Roles for Epoxidation and Detoxification of Coumarin in Determining Species Differences in Clara Cell Toxicity

Jeffrey D. Vassallo*,1, Sarah M. Hicks{dagger}, Stephanie L. Born* and George P. Daston*

* Miami Valley Laboratories, The Procter and Gamble Company, 11810 East Miami River Road, Cincinnati, Ohio 45252; {dagger} University of Wisconsin-Madison, Rennebohm Hall School of Pharmacy, 777 Highland Avenue Madison, Wisconsin 53705

Received June 14, 2004; accepted July 26, 2004


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Coumarin-induced mouse Clara cell toxicity is thought to result from the local formation of coumarin 3,4-epoxide (CE). However, this toxicity is not observed in the rat, indicating species differences in coumarin metabolism. The purpose of the present work was to characterize the in vitro kinetics of coumarin metabolism in mouse, rat, and human whole lung microsomes, and to determine whether species differences in coumarin-induced Clara cell toxicity correlate with coumarin epoxidation or detoxification. In B6C3F1 mouse lung microsomes, coumarin was metabolized to CE, which in the absence of glutathione spontaneously rearranges to o-hydroxyphenylacetaldehyde (o-HPA). The Km and Vmax for o-HPA formation were 155 µM and 7.3 nmol/min/mg protein, respectively. In contrast, the Km and Vmax were 2573 µM and 1.75 nmol/min/mg protein, respectively, in F344 rat lung microsomes. Since the intrinsic clearance through the epoxidation pathway was 69 times higher in the mouse, the epoxidation rate was shown to correlate with species sensitivity to toxicity. To determine whether detoxification reactions contribute to species differences in toxicity, the fate of CE and o-HPA were examined. Detoxification of CE via conjugation with glutathione was evaluated in lung cytosol from mice and rats, and the Km of this reaction was approximately 800 µM in both species, whereas the Vmax was 3.5 and 6 nmol/min/mg protein, respectively, indicating that conjugation is faster in the rat. Oxidation of o-HPA to o-hydroxyphenylacetic acid (o-HPAA) was examined in lung cytosol from mice and rats. The Km of this reaction was approximately 1.5 µM in both species, whereas the Vmax was 0.08 and 0.33 nmol/min/mg protein in mice and rats, respectively, indicating that oxidation is faster in the rat. While the rate of epoxidation correlates with species sensitivity to coumarin, it is likely that Clara cell toxicity is modulated by CE and o-HPA detoxification. In contrast to rodent lung microsomes, bioactivation of coumarin to o-HPA did not occur in 16 different human lung microsomes, which suggests metabolism-dependent toxicity in the human lung is unlikely following low level coumarin exposure.

Key Words: coumarin; lung; mouse; human; Clara cell; glutathione.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Coumarin (1,2-benzopyrone) is a natural product used in perfumes and detergents as a fixative and enhancing agent (Lake, 1999Go). Although coumarin was once a food additive, its use has been restricted because it was found to be hepatotoxic in rats (Hazelton et al., 1956Go). Despite this restriction, human exposure to in foods and its use as a fragrance ingredient (Lake, 1999Go). Additionally, coumarin has been evaluated clinically for chemopreventive properties and for the treatment of lymphedema and venous insufficiency (Egan et al., 1990Go; Lake, 1999Go; Marshall et al., 1994Go).

Coumarin was recently shown to cause acute Clara cell necrosis and an increased incidence of alveolar/bronchiolar adenomas and carcinomas in B6C3F1 mouse lungs, an effect that was not observed in F344 rats (Born et al., 1998Go; NTP, 1993aGo). To date, minimal effort has been directed toward understanding the mechanism by which acute and chronic mouse lung injury occurs, and the human relevance of these lung effects is unknown. However, it appears that the acute and chronic effects of coumarin in the mouse lung are not unique to this chemical. Naphthalene (Mahvi et al., 1977Go; NTP, 1992Go; O'Brien et al., 1985Go), methylene chloride (Foster et al., 1992Go; NTP, 1986Go), trichloroethylene (Fukuda et al., 1983Go; Maltoni et al., 1986Go; NTP, 1990Go; Odum et al., 1992Go), and styrene (Cruzan et al., 1997Go, 2001Go; Green et al., 2001Go) share a similar profile of Clara cell toxicity and an increased incidence of lung tumors in mice. In contrast, none of these chemicals cause Clara cell necrosis or lung tumors in rats (Cruzan et al., 1997Go, 1998Go; Green et al., 2001Go; NTP, 1986Go, 1990Go, 1993aGo, 2000Go), suggesting a mechanism that is unique to the mouse lung.

Since Clara cells are the primary site of xenobiotic metabolism in the rodent lung (Baron and Voigt, 1990Go; Serabjit-Singh et al., 1988Go; Widdicombe and Pack, 1982Go), it is likely that metabolism plays an important role in determining the species-specific responses to coumarin. In fact, it was recently demonstrated that coumarin-induced mouse Clara cell toxicity is dependent on the formation of coumarin 3,4-epoxide (CE) (Born et al., 1998Go). Despite the fact that CE has a short half-life, and its effects cannot be directly examined, its role as a lung toxicant was demonstrated through studies conducted with dihydrocoumarin (DHC), a structural analog of coumarin that is saturated at the 3,4-position, which prohibits epoxide formation. In contrast to coumarin, no Clara cell toxicity or chronic lung effects were observed in mice treated with DHC (Born et al., 1998Go; NTP, 1993bGo).

Coumarin 3,4-epoxide has two possible fates. It can either form a conjugate (CE-SG) with glutathione (GSH) (Huwer et al., 1991Go; Lake, 1984Go; Vassallo et al., 2003Go) or it can rearrange spontaneously to o-hydroxyphenylacetaldehyde (o-HPA) (Born et al., 1997Go). This aldehyde is hepatotoxic and can be further oxidized or reduced to o-hydroxyphenylacetic acid (o-HPAA) or o-hydroxyphenylethanol (o-HPE), respectively (Fig. 1) (Born et al., 2000Go; Fentem et al., 1991Go; Lake et al., 1992Go; Steensma et al., 1994Go). The toxic effects of coumarin in the mouse lung are likely due to the formation of CE and o-HPA as CE-SG, o-HPAA, and o-HPE are considered nontoxic metabolites (Lake et al., 1989Go; Ratanasavanh et al., 1996Go).



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FIG. 1. Metabolic pathways of coumarin. Coumarin is metabolized to coumarin 3,4-epoxide which can either be conjugated with GSH or lose CO2 and rearrange to o-hydroxyphenylacetaldehyde which is subsequently oxidized to o-hydroxyphenylacetic acid or reduced to o-hydroxyphenylethanol.

 
Since Clara cell toxicity is dependent on the formation of CE it was hypothesized that the major determinant of mouse-specific Clara cell toxicity would be either elevated coumarin epoxidation or reduced detoxification of CE and o-HPA relative to that observed in the rat lung. Therefore, the purpose of the present work was to test this hypothesis by determining the kinetic characteristics of these reactions in vitro. Furthermore, to determine the human relevance of these findings, coumarin metabolism was evaluated in human lung samples.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Coumarin and metabolites. Coumarin, o-HPAA and o-HPE were purchased from the Aldrich Chemical Company (Milwaukee, WI). o-Hydroxyphenylacetaldehyde was synthesized by S. L. Born according to the method of Bruce and Creed, and the purity exceeded 97% (Bruce and Creed, 1970Go). CE-SG was metabolically synthesized using mouse liver microsomes and cytosol (Vassallo et al., 2003Go). Stock solutions of coumarin, o-HPAA, o-HPE, CE, and o-HPA were prepared in dimethyl sulfoxide (DMSO), whereas the stock solution of CE-SG was prepared in distilled, deionized water.

Other reagents. GSH, NAD+, dimethyl sulfoxide (DMSO), NADH, and NADP were purchased from the Sigma Chemical Company (St. Louis, MO). Glutathione was prepared in nitrogen-purged water. All other reagents were high-performance liquid chromatography (HPLC) grade or the highest grade available.

Animals. Female B6C3F1 mice (20–25 g) and male F344 rats (210–220 g) were purchased from Charles River Laboratories (Portage, MI). The strain and sex of animals were selected based on the coumarin bioassay data (NTP, 1993aGo). These data indicated liver necrosis was more severe in male rats than female rats, and female mice were more susceptible to coumarin-induced lung tumor formation than male mice. Animals were housed in humidity- and temperature-controlled rooms and allowed free access to a standard lab diet (Purina Laboratory Rodent Chow, Ralston-Purina, St. Louis, MO) and water.

Preparation of rodent microsomes and cytosol. Lung and liver microsomes and lung cytosol were prepared from untreated female B6C3F1 mice (n = 25/pool) and untreated male F344 rats (n = 23/pool). Microsomes and cytosol were prepared via differential centrifugation (Guengerich, 1989Go). Cytosol from mice and rats was processed through a PD-10 desalting column manufactured by Amersham Pharmacia Biotech AB (Uppsala, Sweden) to remove GSH and cofactors. Total protein was determined by the Bradford assay with bovine serum albumin as the standard (Bradford, 1976Go). Microsomes and cytosol were stored at –80°C until time of use.

Human lung microsomes. Human lung microsomes were purchased from Human Biologics International, LLC (Scottsdale, AZ) (HPM236, HPM237, HPM238, HPM239, HPM240, and HPM241), International Institute for the Advancement of Medicine (Exton, PA) (914951L, 1029951L, 827951L, 912951L, and 1016951L), or were a gift from Dr. Thomas Massey's laboratory (Queen's University, Ontario, Canada) to Dr. Stephanie Born (2 DM, 5 DM, 3 GM, 6 GM, 2 HM) (Smith et al., 2001Go). Table 1 describes the donor characteristics of these samples. Detectable levels of cytochrome P-450 (CYP)1A1 catalyzed 7-ethoxyresorufin O-dealkylation (EROD) activity, as determined by each supplier, was found in 10 of these microsomal samples (range 1–31 pmol/min/mg protein), whereas six of these samples had no EROD activity. The absence of CYP1A1 activity in these human samples is consistent with previous findings in which marked interindividual variability in pulmonary CYP1A1 activity exists (Smith et al., 2001Go). Despite the fact that there was variation in CYP1A1 activity the metabolism of coumarin was similar in all human samples.


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TABLE 1 Characteristics of Human Lung Donors

 
Glutathione S-transferase (GST) activity. The GST activity was expressed as the amount of enzyme, in units that catalyzes the conjugation of GSH to 1 µmol of 1-chloro-2,4-dinitrobenzene (CDNB) in 1 min at room temperature in 100 mM potassium phosphate buffer, pH 6.5 (Habig et al., 1974Go; Jakoby, 1981Go).

Coumarin metabolism in microsomes. Coumarin (10–7500 µM) metabolism was studied in a 1 ml lung or liver microsomal reaction mixture containing either 0.25 mg protein/ml for the mouse and rat or 1 mg protein/ml for the human. The microsomal reaction mixture also contained potassium phosphate buffer (100 mM, pH 7.4), EDTA (1 mM), MgCl2 (3 mM), 1% DMSO, and an NADPH regenerating system consisting of glucose 6-phosphate (5 mM) and glucose 6-phosphate dehydrogenase (1 IU/ml). After a 2-min pre-incubation in a 37°C shaking water bath, NADP was added to a final concentration of 1 mM to initiate the 30-min reaction.

Detoxification of coumarin 3,4-epoxide: GSH conjugation, oxidation and reduction reactions. CE-SG formation was evaluated by adding GSH (5 mM) and mouse or rat lung cytosol equal to 1 unit of GST activity to a mouse liver microsomal reaction mixture. Mouse liver microsomes were used because they show a high rate of CE formation and they are in abundant supply. Additionally, they provide a constant source of CE which allows the formation of CE-SG to be compared between species.

Oxidation of o-HPA to o-HPAA was determined in mouse and rat lung cytosol (0.25 mg protein) reaction mixtures without microsomes. The reaction mixtures contained potassium phosphate buffer (100 mM, pH 7.4) and NAD+ (1 mM) in a final volume of 1 ml containing 1% DMSO. After a 2-min pre-incubation in a 25°C shaking water bath, o-HPA (1 to 2000 µM final concentration) was added to initiate the reaction and the samples were incubated for up to 30 min.

Reduction of o-HPA to o-HPE was determined using the same conditions described for the examination of o-HPA oxidation with the exception that 0.5 mg of cytosolic protein was used and NAD+ was replaced by NADH (0.5 mM).

Sample preparation and RP-HPLC analysis of o-HPAA, o-HPE, o-HPA, and CE-SG. All reactions were terminated by the addition of 0.25 ml of ice-cold 15% trichloroacetic acid followed by centrifugation at 16,000 x g for 10 min. The ring-opened coumarin metabolites (o-HPAA, o-HPE and o-HPA) in the supernatant were separated by HPLC and quantified by UV detection at 275 nm (Vassallo et al., 2003Go). CE-SG was quantified at 332 nm using the same HPLC system (Vassallo et al., 2003Go). Linearity of product formation was demonstrated with time, and protein for o-HPAA, o-HPE, o-HPA, and CE-SG, and reaction conditions were adjusted in order to maintain initial rates of reaction. The limit of quantification for the ring-opened metabolites and CE-SG was 10 pmol and 0.5 pmol on column, respectively (Vassallo et al., 2003Go).

Data analysis. The apparent kinetic constants, Km and Vmax, for each reaction were initially evaluated using Michaelis-Menten, Lineweaver-Burk, and Eadie-Hofstee plots. These data suggested that all the reactions could be best described by a single-site model. Curve-fitting of the data was conducted using the Enzyme Kinetics Module (version 1.1) for SigmaPlot 2001 (Chicago, IL), and the best fit lines of the single-site model were generated using the Michaelis-Menten equation. The high-affinity site was described by Km and Vmax and the intrinsic clearance (CLint) was calculated by dividing the maximal rate of metabolism by the Michaelis constant (Vmax/Km).

All analyses were carried out as four independent determinations, and the results are presented as the mean ± SE of the replicates.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The rate of coumarin epoxidation was determined in lung microsomal reaction mixtures in the absence of GSH. Since CE has a half-life of 4 s (Born et al., 1997Go), the rate of epoxidation was determined indirectly by measuring the formation of o-HPA. The highest rate of o-HPA formation, which exceeded 7 nmol/min/mg protein (2 mM coumarin), was observed in mouse lung microsomes, and this was 8 times higher than in rat lung microsomes (Fig. 2). The Km for epoxidation in mice and rats was 155 µM and 2573 µM, respectively, indicating mouse, but not rat, lung microsomes readily form CE (Table 2). This apparent species difference in coumarin epoxidation is best represented by the total intrinsic clearance (CL), with epoxidation proceeding 69 times faster in mouse compared to rat lung microsomes.



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FIG. 2. Michaelis-Menten analysis of coumarin 3,4-epoxidation. The epoxidation of coumarin was determined in mouse, rat and human lung microsomal reaction mixtures. The rate of epoxidation was greatest in the mouse, whereas no CE was formed in 16 different human lung microsomes. These data represent the mean (±SE) of four separate determinations.

 

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TABLE 2 Apparent Kinetic Constants for o-HPA Formation in Lung Microsomes

 
o-Hydroxyphenylacetaldehyde was not detected in lung microsomal reaction mixtures from sixteen different humans, and the absence of detectable levels of o-HPA in human lung samples indicates that CE was not formed. Since o-HPA formation was not detected, human samples were excluded when detoxification reactions were examined.

The kinetics of CE-SG formation in a mouse liver microsomal reaction mixture containing mouse or rat lung cytosol were determined, and these data represent the sum of enzymatic and non-enzymatic conjugation. The average Km for GSH conjugation was approximately 800 µM for mice and rats, and the Vmax was 3.5 nmol/min/mg protein and 6 nmol/min/mg protein for mice and rats, respectively (Fig. 3 and Table 3). In the mouse, the intrinsic clearance for epoxidation was 11 times higher than the intrinsic clearance for GSH conjugation. Therefore, it is likely that a large portion of CE formed would escape GSH conjugation and form o-HPA, a cytotoxic metabolite. In contrast, the intrinsic clearance for GSH conjugation was 12 times higher in the rat than the intrinsic clearance for epoxidation. Consequently, any CE formed in the rat lung would likely be readily detoxified via GSH conjugation.



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FIG. 3. Michaelis-Menten analysis of GSH conjugation of CE. Mouse liver microsomes (0.25 mg protein) were used to metabolize coumarin to CE, and rat and mouse lung cytosol (equal to 1 unit of GST activity) and GSH (5 mM) were added to catalyze GSH conjugation. When CE formation was constant, CE-SG formation was greatest in the rat. These data represent the mean (±SE) of four separate determinations.

 

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TABLE 3 Apparent Kinetic Constants for CE and o-HPA Detoxification in Lung Cytosol

 
Coumarin 3,4-epoxide can also rearrange to o-HPA, and this metabolite is detoxified by one of two routes. It can be oxidized to o-HPAA or reduced to o-HPE. The average Km for oxidation in mice and rats was 1.5 µM (Fig. 4 and Table 3). In contrast, the Km for reduction was approximately 1000 and 400 µM in the mouse and rat, respectively (Fig. 5 and Table 3). These high Km values indicate reduction contributes very little to the overall clearance of o-HPA. Instead, o-HPAA formation is favored, and the Vmax for this reaction was 0.08 and 0.33 nmol/min/mg protein in the mouse and rat, respectively (Fig. 4 and Table 3). Although the Km for the mouse suggests oxidation of o-HPA is a high-affinity reaction, the Vmax indicates the rate of o-HPAA formation may be rate limiting and inadequate to address the rapid formation of o-HPA in the mouse lung. In contrast, detoxification reactions are less important in the rat since the Km for o-HPA formation is 2573 µM, which indicates o-HPA would not form to any appreciable extent.



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FIG. 4. Michaelis-Menten analysis of oxidation of o-HPA to o-HPAA. Oxidation of o-HPA to o-HPAA was determined with mouse and rat lung cytosol (0.25 mg protein). Oxidation of o-HPA was most efficient in the rat. These data represent the mean (±SE) of four separate determinations.

 


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FIG. 5. Michaelis-Menten analysis of reduction of o-HPA to o-HPE. Reduction of o-HPA to o-HPE was determined with mouse and rat lung cytosol (0.5 mg protein). Reduction of o-HPA was similar in the mouse and rat. These data represent the mean (±SE) of four separate determinations.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
This study provides new insight into the role of species-specific metabolism in the sensitivity of mouse Clara cells to coumarin toxicity. The current data indicates that rates of CE formation in mouse and rat whole lung microsomes are predictive of species susceptibility to lung injury in vivo. However, these data also suggest that the balance between the rate of coumarin epoxidation and detoxification is a major determinant of species differences in coumarin-induced Clara cell injury. Furthermore, it may be inferred that species that fail to metabolize coumarin to CE in the lung should be resistant to acute Clara cell toxicity.

The current data support the hypothesis that coumarin-induced Clara cell toxicity in the mouse is mediated by local CE formation. It is unlikely that cytotoxic coumarin metabolites generated in the liver play a critical role in Clara cell toxicity given that CE has a half-life of 4 s (Born et al., 1997Go), and neither CE nor o-HPA have been detected in the circulation of mice or rats following oral coumarin administration (Born et al., 2003Go). This conclusion is further supported by the fact that the maximum concentration of radioactivity in the B6C3F1 mouse lung following an oral dose of 14C-coumarin (200 mg/kg) was 561 µM, and this is greater than the Km (155 µM) for CE formation in mouse lung microsomes (Born et al., 2003Go). Interestingly, whereas similar levels of radioactivity were achieved in the F344 rat lung following oral 14C-coumarin (200 mg/kg) administration, the Km for CE formation is much higher (2.6 mM) in rat lung microsomes (Born et al., 2003Go). Thus, the epoxidation of coumarin is a favorable reaction in the mouse but not the rat, an observation that is consistent with the lack of pulmonary effects in the rat lung. Collectively, these data are consistent with the hypothesis that mouse Clara cell toxicity results from local bioactivation of coumarin.

The localization of coumarin metabolism to the Clara cell is further supported by the fact that CE formation in mouse lung microsomes is predominantly catalyzed by CYP2F2 (Born et al., 2002Go) and that the Clara cell is the sole site of CYP2F expression in the mouse lung (Buckpitt et al., 1995Go; Ritter et al., 1991Go). Similarly, naphthalene and styrene cause acute Clara cell necrosis in the mouse lung through a mechanism attributed to CYP2F2 metabolism (Buckpitt et al., 1995Go; Hynes et al., 1999Go; Nagata et al., 1990Go). In contrast, lung CYP2F expression is lower in the rat than in the mouse (Buckpitt et al., 1995Go), and Clara cell necrosis is not observed in rat lung following exposure to these compounds (Green et al., 2001Go; O'Brien et al., 1985Go).

Cytochrome P-450 2F1, an ortholog of CYP2F2 with approximately 80% homology, has been identified in human lung (Nhamburo et al., 1990Go). However, in this study, coumarin was not metabolized to CE in 16 different human lung microsomal preparations, which suggests that either human CYP2F has a qualitatively different catalytic activity than rodent CYP2F or that the low abundance of CYP2F1 results in negligible CE formation. As with coumarin, human lung microsomes are not particularly effective in metabolizing styrene to styrene oxide (Carlson et al., 2000Go; Filser et al., 2002Go; Nakajima et al., 1994Go), and metabolism of naphthalene to naphthalene oxide is much slower in human than in mouse whole lung microsomes (Buckpitt and Bahnson, 1986Go). Thus, it is hypothesized that the abundance and high activity of CYP2F2 in the mouse Clara cell predisposes this species and cell type to metabolism-dependent toxicity from xenobiotics such as coumarin, naphthalene, and styrene (Born et al., 2002Go; Buckpitt et al., 1995Go; Hynes et al., 1999Go).

Species differences in lung metabolic activity also appear to be driven in part by the significantly higher number of Clara cells within the terminal bronchioles of mice compared to rats and humans (Lumsden et al., 1984Go; Plopper et al., 1980bGo). Furthermore, Clara cells differ morphologically among species; with human cells containing little smooth endoplasmic reticulum (Plopper et al., 1980aGo). Based on metabolic, anatomical, and morphological differences between rodents and humans, and the apparent metabolic requirements for toxicity in the mouse lung, the human lung is unlikely to be susceptible to coumarin-induced Clara cell toxicity.

Although coumarin-induced Clara cell toxicity is dependent on CE formation, it is hypothesized that the balance between coumarin epoxidation and detoxification reactions dictates whether toxicity occurs in individual Clara cells. GSH plays a major role in detoxifying CE in the liver (Huwer et al., 1991Go; Lake, 1984Go; Vassallo et al., 2004Go), and the role of GSH in modulating Clara cell toxicity is based on several studies in which GSH depletion by diethyl maleate or buthionine sulfoximine rendered mice more susceptible to Clara cell toxicants including 4-ipomeanol, dichloroethylene, and naphthalene (Boyd et al., 1981Go; Okine et al., 1985Go; Warren et al., 1982Go). Recently, West et al. (2000b)Go determined that the steady-state GSH varies widely between individual Clara cells, and it was concluded that this heterogeneity is a significant factor in determining the susceptibility of individual Clara cells to naphthalene injury (West et al., 2000bGo). Several investigators have determined that mouse Clara cells become less susceptible to injury, or tolerant, following repeated administration of coumarin, naphthalene or 4-ipomeanol (Born et al., 1999Go; Boyd et al., 1981Go; O'Brien et al., 1989Go), and recent evidence suggests tolerance is mediated largely by changes in GSH regulation (West et al., 2000aGo). Since Clara cell tolerance does not result from decreased CE formation in the mouse lung (Born et al., 1999Go), it is hypothesized that Clara cell tolerance to coumarin develops, at least in part, from an increased ability of cells to detoxify CE via GSH conjugation.

In the absence of GSH, CE spontaneously rearranges to o-HPA (Born et al., 1997Go). This aldehyde is cytotoxic and its detoxification by aldehyde dehydrogenase (ALDH) is a critical determinant of species differences in coumarin-induced hepatotoxicity (Born et al., 2000Go; Vassallo et al., 2004Go). While o-HPA can be detoxified to either o-HPAA or o-HPE by lung cytosol, the in vitro kinetics of these reactions indicates that the lung favors oxidation of the aldehyde. Histochemical localization of ALDH in the mouse and rat lung indicates this activity is predominately located in the Clara cell (Bogdanffy et al., 1986Go; Dragani et al., 1996Go). Therefore, it is reasonable to expect that the in vitro kinetics of o-HPA oxidation qualitatively represent detoxification in the Clara cell.

Despite the low Km (1.5 µM) for o-HPA oxidation, the Vmax for this reaction is 90 times slower than the Vmax for o-HPA formation in mouse lung microsomes. Consequently, if intracellular GSH were depleted, o-HPA oxidation would become a rate-limiting step in detoxification. This conclusion is supported by the fact that while an oral 200 mg/kg/day bolus dose of coumarin caused acute and chronic lung effects in the mouse (Born et al., 1998Go; NTP, 1993aGo), coumarin administered in the diet at 271 mg/kg/day did not result in an increased lung tumor incidence (Carlton et al., 1996Go). In fact, after a 200 mg/kg oral bolus dose of 14C-coumarin the maximum concentration in the mouse lung was 37-times higher than the maximum concentration obtained after dietary dosing at 200 mg/kg (Born et al., 2003Go). Collectively, these data suggest that a 200 mg/kg bolus dose of coumarin overwhelms the detoxification pathways of the mouse lung, namely GSH conjugation and o-HPA oxidation, whereas dietary administration of coumarin generates CE at a level that is within the detoxification capacity of the lung.

The susceptibility of the mouse to acute Clara cell toxicity and lung tumor formation is not unique to coumarin. Naphthalene (Mahvi et al., 1977Go; NTP, 1992Go; O'Brien et al., 1985Go), methylene chloride (Foster et al., 1992Go; NTP, 1986Go), trichloroethylene (Fukuda et al., 1983Go; Maltoni et al., 1986Go; NTP, 1990Go; Odum et al., 1992Go), and styrene (Cruzan et al., 1997Go, 2001Go; Green et al., 2001Go) share a similar profile of acute Clara cell toxicity and an increased incidence of lung tumors in mice, yet none of these chemicals cause Clara cell necrosis or lung tumors in rats (Cruzan et al., 1997Go, 1998Go; Green et al., 2001Go; NTP, 1986Go, 1990Go, 1993aGo, 2000Go). Although the mechanism linking Clara cell toxicity and lung tumor formation remains to be elucidated, it seems that this pattern is mouse-specific, and with regard to coumarin, the formation of the epoxide is required (Born et al., 1998Go; NTP, 1993bGo). Since coumarin is not metabolized to CE in human lung microsomes, it is unlikely that it would cause any toxicity to the human lung.

In summary, this work demonstrates that coumarin-induced mouse Clara cell toxicity is dependent on the formation of coumarin 3,4-epoxide. Furthermore, these data suggest that it is the balance between epoxidation and detoxification that dictates species susceptibility to coumarin-induced Clara cell toxicity. More importantly, CE formation was not detected in human whole-lung microsomes, which indicates that the human lung is unlikely to be susceptible to acute Clara cell toxicity following exposure to levels associated with foods or consumer products (approximately 0.06 mg/kg/day; Lake, 1999Go).


    ACKNOWLEDGMENTS
 
The authors are grateful to Dr. Lois Lehman-McKeeman for her intellectual contributions to the research leading up to and supporting this work.


    NOTES
 

1 To whom correspondence should be addressed at Miami Valley Laboratories, The Procter and Gamble Company, 11810 East Miami River Road, Cincinnati, OH 45252. Fax: (513) 627-1760. E-mail: vassallo.jd{at}pg.com.


    REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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