Rapid Reduction of Intracellular Glutathione in Human Bronchial Epithelial Cells Exposed to Occupational Levels of Toluene Diisocyanate

R. Clark Lantz*,1, Ranulfo Lemus{dagger}, Robert W. Lange{ddagger} and Meryl H. Karol{dagger}

* Department of Cell Biology and Anatomy, The University of Arizona, Tucson, Arizona 85724; {dagger} Department of Environmental and Occupational Health, University of Pittsburgh, Pittsburgh, Pennsylvania 15213; and {ddagger} 3M Pharmaceuticals, Pathology and Toxicology, St. Paul, Minnestoa 55144

Received September 21, 2000; accepted December 4, 2000


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Toluene diisocyanate (TDI) is a recognized chemical asthmogen, yet the mechanism of this toxicity and the molecular reactions involved have not been elucidated. We have previously shown that TDI vapor forms adducts with the apical surface of the respiratory epithelium, and that it colocalizes with ciliary tubulin. In vitro, we have shown rapid reaction of TDI with glutathione (GSH) and transfer of the bisGS-TDI adduct to a sulfhydryl-containing major histocompatibility complex peptide. This study sought to determine if intracellular GSH is altered following exposure to TDI. We used the dye CellTracker Green (chloromethylfluorescein, CMFDA) for detection of glutathione. One-day and 6-day air–liquid cultures of human bronchoepithelial cells (HBE) were exposed to 20–100 ppb TDI vapor for 5, 15, or 30 min. Cells were subsequently imaged using a confocal microscope. Both 1- and 6-day cultures showed a decrease in intensity of the thiol staining as a function of the TDI exposure dose. Doses as low as 20 ppb, the current permissible exposure limit (PEL) to TDI, resulted in rapid (within 5 min) decreases in fluorescence. The decreased fluorescence was not due to cytotoxicity or decrease in either esterase or glutathione-S-transferase activity, enzymes necessary for activation of the fluorescence of CMFDA. The decrease in glutathione levels was verified using anothher fluorescent label, ThioGl TM 1, and cell extracts. In addition, the mucus produced by 6-day air–liquid interface HBE cells in response to TDI exposure appeared to be protective, as HBE cells underlying mucus retained more fluorescence than did cells in the same cultures that were not covered with mucus. These results, along with previous data, strongly suggest that TDI enters pulmonary cells and reacts rapidly with intracellular GSH, and that this can occur at the current PEL of 20 ppb. This rapid reaction suggests the importance of cellular thiols in TDI-induced pulmonary disease.

Key Words: glutathione; toluene diisocyanate (TDI); human bronchial epithelial cells..


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Several low molecular weight compounds are recognized for their ability to induce occupational asthma. Among these, toluene diisocyanate (TDI), a highly reactive chemical used in the manufacture of polyurethane, is the most prevalent (Mapp et al., 1994Go). However, even though numerous studies have been performed, the pathogenic mechanism of TDI asthma remains unclear.

Previous work from our laboratory has sought to define the molecular events that occur in the early phases of the disease by characterizing adducts that are formed by the chemical. In vitro studies have shown that TDI reacts rapidly with glutathione to form a bis (S) adduct at physiological pH and temperature (Day et al., 1997Go). In vivo, TDI was found to bind to at least five proteins in the lavage fluid following inhalation exposure of guinea pigs (Jin et al., 1993Go). TDI was found to localize to the apical surfaces and to the cilia of airway epithelial cells following either in vivo or in vitro exposure (Ebino et al., 1998Go; Karol et al., 1997Go; Lange et al., 1999bGo).

These experiments demonstrate the formation of TDI adducts and interaction of TDI with airway epithelial cells. However, the majority of the research was done at 100–500 ppb, concentrations 5 to 25 times greater than the maximal allowable workplace concentration of 20 ppb.

The aim of the current study was to determine whether exposure of human bronchial epithelial cells to TDI would alter intracellular glutathione concentration. Time- and dose-dependent TDI-induced alterations of glutathione were investigated using realistic doses (20–100 ppb) that could be experienced in the workplace. The results imply a chemical pathway for the toxic effects of TDI and infer the importance of oxidative stress in susceptibility to the toxic effects of this chemical agent.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Human bronchial epithelial (HBE) cell culture.
Primary cultures of HBE cells were kindly provided by Dr. Joseph Pilewski, University of Pittsburgh Medical School. The cells are noncancerous, surgical specimens from lung transplant recipients isolated as previously described (Mette et al., 1993Go). HBE cells were cultured according to Lange et al. (1999a). Briefly, undifferentiated cells were grown submerged in Keratinocyte-Serum Free Medium (K-SFM, Gibco Gaithersburgh, MD) supplemented with epidermal growth factor (5 ng/ml, Gibco), bovine pituitary extract (50 µg/ml, Gibco), Fungizone (1% w/v, Gibco), penicillin (100 U/ml. Gibco)/streptomycin (100 µg/ml, Gibco) and L-glutamine (2 mM, Gibco). Passage 2 HBE cells were detached from culture flasks (0.05% trypsin with EDTA, Gibco) and seeded at 8 x 104 cells/cm2 on 12 or 24 mm diameter, clear TranswellTM tissue culture inserts (Corning-Costar, Grand Island, NY) in K-SFM. Cells were fed with K-SFM in the apical and basal compartments, respectively. The apical medium was removed when HBE cells reached confluence. The basal medium was replaced with a 1:1 mixture of D-MEM (Gibco) and F-12 culture media (Gibco) supplemented with 2% ULTROSER G (BioSepra S.A., France), Fungizone, penicillin, streptomycin, and L-glutamine as above, henceforth referred to as air–liquid interface (ALI) medium. ALI culture favors the development and retention of epithelial features including bioelectric properties, mucin secretion, and ciliagenesis (Gray et al., 1996Go). HBE cells were incubated 1–6 days at the ALI prior to TDI exposure.

Exposure of HBE Cells to TDI and collection.
The TranswellsTM containing the HBE cell monolayers were placed onto a stainless steel sieve held in a 28-cm glass petri dish that contained 1 ml of medium. Cells remained hydrated during the exposure by wicking the medium through the sieve. The petri dishes containing HBE cells were placed on a platform in a 70-liter glass chamber and exposed to 20, 50, or 100 ppb TDI vapor for 5, 15, or 30 min. TDI vapor was generated by passing dried air through a glass impinger containing 4 ml neat TDI as previously described (Jin et al., 1993Go). Real-time chamber atmosphere monitoring was performed using an AutostepTM continuous toxic gas analyzer (Bacharach, Inc., Pittsburgh, PA) with its probe placed ca. 5 cm above the culture dishes. Control, identically cultured TranswellsTM were placed in a similar tank and exposed using PBS in the impinger.

Cell viability.
Cell viability was analyzed using the Live/Dead Reduced Biohazard Viability/Cytotoxicity Kit (Molecular Probes, Eugene, OR). After exposure, cells were washed in PBS. The PBS was removed and cells were incubated in the dilute dye solution for 15 min at room temperature. Dyes were washed from the cells with PBS and cells were fixed with 4% glutaraldehyde for 1 h. Membranes containing the cells were then removed, mounted on glass slides, covered with coverslips, and viewed using a Leica TCS confocal microscope. Two-wavelength analysis was performed to determine the percentage of live cells (green fluorescence) versus dead cells (red fluorescence).

Fluorescence detection of cellular glutathione (GSH).
Cellular GSH levels were analyzed using 5-chloromethylfluorescein diacetate (CMFDA, Molecular Probes, Eugene, OR). After exposure, the TranswellsTM containing the HBE cell monolayers were incubated with prewarmed (37°C) CMFDA-containing medium (10 µM). After 30 min at 37°C, the media was replaced with fresh prewarmed medium, and cells were incubated for an additional 30 min at 37°C. Cells were washed with PBS and fixed with 3.7% paraformaldehyde. Membranes containing cells were removed, mounted on glass slides, and viewed with a Leica TCS confocal microscope.

Quantification of cellular GSH.
Cellular GSH content in HBE cells was estimated using the thiol-specific fluorophore ThioGloTM 1 (Covalent Associates Inc., Woburn, MA) as previously described (Lange et al., 1999aGo; Langmuir et al., 1996Go). Briefly, after TDI exposure, 0.4 ml M-Per mammalian protein extraction reagent (Pierce, Rockford, IL) was added to each TranswellTM and plates were rocked gently for 10 min at ambient temperature. HBE cells were scraped from the TranswellsTM using a rubber policeman, and samples were stored at –70°C for subsequent analysis.

HBE samples were thawed on ice and centrifuged for 10 min at 13,000 rpm. Total protein in the supernatant was determined using the Bradford assay (Bio-Rad, Hercules, CA). For GSH analysis, an aliquot of cell supernatant was added to 50 mM Na2PO4 buffer (pH 7.4) to a final volume of 290 µl in a 96-well plate. Ten microliters of 100 µM ThioGloTM 1 was added. The solution was mixed and the fluorescence at 530 {eta}m was recorded. Fluorescence represented emission from ThioGloTM 1-adducted GSH. Sample measurements were compared to a standard curve of GS-adducted ThioGloTM 1 fluorescence, constructed by the same procedure as described above, with aliquots of 0.1 mM GSH added in place of the cell supernatant.

Analysis of cellular esterase activity.
Cellular esterases were analyzed using 5-(and 6-) carboxyfluorescein succinidimyl ester (CFSE, Molecular Probes, Eugene, OR). After TDI exposure, the cell monolayers were incubated for 1 h at ambient temperature with 5 µM CFSE-containing PBS (pH 7.4). Cells were washed in dye-free buffer before fixing with 3.7% paraformaldehyde. Membranes containing cells were removed and mounted on glass slides for viewing with a Leica TCS confocal microscope.

Analysis of glutathione-S.-transferase (GST) activity.
GST activity toward 1-chloro-2,4-dinitrobenzene (CDNB, Sigma, St. Louis, MO) was determined as described by Habig et al. (1974). Briefly, assays were conducted at 25°C using 1 mM CDNB in 100 mM potassium phosphate at pH 6.5, containing 5 mM GSH (Sigma) and 100 µM EDTA. The absorbance was monitored at 340 {eta}m and the activity was calculated using the {varepsilon}340 {eta}m 9.6 mM–1cm–1. A unit of activity was defined as the amount of enzyme catalyzing the formation of 1 µmole of product per min. Specific activity was defined as the units of enzyme per milligram of protein.

Confocal microscopy.
Images of fluorescently stained cells were obtained using a Leica TCS confocal microscope equipped with a Kr/Ar laser and a Leica inverted scope. Epithelium was imaged using a 40x lens. In order to ensure uniformity for comparison of changes in fluorescence as a function of TDI exposure dose, all samples were analyzed the same day with the same settings on the microscope. In addition, images of control and air-exposed cells were taken at the beginning and end of the microscope session to ensure that there was not significant drift of the confocal during the imaging session. Two HBE cultures were evaluated at each time and dose. Four randomly selected fields in each culture were taken. Intensity levels from each field were used to estimate the average intensity for each time and dose. Images presented in the figures have intensities that are closest to the average intensity for that time and dose. Average field grayscale values were determined using Photoshop. Images were converted to grayscale and average intensity of the field was obtained using the histogram function.

Statistics.
Data were analyzed using a two-factor analysis of variance, with dose and time of exposure being the two factors. Significant differences between groups was determined using a Newman-Keuls analysis with significance set at p < 0.05.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Intracellular GSH Detected by CMFDA
CMFDA is a cell-permeant chloromethyl derivative of fluorescein diacetate. The compound remains nonfluorescent until cleaved by intracellular esterases. Glutathione-S-transferase (GST)-mediated reactions then produce a cell-impermeant product. Because glutathione occurs in high concentrations in cells and GSTs are ubiquitous, CMFDA provides an excellent means for studying intracellular thiols.

GSH levels were evaluated in HBE cultures that had been grown at an air–liquid interface for 1 or 6 days. Previous results had shown that GSH levels were much higher in 1-day cultures compared with 6-day cultures (Lange et al., 1999aGo). Similar results were seen with the CMFDA staining (Fig. 1Go). Fluorescent intensity was much greater in day 1 cultures compared with day 6. Confocal laser power and gain and offset of the photomultiplier tube (PMT) were set so that maximum intensity of day 1 cultures was just below saturation of the PMT. These settings were then used to obtain the images for both day 1 and day 6 cultures. CMFDA can be used under our culture conditions, and the intensity is reflective of the abundance of GSH in the HBE cells.



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FIG. 1. Confocal images comparing fluorescence between 1-day culture and 6-day culture controls using same settings for confocal microscopy. Cultures were labeled to detect thiols with CMFDA. Intensity of fluorescence was much higher in 1-day cultures. Bar: 25 µm.

 
Dose-dependent Reduction of GSH
The effect of TDI exposure on intracellular thiols was determined using CMFDA as the indicator (Figs. 2–4GoGoGo). Cultures that had been at an air–liquid interface for 1 or 6 days were exposed to TDI concentrations of 20 to 100 ppb for up to 30 min. After exposure, cultures were stained with CMFDA as described in Materials and Methods. Images were obtained using confocal microscopy.



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FIG. 2. One-day cultures exposed to TDI (0–100 ppb) for up to 30 min. Cells were labeled with CMFDA to detect cellular thiols. Fluorescent intensity decreased as a function of concentration. Decreases in fluorescence were present at 5 min exposure. No mucus was present in these cultures. All panels were taken with the same confocal settings. Bar: 25 µm.

 


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FIG. 3. Six-day cultures of bronchoepithelial cells. Cells were exposed to various concentrations of TDI (0–100 ppb) for up to 30 min. Cells were labeled with CMFDA to detect the level of thiols in the cells. While little or no mucus was present in the air controls, TDI exposure led to increased presence of mucus covering the cells. Images in this figure were taken over areas of the cultures covered by mucus. Fluorescent intensity decreased as a function of concentration. All panels were taken with the same confocal settings. Bar: 25 µm.

 


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FIG. 4. Six-day cultures of bronchoepithelial cells. Cells were exposed to increasing concentrations of TDI (0–100 ppb) for up to 30 min. Cells were labeled with CMFDA to detect intracellular thiols. In this case images were taken over areas of the cultures that were not covered by mucus. Fluorescent intensity decreased as a function of concentration. In addition, compared to Figure 3Go, decreases in intensity occurred at lower doses. This indicates an apparent protective effect of mucus. All panels were taken with the same confocal settings as those in Figure 3Go. Bar: 25 µm.

 
Because of differences in basal levels of thiols between day 1 and day 6 cultures, comparison of the effects of TDI were made within each culture and not between day 1 and day 6 cultures. Confocal settings for day 1 and day 6 cultures were established to give maximal intensity without saturation of the PMT for air-exposed cells at each culture time. Thereafter, settings were not changed so that comparison of the relative effect of TDI exposure could be assessed. Visual examination and measurement of relative intensities of images from control cultures taken before and after completion of the collection of images for all the TDI exposures were similar, indicating no drift in the confocal during the collection of the images.

Exposure of either day 1 or day 6 cultures led to a dose-dependent decrease in the fluorescent intensity of CMFDA (Figs. 2–4GoGoGo). Air-exposed cells did not show any apparent alteration in intensity up to 30 min of exposure. However, concentrations of TDI as low as 20 ppb for as few as 5 min led to significant loss of fluorescence. Increasing levels of exposure (50 and 100 ppb) led to increased loss of the fluorescence in an apparent dose-dependent manner. Although decreased fluorescence was seen at 5 min, very little additional decrease was seen up to the 30-min exposures. This indicates that the effect of TDI on thiols occurs within the first 5 min of exposure.

Additional differences in the response of the 6-day cultures to TDI exposure were noted. Exposure of cells to 20 ppb TDI or higher led to secretion of mucus that appeared to have a protective effect. The presence of mucus in the samples was apparent when the cells were observed with transmitted light microscopy prior to confocal imaging. Samples were scanned using low magnification transmitted light microscopy to find areas where mucus was either present or absent. At 20 ppb, most of the cells in the 6-day cultures were covered with mucus. Air-exposed controls did not produce any apparent mucus. At a given TDI concentration, higher fluorescence intensity was noted in cells covered with mucus (Fig. 3Go) compared with those that were not covered (Fig. 4Go). Images in Figures 3 and 4GoGo were taken from the same cultures with the same confocal settings. Lower fluorescent intensity was seen for each TDI concentration for the cells that were not covered with mucus.

Measurement of average graylevel intensity for each time and dose verified the visual results (Figs. 5–7GoGoGo). All concentrations of TDI led to significant decreases in fluorescence. A dose-dependent decrease in intensity was noted for each of the cultures, with significant decreases following 20 ppb TDI exposure (Fig. 5Go). Further decreases were seen at 50 ppb. However, 50 and 100 ppb were not different from each other. Significant differences were seen only with dose, not with time of exposure (Figs. 6 and 7GoGo). This verifies our earlier observation that reactions occurred rapidly, within the first 5 min of exposure.



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FIG. 5. Average grayscale intensity levels for HBE cells cultured 1 day in air and exposed to TDI. At all TDI concentrations studied, the average intensity of the CMFDA fluorescence was significantly reduced from air controls. In addition, 50 and 100 ppb exposures were significantly different from 20 ppb. Significant changes in intensity were a function only of TDI concentration. There were no significant differences due to length of exposure. A = significantly different from air controls. B = significantly different from air controls and 20 ppb TDI exposure. (p < 0.001).

 


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FIG. 6. Average grayscale intensity levels for HBE cells cultured 6 days at the air–liquid interface then exposed to TDI. Intensities were measured in cells that were covered with a mucous layer. All levels of exposure were significantly different from each other. Significant changes in intensity were a function only of TDI concentration and not duration of exposure. A = significantly different from all other TDI exposure concentrations. (p < 0.001)

 


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FIG. 7. Average grayscale intensity levels for HBE cells cultured 6 days at the air–liquid interface then exposed to TDI. Intensities were measured in cells that were not covered with a mucous layer. The average intensity of the CMFDA fluorescence was significantly reduced from air controls at all TDI concentrations studied. The percent decrease in intensity was greater in these cells compared with cells in the same cultures that were covered with mucus (Fig. 6Go). No significant differences were seen between any of the TDI-exposed cultures. Significant changes in intensity were a function only of TDI concentration. There were no significant differences as a function of length of exposure. A = significantly different from air controls. (p < 0.001)

 
Comparison of the intensity of the fluorescence for cells covered and not covered by mucus (day 6 cultures) shows that, at least at the lower TDI concentrations, mucus protected the cells from the TDI-induced reduction in fluorescence. Intensity in cells covered with mucus decreased in a dose-dependent manner (Fig. 6Go), with each dose being significantly different from every other dose. Cells that were not covered with mucus showed more rapid decline in fluorescence at lower doses. Again, exposure time did not have an effect on the response seen.

GSH Levels Detected by ThioGlo
We used an independent means to verify that thiols were decreased as a result of TDI exposure. The reaction between GSH in cellular homogenates and ThioGloTM (a maleimide derivative) involves addition of the thiol across the double bond of the maleimide to yield a fluorescent thioether. Intracellular thiol reactions with CMFDA occur via the chloromethyl group of CMFDA reacting with GSH (through what is believed to be a GST-mediated reaction), producing a cell-impermeable thioether. Fluorescence results from cleavage of CMFDA acetate groups by intracellular esterases.

Differentiated cell monolayers were exposed to 20 ppb TDI for 5 or 30 min, and the cellular GSH was quantified using ThioGlo. Results are shown in Figure 8Go. Exposure of 6-day cells resulted in a decrease in GSH to approximately 60% of control levels. Two-factor analysis of variance showed that the decrease was significant, and that there was no difference between the 5- and 30-min exposures. These results are comparable to those found using CMDA for analysis.



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FIG. 8. Reduced glutathione levels in 6-day air cultures of HBE cells. GSH levels were significantly reduced after exposure to 20 ppb TDI for as few as 5 min. There were no significant differences between 5-min and 30-min exposures. A = significantly different from air controls. (p < 0.025)

 
Effect of TDI on Cell Viability, Esterase, and GST Activities
The ability of CMFDA to detect thiols requires active esterases and glutathione-S-transferases. Because cells must be viable to retain their esterase activity, we also examined cell viability to be confident that the decreased CMFDA fluorescence we detected was indeed the result of altered GSH levels. The effect of TDI on cell viability was evaluated using the live-dead assay, which uses alterations in membrane permeability to nucleic acid stains as an index of viability. The live cell dye is permeant to the cell membrane in living cells, whereas the dead cell dye will stain nucleic acids only when the cell membrane has been compromised. Values were obtained from cells covered with mucus. Exposure of 6-day cultures to 20 ppb TDI for 30 min did not result in any decrease in live cell or increase in dead cell staining (Fig. 9Go). Therefore, 20 ppb TDI administered for 30 min was not cytotoxic.



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FIG. 9. Confocal images of 6-day HBE cultures exposed to 20 ppb TDI for 30 min. Cell viability was assessed with live-dead assays in columns 1 and 2. No decrease in live cell fluorescence or increase in dead cells was seen. Esterase activity, measured by fluorescence of CSFE, was also not affected by exposure to TDI. The last column shows TDI-induced decrease in CMFDA fluorescence. All cultures represented in this figure were simultaneously exposed to TDI. Bar: 25 µm.

 
Esterase activity in HBE cells was evaluated using 5-(and 6-)carboxyfluorescein succinidimyl ester (CFSE, Molecular Probes, Eugene, OR). CFSE is a cell-permeant dye that requires esterase activity to activate the fluorophore. Figure 9Go shows that 20 ppb TDI administered for 30 min did not alter the esterase activity. No significant differences in intensity were seen between air and 20 ppb TDI-exposed cells. Therefore, the observed decrease in CMFDA fluorescence following 20 ppb TDI for 30 min (Fig. 9Go) was neither due to cell death nor to decrease in esterase activity.

Lastly, CMFDA fluorescence could be altered by decreases in GST activity. GSTs are required to transfer the thiol to the dye, making the latter impermeable to the cell membrane and thus trapping the dye within the cell. Decreased GST could result in less dye trapped and apparent lower cellular fluorescence. We did not find GST activity to be significantly affected by 20 ppb TDI, even after 30 min of exposure.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The molecular events that occur in the early phases of occupational chemical asthma are not known. We have hypothesized that sensitization is initiated by covalent binding of the chemical to distinct sites on biomacromolecules and that the nature of the initial and subsequent chemical reactions is critical to the sensitization outcome. We have previously detected TDI colocalized with tubulin on the cilia of HBE cells (Lange et al., 1999bGo) and detected TDI-glutathionyl adduct formation in vitro (Day et al., 1997Go), in cell cultures, and in vivo using mice intrabronchially instilled with TDI (Lange et al., 1999aGo). The goal of this study was to investigate the intracellular chemical reactions of TDI using exposure levels occurring in the workplace and focusing on cellular thiols and thiol-dependent enzymes.

The results of this study demonstrate that TDI exposure can alter intracellular glutathione. TDI at the current permissible exposure level (20 ppb) led to significant decreases in GSH concentrations. The decreases occurred within the first 5 min of exposure and remained stable during 30 min of exposure. The responses were dose dependent, with GSH levels decreasing with increasing doses up to 100 ppb. These data, along with our previous results, strongly suggest that TDI can enter pulmonary cells and react rapidly with GSH to substantially lower intracellular concentrations.

GSH levels measured either in cellular extracts or by confocal intensity were found to differ in HBE cells cultured at the air–liquid interface for 1 day versus 6 days. In addition, both methods of analysis indicated that GSH levels decreased after TDI treatment, falling to approximately 60–70% of control levels (Figs. 6 and 8GoGo). With both methods, significant differences were seen only with exposure dose and not with time.

According to the literature, on day 5 HBE cells at the air–liquid interface are flat and poorly differentiated. By 7 days, the cell surface is scattered with microvilli (Kaartinen et al., 1993) and pseudostratification. Secretory cells are visible at day 8 (De Jong et al., 1994Go). Prolongation of the culturing period to 14–21 days at the air–liquid interface increases progressively the number of ciliated cells, maturation of cilia, and goblet-type secretory cells. We have previously reported that in 14- to 21-day cultures, TDI colocalizes to the cilia. In 14- to 21-day cultures, ciliated cells make up approximately 50–60% of the cells present. Experiments performed in our current study were done on 1-day and 6-day air–liquid interface cells. Although we have not examined the cellular phenotypes in our cultures, the literature would suggest that the cells are still relatively undifferentiated at these times. Even so, the presence of mucus in the 6-day cultures suggests that cells capable of producing and secreting mucus are present. It is therefore interesting to note that the decrease in CMFDA fluorescence occurred in all the cells in the culture. This would suggest that in addition to ciliated epithelial cells, TDI also affects mucous cells. Our experiments have shown that mucus is produced in the 6-day HBE cultures in response to TDI exposure, even at 20 ppb. This finding is similar to results we have reported in older cultures (Lange et al., 1999bGo).

Mucus production is a characteristic feature of TDI asthma. TDI exposure can lead to rapid secretion of mucus in differentiated human bronchial epithelial cells (Lange et al., 1999bGo). Our results suggest that mucus is protective to the cells. Cells covered by mucus retained higher levels of glutathione than did those in the same cultures that were not covered by mucus. This protection could be due to thiols in the mucins, as glycosylated and nonglycosylated domains of mucins from tracheobronchial epithelium have been shown to contain thiol residues (Rose et al., 1989Go). The thiols could react with TDI, effectively reducing the amount of TDI gaining intracellular entrance. Although mucous plugs have been associated with TDI-induced deaths, the secretion of mucus in response to TDI may also be a protective response.

Reduction of intracellular GSH can result in oxidative stress, making cells less able to resist cell damage from electrophiles such as TDI. Another consequence can be induction of apoptotic signals and cell death. Cell death was noted following treatment of vascular endothelial cells with inhibitors of glutathione synthesis (Slim et al., 2000Go). Increased cell death would be consistent with results from in vivo studies that have demonstrated loss of airway epithelium and denudation of airway epithelium following exposure to high doses of TDI (Fabbri et al., 1988Go; Gordon et al., 1985Go; Miller et al., 1986Go). Epithelial cell loss would allow increased access of agents, including TDI, to the submucosa, possibly resulting in increased airway reactivity. Loss of epithelium would also reduce the levels of neutral endopeptidase, thus increasing airway concentrations of bronchoconstrictive neuropeptides. In the current study, we did not observe cell death following exposure to 20 ppb TDI (Fig. 9Go), and previously we found that cell membrane integrity was unchanged following exposure of HBE cells to 100 ppb TDI for 30 min (Lange et al., 1999aGo). If reduced GSH levels result in cell death, the cytotoxic events must be occurring past 30 min of exposure or at higher TDI exposure levels.

Reduction of intracellular GSH levels (and inhibition of glutathione-dependent enzymes including glutathione reductase, glutathione-S-transferase, glutamyl transpeptidase, and glutathione peroxidase) leads to altered cellular redox status. Because of their close association with glutathione, these enzymes may be targets of TDI toxicity. Inhibition of glutathione reductase has been shown to occur following exposure to metabolites of methyl isocyanate (Jochheim and Baillie, 1994Go). If similar reactions are occurring with TDI, the result would be significant alteration of the cellular redox status. Indeed, TDI could produce a two-pronged effect, reducing cellular GSH levels by formation of TDI-GSH adducts and inhibition of enzymes responsible for regeneration of reduced glutathione. We have recent evidence that TDI inhibits {gamma}-glutamyl transpeptidase (unpublished data).

Alterations in cellular redox status have been shown to result in altered gene expression (Allen and Tresini, 2000Go). Evidence also indicates that decreased cellular glutathione levels promote expression of the Th2 lymphocyte phenotype that is characteristic of the allergic lung (Peterson et al., 1998Go). If TDI is capable of altering GSH levels in lymphocytes similar to what we have shown in epithelial cells, increased expression of Th2 phenotypes could occur. TDI can directly induce Th2 type responses. Using a local lymph node assay, Vandebriel et al. (2000) have shown that direct application of TDI leads to lymphocyte proliferation and production of Th2 cytokines. The mechanism of this response is not known.

Reduction of glutathione can alter the profile of important inflammatory mediators. Leukotrienes are lipid mediators that have been associated with inflammation and asthma. The primary product of the lipoxygenase metabolic pathway is LTA4, which can be metabolized to either LTB4 (a potent chemoattractant for neutrophils) or LTC4 (O'Byrne, 1997Go). Airway epithelial production of these important mediators has been shown to occur following exposure to ozone (McKinnon et al., 1993Go). The pathway for production of LTC4 requires glutathione and a glutathione transferase enzyme (leukotriene C4 synthase). LTC4 and the subsequently produced cysteinyl leukotrienes are intimately associated with asthma. Reduction of cellular glutathione would shift the production of leukotrienes away from LTC4 toward increased production of LTB4, leading to chronic inflammation (Rouzer et al., 1981Go). Support for this mechanism is the finding that late and dual asthmatic reactions to TDI are associated with an acute inflammatory reaction characterized by increased neutrophils and eosinophils in the BALF. These cellular increases were accompanied by an increase in LTB4 in the BALF. As a majority of subjects with TDI asthma continue to have asthma long after cessation of exposure (Zocca et al., 1990Go), these results suggest that persistent airway inflammation may be involved.

Although we have shown that the immediate short-term effects of TDI are to lower cellular glutathione levels, the chronic effects of exposure are not known. The long-term response of the cells may be to increase, rather than decrease, glutathione levels by inducing {gamma}-glutamylcysteine synthetase, the rate-limiting enzyme responsible for synthesis of glutathione. This type of response (including the immediate decrease in glutathione levels) has been seen with other thiol-reactive compounds such as arsenic and cigarette smoke (Ochi, 1997Go; Rahman et al., 1996Go). However, within 24 h, the amount of glutathione exceeded preexposure levels.

Heterogeneity of glutathione distribution both within cellular compartments and between individual pulmonary cells has been reported (Forkert and Moussa, 1993Go; West et al., 2000Go). Clara cells and type II alveolar epithelial cells appear to have high concentrations of GSH. Exposure to 1,1-dichloroethylene significantly decreased the levels (Forkert and Moussa, 1993Go). Using the same dye used in the current study, West et al. (2000) demonstrated that the highest intensity of labeling within Clara cells colocalizes with mitochondrial indicators. Diffuse labeling was seen throughout the cytoplasm, with little or no staining in the nucleus. Although not the emphasis of this study, our results show a similar pattern of staining. The most intense staining occurred in spheroidal areas of the cells, with less staining in the cytoplasm. The nucleus appears not to be stained. (for example, see Fig. 9Go, thiol staining in air-exposed cells). TDI exposure decreased the staining intensity in both the spheroidal compartments and in the cytoplasm.

As with isolated Clara cells, our results show heterogeneity of intensity between cells. West and colleagues postulate that the heterogeneity of glutathione levels determines the response of individual cells to toxicant-induced oxidative stress. This may be true of bronchial epithelial cells as well and suggests that if heterogeneity exists in situ, cells of the airways may differ in their susceptibility to TDI.

While heterogeneity of fluorescent intensity between cells exists, we used the overall field intensity to evaluate TDI-induced effects. Field intensity is a good indicator of the average cellular response, as within a given exposure and time, average intensity was consistent from field to field. In addition, the intensity levels measured with confocal microscopy were indicative of levels of glutathione measured by other means.

In conclusion, we have shown that TDI stimulates increased mucus secretion and dose-dependent intracellular glutathione reduction in human bronchial epithelial cells. The reduction of GSH is rapid, occurring within the first 5 min of exposure, and occurs at TDI concentrations that are at the current permissible exposure level. Decreased glutathione can result in altered cellular redox status and production of inflammatory mediators that have been associated with TDI-induced asthma. Further elucidation of the chemical pathway pursuant to TDI exposure should contribute valuable information regarding the pathogenesis of TDI asthma and individual susceptibility.


    ACKNOWLEDGMENTS
 
Supported by NIEHS Center Grant #ES06694 and NIEHS #05651.


    NOTES
 
1 To whom correspondence should be addressed. Fax: (520) 626-2097. E-mail: lantz{at}email.arizona.edu. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Allen, R. G., and Tresini, M. (2000). Oxidative stress and gene regulation. Free Radic. Biol. Med. 28, 463–499.[ISI][Medline]

Day, B. W., Jin, R., Basalyga, D. M., Kramarik, J. A., and Karol, M. H. (1997). Formation, solvolysis, and transcarbamoylation reactions of bis(s-glutathionyl) adducts of 2,4- and 2,6-diisocyanatotoluene. Chem. Res. Toxicol. 10, 424–431.[ISI][Medline]

De Jong, P. M., van Sterkenburg, M. A., Hesseling, S. C., Kempenaar, J. A., Mulder, A. A., Mommaas, A. M., Dijkman, J. H., and Ponec, M. (1994). Ciliogenesis in human bronchial epithelial cells cultured at the air-liquid interface. Am. J. Respir. Cell Mol. Biol. 10, 271–277.[Abstract]

Ebino, K., Kramarik, J., Lemus, R., and Karol, M. H. (1998). A mouse model for the study of localized toluene diisocyanate adducts following intrabronchial administration of the chemical: Inflammation and antibody production. Inhal. Toxicol. 10, 503–529.[ISI]

Fabbri, L. M., Danieli, D., Crescioli, S., Bevilacqua, P., Meli, S., Saetta, M. and Mapp, C. E. (1988). Fatal asthma in a subject sensitized to toluene diisocyanate. Am. Rev. Respir. Dispos. 137, 1494–1498.

Forkert, P. G., and Moussa, M. (1993). Temporal effects of 1,1-dichloroethylene on nonprotein sulfhydryl content in murine lung and liver. Drug Metab. Dispos. 21, 770–776.[Abstract]

Gordon, T., Sheppard, D., McDonald, D. M., Distefano, S., and Scypinski, L. (1985). Airway hyperresponsiveness and inflammation induced by toluene diisocyanate in guinea pigs. Am. Rev. Respir. Dis. 132, 1106–1112.[ISI][Medline]

Gray, T. E., Guzman, K., Davis, C. W., Abdullah, L. H., and Nettesheim, P. (1996). Mucociliary differentiation of serially passaged normal human tracheobronchial epithelial cells. Am. J. Respir. Cell. Mol. Biol. 14, 104–112.[Abstract]

Habig, W. H., Pabst, M. J., and Jakoby, W. B. (1974). Glutathione S-transferases: The first enzymatic step in mercapturic acid formation. J. Biol. Chem. 249, 7130–7139.[Abstract/Free Full Text]

Jin, R., Day, B. W., and Karol, M. H. (1993). Toluene diisocyanate protein adducts in the bronchoalveolar lavage of guinea pigs exposed to vapors of the chemical. Chem. Res. Toxicol. 6, 906–912.[ISI][Medline]

Jochheim, C. M., and Baillie, T. A. (1994). Selective and irreversible inhibition of glutathione reductase in vitro by carbamate thioester conjugates of methyl isocyanate. Biochem. Pharmacol. 47, 1197–1206.[ISI][Medline]

Kaartinen. L., Nettesheim, P., Adler, K. B., and Randell, S. H. (1993). Rat tracheal epithelial cell differentiation in vitro. In Vitro Cell Dev. Biol. Anim. 29A, 481–492.

Karol, M. H., Jin, R., and Lantz, R. C. (1997). Immunohistochemical detection of toluene diisocyanate (TDI) adducts in pulmonary tissue of guinea pigs following inhalation exposure. Inhal. Toxicol. 9, 63–83.[ISI]

Lange, R. W., Day, B. W., Lemus, R., Tyurin, V. A., Kagan, V. E., and Karol, M. H. (1999a). Intracellular S-glutathionyl adducts in murine lung and human bronchoepithelial cells after exposure to diisocyanatotoluene. Chem. Res. Toxicol. 12, 931–936.[ISI][Medline]

Lange, R. W., Lantz, R. C., Stolz, D. B., Watkins, S. C., Sundareshan, P., Lemus, R., and Karol, M. H. (1999b). Toluene diisocyanate colocalizes with tubulin on cilia of differentiated human airway epithelial cells. Toxicol. Sci. 50, 64–71.[Abstract]

Langmuir, M. E., Yang, J-R., Le Compte, K. A., and Durand, R. E. (1996). New thiol active fluorophores for intracellular thiols and glutathione measurement. In Fluorescence Microscopy and Fluorescent Probes (Y. Slavik, Ed.), pp. 229–233. Plenum Press, New York.

Mapp, C. E., Saetta, M., Maestrelli, P., Di Stefano, A., Chitano, P., Boschetto, P., Ciaccia, A., and Fabbri, L. M. (1994). Mechanisms and pathology of occupational asthma. Eur. Respir. J. 7, 544–554.[Abstract/Free Full Text]

McKinnon, K. P., Madden, M. C., Noah, T. L., and Devlin, R. B. (1993). In vitro ozone exposure increases release of arachidonic acid products from a human bronchial epithelial cell line. Toxicol. Appl. Pharmacol. 118, 215–223.[ISI][Medline]

Mette, S. A., Pilewski, J., Buck, C. A., and Albelda S. M. (1993). Distribution of integrin cell adhesion receptors on normal bronchial epithelial cells and lung cancer cells in vitro and in vivo. Am. J. Respir. Cell. Mol. Biol. 8, 562–572.[ISI][Medline]

Miller, M. L., Andringa, A., Vinegar, A., Adams, W. D., Cibulas, W., Jr., and Brooks, S. M. (1986). Morphology of tracheal and bronchial epithelium and type II cells of the peripheral lung of the guinea pig after inhalation of toluene diisocyanate vapors. Exp. Lung Res. 11, 145–163.[ISI][Medline]

O'Byrne, P. M. (1997). Leukotrienes in the pathogenesis of asthma. Chest 111, 27S–34S.[Abstract/Free Full Text]

Ochi, T. (1997). Arsenic compound-induced increases in glutathione levels in cultured Chinese hamster V79 cells and mechanisms associated with changes in gamma-glutamylcysteine synthetase activity, cystine uptake and utilization of cysteine. Arch. Toxicol. 71, 730–740.[ISI][Medline]

Peterson, J. D., Herzenberg, L. A., Vasquez, K., and Waltenbaugh, C. (1998). Glutathione levels in antigen-presenting cells modulate Th1 versus Th2 response patterns. Proc. Natl. Acad. Sci. U.S.A. 95, 3071–3076.[Abstract/Free Full Text]

Rahman, I., Smith C. A., Lawson, M. F., Harrison, D. J., and MacNee, W. (1996). Induction of gamma-glutamylcysteine synthetase by cigarette smoke is associated with AP-1 in human alveolar epithelial cells. FEBS Lett. 396, 21–25.[ISI][Medline]

Rose, M. C., Kaufman, B., and Martin, B. M. (1989). Proteolytic fragmentation and peptide mapping of human carboxyamidomethylated tracheobronchial mucin. J. Biol. Chem. 264, 8193–8199.[Abstract/Free Full Text]

Rouzer, C. A., Scott, W. A., Griffith, O. W., Hamill, A. L., and Cohn, Z. A. (1981). Depletion of glutathione selectively inhibits synthesis of leukotriene C by macrophages. Proc. Natl. Acad. Sci. U.S.A. 78, 2532–2536.[Abstract]

Slim, R., Toborek, M., Robertson, L. W., Lehmler, H. J., and Hennig, B. (2000). Cellular glutathione status modulates polychlorinated biphenyl-induced stress response and apoptosis in vascular endothelial cells. Toxicol. Appl. Pharmacol. 166, 36–42.[ISI][Medline]

Vandebriel, R. J., De Jong, W. H., Spiekstra, S. W., Van Dijk, M., Fluitman, A., Garssen, J., and Van Loveren, H. (2000). Assessment of preferential T-helper 1 or T-helper 2 induction by low molecular weight compounds using the local lymph node assay in conjunction with RT-PCR and ELISA for interferon-{gamma} and interleukin-4. Toxicol. Appl. Pharmacol. 162, 77–85.[ISI][Medline]

West, J. A. A., Chichester, H., Buckpitt, A. R., Tyler, N. K., Brennan, P., Helton, C., and Plopper, C. G. (2000). Heterogeneity of Clara cell glutathione. A possible basis for differences in cellular responses to pulmonary cytotoxicants. Am. J. Respir. Cell Mol. Biol. 23, 27–36.[Abstract/Free Full Text]

Zocca, E., Fabbri, L. M., Boschetto, P., Plebani, M., Masiero, M., Milani, G. F., Pivirotto, F., and Mapp, C. E. (1990). Leukotriene B4 and late asthmatic reactions induced by toluene diisocyanate. J. Appl. Physiol. 68, 1576–1580.[Abstract/Free Full Text]