Oxidized Phospholipids Derived from Ozone-Treated Lung Surfactant

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Chem. Res. Toxicol. 2002, 15, 896-906

Articles Oxidized Phospholipids Derived from Ozone-Treated Lung Surfactant Extract Reduce Macrophage and Epithelial Cell Viability Charis Uhlson, Kathleen Harrison, Corrie B. Allen, Shama Ahmad, Carl W. White, and Robert C. Murphy* Division of Cell Biology, Department of Pediatrics, National Jewish Medical and Research Center, 1400 Jackson Street, Denver, Colorado 80206 Received December 28, 2001

Ozone is known to be a highly toxic gas present in the urban air which exerts its effect on pulmonary tissue through its facile chemical reactions with target molecules in the airway. One of the first barriers encountered by ozone is epithelial lining fluid which contains pulmonary surfactant rich in glycerophosphocholine lipids. The reaction of ozone with calf lung surfactant extract was found to result in the production of 1-palmitoyl-2-(9′-oxo-nonanoyl)-glycerophosphocholine (16:0a/9-al-GPCho) as an expected product of the ozonolysis of abundant unsaturated phospholipids containing unsaturated fatty acyl groups with a double bond at carbon-9. This oxidized phospholipid was identified as a biologically active product in that it reduced elicited macrophage viability by necrosis with an ED50 of 6 µM. Further studies of the biological activity of 16:0a/9-al-GPCho revealed that concentrations from 100 to 200 nM initiated apoptosis in pulmonary epithelial-like A549 cells as assessed by TUNEL staining, nuclear size, and caspase-3 activation with loss of viability indicated by reduction of mitochondrial dehydrogenase activity. The release of IL-8, a neutrophil chemokine, from A549 cells was also stimulated by 50-100 nM 16:0a/9-al-GPCho. Exposure of calf lung surfactant to low levels of ozone (62.5, 125, and 250 ppb) for various time periods from 2 to 48 h in a feedback-regulated ozone exposure chamber resulted in a dose- and time-dependent increase in the formation of 16:0a/9-al-GPCho as measured by a specific and sensitive LC/MS/MS assay. The quantity of this biologically active chain-shortened glycerophosphocholine lipid generated even at 125 ppb ozone for 2-4 h (50100 nM) was consistent with this product mediating the toxic effects of ozone on cells in contact with surfactant.

Introduction While ozone serves an important protective role in the terrestrial biosphere, low concentrations of ozone present in the ambient air of the urban environment are known to cause pulmonary toxicity (1). The mechanism by which ozone exerts its toxic effects in lung is of considerable interest and has been the focus of a number of fundamental studies. A concept that has emerged from these studies is the generation of biologically active mediator(s) formed from endogenous constituents present within the thin layer of epithelial lining fluid during chemical reaction with ozone (2, 3). This epithelial lining fluid contains a complex mixture of substances including abundant lipid-soluble components termed surfactant that play a critical role in maintaining proper inflation of the lung (4). Thus, this surfactant-containing layer serves as the first barrier through which ozone must penetrate before exerting its toxic effect. Because of the * Correspondence should be addressed to this author at the Division of Cell Biology, National Jewish Medical and Research Center, 1400 Jackson St., Room K929, Denver, CO 80206. Tel: (303)398-1849, Fax: (303)398-1694, E-mail: [email protected].

chemical reactivity of ozone, this initial barrier is the possible site for the formation of products of endogenous proteins and lipids, which might subsequently mediate the biological response(s) within the lung following exposure with ozone. The chemical reactivity of ozone has been extensively studied for over half a century. These studies include the facile reaction of ozone with lipids containing an isolated double bond (5, 6). A somewhat stable intermediate ozonide can be formed as an initial product which decomposes to aldehydes and hydroxy hydroperoxy products with ultimate cleavage of the carbon-carbon double bond. Ozone also can react with amino acid side chains present in proteins, the most reactive being histidine, tryptophan, methionine, and tyrosine (7). The reaction of ozone with phospholipids has been widely studied, in particular the reaction with 1-palmitoyl-2-oleoyl-glycerophosphocholine, a major phospholipid present in mammalian pulmonary surfactant containing an unsaturated fatty acid acyl substituent (8, 9). Ozone also rapidly reacts with ascorbic acid (10) and glutathione (10, 11), both important antioxidant constituents of surfactant (4).

10.1021/tx010183i CCC: $22.00 © 2002 American Chemical Society Published on Web 06/07/2002

Ozone-Treated Lung Surfactant

Pulmonary surfactant extracted from lung epithelial lining fluid is a complex mixture of organic components containing approximately 85-90% phospholipids and 10% proteins including surfactant proteins (SP-A, SP-B, SP-C, and SP-D) (12). The phospholipid compartment is typically greater than 90% glycerophosphocholine lipids, the most abundant being the saturated, dipalmitoyl glycerophosphocholine, and 1-myristoyl-2-palmitoyl glycerophosphocholine. However, abundant unsaturated phospholipids are present, including 14% 1-palmitoyl-2-oleoylglycerophosphocholine (16:0a/18:1-GPCho) and 18% 1-palmitoyl-2-palmitoleoyl-glycerophosphocholine (12), which would be an expected chemical target for reaction with ozone. There are ether-containing glycerophosphocholine lipids which constitute approximately 2% of the entire phospholipid pool. In addition to phospholipids, cholesterol constitutes approximately 8 mol % of the lipids in pulmonary surfactant. Cholesterol also contains a single carbon-carbon double bond which can be the reactive center for ozone and form several oxidized cholesterol species. It is now becoming widely recognized that intact oxidized phospholipids can exert biological activity and stimulate cellular events. A number of the oxidized phospholipids have been discovered as products formed in oxidized low density lipoprotein (LDL) which exert profound activity in systems relevant to atherosclerosis (13). Oxidized products include very complex phospholipids such as those containing isoprostanes (14) as well as chain-shortened phospholipids which result from cleavage of the fatty acyl carbon chain, typically at the sn-2 position where polyunsaturated fatty acyl moieties predominate within mammalian glycerophospholipids. Chain-shortened ether phospholipids have been identified as oxidized products which exert their biological action through the platelet activating factor receptor (15) and recently as agonists of nuclear regulatory factors including PPARγ (16). In addition to this, recent evidence has suggested that one of the expected products of the major monounsaturated glycerophosphocholine lipid present in lung surfactant, namely, 16:0a/18:1-GPCho, reacts with ozone to form 1-palmitoyl-2-(9′-oxo-nonanoyl)-glycerophosphocholine (16:0a/9-al-GPCho), which can have a multitude of effects on cells such as pulmonary epithelial cells including enhanced release of PGE2 (17), release of IL-8 (17), and activation of phospholipase A2 and other phospholipases (18). While studies have focused on formation of this product from purified glycerophosphocholine substrates, there have been no reports on generation of such biologically active products by exposure of ozone to pulmonary surfactant with potential biological activities in the nanomolar range.

Experimental Procedures Materials. Calf lung surfactant extract (CLSE) was obtained from ONY Inc. (Buffalo, NY) as a sterile, nonpyrogenic suspension used for intratracheal administration to human subjects. This chloroform/methanol extract of calf lung epithelial lining fluid contains phospholipids, neutral lipids, hydrophobic surfactant proteins B and C (SP-B and SP-C), but no preservative, suspended in 0.9% aqueous sodium chloride, pH 5-6. Each milliliter of the suspension contained 35 mg of total phospholipids (26 mg of phosphatidylcholine of which 16 mg was saturated glycerophosphocholine lipids) and 0.65 mg of protein including the 0.26 mg of SP-B. Radiolabeled 1-palmitoyl-2-[1′14C]oleoyl-glycerophosphocholine (56.0 µCi/mmol) was obtained from Amersham Pharmacia Biotech (Piscataway, NJ). Deuterium-

Chem. Res. Toxicol., Vol. 15, No. 7, 2002 897 labeled platelet activating factor (d3-PAF) was made as previously described (19). Lipase from Rhizopus arrhizus (Type XI) was obtained from Sigma Chemical Co. (St. Louis, MO). All reagents and solvents were commercially obtained. Ozonation of Calf Lung Surfactant Extract. Ozone was generated in a commercial apparatus (Supelco, Belefonte, PA) by passing a stream of 2 mL/min oxygen through a high-voltage Tesla coil fitted with a glass sleeve surrounding the high-voltage electrode. A stock solution of CLSE (200 µL) was diluted to 0.5 mL with phosphated-buffered saline (PBS), and ozone was directed through this solution for 2 min at room temperature. Following exposure, the sample was kept at room temperature for 15 min and then diluted with 0.5 mL of H2O and rapidly stirred. Chloroform (1 mL) was added in the sample vortex followed by the addition of 1 mL of methanol. After mixing, the sample was centrifuged for 3 min, and the bottom layer (chloroform) was taken out and subsequently analyzed. HPLC Analysis. Normal phase HPLC was carried out using a Ultramex 5 silica (250 × 4.6 mm) normal phase column (Phenomenex, Rancho Cordova, CA) starting from 50% solvent A (30:40, hexane:2-propanol) and 50% solvent B (30:40:7, hexane:2-propanol:water containing 1 mM ammonium acetate). The column was maintained isocratic for 3 min at 1 mL/min followed by a linear gradient for 10 min to 100% B. It was maintained at 100% B for 40 min. The eluate of the HPLC column was partially directed (20%) into a tandem quadrupole mass spectrometry, and 80% of the sample collected at one fraction per minute. Cell Viability. Elicited mouse peritoneal macrophages or human blood peripheral monocytes were isolated as previously described (20, 21). Cells were suspended at 1 × 106/mL in Hank’s balanced salt solution, pH 7.4. Cells were then brought to 37 °C in a water bath for viability measurements. HPLC fractions (1-2%) were taken from each collected fraction and taken to dryness in a flowing stream of nitrogen. The cell suspension (100 µL) was added to the dried sample and the suspension gently vortexed followed by incubation for 1 h at 37 °C. Cell viability was assessed by exclusion of trypan blue as previously described following the supplier’s protocol (Sigma Chemical Co., St. Louis, MO). Cell viability was determined following 5 min exposure to 0.4% trypan blue prepared in Hank’s balanced salt solution. For human lung epithelial-like A549 cells, cell survival was measured with the MTT assay (22) using the water-soluble tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Sigma). Cells were plated in 96-well half-area tissue culture plates (Costar 3696), and the medium was replaced by 100 µL of a 1:1 serum- and phenol red-free DMEMF-12 medium mixture. Fifty microliters of MTT (4 mg/mL) in the serum- and phenol red-free DMEM-F-12 medium mixture was added, and the plate was incubated for 4 h at 37 °C. The purple formazan crystals thus formed were dissolved in 50 µL of DMSO, and the optical density of the wells on the plate was read at 540 nm with a plate reader. Interleukin-8 (IL-8) release from A549 cells was quantitated using a commercial ELISA (R&D Systems, Minneapolis, MN). Apoptosis Assays. A549 cells were seeded onto 12 mm circular glass coverslips in 24 well plates at 50 000 cells/well. After 24 h, cells were treated with lipids dissolved in a total volume of 1-3 µL of ethanol and suspended in 100 µL of media or media containing 10 mM hydrogen peroxide for 4-6 h in F12K media in the absence of serum. Exposure of 3′-OH termini of cellular DNA, upon DNA fragmentation, was used as one indicator of apoptosis (23). In situ detection of terminal deoxynucleotidyl transferase (TdT)mediated deoxyuridine triphosphate digoxigenin nick end labeling (TUNEL)-positive cells was used as one of three markers of apoptosis in this study. The ApopTag red kit for this assay was purchased from Intergen (Purchase, NY). The indirect detection protocol included in the kit used for fluorescence microscopy detection was used as follows. After treatment, cells were fixed in 1% paraformaldehyde for 10 min. Cells were rinsed, equili-

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brated in buffer, and incubated with TdT enzyme solution for 1 h at 37 °C. The reaction was ended with a 10 min incubation in stop buffer followed by a 30 min room-temperature incubation with rhodamine-conjugated antidigoxigen. After a final rinse in PBS, coverslips were mounted with Prolong antifade (Molecular Probes in Oregon) containing 2 µg/mL Hoechst dye 33258. Slides were examined using an Olympus Vanox-T fluorescent microscope attached to a digital camera (Cooke, Auburn Hill, MI). Images were recorded with Slide Book software (Intelligent Imaging Innovations, Denver, CO). Because shrinkage of nuclei is one indicator of apoptotic cell death, nuclear area was measured using computer-assisted image analysis (23). For each condition, three fields were randomly chosen, and all nuclei on each field were evaluated, allowing area estimations from at least 20 nuclei. Each nucleus was manually traced, and the area was calculated as pixel counts using the software. Caspase-3 activation was assessed in A549 cells using CaspACE FITC-VAD-FMK in situ marker (Promega, Madison, WI) according to the manufacturer’s instructions. Briefly, 10 µM (final concentration of CaspACE FITC-VAD-FMK in situ marker) was added to 1 × 106 cells collected in 1 mL of PBS after treatments. After incubating for 20 min in dark at 37 °C, cells were washed with PBS by centrifugation for 5 min at 300g. Cells were suspended in 1 mL of PBS and observed by flow cytometry as described by (24). Hydrogen peroxide induced apoptosis was used as a positive control, and untreated cell served as the negative control. Propidium iodide staining was used to identify any potential necrotic cells if present (25). Cells bearing phosphatidylserine in the plasma membrane outer leaflet were identified as those binding Alexa Fluor 488labeled annexin V using a Vybrant Apoptosis Detection Kit (Molecular Probes, Eugene, OR). The binding of Alexa Fluorlabeled annexin V to phosphatidylserine on the surface of apoptotic cells closely correlates with the appearance of nuclear and cytoplasmic condensation by light microscopy (26) and the appearance of hypodiploid DNA (27). Briefly, cells were pelleted, resuspended in annexin binding buffer, and incubated with Alexa Fluor annexin V and the fluorescent DNA-binding dye SYTOX Green for 15 min at room temperature in the dark. The samples were then put on ice, and the sample volume was brought to 0.5 mL. Analysis was done on a FACScalibur flow cytometer (Becton Dickinson, San Jose, CA) using the FITC signal detector FL1. Annexin-positive cells were determined as described by the manufacturer. The population separates into three groups: live cells with only a very low level of fluoresence, apoptotic cells with a moderate green fluorescence, and necrotic cells with a high-intensity green fluorescence. A549 cells treated with 2 mM hydrogen peroxide as well as those treated with 1 mM staurosporine were used as positive controls for apoptosis. Annexin V binding to PS on the cell surface previously has been shown to correlate closely with the appearance of cytoplasmic and nuclear condensation by light microscopy and the appearance of hypodiploid DNA (27). DNA degradation was determined by the appearance of a hypodiploid fraction in permeabilized propidium iodide-stained cells. Cells were pelleted, washed once, and resuspended in phosphate-buffered saline (pH 7.4) which was then brought to 80% ethanol. The cells were then held at -20 °C overnight. Following storage, cells were pelleted to remove ethanol at 4 °C and treated with 200 ng/mL RNase A for 30 min at 37 °C and stained with 50 mg/mL propidium iodide. The cells were then incubated at 4 °C for at least 1 h before analysis in the flow cytometer. Analyzing for PI-stained DNA in the red channel, degraded (hypodiploid) DNA could be separated from intact (diploid) DNA due to the appearance of these respective peaks on the left and right sides of a histogram. Mass Spectrometry. Electrospray ionization mass spectrometry was carried out in a tandem quadrupole instrument (PE-Sciex API-III+, Thornhill, Ontario, Canada) typically under positive ion conditions to detect the elution of glycerophosphocholine lipids (28). Tandem mass spectrometry of the detected

Uhlson et al. [M+H]+ ions was carried out with a collision energy of 20 eV (laboratory frame of reference) with specific detection of the product ion m/z 184 specific for glycerophosphocholine lipids. Negative ion electrospray ionization was also performed for structural characterization by tandem mass spectrometry as previously described (29). In these studies, the [M-15]- anion was collisionally activated to generate the characteristic product ions for structural elucidation. The formation of the methoxime derivative of 16:0/9-al-GPCho was carried out with 1 mL of methoxylamine‚HCl (1 µM)/sodium acetate in methanol followed by incubation at room temperature for 30 min. The reacted product was passed through a C18 reversed solid-phase extraction cartridge which had been previously activated with 1 mL of methanol and washed with 1 mL of water. Sample was eluted with 1 mL of methanol, taken to dryness, and resuspended in solvent system A for normal phase HPLC purification. The material was analyzed under positive ion LC/MS and LC/MS/ MS conditions. Quantitative analysis of 16:0/9-al-GPCho was carried out using multiple reaction monitoring conditions to detect the quantity of 16:0/9-al-GPCho ([M+H]+ at m/z 650) relative to the internal standard d3-PAF ([M+H]+ at m/z 527). Collisional activation of each of these phospholipid species resulted in the formation of the characteristic glycerophosphocholine ion at m/z 184 while the d3-PAF yielded the glycerophosphocholine ion at m/z 185 (30). The multiple reaction monitoring LC/MS/MS assay (MRM) was established from the ratio of ion abundance m/z 650f184 from known quantities of synthetic 16:0a/9-al-GPCho mixed with a fixed quantity (38 pmol) of d3-PAF measured by the ion abundance at m/z 527f184. The quantity of 16:0/9-aGPCho employed to generate the standard curve was calculated from the known specific activity of the synthetic 16:0a/9-alGPCho derived from radiolabeled 1-palmitoyl-2-oleoyl-GPC ozonolysis. The standard curve was found to be linear from 25 to 350 pmol injected onto the column to the LC/MS/MS instrument with a correlation coefficient (r2) of 0.995. Synthesis of 16:0a/9-al-GPCho. Synthetic 1-palmitoyl-2(9′-oxo-nonanoyl)-GPCho (16:0a/9-al-GPCho) was made following ozonolysis of [14C]16:0a/18:1-GPCho essentially as previously described (9). The radiolabeled diacyl phospholipid was diluted with unlabeled 16:0a/18:1-GPCho (Avanti Polar Lipids, Alabaster, AL) to a final specific activity of 0.395 µCi/µmol prior to ozonolysis. Final purification of the 16:0a/9-al-GPCho product was carried out by normal phase HPLC described above, and the precise quantity of product obtained was calculated from the radioactivity content determined by scintillation counting techniques using the external standard technique. Ozone Exposure Chamber. Exposure of calf lung surfactant to ozone at precise levels was carried out in a computercontrolled in vitro exposure chamber. Briefly, the exposure system consisted of four identical exposure systems maintained in a single temperature-controlled (37 °C) environmental chamber (Forma Scientific, Marietta, OH) controlled by a single desktop computer. One of these systems is always used for an air control while the other three can be used for exposure of materials or cells to a variety of ozone concentrations. The material to be exposed to each concentration of ozone is placed within a specially designed (31) glass exposure chamber of 3.66 L volume. Mass flow controllers (Sierra Instruments, Monterey, CA) are used to mix filtered dry air with compressed carbon dioxide to achieve a 5% CO2 concentration. Ozone is produced by passing medical-grade compressed oxygen through a coldspark corona discharge ozone generator (model OZ2SS-SS, Ozotech, Yreka, CA). The ozone/oxygen mixture is then mixed with the 5% CO2/air mixture using another mass flow controller. Because the maximum flow of ozone/oxygen into the system is limited to 75 mL/min by the capacity of the oxygen mass flowmeters, and the air/CO2 flow is at least 9 L/min, the greatest contribution of the additional oxygen to the oxygen content of the mixed gases is 0.8%. All plumbing within this system was constructed either of Teflon or of stainless steel to minimize reactivity with the ozone.

Ozone-Treated Lung Surfactant The air/CO2/ozone mixture is directed into the warmed environmental chamber where it is warmed and humidified by passage over a glass water bath containing 1 L of water thermostatically maintained at 48 °C. The warm, humidified gas mixture then is passed to the glass exposure chamber. Gas flow through the chambers for these experiments is maintained at 9 L/min. Effluent gas from the chambers is directed to the facility’s waste air system. Sample lines from each chamber are connected to a manifold system which sequentially selects the line to be sampled through the use of electromagnetic valves under computer control. The gas from the selected line flows to an ultraviolet ozone analyzer (M400A, Advanced Pollution Instrumentation, Inc., San Diego, CA). Ozone values determined by this instrument are converted to an analogue voltage internally, and this voltage was measured by a data acquisition card (PCI-MIO-16E-4, National Instruments, Austin, TX) installed on a Macintosh G3 desktop computer. The ozone data are used by a program written by one of us (C.B.A.) using the LabView software (National Instruments) to provide feedback regulation to the oxygen flowmeters in order to maintain the desired ozone levels within each chamber. The feedback signal is generated through the use of an analogue output card (PCI6703, National Instruments) also installed in the Macintosh G3 that is connected to the flowmeter system. The ozone concentrations achieved using this system are typically maintained within 1% of the desired concentration. The computer program also measures the ozone levels in the room surrounding the exposure apparatus in order to ensure operator safety. In addition, the concentrations of ozone measured in each chamber, the rate of oxygen flow, and relevant time indices are recorded in a digital file for future examination. CLSE (2.3 µmol) phospholipid was diluted to 0.5 mL with PBS and placed in a glass vessel (27.6 cm2 area) with a thickness of 0.02 cm. The air above the surface area was precisely controlled at 0, 62.5, 125, or 250 ppb ozone in air. After exposure, the CLSE was diluted with 1 mL of methanol, 0.5 mL of water; then 1 mL of chloroform was added and the two-phase system vortexed briefly. The lower layer (1 mL) was taken for analysis. An aliquot (0.2%) was diluted into 200 µL of hexane/2-propanol (30: 40) to which 30 pmol of d3-PAF was added as an internal standard. A portion of the sample was analyzed by LC/MS/MS as described above.

Results Analysis of calf lung surfactant extract (CLSE) by LC/ MS under normal phase separation revealed a complex mixture of lipids eluting from the HPLC column (Figure 1A). From the relative abundance of each of the components represented by the total ionization chromatogram (Figure 1A), the first major component corresponded to diradyl glycerophospholipids (retention time 16-21 min) (Figure 1B). Qualitative analysis of the major components present in the mixture could be readily assessed from the molecular ion species [M+H]+ observed during elution of components with retention times 16-21 min. Lysoglycerophospholipids, 16:0a/lyso-GPCho and 18:1a/ lyso-GPCho, were the components eluting with retention times between 26 and 32 min (Figure 1C). Based upon the observed positive ions from the diradyl phosphocholine lipids as well as the collisional activation of the corresponding [M-15]- ions (data not shown), an assessment of the identity of diradyl glycerophospholipids present in CLSE could be obtained (Table 1) and was found to be similar to other mammalian surfactant (32). The molecular species containing palmitate at the sn-1 glycerol position, and an unsaturated fatty acyl group with the first double bond at carbon-9, corresponded to 37% of the total. Exposure of CLSE to 100% oxygen (control experiments) did not alter the phospholipid

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Figure 1. (A) Normal phase HPLC separation and LC/MS total ionization plot of calf lung surfactant following exposure to oxygen for 2 min. (B) Summed electrospray mass spectra (positive ions) for components eluting in area I between 16 and 18 min. (C) Summed electrospray mass spectra (positive ions) for components eluting in area II between 30 and 32 min. Table 1. Molecular Species of Glycerophosphocholine Lipids Present in Calf Lung Surfactant Extract As Determined by LC/MS and LC/MS/MSa total radyl carbon atoms: double bonds 30:1 30:0 32:0 ether 32:1 32:0 34:1 ether 34:2 34:1 36:2 36:1

molecular speciesb 14:0a/16:0 14:0a/16:0 16:0e/16:0 16:0a/16:1 16:0a/16:0 16:0e/18:1 16:0a/18:2 (16:0a/18:1)c 16:0a/18:1 18:1a/18:1 (16:0a/20:2)c 18:0a/18:1

[M+H]+ 704.5 706.5 720.5 732.5 734.5 746.5 758.5 760.5 786.5 788.5

[M+Na]+ 728.5 754.5 756.5

782.5 808.5

relative abundance 2.1 20.5 4.0 16.6 31.7 1.0 5.9 14.5 2.8 1.0

a

Abundances are expressed as percent of total ionization of all major molecular species. Abundances are not corrected for massdependent effects which are assumed to be minimal for the difference in mass for the major molecular species. b Molecular species were determined from MS/MS studies of the corresponding [M-15]- anion. c Minor isobaric component.

molecular species as analyzed by LC/MS, but exposure of CLSE to high concentrations of ozone for a short period of time led to a change in the composition of CLSE as revealed by normal phase LC/MS analysis (Figure 2A). New components of intermediate polarity eluted between the diradyl and lysophospholipids between fractions 20 and 25 as could be readily observed from the [M+H]+ ions present (Figure 2B). The elution of specific compounds could be determined within this region by as-

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Uhlson et al. Table 2. Viability of Monocytes after Exposure to Normal Phase HPLC Fractions (1-2% of the Total HPLC Fraction) of Calf Lung Surfactant Treated with Ozone or Oxygen (Control) HPLC fraction

ozonea

controla

21 22 23 24 25 26 27

85 ( 1 67 ( 8.3 71 ( 5.7 64 ( 6.7 46 ( 7.4 53 ( 9 90 ( 6

98 ( 2 95 ( 4 91 ( 3 93 ( 2 91 ( 3 95 ( 2 95 ( 2

a Average ( SEM (n ) 3-5) unless otherwise indicated using trypan blue exclusion to test for viability.

Figure 2. (A) Normal phase HPLC separation and LC/MS base-peak ionization plot of calf lung surfactant following exposure to ozone for 2 min. The total ionization axis has been expanded 4-fold to reveal new components in area III. (B) Summed electrospray mass spectra (positive ions) for components eluting in area III between 21 and 25 min.

Figure 4. Electrospray tandem mass spectrometric (negative ion) analysis of the major oxidized glycerophosphocholine lipid eluting in the fraction. The abundant negative ion species [M-15]- observed at m/z 634 was collisionally activated to yield the observed product ions consistent with 1-palmitoyl-2-(9′-oxononanoyl)-glycerophosphocholine (16:0a/9-al-GPCho).

Figure 3. Representative experiment of the bioassay of HPLC fractions (1 min per fraction) of calf lung surfactant exposed to ozone or oxygen. (A) Activity of components present in normal phase HPLC fractions from CLSE exposed to ozone as assessed by a reduction in monocyte viability using trypan blue exclusion. (B) Activity of components present in normal phase HPLC from CLSE exposed to oxygen using trypan blue exclusion with peritoneal macrophages.

sessing the time-dependent emergence of specific ions such as m/z 636, 650, and 672 (Figure 2B). Of particular interest was the elution of the components generating m/z 636 and 650 that maximized with a retention time of approximately 24 min on this normal phase separation. The ozone-treated lung surfactant before normal phase HPLC was found to cause a dose-related decrease in monocyte viability when added to cells in a trypan blue

exclusion assay. As little as 10 µL of ozone-treated CLSE caused 75% of the monocytes to die while the same quantity of CLSE exposed to 100% oxygen for the same period had no significant effect on monocyte viability. Testing individual components eluting from the normal phase HPLC separation of both oxygen- and ozonetreated CLSE (Figure 3) for potential exposure effects on either monocyte or macrophage cell viability following short-term exposure (1 h) revealed little effect of CLSE exposure to 100% oxygen on cell viability (Figure 3B). However, there were components present in the ozonetreated CLSE which significantly reduced cell viability of both cell types. One component eluted early in the HPLC run (pooled fraction 4), and a more polar fraction (Figure 3A, fractions 23-26) also contained active compounds. The absolute retention time of the active component in the polar fraction eluting from the normal phase HPLC varied to some extent as expected for normal phase separation involving mobile phases containing water. However, in five separate experiments (Table 2), the most active fractions consistently eluted between fractions 25 and 26 with no activity present in control CLSE exposed to oxygen separated under the same


Figure 5. Representative experiment of the bioassay of HPLC fractions of calf lung surfactant treated with ozone and separated by normal phase HPLC using trypan blue exclusion in peritoneal macrophages. Solid bars correspond to calf lung surfactant treated with ozone as well as lipase from Rhizopus arrhizus. Striped bars indicate calf lung surfactant extract treated with ozone, but not lipase.

conditions. Mass spectrometric analysis of the most active fraction (Figure 2B) revealed elution of an abundant phospholipid with a [M+H]+ ion at m/z 650. However, additional components were also present including those yielding [M+H]+ molecular ion species at at m/z 636 and 672. Detailed analysis of the elution of each of these components with the indicated [M+H]+ ions revealed the elution of only m/z 650 and 636 with a retention time consistent with that of fractions 25-26. Collisional


activation of the corresponding [M-15]- ion for glycerophosphocholine lipids at m/z 634 (fraction 25, Figure 4) revealed abundant product ions at m/z 171 and 255 indicative of an esterified hexadecanoic as well as a 9-carbon aldehyde esterified to the glycerophosphocholine backbone. These ions would be consistent with the structure 1-hexadecanoyl-2-(9′-oxo-nonanoyl)-GPCho, an expected ozonolysis product of 1-hexadecanoyl-2-oleoylGPCho (8, 9). A methoxime derivative was formed from an aliquot of fraction 25, and the corresponding mass spectrum revealed a [M+H]+ at m/z 679 expected for the presence of an aldehyde or ketone in the chain-shortened acyl group. Interestingly, the methoxymated fraction 25 lost all biological activity (data not shown). The presence of the [M+H]+ ion at m/z 636 was consistent with an ether lipid containing a 9-carbon aldehyde at sn-2 [1-O-hexadecyl-2-(9′-oxo-nonanoyl)GPCho] present in the active fraction. The MS/MS analysis of the corresponding [M-15]- anion at m/z 620 revealed a single carboxylate anion at m/z 171. To ascertain if this ether lipid was an active component in fraction 25, an aliquot of the crude phospholipid extract from the ozone-treated CLSE was digested with lipase from Rhizopus arrhizus, which specifically cleaves ester substituents at sn-1 of diacylglycerophosphocholine lipids (33). After normal phase LC/MS analysis, the abundance of m/z 636 was unaltered relative to that observed in the ozonated CLSE not treated with lipase. As expected, the

Figure 6. Effect of 16:0a/9-al-GPCho on viability and IL-8 release by human lung epithelial-like A549 cells. A549 cells were incubated with no additions (control), 16:0a/18:1-GPCho, or 16:0a/9-al-GPCho at the indicated concentrations for 6 h. (A) Cell viability was measured following the incubation using the MTT assay as described under Experimental Procedures, which measures mitochondrial dehydrogenase activity. The upper panel indicates the effect of these compounds on viability of A549 cells incubated with them in the absence of serum. The lower panel indicates effects of the phospholipids in serum-containing incubations. (B) IL-8 release from A549 cells incubated in the absence (upper panel) or presence (lower panel) of serum was quantitated by ELISA as indicated under Experimental Procedures. In all panels, an asterisk indicates a significant difference (p < 0.05) from ‘no additions’ conditions (‘control’) by analysis-of-variance followed by Tukey-Kramer test for multiple comparisons.

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m/z 650 product was reduced over 10-fold by lipase treatment. Comparing the biological activity of lipasetreated ozonated CLSE to that not exposed to lipase resulted in a major loss of activity (Figure 5), consistent with 1-palmitoyl-2-(9′-oxo-nonanoyl)-GPCho as the active component within fraction 25 causing cell death. This experiment was carried out twice. 16:0a/9-al-GPCho Cytotoxicity and IL-8 Release. Synthetic 1-hexadecanoyl-(9′-oxo-nonanoyl)-GPCho made following the ozonolysis of [14C]16:0a/18:1-GPCho of known specific activity was used to assess a precise dose response for the immediate response in cell viability (1 h). A calculated EC50 was 6-7 µM for the immediate cytotoxic effect of this chain-shortened phospholipid. Cytotoxicity due to 16:0a/9-al-GPCho was examined in pulmonary epithelial-like A549 cells exposed for 6 h with serum-containing and non-serum-containing buffer. A different effect of this oxidized phospholipid was observed when these longer exposure times were employed to assess cytotoxicity. As shown in Figure 6A (upper panel), 16:0a/9-al-GPCho caused a concentration-dependent loss of viability as assessed by the activity of mitochondrial dehydrogenases. A concentration range from 50 to 200 nM caused a significant decrease in cell viability as assessed by formation of formazan from MTT in that the highest concentration resulted in approximately 75% cell death (Figure 6A). In contrast, exposure of A549 cells to a normal phospholipid (synthetic 16:0a/18:1-GPCho) at concentrations 1000× higher caused no significant loss in cell viability relative to that detected in controls not exposed to either lipid. When 16:0a/9-al-GPCho was exposed to cells under serum-containing conditions at the same concentrations, a less marked, but nevertheless concentration-dependent cytotoxicity also was observed (Figure 6B). This cytotoxic oxidized phospholipid also caused significant release of IL-8, both under serum-containing and non-serum-containing incubation conditions of A549 cells. In the absence of serum, 16:0a/9-al-GPCho cause a low level of IL-8 release with little evidence of a dose-related increase when going from 50 to 200 nM (Figure 6C), but the level of IL-8 released was significantly lower when A549 cells were exposed to high concentrations of 16:0a/ 18:1-GPCho. In contrast, under serum-containing conditions, 16:0a/9-al-GPCho caused a greater increase in cellular IL-8 release from A549 cells than that observed under non-serum-containing conditions. When cells were exposed to 16:0a/9-al-GPCho concentrations of 100-200 nM, IL-8 release approached that initiated by TNF from 10 to 20 ng/mL. Again, the nonoxidized phospholipid did not cause IL-8 release even when present at 1000 times higher concentrations exposed to A549 cells. IL-8 release did not appear to be related to the cytotoxic effect (see below). Several lines of evidence indicated that the cell death caused by 16:0a/9-al-GPCho at low concentrations during more prolonged exposures occurred by an apoptotic mechanism. The exposure of 16:0a/9-al-GPCho caused a considerable decrease in nuclear size (date not shown). In these studies, nuclear diameter was assessed using a Hoechst dye and quantitated using fluorescence microscopy (23). Nuclear shrinkage was of comparable magnitude to that caused by hydrogen peroxide, an oxidant known to induce apoptosis (Figure 7). A second line of evidence involved studies of activation of caspase-3 initiated by 16:0a/9-al-GPCho. During a 6 h exposure

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Figure 7. Effect of 16:0a/9-al-GPCho on nuclear size in pulmonary epithelial-like A549 cells. Pictured are A549 cells exposed to 21% oxygen (control; panel A), 10 mM hydrogen peroxide (panel B), oxygen-exposed calf-lung surfactant (panel C), 16:0a/18:1-GPCho (60 nM)(panel E), or 16:0a/9-al-GPCho (60 nM; panels D and F) for 6 h. Nuclear staining is with Hoechst dye observed by fluorescence microscopy.

period, 16:0a/9-al-GPCho caused a concentration-dependent increase in activated caspase-3 as detected by a specific antibody and quantitated by flow cytometry (Figure 8) (24). At higher 16:0a/9-al-GPCho concentrations, the level of activated caspase-3 detected decreased concomitantly with a significant increase in the number (25-30%) of propidium iodide-positive (necrotic) cells (Figure 7) (25). Exposure of 16:0a/9-al-GPCho to A549 pulmonary epithelial cells for 6 h caused an intense TUNEL staining of the cells comparable in intensity to that caused by hydrogen peroxide (2 mM). Using another marker of apoptosis, that of annexin V binding to phosphatidylserine on the outer leaflet of the plasma membrane, it was found that exposure to 16:0a/ 9-al-GPCho (60 nM) also caused an increase in annexin V binding to A549 cells comparable to that caused by exposure to classical apoptotic stimuli such as hydrogen peroxide (2 mM) and staurosporine (1 µM). Exposure to equimolar phospholipid exposed to oxygen, but not to ozone, caused no increase in the percentage of annexin V-binding cells (Figure 9). Finally, there was a 16:0a/9al-GPCho concentration-dependent increase in hypodip-


Figure 8. Effect of 16:0a/9-al-GPCho on caspase-3 activation and propidium iodide-positive nuclear staining in human lung epithelial-like A549 cells. A549 cells were incubated for 6 h with the indicated concentrations of 16:0a/9-al-GPCho. Activated caspase-3 in cells (“percent apoptotic”) was measured using binding of a specific antibody detected by flow cytometry as described under Experimental Procedures. Nuclear staining by propidium iodide also was measured by flow cytometry as indicated under Experimental Procedures. Hydrogen peroxide (2 mM) was added to cells as a positive control for apoptotic cell death. An asterisk indicates a significant difference (p < 0.05) from ‘no additions’ conditions (control) by analysis-ofvariance followed by Tukey-Kramer test for multiple comparisons. In addition, both 240 and 480 nM 16:0a/9-al-GPCho yielded propidium iodide positive cell percentages which were significantly different from control conditions.


Figure 10. LC/MS/MS assay for 16:0a/9-al-GPCho present in calf lung surfactant extract exposed to the indicated concentrations of ozone. The internal standard (d3-PAF) was added to each sample, and the response for the transition m/z 650f184 from 16:0a/9-al-GPCho was normalized to the transition m/z 527f185 from the d3-PAF internal standard. Elution of the target oxidized phospholipid at 28 min is indicated in three separate LC/MS/ MS analyses at 0, 62.5, 125, and 250 ppb ozone concentration exposed to calf lung surfactant for 2 h.

Figure 11. Formation of 16:0a/9-al-GPCho (pmol) as measured by LC/MS/MS in calf lung surfactant (0.5 mL) exposed to ozone at various concentrations for 2(&), 4 (]), and 48 (O) h.

Figure 9. Effect of 16:0a/9-al-GPCho on annexin V binding to phosphatidylserine in the plasma membrane outer leaflet of pulmonary epithelial-like A549 cells. A549 cells were incubated with 16:0a/9-al-GPCho for 6 h. Column 1 indicates A549 cells not exposed to lipids, column 2 indicates those exposed to nonoxidized lipids (16:0a/18:1-GPCho), column 3 indicates those exposed to 16:0a/9-al-GPCho (60 nM), and columns 4 and 5 indicate apoptotic controls. N ) 3 determinations for each condition except for columns 4 and 5 where the mean of 2 determinations is shown for reference. The asterisk indicates a significant difference (p < 0.05) from control. Data are mean ( SEM. The error bars for columns 1 and 2 are too small to be visualized.

loid (degraded) DNA relative to diploid (intact) DNA. Specifically, of the total DNA measured in windows for diploid and hypodiploid DNA, 62% was diploid and 32% was hypodiploid at a 16:0a/9-al-GPCho concentration of 30 nM. At a 16:0a/9-al-GPCho concentration of 60 nM, 18% was diploid and 82% was hypodiploid. Gating for these experiments was based on apoptotic controls obtained by exposure to staurosporine (0.5-1.0 µM). Together these five lines of evidence, nuclear shrinkage, caspase-3 activation, positive TUNEL staining, annexin V binding to externalized phosphatidylserine, and appearance of hypodiploid DNA, favor a cell death mechanism of apoptosis at lower concentrations (50-200 nM) of 16:0a/9-al-GPCho. By contrast, at higher concentra-

tions, the cytotoxicity frequently involved necrosis. The initial screening of the biological activity of oxidized phospholipids derived from calf lung surfactant likely involved detection of components leading to this necrotic event. While the experiments described above involved the formation of biologically active glycerophospholipids derived from CLSE and the identification of a specific component initializing cell death, the question remained as to whether low concentrations of ozone, relevant to ozone toxicity (0.1-0.25 ppm ozone), in man could cause a generation of nanomolar quantities of 16:0a/9-al-GPCho observed as a biologically active product derived from CLSE. In separate experiments, CLSE (0.5 mL) was exposed as a thin aqueous layer (0.2 mm thickness) for times up to 48 h at various concentrations of ozone in a controlled exposure chamber. For example, the quantity of 16:0/9-al-GPCho formed within CLSE after exposure to ozone for 2 h (Figure 10) was determined using a quantitative LC/MS/MS mass spectrometric assay, based on the abundance of 16:0/9-al-GPCho (as detected by multiple reaction monitoring of the collision-induced decomposition of m/z 650f184) to that of the added internal standard d3-PAF (detected by the MRM ion m/z 524f187). Both target compounds closely eluted on normal phase HPLC. Exposure of CLSE even at 62.5 ppb for 2 h was found to yield 40 pmol of 16:0/9-al-GPCho (80 nM in the surfactant sample) (Figure 10). Furthermore, a graded increase in the quantity of chain-shortened phospholipid was observed when the concentration of ozone was increased from 62.5 to 250 ppb for 2 h (Figure 11). Also, there was a linear increase in 16:0a/9-al-GPCho when CLSE was exposed to each ozone concentration for 4 h (Figure 11). However, exposure of ozone to CLSE for 48

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h did not lead to a linear increase in 16:0a/9-al-GPCho, nor a 10-20-fold increase in this oxidized phospholipid when compared to quantities formed at each concentration of ozone for shorter time periods. These quantitative observations suggest further reactions of the initially formed 16:0a/9-al-GPCho during the extended exposure of CLSE to ozone.

Discussion Chemical reactivity is likely the fundamental property of ozone which accounts for toxicity of this important environmental pollutant. The mechanisms involved in toxicity either at the cellular or at the tissue level are expected to be quite complex, in part due to the large number of molecules which can be targets of covalent modification by ozone and the diversity of effects each product could exert. A primary focus of these studies concerned the reactivity of ozone with phospholipids present in lung surfactant extracts with the formation of products that could alter cell viability. The reaction of ozone even with a pure phospholipid containing a singly unsaturated fatty acyl group is complex with a number of products that can emanate from the initial cyclic molozonide formed by the covalent addition of ozone across a single carbon-carbon double bond. Possible products can have functional groups at the original carbon-carbon double bond of the fatty acyl substituent, including hydroxy hydroperoxy (6, 9), aldehyde (5, 6, 9), and carboxylic acids (34) after further oxidation of intermediates. Intact ozonides are also sufficiently stable to be isolated and even studied by LC/MC techniques (35). In addition, epithelial lining fluid which contains the lipophilic lung surfactant is also a complex mixture of both major and minor phospholipid components secreted by Type II alveolar cells. While an important physiological role played by this hydrophobic film is that of reducing the surface tension and protecting the alveolus from collapse during the process of normal respiration, lipids and proteins present in surfactant also play an important role in protecting the air spaces from toxic substances. Considering only the major glycerophosphocholine lipids, several unsaturated fatty acyl substituted molecular species are reasonably abundant in surfactant, and these phospholipids have long been considered to be the primary targets of ozone (7-9). Furthermore, ozone has limited aqueous solubility, and this feature, combined with a facile reactivity of ozone with components within lung lining fluid, has been used as argument to suggest that ozone can unlikely penetrate completely through the layer of surfactant and, thus, the formation of products which could mediate the toxicity of ozone are likely to originate in the surfactant layer itself (36). Not only are unsaturated phospholipids present in this layer, but also are ascorbate acid, glutathione, and proteins, which undoubtedly react with ozone, but have been assumed to yield few toxic products (37). Exposure of CLSE to ozone led to the formation of a complex mixture of products as assessed by liquid chromatography/mass spectrometry (LC/MS) techniques. The identification of 1-palmitoyl-2-(9′-oxo-nonanoyl)-GPCho (16:0a/9-al-GPCho) was not unexpected considering the occurrence of glycerophosphocholine lipids containing oleate (18:1[n-9]) and palmitoleate (16:1[n-9]) esterified at the sn-2 position. However, it was unexpected that this major product was also the compound which was found to cause cellular apoptosis at nanomolar concentrations.

Uhlson et al.

The mass spectrometric assay used to measure the formation of 16:0a/9-al-GPCho was modified in a straightforward manner into a quantitative assay using an internal standard (d3-PAF) to assess the abundance of the ions measured by multiple reaction monitoring specific for 16:0a/9-al-GPCho. This assay was both highly sensitive as well as specific for the formation of this unique oxidized glycerophosphocholine lipid. The LC/MS/ MS assay had sufficient sensitivity to detect the existence of endogenous 16:0a/9-al-GPCho in CLSE as well as formation of additional oxidized glycerophosphocholine lipid when as little as 62.5 ppb of ozone was exposed to a thin layer of CLSE suspended in aqueous buffer for a period of only 2 h. Formation of 16:0a/9-al-GPCho within the CLSE was found to be dependent not only on the concentration of ozone in the exposure chamber but also on the duration of exposure to ozone. When the CLSE was exposed to ozone at 125 ppb, a concentration set by the Environmental Protection Agency as the limit for 8 h exposure (38), there was sufficient 16:0a/9-al-GPCho present (140 nM) that would elicit a response at least with either isolated macrophages or A549 cells in culture. While the increase in 16:0a/9-al-GPCho observed when changing incubation time from 2 to 4 h resulted in an essential doubling of the observed product, much longer incubation times (48 h) did not correspondingly lead to a 10- and 20-fold increase in the quantity of 16:0a/9-alGPCho. There are likely several reasons for this observation. There is certainly a limited pool of precursor diacyl phospholipids containing an unsaturation at carbon-9, which could form this product. However, it is more likely that subsequent reactions are taking place at a slower time rate than the initial formation of the oxidized glycerophosphocholine lipid. The newly formed phospholipid aldehyde could react with other constituents present in the CLSE including lysine residues on SP-B, as well as undergo further oxidative degradation, possibly even into the 9-carboxylic acid phospholipid analogue. One particularly interesting product was the ether phospholipid 1-O-hexadecyl-2-(9′-oxo-nonanoyl)-GPCho (16:0e/9-al GPCho) which was present in substantially lower levels, but definitely present as an ozonolysis product found within CLSE. It was clear that this particular ether glycerophospholipid was not responsible for the increase in macrophage apoptosis in those purified fractions of phospholipids found to contain biologically active products. However, it is possible that this oxidized phospholipid or subsequent metabolite could exert other biological effects not measured in this study. A closely related chain-shortened ether phospholipid was reported to exert effects on the PPARγ receptor that can regulate protein expression (16). An oxidized ether phospholipid product also has been reported in oxidized LDL to activate the platelet activating factor receptor (39). Another closely related aldehydic phospholipid, 1-palmitoyl-2-(5′-oxo-pentanoyl)-GPC (16:0a/5-oxo-GPC), was recently found to increase binding of monocytes to endothelial cells with an ED50 of 7-8 µM (40), a dose response effect significantly higher than the potency of 16:0a/9al-GPCho reported here which induces cellular apoptosis. Previous investigations that studied the pharmacological action of 16:0a/9-al-GPCho revealed a host of activities including increased production of PGE2 (17), activation of phospholipase A2 (18), and possibly the release of platelet activating factor (41), all activities consistent with this oxidized phospholipid being proinflammatory.


The potency of 16:0a/9-al-GPCho for these activities may appear to be somewhat high (5-10 µM); however, it is clear from the quantitative studies of the production of this oxidized phosphocholine lipid in CLSE exposed to ozone at low levels (62.5-250 ppb), that nanomolar levels (50-150 nM) of this lipid product, relevant to a proapoptotic activity, can be achieved or even exceeded in the CLSE. While the situation present within epithelial lining fluid may be somewhat different due to the concentrations of phospholipids in the fluid, it is quite possible that a sufficient concentration of 16:0a/9-alGPCho could be formed to exert biological effects on neighboring cells that are bathed by the epithelial lining fluid. It remains to be established what dynamics, in terms of metabolism and clearance of this biologically active oxidized phospholipid, come to bear within the intact lung when this active oxidized phospholipid is formed, but the evidence suggests that this molecule could play an important role in mediating the effects of ozone in pulmonary toxicity. The neutrophil chemokine IL-8 is a principal chemokine expressed in primate distal airway epithelial cells after acute exposure to ozone (42), and 16:0a/9-al-GPCho at relatively low concentrations (50-200 nM) can stimulate release of IL-8 from A549 cells. Acute ozone exposure in adult humans causes the appearance of neutrophils and sloughed epithelial cells in the bronchoalveolar lavage fluid (BALF) within 6 h. The proportion of neutrophils in BALF has been correlated positively with the IL-8 measured in the peripheral airways (43). Airway inflammation may be exaggerated at lower concentrations of ozone (44) or be more severe in asthmatic subjects (45), and the possibility exists that 16:0a/9-al-GPCho may mediate proinflammatory responses of pulmonary tissue. Phospholipids, ascorbate, and larger molecular weight constituents can be reaction targets for ozone in the lung epithelial lining fluid (46). Direct exposure to low concentrations of ozone (0.1 ppm) to pulmonary epitheliallike A549 cells, which can produce and store surfactant phospholipids, can cause IL-8 release (47). Interestingly, one previous report indicated that 16:0/9-al-GPCho did not cause IL-8 release from pulmonary epithelial cells (17). Our studies employed significantly lower concentrations of 16:0/9-al-GPCho than those used in that study (50-200 nM rather than 10 µM). Differences in cell type and/or other experimental conditions, such as the presence or absence of serum, also could have accounted for differences in our findings from those in that report. Apoptosis also has been described within 6 h following ozone exposure (0.4 ppm) of airway cells in vivo in humans (48). Adducts of another aldehyde, 4-hydroxynonenal, were found in BALF, as were apoptotic bodies among the lung cells obtained by lavage. However, most of these cells may have been macrophages. Our study indicates that a different aldehyde, 16:0/9-al-GPCho, which we have shown to be a principal product of the reaction of ozone with lung surfactant, can cause both IL-8 release and apoptosis in human lung epithelial-like cells. Three different lines of evidence, including the detection of nuclear shrinkage, the presence of activated caspase-3, and the presence of free 3′-OH termini in cellular DNA, indicated that apoptosis was the principal cell death process activated by exposure to lower concentrations of 16:0/9-al-GPCho. These mechanisms may contribute to airway injury and inflammation in asthmatics and other susceptible patients.


Acknowledgment. This work was supported, in part, by a grant from the Environmental Protection Agency (R825702). We acknowledge the excellent technical assistance of Kelly Schneider and Anita C. Holland in performing the fluorescence microscopy studies in operation of the ozone exposure chamber. We are grateful to Dr. Bruce Holm for the gift of Infasurf, the calf lung surfactant preparation used in these studies.

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Oxidized Phospholipids Derived from Ozone-Treated Lung Surfactant

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