Acetaldehyde-mediated cilia dysfunction in bovine bronchial epithelial cells JOSEPH H. SISSON, DEAN J. TUMA, AND STEPHEN I. RENNARD Pulmonary Section and Liver Study Unit, Departments of Medicine and Biochemistry, University of Nebraska Medical Center, Omaha, Nebraska 681054 065

SIMON, (JOSEPH H., DEAN NARD. Acetaldehcyde-mediated

J. TUMA, AND STEPHEN I. RENcilia dLysfunction in bovine bronchial epithelial cells. Am. J. Physiol. 260 (Lung Cell. Mol. Physiol. 4): L29-L36, 1991.-Acetaldehyde, which is present in significant concentrations in cigarette smoke and is elevated during alcohol ingestion, has been demonstrated to impair mucociliary clearance of the lung. Acetaldehyde is also known t,o impair protein function through the formation of acetaldehyde-protein adducts. We hypothesized that acetaldehyde impairs bronchial epithelial cilia motion by inhibiting cilia dynein adenosinetriphosphatase (ATPase) activity through the formation of acetaldehyde adducts with cilia proteins. Acetaldehyde induced concentrationand time-dependent slowing of cilia beating and cilia-derived dynein ATPase activity in primary cultures and isolated axonemes of bovine airway epithelial cells. Cilia slowing and ATPase inhibitory effects were also observed with related aldehydes but not with ethanol. Acetaldehyde binding, assessed by gel electrophoresis using [‘“Cl acetaldehyde, was demonstrated to occur with the dynein heavy chains and with tubulin and closely paralleled ATPase inhibition. We conclude that acetaldehyde directly impairs bronchial cilia function causing slowing of cilia beating, inhibits cilia dynein ATPase activity, and binds to cilia proteins critical for motion including dynein and tubulin. These data suggest that acetaldehyde-induced cilia dysfunction may be related to direct cilia ATPase inactivation and adduct formation with cilia dynein and tubulin. This may be an important mechanism by which airway host defenses are impaired in clinical settings where acetaldehyde exposure occurs, e.g., with cigarette smoking and alcohol ingestion. smoke; ethanol;

mucociliary

clearance;

dynein;

axoneme

SMOKERS and alcoholics have an increased incidence of pulmonary infections (2, 4, 17, 20). The causesof these infections are thought to be due to altered host defenses related to smoke and alcohol toxicity. An important airway defense function that is impaired in these clinical settings is the mucociliary clearance system (1, 12, 35). The mechanism of mucociliary impairment in smokers and alcoholics is not known and is probably multifactorial in origin. One possible mechanism of mucociliary impairment common to both of these circumstances is aldehyde exposure. Aldehydes are highly reactive molecules that have been recognized as important toxins in biological systems (1315, 25,30). Several aldehydes have been identified in the vapor phase of cigarette smoke, including propionaldehyde, butyraldehyde, isobutyraldehyde and acetaldehyde (26, 38). Acetaldehyde is of unique importance among CIGARETTE

1040-0605/91

$150

Copyright

aldehydes for several reasons. First, acetaldehyde is present in cigarette smoke in the greatest concentration compared with other aldehydes (26). Second, it is produced in biologically significant quantities from the metabolism of ethanol. Third, acetaldehyde has been demonstrated to covalently bind to numerous proteins, resulting in protein and cellular dysfunction (6, 7, 34). In this context, we investigated the effects of acetaldehyde on bronchial epithelial cilia. Our studies demonstrate that acetaldehyde directly impairs airway cilia motion, inhibits axonemal dynein adenosinetriphosphatase (ATPase) activity, and binds to axonemal proteins critical for cilia motion including dynein and tubulin. These effects appear to be related to the aldehyde part of the molecule, since other aldehydes, but not ethanol, had similar effects. METHODS

Preparation and culture of ciliated cells. Ciliated bovine bronchial epithelial cells were obtained from fresh bronchi using a modification of the method of Wu and Smith (37). Fresh bovine lungs were obtained from the local slaughterhouse, and the second- and third-order bronchi were dissected from the lung tissue. The bronchi were incubated overnight at 4°C in 0.1% protease solution in medium 199 (M199; GIBCO Laboratories, Grand Island, NY) conta ining penicillin, streptomycin, and amphotericin B. On the following day the bronchi al lu mina were rinsed repeatedly with Ml99 containing 10% fetal calf serum (Biofluids, Rockville, MD), and the resulting cell solution was passed through a 100~pm mesh screen. The ciliated cell aggregates retained on the mesh were removed by gentle scraping. Aliquots of the aggregate suspension were placed onto Petri dishes precoated with type I collagen gel matrix (Vitrogen 100, Collagen Corp., Palo Alto, CA) mixed 1:l with Ml99. The ciliated cell aggregates adhered to the collagen matrix and formed incomplete monolayers within 48 h of plating. Direct inspection under phase-contrast microscopy demonstrated that cells prepared in this fashion were predominantly ciliated cells and could be maintained in an actively beating form in culture for l-2 wk. Cilia beat frequency determination. Actively beating ciliated cells were observed, and their motion was quantified by measuring cilia beat frequency using phasecontrast microscopy and videotape analysis. Ciliated cells in culture were maintained at a constant tempera-

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ture (24 t 05°C) by a thermostatically controlled stage. All observations were recorded for review using a Panasonic WV-D5000 videocamera and a Panasonic AG-1950 videotape recorder. Cilia beat frequency was quantified by observing the recording of a beating cilium or tuft of adjacent cilia and counting the number of frames required per cycle for at least 10 cycles during slow-motion playback (36). Preparation of isolated cilia. Tracheal cilia were obtained using the method of Hastie et al. (16). Briefly, fresh bovine trachea obtained from a local slaughterhouse were dissected from the lung parenchyma and clamped at both ends, and the lumen was filled with a solution containing 20 mM tris( hydroxymethyl)aminomethane (Tris) l HCl (pH 7.4), 50 mM NaCl, 10 mM CaCIS, 1 mM EDTA, 7 mM 2-mercaptoethanol, 0.1% Triton X-100, and 1 mM trypsin soybean inhibitor. The trachea was shaken vigorously for 1 min, and the solution was collected and spun at 1,500 g for 2 min to remove cellular debris. The cilia remained in suspension and were collected by centrifugation of the supernatant at 12,000 g for 5 min. The cilia pellet was resuspended in a buffer (resuspension buffer) containing 20 mM Tris . HCl (pH S.O), 50 mM KCl, 4 mM MgCIZ, 0.5 mM EDTA, 1 mM 1,4-dithiothreitol, and 10 mM soybean trypsin inhibitor and stored at 4°C. Cilia preparations were used within 24 h of collection. Fresh cilia obtained in this manner were capable of reactivation when incubated with fresh ATP (5 mM) and demonstrated typical cilia beating assessed by phase-contrast microscopy. The presence of intact demembranated axonemes in these preparations was also confirmed by electron microscopy. Measurement of cilia motion in isolated ATP-activated axonemes. Isolated demembranated axonemes, prepared as described, were diluted to a final concentration of 1.0 mg/ml total protein (Lowry method) in resuspension buffer. Fresh axoneme suspension (25 ~1) was placed on a simple perfusion chamber prepared by placing two parallel lines of Corning vacuum grease on a glass microscope slide and applying a glass cover slip. Resuspension buffer containing ATP (25 ~1) was added to the chamber so that final concentration of ATP was 5 mM. All experiments were performed at a constant temperature (24 t O.5”C) by a thermostatically controlled stage, and all reagents and cilia were equilibrated to 24°C before each experiment. The axonemes began to beat rapidly, as assessedby phase-contrast microscopy. After a baseline period of 5 min the beating activity was recorded on videotape for 1 min. Acetaldehyde (50 ~1 of O-1,000 ,uM in resuspension buffer) was added to the chamber and recording was resumed for an additional 5 min. The axoneme motion was qualitatively assessedby comparing the motion of acetaldehyde-exposed axonemes to that of buffer-exposed axonemes. Because the demembranated axonemes do not attach firmly to the glass chamber, beat frequency could not be measured with this technique. Therefore the changes in motion were visually graded 1 min after exposure by two different observers upon separate review of the videotape on a scale of 4 to 0 as follows: 4, no change; 3, detectable but slight slowing; 2, moderate slowing; 1, marked slowing, but motion still detectable; 0, complete cessation of all motion.

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ATPase activity assay. ATPase activity was determined by measuring the liberation of “‘P-labeled inorganic phosphate from [$‘P]ATP (Amersham, Arlington Heights, IL; Ref. 23). The assay solution contained 20 mM Tris HCl (pH 8.0), 4 mM MgCl,, and 0.1 mM [@“PI ATP (2,000 cpm/nmol). Samples containing 50 ~1 of cilia in suspension buffer (5-20 pug protein) were preincubated at 37°C for 5 min, and the reaction was initiated by adding 200 ~1 of assay solution. After a l5min incubation at 37OC, the reaction was terminated by the addition of 100 ,ul of 10 mM silicotungstic acid. Liberated ““P-labeled inorganic phosphate was assayed using the method of Stone et al. (32). Preliminary experiments demonstrated that the ATPase activity present in cilia was inhibited by vanadate but not by ouabain consistent with a dyneinlike ATPase (3, 29). Acetaldehyde binding assay. Cilia preparations diluted to -1 mg (total protein)/ml with resuspension buffer were incubated with appropriate dilutions of [ l,2-‘“Clacetaldehyde (American Radiolabeled Chemicals, St. Louis, MO) at 37°C for l-4 h with constant agitation. The reaction vessels were polyethylene and were sealed to minimize the loss of volatile radioactivity. After incubation, the samples were exhaustively dialyzed and protein-bound radioactivity was determined as described by Donohue et al. (7). Gel electrophoresis. Dialyzed reaction mixtures were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using the modified Laemmli method (18) and stained with Coomassie Blue. The dynein heavy-chain region and the tubulin subunits were easily resolved with this system and were cut out and solubilized using 0.2 ml of 30% H,O, at 75°C. Thiourea (0.5 ml of 2%) was added to each sample directly into the scintillation vial containing Aquasol (Du Pont-New England Nuclear, Boston, MA) neutralized with acetic acid, and radioactivity was determined. Statistical analysis. The results are expressed as means t SE. They were analyzed by multiple comparison (Scheffe type). Significance of concentration and time relationships were calculated by analysis of variance. RESULTS

Acetaldehyde slowed cilia beating in primary cultures of ciliated bovine bronchial epithelial cells in a concentration-dependent manner with significant slowing observed at concentrations of acetaldehyde as low as 15-30 ,uM and complete abrogation of cilia motion seen at concentrations of acetaldehyde >250 PM (Fig. IA). The time course of acetaldehyde-induced cilia slowing was rapid with maximal slowing occurring by 3 min after incubation with acetaldehyde (Fig. 1B). Additional beating experiments were performed in a cell-free system with demembranated axonemes to determine whether the acetaldehyde-induced cilia slowing effects observed in whole cells required cell membrane and/or cytosolic elements. Isolated ATP-reactivated demembranated axonemes, when exposed to acetaldehyde, were detectably slowed at 30 PM acetaldehyde within seconds after exposure and were immediately and completely paralyzed when exposed to concentrations of acetaldehyde ~125 PM (Fig. 1C).

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Cilia dynein ATPase activity is critical to normal cilia motion. Since aldehydes are known to impair specific protein functions, the effects of acetaldehyde on cilia ATPase activity was assessed in isolated axonemes. Acetaldehyde inhibited cilia ATPase activity in both a concentrationand time-dependent fashion. Significant acetaldehyde-induced ATPase inhibition occurred at a concentration ~30 pM, and maximal inhibition occurred with 500 ,uM (Fig. ZA). When incubated at 37°C with a high concentration of acetaldehyde (500 PM), cilia ATPase activity inhibition was rapid with 67% inhibition observed by 2 min and was maximal at 89% inhibition by 60 min (Fig. 2B). Because acetaldehyde-induced protein dysfunction has been linked to acetaldehyde-protein adduct formation in other biological systems (14, 31), experiments were performed to assess the ability of acetaldehyde to bind to specific cilia proteins. 14C-labeled acetaldehyde, incubated with isolated axonemes, irreversibly bound to mul-

pM

3

(minutes)

FIG. 1. Effect of acetaldehyde on cilia beat frequency of 72-hcultured bovine bronchial epithelial cells. Immediately before each experiment medium (Ml99 containing 10% serum) was removed. A: cilia beat frequency measured after a 3-min incubation with various concentrations of acetaldehyde (O-1,000 PM). Each point represents means t SE of 4 different experiments. Beat frequency in each experiment was derived from mean of 5 different cells from different areas of each plate. * Significantly different in magnitude of slowing compared with medium-treated cells (P < 0.05). H: cilia beat frequency determined after acetaldehyde exposure every 0.5 min until a stable baseline value was reached. Values for time 0 were measured immediately before acetaldehyde or medium addition. Beat frequency in each experiment was derived from mean of 5 different cells from different areas of each plate. These data are representative of 3 experiments. C: beating score determined on fresh demembranated ATP-reactivated axonemes incubated with various concentrations of acetaldehyde (O-l,000 PM). Beating score was determined after a lmin observation period after exposure by comparing motion observed in buffer-exposed axonemes with those of acetaldehyde-exposed axonemes (4, no change; 3, detectable but slight slowing; 2, moderate slowing; 1, marked slowing but motion still detectable; 0, complete cessation of all motion). These values are representative of 3 separate experiments on different fresh cilia preparations.

tiple axonemal proteins when analyzed by SDS-PAGE and autoradiography (Fig. 3A). The binding was most intense in the regions that comigrated with purified tubulin standards. Significant binding was also observed in the high-molecular-mass region (>300 kDa) where the dynein heavy chains are known to migrate (16, 23). Densitometric analysis of the relative autoradiogram and protein staining intensities suggest that acetaldehyde binding to the dynein heavy-chain region was approximately twice that seen to the tubulin regions when normalized for protein mass (data not shown). Acetaldehyde binding was also quantified by directly counting the radioactivity of Coomassie Blue-staining regions (Fig. 3B). The tubulin region and the dynein heavy-chain regions contained three to five times the 14C-labeled acetaldehyde compared to a nonprotein-staining background region. To examine the relationship between acetaldehydeinduced ATPase inhibition and acetaldehyde-protein ad-

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benzaldehyde with slowing of beat frequency seen at low concentrations (15-30 PM) and complete abrogation of cilia beating occurring at higher concentrations (250-500 PM; Fig. 5A). Cilia ATPase activity was also inhibited by all of the aldehydes tested (Fig. 5B). In contrast, ethanol, in equimolar concentrations, had no effect on either ciliated-cell beating or cilia ATPase activity (Fig. 5, A and B).

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FIG. 2. Effect, of acetaldehyde on cilia ATPase activity. A: results of’G()-min preincubation with increasing concentrations of acetaldehyde on cilia ATPase activity. Data are expressed as 5% of control and represent mean + SE for 7 experiments on cilia obtained from different tracheas. * Significantly different in magnitude of inhibition compared with buffer alone (P < 0.05). R: effect of increasing incubation time on cilia ATPase activity exposed to acetaldehyde (500 PM). Data are expressed as % of control and represent means t SE for 3 experiments on cilia obtained from different tracheas. All ATPase experiments were performed at 37°C. * Significantly different in magnitude of inhibition compared with buffer alone (P < 0.05).

duct formation, parallel experiments were performed to compare 14C-labeled acetaldehyde binding to the dynein heavy chains with ATPase inhibition over a range of acetaldehyde concentra .tions. Acetaldehyde binding to the dynein heavy-chain region increased in a concentration-dependent fashion that closely paralleled cilia ATPase inhibition (Fig. 4). To determine whether the cilia impairment and ATPase inhibition observed were specific to acetaldehyde, other aldehydes and ethanol were compared with acetaldehyde in the ciliated cell beating and cilia ATPase assays. Cilia beating was progressively depressed by propionaldehyde, butyraldehyde, isobutyraldehyde, and

The mechanisms by which pulmonary host defenses are impaired during ethanol ingestion and cigarette smoking are poorly understood and probably multifactorial in origin. Both alcohol ingestion and smoking have been associated with altered mucociliary airway clearance and an increased incidence of pulmonary infections (2,4, 17, 20). Normal mucociliary clearance is dependent on the interaction of mucus and fluid secreted by secretory epithelial cells and the coordinated beating of cilia by ciliated epithelial cells. A number of biologically active compounds implicated in the pathogenesis of smokingand alcohol-related illnesses are known to alter mucociliary clearance, but the mechanism(s) are unknown. Acetaldehyde has been shown to slow mucociliary clearance in isolated rabbit tracheal strips (5), but direct acetaldehyde effects on cilia motility and acetaldehyde interactions with cilia proteins have not been reported. It is quite likely that the airways are exposed to acetaldehyde in a number of important clinical settings. In humans acetaldehyde exposure occurs during at least two important conditions associated with altered pulmonary host defenses, namely, alcohol ingestion and smoking. During alcohol ingestion, acetaldehyde can reach the airways by several potential pathways. One route is through diffusion into the air space from the vascular compartment either from the hepatic vein and pulmonary capillaries or by way of the bronchial circulation (27). An alternative mechanism by which acetaldehyde may reach the airways during ethanol ingestion is from the local conversion of circulating ethanol to acetaldehyde by either microsomes present in lung tissue (28) or by bacteria present in the upper airway (24). During cigarette smoking the airways are exposed directly to acetaldehyde, which is present in the vapor phase of cigarette smoke (24). In either case, acetaldehyde is likely to reach the airway epithelium and may react with the ciliated cell. Aldehydes are of interest in biological systems, since they are highly reactive compounds due to their ability to form Schiff bases (or unstable adducts) and stable adducts with nucleophiles such as amino groups of proteins. Acetaldehyde, for example, has been demonstrated to bind to a number of important proteins including transport proteins such as albumin and hemoglobin (7, 31), cytoskeletal proteins such as tubulin and erythrocyte membrane proteins (9, 34), and enzymes such as RNase and glucose-6-phosphate dehydrogenase (21, 22). Importantly, acetaldehyde binding has been linked to protein dysfunction under physiological conditions identified with acetaldehyde exposure (19). Our results demonstrate that acetaldehyde impairs

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ACETALDEHYDE-MEDIATED

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FIG. 3. Acetaldehyde binding to cilia proteins. A: electrophoretic and autoradiotraphic analysis of acetaldehydelabeled cilia proteins. Fresh demembranated cilia diluted to 1 mg (total protein)/ml in resuspension buffer were incubated with 5 mM [1,2-‘4C]acetaldehyde at 37°C for 60 min. Protein (30 mg) was electrophoresed in a 6.8% SDS gel under denaturing conditions. Eletrophoretic pattern was assessed by both Coomassie Brilliant Blue stain (total protein) and by autoradiography (acetaldehyde binding) on same sample. Molecular mass standards (kDa) are indicated on left; protein staining regions cut out for quantitative analysis (bands l-7) are indicated on right. B: quantitative acetaldehyde binding. Protein staining regions (bands l-7) and a nonprotein staining region (background) were cut from gel, and radioactivity was determined. Dynein heavy-chain, a-tubulin, and fi-tubulin regions are indicated by solid columns (bands 1. 4. and 5. respectively). Stippled columns (bands 2, 3, 6, and 7) represent other unidentified cilia protein regions. * Band number refers to gel regions indicated in A

cilia function by slowing or stopping cilia beating in whole intact cells in culture. Several potential mechanisms involving acetaldehyde interactions with nonaxonemal cellular elements could be invoked to explain this observation. First, cilia motion is closely associated with intracellular calcium concentrations (ll), and the rapid ciliostasis induced by acetaldehyde in intact cells could be caused by fluxes in intracellular calcium. Second, calmodulin is thought to be a prominent candidate as a signal transducer in axonemal motion (33) and may be altered by acetaldehyde. Finally, correlations between adenosine 3’,5’-cyclic monophosphate (CAMP) levels and axoneme motility have been demonstrated by others (ll), and acetaldehyde may alter phosphorylases that regulate intracellular CAMP levels. Although our experiments do not completely exclude these possibilities, we think it is unlikely that these mechanisms are operative as major causes of cilia impairment, since acetaldehyde also caused rapid ciliostasis in a cell-free system using isolated demembranated ATP-activated axonemes. This leads us to suspect that acetaldehyde may interact directly with axonemal dynein and/or tubulin, since dynein is the “motor molecule” of the axoneme and cilia motion is caused by the interaction of dynein with tubulin (in

the form of microtubules). To explore the possibility of a direct alteration of cilia proteins necessary for axonemal motility by acetaldehyde, we assessed the effects of acetaldehyde on the ATPase function of cilia dyneins. In a manner very similar to the observed cilia motion impairment, acetaldehyde induced a time- and concentration-dependent inhibition of ATPase activity in isolated axonemes. At the lowest concentration of acetaldehyde in which ATPase activity was significantly decreased from control conditions (30 PM), the degree of ATPase inhibition (10% inhibition) was less pronounced than the degree of cilia slowing (50% slowing). Several possibilities exist that could account for this difference. First, multiple dynein ATPase isoforms exist within axonemes (10) for which the relationship between beat frequency and cilia ATPase activity has not been established. Partial inhibition of the cilia1 ATPase activity may inactivate the entire cilium. It is possible, for example, that at low concentrations acetaldehyde preferentially inhibits axonemal ATPase isoforms more critical for motility than other isoforms so that the degree of total ATPase inhibition would not correlate linearly with the degree of cilia slowing. Second, there could be variations in the

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IW:. 4. Comparison of acetaldehyde binding to dynein with acetaldehyde-induced cilia ATPase inhibition. In parallel experiments with same fresh cilia preparation ATPase inhibition (filled circles) and [ 1,2I ‘C Jacetaldehyde binding to dynein heavy-chain region (open circles) were determined over a broad range of acetaldehyde concentrations. Both determinations were made after a 60-min preincuhation with acetaldehyde at 37°C. Binding to dynein heavy-chain region was assessed by gel electrophoresis as described (Fig. X?).

sensitivity of acetaldehyde between different fresh cilia preparations. Third, there could be contamination of nondynein ATPases unrelated to motility that are measured in the ATPase assay. Although our preliminary experiments showed that the majority of the ATPase activity measured with this assay is vanadate sensitive (>90% inhibition; data not shown), vanadate sensitivity is not routinely performed on cilia from each fresh preparation. Despite these small differences in the degrees of cilia slowing vs. ATPase inhibition at low concentrations of acetaldehyde, there was a similar concentration-dependent response in both assays. Since aldehyde-protein adduct formation has been linked to protein dysfunction in other models of aldehyde toxicity (14), we determined whether the cellular and biochemical impairment observed in respiratory cilia was related to acetaldehyde adduct formation with proteins involved in cilia motility. We found that acetaldehyde was capable of binding to multiple proteins present in the cilia axoneme. Acetaldehyde binding did not appear, however, to occur to all axonemal proteins but was most prominent to the dynein heavy-chain region and to tubulin. This is significant because the dynein complex directs the hydrolysis of ATP in the axoneme altering its attachment with microtubules and causing axonemal bending. Importantly, acetaldehyde binding to the dynein heavy-chain region closely paralleled cilia ATPase inhibition. It therefore appears that proteins that are very critical to the motor function of cilia are also those in which acetaldehyde appears to have a high binding potential. This suggests that dynein and tubulin might be particularly susceptible to acetaldehyde toxicity. The rapid time course of acetaldehyde-induced impairment of cilia motion and ATPase activity cannot be explained on the basis of stable adduct formation alone.

B

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FIG. 5. Comparison of various aldehydes and ethanol with acetaldehyde on cilia beating and cilia ATPase activity. Effects of acet,aldehyde (filled circles), ethanol (filled triangles), propionaldehyde (open circles), butyraldehyde (open triangles), isobutyraldehyde (filled diamonds), and benzaldehyde (open squares) determined on cilia beating over a range of concentrations. A: cilia beat frequency was measured as described. Beat frequency in each experiment was derived from mean of 5 different cells from different areas of each plate. H: cilia ATPase activity was measured as described. Activity represents mean of 2 separate determinations in t,he same experiment.

The generation of unstable acetaldehyde adducts, in the form of Schiff-base intermediates, is felt to precede the formation of stable acetaldehyde adducts and can occur seconds after exposure to acetaldehyde (34). Although these experiments were not specifically designed to differentiate between stable and unstable adduct formation, we think that unstable adduct formation would best explain the rapid time course of the ciliostatic and ATPase effects we observed. The threshold concentrations of acetaldehyde-induced impairment in these studies was -30 ,uM for ATPase inhibition and ciliostasis. This concentration is comparable to the acetaldehyde concentration that has been rex>orted in the vax>or Dhase of cigarette smoke (50 PM:

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ACETALDEHYDE-MEDIATED

Ref. 24). It is also possible that these concentrations of acetaldehyde might be encountered by airway epithelial cells in individuals ingesting ethanol. Acetaldehyde levels as high as 50 PM have been reported in the venous blood of drinking alcoholics in the older literature, but most investigators now agree that peripheral venous levels rarely exceed 1-5 PM (8). Acetaldehyde levels encountered by the lungs, however, are probably higher than peripheral venous levels, since hepatic venous blood levels can be as high as 30 PM and a significant arteriovenous drop in acetaldehyde levels has been observed across the pulmonary circulation (27). Additional evidence suggests that ethanol can be converted to acetaldehyde by microsomes present in lung tissue and by microorganisms present in the upper respiratory tract (24, 28). In addition to the actual molar concentration of acetaldehyde present during a given circumstance, it must also be considered that the stable adducts formed are covalent in nature and are t herefore irreversibly bound to the respective protein. This implies that even submicromolar concentrations of acetaldehyde might cause significant protein and cellular dysfuncti on due to the cumulative binding tha t may occur with repeated lowdose exposure. Chronic ethanol ingestion and cigarette smoking would very likely result in cumulative dysfunction if the rate of adduct formation exceeded the ability of the cells to replac se damaged adducted proteins. We cone lude that acetaldehy rde slows axoneme motility in airway epithelial cells and in isolated axonemes in vitro, inhibits ciliary dynein ATPase activity in isolated axonemes, and binds to ciliary proteins in a manner comparable to the ATPase inhibition. These effects appear to be specifically related to the aldehyde portion of the molecule. These findings suggest that acetaldehydeinduced ciliostasis is related to dynein ATPase inactivation, which may be caused, at least in part, by binding to dynein and other critical cilia proteins. This may be one important mechanism by which airway host defenses are impaired in clinical settings where acetaldehyde exposure occurs including alcohol ingestion and cigarette smoking.

CILIA

4.

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19. The aut,hors thank Drs. Richard B. Jennett and Michael F. Sorrel1 fi)r their helpful ideas and discussions in the early phase of these studies and Kathryn 1~. Leise and and Abbas Safari-Fard for their valuable technical assistance. This work was supported by a grant from the Nebraska Cancer and Smoking Disease Research Program. It was presented in part at the Annual Meeting of the American Thoracic Society, Cincinnati, OH, May 1989. Address for reprint requests: J. H. Sisson, Dept. of Medicine, Pulmonary Sect., University of Nebraska Medical Center, 42nd and Dewey Ave., Omaha, NE 68105-1065. Received

20 .July 1990; accepted

in f’inal form

27 July

20.

21.

22.

1990. 23.

REFERENCES E., H. T. PETERSON, D. E. BOHNING, AND M. Short-term effects of cigarette smoking on bronchial clearance in humans. Arch. Environ. Health 30: 361-367, 1975. 2. BLAKE, G. FL, T. D. ABEU,, ANI) W. G. STANI,EY. Cigarette smoking and upper respiratory infection among recruits in basic combat training. Ann. Intern. Med. 109: 198-202, 1988. 3. BOWMAN, B. ,J., S. E. MAINZER, K. E. ALLEN, AND C. W. SLAYMAN. 1. AI,RERT, LIPPMANN.

R.

24.

25.

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Acetaldehyde-mediated cilia dysfunction in bovine bronchial epithelial cells.

Acetaldehyde, which is present in significant concentrations in cigarette smoke and is elevated during alcohol ingestion, has been demonstrated to imp...
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