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γ-Glutamyl transpeptidase null mice fail to develop tolerance to coumarin-induced Clara cell toxicity
Food and Chemical Toxicology 48 (2010) 1612–1618
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c-Glutamyl transpeptidase null mice fail to develop tolerance to coumarin-induced Clara cell toxicity Jeffrey D. Vassallo a,*, Rhonda S. Kaetzel b, Stephanie L. Born a, Cindy L. Lewis a, Lois D. Lehman-McKeeman a, Donald J. Reed c a b c
Miami Valley Innovation Center, The Procter and Gamble Company, 11810 East Miami River Road, Cincinnati, OH 45252, United States Exponent, 15375 SE 30th Place, Suite 250, Bellevue, WA 98007, United States Oregon State University, 1037 ALS, Corvallis, OR 97331, United States
a r t i c l e
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Article history: Received 25 November 2009 Accepted 22 March 2010
Keywords: Coumarin Glutathione Knockout mouse Glutamyl transpeptidase Tolerance Clara cell
a b s t r a c t Coumarin was used as a model Clara cell toxicant to test the hypothesis that tolerance to injury requires increased c-glutamyl transpeptidase (GGT) activity. Wildtype (GGT+/+) and GGT-deficient (GGT/) mice on a C57BL/6/129SvEv hybrid background were dosed orally with corn oil (vehicle) or coumarin (200 mg/ kg). In vehicle-treated mice, Clara cell secretory protein (CC10) expression was distributed throughout the bronchiolar epithelium. After one dose of coumarin, CC10 expression was dramatically reduced and the bronchiolar epithelium was devoid of Clara cells in GGT+/+ and GGT/ mice. In wildtype mice, 9 doses of coumarin produced tolerance, characterized as a renewed bronchiolar epithelium with Clara cells expressing CC10 along with a 40% increase in total glutathione (GSH) and a 7-fold increase in GGT activity in the lung. In contrast, tolerance was not observed in GGT/ mice. To assess whether changes in whole lung levels of GSH and GGT activity reflect Clara cell specific changes an enriched population of cells was isolated from female wildtype B6C3F1 mice made tolerant to coumarin. Compared to Clara cells from control mice, GSH and GGT activity increased 3- and 13-fold, respectively. Collectively, these data suggest Clara cell tolerance to coumarin toxicity requires increased GGT activity favoring enhanced GSH synthesis. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Coumarin (1,2-benzopyrone), a natural product found in a variety of plants, is widely used in consumer products as a fragrance ingredient (Lake, 1999). Coumarin has also been evaluated clinically for the treatment of certain malignancies (Egan et al., 1990; Lake, 1999; Marshall et al., 1994). While toxicity in humans is rare, it is well recognized that coumarin is a rat hepatotoxicant. Coumarin is also acutely toxic to the mouse lung, where it selectively targets the non-ciliated bronchiolar epithelial cells, or Clara cells (Born et al., 1998). Additionally, chronic coumarin administration increased the incidence of lung tumors (alveolar/bronchiolar adenomas and carcinomas) in B6C3F1 mice, an effect not observed in F344 rats (NTP, 1993). Other agents known to cause Clara cell necrosis acutely and lung tumors in mice after chronic exposure include naphthalene (Mahvi et al., 1977; NTP, 1992; O’Brien et al., 1985), methylene chloride (Foster et al., 1992; NTP, 1986),
* Corresponding author. Address: Bristol-Myers Squibb, Route 206 and Provinceline Road, Princeton, NJ 08540, United States. Tel.: +1 609 252 7112; fax: +1 609 252 7046. E-mail addresses: [email protected] (J.D. Vassallo), [email protected] (R.S. Kaetzel), [email protected] (D.J. Reed). 0278-6915/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2010.03.034
trichloroethylene (Fukuda et al., 1983; Maltoni et al., 1986; NTP, 1990; Odum et al., 1992), and styrene (Cruzan et al., 1997, 2001; Green et al., 2001). These chemicals do not cause Clara cell necrosis or lung tumors in rats, suggesting a mechanism that may be unique to the mouse lung (Cruzan et al., 1997, 1998; Green et al., 2001; NTP, 1986, 1990, 1993, 2000). As the most metabolically-active cell type in the rodent lung, Clara cells are uniquely susceptible to xenobiotic-mediated toxicity (Serabjit-Singh et al., 1988; Widdicombe and Pack, 1982). Cytochrome P450 2F2 (CYP2F2), which is highly expressed in mouse Clara cells catalyzes the formation of reactive epoxide metabolites that contribute to naphthalene-, styrene- and coumarin-induced Clara cell toxicity (Born et al., 2002; Buckpitt et al., 1995; Green et al., 2001; Hynes et al., 1999; Nagata et al., 1990; O’Brien et al., 1985; Ritter et al., 1991). Naphthalene-, styrene-, and coumarin-derived epoxides are detoxified by glutathione (GSH) conjugation (Huwer et al., 1991; Lake, 1984; Pacifici et al., 1981; Warren et al., 1982). However, in the lung, steady-state GSH levels vary widely between individual Clara cells, and it has been demonstrated that this heterogeneity is a significant factor in determining the susceptibility of individual Clara cells to naphthalene toxicity (West et al., 2000a,b). A common feature of Clara cell toxicants is that following repeated exposure, Clara cells become refractory or tolerant to
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toxicity. This phenotype has been described for several chemicals, including naphthalene, coumarin, methylene chloride, trichloroethylene and 4-ipomeanol (Born et al., 1999; Boyd et al., 1981; Foster et al., 1992; O’Brien et al., 1989; Odum et al., 1992), and Clara cells tolerant to coumarin exhibit cross-resistance to naphthalene (Born et al., 1999). Although the exact mechanisms leading to the development of mouse Clara cell tolerance have yet to be established, it is generally recognized that tolerance is an adaptive response. It was originally suggested that Clara cell tolerance results from a reduction in metabolic activation (Foster et al., 1992; O’Brien et al., 1989; Odum et al., 1992). However, subsequent studies have determined that CYP2F2 expression levels and epoxidation rates are similar in lung fractions from control mice and mice made tolerant to naphthalene or coumarin (Born et al., 1999; West et al., 2002). In contrast, experiments focused on detoxification pathways have demonstrated that tolerance to naphthalene is associated with elevated GSH levels as well as an increase in c-glutamylcysteine synthetase (GCS) activity in mouse lung terminal bronchioles (West et al., 2002). Although GCS is rate-limiting in GSH synthesis, c-glutamyl transpeptidase (GGT), located on the outer surface of the plasma membrane, plays an important role in the extracellular hydrolysis of GSH that is required to provide the amino acids for GSH synthesis. GGT has been used as a marker of Clara cell injury in bronchiolar alveolar lavage fluid (Day et al., 1990) and is also markedly elevated in the lungs from B6C3F1 mice made tolerant to coumarin (Vassallo et al., 2000). In light of these data, and the fact that GGT plays an important role in intracellular GSH synthesis, the purpose of this work was to test the hypothesis that Clara cell tolerance to coumarin requires an increased level of GGT activity. For this work, GGTdeficient (GGT/) mice generated on a C57BL/6/129SvEv hybrid background were used as a model to determine whether tolerance to coumarin developed in the absence of GGT. Additional studies were conducted to determine if the coumarin-induced increase in GGT activity was specific to Clara cells isolated from B6C3F1 mice. 2. Materials and methods 2.1. Chemicals Coumarin was purchased from the Sigma–Aldrich Chemical Company and the purity exceeded 99% (Milwaukee, WI). Corn oil was purchased from Acros Organics (Geel, Belgium). All other reagents were HPLC grade or the highest grade available. 2.2. Animals Breeder pairs of heterozygous mice (GGT+/) (19–30 g) were previously generated on a C57BL/6/129SvEv hybrid background (Lieberman et al., 1996). Mice were bred and offspring were genotyped by polymerase chain reaction as previously reported (Will et al., 2000). Wildtype (GGT+/+) (18–32 g) and age-matched GGT-deficient (GGT/) (16–26 g) mice of both sexes were provided N-acetylcysteine (NAC) ad libitum (10 mg/ml, pH 7 in drinking water) at weaning and throughout the experimental period. Supplementation of NAC in the drinking water helps to restore GSH and cysteine levels and improves the health and longevity of these animals (Lieberman et al., 1996). Female B6C3F1 mice (19–25 g) were purchased from Harlan Sprague Dawley, Inc. (Indianapolis, IN). Female B6C3F1 mice were selected because previous work showing Clara cell toxicity and tolerance were demonstrated in this sex and strain (Born et al., 1998, 1999). All mice were housed in a temperature- and humidity-controlled environment with 12-h light/dark cycles. Animals were allowed rodent chow (Purina Laboratory Rodent Chow; Ralston-Purina, St. Louis, MO) and water ad libitum throughout the studies. All animal procedures were approved by The Procter and Gamble Company (AALAC accredited) and Oregon State University’s Lab Animal Resources facility Institutional Animal Care and Use Committee (IACUC). 2.3. In vivo assessment of coumarin-induced Clara cell toxicity and tolerance Preliminary studies with C57BL/6/129SvEv mice indicated that this strain develops coumarin-induced Clara cell toxicity and tolerance similar to B6C3F1 mice (data not shown) (Born et al., 1998, 1999). GGT+/+ and GGT/ mice, randomized by body weight into treatment groups (n = 7–8), were dosed orally by gavage with corn
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oil (vehicle) or coumarin (200 mg/kg; 5 ml/kg). This dose was selected because it has been previously shown to cause coumarin-induced lung toxicity and tolerance (Born et al., 1998, 1999). To evaluate acute toxicity, mice received a single dose of coumarin and the biochemical and histological changes were evaluated 24 h after dosing. To produce tolerance, mice were given a total of 9 doses of coumarin with a protocol similar to the NTP bioassay. Mice were dosed Monday–Friday (days 1–5), not dosed Saturday–Sunday with dose days 6–9 representing the 2nd week of dosing (Monday–Thursday). Biochemical and histological changes were evaluated 24 h after the last dose as previously described (Born et al., 1999). Separate groups of mice were used for biochemical, histopathological and immunohistochemical assessments. 2.4. Tissue collection for biochemical assessment Under sodium pentobarbital anesthesia (50 mg/kg; ip) mice were exsanguinated. Two small pieces of lung (<100 mg) were removed and homogenized with either five volumes of 5% sulfosalicylic acid for total glutathione analysis or five volumes of Tris–HCl buffer (0.1 M, pH 8.5) for GGT analysis. Total protein in the BALF and lung homogenates was determined by the Bradford assay with bovine serum albumin as the standard following solubilization in 0.1 N NaOH (Bradford, 1976). All samples were stored at 80 °C until time of use. 2.5. Tissue collection for histopathological assessment Mice were exsanguinated under sodium pentobarbital (50 mg/kg; ip) anesthesia, after which the lungs, with the trachea and heart attached, were removed and perfusion fixed using Karnovsky’s fixative at a constant pressure of 25 cm via the trachea. For histopathology, serial sections from lung blocks were cut at 5 lm thickness, stained with hematoxylin and eosin (H&E) and examined by light microscopy (magnification 60). 2.6. Tissue collection for immunohistochemical assessment Lungs were fixed by inflation with 1% paraformaldehyde. After immersion in 1% paraformaldehyde for 1 h, the tissues were transferred to 70% ethanol for processing overnight and embedding in paraffin the next day. Paraffin sections (5 lm thick) were stained for Clara cell secretory protein (CC10) using the avidin–biotin-peroxidase complex (ABC) method (Vector Laboratories, Burlingame, CA). All tissues were blocked for endogenous peroxidase activity for 10 min in 3% hydrogen peroxide, rinsed, and then blocked for proteins by incubating for 20 min in a Universal Blocking Reagent (Biogenex, San Ramon, CA). For CC10 staining, the primary antibody was an anti-rat CC10 antiserum (Singh and Katyal, 1984; kindly provided by Dr. Gurmukh Singh, University of Pittsburgh). The antiserum was diluted 1:10,000 and allowed to incubate on the slides for 1 h at room temperature. Following incubation with the primary antibody, the Elite Rabbit kit (Vector Laboratories) for the secondary antibody and the ABC steps were followed as per the manufacturer’s directions. Both stains were developed for 5 min using Vector’s 3,30 -diaminobenzidine kit with nickel enhancement. The sections of CC10 were counterstained with Mayer’s hematoxylin (Biogenex, San Ramon, CA). 2.7. Assessment of GSH and GGT activity in isolated Clara cells Female B6C3F1 mice, randomized by body weight into treatment groups (n = 4), were dosed orally by gavage with corn oil (vehicle) or coumarin (200 mg/kg) (5 ml/ kg). To induce tolerance, mice received 9 doses of coumarin (5 days/week), and Clara cells were isolated 24 h after the last dose (Born et al., 1999). This dose was selected because it has been previously shown to cause coumarin-induced lung toxicity and tolerance (Born et al., 1998, 1999). Clara cells were isolated according to the method of Bolton et al. (1993) and modified by replacing elastase with dispase. A Nycodenz gradient was used according to the method of Viscardi et al. (1992) instead of IgG panning (Ford and Rickwood, 1982). Cells were collected and counted on a hemocytometer. Cell viability and Clara cell purity were determined by erythrosine B exclusion and nitro blue tetrazolium staining, respectively (Devereux and Fouts, 1980). Following isolation, Clara cells were centrifuged at 700g for 10 min. The medium was removed and the cells were resuspended in 0.5 ml of 0.9% saline. This process was repeated two more times and following the third spin, the cells were resuspended in either 5% sulfosalicylic acid (106 cells/ml) for GSH analysis or Tris–HCl (100 mM, pH 8.5) (106 cells/0.1 ml) for GGT analysis. Following a brief vortex and sonication (10 s), the cells were either analyzed for protein and GGT activity or they were centrifuged at 14,000 rpm for 10 min and the supernatant was analyzed for protein and GSH. Total protein was determined by the Bradford assay with bovine serum albumin as the standard following solubilization in 0.1 N NaOH (Bradford, 1976). 2.8. Total glutathione determination Total glutathione, representing reduced GSH, oxidized glutathione (GSSG), and glutathione–protein mixed disulfides, was quantified in whole lung homogenates
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and BALF by HPLC as the fluorescent monobromobimane derivative following reduction with sodium borohydride (Slordal et al., 1993). Sulfosalicylic acidtreated samples were mixed vigorously, centrifuged, and then 50 ll of the supernatant was transferred to a tube containing 50 ll of an internal standard (glutathione ethyl ester), 20 ll of NaBH4, 900 ll of Tris and 50 ll of a 0.25% monobromobimane solution. Following a 15 min incubation at room temperature, samples were extracted with 1 ml of methylene chloride, after which a 200 ll aliquot was acidified with 100 ll of 1 N HCl. Chromatography was performed at 40 °C on a 75 mm 4.6 mm ID Zorbax SB C18 column with 3.5 lm particle size at a flow rate of 2 ml/min. Solvent A was composed of 0.2% trifluoroacetic acid (TFA) in 95:5 water:acetonitrile; solvent B contained the same acid modifier in 100% acetonitrile. A 10 ll sample was injected onto the column under initial conditions (100% solvent A) and a linear gradient was performed from 100% to 85% solvent A and 0% to 15% solvent B in 10 min using a Waters 2690 separation model HPLC with a Waters 2487 dual channel wavelength detector (Waters, Milford, MA). The column was allowed to equilibrate under initial mobile phase conditions for 6 min between injections. Total glutathione and the internal standard, monitored at an excitation wavelength of 380 nm and an emission wavelength of 465 nm, had retention times of approximately 5 and 8.5 min, respectively. Total glutathione quantification was based on an external standard curve with GSH.
2.9. GGT activity GGT was measured fluorometrically with L-c-Glu-7-amino-4-methyl-coumarin as a substrate (Smith et al., 1979). A sample volume of 100 ll, containing protein from control mice (lung = 50 lg) or mice made tolerant to coumarin (lung = 10 lg) was transferred to a 200 ll solution containing a final concentration of 0.1 M ammediol/HCl, 20 mM diglycine, 0.1% Triton X-100, and 26 lM L-c-Glu-7-amino4-methyl-coumarin (pH 8.5). Following incubation for 45 min in a 37 °C shaking water bath, the reaction was terminated by the addition of 1.7 ml of ice-cold 0.05 M glycine buffer (pH 10.4). The samples were mixed, transferred to a 96-well microtitre plate, and analyzed on a fluorometer (Perkin Elmer LS50B with a plate reader) with an emission wavelength and slit width setting of 440 and 15.0 nm, respectively, and an excitation wavelength and slit width setting of 370 and 2.5 nm, respectively. GGT activity was calculated as pmol of 7-amino-4-methylcoumarin (AMC)/min/mg protein based on a standard curve of AMC. 2.10. Statistics Data were analyzed by a Student’s t-test or ANOVA followed by a multiple comparison test (Fischer’s PLSD) using StatView (SAS Institute, Cary, NC) at a 5% significance level (p < 0.05).
Fig. 1. Coumarin-induced Clara cell toxicity and tolerance in GGT+/+ and GGT/ mice. Mice were dosed orally with corn oil or coumarin (200 mg/kg) for 1 or 9 days (5 days/ week) and lung histology was evaluated 24 h after the last dose. Normal terminal bronchioles from GGT+/+ (A) and GGT/ (B) mice shows Clara cells with apical caps (black arrows). A single dose of coumarin caused exfoliation of the bronchiolar epithelium in GGT+/+ (C) and GGT/ (D) mice. Necrotic Clara cells are sloughed off in the bronchiole lumen (black arrow) whereas the ciliated epithelial cells are unaffected by the treatment (grey arrow). Clara cells repopulated the bronchiolar epithelium (black arrow) following repeated coumarin administration in GGT+/+ mice (E) whereas the bronchiolar epithelium remained devoid of Clara in the GGT/ mice (F), indicating tolerance did not develop. In the absence of Clara cells, the epithelium appears thicker and more disorganized compared to the control.
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3. Results The histomorphology of terminal bronchioles from mice following single or repeated coumarin administration (200 mg/kg) is illustrated in Fig. 1. The terminal bronchioles from vehicle-treated GGT+/+ and GGT/ mice were similar, with Clara cells and ciliated cells lining the epithelium in an evenly distributed and alternating manner (Fig. 1A and B). Following a single oral dose of coumarin, Clara cells were almost completely ablated from the terminal bronchioles in GGT+/+ and GGT/ mice (Fig. 1C and D). Repeated administration of coumarin led to the development of tolerance in GGT+/+ mice and this phenotype was characterized by a renewing of the bronchiolar epithelium with apical capped Clara cells (Fig. 1E), although the epithelium appeared somewhat thickened and disorganized. In contrast, the bronchiolar epithelium remained
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devoid of Clara cells in GGT/ mice indicating that tolerance, following repeated coumarin administration, did not develop (Fig. 1F). Collectively, these data are consistent with the development of coumarin-induced Clara cell toxicity and tolerance in B6C3F1 mice, indicating that there are no strain differences (Born et al., 1998, 1999). To demonstrate that coumarin selectively targets the Clara cell, a marker of Clara cell differentiation (CC10) was evaluated by immunohistochemical detection in mice following single or repeated coumarin administration (Fig. 2). In vehicle-treated GGT+/+ and GGT/ mice, expression of CC10 was intense and evenly distributed throughout the bronchiolar epithelium (Fig. 2A and B), indicating that GGT deficiency does not affect CC10 expression. Following a single dose of coumarin, CC10 expression was dramatically reduced, and this is consistent with
Fig. 2. CC10 immunohistochemistry of Clara cells in GGT+/+ and GGT/ mice. Mice were dosed orally with corn oil or coumarin (200 mg/kg) for 1 or 9 doses (5 days/week) and CC10 immunohistochemistry was evaluated 24 h after the last dose. Terminal bronchioles from normal GGT+/+ (A) and GGT/ (B) mice shows intense and evenly distributed Clara cell-specific CC10 expression (black arrows). Consistent with Clara cell necrosis, a single dose of coumarin caused a significant diminution in CC10 expression in both GGT+/+ (C) and GGT/ (D) mice. Despite the absence of apical capped Clara cells, positively stained cells appear in localized foci along the bronchiolar epithelium (black arrow). Following repeated coumarin administration, a significant increase in CC10 expression was noted in GGT+/+ mice (E). In contrast, CC10 expression in apical capped Clara cells did not return in GGT/ mice (F). However, positively stained cells appear in localized foci along the bronchiolar epithelium (black arrow).
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Table 1 Pulmonary total glutathione and GGT activity following single or repeated dosing of coumarin.a Mouse +/+
GGT
GGT/
Treatment b
Control Coumarin Coumarin Controlb Coumarin Coumarin
Days of dosing
Total glutathione (nmol/mg protein)
GGT (pmol/min/mg protein)
9 1 9 9 1 9
8.0 ± 0.4 10.9 ± 1.1* 11.0 ± 1.0* 7.4 ± 1.4 5.7 ± 0.9 7.3 ± 0.7
40.2 ± 7.8 60.3 ± 8.2 260.5 ± 13.5* –c –c –c
a GGT+/+ and GGT/ mice were dosed with corn oil or coumarin (200 mg/kg) for 1 or 9 days (5 days/week). Total glutathione and GGT activity were determined 24 h after the last dose. Data represent the mean and SE of 5–6 mice per group. b Total glutathione and GGT activity were similar in mice dosed with corn oil for 1 (data not shown) or 9 days. c – Denotes no detectable GGT activity in GGT/ mice. * Statistically significant at p < 0.05.
Table 2 Total glutathione and GGT activity in Clara cells from B6C3F1 mice following repeated dosing of coumarin.a Treatment
Cell viability (%)b
Clara cell purity (%)c
Total glutathione (nmol/mg protein)
GGT (pmol/min/mg protein)
Control Coumarin
90.6 ± 0.6 92.2 ± 3.4
62.3 ± 7.4 61.9 ± 5.8
3.2 ± 1.0 10.5 ± 2.5*
22.8 ± 7.3 287.5 ± 39.2*
a B6C3F1 mice were dosed with corn oil or coumarin (200 mg/kg) for 9 days (5 days/week). Total glutathione and GGT activity were analyzed 24 h after the last dose in a Clara cell enriched fraction. Data represent the mean and SE of three separate isolations. b Determined by erythrosine B exclusion. c Determined by staining with nitro blue tetrazolium. * Statistically significant at p < 0.05.
the loss of viable Clara cells from the bronchiolar epithelium (Fig. 2C and D). With repeated coumarin administration, tolerance to toxicity resulted in a significant increase in CC10 expression in GGT+/+ mice whereas expression remained reduced in GGT/ mice (Fig. 2E and F). Total glutathione levels were similar in GGT+/+ and GGT/ mice supplemented with NAC in the drinking water (Lieberman et al., 1996). Table 1 illustrates that pulmonary total glutathione levels in control GGT+/+ and GGT/ mice were similar. The level of total glutathione in perfused whole lung homogenates from GGT+/+ mice was increased by 40% 24 h after a single dose of coumarin and remained elevated as Clara cell tolerance developed following repeated coumarin administration (Table 1). The development of tolerance in GGT+/+ mice was also associated with a 7-fold increase in GGT activity in whole lung homogenates. In contrast, the level of total glutathione in whole lung homogenates from GGT/ mice did not increase following single or repeated coumarin administration. To assess whether changes in whole lung levels of GSH and GGT activity reflect specific changes in tolerant Clara cells, an enriched population of these cells was isolated from B6C3F1 mice treated with corn oil or coumarin for 9 doses. Compared to Clara cells from vehicle-treated mice, there was a 3- and 13-fold increase in GSH and GGT activity, respectively in the enriched fractions of Clara cells (Table 2).
4. Discussion GGT plays a significant role in GSH regulation and is markedly increased in response to a variety of lung oxidant injuries (Zhang and Forman, 2009). As such, the GGT/ mouse has been used as a model to investigate the role of GSH regulation in protecting the lung from hyperoxia- and bleomycin-induced injury (Barrios et al., 2002; Klings et al., 2009; Pardo et al., 2003). Similarly, the GGT/ mouse is an ideal model for investigating whether tolerance to coumarin-induced Clara cell toxicity, which is associated with an increase in GGT and GSH, could develop without GGT. In
the absence of GGT, mice exhibit a severe glutathionuria resulting from the inability to hydrolyze and recycle GSH in renal proximal tubules. Consequently, cysteine deficiency develops and leads to a reduction in GSH synthesis and a chronic intracellular GSH deficiency in most tissues, including Clara cells (Jean et al., 2003). While this deficit results in a reduced life-span, supplementation of NAC in the drinking water helps to restore GSH and cysteine levels and improves the health of these animals (Lieberman et al., 1996). It has been shown that Clara cell tolerance to naphthalene-induced toxicity is an adaptive cellular response associated with an induction in GCS activity which favors increased GSH synthesis (West et al., 2000a, 2002). While GGT activity was not measured in the aforementioned studies, the current data suggest that Clara cell tolerance requires GGT. GGT+/+ mice showed a significant increase in pulmonary total glutathione associated with the development of Clara cell tolerance, whereas GGT/ mice were unable to upregulate GSH formation, exhibited severe lung toxicity, and showed no evidence of Clara cell tolerance. Given that GCS is dependent on the extracellular availability of cysteine in maintaining intracellular GSH homeostasis via regulation of the c-glutamyl cycle, it is likely that a common mechanism of Clara cell tolerance exists, and is coordinated with an increase in both GGT and GCS activity which favors increased GSH synthesis. Since GSH conjugation to coumarin 3,4-epoxide is a non-enzymatic detoxification reaction, the increased synthesis of GSH could help protect the Clara cell from this reactive metabolite (Vassallo et al., 2004a,b). This hypothesis is consistent with the fact that susceptibility of Clara cells to naphthalene toxicity is dependent on GSH levels (West et al., 2000a,b). While a comprehensive assessment of the molecular changes associated with tolerance to coumarin has not been reported, this adaptive phenotype, favoring increased GGT activity, may represent an early preneoplastic change as chronic exposure to naphthalene or coumarin is associated with an increased incidence of mouse lung tumors (NTP, 1993, 2000). It is generally recognized that increased GGT expression is a marker of preneoplastic foci in the liver during chemical carcinogenesis (Pitot et al., 1985),
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and GGT is simultaneously increased in the bronchiolar epithelium in concert with tumor development resulting from exposure to 3methylcholanthrene in the rat lung (He, 1991). Establishing a causal association between increased GGT expression and preneoplastic changes in the lung will require additional studies. However, this work lays the foundation that Clara cell toxicity is an adaptive response requiring an increase in pulmonary GGT activity. This in turn is likely to favor increased synthesis of GSH and may ultimately provide a selective growth advantage to the affected cells. Conflict of Interest The authors declare that there are no conflicts of interest. Acknowledgements This work was carried out at The Procter and Gamble Company (JDV, SLB, CLL, LDLM) and Oregon State University (RSK and DJR) and was supported in part by the Research Institute for Fragrance Materials and by the following NIH grants: ES-00040, ES-00210, and ES-01978. The authors would like to thank Dr. Michael Lieberman for providing the GGT+/ breeder mice used for these studies. The technical assistance provided by Marda Brown, Tamara Fraley, LouAnn Amberg and Bill Amberg in the breeding, screening and maintenance of the GGT-deficient colony was also greatly appreciated. References Barrios, R., Shi, Z.-Z., Kala, S.V., Wiseman, A.L., Welty, S.E., Kala, G., Bahler, A.A., Ou, C.-N., Lieberman, M.W., 2002. Oxygen-induced pulmonary injury in c-glutamyl transpeptidase-deficient mice. Lung 179 (5), 319–330. Bolton, J.L., Thompson, J.A., Allentoff, A.J., Miley, F.B., Malkinson, A.M., 1993. Metabolic activation of butylated hydroxytoluene by mouse bronchiolar Clara cells. Toxicol. Appl. Pharmacol. 123 (1), 43–49. Born, S.L., Fix, A.S., Caudill, D., Lehman-Mckeeman, L.D., 1998. Selective Clara cell injury in mouse lung following acute administration of coumarin. Toxicol. Appl. Pharmacol. 151 (1), 45–56. Born, S.L., Fix, A.S., Caudill, D., Lehman-Mckeeman, L.D., 1999. Development of tolerance to Clara cell necrosis with repeat administration of coumarin. Toxicol. Sci. 51 (2), 300–309. Born, S.L., Caudill, D., Fliter, K.L., Purdon, M.P., 2002. Identification of the cytochromes P450 that catalyze coumarin 3,4-epoxidation and 3hydroxylation. Drug Metab. Dispos. 30 (5), 483–487. Boyd, M.R., Burka, L.T., Wilson, B.J., Sastry, B.V., 1981. Development of tolerance to the pulmonary toxin, 4-ipomeanol. Toxicology 19 (2), 85–100. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal. Biochem. 72, 248–254. Buckpitt, A., Chang, A., Weir, A., Van Winkle, L., Duan, X., Philpot, R., Plopper, C., 1995. Relationship of cytochrome P450 activity to Clara cell cytotoxicity. IV. Metabolism of naphthalene and naphthalene oxide in microdissected airways from mice, rats, and hamsters. Mol. Pharmacol. 47 (1), 74–81. Cruzan, G., Cushman, J.R., Andrews, L.S., Granville, G.C., Miller, R.R., Hardy, C.J., Coombes, D.W., Mullins, P.A., 1997. Sub-chronic inhalation studies of styrene in CD rats and CD1 mice. Fundam. Appl. Toxicol. 35 (2), 152–165. Cruzan, G., Cushman, J.R., Andrews, L.S., Granville, G.C., Johnson, K.A., Hardy, C.J., Coombs, D.W., Mullins, P.A., Brown, W.R., 1998. Chronic toxicity/oncogenicity study of styrene in CD rats by inhalation exposure for 104 weeks. Toxicol. Sci. 46 (2), 266–281. Cruzan, G., Cushman, J.R., Andrews, L.S., Granville, G.C., Johnson, K.A., Bevan, C., Hardy, C.J., Coombes, D.W., Mullins, P.A., Brown, W.R., 2001. Chronic toxicity/ oncogenicity study of styrene in CD-1 mice by inhalation exposure for 104 weeks. J. Appl. Toxicol. 21 (3), 185–198. Day, B.J., Carlson, G.P., DeNicola, D.B., 1990. Gamma-glutamyl transpeptidase in rat bronchoalveolar lavage fluid as a probe of 4-ipomeanol and alphanaphthylthiourea-induced pneumotoxicity. J. Pharmacol. Methods 24, 1–8. Devereux, T.R., Fouts, J.R., 1980. Isolation and identification of Clara cells from rabbit lung. In Vitro 16 (11), 958–968. Egan, D., O’Kennedy, R., Moran, E., Cox, D., Prosser, E., Thornes, R.D., 1990. The pharmacology, metabolism, analysis, and applications of coumarin and coumarin-related compounds. Drug Metab. Rev. 22 (5), 503–529. Ford, T.C., Rickwood, D., 1982. Formation of isotonic nycodenz gradients for cell separations. Anal. Biochem. 124 (2), 293–298. Foster, J.R., Green, T., Smith, L.L., Lewis, R.W., Hext, P.M., Wyatt, I., 1992. Methylene chloride – an inhalation study to investigate pathological and biochemical
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National Toxicology Program, 2000. Toxicology and Carcinogenesis Studies of Naphthalene in F344/N Rats (Inhalation Studies), NTP TR 500. US Department of Health and Human Services, Public Health Service, National Institutes of Health. O’Brien, K.A., Smith, L.L., Cohen, G.M., 1985. Differences in naphthalene-induced toxicity in the mouse and rat. Chem.-Biol. Interact. 55 (1–2), 109–122. O’Brien, K.A., Suverkropp, C., Kanekal, S., Plopper, C.G., Buckpitt, A.R., 1989. Tolerance to multiple doses of the pulmonary toxicant naphthalene. Toxicol. Appl. Pharmacol. 99 (3), 487–500. Odum, J., Foster, J.R., Green, T., 1992. A mechanism for the development of Clara cell lesions in the mouse lung after exposure to trichloroethylene. Chem.-Biol. Interact. 83 (2), 135–153. Pacifici, G.M., Boobis, A.R., Brodie, M.J., McManus, M.E., Davies, D.S., 1981. Tissue and species differences in enzymes of epoxide metabolism. Xenobiotica 11 (2), 73–79. Pardo, A., Ruiz, V., Arreola, J.L., Ramirez, R., Cisneros-Lira, J., Gaxiola, M., Barrios, R., Kala, S.V., Lieberman, M.W., Selman, M., 2003. Bleomycin-induced pulmonary fibrosis is attenuated in gamma-glutamyl transpeptidase-deficient mice. Am. J. Respir. Crit. Care Med. 167 (6), 925–932. Pitot, H.C., Glauert, H.P., Hanigan, M., 1985. The significance of selected biochemical markers in the characterization of putative initiated cell populations in rodent liver. Cancer Lett. 29 (1), 1–14. Ritter, J.K., Owens, I.S., Negishi, M., Nagata, K., Sheen, Y.Y., Gillette, J.R., Sasame, H.A., 1991. Mouse pulmonary cytochrome P-450 naphthalene hydroxylase: cDNA cloning, sequence, and expression in Saccharomyces cerevisiae. Biochemistry 30 (48), 11430–11437. Serabjit-Singh, C.J., Nishio, S.J., Philpot, R.M., Plopper, C.G., 1988. The distribution of cytochrome P-450 monooxygenase in cells of the rabbit lung: an ultrastructural immunocytochemical characterization. Mol. Pharmacol. 33 (3), 279–289.
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Singh, G., Katyal, S.L., 1984. An immunologic study of the secretory products of rat Clara cells. J. Histochem. Cytochem. 32 (1), 49–54. Slordal, L., Andersen, A., Dajani, L., Warren, D.J., 1993. A simple HPLC method for the determination of cellular glutathione. Pharmacol. Toxicol. 73 (2), 124–126. Smith, G.D., Ding, J.L., Peters, T.J., 1979. A sensitive fluorimetric assay for c-glutamyl transferase. Anal. Biochem. 100 (1), 136–139. Vassallo, J., Curry, S., Purdon, M., Lewis, C., Fix, A., Lehman-McKeeman, L.D., 2000. Elevated glutathione (GSH) pools and gamma glutamyl transpeptidase (GGT) activity in coumarin-induced Clara cell tolerance. The Toxicologist 54. Vassallo, J.D., Hicks, S.M., Born, S.L., Daston, G.P., 2004a. Roles for epoxidation and detoxification of coumarin in determining species differences in Clara cell toxicity. Toxicol. Sci. 82 (1), 26–33. Vassallo, J.D., Hicks, S.M., Daston, G.P., Lehman-McKeeman, L.D., 2004b. Metabolic detoxification determines species differences in coumarin-induced hepatotoxicity. Toxicol. Sci. 80 (2), 249–257. Viscardi, R.M., Ullsperger, S., Resau, J.H., 1992. Reproducible isolation of type II pneumocytes from fetal and adult rat lung using nycodenz density gradients. Exp. Lung Res. 18 (2), 225–245. Warren, D.L., Brown, J.R., Buckbpitt, A.R., 1982. Evidence for cytochrome P450 mediated metabolism in the bronchiolar damage by naphthalene. Chem.-Biol. Interact. 40 (3), 287–303.
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c-Glutamyl transpeptidase null mice fail to develop tolerance to coumarin-induced Clara cell toxicity Jeffrey D. Vassallo a,*, Rhonda S. Kaetzel b, Stephanie L. Born a, Cindy L. Lewis a, Lois D. Lehman-McKeeman a, Donald J. Reed c a b c
Miami Valley Innovation Center, The Procter and Gamble Company, 11810 East Miami River Road, Cincinnati, OH 45252, United States Exponent, 15375 SE 30th Place, Suite 250, Bellevue, WA 98007, United States Oregon State University, 1037 ALS, Corvallis, OR 97331, United States
a r t i c l e
i n f o
Article history: Received 25 November 2009 Accepted 22 March 2010
Keywords: Coumarin Glutathione Knockout mouse Glutamyl transpeptidase Tolerance Clara cell
a b s t r a c t Coumarin was used as a model Clara cell toxicant to test the hypothesis that tolerance to injury requires increased c-glutamyl transpeptidase (GGT) activity. Wildtype (GGT+/+) and GGT-deficient (GGT/) mice on a C57BL/6/129SvEv hybrid background were dosed orally with corn oil (vehicle) or coumarin (200 mg/ kg). In vehicle-treated mice, Clara cell secretory protein (CC10) expression was distributed throughout the bronchiolar epithelium. After one dose of coumarin, CC10 expression was dramatically reduced and the bronchiolar epithelium was devoid of Clara cells in GGT+/+ and GGT/ mice. In wildtype mice, 9 doses of coumarin produced tolerance, characterized as a renewed bronchiolar epithelium with Clara cells expressing CC10 along with a 40% increase in total glutathione (GSH) and a 7-fold increase in GGT activity in the lung. In contrast, tolerance was not observed in GGT/ mice. To assess whether changes in whole lung levels of GSH and GGT activity reflect Clara cell specific changes an enriched population of cells was isolated from female wildtype B6C3F1 mice made tolerant to coumarin. Compared to Clara cells from control mice, GSH and GGT activity increased 3- and 13-fold, respectively. Collectively, these data suggest Clara cell tolerance to coumarin toxicity requires increased GGT activity favoring enhanced GSH synthesis. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Coumarin (1,2-benzopyrone), a natural product found in a variety of plants, is widely used in consumer products as a fragrance ingredient (Lake, 1999). Coumarin has also been evaluated clinically for the treatment of certain malignancies (Egan et al., 1990; Lake, 1999; Marshall et al., 1994). While toxicity in humans is rare, it is well recognized that coumarin is a rat hepatotoxicant. Coumarin is also acutely toxic to the mouse lung, where it selectively targets the non-ciliated bronchiolar epithelial cells, or Clara cells (Born et al., 1998). Additionally, chronic coumarin administration increased the incidence of lung tumors (alveolar/bronchiolar adenomas and carcinomas) in B6C3F1 mice, an effect not observed in F344 rats (NTP, 1993). Other agents known to cause Clara cell necrosis acutely and lung tumors in mice after chronic exposure include naphthalene (Mahvi et al., 1977; NTP, 1992; O’Brien et al., 1985), methylene chloride (Foster et al., 1992; NTP, 1986),
* Corresponding author. Address: Bristol-Myers Squibb, Route 206 and Provinceline Road, Princeton, NJ 08540, United States. Tel.: +1 609 252 7112; fax: +1 609 252 7046. E-mail addresses: [email protected] (J.D. Vassallo), [email protected] (R.S. Kaetzel), [email protected] (D.J. Reed). 0278-6915/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2010.03.034
trichloroethylene (Fukuda et al., 1983; Maltoni et al., 1986; NTP, 1990; Odum et al., 1992), and styrene (Cruzan et al., 1997, 2001; Green et al., 2001). These chemicals do not cause Clara cell necrosis or lung tumors in rats, suggesting a mechanism that may be unique to the mouse lung (Cruzan et al., 1997, 1998; Green et al., 2001; NTP, 1986, 1990, 1993, 2000). As the most metabolically-active cell type in the rodent lung, Clara cells are uniquely susceptible to xenobiotic-mediated toxicity (Serabjit-Singh et al., 1988; Widdicombe and Pack, 1982). Cytochrome P450 2F2 (CYP2F2), which is highly expressed in mouse Clara cells catalyzes the formation of reactive epoxide metabolites that contribute to naphthalene-, styrene- and coumarin-induced Clara cell toxicity (Born et al., 2002; Buckpitt et al., 1995; Green et al., 2001; Hynes et al., 1999; Nagata et al., 1990; O’Brien et al., 1985; Ritter et al., 1991). Naphthalene-, styrene-, and coumarin-derived epoxides are detoxified by glutathione (GSH) conjugation (Huwer et al., 1991; Lake, 1984; Pacifici et al., 1981; Warren et al., 1982). However, in the lung, steady-state GSH levels vary widely between individual Clara cells, and it has been demonstrated that this heterogeneity is a significant factor in determining the susceptibility of individual Clara cells to naphthalene toxicity (West et al., 2000a,b). A common feature of Clara cell toxicants is that following repeated exposure, Clara cells become refractory or tolerant to
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toxicity. This phenotype has been described for several chemicals, including naphthalene, coumarin, methylene chloride, trichloroethylene and 4-ipomeanol (Born et al., 1999; Boyd et al., 1981; Foster et al., 1992; O’Brien et al., 1989; Odum et al., 1992), and Clara cells tolerant to coumarin exhibit cross-resistance to naphthalene (Born et al., 1999). Although the exact mechanisms leading to the development of mouse Clara cell tolerance have yet to be established, it is generally recognized that tolerance is an adaptive response. It was originally suggested that Clara cell tolerance results from a reduction in metabolic activation (Foster et al., 1992; O’Brien et al., 1989; Odum et al., 1992). However, subsequent studies have determined that CYP2F2 expression levels and epoxidation rates are similar in lung fractions from control mice and mice made tolerant to naphthalene or coumarin (Born et al., 1999; West et al., 2002). In contrast, experiments focused on detoxification pathways have demonstrated that tolerance to naphthalene is associated with elevated GSH levels as well as an increase in c-glutamylcysteine synthetase (GCS) activity in mouse lung terminal bronchioles (West et al., 2002). Although GCS is rate-limiting in GSH synthesis, c-glutamyl transpeptidase (GGT), located on the outer surface of the plasma membrane, plays an important role in the extracellular hydrolysis of GSH that is required to provide the amino acids for GSH synthesis. GGT has been used as a marker of Clara cell injury in bronchiolar alveolar lavage fluid (Day et al., 1990) and is also markedly elevated in the lungs from B6C3F1 mice made tolerant to coumarin (Vassallo et al., 2000). In light of these data, and the fact that GGT plays an important role in intracellular GSH synthesis, the purpose of this work was to test the hypothesis that Clara cell tolerance to coumarin requires an increased level of GGT activity. For this work, GGTdeficient (GGT/) mice generated on a C57BL/6/129SvEv hybrid background were used as a model to determine whether tolerance to coumarin developed in the absence of GGT. Additional studies were conducted to determine if the coumarin-induced increase in GGT activity was specific to Clara cells isolated from B6C3F1 mice. 2. Materials and methods 2.1. Chemicals Coumarin was purchased from the Sigma–Aldrich Chemical Company and the purity exceeded 99% (Milwaukee, WI). Corn oil was purchased from Acros Organics (Geel, Belgium). All other reagents were HPLC grade or the highest grade available. 2.2. Animals Breeder pairs of heterozygous mice (GGT+/) (19–30 g) were previously generated on a C57BL/6/129SvEv hybrid background (Lieberman et al., 1996). Mice were bred and offspring were genotyped by polymerase chain reaction as previously reported (Will et al., 2000). Wildtype (GGT+/+) (18–32 g) and age-matched GGT-deficient (GGT/) (16–26 g) mice of both sexes were provided N-acetylcysteine (NAC) ad libitum (10 mg/ml, pH 7 in drinking water) at weaning and throughout the experimental period. Supplementation of NAC in the drinking water helps to restore GSH and cysteine levels and improves the health and longevity of these animals (Lieberman et al., 1996). Female B6C3F1 mice (19–25 g) were purchased from Harlan Sprague Dawley, Inc. (Indianapolis, IN). Female B6C3F1 mice were selected because previous work showing Clara cell toxicity and tolerance were demonstrated in this sex and strain (Born et al., 1998, 1999). All mice were housed in a temperature- and humidity-controlled environment with 12-h light/dark cycles. Animals were allowed rodent chow (Purina Laboratory Rodent Chow; Ralston-Purina, St. Louis, MO) and water ad libitum throughout the studies. All animal procedures were approved by The Procter and Gamble Company (AALAC accredited) and Oregon State University’s Lab Animal Resources facility Institutional Animal Care and Use Committee (IACUC). 2.3. In vivo assessment of coumarin-induced Clara cell toxicity and tolerance Preliminary studies with C57BL/6/129SvEv mice indicated that this strain develops coumarin-induced Clara cell toxicity and tolerance similar to B6C3F1 mice (data not shown) (Born et al., 1998, 1999). GGT+/+ and GGT/ mice, randomized by body weight into treatment groups (n = 7–8), were dosed orally by gavage with corn
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oil (vehicle) or coumarin (200 mg/kg; 5 ml/kg). This dose was selected because it has been previously shown to cause coumarin-induced lung toxicity and tolerance (Born et al., 1998, 1999). To evaluate acute toxicity, mice received a single dose of coumarin and the biochemical and histological changes were evaluated 24 h after dosing. To produce tolerance, mice were given a total of 9 doses of coumarin with a protocol similar to the NTP bioassay. Mice were dosed Monday–Friday (days 1–5), not dosed Saturday–Sunday with dose days 6–9 representing the 2nd week of dosing (Monday–Thursday). Biochemical and histological changes were evaluated 24 h after the last dose as previously described (Born et al., 1999). Separate groups of mice were used for biochemical, histopathological and immunohistochemical assessments. 2.4. Tissue collection for biochemical assessment Under sodium pentobarbital anesthesia (50 mg/kg; ip) mice were exsanguinated. Two small pieces of lung (<100 mg) were removed and homogenized with either five volumes of 5% sulfosalicylic acid for total glutathione analysis or five volumes of Tris–HCl buffer (0.1 M, pH 8.5) for GGT analysis. Total protein in the BALF and lung homogenates was determined by the Bradford assay with bovine serum albumin as the standard following solubilization in 0.1 N NaOH (Bradford, 1976). All samples were stored at 80 °C until time of use. 2.5. Tissue collection for histopathological assessment Mice were exsanguinated under sodium pentobarbital (50 mg/kg; ip) anesthesia, after which the lungs, with the trachea and heart attached, were removed and perfusion fixed using Karnovsky’s fixative at a constant pressure of 25 cm via the trachea. For histopathology, serial sections from lung blocks were cut at 5 lm thickness, stained with hematoxylin and eosin (H&E) and examined by light microscopy (magnification 60). 2.6. Tissue collection for immunohistochemical assessment Lungs were fixed by inflation with 1% paraformaldehyde. After immersion in 1% paraformaldehyde for 1 h, the tissues were transferred to 70% ethanol for processing overnight and embedding in paraffin the next day. Paraffin sections (5 lm thick) were stained for Clara cell secretory protein (CC10) using the avidin–biotin-peroxidase complex (ABC) method (Vector Laboratories, Burlingame, CA). All tissues were blocked for endogenous peroxidase activity for 10 min in 3% hydrogen peroxide, rinsed, and then blocked for proteins by incubating for 20 min in a Universal Blocking Reagent (Biogenex, San Ramon, CA). For CC10 staining, the primary antibody was an anti-rat CC10 antiserum (Singh and Katyal, 1984; kindly provided by Dr. Gurmukh Singh, University of Pittsburgh). The antiserum was diluted 1:10,000 and allowed to incubate on the slides for 1 h at room temperature. Following incubation with the primary antibody, the Elite Rabbit kit (Vector Laboratories) for the secondary antibody and the ABC steps were followed as per the manufacturer’s directions. Both stains were developed for 5 min using Vector’s 3,30 -diaminobenzidine kit with nickel enhancement. The sections of CC10 were counterstained with Mayer’s hematoxylin (Biogenex, San Ramon, CA). 2.7. Assessment of GSH and GGT activity in isolated Clara cells Female B6C3F1 mice, randomized by body weight into treatment groups (n = 4), were dosed orally by gavage with corn oil (vehicle) or coumarin (200 mg/kg) (5 ml/ kg). To induce tolerance, mice received 9 doses of coumarin (5 days/week), and Clara cells were isolated 24 h after the last dose (Born et al., 1999). This dose was selected because it has been previously shown to cause coumarin-induced lung toxicity and tolerance (Born et al., 1998, 1999). Clara cells were isolated according to the method of Bolton et al. (1993) and modified by replacing elastase with dispase. A Nycodenz gradient was used according to the method of Viscardi et al. (1992) instead of IgG panning (Ford and Rickwood, 1982). Cells were collected and counted on a hemocytometer. Cell viability and Clara cell purity were determined by erythrosine B exclusion and nitro blue tetrazolium staining, respectively (Devereux and Fouts, 1980). Following isolation, Clara cells were centrifuged at 700g for 10 min. The medium was removed and the cells were resuspended in 0.5 ml of 0.9% saline. This process was repeated two more times and following the third spin, the cells were resuspended in either 5% sulfosalicylic acid (106 cells/ml) for GSH analysis or Tris–HCl (100 mM, pH 8.5) (106 cells/0.1 ml) for GGT analysis. Following a brief vortex and sonication (10 s), the cells were either analyzed for protein and GGT activity or they were centrifuged at 14,000 rpm for 10 min and the supernatant was analyzed for protein and GSH. Total protein was determined by the Bradford assay with bovine serum albumin as the standard following solubilization in 0.1 N NaOH (Bradford, 1976). 2.8. Total glutathione determination Total glutathione, representing reduced GSH, oxidized glutathione (GSSG), and glutathione–protein mixed disulfides, was quantified in whole lung homogenates
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and BALF by HPLC as the fluorescent monobromobimane derivative following reduction with sodium borohydride (Slordal et al., 1993). Sulfosalicylic acidtreated samples were mixed vigorously, centrifuged, and then 50 ll of the supernatant was transferred to a tube containing 50 ll of an internal standard (glutathione ethyl ester), 20 ll of NaBH4, 900 ll of Tris and 50 ll of a 0.25% monobromobimane solution. Following a 15 min incubation at room temperature, samples were extracted with 1 ml of methylene chloride, after which a 200 ll aliquot was acidified with 100 ll of 1 N HCl. Chromatography was performed at 40 °C on a 75 mm 4.6 mm ID Zorbax SB C18 column with 3.5 lm particle size at a flow rate of 2 ml/min. Solvent A was composed of 0.2% trifluoroacetic acid (TFA) in 95:5 water:acetonitrile; solvent B contained the same acid modifier in 100% acetonitrile. A 10 ll sample was injected onto the column under initial conditions (100% solvent A) and a linear gradient was performed from 100% to 85% solvent A and 0% to 15% solvent B in 10 min using a Waters 2690 separation model HPLC with a Waters 2487 dual channel wavelength detector (Waters, Milford, MA). The column was allowed to equilibrate under initial mobile phase conditions for 6 min between injections. Total glutathione and the internal standard, monitored at an excitation wavelength of 380 nm and an emission wavelength of 465 nm, had retention times of approximately 5 and 8.5 min, respectively. Total glutathione quantification was based on an external standard curve with GSH.
2.9. GGT activity GGT was measured fluorometrically with L-c-Glu-7-amino-4-methyl-coumarin as a substrate (Smith et al., 1979). A sample volume of 100 ll, containing protein from control mice (lung = 50 lg) or mice made tolerant to coumarin (lung = 10 lg) was transferred to a 200 ll solution containing a final concentration of 0.1 M ammediol/HCl, 20 mM diglycine, 0.1% Triton X-100, and 26 lM L-c-Glu-7-amino4-methyl-coumarin (pH 8.5). Following incubation for 45 min in a 37 °C shaking water bath, the reaction was terminated by the addition of 1.7 ml of ice-cold 0.05 M glycine buffer (pH 10.4). The samples were mixed, transferred to a 96-well microtitre plate, and analyzed on a fluorometer (Perkin Elmer LS50B with a plate reader) with an emission wavelength and slit width setting of 440 and 15.0 nm, respectively, and an excitation wavelength and slit width setting of 370 and 2.5 nm, respectively. GGT activity was calculated as pmol of 7-amino-4-methylcoumarin (AMC)/min/mg protein based on a standard curve of AMC. 2.10. Statistics Data were analyzed by a Student’s t-test or ANOVA followed by a multiple comparison test (Fischer’s PLSD) using StatView (SAS Institute, Cary, NC) at a 5% significance level (p < 0.05).
Fig. 1. Coumarin-induced Clara cell toxicity and tolerance in GGT+/+ and GGT/ mice. Mice were dosed orally with corn oil or coumarin (200 mg/kg) for 1 or 9 days (5 days/ week) and lung histology was evaluated 24 h after the last dose. Normal terminal bronchioles from GGT+/+ (A) and GGT/ (B) mice shows Clara cells with apical caps (black arrows). A single dose of coumarin caused exfoliation of the bronchiolar epithelium in GGT+/+ (C) and GGT/ (D) mice. Necrotic Clara cells are sloughed off in the bronchiole lumen (black arrow) whereas the ciliated epithelial cells are unaffected by the treatment (grey arrow). Clara cells repopulated the bronchiolar epithelium (black arrow) following repeated coumarin administration in GGT+/+ mice (E) whereas the bronchiolar epithelium remained devoid of Clara in the GGT/ mice (F), indicating tolerance did not develop. In the absence of Clara cells, the epithelium appears thicker and more disorganized compared to the control.
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3. Results The histomorphology of terminal bronchioles from mice following single or repeated coumarin administration (200 mg/kg) is illustrated in Fig. 1. The terminal bronchioles from vehicle-treated GGT+/+ and GGT/ mice were similar, with Clara cells and ciliated cells lining the epithelium in an evenly distributed and alternating manner (Fig. 1A and B). Following a single oral dose of coumarin, Clara cells were almost completely ablated from the terminal bronchioles in GGT+/+ and GGT/ mice (Fig. 1C and D). Repeated administration of coumarin led to the development of tolerance in GGT+/+ mice and this phenotype was characterized by a renewing of the bronchiolar epithelium with apical capped Clara cells (Fig. 1E), although the epithelium appeared somewhat thickened and disorganized. In contrast, the bronchiolar epithelium remained
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devoid of Clara cells in GGT/ mice indicating that tolerance, following repeated coumarin administration, did not develop (Fig. 1F). Collectively, these data are consistent with the development of coumarin-induced Clara cell toxicity and tolerance in B6C3F1 mice, indicating that there are no strain differences (Born et al., 1998, 1999). To demonstrate that coumarin selectively targets the Clara cell, a marker of Clara cell differentiation (CC10) was evaluated by immunohistochemical detection in mice following single or repeated coumarin administration (Fig. 2). In vehicle-treated GGT+/+ and GGT/ mice, expression of CC10 was intense and evenly distributed throughout the bronchiolar epithelium (Fig. 2A and B), indicating that GGT deficiency does not affect CC10 expression. Following a single dose of coumarin, CC10 expression was dramatically reduced, and this is consistent with
Fig. 2. CC10 immunohistochemistry of Clara cells in GGT+/+ and GGT/ mice. Mice were dosed orally with corn oil or coumarin (200 mg/kg) for 1 or 9 doses (5 days/week) and CC10 immunohistochemistry was evaluated 24 h after the last dose. Terminal bronchioles from normal GGT+/+ (A) and GGT/ (B) mice shows intense and evenly distributed Clara cell-specific CC10 expression (black arrows). Consistent with Clara cell necrosis, a single dose of coumarin caused a significant diminution in CC10 expression in both GGT+/+ (C) and GGT/ (D) mice. Despite the absence of apical capped Clara cells, positively stained cells appear in localized foci along the bronchiolar epithelium (black arrow). Following repeated coumarin administration, a significant increase in CC10 expression was noted in GGT+/+ mice (E). In contrast, CC10 expression in apical capped Clara cells did not return in GGT/ mice (F). However, positively stained cells appear in localized foci along the bronchiolar epithelium (black arrow).
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Table 1 Pulmonary total glutathione and GGT activity following single or repeated dosing of coumarin.a Mouse +/+
GGT
GGT/
Treatment b
Control Coumarin Coumarin Controlb Coumarin Coumarin
Days of dosing
Total glutathione (nmol/mg protein)
GGT (pmol/min/mg protein)
9 1 9 9 1 9
8.0 ± 0.4 10.9 ± 1.1* 11.0 ± 1.0* 7.4 ± 1.4 5.7 ± 0.9 7.3 ± 0.7
40.2 ± 7.8 60.3 ± 8.2 260.5 ± 13.5* –c –c –c
a GGT+/+ and GGT/ mice were dosed with corn oil or coumarin (200 mg/kg) for 1 or 9 days (5 days/week). Total glutathione and GGT activity were determined 24 h after the last dose. Data represent the mean and SE of 5–6 mice per group. b Total glutathione and GGT activity were similar in mice dosed with corn oil for 1 (data not shown) or 9 days. c – Denotes no detectable GGT activity in GGT/ mice. * Statistically significant at p < 0.05.
Table 2 Total glutathione and GGT activity in Clara cells from B6C3F1 mice following repeated dosing of coumarin.a Treatment
Cell viability (%)b
Clara cell purity (%)c
Total glutathione (nmol/mg protein)
GGT (pmol/min/mg protein)
Control Coumarin
90.6 ± 0.6 92.2 ± 3.4
62.3 ± 7.4 61.9 ± 5.8
3.2 ± 1.0 10.5 ± 2.5*
22.8 ± 7.3 287.5 ± 39.2*
a B6C3F1 mice were dosed with corn oil or coumarin (200 mg/kg) for 9 days (5 days/week). Total glutathione and GGT activity were analyzed 24 h after the last dose in a Clara cell enriched fraction. Data represent the mean and SE of three separate isolations. b Determined by erythrosine B exclusion. c Determined by staining with nitro blue tetrazolium. * Statistically significant at p < 0.05.
the loss of viable Clara cells from the bronchiolar epithelium (Fig. 2C and D). With repeated coumarin administration, tolerance to toxicity resulted in a significant increase in CC10 expression in GGT+/+ mice whereas expression remained reduced in GGT/ mice (Fig. 2E and F). Total glutathione levels were similar in GGT+/+ and GGT/ mice supplemented with NAC in the drinking water (Lieberman et al., 1996). Table 1 illustrates that pulmonary total glutathione levels in control GGT+/+ and GGT/ mice were similar. The level of total glutathione in perfused whole lung homogenates from GGT+/+ mice was increased by 40% 24 h after a single dose of coumarin and remained elevated as Clara cell tolerance developed following repeated coumarin administration (Table 1). The development of tolerance in GGT+/+ mice was also associated with a 7-fold increase in GGT activity in whole lung homogenates. In contrast, the level of total glutathione in whole lung homogenates from GGT/ mice did not increase following single or repeated coumarin administration. To assess whether changes in whole lung levels of GSH and GGT activity reflect specific changes in tolerant Clara cells, an enriched population of these cells was isolated from B6C3F1 mice treated with corn oil or coumarin for 9 doses. Compared to Clara cells from vehicle-treated mice, there was a 3- and 13-fold increase in GSH and GGT activity, respectively in the enriched fractions of Clara cells (Table 2).
4. Discussion GGT plays a significant role in GSH regulation and is markedly increased in response to a variety of lung oxidant injuries (Zhang and Forman, 2009). As such, the GGT/ mouse has been used as a model to investigate the role of GSH regulation in protecting the lung from hyperoxia- and bleomycin-induced injury (Barrios et al., 2002; Klings et al., 2009; Pardo et al., 2003). Similarly, the GGT/ mouse is an ideal model for investigating whether tolerance to coumarin-induced Clara cell toxicity, which is associated with an increase in GGT and GSH, could develop without GGT. In
the absence of GGT, mice exhibit a severe glutathionuria resulting from the inability to hydrolyze and recycle GSH in renal proximal tubules. Consequently, cysteine deficiency develops and leads to a reduction in GSH synthesis and a chronic intracellular GSH deficiency in most tissues, including Clara cells (Jean et al., 2003). While this deficit results in a reduced life-span, supplementation of NAC in the drinking water helps to restore GSH and cysteine levels and improves the health of these animals (Lieberman et al., 1996). It has been shown that Clara cell tolerance to naphthalene-induced toxicity is an adaptive cellular response associated with an induction in GCS activity which favors increased GSH synthesis (West et al., 2000a, 2002). While GGT activity was not measured in the aforementioned studies, the current data suggest that Clara cell tolerance requires GGT. GGT+/+ mice showed a significant increase in pulmonary total glutathione associated with the development of Clara cell tolerance, whereas GGT/ mice were unable to upregulate GSH formation, exhibited severe lung toxicity, and showed no evidence of Clara cell tolerance. Given that GCS is dependent on the extracellular availability of cysteine in maintaining intracellular GSH homeostasis via regulation of the c-glutamyl cycle, it is likely that a common mechanism of Clara cell tolerance exists, and is coordinated with an increase in both GGT and GCS activity which favors increased GSH synthesis. Since GSH conjugation to coumarin 3,4-epoxide is a non-enzymatic detoxification reaction, the increased synthesis of GSH could help protect the Clara cell from this reactive metabolite (Vassallo et al., 2004a,b). This hypothesis is consistent with the fact that susceptibility of Clara cells to naphthalene toxicity is dependent on GSH levels (West et al., 2000a,b). While a comprehensive assessment of the molecular changes associated with tolerance to coumarin has not been reported, this adaptive phenotype, favoring increased GGT activity, may represent an early preneoplastic change as chronic exposure to naphthalene or coumarin is associated with an increased incidence of mouse lung tumors (NTP, 1993, 2000). It is generally recognized that increased GGT expression is a marker of preneoplastic foci in the liver during chemical carcinogenesis (Pitot et al., 1985),
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and GGT is simultaneously increased in the bronchiolar epithelium in concert with tumor development resulting from exposure to 3methylcholanthrene in the rat lung (He, 1991). Establishing a causal association between increased GGT expression and preneoplastic changes in the lung will require additional studies. However, this work lays the foundation that Clara cell toxicity is an adaptive response requiring an increase in pulmonary GGT activity. This in turn is likely to favor increased synthesis of GSH and may ultimately provide a selective growth advantage to the affected cells. Conflict of Interest The authors declare that there are no conflicts of interest. Acknowledgements This work was carried out at The Procter and Gamble Company (JDV, SLB, CLL, LDLM) and Oregon State University (RSK and DJR) and was supported in part by the Research Institute for Fragrance Materials and by the following NIH grants: ES-00040, ES-00210, and ES-01978. The authors would like to thank Dr. Michael Lieberman for providing the GGT+/ breeder mice used for these studies. The technical assistance provided by Marda Brown, Tamara Fraley, LouAnn Amberg and Bill Amberg in the breeding, screening and maintenance of the GGT-deficient colony was also greatly appreciated. References Barrios, R., Shi, Z.-Z., Kala, S.V., Wiseman, A.L., Welty, S.E., Kala, G., Bahler, A.A., Ou, C.-N., Lieberman, M.W., 2002. Oxygen-induced pulmonary injury in c-glutamyl transpeptidase-deficient mice. Lung 179 (5), 319–330. Bolton, J.L., Thompson, J.A., Allentoff, A.J., Miley, F.B., Malkinson, A.M., 1993. Metabolic activation of butylated hydroxytoluene by mouse bronchiolar Clara cells. Toxicol. Appl. Pharmacol. 123 (1), 43–49. Born, S.L., Fix, A.S., Caudill, D., Lehman-Mckeeman, L.D., 1998. Selective Clara cell injury in mouse lung following acute administration of coumarin. Toxicol. Appl. Pharmacol. 151 (1), 45–56. Born, S.L., Fix, A.S., Caudill, D., Lehman-Mckeeman, L.D., 1999. Development of tolerance to Clara cell necrosis with repeat administration of coumarin. Toxicol. Sci. 51 (2), 300–309. Born, S.L., Caudill, D., Fliter, K.L., Purdon, M.P., 2002. Identification of the cytochromes P450 that catalyze coumarin 3,4-epoxidation and 3hydroxylation. Drug Metab. Dispos. 30 (5), 483–487. Boyd, M.R., Burka, L.T., Wilson, B.J., Sastry, B.V., 1981. Development of tolerance to the pulmonary toxin, 4-ipomeanol. Toxicology 19 (2), 85–100. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal. Biochem. 72, 248–254. Buckpitt, A., Chang, A., Weir, A., Van Winkle, L., Duan, X., Philpot, R., Plopper, C., 1995. Relationship of cytochrome P450 activity to Clara cell cytotoxicity. IV. Metabolism of naphthalene and naphthalene oxide in microdissected airways from mice, rats, and hamsters. Mol. Pharmacol. 47 (1), 74–81. Cruzan, G., Cushman, J.R., Andrews, L.S., Granville, G.C., Miller, R.R., Hardy, C.J., Coombes, D.W., Mullins, P.A., 1997. Sub-chronic inhalation studies of styrene in CD rats and CD1 mice. Fundam. Appl. Toxicol. 35 (2), 152–165. Cruzan, G., Cushman, J.R., Andrews, L.S., Granville, G.C., Johnson, K.A., Hardy, C.J., Coombs, D.W., Mullins, P.A., Brown, W.R., 1998. Chronic toxicity/oncogenicity study of styrene in CD rats by inhalation exposure for 104 weeks. Toxicol. Sci. 46 (2), 266–281. Cruzan, G., Cushman, J.R., Andrews, L.S., Granville, G.C., Johnson, K.A., Bevan, C., Hardy, C.J., Coombes, D.W., Mullins, P.A., Brown, W.R., 2001. Chronic toxicity/ oncogenicity study of styrene in CD-1 mice by inhalation exposure for 104 weeks. J. Appl. Toxicol. 21 (3), 185–198. Day, B.J., Carlson, G.P., DeNicola, D.B., 1990. Gamma-glutamyl transpeptidase in rat bronchoalveolar lavage fluid as a probe of 4-ipomeanol and alphanaphthylthiourea-induced pneumotoxicity. J. Pharmacol. Methods 24, 1–8. Devereux, T.R., Fouts, J.R., 1980. Isolation and identification of Clara cells from rabbit lung. In Vitro 16 (11), 958–968. Egan, D., O’Kennedy, R., Moran, E., Cox, D., Prosser, E., Thornes, R.D., 1990. The pharmacology, metabolism, analysis, and applications of coumarin and coumarin-related compounds. Drug Metab. Rev. 22 (5), 503–529. Ford, T.C., Rickwood, D., 1982. Formation of isotonic nycodenz gradients for cell separations. Anal. Biochem. 124 (2), 293–298. Foster, J.R., Green, T., Smith, L.L., Lewis, R.W., Hext, P.M., Wyatt, I., 1992. Methylene chloride – an inhalation study to investigate pathological and biochemical
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