* Department of Anatomy, Physiology, and Cell Biology, University of California, Davis, California 95616; Department of Molecular Biosciences, University of California, Davis, California 95616
Received June 15, 2004; accepted August 11, 2004
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ABSTRACT |
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Key Words: glutathione (GSH); respiratory toxicity; naphthalene; P450 metabolism; high-performance liquid chromatography; HPLC.
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INTRODUCTION |
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We hypothesized that NA acts directly on respiratory tissues to cause the loss of GSH in animals exposed to NA. To test this hypothesis, we exposed mice to NA vapor, which when inhaled reaches the respiratory tract directly, avoiding first-pass hepatic metabolism. Following a single NA inhalation exposure (24 h duration), GSH levels and cytotoxicity were monitored 824 h post-exposure. In addition to measuring liver and lung GSH levels, we surgically isolated defined regions of the respiratory tract (i.e., intrapulmonary conducting airways and nasal ethmoid turbinates) by microdissection, for GSH analysis. The respiratory architecture is complex, with nearly 50 cellular phenotypes organized into intricate branching patterns (Harkema, 1992; Plopper et al., 1998
). Isolating intrapulmonary airways and olfactory epithelium allowed us to measure GSH levels within samples enriched for target cells, e.g., Clara cells represent 4% of the lung by mass but 50% of the airway epithelium (Plopper et al., 1997
). We utilized two concentrations of NA vapor (1.5 and 15 ppm) anticipated to show different degrees of injury based on a prior inhalation dose-response study (West et al., 2001
). These concentrations bracket the current human workplace exposure limit of 10 ppm for 8 h (OSHA, 2003
).
Half of the mice in each treatment group were given the GSH depleting agent diethylmaleate (DEM) 1 hour prior to the start of NA exposure. DEM produces a rapid, yet transient, loss of GSH through enzymatic conjugation of DEM to GSH (Boyland and Chasseaud, 1967). With less GSH available, more of the reactive NA metabolites would persist within target cells, potentially increasing severity and onset of toxicity. Earlier studies had demonstrated that depletion of GSH with DEM can increase the severity of lung injury and increase protein adduct formation 6-fold in mice given intraperitoneal doses of NA (Warren et al., 1982
). However, reactive NA metabolites formed in the liver are capable of reaching the lungs via the bloodstream (Buckpitt and Warren, 1983
), raising the possibility that increased protein adducts reflect changes in hepatic metabolism and detoxification of NA. Inhalation delivers NA directly to the lung, minimizing the role of the liver as a source of circulating metabolites targeting the lung. In order to better understand how GSH loss affects NA metabolism and adduct formation within the respiratory tract, intrapulmonary airway segments were isolated from DEM treated animals by microdissection and then incubated with [14C]-labeled naphthalene in vitro.
Using NA inhalation and isolated airways, this study addressed the following questions: (1) does exposure to inhaled NA deplete respiratory tract GSH directly; (2) can the liver supply sufficient GSH to maintain respiratory GSH pools during inhaled toxicant exposure; and (3) does GSH loss increase NA covalent binding within airways exposed to NA in culture?
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METHODS |
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Animals and treatment. Outbred male NIH Swiss mice, aged 710 weeks, were purchased from Harlan Laboratories (San Diego, CA). Animals were allowed free access to food and water and were not fasted before treatments. They were housed in cages on sterile paper fiber bedding in HEPA-filtered isolation chambers at the University of California, Davis, for at least 5 days before use. Mice were exposed to filtered air (control) or naphthalene vapor in glass metabolism cages (BioServ, Inc., Frenchtown, NJ) as described previously (West et al., 2001). Air volume through the chamber was maintained at 1.0 liter min1 resulting in a complete change of air every 5 min. Samples of chamber air were extracted into acetonitrile (80% extraction efficiency), and the naphthalene concentration was determined by absorbance at 219 nm (extinction coefficient of 8,896 M1 cm1). Exposures were conducted between 8:00 a.m. and noon to minimize diurnal fluctuations in GSH levels and lasted 2 or 4 h. Samples were collected for GSH analysis and histopathology immediately after exposure to naphthalene vapor or after allowing the mice to recover in fresh air for 424 h as indicated. Half of the mice were given the GSH depleting agent diethylmaleate (1 g/kg i.p. dose; 1.0 M solution in corn oil) 1 hour prior to starting exposure; a dose and time combination that would produce maximal GSH depletion in the lung and liver (unpublished results) before the naphthalene vapor exposure was started. An equivalent volume of corn oil was given to animals not treated with DEM (NA only animals), as a control for the DEM/corn oil treatment. Animals were euthanized with an overdose of pentobarbital sodium.
HPLC analysis of GSH. A previously described method (Lakritz et al., 1997) was employed for the quantitation of GSH in tissues. Briefly, acidic tissue homogenates were subjected to reverse-phase HPLC coupled with electrochemical detection to directly measure reduced GSH. The response was linear from 1.6 to 3200 pmol of GSH (R2 = 0.995). GSH levels were measured in liver, lung, proximal (near the trachea) and distal (near the alveoli) intrapulmonary airways, and in nasal ethmoid turbinates. Intrapulmonary airways were isolated by microdissection procedures described in detail by Plopper et al. (1991) with the modification that the lungs were perfused free of blood with ice-cold 1 mM EDTA/saline while still in the chest. To isolate nasal tissue, the head was split along the nasal septum with Teflon (PTFE)-coated razor blades, and the ethmoid turbinates were physically removed under a dissecting microscope with forceps. At least 4 animals were evaluated at each time point for each treatment. GSH measurements were normalized to protein levels (Lowry et al., 1951
).
High-resolution histopathology. A minimum of 3 mice were compared for each treatment at each time point. Lungs were inflated in situ via a tracheal cannula with 4% formaldehyde, buffered to pH 7 with phosphate at 30 cm water pressure. Nasal tissue was fixed by forcing fixative through the nose with a syringe via a cannula inserted into the nasopharanx followed by immersion in the fixative for at least 3 days. Blocks of fixed lung tissue (35 mm thick) were sampled in orientations both parallel and perpendicular to the main airway path. Careful sampling was necessary because inhaled toxicants do not produce uniform injury patterns, because of a combination of airflow dynamics (e.g., laminar vs turbulent) and the complex three-dimensional branching pattern of the airways, which causes localized differences in dose (Overton et al., 1989; Plopper et al., 1998
). The lateral side of the first airway generation of the left lobe and the terminal bronchioles were selected to represent the proximal and distal airways, respectively. These two regions were examined separately for injury (and for GSH levels) because studies have shown differential susceptibility of these regions to NA; and this may be due to phenotypic differences in the Clara cell populations in proximal compared to distal regions (Plopper et al., 1992
; West et al., 2001
). Careful sampling of nasal tissue was also required, and four sagittal sections were prepared along the length of the nose for embedding. Prior to sampling, the fixed nasal tissues were de-calcified by immersion in 13% formic acid for 4 days (Eggert and Germain, 1979
), followed by extensive rinsing with water. Areas of ethmoid turbinate with high amounts of olfactory epithelium (relative to respiratory epithelium) were chosen for imaging. All tissues were embedded in glycol methacrylate resin. Two-micron thick sections were stained with methylene blue/azure II, and fields were recorded at 256x magnification on an Olympus BX41 microscope (Olympus International, Melville, NY) equipped with a Q-imaging cooled CCD-camera (Burnaby, B.C., Canada).
Naphthalene metabolism. Mice were either treated with diethylmaleate (1 g/kg; 1.0 M corn oil solution) or an equivalent volume of corn oil. One hour later, animals were euthanized and intrapulmonary airways were isolated by microdissection. Following isolation, airways were allowed to recover in Waymouth's media for 30 min in a shaking water bath at 37°C in 2 ml Teflon (PTFE) microcentrifuge tubes (Upchurch Scientific, Oak Harbor, WA). At the end of the 30-min incubation a small portion of each sample was removed to measure tissue GSH levels as described above. To the remaining tissues, each in 500 µl of media, was added 1 µl of 20 mM [14C]-naphthalene in methanol (50 mCi/mmol), to reach a final naphthalene concentration of 0.2 mM. [Note: 0.2 mM is well above the Km of CYP2F2 (3µM), the predominant cytochrome P450 monooxygenase isozyme responsible for naphthalene metabolism, effectively making these incubations saturated with naphthalene.] After 60 min at 37°C, 1 ml of acetone was added to quench the reactions before storing the incubation mixtures overnight at 20°C. Tissues were separated from media by centrifugation at 14,000 x g for 15 min. Unmetabolized naphthalene is volatile, whereas the metabolites are not, so the resulting tissue pellets were dried under reduced pressure. Once dry, they were washed exhaustively with acetone, dried a second time, and dissolved in 1 N NaOH for analysis by liquid scintillation counting. The incubation media was also dried under reduced pressure before dissolving it in 50/50 methanol/water for analysis of water-soluble NA metabolites by HPLC. Aliquots were taken for liquid scintillation counting, and an aliquot of the dissolved tissue fraction was used to determine the protein content (Lowry et al., 1951) using BSA as a standard.
HPLC analysis of metabolites. Individual naphthalene metabolites from the incubation media were separated by reverse phase HPLC as described (Lakritz et al., 1996) with the minor modification that the mobile phase consisted of buffer A (0.6% triethylamine-phosphate, pH 3.1) developed against pure acetonitrile. The gradient was generated as follows: starting with 98% buffer A and 2% acetonitrile, moving to 11% acetonitrile by 60 min, 85% acetonitrile by 90 min, and 100% acetonitrile from 95 to 110 min, and ending at 120 min. Metabolite standards were prepared (see below) containing diastereomeric mixtures of the glutathionyl-NA, cysteinyl-glycyl-NA, cysteinyl-NA, and N-acetyl-cysteine-NA conjugates of naphthalene epoxide (e.g., 1,2-dihydro-1-hydroxy-2-cysteinyl-naphthalene), as well as naphthalene and naphthalene dihydrodiol. Samples were spiked with the mixture of standards before HPLC analysis, and the elution order of the metabolites was determined by monitoring the absorbance at 260 nm. Eluate fractions were collected every minute, and the radioactivity was determined by liquid scintillation counting. Radioprofiles were matched with UV absorbance traces from HPLC so that the amounts of the individual metabolites could be calculated; they are presented as nmoles of metabolite formed per mg protein per 60 min.
Standards were prepared by reacting racemic NA-epoxide [0.2 mmol; prepared as described by (Yagi and Jerina, 1975)] with 0.1 mmoles of the appropriate thiol compound (e.g., GSH, cysteine, etc.) in 5 ml of 0.1 M sodium phosphate buffer at pH 8.5 under an argon atmosphere. Unreacted epoxide was extracted into diethyl ether, and the aqueous phase was loaded onto a styrene divinylbenzene solid phase extraction column pre-equilibrated with 2% acetic acid. The column was washed with 10 volumes of 2% acetic acid, and the thiol derivatives were eluted with 75% acetonitrile before lyophilization. The purity and structure of the individual conjugates was checked by HPLC (described above) and electrospray mass-spectrometry after loop injection in 0.1% formic acid/50% acetonitrile in negative ion mode (VG Quattro BQ, Fisons Instrument, Atrincham, England). An equimolar mixture was prepared in methanol from the individual conjugates, NA and NA diol, and stored at 80°C until used.
Statistical analysis. All data are reported as the mean ± 1 standard deviation. Statistical differences were determined by one-way analysis of variance (ANOVA) and the Bonfferoni-Dunn post-hoc testing method for pairwise comparisons at p < 0.05 significance level.
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RESULTS |
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In contrast with the alterations in airway epithelium after exposure to 15 ppm NA, the lower dose of 1.5 ppm resulted in no observable effects on Clara cells at either airway level (Fig. 6A and 6C). However, in animals exposed to both DEM and 1.5 ppm NA, Clara cell necrosis was apparent in the proximal airways (Fig. 6B) and initial signs of NA-induced injury (i.e., swelling) were evident in the distal airways (Fig. 6D). In the olfactory epithelium, 1.5 ppm NA caused some cell loss (Fig. 6E), and DEM pretreatment greatly increased the severity of the injury, with large portions of the ethmoid turbinates being devoid of epithelium (Fig. 6F).
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DISCUSSION |
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Previous studies have shown that inhaled NA is toxic to the respiratory tract (National Toxicology Program, 2000; West et al., 2001
), but this is the first study correlating GSH loss with inhaled NA respiratory toxicity. Toxicity was only apparent in tissues severely depleted of GSH (1.5 ppm NA + DEM or 15 ppm NA ± DEM) suggesting that substantial GSH loss must occur for injury to take place. The intrapulmonary airways lost the greatest percentage of GSH (Fig. 1B-C) and were the most extensively injured (Fig. 34), whereas the liver maintained more GSH and is not susceptible to injury (O'Brien et al., 1985
; Plopper et al., 1992
; Shopp et al., 1984
). It should be noted that in mice exposed to NA alone, hepatic GSH never dropped below 57.6 nmol/mg protein, substantially higher than lowest levels measured in the lung and airways, 9.7 and 1.0 nmol/mg protein, respectively (Fig. 1). Therefore, even though the liver did experience a decrease in GSH levels, the absolute concentration of GSH within the liver may have always been high enough to protect the cells from injury. Additionally, it should be noted that when DEM is given by itself, hepatic and pulmonary GSH levels can drop by 5090% within 1 h, recovering to steady-state values within 26 h (Deneke et al., 1985
; Gerard-Monnier et al., 1992
). Van Winkle et al. have found that soon after Clara cells are exposed to NA the endoplasmic reticulum and mitochondria dilate, increasing cell volume about 50% (Van Winkle et al., 1995
; Van Winkle et al., 1999
). Epithelial swelling was evident at the end of the 15 ppm NA 4 hour exposure (Fig. 3B and 4B). However, at the same time point the Clara cells were smaller and denser than normal in animals pretreated with DEM (Fig. 3C and 4C), resembling the later stages of NA-induced Clara cell death. This suggests that DEM treatment is accelerating the injury process by removing GSH needed for NA detoxification.
The olfactory epithelium was more sensitive to NA inhalation than the intrapulmonary airways, with injury developing from exposure to 1.5 ppm for 2 h. GSH depletion with DEM greatly enhanced NA injury to the olfactory epithelium (Fig. 5 and 6EF), however, it was not clear if exposure to NA alone caused GSH losses in the nasal tissues. Because we were unable to clear the nasal capillaries of blood during tissue collection, our GSH measurements might be skewed by the high content of GSH found in red blood cells (2.4 mM; (Anderson et al., 1985)). Chronic inhalation of NA vapors has been shown to induce olfactory neuroblastomas in rats in a concentration dependent manner (National Toxicology Program, 2000
), prompting the listing of NA as a probable human carcinogen (National Toxicology Program, 2003
). The current OSHA limits for human exposure to NA are 10 ppm TWA for 8 h with a standard threshold of 15 ppm (OSHA, 2003
). Given that 1.5 ppm NA is much lower than the current exposure limit and that it produced significant injury (Fig. 6E), our results may raise concerns over the adequacy of current limits, although it has not been established how good of a model mice are for NA respiratory toxicity in humans.
The data in this study reinforce the concept that GSH loss sensitizes cells to injury from bioactivated toxicants such as NA by allowing more adducts to form. In DEM treated animals the levels of protein adducts nearly doubled (0.33 to 0.65 nmol/mg protein) but was countered by a large decrease in water-soluble metabolites (decreasing from 8.88 to 4.59 nmol/mg protein/60 min). The result was a net decrease in total NA metabolism (43%). By quantifying individual NA metabolites, we were able to determine that an overall decrease in metabolism was occurring, not simply a shift in the types of metabolites formed. However, the total decrease in metabolism was smaller (26%) when determined by summing the individual metabolites resolved by HPLC when compared with analyzing all of the metabolites before separation (43%; compare Fig. 7 with Table 1). A decrease NA-GSH conjugates might be anticipated given the decreased availability of GSH in tissues isolated from DEM treated animals, but it was surprising that NA diol, a product of epoxide hydrolase, was also decreased by 45%. There was a slight increase in a group of unidentified metabolites (Table 1), which may represent particularly toxic intermediates (e.g., naphthoquinones). Regardless of the nature of these metabolites, the number of NA adducts formed nearly doubled in airways depleted of GSH with DEM and may be responsible for the accelerated injury seen in DEM treated animals.
A few reports have questioned the use of DEM as a GSH depleting agent on the basis that it might produce changes in cellular and toxicant metabolism not solely related to GSH depletion (Costa and Murphy, 1986; Krack et al., 1980
; Reiter and Wendel, 1982
). However, it was also noted that preparations of DEM can be contaminated with up to 20% diethylfumarate (Plummer et al., 1981
), which can inhibit enzymes of the citric acid cycle. It is unclear how DEM inhibits NA metabolism but it does not likely involve contaminants, as we used the highest purity DEM available (98.4% by gas chromatography). Severe GSH loss has the potential to effect toxicant metabolism by several means, e.g., decreased pools of NADPH available to drive cytochrome P450 monooxygenase activity, or changes in enzyme structure, such as glutathionylation, which can occur with a drop in the GSH:GSSG ratio (Pompella et al., 2003
; Schafer and Buettner, 2001
). Whatever the cause, it is clear that DEM inhibits NA metabolism in airways, and perhaps it does so indirectly by causing GSH depletion.
There may be other factors contributing to NA toxicity that we were unable to evaluate here. For example, we did not examine what role lung-lining fluid plays in NA inhalation toxicity, even though it is very rich in GSH and other antioxidants (Cross et al., 1994). Further, we were not able to precisely measure the biochemical state within target cell types explicitly; rather we used tissues enriched for target cells. Microdissected airways are a rich source of Clara cells, but they also contain ciliated and basal cells, fibroblasts, and large amounts of extracellular matrix protein (e.g., collagen). As our measurements were scaled against protein levels, there may not be sufficient experimental resolution to accurately describe the GSH levels and covalent binding within Clara cells. Unfortunately, isolated Clara cells have not been successfully maintained in culture in a differentiated state. Airways are the best current method for conducting in vitro incubations.
This study represents an important step in defining the relationship between the status of the intracellular GSH pool and P450-mediated cytotoxicity in Clara cells and nasal olfactory epithelium. GSH loss in the respiratory tract is due to the direct effect of the bioactivated cytotoxicant NA on target tissues and is not dependent on the hepatic supply of GSH or its precursors. Apparently, the reservoir of GSH in the liver is not available to balance loss in the respiratory tract within a time frame sufficient to modulate or prevent injury. GSH depletion by DEM enhances the inhaled NA respiratory tract toxicity and increases covalent binding of reactive NA metabolites to protein.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed at Department of Molecular Biosciences, University of California, 1311 Haring Hall, Davis, CA 95616. Fax: (530) 752-4698. E-mail: aphimister{at}ucdavis.edu.
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