Generation of glycolaldehyde from guinea pig airway epithelial monolayers exposed to nitrogen dioxide and its effects on sodium pump activity.

Pulmonary injury from nitrogen dioxide (NO2) may in part be related to the generation of aldehydic compounds, which bind with cellular proteins and subsequently impair or inhibit cell function. We examined the generation of aldehydes from guinea pig tracheobronchial epithelial (GPTE) cell monolayers exposed to NO2. With the use of dinitrophenylhydrazine (DNP) to derivatize aldehydic compounds, glycolaldehyde, a two carbon alpha-hydroxyaldehyde, was identified in elevated levels in the basolateral fluid from monolayers exposed to NO2. DNP-glycolaldehyde levels were 81.2 +/- 2.7 and 234.0 +/- 42.6 nM in response to a 1-hr exposure to 1 and 5 ppm NO2, respectively, as compared to an air-control value of 20.3 +/- 6.8 nM. Taking into account dilution and reactivity, cellular glycolaldehyde levels could have reached as high as 3 mM for the 60-min exposure period (i.e., 0.05 mM/min). The effects of exogenous glycolaldehyde on GPTE ouabain-sensitive basolateral 86Rb uptake (an index of Na+,K(+)-ATPase activity) were examined and compared with the actions of NO2 exposure. Bolus addition of glycolaldehyde to the basolateral fluid at concentrations > or = 5 mM led to an inhibition of ouabain-sensitive 86Rb uptake, while lower concentrations had no effect. the effects of exogenous glycolaldehyde differ from NO2 exposure, which led to a sustained elevation of ouabain-sensitive 86Rb uptake with presumed generation of glycolaldehyde at a continuous low level. Glycolaldehyde does not appear to play a significant role in the acute alterations of sodium pump activity, suggesting that the NO2-induced changes in Na+,K(+)-ATPase activity of GPTE monolayers probably are further mediated by other lipid peroxidation products/oxidation processes yet to be identified.

Pulmonary injury firom nitrogen dinride (NO2 ) may in part be related to the aton ofaidehydic coMpo-uns which bi w cllular p d sub impair or inhibit .el function.' Wthe g e nfae S p pihei (GPTE):-ell.'monoIayers exposed' so 'NO2. Wish ib use of diniuoph razine.(DNP. to derivatize aldehydic compound, a a t bon l e,was id fied in elevated levels in the lal fluid from monolaye exposed to N02 DNP-gycolaldehydelevels were 81l.2t 2.7 and 234.0 42.6niMiponse to al-hre eto 1 and5 ppm NO2, r to: an air-co`trolvalueof--3 ± 6 into acc unt dilution adrecivt cellular: gccadyelvlsoudhve rahdabg a s Mfrthe idation processes yet to be delAL y wordalir epitheums glycolaidehyde, nitrogen dioxide, oubisenstive 86Rb p . Eniron Health Prp 104:852856 (15 6) Nitrogen dioxide (NO2) is a free radical formed by combustion processes in air. An increased incidence of airway inflammation and reactivity are associated with exposure to elevated indoor levels of this reactive gas (1)(2)(3). The toxicity of NO2 has been attributed to the oxidation of cellular constituents and generation of reactive compounds that initiate cellular injury (4). Free radical oxidation of cellular constituents such as polyunsaturated fatty acids (PUFA) can lead to the generation of aldehydic compounds. Oxidation of sugars may also be an important source of aldehydic compounds (5). These aldehydes may bind with essential cellular proteins to impair or inhibit activity, ultimately leading to adverse effects on cell function and viability. In the present study, we have examined the effects of NO2, a potent oxidant gas, on generation of aldehydic compounds from airway epithelial monolayers and the actions of a predominant aldehydic product on Na+,K+-ATPase (sodium pump), an enzyme that is vital to cellular function and viability.
The predominant reaction of NO2 may be to react with PUFA by abstraction of an allylic hydrogen (6,7). The epithelium lining the lung airways, which possesses a significant content of PUFA (8), represents one of the first major targets of NO2 and receives a large burden of the exposure (9)(10)(11). The mucin layer overlying airway epithelial cells also possesses a significant content of PUFA, as well as other compounds such as sugars, which may potentially interact with this gas (12). Lipid radicals, generated by hydrogen abstraction, in the presence of molecular oxygen initiate free radical chain autoxidation of membrane PUFA (5-7). The first product of autoxidation is a lipid hydroperoxide. These hydroperoxides may decompose to form a number of products, including reactive aldehydes (13). Aldehydes have longer half lives and possess significant cytotoxicity (13).
Cytotoxic effects attributed to aldehydes include depletion of intracellular glutathione; decreased protein thiols; onset of lipid peroxidation; disturbance of calcium homeostasis; inhibition of DNA, RNA, and protein synthesis; and inhibition of cellular respiration and glycolysis (13)(14)(15). Oxidation products derived from either the mucin layer or the apical airway epithelial cell plasma membrane may bind with cellular proteins and subsequently impair or inhibit cellular function.
In the present study, we have examined the generation of aldehydic compounds from guinea pig tracheobronchial epithelial (GPTE) monolayers exposed to NO2 levels associated with indoor exposure. GPTE cells were cultured in an air interface to form tight monolayers that actively transport ions and solutes vectorially (16)(17)(18)(19). These cell monolayers were acutely exposed for 1 hr to NO2 (1 or 5 ppm) and the release of aldehydes into the basolateral fluid was examined. We have identified glycolaldehyde, a two carbon a-hydroxyaldehyde, released into the extracellular medium from GPTE monolayers exposed to NO2. We then evaluated the effects of exogenous glycolaldehyde on epithelial Na+,K+-ATPase activity through measurements of ouabain-sensitive 86Rb uptake and specific 3H-ouabain binding and compared its actions with those observed for NO2 exposure.
Glycolaldehyde dimer and ouabain were obtained from Sigma Chemical Co. (St. Louis, MO). PC-1 (serum-free defined culture medium) was obtained from Hycor Biomedical (Irvine, CA). 2,4-dinitrophenylhydrazine (DNP), acetaldehyde, and trans-2-nonenal were obtained from Aldrich Chemical Co. (Milwaukee, WI). NO2 in air at levels of 1 or 5 ppm and compressed air (NOX <0.1 ppm) were obtained from Airgas (Los Angeles, CA). NO2 levels in air were certified to be of ±5% analytical accuracy for a period of six months from the date of production. For some experiments, NO2 levels in air were verified with an NOX analyzer, Model 1150SP (Interscan Corp. Chatsworth, CA). Other reagents were obtained from standard commercial sources.

Exposure to Nitrogen Dioxide
Monolayers with similar spontaneous potential difference (SPD) and transepithelial resistance (RTE) values, as measured with the use of a Millicell ERS (Millipore, Bedford, MA), were paired into air or NO2 exposure groups prior to actual exposure (18). Confluent GPTE monolayers in 12mm Transwells were rinsed twice with 2 ml (0.5 ml added to the apical side and 1.5 ml to the basolateral side) of Krebs Ringers phosphate-buffered HEPES solution (KRPH) composed of 125 mM NaCl, 5 mM KCl, 1.0 mM MgSO4, 8 (18,19). Following the two rinses, KRPH (0.9 ml) was added to the basolateral side of the monolayer, while the apical side was left fluid free to facilitate contact with air or NO2. Monolayers in 6.5-mm Transwells were rinsed twice with 1.25 ml (0.25 ml added to the apical side and 1.0 ml to the basolateral side) of KRPH and then 0.4 ml was added to the basolateral side alone. Monolayers were allowed to equilibrate in KRPH for 60 min at 370C (18,19). GPTE monolayers were then exposed to NO2 (1 or 5 ppm) in air or air alone using previous-ly described procedures (18,19). Nominal NO2 levels rather than actual levels have been provided; exposure time was 1 hr.

Identification and Measurement of DNP-carbonyl Compounds
Basolateral fluid or apical rinse fluid containing products liberated from exposed GPTE monolayers were derivatized with DNP using procedures previously described by Eserbauer et al. (21) and Poli et al. (22), as modified by Robison et al. (23). DNPcarbonyl compounds were separated into classes by thin layer chromatography, and individual aldehydes were quantitated through HPLC separation in conjunction with a Perkin Elmer LC 235 UV diode array detector (Perkin Elmer, Norwalk, CT). The HPLC eluate was monitored at 235 and 365 nm. Known amounts of 2decadienal dinitrophenylhydrazone were added as an internal standard for quantitation of DNP-derivatized carbonyl compounds. The concentrations of DNP derivatives were estimated by UV analysis at 365 nm using £ = 2.8 x 104 M1lcm-1 at 365 nm

Effect of Glycolaldehyde on Airway Epithelial Bioelectric Properties
Airway epithelial viability in response to glycolaldehyde treatment was evaluated through measurements of bioelectric properties (short circuit current and RTE). Monolayers were mounted in Ussing chambers (Precision Instrument Design, Tahoe City, CA) and allowed to equilibrate for 15 min in 10 ml of KRPH on both sides (18). Glycolaldehyde was added to either the apical or basolateral bathing fluid at concentrations ranging from 0.1 to 20 mM. Short circuit current (SCC) was measured with an automatic voltage/current clamp (DVC-1000; World Precision Instruments, Sarasota, FL) (18). The monolayer was continuously short circuited except for a brief period every 30 sec when a constant voltage pulse (dV = 2 mV) was imposed for 3 sec to yield a current response (dI) (18). RTE was estimated from the relation of dV/dI and SPD estimated from RTE X SCC (18). Bioelectric properties were monitored for 30 min following addition of glycolaldehyde.

Measurement ofAirway Epithelial
Ouabain-sensitive 86Rb Uptake To estimate the Na+,K`-ATPase activities of glycolaldehyde-treated airway epithelial monolayers, we measured 86Rb (K+ surro-gate) uptake into epithelial cells from the basolateral fluid in the presence and absence of 1 mM ouabain in the basolateral fluid using procedures previously described by Robison and Kim (19) and Kim and Suh (24). GPTE monolayers, in 6.5-mm Transwells, were treated basolaterally with glycolaldehyde (1 to 20 mM) or the vehicle control (water) for 15 min. After the 15min treatment, monolayers were separated into two groups. Ouabain (1 mM) was added to the basolateral fluid of one group of monolayers for a 30-min incubation at 37°C, while the other group of monolayers received an equal volume of vehicle (i.e., 25 pl water) added to the basolateral KRPH (400 pl) (19). Following ouabain or vehicle treatment, 86RbCl was added to the basolateral fluid (final concentration 10 pCi/ml) and incubated for 5, 15, or 30 min (19). Glycolaldehyde was present in the basolateral fluid throughout the ouabain treatment and 86Rb uptake periods. Monolayers were washed, solubilized, and counted for 86Rb activity as previously described (19). Apparent Na+,K+-ATPase activity was expressed as ouabain-sensitive 86Rb uptake (nanomoles per mg protein per 5, 15, or 30 min). Cell protein was measured using the BCA protein assay (Pierce, Rockford, IL).

Measurement of Specific 3H-Ouabain Binding
With the use of previously described procedures by Robison and Kim (19), specific 3H-ouabain binding was used to measure alterations of density and affinity of Na+,K+-ATPase on the airway epithelial basolateral cell surface in response to glycolaldehyde treatment. Monolayers were treated with glycolaldehyde (0 or 20 mM) for 15 min; basolateral fluid was removed and replaced with 3H-ouabain binding assay buffer which was composed of 250 mM sucrose, 3 mM MgSO4, 3 mM Na2HPO4, 1 mM Na3VO4, 10 mM Tris-HCl, and 0.5% BSA at pH 7.2 and containing 3H-ouabain at concentrations ranging from 0.125 to 2 pM (19,(25)(26)(27). Monolayers, in 6.5-mm Transwells, were allowed to bind 3Houabain for 2 hr at 37°C. Nonspecific binding was determined in the presence of excess (1,000 x) unlabeled ouabain at each radiolabeled ouabain concentration (19). Monolayers were washed, solubilized, and counted as previously described, and alterations of ouabain binding were determined by Scatchard analysis (19).

Statistical Analysis
Data are expressed as the mean ± standard error of the mean (SEM). NO2 and air or glycolaldehyde and control treatment groups were compared using analyses of variance Environmental Health Perspectives * Volume 104, Number 8, August 1996

Results
Generation of Carbonyl Compounds in Response to NO2 Exposure HPLC separation of zone 2 carbonyl compounds indicated that a major aldehydic compound released into the basolateral fluid from N02-exposed GPTE monolayers was the Di-DNP derivative of glycolaldehyde (Fig. 1). Derivatization with DNP, TLC separation, and HPLC retention time of this compound generated from GPTE monolayers are consistent with previous identification of glycolaldhyde in this laboratory using identical techniques and confirmation of structure with the use of gas chromatography/mass spectrometry (23,28). The Di-DNP derivative of glycolaldehyde was present as two peaks, the first with a retention time of 31.8 min, which was the oxidized form that has lost two hydrogens, and the second at 33.3 min, which was the reduced form (23). A DNP derivative of authentic glycolaldehyde was found to co-elute with these two peaks obtained from an extract of the baso-lateral fluid treated with DNP. The oxidized form of glycolaldehyde was observed in air controls; however, the reduced form was not present. The DNP derivative of decadienal, with a retention time at 47.2 min, was added as an internal standard for quantitation. Glycolaldehyde levels were significantly increased in response to a 1-hr exposure to 1 or 5 ppm NO2 as compared with air controls (Fig. 2). Release of glycolaldehyde toward the apical side was below the detection limit.

Effect of Glycolaldehyde on Bioelectric Properties
The effects of exogenous glycolaldehyde on GPTE bioelectric properties (e.g., SCC and RTE) were evaluated (Table 1). GPTE monolayers mounted in Ussing chambers were bathed with KRPH on both sides, and glycolaldehyde (2 to 20 mM) was added to the basolateral fluid. Bolus addition of glycolaldehyde at concentrations <1 mM had minimal effects on the SCC or RTE.
Effect of Glycolaldehyde on Ouabain-sensitive 86Rb Uptake Treatment of the basolateral aspect of GPTE monolayers with 20 mM glycolaldehyde transiently increased ouabain-sensitive 8'6Rb uptake at 5 min; however, at 15 and 30 min, uptake was significantly decreased below control levels (Fig. 3). Concentrations  of 5 or 10 mM did not alter 86Rb uptake at 5 min in comparison with control; however, the uptake at 30 min was significantly decreased.
Effect of Glycolaidehyde on Nae,K+-ATPase Density and Affinity The specific 3H-ouabain binding capacity (Bna) and binding constant (K) for basolateral Na+,K+-ATPase were measured in response to basolateral glycolaldehyde treatment. A representative Scatchard plot for a 15-min treatment of GPTE monolayers with 0 or 20 mM glycolaldehyde is shown in Figure 4.

Discussion
In the present study, a significant generation of aldehydes from airway epithelial monolayers exposed to NO2 was detected. Glycolaldehyde, an ax-hydroxyaldehyde, was a major aldehydic product generated from exposure of GPTE monolayers to NO2. Glycolaldehyde was also identified as a major aldehydic product released from alveolar macrophages exposed to NO2 (23,28). Preliminary studies using 3Harachidonic acid-labeled monolayers suggest that glycolaldehyde may be derived from cellular lipids. A recent report has identified a number of a-hydroxyaldehydes derived from peroxidation of arachidonic acid (29). Glycolaldehyde may be formed from PUFA through a similar oxidative mechanism. Another possible route for the formation of glycolaldehyde may be through the decomposition of sugars (5,30,31); however, the reactivity of NO2 with sugars is known to be much slower than that found with PUFA. Glycolaldehyde is reactive with proteins, rapidly forming Schiff base adducts with amino groups (14,15). These Schiff base adducts can undergo Amadori rearrangement to yield a new aldehyde moiety that can form a Schiff base adduct with another amino group (14). This process can modify the physical properties of proteins as well as generate intra-and intermolecular crosslinking in or between proteins, respectively (14). Basolateral glycolaldehyde concentrations estimated in the present studies may be lower than cellular concentrations due to diffusion of this compound into the larger volume of the extracellular medium, as well as losses due to binding with cellular components, metabolism, and volatility (23). In our preliminary studies, treatment of GPTE monolayers with exogenous hexanal yielded a recovery of only 17%. Based upon a measure of approximately 5 x 105 cells per confluent monolayer, the total cellular volume is estimated to be about 0.5 pi. Thus, the cellular glycolaldehyde concentration for a 1-hr exposure to 5 ppm NO2 was calculated to reach a maximum of approximately 3 mM. Assuming continuous generation of glycolaldehyde during the 60-min exposure, the production rate could have been as much as 0.05 mM/min.
The effect of exogenous glycolaldehyde on GPTE ouabain-sensitive basolateral Na+,K+-ATPase was examined using approximated cellular concentrations of this compound. Treatment with 20 mM glycolaldehyde, transiently stimulated ouabainsensitive 86Rb uptake at  uptake was inhibited at 30 min with concentrations .5 mM. Changes of airway epithelial bioelectric properties (RTE and SCC) in response to glycolaldehyde treatment were small and do not suggest a major loss of cell viability. The inhibition of ouabain-sensitive 86Rb uptake in response to glycolaldehyde treatment was not associated with any alterations of ouabain binding characteristics, suggesting that aldehyde binding directly to Na+,K+-ATPase was either not involved in the observed changes of activity or occurs at sites distant from ouabain binding. These data indicate that the effects of exogenous glycolaldehyde differ from that observed with a 1-hr exposure to 1 or 5 ppm NO2 where a twofold increase of ouabain-sensitive 86Rb uptake was observed for 2 hr postexposure (19) and glycolaldehyde was presumably generated at a continuous low level. The dynamics associated with bolus addition of glycolaldehyde appear to be quite different from those of cellular glycolaldehyde generation in response to NO2 exposure. These differences may include alterations in access to essential cellular targets, binding to nonessential targets such as the collagen matrix, and metabolism by various cellular enzymes such as aldehyde dehydrogenase (32,33). These results with exogenous glycolaldehyde may suggest that other lipid peroxidation products are probably mediating the actions of this reactive gas at levels associated with elevated indoor exposure.
Environmental Health Perspectives * Volume 104, Number  Glycolaldehyde may have significant long-term pathological consequences to cell function and viability with regard to the formation of glycation products (34). Through a process known as glycation, protein crosslinking occurs nonenzymatically and involves reaction of a compound such as glycolaldehyde with the amino group of a protein (34). These reactive Amadori products may then bind with amino groups on other proteins to form advanced glycation end products (AGEs) through intermolecular crosslinks (34). This process may interfere with cell-to-cell attachment as well as cell adhesion to the basement membrane, potentially resulting in significant disruption of the epithelial barrier and development of increased airway reactivity.
In summary, we have found that glycolaldehyde, an aldehydic compound, is generated in elevated levels from airway epithelial monolayers acutely exposed to NO2, in the range associated with elevated indoor levels. Exogenous glycolaldehyde does not appear to play a significant role in the previously observed N02-induced enhancement of airway epithelial Na+,K+-ATPase activity, suggesting that the alteration of pump activity is most likely mediated by other lipid peroxidation products yet to be identified. Glycolaldehyde may have significant long-term pathological consequences with regard to disruption of the epithelial barrier through its known reactivity with cellular proteins.