Effect of Fatty Acid Profiles on the Susceptibility of Cultured Rabbit Tracheal Epithelial Cells to Hyperoxic Injury Phyllis A. Dennery, Constance M. Kramer, and Stephen E. Alpert Department of Pediatrics, Divisions of Neonatology and Pediatric Pulmonology, Case Western Reserve University, Cleveland, Ohio

To investigate the role of cellular fatty acid content on the susceptibility of airway epithelial cells to hyperoxic injury, monolayer cultures of rabbit tracheal epithelial (TE) cells were grown to confluence in serum-free media with or without a commercial mixture of cholesterol esters and phospholipid-rich lipoproteins (Excyte III, Miles-Pentex, Kankakee, IL) in conjunction with arachidonic acid complexed to BSA. Monolayer cultures were then exposed to control (5 % CO 2/air) or hyperoxic atmospheres (95 % oxygen/5 % CO 2) for 2 h using an in vitro system in which cells were maintained at a gas-liquid interface analogous to in vivo conditions. Hyperoxic injury was assessed by cell viability (trypan blue exclusion) and by the generation of lipid peroxides measured as thiobarbituric acid (TBA) reactive substances. Changes in TE cell and cell culture effluent fatty acid content induced by exposure to control or hyperoxic atmospheres were analyzed by gas chromatography. TE cells grown in lipid-unsupplemented media had fatty acid profiles characteristic of essential fatty acid deficiency, whereas the fatty acid content of lipidsupplemented TE cells more closely resembled those of acutely recovered TE cells. Lipid-unsupplemented cells were more susceptible to hyperoxic injury as demonstrated by decreased viability and increased production of TBA-reactive substances compared to cells maintained in lipid-supplemented media. In both lipid-supplemented and unsupplemented cells, hyperoxic exposure was associated with a decreased relative cellular content of the monounsaturated and polyunsaturated fatty acids (PUFA) and an increased content of saturated fatty acids. The fatty acid content of cell culture effluents from lipid-supplemented and unsupplemented cells contained little to no PUFA and this did not change after exposures. Our observations suggest that differences in hyperoxia-induced cytotoxicity between the lipid-supplemented and unsupplemented TE cells may be due to varying susceptibilities of constituent membrane fatty acids to lipid peroxidation and provide further evidence for a protective effect of PUFA against hyperoxic injury.

Numerous in vivo and in vitro studies have established that lung injury from hyperoxic exposure is associated with lipid peroxidation, and have documented the importance of cellular antioxidant enzymes in protecting against oxidant injury (1-4). Recent studies, however, also indicate that the composition of lung membrane lipids may influence susceptibility to hyperoxia (5-8). Sosenko and associates demonstrated increased survival and decreased histologic evidence of lung injury in neonatal rats whose lungs had an increased relative content of polyunsaturated fatty acids (PUFA) achieved Key Words: cultured tracheal epithelium, hyperoxia, fatty acid profiles (Received in original form December 12, 1989 and in revised form March 8, 1990) Address correspondence to: Phyllis A. Dennery, M.D., Division of Neonatology, Stanford University Medical Center, Stanford, CA 94305. Abbreviations: arachidonic acid, AA; Hanks' balanced salt solution, HBSS; malondialdehyde, MDA; polyunsaturated fatty acids, PUPA; thiobarbituric acid, TBA; tracheal epithelial, TE. Am. J. Respir. Cell Mol. BioI. Vol. 3. pp. 137-144, 1990

through dietary manipulations (6, 7). Increased survival could not be attributed to changes in lung antioxidant enzyme activities or total surfactant content (6). In vitro studies with cultured porcine endothelial cells also suggest that alterations in cellular fatty acid composition can alter susceptibility to hyperoxic injury (8). Precisely how alterations in fatty acid content, and in particular PUFA content, affect hyperoxic injury is not certain. Proposed mechanisms include differences in the susceptibility of constituent membrane fatty acids to peroxidation and/or "expendable" pools of fatty acids in non-membrane-associated lipids, which serve as scavengers of free radicals and thereby protect membrane fatty acids from oxidation (9-11). We have previously reported that cultured human and rabbit tracheal epithelial (TE) cells grown in serum-free media have abnormal fatty acid profiles characteristic of essential fatty acid deficiency, and that supplementation of culture media with exogenous lipids can restore the fatty acid composition of the cultured cells to more closely resemble that of native epithelium (12). In the present study, we sought to

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determine whether these differences in TE cell fatty acid content affect the susceptibility of the cultured cells to hyperoxic injury. Monolayers of rabbit TE cells were exposed to control and hyperoxic atmospheres by employing an in vitro system in which cells are maintained at a gasliquid interface to simulate in vivo conditions. Hyperoxic injury was assessed by determining cell viability and by the production of lipid peroxides measured as thiobarbituric acid (TBA)-reactive substances. Since previous studies have demonstrated alterations in the lipid composition in lungs and lung lavage fluid of animals (13, 14), and in cultured cells and effluent media after exposure to oxidant gases (8, 15), we also assessed changes in fatty acid profiles of TE cells and cell culture effluents following control or hyperoxic exposures.

Materials and Methods Isolation and Culture of TE cells Monolayer cultures of rabbit TE cells were established by minor modifications of the methods of Wu and Smith (16). New Zealand White rabbits were sacrificed by CO 2 narcosis, the trachea excised, and epithelial cells recovered following intraluminal digestion with 0.1% protease (type XIV; Sigma Chemical Co, St. Louis, MO) at 4 C for 16 to 24 h as described. The free epithelial cells were plated at a density of 4 X 105 cells/dish on collagen-coated (Vitrogen 100 [7 p,g/cm2] ; Collagen Corporation, Palo Alto, CA) modified 35-mm glass petri dishes in 2 ml serum-free Ham's F12 media (GIBCO, Grand Island, NY) containing 7.5 ""g/ml insulin, 2.5 p,g/ml transferrin, l""M hydrocortisone, 20 ng/ml epidermal growth factor, 15 ""g/ml endothelial cell growth supplement, 15 ng/ml cholera toxin, 0.1 p,M retinoic acid, 15 mM Hepes buffer, and antibiotics (20 p,g/ml gentamicin, 50 V/ml penicillin, and 50 p,g/ml streptomycin). Growth factors and reagents were purchased from Sigma, except gentamicin (Whittaker Bioproducts, Walkersville, MD). Growth medium was changed initially at 24 h and every 48 h thereafter, and confluent monolayers were achieved in 7 to 10 d. The epithelial nature of the cultured cells has been confirmed by uniform staining with antikeratin antibody and by their ability to repopulate denuded tracheal grafts with re-expression of a mucociliary epithelium (17). 0

Lipid Supplementation of Growth Media Serum-free growth media were further supplemented in some cultures with 0.1% Excyte ill (Miles-Pentex), a mixture of cholesterol esters and phospholipid-rich lipoproteins, and with 1 ""M (300 ng/ml) arachidonic acid (AA) (Cayman Chemical, Ann Arbor, MI) complexed to fatty acid-free BSA (A-6003; Sigma). The AA-BSA complex was prepared at a molar ratio of 3.3:1 in 0.1 N sodium hydroxide at room temperature as described by Spector and colleagues (18). Exogenous AA was used in conjunction with Excyte III because this commercial preparation provides primarily linoleic acid (18:2w6) and contains only negligible amounts of AA (Production Information, Miles-Pentex), and previous studies in our laboratory have indicated that this combination of exogenous lipids produces a fatty acid profile in cultured rabbit TE cells more similar to that of acutely recovered native TE cells (12).

Hyperoxic Exposure A system modified from those described by Rasmussen (19) and Guerrero (20) allowed for exposure of TE cells to hyperoxic atmospheres at a gas-liquid interface analogous to in vivo conditions with only a thin film of fluid overlying the cells (Figure 1, upper panel). The system provides for maintenance of viability during exposure without imposing an appreciable diffusion barrier to gaseous irritants. A hyperoxic atmosphere of approximately 90 % oxygen was achieved with a 5 % CO 2/95 % O2 commercial cylinder (Figure 1, lower panel). Gas flow was regulated at 2 liters/min by a rotameter. Prior to entry into the chamber, the gas stream was filtered (Filinert 13060 [2 ""m]; Nucleopore Corp., Pleasanton, CA), warmed to 37 0 C and humidified. Oxygen concentrations in the chamber were monitored with a Ventronics oxygen analyzer (model 5575; Hudson Co., Temecula, CA). To obviate oxidation of growth media components, Hanks' balanced salt solution (HBSS) was used during the exposures. Preliminary experiments indicated that TE cultures could be maintained without physical disruption of the monolayers or loss of cell viability in the humidified 5 % CO 2/air environment of the chamber for more than 2 h by flooding the culture dishes with HBSS (2 ml, 20-s delivery) every 15 min to renew the thin layer of fluid overlying the cells. For exposure, the culture dishes were positioned in the chamber covered with HBSS. When the concentration of oxygen had stabilized, the HBSS was drained off and the exposure period begun. Monolayer cultures established from the same animal were exposed to control (5 % CO 2/air) or hyperoxic atmospheres for 2 h using the protocol described above. HBSS effluents from companion TE mono layers were collected as a single sample in acid-washed glass test tubes at the start and at the completion of exposures. To minimize the potential effects of growth cycle and cell density on susceptibility to oxygen, the cultures were exposed on days when they first reached confluence as assessed by visual inspection. Extraction of Lipids from TE Cells and HBSS Effluents and Analysis of Fatty Acid Profiles Following control or hyperoxic exposures, TE cells were removed from the culture dishes by a brief digestion with 0.5 % trypsin-EDTA (GIBCO) , and the washed, recovered cell pellets from two similarly exposed cultures were pooled and resuspended in 0.5 ml HBSS. Cell viability (0.04 % trypan blue dye exclusion) and number were determined by hemocytometer. Freshly isolated rabbit TE cells were recovered by enzymatic digestion as described. A Percoll density gradient was employed to separate TE cells from contaminating erythrocytes (21) and thus to avoid contribution of the erythrocyte membranes in the analysis of fatty acid profiles. Lipids from cell pellets and HBSS effluents were extracted with chloroform-methanol (2:1) at a ratio of 4:1 with the sample. Fatty acid methyl esters were prepared (22) and analyzed on a Hewlett-Packard 5890A gas chromatograph equipped with a splitless injector, a 12-m, 0.2-mm ID HP-l column (Hewlett-Packard, Avondale, PA), a flame ionization detector, and a Hewlett-Packard model 3392 integrator. The carrier gas was helium at 15 psi and the column temperature was programmed from 100 0 C to 260 0 C at 100 Cimino Retention

Dennery, Kramer, and Alpert : Fatty Acid Profiles and Hyperoxic Injury in Cultured Tracheal Epithelial Cells

139

..::. ~~.'.,;

.:: "

37·lncubalor

t

Figure 1. Hyperoxic exposure system. A stainless steel "Rochester chamber" (vol, I ft3) enclosed in a 370 C incubator ensures uniform gas flow in the central portion of the chamber where the culture dishes are positioned on a rocker platform. Upper panel: Media supply and collection . Medium (HBSS) is delivered to each dish by a multichannel peristaltic pump. Immediately after delivery, the platform tilts, draining the media via an exit port parallel to the floor of the dish, leaving only a film of fluid adherent to the cells. A programmable two-channel timer coordinates delivery of HBSS with activation of the rocker platform. Lower panel: Hyperoxic atmosphere. A glass plate secured against the front face of the chamber creates a closed airtight system. Gas stream filtration and humidification and monitoring of oxygen concentration are described in text.

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TABLE 1

pane; Sigma) and reproducible measurements of TBA-reactive substances from TE cells recovered from three culture dishes (approximately 7.5 x 105 cells). The TBA reaction mixture consisted of 0.1 ml disrupted cells in 1.15% KCI, 0.1 m18.1% SDS, 0.75 ml20% acetic acid solution (pH 3.5),0.75 ml 0.8% aqueous TBA solution (Fisher Scientific, Pittsburgh, PA), and 0.3 ml distilled water. The mixture was heated at 95 0 C for 60 min in tightly capped tubes. After cooling, 0.1 ml of distilled water was added. The samples were then extracted with 2.5 ml n-butanol:pyridine (15:1; both from Fisher) and centrifuged (1,000 x g for 20 min) to separate the phases and remove cellular debris. The organic phase was analyzed (excitation: 515 nm; emission: 553 nm) with an AmincoBowman spectrofluorometer (model JH8960; American Instruments, Silver Spring, MD). TBA-reactive substances were expressed as nMol MDA/I0 6 cells by extrapolation from external standard curves.

Fatty acid composition offreshly isolated and cultured rabbit TE cells: effect of supplementation of culture media with exogenous lipids Culture Media Supplements Fatty Acids

14:0 16:0 16:1w7 18:0 18:1w9 18:2w6 18:3w6 20:4w6 22:4,5w6

Freshly Isolated

1.2 23.6 1.6 22.4 24.9 15.4 0.5 7.9 2.3

nt

Lipidsupplemented

None

± 0.9* ± 6.4 ± 0.9 ± 3.0 ± 2.1 ± 5.4 ± 0.8 ± 3.1 ± 1.9

6.4 33.5 17.8 7.3 34.2 1.5

± 1.3t ± 1.6t ± 0.8 t ± 0.9t ± 1.It ± OAt

3.7 30.0 6.6 18.9 21.9 11.6 2.2 3.8 0.7

NO

0.2 ± 0.7 t NO

6

± 1.8 ± 8.0 ± 2.7 ± 6.7 ± 11.0 ± 6.2 ± 0.5

±

1.8t

± 1.2 16

4

* Mean ± SD expressed as weight % of fatty acid methyl esters detected by gas chromatography. t p < 0.05 vs. freshly isolated cells. t n = Number of tracheas from which TE cells were isolated or cultures established. ND = Not detected.

Statistical Analysis Cell viability, production of TBA-reactive substances, and changes in the relative content of fatty acids after control or hyperoxic exposures were assessed by analysis of variance of single measures using the Statview 512 statistical software program of the Macintosh SE computer. Significance was considered at a P value of less than 0.05.

times of sample peaks were compared to those of authentic fatty acid methyl esters of 8- to 22-carbon chain length (PUFA 2 and GLC-70; Supelco, Bellefonte, PA). Relative weight percents were calculated from the integrated areas of identified peaks. More than 95 % of the total integrated sample peaks were identified.

Results Effect of Lipid Supplementation on Fatty Acid Composition of Cultured TE Cells The fatty acid content of rabbit TE cells grown in serum-free media without exogenous lipid supplementation differed markedly from that of acutely recovered native epithelium (Table 1). The pattern of fatty acid composition of the lipidunsupplemented TE cells (increased palmitoleic [16:1w7] and oleic [18:1w9] and decreased linoleic [18:2w6] and AA [20:4w6]) is characteristic of essential fatty acid deficiency (25). Addition of exogenous lipids (0.1% Excyte III and 1 JLM AA) to the culture media partially corrected these abnormalities and restored the fatty acid profiles of the cultured cells to more closely resemble that of acutely recovered native epithelium.

Measurement of Lipid Peroxides The lipid peroxide content of TE cells was determined after control or hyperoxic exposures by measurement of TBAreactive substances as described by Ohkawa and associates (23). The relative volumes of the assay were proportionally reduced, and fluorescent spectrophotometry, rather than absorbance at 532 nm, employed to increase the sensitivity of the assay (24). These modifications permitted detection of subnanomolar quantities of malondialdehyde (MDA) produced from an external standard (l,1,3,3-tetramethoxypro-

TABLE 2

Viability and lipid peroxide content of lipid-supplemented and unsupplemented rabbit TE cells exposed to 5 % CO2/air or hyperoxic atmospheres TE Cell Culture Conditions Lipid-unsupplemented

Lipid-supplemented

Exposure conditions

Unexposed

5% CO2/air exposure

Hyperoxic exposure

Cell viability (%)

91.8 ± 2.4

82.9 ± 1.9*

54.8 ± 4.8 t 0.56 ± .14 t

0.04 ± 0.1

Lipid Peroxides (nMol MOA/106 cells)

4



* P < 0.05

vs. vs. t p < 0.05 vs. § n = Number t

p

< 0.05

11

9

Unexposed

5% COiair exposure

± 2.3

92.9 ± 1.3t

73.3 ± 2.4tt

0.06 ± .01t

0.26 ± .05tt

8

8

94.7

4

unexposed controls grown in similar culture conditions. Values are mean ± SD. 5% CO 2/air exposed controls grown in similar cell culture conditions. Values for duplicate samples varied by less than 10%. similarly exposed un supplemented TE cells. of trachea from which cultures were established.

Hyperoxic exposure

Dennery, Kramer, and Alpert: Fatty Acid Profiles and Hyperoxic Injury in Cultured Tracheal Epithelial Cells

TABLE 3

Effect of 5% COz/air or hyperoxic exposure on fatty acid profiles of lipid-supplemented and unsupplemented rabbit TE cells TE Cell Culture Conditions Lipid-unsupplemented Fatty Acids

5% CO 2/Air Exposure

14:0 5.8 ± 0.7* 16:0 30.2 ± 2.8 16:1w7 9.8 ± 1.4 18:0 12.5 ± 1.5 18:1w9 39.1 ± 2.9 18:2w6 1.2 ± 0.3 20:4w6 0.5 ± 0.2 n§

11

Hyperoxic Exposure

8.7 38.9 7.6 31.1 14.1 1.0 0.3

± ± ± ± ± ± ±

0.9t 2.4t 1.9 2.6t 2.5t 0.2 0.1

Lipid-supplemented 5% CO2/Air Exposure

2.9 27.6 4.2 23.2 23.7 12.5 5.4

9

± ± ± ± ± ± ±

Hyperoxic Exposur e

0.9* 5.5 ± OAtt 2.7 39.1 ± 3.1t 0.2* 3.8 ± 0.8 3.1* 32.9 ± l.4t 2.6* 13.1 ± 2.9 2.3* 5.1 ± 1.0tt 0.6* 0.3 ± 0.2t

8

8

* Mean ± SD expressed as wt %. Relative content of l8 :3w6 and 22:4 ,5w6 was less than 2 %. t P < 0.05 vs. 5 % C0 2/air exposed controls grown in similar cell culture conditions. :I P < 0.05 vs. similarly exposed unsupplemented TE cells. § n = Number of tracheas from which cell cultures were established.

Response of Cultured TE Cells to Control and Hyperoxic Exposures TE cell viability and number. Table 2 summarizes the viability of lipid-supplemented and unsupplemented TE cells recovered from unexposed mono layers and following control or hyperoxic exposure. Compared to unexposed controls, 5 % COz/air exposure did not affect the viability of lipidsupplemented TE mono layers but did result in decreased viability in unsupplemented TE cultures. The observed decrease in viability of unsupplemented TE cells following 5%

COz/air was significant in comparison to similarly exposed lipid-supplemented cultures. Hyperoxic exposure resulted in a decrease in viability of both lipid-supplemented and unsupplemented cultures, but the decreased viability was significantly greater in lipid-un supplemented cultures (Table 2). The number of TE cells recovered from the culture dishes did not change following control or hyperoxic exposure in either lipid-supplemented or unsupplemented cultures, and in preliminary experiments, photomicrographs obtained preand post-exposure documented that TE cells were not dislodged from the monolayers during the exposures (data not shown). Lipid peroxidation. Thiobarbiturate-reactive substances increased in both lipid-supplemented and unsupplemented TE cells following hyperoxic exposure compared to those detected in respective cultures exposed to 5 % COz/air (Table 2). However, the production ofTBA-reactive substances following hyperoxic exposure was significantly greater in lipidunsupplemented cells. Fatty acid content of TE cells. Table 3 shows the relative fatty acid content of lipid-supplemented and lipid-unsupplemented TE cells after exposure to control or hyperoxic atmospheres. Exposure to 5 % COz/air did not significantly alter the fatty acid profiles of either lipid-supplemented or unsupplemented TE cells (Tables 1 and 3). In contrast, hyperoxic exposure caused significant changes in the fatty acid composition of both lipid-supplemented and lipid-unsupplemented TE cells . Figure 2 illustrates the changes in saturated (14:0, 16:0, and 18:0), monounsaturated (16:lw7, 18:1w9), and PUFA (l8:2w6 and 20:4w6) following control or hyperoxic exposure. In both lipid-supplemented and lipid-unsupple mented cultures, hyperoxic exposure resulted in a decreased relative content of monounsaturated and polyunsaturated fatty acids and an increased relative content of saturated fatty acids. In the lipid-supplemented cells, total cellular content

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LIPID SUPPLEMENTED

100

-0

(j)

80

*

*



o

Post 5%C02/alr exposure.

~ Post hyperoxlc exposure.

+J

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rabbit TE cells exposed to 5 % COz/air or hyperoxic atmospheres. * = p < 0.05 for 5 % COz/air vs. hyperoxic exposure. t = Values expressed as mean

± SD.

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142

AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 3 1990

TABLE 4

Effect of 5 % COz/air or hyperoxic exposure on fatty acid content of HBSS efJiuents from lipid-supplemented and lipid-unsupplemented rabbit TE cultures TE Cell Culture Conditions Lipid-unsupplemented

Fatty Acids

14:0 16:0 16:1w7 18:0 18:1w9 18:2w6 20:4w6

Post 5% COz/Air Exposure

Pre 5% COz/Air Exposure

7.4±1.1* 51.1 ± 2.9 4.2 ± 1.1 24.2 ± 1.4 16.5 ± 2.5 1.2 ± 0.5 0

n~

11.5 52.8 2.8 22.7 8.1 0.1 6

± ± ± ± ± ± 0

1.9 1.8 0.9 1.3 1.9§ 0.1

Lipid-supplemented

Pre Hyperoxic Exposure

4.1 47.6 1.3 26.0 18.4 0.8

± ± ± ± ± ± 0

Post Hyperoxic Exposure

2.3 4.3 0.8 2.3 4.8 0.6

4.6 57.9 1.5 28.5 9.0 1.1

Pre 5% COz/Air Exposure

± 1.7 ± 3.6 ± 1.0 ± 2.2 ± 2.0§ ±0.7 0

10.8 52.8 2.8 20.7 5.6 1.4 1.5

± 3.2 ± 1.8 ± 0.9 ± 2.5 ± l.4t ± 0.4 ± 0.9t

6

6

Post 5% COz/Air Exposure

Pre Hyperoxic Exposure

5.5±4.1 53.3 ± 5.1 1.9±0.1 22.9 ± 0.8 5.6 ± 0.6 1.1 ±0.4 1.6 ± 1.6t

13.6 ± 8.8t 49.5 ± 5.6 1.1 ± 0.5 18.7 ± 1.9t 7.7 ± 2.0t 2.7±2.lt 1.0 ± 0.9t

Post Hyperoxic Exposure

18.4 50.6 1.4 17.2 8.3 2.3

± ± ± ±

3.9t 3.7 0.8 1.6t ± 2.4 ± 1.1+ 0

4

* Mean ± SD expressed as wt %. Relative content of 18:3w6 and 22:4,5w6 was less than 2%. < 0.05 vs. similarly exposed unsupplemented TE cells. t p < 0.05 vs. 5 % COz/air exposed controls grown in similar cell culture conditions. § P < 0.05 vs. pre-exposure. t p

~ n = Number of tracheas from which cell cultures were established.

oflinoleic acid (18:2w6) and AA (20:4w6) was dramatically reduced. Fatty acid content of HBSS effluents. Table 4 summarizes the results of analysis of the lipid content of HBSS cell culture effluents. Saturated fatty acids predominated in both lipid-supplemented and lipid-unsupplemented effluent media collected at the start of exposures. The relative content of these fatty acids in HBSS effluents was not significantly altered by control or hyperoxic exposure. Monounsaturated fatty acids were present in low quantities in culture effluents from both lipid-supplemented and lipid-unsupplemented cultures. The relative content of palmitoleic acid (18:lw9) was significantly higher in unsupplemented cells compared to supplemented cells and decreased after both control and hyperoxic exposure in unsupplemented cultures. The relative content of PUPA in cell culture effluents was small (less than 3 %). In unsupplemented cells, AA was not detected in culture effluents whereas the small quantity of AA in effluent media from supplemented cells was no longer detectable after hyperoxic exposure.

Discussion Previous whole-animal exposure studies have shown a relationship between lung fatty acid content and survival following exposure to hyperoxic atmospheres. Kehrer and Autor (5) demonstrated that adult rats maintained on a diet rich in PUPA had increased lung triglyceride PUPA content and that these animals tolerated exposure to hyperoxia better than rats fed a standard diet, and dramatically better than animals fed a diet rich in saturated fatty acids. Sosenko and associates (6) have reported similar observations on dietary manipulation of lung fatty acid composition, lung PUPA content, and susceptibility to hyperoxic injury. Newborn rats of dams fed a diet rich in PUPA had increased lung phospholipid and triglyceride content of linoleic acid (18:2w6) and AA (20:4w6), and an increased PUPA/saturated fatty acid ratio compared to offspring of dams fed a regular diet or one low in PUPA.

Rat pups with increased lung PUPA content had a marked increase in survival following 7 d ofhyperoxic exposure. The observation that lung antioxidant enzyme activities and surfactant content were not different in the groups led the investigators to speculate that the differences in survival might be due to varying susceptibilities of membrane fatty acids to lipid peroxidation. More recent studies by these investigators have shown that supplementing the diets of lactating dams with a commercial fat emulsion (Intralipid't) increased the lung PUPA content of the nursing pups and led to protection against hyperoxic injury as assessed by survival and by decreased histologic lung injury (7). In our study, cultured rabbit TE cells grown in media supplemented with exogenous lipids had an increased PUPA content compared to cells maintained in unsupplemented serum-free media. Lipid-supplemented cells demonstrated superior survival compared to similarly exposed unsupplemented TE cells. No decrease in viability was observed following 5 % CO 2/air in the lipid-supplemented cells, which argues that the exposure system per se was not responsible for the dramatic decrease in viability seen after hyperoxic exposure. However, the cells were maintained in a simple salt solution during exposures rather than in nutrient media and this may have contributed to some of the decreased viability over the course of the 2-h exposure. Although the relative PUPA content of the lipid-supplemented cells was somewhat less than that of acutely recovered TE cells, in general, the fatty acid profiles of the lipid-supplemented TE cells were more similar to that of native epithelium. Our observations are consistent with the cited in vivo studies and provide additional evidence for a protective effect of cellular PUPA content against the toxic effects of hyperoxia. In vitro studies have also demonstrated that changes in cellular fatty acid composition may influence the susceptibility of cultured cells to oxidant injury. Block (8) reported that short-term incubation of cultured porcine endothelial cells with cis-vaccenic acid (18:1dl1) increased the content of this

Dennery, Kramer, and Alpert: Fatty Acid Profiles and Hyperoxic Injury in Cultured Tracheal Epithelial Cells

fatty acid in various lipid fractions, resulting in a greater number of double bonds in neutral lipids and a decrease in the relative double-bond content of membrane-associated phospholipids. Following hyperoxic exposure (66 h, 95 % oxygen), release of intracellular lactic dehydrogenase was significantly reduced in the vaccenic acid-supplemented cells. As initially proposed by Dormandy (11), it was speculated that PUFA in non-membrane-associated neutral lipids served as an expendable fatty acid pool to scavenge free radicals, thus protecting membrane PUFA from hyperoxic injury. We did not determine which lipid fractions were modified by exogenous lipid supplementation and, in particular, which lipid fractions were enriched with PUFA. Thus, in accounting for the increased survival of lipid-supplemented cells, we cannot distinguish the possible contribution of "nonessential" expendable fatty acid pools in protecting membrane fatty acids against free radical injury from the known varying susceptibility of different fatty acids to lipid peroxidation (9-11). Our studies suggest an association between decreased production of lipid peroxides and increased TE cell viability after hyperoxic exposure. These observations are consistent with the concept that lipid peroxidation is primarily responsible for cytotoxicity following exposure to oxidant gases (26). Thiobarbituric-reactive substances have been detected in lung homogenates of animals exposed to ozone (13, 27) or hyperoxia (28), and in cultured porcine endothelial cells following in vitro hyperoxic exposure (4). Although TBAreactive substances may not be the most direct measure of the products of lipid peroxidation, this assay does provide a general index of lipid peroxidation (11,29). Our observation of decreased production of TBA-reactive substances after hyperoxic exposure in lipid-supplemented TE cells with a more normal PUFA content supports the concept that PUFA may exert a protective effect against lipid peroxidation. In the study of Block (8), the relative percentage of cellular PUFA content did not change dramatically after hyperoxic exposure. Similarly, there was no significant change in the PUFA content of lipid-unsupplemented cells following hyperoxic exposure, and yet a marked increase in TBA-reactive substances was observed. Conjugated double bonds are required to generate lipid peroxides and other compounds measured as TBA-reactive substances (11, 29). Oxidation of even a small percentage of PUFA might be sufficient to generate the picomolar quantities of TBA-reactive substances detected, but the source ofincreased TBA-reactivesubstances in our study is not certain. Antioxidant enzymes are important determinants of hyperoxic injury (2-4). In the present study, antioxidant enzyme activity in the cultured TE cells was not measured, and it is conceivable that exogenous lipid supplementation may have altered cellular antioxidant enzyme content. However, changes in fatty acid composition from incorporation ofliposomes did not change the superoxide dismutase content of cultured endothelial cells (4) and, as previously reviewed, antioxidant enzyme activity in lung homogenates was not altered by dietary manipulations that caused changes in lung fatty acid content (6). Thus, it cannot be assumed a priori that addition. of exogenous lipids to cell culture media affected cellular antioxidant enzyme activity in our study. Hyperoxic exposure can also increase lung antioxidant enzyme

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activity and expression of specific mRNA encoding for antioxidant enzymes (30, 31). However, the length of hyperoxic exposure in our studies was brief (2 h), making antioxidant enzyme induction an unlikely explanation for the marked differences in survival observed between lipid-supplemented and lipid-un supplemented TE cell cultures. Changes in the relative fatty acid content of the lungs of animals exposed to ozone (13, 32) and hyperoxia (6) have been documented. Following 90 d of ozone exposure, the relative content of monounsaturated fatty acid and PUFA decreased and the saturated fatty acid content increased in the lungs of monkeys (13). Decreases in the PUFA content and unsaturation index were reported in triglycerides and phospholipids of porcine endothelial cells exposed in vitro to hyperoxic atmospheres for 24 or 48 h (8). We observed similar qualitative changes in cellular fatty acid content after hyperoxic exposure (decreases in the relative content of polyunsaturated and monounsaturated fatty acids and increases in saturated fatty acids) and demonstrated that these changes occurred even after a short-term (2 h) hyperoxic exposure. The greater contact with gaseous atmospheres afforded by' our exposure system may account for the more rapid appearance of altered fatty acid profiles in our study. Other investigators have reported changes in the lipid composition of lung lavage fluid and of cell culture effluents following exposure to oxidant gases (14, 15, 33). We were particularly interested to see if cellular AA was released into TE cell effluents after hyperoxic exposure, analogous to the observations of increased AA in lavage fluid from ozoneexposed rats (14) and release of (14C]AA into effluent media from hyperoxic-exposed fetal rat lung cell monolayers (15). The only significant alteration in fatty acid content of TE cell culture effluents observed was a decrease in the relative content of 18:1w9 in effluent media from lipid-un supplemented cells, noted after both control or hyperoxic exposure. While the cellular content of AA decreased after hyperoxic exposure in lipid-unsupplemented cells, increased release of AA into effluent media was not detected. In the present report, we did not assess the release of AA metabolites into cell culture effluents, but in parallel studies, we have observed evidence of activation of the AA cascade following hyperoxic exposure by high-pressure liquid chromatographic analysis of HBSS effluents (34). It is also possible that free AA released into effluent media following hyperoxic exposure was oxidized to shorter-chain fatty acids, as was reported recently by Rabinowitz (32) in the lungs of ozoneexposed rats. Increased content of AA in lung cells could conceivably augment hyperoxic injury by providing increased unsaturated double bond sites for free radical attack and/or by providing substrate for the production of proinflammatory eicosanoid metabolites. Following hyperoxic exposure, Freeman and coworkers (4) reported increased release of lactic dehydrogenase and increased production of TBAreactive substances in cultured porcine endothelial cells with increased AA content relative to control cells. However, in the study by Sosenko and associates (6), there was no difference in the relative content of AA in membrane-associated phospholipids in the lungs of the rat pups from the low- and high-PUFA diet groups despite marked differences in survival to hyperoxic exposure. Thus the role of increased cellu-

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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 3 1990

lar AA content in hyperoxic injury is not clear from the present studies and may vary among cell types, depending not only on the array of AA metabolites generated but also on complex intracellular regulatory actions of these compounds (35). We observed that rabbit TE cells grown in serum-free lipid-unsupplemented media had decreased PUFA content and fatty acid profiles characteristic of essential fatty acid deficiency. Exposure of these cells to 5 % CO 2/air was associated with decreased viability whereas similar exposure did not affect the viability of lipid-supplemented TE cells whose fatty acid profiles were more similar to those of native epithelium. These observations may be relevant to sick newborn infants requiring supplemental oxygen in the first days of life. When deprived of dietary lipids, neonates can develop essential fatty acid deficiency (37) and thus might be predisposed to airway epithelial injury even in normoxic atmospheres. However, the use of intravenous lipid emulsions in human neonates with increased oxygen requirements remains controversial, with some recent studies showing detrimental effects (38, 39). We have demonstrated that rabbit TE cells grown in serum-free media are more susceptible to hyperoxic injury than are cells whose total cellular fatty acid profiles have been "normalized" by supplementation of culture media with exogenous lipids. These observations are not unique to rabbit TE cells. In preliminary studies with cultured human TE cells, we have demonstrated increased lipid peroxidation and similar but less dramatic changes in cell viability after hyperoxic exposure (40). Our observations support in vivo studies which suggest that the PUFA content of lung tissues may influence susceptibility to hyperoxic injury. Acknowledgments: The authors thank Dr. Robert M. Kliegman of the Neonatal Metabolism Laboratory at Rainbow Babies and Children's Hospital for providing the investigators access to gas chromatography equipment. This work was supported in part by Grant Z0779C-l from the Cystic Fibrosis Foundation.

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Effect of fatty acid profiles on the susceptibility of cultured rabbit tracheal epithelial cells to hyperoxic injury.

To investigate the role of cellular fatty acid content on the susceptibility of airway epithelial cells to hyperoxic injury, monolayer cultures of rab...
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