Evidence for cytochrome P-450 mediated metabolism in the bronchiolar damage by naphthalene

Chem.-Biol. Interactions, 40 (1982) 287--303 Elsevier/North-Holland Scientific Publishers Ltd.

287

EVIDENCE FOR CYTOCHROME P-450 MEDIATED METABOLISM IN THE BRONCHIOLAR DAMAGE BY NAPHTHALENE*

D.L. WARREN, D.L. BROWN, JR. and A.R. BUCKPITT**

Department of Community and Environmental Medicine and Southern Occupational Health Center, University of California, Irvine, CA 92717 (U.S.A.) (Received September 2nd, 1981) (Revision received January 29th, 1982) (Accepted February 5th, 1982)

SUMMARY

Intraperitoneal administration of the volatile hydrocarbon, naphthalene, resulted in severe bronchiolar epithelial cell necrosis in mice, while hepatic or renal necrosis was not observed. Pulmonary damage and mortality by naphthalene were increased by prior treatment with diethyl maleate and decreased by prior treatment with piperonyl butoxide (1600 mg/kg). SKF 525A pretreatment had no effect on naphthalene-induced pulmonary damage. Administration of [14C] naphthalene resulted in the covalent binding of radiolabel to tissue macromolecules. Highest levels of binding occurred in lung, liver and kidney. Levels of covalent binding reached a maximum 2--4 h after treatment and corresponded to rapid glutathione depletion in lung and liver. Covalent binding was dose-dependent and showed a threshold between 200 and 400 mg/kg which coincided with almost total depletion of tissue glutathione levels. Covalent binding of reactive metabolites was increased 3--4-fold by prior treatment with diethyl maleate, and was decreased 3--4-fold by pretreatment with piperonyl butoxide. These studies support the view that naphthalene-induced pulmonary damage is mediated by the cytochrome P-450
INTRODUCTION

A growing number of chemicals are known to be metabolized by the cytochrome P-450 monooxygenase enzymes to highly reactive, electrophilic intermediates which have been implicated in carcinogenesis, mutagenesis *Supported by Air Force Contract #AF 33615-80-C-0512. **Author to whom correspondence should be sent.

288 and/or cytotoxicity. Although lung quantitatively contains lower levels of monooxygenase activity in comparison to liver, there is now substantial evidence suggesting that xenobiotic bioactivation plays a role in chemicalinduced pulmonary damage [1]. The sensitivity of the lung to damage by chemicals requiring metabolic activation may stem, in part, from the apparent high degree of cellular localization of c y t o c h r o m e P-450 monooxygenases. A number of recent studies, utilizing a variety of experimental approaches, indicate that the non~iliated bronchiolar epithelial cell (Clara cell) is a major locus of pulmonary cytochrome P-450 monooxygenase activity and indeed this cell t y p e appears to be an important target cell for a variety of cytotoxic and carcinogenic chemicals requiring metabolic activation [2--6]. Intraperitoneal administration of a number of aromatic hydrocarbons, including bromobenzene, chlorobenzene and naphthalene, has been shown to cause necrosis of pulmonary bronchiolar epithelial cells in mice [7]. Electron microscopic studies showing the naphthalene preferentially damaged Clara cells [8] and studies showing that this hydrocarbon is metabolized in vivo [9] and in vitro [10] to a number of reactive, potentially cytotoxic intermediates suggested that metabolism of naphthalene by the cytochrome P-450 monooxygenases may be important in mediating the pulmonary damage. The studies described in this report were done to explore the possibility that naphthalene is metabolized by c y t o c h r o m e P-450 monooxygenases in vivo to reactive intermediates and to determine whether the formation and subsequent covalent binding of these metabolites could be correlated with target tissue damage. MATERIALS AND METHODS Animals Male Swiss Webster mice weighing 20--25 g were purchased from Simonsen Breeding Labs, Gilroy, CA. All animals were housed over hardwood bedding and were allowed free access to f o o d and water. Animals were not used sooner than 5 days after receipt from the supplier. Chemicals Reduced glutathione was purchased from Calbiochem, La Jolla, CA; piperonyl butoxide from Chemical Dynamics Corporation, South Plainfield, NJ; diethyl maleate from Aldrich Chemical Company, Milwaukee, WI. SKF 525A (~99.8% pure by high pressure liquid chromatography on a C1~ reverse phase column using 65% methanol/water as the mobile phase. [~4C]Naphthalene was diluted with unlabelled c o m p o u n d to give final specific activities ranging between 70 and 1500 dpm/nmol. Drug treatments. With the exception of SKF 525A, which was dissolved

289 in 0.9% saline, all compounds were dissolved in corn oil such that 0.1 ml was administered intraperitoneally per 10 g b o d y wt. to achieve the desired dose. Control animals received vehicle only. Pretreatments (SKF 525A, piperonyl butoxide or diethyl maleate) were given 30 min prior to the administration of naphthalene. Histopathology. Animals were sacrificed by pentobarbital overdose 24 h after the administration of naphthalene. The trachea was ligated, and the lungs and heart were removed en bloc for fixation in 10% buffered formalin. A 3--5-ram section of liver was cut with a razor blade and was transferred with the kidneys to buffered formalin. All tissues were paraffin embedded, sectioned 5--6 pm and stained with hematoxylin and eosin.

Assay for reduced glutathione Animals were sacrificed by cervical dislocation, lungs were thoroughly perfused with i c e , o l d heparinized saline and tissues were quickly removed and frozen at - 8 5 ° C . All tissues were homogenized in 0.1 M phosphate buffer (pH 7.4) and an aliquot of the homogenate was vortexed with ice-cold 4% sulfosalicylic acid. The samples were centrifuged at 4°C for 30 min at 3000 X g and an aliquot of the supernatant was taken for non-protein sulfhydryl determination by the method of Ellman [11]. Absorbance at 412 nm of authentic glutathione standards was linear between 10 and 200 pg/tube.

Covalent binding determination The protein precipitate remaining in the centrifuge tubes after sulfosalicylic acid precipitation was washed exhaustively with ethanol/ether (3 : 1) and methanol until no {urther radioactivity could be detected in the washes. The protein pellet remaining after the final wash was dissolved in 1.0 N NaOH, an aliquot was removed for protein determination [12] and a further aliquot was added to 5 ml ACS (Amersham/Searle) for the determination of radioactivity. All samples were counted in a Beckman 3150 T scintillation counter for 20 min. Counts were corrected for quench by internal standardization with [ 14C/toluene standard. Covalent binding was expressed as nmol bound/ mg protein. To demonstrate that radioactivity remaining in the washed macromolecular pellet was covalently bound, an aliquot of the NaOH digest was spotted on a Silica Gel G plate and successively eluted with chloroform, ethyl acetate and methanol. Solvent was allowed to evaporate between each step. Sections of the plate (0.5 cm) were scraped into liquid scintillation vials and the radioactivity was shown to remain with the protein at the origin. In a d d i t i o n , the protein digest from several liver samples was combined and sufficient 10% trichloroacetic acid was added to precipitate the macromolecules. After centrifugation, the supernatant was discarded and the precipitate was redissolved in 1.0 N NaOH. After an aliquot of the digest was taken for determination of cpm/mg protein, the process of precipitation and redisSolution was repeated 3 more times. The specific activity of the digest remained constant throughout this process.

290

Separation of subcellular fractions. Microsomal, nuclear, mitochondrial and cytosolic fractions were prepared from lung, liver and kidney by discontinuous sucrose gradient centrifugation as described by Mazel [13]. Each subfraction was washed and repelleted at least twice more than described in the original procedure. RESULTS

Lethality and target tissue necrosis by naphthalene The 24-h LDs0 of naphthalene, administered intraperitoneally to male Swiss Webster mice, is 380 mg/kg (350--413 mg/kg, 95% confidence limits, n = 65) as calculated by the method of Lichtfield and Wilcoxon [14]. Deaths occurred in the first 24 h; those animals surviving the first 24 h were held an additional 6 days with no further deaths. Histologic examination of lungs from animals treated 24 h earlier with 200 mg/kg naphthalene revealed mild to moderate disruption of the normal cuboidal appearance of the bronchiolar epithelium. At higher doses of naphthalene, there was extensive necrosis of bronchial and bronchiolar epithelial cells, and large numbers of exfoliated epithelial cells were observed in the bronchiolar lumen. Tissue necrosis was not observed in either liver or kidney 24, 48 or 72 h after the administration of naphthalene in doses as high as 375 mg/kg. These results confirm and extend earlier reports on the bronchiolar damage b y naphthalene [7,8,15]. Covalent binding of reactive metabolites to tissue macromolecules in vivo Four hours after the intraperitoneal administration of [14C]naphthalene (200 mg/kg, 250 dpm/nmol), radiolabel was b o u n d covalently to macromolecules in several tissues (Fig. 1). Covalent binding levels were highest in tissues which contain c y t o c h r o m e P-450 monooxygenase activity, such as lung, liver and kidney, and were much lower in tissue like muscle where monooxygenase activity is undetectable. The tissue specificity for covalent binding of radioactivity did n o t parallel the selectivity of tissue damage by naphthalene. Time course for covalent binding and depletion of reduced glutathione Tissues from groups of four mice each, sacrificed at times ranging from 15 min to 8 h after the administration of [14C]naphthalene (200 mg/kg, 288 dpm/nmol), were assayed for glutathione and covalently b o u n d naphthalene metabolites. Animals serving as glutathione controls were treated with vehicle only and sacrificed 1 h (control for 1- and 2-h time points) or 6 h (control for 4- and 8-h time points) later. The data in Fig. 2 show that intraperitoneal administration of 200 mg/kg naphthalene caused significant depletion of pulmonary and hepatic but not renal glutathione. The levels of reduced giutathione in the lung 'decreased to 44% of control at 4 h but returned to nearly 80% of the control levels after 8 h. Hepatic glutathione levels decreased to 17% of control levels at 2 h and gradually returned to

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control values at 8 h. In contrast, renal non-protein sulfhydryl levels were decreased only slightly after a 200 mg/kg dose of naphthalene. The levels of covalently bound naphthalene metabolites rose to a maximum between 2 and 4 h and inversely paralleled the decrease in glutathione content in lung and liver. Similar to the results presented in Fig. 1, binding levels were highest in liver, with lower levels being found in kidney and lung, respectively. Negligible covalent binding was observed in muscle.

Dose response for covalent binding and glutathione depletion Groups of 3--4 mice were treated intraperitoneally with doses of [~4C]naphthalene ranging from 25 to 600 mg/kg and sacrificed 4 h later for the determination of reduced glutathione levels and covalently bound naphthalene metabolites. Specific activities of the dose solutions were 1359 dpm/ nmol (25, 50 mg/kg), 471 d p m / n m o l (100, 200 mg/kg) and 117 d p m / n m o l (400, 600 mg/kg). Increasing doses of naphthalene from 25 to 200 mg/kg resulted in a moderate decrease in tissue glutathione levels in lung and liver but not kidney (Fig. 3). As the dose increased from 200 to 400 mg/kg, there was a substantial drop in tissue glutathione levels in lung, liver and kidney. Glutathione levels in all three tissues were less than 10% of control levels at the 600 mg/kg dose. While the covalent binding of reactive naph-

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296 of four to five mice were administered piperonyl butoxide (1600 mg/kg), SKF 525A (25 mg/kg) or corn oil intraperitoneally followed 30 min later by either [~4C]naphthalene (115 dpm/nmol, 400 mg/kg) or unlabelled naphthalene (400 mg/kg). Animals receiving doses of [14C]naphthalene were sacrificed 4 h later for covalent binding and glutathione determination, while animals receiving unlabelled hydrocarbon were sacrificed by pentobarbital overdose 24 h later for histopathology. Light micrographs showing sections of terminal bronchiolar airways from mice treated with either corn oil alone or corn oil, SKF 525A or piperonyl butoxide plus 400 mg/kg naphthalene are shown in Fig. 4 (A--D, respectively). Intraperitoneal administration of 400 mg/kg naphthalene caused extensive disruption, necrosis and exfoliation of cells of the bronchiolar epithelium

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297 which was not prevented by pretreatment with SKF 525A put was decreased substantially by prior administration of piperonyl butoxide. Piperonyl butoxide pretreatment also decreased the 24-h mortality by napthalene. Of five mice treated with corn oil plus 500 mg/kg naphthalene, none survived while all five mice treated with piperonyl butoxide prior to 500 mg/kg naphthalene lived. The data in Fig. 5 show the effects of prior treatment with SKF 525A or piperonyl butoxide on glutathione depletion and the covalent binding of reactive metabolites after a 400-mg/kg dose of naphthalene. Consistent with previous data, 400-mg/kg doses of naphthalene produced substantial depletion of glutathione in lung, liver and kidney. Pretreating mice with SKF 525A did not block the glutathione depletion caused by 400 mg/kg naphthalene. In contrast, pulmonary, hepatic and renal glutathione levels in mice treated with piperonyl butoxide and naphthalene were significantly higher than in corn oil plus naphthalene-treated animals. The covalent binding of reactive metabolites to tissue macromolecules in lung, liver and kidney was decreased to 50% and 25% of the levels in vehicle pretreated animals by SKF 525A and piperonyl butoxide pretreatment, respectively.

Effect of diethyl maleate pretreatment Further evidence suggesting that glutathione modulates the bronchiolar damage by naphthalene and the covalent binding of reactive naphthalene metabolites was provided by experiments in which mice were pretreated with diethyl maleate. Prior treatment with diethyl maleate resulted in a substantial increase in naphthalene induced bronchiolar damage (Fig. 6) and in lethality 24 h after treatment with naphthalene. Extensive sloughing and exfoliation of bronchiolar epithelial cells occurred in lungs of all 5 mice treated with diethyl maleate prior to 50 mg/kg naphthalene whereas lungs of mice (n = 5) treated with vehicle prior to 50 mg/kg naphthalene were indistinguishable from controls. Within 24 h after treatment with 300 mg/kg naphthalene, one out of 5 mice pretreated with corn oil and all 5 mice pretreated with diethyl maleate died. Pretreatment with diethyl maleate also markedly increased the covalent binding of reactive metabolites to tissue macromolecules in lung, liver and kidney 4 h after administration of 200 mg/kg [14C]naphthalene (Fig. 7). The ratio of bound metabolites in lung, liver and kidney was similar in vehicle pretreated versus diethyl maleate pretreated animals.

Covalent binding to subcellular fractions To determine whether there were differences in the binding to macromolecules isolated from subcellular fractions of target vs. non-target tissues in the mouse, 15 mice were treated intraperitoneally with 400 mg/kg [14C]naphthalene (70 dpm/nmol) and sacrificed 4 h later. Binding to macromolecules in various subcellular fractions is shown in Table I. Binding levels were highest in microsomal and supernatant fractions and lower in the nuclear and mitochondrial fractions.

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TABLE I COVALENT BINDING OF RADIOLABEL FROM [14C]NAPHTHALENE IN SUBCELLULAR FRACTIONS OF MOUSE TISSUES Values are the average of 2 determinations from pooled tissues of 15 animals. Values in parenthesis indicate the covalent binding as a percentage of that in the homogenate. Subcellular fraction

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DISCUSSION T h e results o f t h e p r e s e n t s t u d y are c o n s i s t e n t w i t h t h e view t h a t c y t o c h r o m e P - 4 5 0 < l e p e n d e n t m e t a b o l i c a c t i v a t i o n o f n a p h t h a l e n e plays a role in t h e b r o n c h i o l a r d a m a g e elicited b y this h y d r o c a r b o n in mice. These studies have s h o w n t h a t n a p h t h a l e n e is m e t a b o l i z e d in vivo t o a reactive m e t a b o l i t e or

300 metabolites which covalently bind to tissue macromolecules. Binding was consistently greater in tissues with high cytochrome P-450 monooxygenase activity than in tissues which do not contain detectable activity (Fig. 1). Maximal levels of covalent binding were achieved well before the onset of detectable signs of bronchiolar damage [8] (Fig. 2). Covalent binding was dose-dependent and showed a marked dose threshold which depended u p o n substantial depletion of tissue glutathione stores (Fig. 3). This threshold is similar to that observed for acetaminophen or bromobenzene [16]. Lung damage at the 200 mg/kg dose of naphthalene was mild; swelling occurred in a few cells of the distal bronchiolar airways and only small numbers of cells were sloughed into the airway lumen. The 300 and 400 mg/kg doses of naphthalene caused extensive disruption and exfoliation of bronchiolar epithelial cells. Thus, the severity of bronchiolar damage roughly correlated with the extent of reactive metabolite binding in the lung. Pretreatments which altered the bronchiolar damage by naphthalene altered the covalent binding in the lung as well. For example, prior treatment with piperonyl butoxide, but not SKF 525A, decreased lung damage (Fig. 4). Likewise, piperonyl butoxide pretreatment inhibited covalent binding to a much greater extent than did SKF 525A pretreatment (Fig. 5). These results also are consistent with data from previous in vitro studies [10] showing that the formation of reactive intermediates from naphthalene is dependent u p o n c y t o c h r o m e P-450 mediated metabolism. In addition to the parallel association between pulmonary covalent binding and bronchiolar necrosis which was demonstrated with the monooxygenase inhibitors, a clear correlation between binding and necrosis was shown in animals pretreated with diethyl maleate. Covalent binding and lung damage were increased substantially by prior diethyl maleate-induced depletion of glutathione (Figs. 6 and 7). Although the extent of covalent binding of reactive metabolites in the lung appears to correlate with bronchiolar damage, the tissue selectivity for damage by naphthalene is not reflected in the preferential arylation of tissue macromolecules in the lung. At all doses studied, covalent binding was higher in non-target tissues (liver and kidney) than it was in the lung. There are several possible explanations for this lack of tissue specificity for covalent binding which would still be consistent with a role for reactive metabolites in bronchiolar damage. Due to the high degree of cellular localization of pulmonary cytochrome P-450, the formation and covalent binding of reactive naphthalene metabolites may occur in a very small portion of total lung cells. Thus, if covalent binding could be expressed on the basis of nanomoles bound per cell, it is possible that the specific activity of binding in bronchiolar cells would exceed that in hepatic or renal cells. Autoradiographic studies may be necessary to address this point. Furthermore, metabolism of naphthalene may result in the formation of several different reactive species {such as the 1,2 oxide, 1,2:3,4
301 molecules critical to the survival of the cell. Thus, the nature of reactive metabolites produced in different tissues could determine the tissue specificity of damage. A further possibility is that the macromolecular targets to which reactive metabolites of naphthalene bind differ in target and nontarget tissues. At present the cell constituent-metabolite interaction(s) critical to the death of the cell has not been identified for any of the hepatoor pulmonary toxins requiring metabolic activation. (See discussions by Gillette [ 16 ] ). The apparent interrelationship between the generation of reactive metabolites, the depletion of reduced glutathione and the bronchiolar damage by naphthalene noted in this study is similar to that observed with many hepatic and pulmonary toxicants [15,18]. The time course and dose response studies showed an inverse relationship between covalent binding and glutathione levels. The marked decrease in the covalent binding of reactive metabolites in animals pretreated with piperonyl butoxide was reflected in significantly higher tissue glutathione levels in comparison to vehicle pretreated animals given 400 mg/kg naphthalene (Fig. 5). Moreover, the dose response and SKF 525A pretreatment studies suggested that reactive naphthalene metabolites preferentially form conjugates with glutathione; only after the supply of tissue glutathione was exhausted did significant covalent binding occur. These results contrast with those observed with the bronchiolar cytotoxin, 4-ipomeanol, where there is no apparent threshold for covalent binding [19]. Although glutathione modulates the covalent binding and the lung damage of this furan derivative, reactive 4-ipomeanol metabolites do not appear to form glutathione adducts preferentially. Thus, the differences in the mechanism by which glutathione modulates the toxicity of naphthalene and 4-ipomeanol may be related to differences in the ability of the glutathione transferases to catalyze glutathione adduct formation with the electrophiles generated during their metabolism (cf. Discussion in Refs. 20 and 21). That glutathione plays an important role in the detoxification of naphthalene metabolites is supported b y the studies on the effects of diethyl maleate pretreatment. An additional question which cannot be answered by these studies is whether metabolites that are b o u n d in the lung and/or appear to be related to the bronchiolar necrosis are generated in situ in pulmonary tissue or arise via metabolism in the liver. Several lines of evidence suggest that the liver could play an active role in either or both of these processes. The data showing that pulmonary glutathione is nearly totally depleted by 400- and 600-mg/kg doses of naphthalene indicates that electrophilic metabolites are reaching a majority of glutathione containing cells in the lung. Whether these electrophilic metabolites are generated within the apparently few c y t o c h r o m e P-450 containing cell types in the lung or in other tissues is not known, but it seems that they are capable of crossing cell membranes. The possibility that 1-naphthol, formed by hepatic monooxygenases, could circulate to the lung and undergo metabolism to reactive intermediates must be considered, since Hesse and Metzger [10] have recently shown that

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covalently bound metabolites from naphthalene arise from the secondary metabolism of 1-naphthol. Further studies will be needed to determine the relative roles of the lung and liver in the formation of naphthalene metabolires which are reactive and/or initiate the lung damage. ACKNOWLEDGEMENT

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