Electrospray ionization and tandem mass spectrometry of eicosanoids.
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Cannabinoid Receptor Modulators↗
Biomedical subjects
Robert C Murphy
Publications and source records attributed to Robert C Murphy.
Ultraviolet B radiation generates platelet-activating factor-like phospholipids underlying cutaneous damage.
Ultraviolet B light (UVB) causes cutaneous inflammation and cell death, but the agents responsible are not defined. These studies examined the role of the platelet-activating factor (PAF) signaling system in UVB-mediated effects. Expression of the PAF receptor in the PAF receptor-negative epidermoid cell line KB augmented apoptosis in response to UVB irradiation. Overexpression of the PAF receptor in primary human keratinocytes also enhanced UVB-mediated apoptosis in vitro, and it enhanced apoptosis in an in vivo model of human keratinocytes grafted onto severe combined immune-deficient (SCID) mice. To define the mechanism by which UVB activates the PAF receptor, we used mass spectrometry to demonstrate significant amounts of the C4 PAF analogs 1-alkyl-2-(butanoyl and butenoyl)-sn-glycero-3-phosphocholine, as well as native PAF in an epidermal cell line after UVB irradiation. Supplementing the cells with the precursor phospholipid 1-hexadecyl-2-arachidonoyl-sn-glycero-3-phosphocholine (HAPC) increased the amount of C4 PAF analogs recovered after UVB exposure. We irradiated HAPC directly and found, even in the absence of a photosensitizer, fragmentation to C4-PAF receptor ligands. We conclude UVB photo-oxidizes cellular phospholipids, creating PAF analogs that stimulate the PAF receptor to induce further PAF synthesis and apoptosis. PAF signaling may participate in the cutaneous inflammation that occurs during photo-aggravated dermatoses.
Animals↗
Prostaglandin signaling in the renal collecting duct: release, reuptake, and oxidation in the same cell.
Prostaglandins mediate autacrine and paracrine signaling over short distances. We used the renal collecting duct as a model system to test the hypothesis that local control of prostaglandin signaling is achieved by expressing inactivation in the same cell as synthesis. Immunocytochemical studies demonstrated that renal collecting ducts in situ express the prostaglandin (PG) synthesis enzyme, cyclooxygenase-1 (COX-1), as well as both components of prostaglandin metabolic inactivation, i.e. the prostaglandin uptake carrier prostaglandin transporter (PGT) and the enzyme 15-hydroxyprostaglandin dehydrogenase. We characterized this system further using the collecting duct cell line Madin-Darby canine kidney (MDCK), which retains COX-2 and prostaglandin dehydrogenase expression but which has lost PGT expression. When we reintroduced PGT, it was correctly sorted to the apical membrane where it altered the sidedness of prostaglandin E2 (PGE2) release, a process we call "vectorial release via sided reuptake." Importantly, although COX-2 and prostaglandin dehydrogenase are expressed in the same MDCK cell, they must be compartmentalized because even in the presence of excess dehydrogenase newly synthesized PGE2 is released largely un-oxidized. However, when PGE2 undergoes first release and then PGT-mediated reuptake, significant oxidation takes place, suggesting that PGT imports PGE2 into the prostaglandin dehydrogenase compartment. Our data are consistent with a new model that offers significant new mechanisms for the fine control of eicosanoid signaling.
Animals↗
Quantitation of cardiolipin molecular species in spontaneously hypertensive heart failure rats using electrospray ionization mass spectrometry.
Electrospray ionization mass spectrometry has previously been used to probe qualitative changes in the phospholipid cardiolipin (CL), but it has rarely been used in a quantitative manner. We assessed changes in the amount of individual molecular species of cardiac CL present in a model of congestive heart failure using 1,1',2,2'-tetramyristoyl cardiolipin as an internal standard. There was a linear relationship between the ratio of the negative molecular ion ([M-H]-) current from four different CL reference standards and the [M-H]- from the internal standard, as a function of the concentration of CL molecular species. Therefore, this internal standard can be used to quantitate many naturally occurring CL molecular species over a wide range of CL concentrations. Using this method, changes to individual molecular species of CL in failing hearts from male spontaneously hypertensive heart failure rats were examined. CL isolated from cardiac mitochondria was compared with left ventricular tissue to demonstrate the feasibility of extracting and quantitating CL from either mitochondrial or tissue samples. The acyl chain composition of individual CL molecular species was identified using tandem mass spectrometry. In animals with heart failure, the major cardiac CL species (tetralinoloyl) decreased significantly, whereas other minor CL species were significantly increased.
Animals↗
A comprehensive classification system for lipids.
Lipids are produced, transported, and recognized by the concerted actions of numerous enzymes, binding proteins, and receptors. A comprehensive analysis of lipid molecules, "lipidomics," in the context of genomics and proteomics is crucial to understanding cellular physiology and pathology; consequently, lipid biology has become a major research target of the postgenomic revolution and systems biology. To facilitate international communication about lipids, a comprehensive classification of lipids with a common platform that is compatible with informatics requirements has been developed to deal with the massive amounts of data that will be generated by our lipid community. As an initial step in this development, we divide lipids into eight categories (fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, prenol lipids, saccharolipids, and polyketides) containing distinct classes and subclasses of molecules, devise a common manner of representing the chemical structures of individual lipids and their derivatives, and provide a 12 digit identifier for each unique lipid molecule. The lipid classification scheme is chemically based and driven by the distinct hydrophobic and hydrophilic elements that compose the lipid. This structured vocabulary will facilitate the systematization of lipid biology and enable the cataloging of lipids and their properties in a way that is compatible with other macromolecular databases.
Database Management Systems↗
Analysis of cell membrane aminophospholipids as isotope-tagged derivatives.
Glycerophosphoethanolamine (GPEtn) and glycerophosphoserine (GPSer) lipids were reacted with a multiplexed set of differentially isotopically enriched N-methylpiperazine acetic acid N-hydroxysuccinimide ester reagents, which place isobaric mass labels at a primary amino group. The resulting derivatized aminophospholipids were isobaric and chromatographically indistinguishable but yielded positive reporter ions (m/z 114 or 117) after collisional activation that could be used to identify and quantify individual members of the multiplex set. The chromatographic and mass spectrometric response of N-methylpiperazine amide-tagged aminophospholipids was probed using glycerophosphoethanolamine and glycerophosphoserine lipid standards. The [M+H]+ of each tagged aminophospholipid shifted 144 Da, and during collision-induced dissociation the major fragmentation ion was either m/z 114 or 117. This mode of detecting aminophospholipids was useful for an unbiased analysis of plasmalogen GPEtn lipids. Molecular species information on the esterified fatty acyl substituents was obtained by collisional activation of the [M-H]- ions. The isotope-tagged reagents were used to assess changes in the distribution of GPEtn lipids after exposure of liposomes made from phospholipids extracted from RAW 264.7 cells to Cu2+/H2O2 to illustrate the ability of these reagents to aid in the mass spectrometric identification of aminophospholipid changes that occur during biological stimuli.
Animals↗
Direct qualitative analysis of triacylglycerols by electrospray mass spectrometry using a linear ion trap.
Triacylglycerols (TAGs) isolated from a biological sample provide a challenge for mass spectrometric analysis because of the complexity of naturally occurring TAGs, which may contain different fatty acyl substituents resulting in a large number of molecular species having the identical elemental composition. We have investigated the use of mass spectrometry to obtain unambiguous information as to the individual TAG molecular species present in a complex mixture of triacylglycerols using a linear ion trap mass spectrometer. Ammonium adducts of TAGs, [M+NH4]+, were generated by electrospray ionization, which permitted the molecular weight of each TAG molecular species to be determined. The mechanisms involved in the decomposition of the [M+NH4]+ and subsequent fragment ions were investigated using deuterium labeling, MS/MS, and MS3 experiments. Collision induced decomposition of [M+NH4]+ ions resulted in the neutral loss of NH3 and an acyl side-chain (as a carboxylic acid) to generate a diacyl product ion. MS/MS data were used to identify each acyl group present for a given [M+NH4]+ ion, and this information could be combined with molecular weight data to identify possible TAG molecular species present in a biological extract. Subsequent MS3 experiments on the resultant diacyl product ions, which gave rise to acylium (RCO+) and related ions, enabled unambiguous TAG molecular assignments. These strategies of MS, MS/MS, and MS3 experiments were applied to identify components within a complex mixture of neutral lipids extracted from RAW 264.7 cells.
Animals↗
Mass spectrometry advances in lipidomica: collision-induced decomposition of Kdo2-lipid A.
Mass spectrometry has made significant advances in the analysis of lipid substances, both simple and complex present in extracts of eukaryotic and prokaryotic cells. The development of the ionization techniques of electrospray ionization and matrix-assisted laser desorption ionization (MALDI) have both been applied to the analysis of lipids. The example of the types of structural information that can be obtained from MALDI-TOF tandem mass spectrometry is exemplified by the analysis of Kdo2-lipid A, a complex lipopolysaccharide known to activate toll-like 4 receptors on mammalian cells. Analysis of Kdo2-lipid A obtained from an Escherichia coli WBB06 was found to generate an abundant [M-H]- ion at m/z 2236.4 and a more abundant carbon-13 isotope at m/z 2237.4. Furthermore, collisional activation of the lipid A portion of the molecule at m/z 1796.3 resulted in a series of ions corresponding to the loss of all four fatty acyl groups as neutral carboxylic acids. An altogether different challenge of mass spectrometry applied to the area of lipid analysis is that of quantitative analysis. Two rather different requirements have emerged. One with high precision and accuracy for the measurement of relatively few lipid species that are produced at very low concentrations and typically interact with specific receptor proteins. A rather different challenge is that for the analysis of abundant lipid classes, which are composed of multiple molecular species that can approach several hundred under certain circumstances.
Endotoxins↗
Epidermal peroxisome proliferator-activated receptor gamma as a target for ultraviolet B radiation.
Ultraviolet B radiation (UVB) is a pro-oxidative stressor with profound effects on skin in part through its ability to stimulate cytokine production. Peroxisome proliferator-activated receptor gamma (PPAR gamma) has been shown to regulate inflammatory processes and cytokine release in various cell types. Since the oxidized glycerophospholipid 1-hexadecyl-2-azelaoyl glycerophosphocholine (azPC) has been shown to be a potent PPAR gamma agonist, this study was designed to assess whether the PPAR gamma system is a target for UVB irradiation and involved in UVB-induced inflammation in epidermal cells. The present studies demonstrated the presence of PPAR gamma mRNA and functional protein in human keratinocytes and epithelial cell lines HaCaT, KB, and A431. The treatment of epidermal cells with the PPAR gamma-specific agonist ciglitazone or azPC augmented cyclooxygenase-2 expression and enzyme activity induced by phorbol 12-myristate-13-acetate or interleukin-1 beta. Lipid extracts from the cell homogenate of UVB-irradiated, but not control, cells contained a PPAR gamma-agonistic activity identified by reporter assay, and this activity up-regulated cyclooxygenase-2 expression induced by phorbol 12-myristate-13-acetate. Subjecting purified 1-hexadecyl-2-arachidonoyl-glycerophosphocholine to UVB irradiation generated a PPAR gamma-agonistic activity, among which the specific PPAR gamma agonist azPC was identified by mass spectrometry. These findings suggested that UVB-generated PPAR gamma-agonistic activity was due to the free radical mediated non-enzymatic cleavage of endogenous glycerophosphocholines. Treatment with the specific PPAR gamma antagonist GW9662 or expression of a dominant-negative PPAR gamma mutant in KB cells inhibited UVB-induced epidermal cell prostaglandin E(2) production. These findings suggested that UVB-generated PPAR gamma activity is necessary for the optimal production of epidermal prostaglandins. These studies demonstrated that epithelial cells contain a functional PPAR gamma system, and this system is a target for UVB through the production of novel oxidatively modified endogenous phospholipids.
Animals↗
Surfactant protein A binds Mycoplasma pneumoniae with high affinity and attenuates its growth by recognition of disaturated phosphatidylglycerols.
Surfactant Protein A (SP-A) is an abundant, multifunctional lectin that resides within the bronchoalveolar compartment of the lung and plays an important role in the innate immunity of the organ. Mycoplasma pneumoniae is a human pathogen that resides in the same compartment as SP-A, and we examined the interaction between the two. Preparations of human and rat SP-A recognized the mycoplasma with high affinity in the presence of Ca(2+), exhibiting apparent K(')(d) values in the nanomolar range. Membranes prepared from the microbe also bound human and rat SP-A with similar characteristics and affinity to the intact cells. The ligand for SP-A was insensitive to proteolysis. Lipid extracts prepared from the mycoplasma, bound SP-A with high affinity when examined by ligand blot analysis. These lipid extracts were also potent competitive inhibitors (IC(50) = 0.2 nM) of human SP-A binding to mycoplasma membranes. The major lipid ligands for the protein identified by mass spectrometry are a group of disaturated phosphatidylglycerols. The addition of SP-A to cultures of M. pneumoniae markedly attenuated the growth of the organism assessed by colony formation, metabolic activity, and DNA replication. The bacteriostatic effects of SP-A were reversed by dipalmitoylphosphatidylglycerol. These findings demonstrate that human SP-A can play a direct role in antibody-independent immunity to M. pneumoniae by interacting with lipid ligands expressed on the surface of the organism and implicate SP-A in the immediate host response to the bacteria.
Animals↗
Targeted over-expression of mPGES-1 and elevated PGE2 production is not sufficient for lung tumorigenesis in mice.
There is a significant body of evidence suggesting that enzymes involved in arachidonic acid metabolism and their eicosanoid products play a role in various cancers, having both pro- and antitumorigenic effects. The goal of this study was to further define the role microsomal prostaglandin E synthases (mPGES-1) play in lung tumorigenesis. Transgenic mice were created with targeted over-expression of human mPGES-1 in the alveolar and airway epithelial cells using an SP-C promoter driven construct. Transgene positive (mPGES-1+) mice were shown to significantly over-express functional mPGES-1 in the lung and more specifically in alveolar type II cells. To study the effects of mPGES-1 over-expression in lung tumor formation, mice were exposed to a complete carcinogen protocol with a single injection of urethane or an initiation/promotion model with a single injection of 3-methylcholanthrene (MCA) followed by multiple injections of butylated hydroxytoluene (BHT). mPGES-1+ mice did not show a significant difference in tumor multiplicity or tumor size at 10, 16, 19 or 30 weeks after urethane injection compared with mPGES-1- mice. No significant difference was seen in tumor incidence, multiplicity or size at 19 weeks after treatment with MCA/BHT. Western blots verified that mPGES-1 expression was increased in tumors versus uninvolved tissue of both mPGES-1+ and mPGES-1- mice with overall expression being significantly higher in mPGES-1+ mice. Cyclooxygenase-2 levels were elevated in tumors in both groups. From these studies we conclude that over-expression of mPGES-1 and highly elevated PGE2 production are not sufficient to induce lung tumors.
Animals↗
Mass spectrometric quantitation of deoxyguanosine and leukotriene A4-deoxyguanosine adducts of DNA.
An assay was developed using electrospray ionization negative ion tandem mass spectrometry (MS) to identify and quantitate the major product in the reaction of leukotriene A(4) (LTA(4)) with deoxyguanosine (dGuo). A second quantitative assay was established using the same separation and detection techniques to determine the amount of dGuo isolated from enzymatically processed DNA. The amount of LTA(4)-dGuo adduct could then be analytically determined in DNA samples and normalized to the amount of dGuo that had been simultaneously derived from the DNA sample. Stable isotope-labeled internal standards used for these quantitative assays were readily synthesized from isotopically labeled [(15)N(5)(13)C(10)]deoxyguanosine triphosphate and analyzed for isotopic purity using MS. A comparison of fragment ions formed from stable isotope analogs of dGuo revealed the loss of deoxyribose and secondarily the loss of a series of stable neutral small molecules in a fashion similar to patterns described previously for the collisional fragmentation of protonated guanine determined by positive ion fast atom bombardment/MS/MS. The combined quantitative assays were used for the determination of the amount of endogenously formed LTA(4)-dGuo adducts observed in DNA when isolated human neutrophils that had been incubated with arachidonic acid were stimulated with calcium ionophore to initiate leukotriene biosynthesis.
Arachidonic Acid↗
Fatty acid binding proteins stabilize leukotriene A4: competition with arachidonic acid but not other lipoxygenase products.
Leukotriene A(4) (LTA(4)) is a chemically reactive conjugated triene epoxide product derived from 5-lipoxygenase oxygenation of arachidonic acid. At physiological pH, this reactive compound has a half-life of less than 3 s at 37 degrees C and approximately 40 s at 4 degrees C. Regardless of this aqueous instability, LTA(4) is an intermediate in the formation of biologically active leukotrienes, which can be formed through either intracellular or transcellular biosynthesis. Previously, epithelial fatty acid binding protein (E-FABP) present in RBL-1 cells was shown to increase the half-life of LTA(4) to approximately 20 min at 4 degrees C. Five FABPs (adipocyte FABP, intestinal FABP, E-FABP, heart/muscle FABP, and liver FABP) have now been examined and also found to increase the half-life of LTA(4) at 4 degrees C to approximately 20 min with protein present. Stabilization of LTA(4) was examined when arachidonic acid was present to compete with LTA(4) for the binding site on E-FABP. Arachidonate has an apparent higher affinity for E-FABP than LTA(4) and was able to completely block stabilization of the latter. When E-FABP is not saturated with arachidonate, FABP can still stabilize LTA(4). Several lipoxygenase products, including 5-hydroxyeicosatetraenoic acid, 5,6-dihydroxyeicosatetraenoic acid, and leukotriene B(4), were found to have no effect on the stability of LTA(4) induced by E-FABP even when present at concentrations 3-fold higher than LTA(4).
Animals↗
Recombinant human activated protein C reduces human endotoxin-induced pulmonary inflammation via inhibition of neutrophil chemotaxis.
Recombinant human activated protein C (rhAPC) is a natural anticoagulant with potentially important anti-inflammatory properties. In humans with severe sepsis, rhAPC treatment reduces mortality, but mechanisms responsible have not been well characterized. Accumulation of activated neutrophils in the lungs and other organs during severe infection contributes to sepsis-induced organ dysfunction, including acute inflammatory lung injury. Because neutrophils express an APC receptor, we hypothesized that immunomodulatory effects of rhAPC occur, in part, via modulation of neutrophil responses. To examine this issue, we performed a double-blinded, placebo-controlled study of rhAPC in a human model of endotoxin-induced pulmonary inflammation. Administration of rhAPC significantly reduced leukocyte accumulation to the airspaces, independent of pulmonary cytokine or chemokine release. Neutrophils recovered from bronchoalveolar lavage fluid of volunteers receiving rhAPC demonstrated decreased chemotaxis ex vivo. Decreased neutrophil chemotaxis following exposure to rhAPC was confirmed in vitro. No differences were detected in gene expression, kinase activation, cytokine release, cell survival, or apoptosis of neutrophils recovered in the presence or absence of rhAPC. These studies demonstrate that rhAPC reduces both endotoxin-induced accumulation of leukocytes in the airspaces and neutrophil chemotaxis. These rhAPC-induced effects on neutrophil function may represent a mechanism by which rhAPC improves survival in patients with sepsis.
Adolescent↗
Ozone exposure in vivo and formation of biologically active oxysterols in the lung.
Ozone toxicity in the lung is thought to be mediated by products derived from the reaction of ozone with components of the lung epithelial lining fluid. Cholesterol is an abundant component of this epithelial lining fluid, and it is susceptible to ozonolysis, yielding several stable products including 3beta-hydroxy-5-oxo-5,6-secocholestan-6-al and 5beta,6beta-epoxycholesterol. Both 5beta,6beta-epoxycholesterol and its metabolite, cholestan-6-oxo-3,5-diol, have been shown to cause cytotoxicity in vitro, suggesting that they may be potential mediators of ozone toxicity in vivo. An ozone-sensitive mouse strain, C57BL/6J, was exposed to varying concentrations of ozone (0.5-3.0 ppm), and subsequently the levels of these cholesterol ozonolysis products were quantitated by electrospray ionization mass spectrometry in bronchoalveolar lavage fluid, lavaged cells, and lung homogenate. An ozone dose-dependent formation of these biologically active oxysterols was observed in vivo, supporting a role for these compounds in ozone toxicity. Since the 5beta,6beta-epoxycholesterol metabolite, cholestan-6-oxo-3,5-diol, was isobaric with other cholesterol ozonolysis products, 3beta-hydroxy-5-oxo-5,6-secocholestan-6-al and its aldol condensation product, 3beta-hydroxy-5beta-hydroxy-B-norcholestan-6beta-carboxaldehyde, detailed mass spectral analysis using electron impact ionization was utilized to differentiate these isobaric cholesterol ozonolysis products. The specific detection of cholestan-6-oxo-3,5-diol in lung homogenate after ozone exposure established formation of 5beta,6beta-epoxycholesterol within the lung after exposure to 0.5 ppm ozone.
Animals↗
Phosphatidylcholine and N-methylated phospholipids are nonessential in Saccharomyces cerevisiae.
Phosphatidylcholine (PtdCho) is the most abundant phospholipid in numerous eukaryotes and is generally thought to be essential for membrane structure and cellular function. We designed a specific test of this idea by using genetic and biochemical manipulation of yeast. Yeast mutants (pem1 pem2Delta) lacking the phosphatidylethanolamine (PtdEtn) methyltransferase enzymes require choline for growth and cannot make N-methylated phospholipids. When these strains are grown on a glucose carbon source supplemented with 20 mm propanolamine (Prn), the PtdCho level declines precipitously to the limits of detection (<0.6%), and the hexagonal phase-forming, primary amine-containing lipids, PtdEtn and PtdPrn, constitute approximately 60% of the total phospholipid content of the cell. When the lipids were analyzed by mass spectrometry, there was no compensatory shift in unsaturation of the PtdEtn and PtdPrn toward more bilayer-forming species. Thus the majority of the cellular amino phospholipids remained hexagonal phase-forming. The pem1 pem2Delta cells will also grow without choline, in the presence of Prn, on nonfermentable carbon sources (requiring functional mitochondria) and accumulate nearly 70% of their phospholipid as hexagonal phase-forming types. These data provide compelling evidence that the functions of PtdCho and N-methylated lipids in membranes are nonessential in Saccharomyces cerevisiae.
Choline↗
Lipid rafts regulate lipopolysaccharide-induced activation of Cdc42 and inflammatory functions of the human neutrophil.
Lipid rafts are cholesterol-rich membrane microdomains that are thought to act as coordinated signaling platforms by regulating dynamic, agonist-induced translocation of signaling proteins. They have been described to play a role in multiple prototypical cascades, among them the lipopolysaccharide pathway, and to host multiple signaling proteins, including kinases and low molecular weight G-proteins. Here we report lipopolysaccharide-induced activation of the Rho family GTPase Cdc42, and we show its activation in the human neutrophil to be mediated by a p38 mitogen-activated protein kinase-dependent mechanism. Subcellular fractionation reveals that lipopolysaccharide induces translocation of Cdc42 to lipid rafts, where it and p38 are both found to be activated. By contrast, lipopolysaccharide causes translocation of Rac from the polymorphonuclear leukocyte (PMN) rafts and does not induce its activation. With the use of methyl-beta-cyclodextrin, a cholesterol-depleting agent that reversibly disrupts rafts, we confirm an important regulatory role for rafts in the activation state of p38 and Cdc42 and in the Rho GTPase-dependent functions superoxide anion production and actin polymerization. Methyl-beta-cyclodextrin induces activation of p38 and Cdc42, but not Rac, in the nonstimulated PMN, yet inhibits subsequent lipopolysaccharide-induced activation of p38 and Cdc42. In parallel, methyl-beta-cyclodextrin primes the human PMN for subsequent superoxide release triggered by the formylated bacterial tripeptide formyl-Met-Leu-Phe, and induces actin polymerization in a subcellular distribution distinct from that induced by lipopolysaccharide. In sum, these findings provide evidence for an important regulatory role of cholesterol in both transmission of the lipopolysaccharide signal and the inflammatory phenotype of the human neutrophil.
Actins↗
Formation of biologically active oxysterols during ozonolysis of cholesterol present in lung surfactant.
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 3beta-hydroxy-5-oxo-5,6-secocholestan-6-al, 5-hydroperoxy-B-homo-6-oxa-cholestan-3beta,7a-diol, and 5beta,6beta-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 5beta,6beta-epoxycholesterol, which is only a minor product during ozonolysis of cholesterol in solution. A dose-dependent formation of 5beta,6beta-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-3beta,5alpha,6beta-triol, and more abundant levels of an unexpected metabolite, cholestan-6-oxo-3beta,5alpha-diol. Both 5beta,6beta-epoxycholesterol and cholestan-6-oxo-3beta,5alpha-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.
Animals↗