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• W2034991121 abstract "Exposure of the lung to concentrations of ozone found in ambient air is known to cause toxicity to the epithelial cells of the lung. Because of the chemical reactivity of ozone, it likely reacts with target molecules in pulmonary surfactant, a lipid-rich material that lines the epithelial cells in the airways. Phospholipids containing unsaturated fatty acyl groups and cholesterol would be susceptible to attack by ozone, which may lead to the formation of cytotoxic products. Whereas free radicalderived oxidized cholesterol products have been frequently studied for their cytotoxic effects, ozonized cholesterol products have not been studied, although they could reasonably play a role in the toxicity of ozone. The reaction of ozone with cholesterol yielded a complex series of products including 3β-hydroxy-5-oxo-5,6-secocholestan-6-al, 5-hydroperoxy-B-homo-6-oxa-cholestan-3β,7a-diol, and 5β,6β-epoxycholesterol. Mass spectrometry and radioactive monitoring were used to identify the major cholesterol-derived product during the reaction of 2 ppm ozone in surfactant as 5β,6β-epoxycholesterol, which is only a minor product during ozonolysis of cholesterol in solution. A dose-dependent formation of 5β,6β-epoxycholesterol was also seen during direct exposure of intact cultured human bronchial epithelial cells (16-HBE) to ozone. Studies of the metabolism of this epoxide in lung epithelial cells yielded small amounts of the expected metabolite, cholestan-3β,5α,6β-triol, and more abundant levels of an unexpected metabolite, cholestan-6-oxo-3β,5α-diol. Both 5β,6β-epoxycholesterol and cholestan-6-oxo-3β,5α-diol were shown to be cytotoxic to cultured 16-HBE cells. A possible mechanism for cytotoxicity is the ability of these oxysterols to inhibit isoprenoid-based cholesterol biosynthesis in these cells. Exposure of the lung to concentrations of ozone found in ambient air is known to cause toxicity to the epithelial cells of the lung. Because of the chemical reactivity of ozone, it likely reacts with target molecules in pulmonary surfactant, a lipid-rich material that lines the epithelial cells in the airways. Phospholipids containing unsaturated fatty acyl groups and cholesterol would be susceptible to attack by ozone, which may lead to the formation of cytotoxic products. Whereas free radicalderived oxidized cholesterol products have been frequently studied for their cytotoxic effects, ozonized cholesterol products have not been studied, although they could reasonably play a role in the toxicity of ozone. The reaction of ozone with cholesterol yielded a complex series of products including 3β-hydroxy-5-oxo-5,6-secocholestan-6-al, 5-hydroperoxy-B-homo-6-oxa-cholestan-3β,7a-diol, and 5β,6β-epoxycholesterol. Mass spectrometry and radioactive monitoring were used to identify the major cholesterol-derived product during the reaction of 2 ppm ozone in surfactant as 5β,6β-epoxycholesterol, which is only a minor product during ozonolysis of cholesterol in solution. A dose-dependent formation of 5β,6β-epoxycholesterol was also seen during direct exposure of intact cultured human bronchial epithelial cells (16-HBE) to ozone. Studies of the metabolism of this epoxide in lung epithelial cells yielded small amounts of the expected metabolite, cholestan-3β,5α,6β-triol, and more abundant levels of an unexpected metabolite, cholestan-6-oxo-3β,5α-diol. Both 5β,6β-epoxycholesterol and cholestan-6-oxo-3β,5α-diol were shown to be cytotoxic to cultured 16-HBE cells. A possible mechanism for cytotoxicity is the ability of these oxysterols to inhibit isoprenoid-based cholesterol biosynthesis in these cells. Human exposure to 0.2 ppm levels of ozone in ambient air has been shown to cause numerous pulmonary effects such as increased airway inflammation and decreased pulmonary function (1Aris R.M. Christian D. Hearne P.Q. Kerr K. Finkbeiner W.E. Balmes J.R. Am. Rev. Respir. Dis. 1993; 148: 1363-1372Crossref PubMed Scopus (269) Google Scholar, 2Balmes J.R. Chen L.L. Scannell C. Tager I. Christian D. Hearne P.Q. Kelly T. Aris R.M. Am. J. Respir. Crit. Care Med. 1996; 153: 904-909Crossref PubMed Scopus (134) Google Scholar). Studies of ozone in animals using up to 3 ppm ozone have been shown to cause increased airway hyperresponsiveness and epithelial cell death. It has been hypothesized that the very high chemical reactivity of ozone limits the distribution of this gas in the pulmonary system, preventing direct exposure to the cellular components of the lung. In part ozone may react with the various components of the epithelial cell lining fluid in the lung, also known as pulmonary surfactant, which includes proteins, lipids, and single electron antioxidant agents such as ascorbic acid (3Uppu R.M. Cueto R. Squadrito G.L. Pryor W.A. Arch. Biochem. Biophys. 1994; 319: 257-266Crossref Scopus (65) Google Scholar, 4Kanofsky J.R. Sima P.D. Arch. Biochem. Biophys. 1995; 316: 52-62Crossref PubMed Scopus (56) Google Scholar, 5Connor L.M. Ballinger C.A. Albrecht R.B. Postlethwait E.M. Am. J. Physiol. 2004; 286: L1109-L1178Crossref Scopus (52) Google Scholar). Because of the very high reactivity of ozone with lipids containing double bonds, considerable emphasis has been placed on the reaction of ozone with lipid compounds in the lungs and the possibility that the adverse effects of ozone are mediated by lipid-ozonized products (6Pryor W.A. Squadrito G.L. Friedman M. Free Rad. Biol. Med. 1995; 19: 934-941Crossref Scopus (275) Google Scholar). Evidence in support of this theory has been accumulating with the identification of biologically active phospholipids (7Kafoury R.M. Pryor W.A. Squadrito G.L. Salgo M.G. Zou X. Friedman M. Am. J. Respir. Crit. Care Med. 1999; 160: 1934-1942Crossref PubMed Scopus (87) Google Scholar) such as 1-hexadecanoyl-2-(9-oxo-nonanoyl)-glycerophosphocholine, found following ozone exposure to lung surfactant (8Uhlson C. Harrison K. Allen C.B. Ahmad S. White C.W. Murphy R.C. Chem. Res. Toxicol. 2002; 15: 896-906Crossref PubMed Scopus (77) Google Scholar). This oxidized phospholipid that eluted as a somewhat polar product on normal phase HPLC 1The abbreviations used are: HPLC, high pressure liquid chromatography; RP-HPLC, reversed phase HPLC; α-epoxide, 5α,6α-epoxycholesterol; β-epoxide, 5β,6β-epoxycholesterol; 3,5,6-triol, cholestan-3β,5α,6β-triol; seco-sterol, 3β-hydroxy-5-oxo-5,6-secocholestan-6-al; 6-oxo-3,5-diol, cholestan-6-oxo-3,5-diol; BAL, bronchoalveolar lavage; BSTFA, Bis(trimethylsilyl) fluoroacetamide; 16:0a/18:1-GPCho, 1-palmitoyl-2-oleoyl-glycerophosphocholine; 16:0a/9al-GPCho, 1-palmitoyl-2-(9′-oxo-nonanoyl)-glycerophosphocholine; d6-16:0a/16:0-GPCho, d6-dipalmitoylglycerophosphocholine; TMS, trimethylsilyl; HBE, human bronchial epithelial cells; LC/MS, liquid chromatography/mass spectrometry. was found to initiate apoptotic death in monocytes and macrophages. However, a relatively nonpolar component was also found to elute from this normal phase HPLC separation that was also cytotoxic, and preliminary data suggested that several oxidized neutral lipid products were present in this fraction. Cholesterol is the most abundant neutral lipid present in pulmonary surfactant, and this molecule has a double bond that would be susceptible to attack by ozone (3Uppu R.M. Cueto R. Squadrito G.L. Pryor W.A. Arch. Biochem. Biophys. 1994; 319: 257-266Crossref Scopus (65) Google Scholar, 10Bailey P.S. Chem. Rev. 1958; 58: 925-1010Crossref Scopus (482) Google Scholar). Although there has been some controversy about the exact chemical structure of the major ozonolysis product when cholesterol is ozonized in solution at high ozone concentrations (>0.1%) (11Gumulka J. Smith L.L. J. Am. Chem. Soc. 1983; 105: 1972-1979Crossref Scopus (64) Google Scholar, 12Jaworski K. Smith L.L. J. Org. Chem. 1988; 53: 545-554Crossref Scopus (42) Google Scholar, 13Paryzek Z. Martynow J. Swoboda W. J. Chem. Soc. Perkin Trans. 1990; 1: 1222-1224Crossref Scopus (27) Google Scholar, 14Paryzek Z. Rychlewska U. J. Chem. Soc. Perkins Trans. 1997; 2: 2313-2318Crossref Scopus (11) Google Scholar), electrospray tandem mass spectrometry was recently used to characterize this chemically reactive cholesterol ozonolysis product as 5-hydroperoxy-B-homo-6-oxa-cholestane-3β,7a-diol (Scheme 1) (15Pulfer M.K. Harrison K.A. Murphy R.C. J. Am. Soc. Mass Spectrom. 2004; 15: 194-202Crossref PubMed Scopus (17) Google Scholar). Reduction of 5-hydroperoxy-B-homo-6-oxacholestane-3β,7a-diol has been shown to primarily yield 3β-hydroxy-5-oxo-5,6-secocholestan-6-al (5,6-seco-sterol) (11Gumulka J. Smith L.L. J. Am. Chem. Soc. 1983; 105: 1972-1979Crossref Scopus (64) Google Scholar, 12Jaworski K. Smith L.L. J. Org. Chem. 1988; 53: 545-554Crossref Scopus (42) Google Scholar, 13Paryzek Z. Martynow J. Swoboda W. J. Chem. Soc. Perkin Trans. 1990; 1: 1222-1224Crossref Scopus (27) Google Scholar). In fact, this reduced product has been detected in lung homogenate and broncheoalveolar lavage fluid of rats exposed to ozone, and it has thus been suggested as a biomarker for ozone exposure (16Pryor W.A. Wang K. Bermudez E. Biochem. Biophys. Res. Commun. 1992; 188: 613-618Crossref Scopus (48) Google Scholar). The recent observation of 5,6-seco-sterol in arterial plaques has provided evidence that the formation of ozone may also occur as the result of normal biochemical events taking place in vivo during an inflammatory response (17Wentworth Jr., P. Nieva J. Takeuchi C. Galve R. Wentworth A.D. Dilley R.B. DeLaria G.A. Saven A. Babior B.M. Janda K.D. Eschenmoser A. Lerner R.A. Science. 2003; 302: 1053-1056Crossref PubMed Scopus (240) Google Scholar). It is important to consider that the interaction between ozone and cholesterol has primarily been studied in organic solvents with high levels of ozone, where 5-hydroperoxy-B-homo-6-oxa-cholestane-3β,7a-diol is the major product (11Gumulka J. Smith L.L. J. Am. Chem. Soc. 1983; 105: 1972-1979Crossref Scopus (64) Google Scholar, 14Paryzek Z. Rychlewska U. J. Chem. Soc. Perkins Trans. 1997; 2: 2313-2318Crossref Scopus (11) Google Scholar, 15Pulfer M.K. Harrison K.A. Murphy R.C. J. Am. Soc. Mass Spectrom. 2004; 15: 194-202Crossref PubMed Scopus (17) Google Scholar). However, environmentally relevant concentrations of ozone acting on lipid cellular membranes or in lipid-rich pulmonary surfactant could involve different chemistry, because of the ordered nature of the lipid bilayer, yielding alternative products. Isolated bronchoalveolar lavage (BAL) fluid was exposed in vitro to precise levels of ozone in a carefully controlled ozone chamber to study the formation of cholesterol-derived ozonolysis products. This revealed the formation of 5β,6β-epoxycholesterol (β-epoxide) (Scheme 1) as a more abundant product than 5-hydroperoxy-B-homo-6-oxa-cholestan-3β,7a-diol in this system. The ability of this compound and its cellular metabolites to cause cytotoxicity and to inhibit cholesterol synthesis in cultured human bronchial epithelial cells was subsequently studied. Materials—Cholesterol (>99%) was purchased from Sigma. Radioactive [4-14C]cholesterol (45–60 mCi/mmol dissolved in ethanol) was purchased from PerkinElmer Life Sciences. Radioactive [1-14C]acetate (56.6 mCi/mmol dissolved in ethanol) was purchased from PerkinElmer Life Sciences. Stable isotope labeled 2,2,3,4,4,6-d6-cholesterol (98% atom %, excess d6) was purchased from Cambridge Isotope Laboratories (Andover, MA). Solvents, cell culture media, and culture plates were purchased from Fisher. Bis(trimethylsilyl)fluoroacetamide (BSTFA) and trypan blue dye (0.4%) were purchase from Sigma. Bis(trimethyl-d9-silyl)acetamide (99%) was purchased from Isotech (Miamisburg, OH). Rat lung lavage fluid was provided by Dennis Voelker (National Jewish Medical and Research Center, Denver, CO). Identification of Cholesterol Ozonolysis Products in Lung Surfactant—Bronchoalevolar lavage fluid from rats was concentrated by centrifugation at 20,000 × g for 1 h in 5 mm CaCl2 (18Suwabe A. Mason R.J. Voelker D.R. Arch. Biochem. Biophys. 1996; 327: 285-291Crossref PubMed Scopus (49) Google Scholar). The pellet was resuspended in phosphate-buffered saline to a final cholesterol concentration of 30 μg/ml cholesterol. For identification of cholesterol metabolites, 2.5 μl of tracer [14C]cholesterol was added to 1 ml of the lavage fluid for a final concentration of 200 nCi/ml (0.2% ethanol). Experiments were carried out in triplicate by ozonolysis of 100 μl of this labeled lavage fluid in 35-mm diameter tissue culture multiwell plates. The plates were exposed to various concentrations of ozone using a computer-controlled in vitro ozone exposure chamber. This system was capable of delivering precise concentrations of ozone from 0.1 to 10 ppm in humidified air, as described previously (8Uhlson C. Harrison K. Allen C.B. Ahmad S. White C.W. Murphy R.C. Chem. Res. Toxicol. 2002; 15: 896-906Crossref PubMed Scopus (77) Google Scholar). After ozonolysis, the samples in each well were diluted with 2 ml of water, transferred to glass tubes, and wells were washed with 2 ml of methanol. To the transferred samples, 3 ml of methylene chloride was added and lipids extracted essentially as described by Bligh and Dyer (19Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42878) Google Scholar). After drying the extract with a stream of dry nitrogen, the products were dissolved in 100 μl of ethanol and then injected onto a C18 (250 × 4.6 mm) reversed phase column (Phenomenex, Torrance, CA) at a flow rate of 1 ml/min. Solvent A was methanol/water/acetonitrile (v/v/v, 60:20:20) with 1 mm ammonium acetate; solvent B was methanol with 1 mm ammonium acetate. The gradient ran from 50 to 100% solvent B in 20 min and stayed at 100% solvent B for 20 min. Radioactive monitoring coupled with mass spectrometry was used to detect the products of cholesterol ozonolysis as described previously (15Pulfer M.K. Harrison K.A. Murphy R.C. J. Am. Soc. Mass Spectrom. 2004; 15: 194-202Crossref PubMed Scopus (17) Google Scholar). Quantitation of Epoxycholesterol, Cholesterol, and Phospholipids—Both the α and β isomers of 5,6-epoxycholesterol were synthesized based on the method of Sevanian et al. (20Sevanian A. Berliner J. Peterson H. J. Lipid Res. 1991; 32: 147-155Abstract Full Text PDF PubMed Google Scholar) with slight modifications. Briefly, 19.1 mg of chloroperoxybenzoic acid was added to 38.6 mg of cholesterol in 12 ml of methylene chloride and stirred overnight at 4 °C. Deuterated [2,2,3,4,4,6-2H6]cholesterol was used for synthesis of deuterated 5,6-epoxycholesterol. The solution was washed three times with water and then with a saturated salt solution. Finally, the solvent was evaporated using a roto-evaporator, and the product was resuspended in ethanol. Separation of the α and β isomers was achieved using RP-HPLC on an Alltech column (250 × 10.0 mm, C18; Deerfield, IL) at a flow rate of 4 ml/min with the gradient described above. Fractions were collected (1 min) and dried under vacuum; after weighing, the fractions were derivatized with BSTFA and analyzed by gas chromatography/mass spectrometry to determine purity of the reference standards (>95%). Quantitation of both cholesterol and β-epoxide was carried out using stable isotope dilution mass spectrometry. Deuterated β-epoxide (40 ng in 25 μl) was added to each point of the standard curve and to all samples before extraction with iso-octane. An identical protocol was used to determine total cholesterol. Samples (10% aliquot) were introduced onto the mass spectrometer using a 150 × 1.0-mm Columbus C18 column (Phenomenex, Rancho Palos Verdes, CA). The solvent system used was the same as for identification of cholesterol ozonolysis products, except a modified gradient was used, with 75–100% solvent B for 10 min followed by 100% solvent B for 20 min. Multiple reaction monitoring analysis was carried out on a Sciex API-2000 mass spectrometer (PerkinElmer Life Sciences). Positive ion mode was used with an ion spray voltage of 4500 V, declustering potential of 40 V, focusing potential 350 V, and collision energy of 12 V. Nitrogen was used in the collision cell with a collision gas thickness of 2.17 × 1015 mol/cm2. The transitions monitored were m/z 420 → 385 for α-epoxide and β-epoxide, m/z 426 → 391 for d6-β-epoxide, m/z 404 → 369 for cholesterol, m/z 436 → 383 for 5,6-seco-sterol and 6-oxo-3,5-diol, and m/z 438 → 385 for 3,5,6-triol. The dwell time for each transition was 800 ms. The standard curve was linear for the range tested, from 0.625 to 320 ng of 5β,6β-epoxycholesterol and from 100 ng to 6.4 μg of cholesterol. Quantitation of 1-palmitoyl-2-oleoyl-glycerophosphocholine (16:0a/18:1-GPCho) and 1-palmitoyl-2-(9′-oxo-nonanoyl)-glycerophosphocholine (16:0a/9al-GPCho) in 75-μl aliquots of rat BAL exposed to 0.2 ppm ozone was achieved by addition of 20 ng of d3-platelet activating factor and 320 ng d6-dipalmitoyl-glycerophosphocholine (d6-16:0a/16:0-GP-Cho) at the same time as the cholesterol and β-epoxide internal standards. After neutral lipid extraction of the samples with iso-octane (see below), 2 ml of methylene chloride was added to the remaining H2O/MeOH layer to extract the phospholipids. The organic layer was dried under nitrogen and solvated in 500 μl of normal phase solvent A (hexane/isopropyl alcohol/H2O, 30:40, v/v) for injection onto a 150 × 1.0-mm Prodigy silica column (Phenomenex; Rancho Palos Verdes, CA). Initial conditions were 75% solvent B (hexane/isopropyl alcohol/H2O, 30:40:7, v/v/v, 1% ammonium acetate). The gradient increased to 100% solvent B over 6 min and stayed constant for another 30 min. The mass spectrometer was run in positive ion mode with a higher declustering potential (100 V) and a higher collision energy (40 V) than used when monitoring cholesterol-derived products. The transitions monitored for the phospholipids were m/z 527 → 184 for d3-platelet activating factor, m/z 650 → 184 for 16:0a/9al-GPCho, m/z 740 → 184 for d6-16:0a/16:0-GPCho, and m/z 760 → 184 for 16:0a/18:1-GPCho. The standard curve was linear for the range tested, 20–640 ng of 16:0a/9al-GPCho and 80 to 2.56 μg of 16:0a/18:1-GPCho. Cell Ozonolysis, Cytotoxicity, and Inhibition of Cholesterol Synthesis—The 16 HBE 14o- (16-HBE) human bronchial epithelial cell line (21Gruenert D.C. Basbaum C.B. Welsh M.J. Finkbeiner W.E. Nadel J.A. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 5951-5955Crossref PubMed Scopus (175) Google Scholar) was maintained in Dulbecco's modified Eagle's medium with 10% fetal bovine serum and 1% penicillin/streptomycin on uncoated 6- or 24-well culture dishes. Cells were grown at 37 °C in a 95% air, 5% CO2 incubator with 100% humidity. For ozonolysis, cells were grown to ∼50% confluency in 6-well plates. Before ozonolysis the media were removed, and cells were washed with phosphate-buffered saline. A small volume (300 μl) of Hanks' buffered saline solution was added to each well to keep the cells from drying out during ozone exposure. After exposure cells were trypsinized, and the lipids were extracted as described above. Trypan blue exclusion was used to measure cytotoxicity (22Ghelli A. Porcelli A.M. Zanna C. Rugolo M. Arch. Biochem. Biophys. 2002; 402: 208-217Crossref PubMed Scopus (25) Google Scholar). 16-HBE cells were grown in 24-well plates to >50% confluency (2 × 105 cells) and then washed and covered with 1 ml of serum-containing media per well. Ethanolic solutions of oxysterol or corresponding vehicle were added to cell media (0.5% ethanol final concentration), and the cells were incubated at 37 °C for the duration of the exposure. At the indicated time points, cells were trypsinized, and a small aliquot was diluted 1:1 with a 0.4% trypan blue solution (final concentration 0.2%). At least 100 cells were counted per sample using a hemocytometer, and the percentage that incorporated the dye was calculated. No effect of vehicle was observed. For studies of protection by exogenous cholesterol, 1, 3, 10, or 30 μm cholesterol in ethanol was added immediately prior to treatment with oxysterol. For studies of inhibition of cholesterol synthesis, the incorporation of radiolabeled acetate into cholesterol and neutral lipids was determined by modification of the method of Kandutsch and Chen (23Kandutsch A.A. Chen H.W. J. Biol. Chem. 1973; 248: 8408-8417Abstract Full Text PDF PubMed Google Scholar). Briefly, cells at 50% confluency in 6-well dishes were washed, and fresh media (3 ml) was added. Cells were treated with the indicated concentrations of oxysterols in ethanol for 24 h and then 7 μCi of [14C]acetate was added for 3 h (final ethanol concentration 0.3%). Cells were trypsinized and lipids were extracted from the media and cells with 3 volumes of iso-octane. The extract was dried with anhydrous magnesium sulfate to remove residual water-soluble radioactive components. The iso-octane was then dried under N2 and resuspended in 100 μl of methylene chloride for TLC chromatography using Silica Gel G (Analtech; Newark, DE) activated for 2 h at 110 °C. The TLC solvent system employed was 99.5% ether with 0.5% ammonium acetate in which cholesterol and neutral lipids had an Rf of 0.61 and 0.83, respectively. The radioactive signal was quantitated by integration of the radioactive signal for the peak at each Rf using a Bioscan system 200 imaging scanner with Win-scan software (Bioscan, Washington, D. C.). Structural Characterization of the Metabolites—Three 10-cm diameter plates of 16-HBE cells (∼30 million cells) were treated with 3 μm β-epoxide overnight (final ethanol concentration 0.03%). Samples were pooled, and the lipids were extracted as described above prior to injection on an RP-HPLC column flowing at 1 ml/min with 50-μl eluate split to the mass spectrometer to monitor for the metabolite transition (m/z 436 → 383), and the remaining effluent directed to a fraction collector (1-min fractions). A portion of the HPLC fraction containing the metabolite was infused into the API III+ instrument (Applied Biosystems, Thornhill, Ontario, Canada) for tandem mass spectrometric analysis, and the rest was dried under vacuum. The same method was used to acquire deuterated metabolites (cells were treated with deuterated β-epoxide under identical conditions). In order to characterize the metabolites by electron ionization (70 eV) mass spectrometry, an aliquot of the metabolite or its deuterated analog was derivatized by addition of 50 μl of acetonitrile and 50 μl of BSTFA followed by a 15-min heating to 65 °C for 15 min. An aliquot (2 μl) of each derivatization solution was analyzed by a gas chromatographmass spectrometer using electron ionization at +70 eV (Trace 2000, Thermo-Finnigan, San Jose, CA). The temperature gradient ran from 150 to 260 °C at 20 °C/min and 260–310 °C at 4 °C/min on a 30-meter DB-1 column (Phenomenex, Torrance, CA) with a 0.25 mm inner diameter and a 0.25-μm stationary film thickness. Sodium borohydride reduction of α-epoxide, β-epoxide, and the unknown metabolite was carried out by adding an excess of NaBH4 to the oxysterol dissolved in ethanol for 2 h followed by iso-octane extraction. Epoxide hydrolysis to vicinal diols of α-epoxide, β-epoxide, and the unknown metabolite was carried out by treating 5 μg of the oxysterol with 30 μl of perchloric acid in 0.5 ml of tetrahydrofuran/H2O/acetone (v/v/v, 4:1:0.5) and stirring for 4 h at room temperature (24Fieser L.F. Fieser M. Reagents for Organic Synthesis. Wiley Interscience, New York1959: 796Google Scholar). For large scale synthesis of cholestane-3,5,6-triol (3,5,6-triol), 10 mg of β-epoxide was treated with 0.5 ml of perchloric acid in 4 ml of tetrahydrofuran/H2O/acetone (v/v/v, 4:1:0.5). The lipids were extracted with methylene chloride, and the organic layer was washed three times with water. The 3,5,6-triol was purified by RP-HPLC as described above for β-epoxide. Cholestan-6-oxo-3,5-diol (6-oxo-3,5-diol) was synthesized by the method of Fieser and Rajagopalan (25Fieser L.F. Rajagopalan S. J. Am. Chem. Soc. 1949; 71: 3938-3941Crossref Scopus (140) Google Scholar). Briefly, 10 mg of cholestanetriol was dissolved in 4.5 ml of ether, 750 μl of methanol, and 750 μl of water. N-Bromosuccinimide (108 mg) was added, and the reaction was stirred for 3 h at room temperature. The solution was diluted with water and extracted with methylene chloride. The organic fraction was dried under vacuum and purified by RP-HPLC. The resulting product was derivatized with BSTFA and characterized by gas chromatography/mass spectrometry. Treatment of diluted calf lung surfactant extract with high concentrations of ozone was reported previously (8Uhlson C. Harrison K. Allen C.B. Ahmad S. White C.W. Murphy R.C. Chem. Res. Toxicol. 2002; 15: 896-906Crossref PubMed Scopus (77) Google Scholar) to generate several classes of biologically active products, two of which had very different polarity when separated by normal phase chromatography. When analyzed by LC/MS, the early eluting normal phase fraction that reduced monocyte viability was found to generate abundant positive ions at m/z 383, 401, 419, 435, and 452 (data not shown). These ions were consistent with various neutral lipids, including oxidized cholesterol, suggesting that cholesterol within pulmonary surfactant could be transformed into biologically active metabolites that mediated the toxicity of ozone. In order to determine unambiguously whether components in lung surfactant could in fact be derived from cholesterol reacting with relevant concentrations of ozone, a trace amount of [14C]cholesterol was added to isolated rat BAL fluid, and the solution was exposed to 2.0 ppm ozone in a controlled chamber for 4 h. Lipids were then extracted and chromatographed by reversed phase HPLC, and the effluent was analyzed by on-line mass spectrometry and radioactive scintillation detection. Two major radioactive products were observed along with unreacted cholesterol (Fig. 1). The least lipophilic component (peak A) eluted at 16 min and generated an abundant ion at m/z 511 (negative ion mode) and at m/z 452 (positive ion mode). This component was identified as 5-hydroperoxy-B-homo-6-oxa-cholestan-3,7a-diol based on collision-induced dissociation properties and RP-HPLC retention time in comparison with a previously identified product following the exposure of cholesterol to ozone in tetrahydrofuran and water (15Pulfer M.K. Harrison K.A. Murphy R.C. J. Am. Soc. Mass Spectrom. 2004; 15: 194-202Crossref PubMed Scopus (17) Google Scholar). The compound that eluted at 21 min (peak B) generated an abundant positive ion at m/z 420 by electrospray ionization that corresponded to an ammonium ion adduct of cholesterol with the addition of one oxygen atom, which agreed with a previously identified product of cholesterol ozonolysis, namely 5,6-epoxycholesterol (11Gumulka J. Smith L.L. J. Am. Chem. Soc. 1983; 105: 1972-1979Crossref Scopus (64) Google Scholar). This oxidized cholesterol product had been studied previously (26Sevanian A. Mead J.F. Stein R.A. Lipids. 1979; 14: 634-643Crossref PubMed Scopus (130) Google Scholar) because it was observed to form during a very different type of oxidative stress, namely free radical-mediated cholesterol peroxidation. Synthesis of both epoxide stereoisomers had been described previously (20Sevanian A. Berliner J. Peterson H. J. Lipid Res. 1991; 32: 147-155Abstract Full Text PDF PubMed Google Scholar), which provided a facile means to synthesize deuterated 5α,6α-epoxycholesterol (α-epoxide) as well as deuterated β-epoxide. Each isomer was added in separate experiments to pulmonary surfactant after exposure to relatively low concentrations of ozone, and it was found that the radioactive peak eluting at 21 min was in fact only one of the epimers, namely 5β,6β-epoxycholesterol based on co-elution of the deuterated β-epoxide with peak B (data not shown). Deuterated β-epoxide was subsequently used as a mass spectrometry internal standard to facilitate quantitation of both isomers of epoxycholesterol in rat BAL treated with ozone. When BAL was exposed for 4 h to increasing levels of 0.2, 0.5, and 1.0 ppm ozone, a dose-dependent formation of β-epoxide was observed (Fig. 2). Up to 200 ng of β-epoxide was observed to form under these conditions, whereas ∼3 μg of unreacted cholesterol remained in the surfactant. The β isomer was formed in preference to the α isomer at all ozone concentrations studied with a ratio of ∼5:1. Samples of rat BAL exposed to filtered air had low but detectable levels of β-epoxide; however, α-epoxide was not detected. Phospholipids that contain a double bond in a fatty acyl chain are also an abundant component of pulmonary surfactant, and therefore it was of interest to determine the abundance of cholesterol-derived ozonolysis products relative to phospholipid-derived ozonolysis products. Aliquots of surfactant containing 3.3 nmol of 16:0a/18:1-GPCho and 5.6 nmol of cholesterol contained 66 pmol of β-epoxide and 10 pmol of the phospholipid ozonolysis product; 16:0a/9al-GPCho was studied previously (8Uhlson C. Harrison K. Allen C.B. Ahmad S. White C.W. Murphy R.C. Chem. Res. Toxicol. 2002; 15: 896-906Crossref PubMed Scopus (77) Google Scholar). After exposure to 0.2 ppm ozone for 4 h, the level of 16:0a/9al-GPCho increased to 42 pmol and β-epoxide increased to 184 pmol. Therefore, there was a 3–4-fold increase in the levels of both products following ozonolysis, suggesting that cholesterol is a relevant target for oxidation by ozone and that cholesterol ozonolysis products are formed in similar abundance to phospholipid-derived ozonolysis products. The pulmonary surfactant layer in some areas of the lung may be sufficiently thin to permit direct exposure of underlying epithelial cells to ozone present in inspired air. This would be especially true for cells in the alveoli where surfactant sits mainly in the junctions between cells (27Weibel" @default.
• W2034991121 created "2016-06-24" @default.
• W2034991121 creator A5037222936 @default.
• W2034991121 creator A5060167307 @default.
• W2034991121 date "2004-06-01" @default.
• W2034991121 modified "2023-10-10" @default.
• W2034991121 title "Formation of Biologically Active Oxysterols during Ozonolysis of Cholesterol Present in Lung Surfactant" @default.
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