Our previous in vitro studies characterized the bifunctional electrophiles ONE and 4-hydroperoxy-2(E)-nonenal as major products arising from the homolytic decomposition of 5-LO-derived 5(S)-HpETE (32Jian W. Lee S.H. Arora J.S. Silva Elipe M.V. Blair I.A. Chem. Res. Toxicol. 2005; 18: 599-610Crossref PubMed Scopus (24) Google Scholar). Reactions with DNA resulted in the formation of etheno-2′-deoxyguanosine (ϵdGuo) from 4-hydroperoxy-2(E)-nonenal and heptanone-ϵdGuo (HϵdGuo) from ONE (Scheme 1). 5,8-Dioxo-6-octenoic acid, a bifunctional electrophile from the carboxyl terminus of 5(S)-HpETE, gave rise to the novel DNA-adduct carboxypentanone-ϵdGuo (CPϵdGuo)-adduct as shown in Scheme 1. Previous studies have demonstrated that lipid hydroperoxides generated by COX-2 could lead to the formation of endogenous DNA adducts in epithelial cells (6Blair I.A. J. Biol. Chem. 2008; 283: 15545-15549Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). Cellular 5-LO, like COX-2, synthesizes lipid hydroperoxides on the nuclear membrane. Therefore, it is highly possible that 5-LO could also mediate the formation of lipid hydroperoxide-derived endogenous DNA adducts in cells. CESS is a human lymphoblastic cell line that expresses both 5-LO and FLAP, and they have been used as a model for inflammatory cells to examine the role of 5-LO metabolites in signal transduction (33Schulam P.G. Shearer W.T. J. Immunol. 1990; 144: 2696-2701PubMed Google Scholar, 34el Makhour-Hojeij Y. Baclet M.C. Chable-Rabinovitch H. Beneytout J.L. Cook J. Prostaglandins. 1994; 48: 21-29Crossref PubMed Scopus (16) Google Scholar). In the present study, CESS cells provided an ideal model to elucidate the relationship of 5-LO mediated-lipid peroxidation and DNA-adduct formation in a cellular setting. Stable isotope dilution chiral LC-electron capture (EC) APCI/MRM/MS (13Lee S.H. Williams M.V. DuBois R.N. Blair I.A. Rapid Commun. Mass Spectrom. 2003; 17: 2168-2176Crossref PubMed Scopus (156) Google Scholar) was used to monitor the concomitant formation of lipid hydroperoxides in the presence of different enzyme stimulator or inhibitors. DNA-adduct formation in the same cells was measured by a stable isotope dilution APCI/MRM/MS method. The powerful tool of chiral lipid analysis enabled us to dissect the complicated lipid peroxidation pathways and to correlate them with endogenous DNA-adduct formation. The results demonstrated that 5-LO-mediated lipid peroxidation could cause HϵdGuo formation in cells. This novel finding provided additional explanation for the previous observation that increased 5-LO activity was associated with cancers and cardiovascular diseases (24Gupta S. Srivastava M. Ahmad N. Sakamoto K. Bostwick D.G. Mukhtar H. Cancer. 2001; 91: 737-743Crossref PubMed Scopus (197) Google Scholar, 25Hennig R. Ding X.Z. Tong W.G. Schneider M.B. Standop J. Friess H. Büchler M.W. Pour P.M. Adrian T.E. Am. J. Pathol. 2002; 161: 421-428Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 26Chen X. Sood S. Yang C.S. Li N. Sun Z. Curr. Cancer Drug Targets. 2006; 6: 613-622Crossref PubMed Scopus (56) Google Scholar). CESS cells was obtained from ATCC (Manassas, VA). RPMI 1640, fetal bovine serum, and penicillin-streptomycin were supplied by Invitrogen. BCA protein assay reagent was obtained from Pierce. Pre-cast 7% NuPAGE Novex Tris acetate gels and 0.45-μm nitrocellulose membranes were obtained from Invitrogen. 5-LO polyclonal antiserum, 15-LO (rabbit reticulocyte) polyclonal antibody, and COX-2 (murine) polyclonal antibody were obtained from Cayman Chemical Co. (Ann Arbor, MI). COX-1 polyclonal antibody (sc-1752) was from Santa Cruz Biotechnology Inc (Santa Cruz, CA). Horseradish peroxidase-conjugated mouse anti-goat/sheep antibody and horseradish peroxidase-conjugated goat anti-rabbit antibody was from Sigma-Aldrich. The enhanced chemiluminescence (ECL) Western blotting reagent was supplied by Amersham Biosciences. 13(R)-HODE, 13(S)-HODE, 5(R)-HETE, 5(S)-HETE, 8(R)-HETE, 8(S)-HETE, 11(R)-HETE, 11(S)-HETE, 12(R)-HETE, 12(S)-HETE, 15(R)-HETE, 15(S)-HETE, LTB4, PGE2, PGD2, PGF2α, 13(S)-[2H4]HODE, 5(S)-[2H8]HETE, 12(S)-[2H8]HETE, 15(S)-[2H8]HETE, [2H4]LTB4, [2H4]PGE2, [2H4]PGD2, [2H4]PGF2α and MK886 were purchased from Cayman Chemical Co. (Ann Arbor, MI). Ammonium acetate, aspirin, calcium ionophore A23187, diisopropylethylamine, DMSO, EDTA, glycine, magnesium chloride, MOPS, Nonidet P-40, phenylmethanesulfonyl fluoride, protease mixture, sodium chloride, Tris hydrochloride, and zinc chloride were obtained from Sigma-Aldrich. DNA extraction kit was purchased from Wako Chemicals USA, Inc. (Richmond, VA). DNase I, phosphodiesterase I, and shrimp alkaline phosphatase were purchased from Calbiochem, Worthington Biochemical Corp. (Lakewood, NJ), and Roche Applied Science, respectively. Costar 0.2-μm microcentrifuge filter was provided by Corning Inc. (Corning, NY). LC-18 and LC-Si solid phase extraction tubes (1 g, 6 ml) were from Supelco (Bellefonte, PA). Acetonitrile, hexane, HPLC grade water, isopropyl alcohol, methanol, and methylene chloride were obtained from Fisher. Gases were supplied by BOC Gases (Lebanon, NJ). CESS cells were incubated in RPMI 1640 (10 ml) containing 10% fetal bovine serum, 100 units/ml of penicillin, and 100 μg/ml of streptomycin at 37 °C in an atmosphere of 5% CO2. After the cells reached the density of 1 × 106/ml, the complete RPMI 1640 was removed and replaced with serum-free RPMI 1640 for treatment. 1 × 107 cells were treated with vitamin C (dissolved in water, final concentration of 1.0 mm) and/or A23187 (dissolved in DMSO, final concentration of 1.0 μm). Cells were incubated for 1 h, and the medium was collected for lipid analysis. The cells were then re-suspended in complete RPMI 1640 and added with vitamin C and/or A23187 again. The cells were incubated till 24 h and collected for DNA extraction. For the treatment of MK886 (dissolved in ethanol, final concentration of 1.0 μm) and aspirin (dissolved in ethanol, final concentration of 200.0 μm), the procedure was the same as above except that the cells were additionally pretreated with MK886 or aspirin for 2 h. Expressions of 5-LO, COX-1, COX-2, 15-LO in the cells were determined by Western blot analysis. Briefly, the cells were washed twice with ice-cold phosphate-buffered saline and suspended in lysis buffer containing 50 mm Tris (pH 8.0), 5 mm EDTA, 150 mm NaCl, 0.5% Nonidet P-40, 0.5 mm phenylmethanesulfonyl fluoride, and 1 × protease mixture for mammalian tissue. The cells were lysed on ice for 20-min and centrifuged at 10,000 × g for 10 min. The supernatant was collected, and the protein concentration was determined by BCA protein assay reagent. Cellular protein (50 μg) was loaded on pre-cast 7% NuPAGE Bis-Tris gels and then transferred to 0.45 μm nitrocellulose membranes. The blots were blocked with 5% nonfat milk in Tris-buffered saline (100 mm Tris (pH 7.5), 150 mm NaCl) containing 0.1% Tween 20 and then incubated with primary antibodies followed by reaction with secondary antibody. Protein bands were visualized with the ECL reagent. A portion of cell culture medium (3 ml) was collected for lipid extraction. A mixture of internal standard containing 1.0 ng of each of following compounds was added to each sample: 13(S)-[2H4]HODE, 5(S)-[2H8]HETE, 12(S)-[2H8]HETE, 15(S)-[2H8]HETE, [2H4]LTB4, [2H4]PGE2, [2H4]PGD2, [2H4]PGF2α. The pH of the samples was adjusted to 3.0 with hydrochloric acid. Lipids were extracted with diethyl ether (4 ml × 2), and the organic layer was evaporated to dryness under nitrogen. Lipids were dissolved in 300 μl of acetonitrile containing PFB-Br (10:300, v/v) and diisopropylethylamine (5:300, v/v) and heated at 60 °C for 1 h. The solution was evaporated to dryness under nitrogen and re-dissolved in 100 μl of hexane/ethanol (97:3, v/v). A portion of the solution (20 μl) was subjected to normal phase chiral LC/ECAPCI/MRM/MS analysis. Calibration curves were obtained by spiking 3 ml of RPMI 1640 with authentic lipid standards. The internal standards were then added, and the standard solutions underwent the same extraction and analytical procedure as for the samples. Calibration curves were calculated with a linear regression analysis of peak area ratios of authentic standards against internal standards. Production of lipid metabolites from the cells was calculated by interpolation from the calibration curve and expressed as pmol/107 cells. DNA from 1 × 107 cells was extracted with Wako DNA extraction kit which employed sodium iodide as a chaotropic agent, and the procedure was described in the manufacturer's instructions. Typically, 600–800 μg of DNA was obtained. The DNA was dissolved in 1 ml of 10 mm Tris/100 mm MgCl2 (pH 7.4). DNase I (556 units in 10 μl of 10 mm MOPS/100 mm MgCl2) was added, and the samples were incubated at 37 °C for 1.5 h. The pH of the sample was then adjusted to 9.0 with 150 μl of 0.2 m glycine buffer (adjust pH to 10.0 with NaOH). Phosphodiesterase I (100 units in 10 μl of water) was added and incubated at 37 °C for 2 h. At the end of incubation, 150 μl of 50 mm Tris-HCl (pH 7.4) was added along with 30 units (30 μl) of shrimp alkaline phosphatase and 150 μl of 10 × shrimp alkaline phosphatase reaction buffer provided by the manufacturer. The incubation was continued at 37 °C for 2 h, and the sample was then filtered through Costar 0.2-μm microcentrifuge filters. An aliquot (50 μl) was taken for LC/UV analysis of the base content. Quantitation of DNA bases was carried out by constructing standard curves of known amount of bases. The remaining sample solution was added with 20.0 ng of 15N5-HϵdGuo as internal standards and applied to LC-18 solid phase extraction tube (1 g, 6 ml), which was preconditioned with acetonitrile (18 ml) followed with water (18 ml). For the calibration curve, a certain amount of authentic HϵdGuo (0, 0.05, 0.10, 0.20, 0.50, 1.00, 2.00, 5.00 ng of each) was mixed with internal standards and subjected to the same procedure as that of samples from this step. The cartridge was then washed with 4 ml of water and 1 ml of methanol/water (5:95, v/v). The DNA adducts were eluted with 6 ml of acetonitrile/water (1:1, v/v). The eluate was evaporated to dryness under nitrogen and then dissolved in 300 μl of acetonitrile containing PFB-Br (20:300, v/v) and diisopropylethylamine (4:300, v/v) and incubated at room temperature for 1.5 h. The solution was evaporated to dryness under nitrogen and dissolved in 100 μl of methanol/methylene chloride (25:75, v/v). The solution was applied to LC-Si solid phase extraction tube (1 g, 6 ml), which was preconditioned with methylene chloride (12 ml). The cartridge was then washed with 3 ml of methanol/methylene chloride (1:99, v/v) and 1 ml of methanol/methylene chloride (5:95, v/v). The derivatized DNA adducts were eluted with 6 ml of methanol/methylene chloride (25:75, v/v). The eluate was evaporated to dryness under nitrogen and then dissolved in 100 μl of acetonitrile, water (80:20, v/v). A portion of the solution (20 μl) was subjected to reverse phase LC/APCI/MRM/MS analysis. Calibration curves were calculated with a linear regression analysis of peak area ratios of authentic standards against internal standards. DNA adduct levels were calculated by interpolation from the calibration curve and normalized with the total base content in each sample. Chromatography was performed using a Waters Alliance 2690 HPLC system (Waters Corp., Milford, MA). A Chiralpak AD-H column (250 × 4.6-mm; inner diameter, 5 μm; Daicel Industries, Ltd., Tokyo, Japan) was employed for system 1. Solvent A was hexane, and solvent B was methanol/isopropanol (1:1, v/v). The flow rate was 1.0 ml/min, and the gradient was as follows: 2% B at 0 min, 2% B at 3 min, 3.6% B at 11 min, 8% B at 15 min, 8% B at 27 min, 50% B at 30 min, 50% B at 35 min, 2% B at 37 min, and 2% B at 45 min. Gradient elution was conducted in linear mode, and the separation was performed at 30 °C. A post-column addition of 0.75 ml methanol/min was used. LC/UV chromatography was conducted using gradient system 2 on a Hitachi l-7100 Pump equipped with Hitachi l-7400 UV detector (Hitachi, San Jose, CA). The separation employed a Phenomenex Synergi polar RP column (250 × 4.6-mm inner diameter, 4 μm). Solvent A was 5 mm ammonium acetate in water, and solvent B was 5 mm ammonium acetate in acetonitrile. The flow rate was 1.0 ml/min, and the gradient was as follows: 6% B at 0 min, 6% B at 3 min, 20% B at 9 min, 20% B at 10 min, 80% B at 12 min, 80% B at 15 min, 6% B at 17 min, and 6% B at 25 min. Gradient elution was conducted in linear mode, and the separations were performed at ambient temperature. The amount of bases was calculated by interpolation from the calibration curves constructed with authentic bases. LC-APCI/MS analyses were conducted using gradient system 3 on Hitachi l-2200 Autosampler equipped with Hitachi l-2130 Pump (Hitachi, San Jose, CA). The separation employed a XTerra C18 column (250 × 4.6-mm; inner diameter, 5 μm; Waters). Solvent A was 5 mm ammonium acetate in water, and solvent B was 5 mm ammonium acetate in acetonitrile. The flow rate was 1.0 ml/min, and the gradient was as follows: 6% B at 0 min, 6% B at 3 min, 20% B at 9 min, 20% B at 13 min, 40% B at 22 min, 70% B at 27 min, 80% B at 28 min, 80% B at 32 min, 6% B at 34 min, and 6% B at 42 min. Gradient elution was conducted in linear mode, and the separations were performed at ambient temperature. A TSQ Quantum Ultra AM triple quadrupole mass spectrometer (Thermo Fisher Scientific, San Jose, CA) equipped with an APCI source was used in the ECAPCI negative ion mode. Operating conditions for the instrument was as follows: vaporizer temperature at 450 °C, heated capillary temperature at 250 °C, with the corona discharge needle set at 30 μA. The sheath gas (nitrogen), auxiliary gas (nitrogen), and ion sweep gas (nitrogen) were 25, 3, and 3 (arbitrary units), respectively. Source collision-induced dissociation energy was 10 eV. Collision-induced dissociation was performed using argon as the collision gas in the radio frequency-only quadrupole. Targeted LC-ECAPCI/MRM/MS analysis was conducted on PFB derivatives. MRM transition for the following 16 lipids and 8 heavy isotope analog internal standards were monitored: 13(R)- and 13(S)-HODE-PFB, m/z 295 → 195 (collision energy, 18 eV); 13(S)-[2H4]HODE-PFB, m/z 299 → 198 (collision energy, 18 eV); 5(R)- and 5(S)-HETE-PFB, m/z 319 → 115 (collision energy, 15 eV); 5(S)-[2H8]HETE-PFB, m/z 327 → 116 (collision energy, 15 eV); 8(R)- and 8(S)-HETE-PFB, m/z 319 → 155 (collision energy, 16 eV); 11(R)- and 11(S)-HETE-PFB, m/z 319 → 167 (collision energy, 16 eV); 12(R)- and 12(S)-HETE-PFB, m/z 319 → 179 (collision energy, 14 eV); 12(S)-[2H8]HETE-PFB, m/z 327 → 184 (collision energy, 14 eV); 15(R)- and 15(S)-HETE-PFB, m/z 319 → 219 (collision energy, 13 eV); 15(S)-[2H8]HETE-PFB, m/z 327 → 226 (collision energy, 13 eV); LTB4-PFB, m/z 335 → 195 (collision energy, 18 eV); [2H4]LTB4-PFB, m/z 339 → 197 (collision energy, 18 eV); PGE2-PFB, m/z 351 → 271 (collision energy, 18 eV); [2H4]PGE2-PFB, m/z 355 → 275 (collision energy, 18 eV); PGD2-PFB, m/z 351 → 271 (collision energy, 18 eV); [2H4]PGD2-PFB, m/z 355 → 275 (collision energy, 18 eV); PGF2α-PFB, m/z 353 → 309 (collision energy, 18 eV); [2H4]PGF2α-PFB, m/z 357 → 313 (collision energy, 18 eV). Standard curves were constructed in the range of 0.20–200.00 pmol/107 cells for 5(S)-HETE and LTB4 and in the range of 0.02–20.00 pmol/107 cells for the other compounds. Concentrations of lipids in the study samples were determined by interpolation from the standard curv" @default.
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• W2103708921 date "2009-06-01" @default.
• W2103708921 modified "2023-10-15" @default.
• W2103708921 title "5-Lipoxygenase-mediated Endogenous DNA Damage" @default.
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• W2103708921 doi "https://doi.org/10.1074/jbc.m109.011841" @default.
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