Relationship of naphthalene and 2-methylnaphthaline metabolism to pulmonary bronchiolar epithelial cell necrosis

Pharmac. Ther. Vol. 41, pp. 39~410, 1989

0163-7258/89 $0.00 + 0.50 Copyright © 1989 Pergamon Press pie

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Specialist Subject Editors: T. E. GRAMand M. BOYD

RELATIONSHIP OF NAPHTHALENE AND 2-METHYLNAPHTHALENE METABOLISM TO PULMONARY BRONCHIOLAR EPITHELIAL CELL NECROSIS ALAN R. BUCKPITT* and RONALDB. FRANKLINt *Occupational and Environmental Health Unit, Northern California Occupational Health Center and Department of Pharmacology and Toxicology, School of Veterinary Medicine, University of California, Davis, CA 95616, U.S.A. f Drug Metabolism and Disposition, Lilly Research Laboratories, Eli Lilly and Co., Indianapolis, IN 46285, U.S.A.

1. INTRODUCTION Non-oncogenic pulmonary diseases were the fifth leading cause of death in the United States in 1979 and were a major factor in morbidity and disability (Whittenberger, 1985). In addition, pulmonary cancer is a leading cause of cancer related deaths in both males and females (Loeb et al., 1984). While cigarette smoking is thought to contribute substantially to various lung diseases, additional environmental factors, including exposure to various chemicals in industry and in air and water are likely to play a role in these disease processes. For a number of reasons, identification of specific environmental factors contributing to pulmonary diseases in humans is difficult and many remain unknown. A significant element in the difficulty of estimating the risk of exposure to a given chemical stems from the uncertainty of extrapolating results derived in animals to the human and from the potential differences in the spectrum of toxicity of particular chemicals, when the lung is exposed to a compound via the vasculature vs by inhalation. While the question of species differences in sensitivity of toxic and/or carcinogenic effects is germane to agents that damage other tissues such as liver or kidney, it is particularly important with pulmonary toxic chemicals since there are major differences in sensitivity of rodent species to a number of these agents. For instance, the respiratory tract of hamsters but not rats is highly susceptible to both diethylnitrosamine and nitrosoheptamethyleneimine (ReznikSchuUer and Reznik, 1979; Reznik-Schuller and Lijinski, 1979). Likewise, mice are far more sensitive than rats to acute bronchiolar epithelial cell injury by dichloroethylene (Chieco et al., 1981; Krijgsheld et al., 1984), various low molecular weight aromatic hydrocarbons such as bromobenzene (Reid et al., 1973) naphthalene (Reid et al., 1973; O'Brien et al., 1985) and 2-methyl-naphthalene (Griffin et al., 1982) and butylated hydroxytoluene (Kehrer and Witschi, 1980) than are rats. Thus, this review will concentrate on the recent efforts of our laboratory and others to examine the underlying processes critical to the bronchiolar toxicity of naphthalene and 2-methyl-naphthalene, and our current approaches in developing methodology appropriate for examining metabolism, cytotoxicity and the possible interrelationship of these two processes in lungs from both rodents and human and nonhuman primates. In addition, this review will cover recent studies bearing on the role of in situ vs extrapulmonary metabolism in the lung toxicity produced by these agents. 2. ENVIRONMENTAL IMPORTANCE/OCCURRENCE Naphthalene is a commercially important precursor in the manufacture of such products as phthalic anhydride, carbaryl, 2-naphthol, synthetic tanning agents, surfactants and 393

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A.R. BUCKPITTand R. B. FRANKLIN

organic intermediates. There are two major sources of manufacture, namely coal tar naphthalene from coal tar feedstocks and petroleum naphthalene from aromatic petroleum refinery streams. These two processes result in an annual production of approximately 650 million pounds (Chemical Economics Handbook, 1981; Kirk and Othmer, 1981). It may be released into the environment in gaseous or particulate form and find its way into ambient water (up to 2 mg/1), sewage plant effluents (up to 22 mg/1) and even drinking water supplies (up to 1.4 mg/1; see Sittig, 1980). Various combustion processes account for the high levels of naphthalene and 2-methylnaphthalene in ambient air. Indeed, naphthalene and 2-methylnaphthalene constitute the major portion of the polyaromatics in ambient air (Arey et al., 1987). A further major source of human exposure to naphthalene and 2-methylnaphthalene is mainstream and sidestream cigarette smoke. Quantities of naphthalene as high as 50 #g have been reported in the sidestream smoke from a single cigarette (Schmeltz et al., 1976). Methylnaphthalenes also have been used extensively as chemical intermediates, in the dye industry, as heat transfer fluids and in pesticide and fungicide formulations. Like naphthalene, methylnaphthalenes have been measured in drinking water and ambient air as well as in automobile exhaust gases and coal-combustion processes (see Franklin, 1987a). Naphthalene and 2-methylnaphthalene are appreciably soluble in water and there is serious concern regarding the toxicity of these two agents in marine organisms. Oil spills, leakages from industrial facilities, and waste from old coal gasification plants are primary sources for environmental pollution with these two hydrocarbons. The abundant literature on this topic will not be further addressed in this review. Additional references for this topic as well as literature dealing with the extensive microbial metabolism of the naphthalenes may be found in other reviews (for example, Franklin, 1987a,b). 3. TOXICOLOGY OF NAPHTHALENE The general toxicology of naphthalene in CD-1 mice has been evaluated in 14- and 90-day studies at doses up to 1 the LDs0 (Shopp et al., 1984). No immunotoxicity or drug-induced hemolysis and little pathology was observed in these investigations. Five to ten percent mortality and slight depression in body weight was observed after administration of naphthalene at the highest dose tested (267 mg/kg). No mortality was reported in the 90-day study at doses as high as 133 mg/kg and there were virtually no changes in organ weights. Data on the subchronic toxicity of 2-methylnaphthalene are not available. The acute toxicity of both naphthalene and 2-methylnaphthalene has been well characterized in a number of rodent species. The oral LDs0 for naphthalene ranges from approximately 350 mg/kg in the mouse (Plasterer et al., 1985) to 2200 mg/kg in the rat (Gaines, 1969). Additional discussion of the ocular and hematotoxicity of naphthalene can be found in the recent reviews by Franklin (1987a,b). Much of the recent research on both naphthalene and 2-methylnaphthalene has focussed on the highly selective lesions which are observed in the lung (Reid et al., 1973; Mahvi et al., 1977; Tong et al., 1981; Griffin et al., 1981, 1982, 1983; Warren et al., 1982) and, at higher doses, in the kidney (O'Brien et al., 1985; Griffin et al., 1983) of the mouse after intraperitoneal administration. All mouse strains tested appear to respond similarly to these compounds although differences in the dose required for the acute lethality are apparent. Bronchiolar necrosis also is observed in mice exposed to naphthalene vapor by nose-only inhalation for 4 hr at concentrations of 100 #g/l and above (Buckpitt et al., 1982). In contast, pulmonary toxicity is not observed in rats (O'Brien et al., 1985; Buckpitt, unpublished). The species and tissue selective toxicity of naphthalene and 2-methylnaphthalene have provided a basis from which to study critical biochemical and metabolic events leading to cellular injury and the remainder of this review will focus on these aspects. The Clara cell of the bronchiolar epithelium is the primary target cell for low doses of naphthalene and 2-methylnaphthalene. Alterations in the morphology of Clara cells are observed as early as 6 hr after the administration of 64 mg/kg naphthalene, i.p. (Mahvi

Relationship of naphthalene and 2-methylnaphthalene

395

et al., 1977; Rasmussen et al., 1986). At later times (24, 48 hr) and at higher doses the

severity of injury to Clara cells increases and ciliated cells also are affected. No ultrastructural alterations have been noted in alveolar Type I or Type II cells at any dose or time period after naphthalene administration. Furthermore, these alveolar cells do not become labelled in animals injected with 3H-thymidine. In comparison, bronchiolar epithelial cells were heavily labelled by 3H-thymidine up to 7 days after 256 mg/kg naphthalene and this correlated with continuing signs of injury to the epithelium (Rasmussen et al., 1986; Tong et al., 1981). The effects of single doses of naphthalene on the morphology of the bronchiolar epithelium contrast sharply with the results of experiments using multiple treatments. Experimental protocols in which naphthalene was administered daily for 7 days at doses of 50, 100 or 200 mg/kg/day failed to reveal significant alterations in the morphology of the lung in comparison to the control. Moreover, the 200 mg/kg/day x 7 dose provided significant protection against 300 mg/kg, administered on Day 8. In comparison, administration of a single 300 mg/kg dose resulted in substantial denudation of the bronchiolar epithelium in animals receiving pretreatments with corn oil (Buckpitt et al., 1988). The degree of protection provided by 7 daily doses of 200 mg/kg declined gradually as the time between the last 200 mg/kg pretreatment dose and the challenge dose was lengthened from 1 to 6 days. The data of Gram and his colleagues (Tong et al., 1981) showing decreases in murine cytochrome(s) P-450 monooxygenase activity following a pulmonary toxic dose of naphthalene suggested that decreases in metabolic activation of naphthalene may be the underlying basis for the tolerance observed after multiple doses of naphthalene. There appears to be a rough correlation between induction of tolerance and a marked and selective decrease in pulmonary microsomal metabolism of naphthalene to the 1R,2S-epoxide enantiomer (Buckpitt, 1988). As discussed in detail later, there is a strong correlation between the rates of formation of 1R,2S-naphthalene oxide and tissue selective toxicity. Thus, the finding of a selective decrease in the formation of one of the enantiomeric epoxides provides support for the concept that high rates of formation of this enantiomer are a critical step in the toxic response of the bronchiolar epithelial cells. Much additional work is needed, however, to determine the precise relationship between altered metabolic capacity and development of tolerance in the lung as well as examination of other possible mechanisms for this phenomenon. The rapid development of tolerance noted in our studies may explain the lack of observable lung pathology in the subchronic studies at doses that produce noticeable effects when given in a single administration (Shopp et al., 1984). 2-Methylnaphthalene is acutely less toxic than naphthalene. Doses as high as 800 mg/kg can be administered intraperitoneally to mice with no mortality. Even at 1 g/kg, only 20% mortality was observed (Griffin et al., 1981). In comparative studies, the pulmonary lesion resulting from intraperitoneal administration of naphthalene and 2-methylnaphthalene is similar in severity and time course (Rasmussen et al., 1986). Labelling of bronchiolar epithelial cells by 3H-thymidine is increased markedly at 72 hr but is near control levels at 7 days. As with naphthalene no liver or alveolar lesions were observed after administration of 2-methylnaphthalene. However, at higher doses renal injury is evident (Griffin et al., 1983). 4. METABOLISM AND RELATIONSHIP TO TOXICITY 4.1. OVERVmW Knowledge of pathways in aromatic hydrocarbon metabolism dates back to the late part of the 19th century when phenol was isolated from the urine of dogs and humans treated with benzene (SchuRzen and Naunyn, 1867--cited in Conti and Bickel, 1977). A combination of their ubiquity in the environment and their potential importance as human toxicants has made aromatic hydrocarbons a very widely studied group of compounds. The metabolic pathways for many of the hydrocarbons are well worked out including the stereochemistry of epoxidation as well as the enantioselectivity of the

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A . R . BUCKPITT a n d R. B. FRANKLIN

detoxication enzymes, epoxide hydrolases and glutathione transferases. There is good evidence for the larger polycyclic aromatic hydrocarbons that the enantiomeric epoxides and diol epoxides differ markedly in their potency as mutagens and carcinogens (for a review see Thakker et al., 1985; Yang, 1988). Correlative evidence suggests that one of the underlying reasons for the selective injury caused by naphthalene to the bronchiolar epithelial cells of the mouse may be the high degree of stereoselectivity with which this substrate is epoxidated in mouse lung. Far less is known about the stereochemistry of 2-methylnaphthalene metabolism. Regioisomeric dihydrodiols are formed, presumably through epoxides, but attempts to correlate the target tissue selectivity with the formation of a particular metabolite have not been successful. The primary metabolic pathways for naphthalene and 2-methylnaphthalene are shown in Figs 1 and 2. The concept that metabolic activation was related to Clara cell necrosis resulting from administration of either naphthalene or 2-methylnaphthalene was a logical extension of the studies indicating that the Clara cell is an important locus of cytochrome P-450 monooxygenase activity (Serabjit-Singh et al., 1980; Devereux et al., 1981) and that this cell type is particularly susceptible to toxicants requiring metabolic activation (Boyd, 1977, 1980; Kehrer and Kacew, 1985). Early studies showing that piperonyl butoxide blocked naphthalene-induced bronchiolar necrosis supported the involvement of cytochrome P-450 dependent metabolism in the lung lesion (Warren et al., 1982). The dose-dependent depletion of glutathione and exacerbation of lung injury in diethylmaleate pretreated mice also were consistent with a role of metabolic activation and the formation of reactive GLUTATHION(CONJUGATE

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FIG. 1. Major biotransformation pathways of naphthalene in mammalian species.

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Relationship of naphthalene and 2-methylnaphthalene

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metabolites in naphthalene lung injury. However, as discussed in detail later in this chapter, the interrelationships between metabolic activation and toxicity for naphthalene are not as clearly defined as they are for lung toxicants like 4-ipomeanol. These interrelationships between metabolism, covalent binding and toxicity are even less certain with 2-methylnaphthalene. In this case, none of the drug metabolism inhibitors tested were capable of altering the severity of the bronchiolar lesion (Griffin et al., 1982, 1983; Buckpitt et al., 1984b). The remainder of this review will provide a detailed description of the current state of knowledge of naphthalene and 2-methylnaphthalene metabolism and will focus primarily on those pathways of metabolism that may be related to the lung lesion.

4.2. NAPHTHALENE

The microsomal metabolism of naphthalene in rabbit and rat liver results in the formation of 1,2-dihydro-l,2-dihydroxynaphthalene (dihydrodiol) and 1-naphthol (Mitoma et al., 1956; Booth and Boyland, 1958). 1-Naphthol is not a metabolite of the dihydrodiol in in vitro incubations (Booth and Boyland, 1958) but apparently can arise from the diol in vivo (Boyland and Wiltshire, 1953). The suggestion that the 1,2-epoxide may be an intermediate in the formation of 1-naphthol, the dihydrodiol and glutathione conjugates was first proposed in 1950 (Boyland, 1950; Booth et al., 1960; Boyland and Sims, 1960). Later, Jerina et al. (1970) provided definitive evidence for the epoxide as an obligate intermediate in the in vitro formation of 1-naphthol, the dihydrodiol and glutathione conjugates. While the chemical reactivity of the epoxide and the knowledge that other aromatic hydrocarbon epoxides are cytotoxic, mutagenic and/or carcinogenic has made the 1,2-oxide a prime candidate as an intermediate in naphthalene-induced bronchiolar necrosis, there are several other reactive naphthalene metabolites including diepoxides and quinones which must be considered as well. As with other aromatic hydrocarbons that are epoxidized by the P-450 monooxygenases, different isozymes show differences in the stereochemistry of epoxidation with naphthalene. Formation of the 1R,2S-naphthalene oxide enantiomer predominates (73 % of the total) in incubations containing cytochrome P-450c, the major isozyme induced by treatment of rats with 3-methylcholanthrene (Van Bladeren et al., 1984). In contrast, the formation of the I S,2-enantiomer was favored in incubations of naphthalene with cytochrome P-450b (one of the P-450 isozymes isolated from rats after treatment with

398

A . R . BUC~PITT and R. B. FRANr,~I~q

phenobarbital--Van Bladeren et al., 1985). The three major metabolic pathways available to the metabolically formed epoxides are hydration by epoxide hydrolases, conjugation by the glutathione transferases and in in vitro systems, spontaneous rearrangement of the epoxide to 1- (95%) and 2-naphthol (5%). Metabolism of naphthalene 1,2-epoxide to a 1,2,3,4-naphthalene diepoxide has been suggested by data on the sulfur derivatives of the compound excreted in the urine (Homing et al., 1980; Stillwell et al., 1982). Epoxide hydrolases show regioselectivity of attack on the naphthalene oxide enantiomers. Hydration of the 1R,2S-enantiomer occurs almost exclusively by attack at the C-2 position to form the 1R,2R-dihydrodiol. In contrast, approximately 40% of the 1S,2R-epoxide is converted to the 1R,2R-diol indicating that a significant portion of the attack occurs at C-1 (Van Bladeren et al., 1985). Four diastereomeric glutathione conjugates are possible from naphthalene 1,2-oxides; three have been isolated by HPLC and have been identified by mass spectrometry and N M R spectroscopy (Buckpitt et al., 1987). The 1R,2S-epoxide appears to form a single glutathione adduct attached at C-2 of the dihydronaphthalene ring, while in an analogous fashion to epoxide hydrolase catalyzed hydration of the 1S,2R-epoxide, nucleophilic attack occurs at both the C-1 and C-2 carbons of the 1S,2R-naphthalene oxide enantiomer to form a mixture of diastereomeric glutathione adducts in approximately equal amounts. The ability to trap naphthalene oxides and to measure their formation in a near quantitative fashion in vitro has been used to determine whether differences in the stereoselectivity of naphthalene epoxidation could be an underlying reason for tissue and species specific cytotoxicity. As will be discussed later, there are virtually no differences in the levels of covalently bound metabolites in the lung, a target tissue and in the liver, a nontarget tissue in vivo in the mouse and thus the organoselective toxicity could not be attributed solely to the more rapid formation of reactive metabolites in the sensitive tissue. If reactive metabolites were involved at all, their differing chemical natures and/or the macromolecular binding sites may determine the organoselective toxicity. The first possibility, namely that the chemical nature of reactive metabolites was different has been tested by monitoring glutathione conjugates formed in microsomal incubations from target and nontarget tissue, in incubations of hepatocytes and in bronchial airways blunt dissected from the mouse. Use of glutathione as a trapping agent has considerable precedence in the literature and was based on the in vivo data that strongly supported a role of this tripeptide in protecting the lungs from naphthalene-induced bronchiolar injury (Warren et al., 1982; Buckpitt and Warren, 1983). Conditions for the assay of microsomal naphthalene epoxidation including time, microsomal protein content, concentration of reduced glutathione and amount of glutathione transferase (semipurified by affinity chromatography) were established to yield linear reaction rates and to generate conjugate at maximum velocity. The rate of formation of naphthalene glutathione adducts is low at pH 7.4 in the absence of glutathione transferases (Buckpitt et al., 1984a); the addition of 10 CDNB units of glutathione transferase catalyzes the reaction at near maximal velocity in incubations of naphthalene with mouse lung or liver microsomes (Buckpitt, unpublished). Under these conditions approximately 85% of naphthalene metabolism in mouse lung or liver microsomal incubations can be accounted for by dihydrodiol or the three glutathione conjugates. Less than 0.3% becomes bound covalently and approximately 14.5% is converted to 1-naphthol. Using these assay conditions we have found that the rate of metabolism of naphthalene to conjugates and dihydrodiol is very similar in mouse lung and liver microsomal incubations but the stereochemistry of epoxidation is substantially different. Mouse liver microsomal metabolism is not stereoselective for naphthalene; conjugates 1, 2 and 3 are formed in a ratio of 1:2:1 (conjugates are named in order of their elution from the HPLC and are: conjugate 1--trans-l-(S)-hydroxy-2-(S)-glutathionyl-l,2-dihydronaphthalene, conjugate 2--trans-l-(R)-hydroxy-2-(R)-glutathionyl1,2-dihydronaphthalene and conjugate 3--trans-l-(R)-glutathionyl-2-(R)-hydroxy-l,2dihydronaphthalene). However, metabolism of naphthalene by mouse lung microsomes is highly stereoselective. The ratio of adducts is 1: 20:1 at high concentrations of naphthalene (1 mM), indicating a 1:10 stereopreference in the formation of the 1R,2S-epoxide

Relationship of naphthalene and 2-methylnaphthalene

399

enantiomer (Buckpitt et al., 1987). The stereoselectivity becomes even more pronounced at lower naphthalene concentrations. For example at 0.015 mM concentrations of naphthalene in mouse lung microsomal incubations, the ratio of formation of the 1R,2S- to 1S,2R-enantiomers is 30:1 (Buckpitt, unpublished). The correlation between high rates of formation of the 1R,2S-epoxide and the sensitivity of tissue to naphthalene cytotoxicity holds in the case of the rat and hamster. Not only are the total rates of metabolism in lung microsomes from these two species substantially lower, but there is little or no stereoselectivity in epoxidation. Similar results were obtained with primate (Buckpitt, unpublished) and human lung microsomes (Buckpitt and Bahnson, 1986). Some caution must be exercised in interpretation of the data in humans and nonhuman primates. Microsomal epoxide hydrolase is substantially more active in these tissues and may, by competing for available epoxide, alter the final glutathione conjugate ratios observed. In fact, studies with purified epoxide hydrolase have indicated that the I S,2R-epoxide enantiomer is a better substrate than the 1R,2S-enantiomer (Van Bladeren et al., 1985). Nevertheless, the comparison of mouse with rat and hamster lung leads to markedly different levels of activity and markedly different patterns of metabolites. Data obtained with microsomal incubations have been supported by recent studies in more intact systems. Mouse hepatocytes metabolize naphthalene to form adducts in a ratio of 1:2:1 (that is, a 1 : 1 ratio of epoxide enantiomers) (Richieri and Buckpitt, 1987). In contrast, stereoselective epoxidation is maintained in incubations of target cell populations (tracheobronchial airways blunt dissected from the mouse) incubated in the presence of large amounts of glutathione and glutathione transferase (ratio of 30:1) (Suverkropp et al., 1988). If the hypothesis that the stereoselective epoxidation of naphthalene plays a critical role in determining target tissue and cell selectivity is correct, then either the enantiomeric epoxides must be differentially cytotoxic or the availability of enzymes capable of hydrating or conjugating the 1R,2S-enantiomer must be at low levels in target cells (mouse lung) and at adequate levels in mouse liver. Our preliminary work with glutathione transferase isozymes shows marked differences in catalytic activity of the various proteins with (+)-naphthalene oxide (Morin, personal communication, 1988). Studies are currently underway to examine the localization of glutathione transferase in mouse lung and to attempt to determine whether Clara cells lack the isozymes necessary for the rapid conjugation of 1R,2S-naphthalene oxide. The alternative explanation, namely that target cells simply produce far more epoxide per cell than nontarget cells is still a viable possibility and cannot be excluded with the data that we presently have. In addition, if naphthalene toxicity in the lung is tied to enantiomeric epoxide formation, a mechanism other than epoxide formation must be invoked to explain the renal lesion by the compound. While the localization of the lesion (i.e. proximal tubular cells) is consistent with the presence of P-450 monooxygenases in this cell type (Dees et al., 1982), naphthalene is very slowly metabolized by renal microsomal preparations by either P-450 or arachidonic acid dependent mechanisms (Buckpitt et al., 1986). As will be discussed later, circulating reactive metabolites may be involved in the renal injury. Alternatively, mechanisms such as metabolism of cysteine conjugates by C-S-lyases or further metabolic activation of phenolic or dihydrodiol metabolites of naphthalene by either monooxygenases or prostaglandin synthases must be considered. The glutathione adducts formed from naphthalene are primarily excreted as mercapturic acids (38% and 65% of a dose is excreted as N-acetylcysteine conjugates by naphthalene treated mice and rats, respectively) (Stillwell et al., 1982; Chen and Dorough, 1979). Using nonspecific methods for determining thioether excretion, Summer et al. (1979) have reported little mercapturic acid in the urine of chimpanzees treated with up to 200 mg/kg naphthalene. This apparent low rate of turnover to the mercapturate may reflect relatively slow conversion of naphthalene to the epoxide in the primate and/or very rapid transformation of the epoxide to dihydrodiol. Our laboratories have been examining the possibility that urinary mercapturic acid isomers could be used to assess the activity and stereoselectivity of naphthalene metabolism in vivo. Administration of diastereomeric glutathione conjugates results in excretion of N-acetylcysteine derivatives in which the J.P.T. 41/1-2--Z

400

A . R . BUCKPITTand R. B. FRANKLIN

stereochemistry has been retained. Small amounts of cysteine adduct are excreted after administation of glutathione adducts derived from 1S,2R-naphthalene oxide. Corresponding administration of the 1R,2S-oxide derived adduct results in very little ( < 1%) cysteine adduct in the urine (Buonarati and Buckpitt, 1988). In addition to a mercapturic acid, administration of adduct 3, where glutathione is attached at the benzylic carbon of naphthalene, results in the excretion of two as yet unidentified metabolites which represent 29-43% of the urinary radioactivity. In addition to mercapturates, small amounts of several other sulfur containing metabolites are excreted after i.p. administration of naphthalene including methylthio, mercaptolactic and mercaptoacetic acid derivatives (Stillwell et al., 1982). Although 1-naphthol is a primary rearrangement product derived directly from naphthalene oxide in vitro, Bakke et al. (1985) have recently presented evidence that 1-naphthol is derived primarily through the intestinal microfloral metabolism of biliary glutathione conjugates in vivo. Treatment of germ free animals with naphthalene results in the excretion of only trace quantities of 1-naphthol or glucuronide conjugates derived therefrom. Likewise, ligation of the bile duct decreases 1-naphthol excretion in comparison to control. In these studies, conventional animals excreted approximately 5% of a dose as 1-naphthol or glucuronide derivatives. 1-Naphthol and the glucuronide conjugates are quantitatively significant metabolites in the conventional animals treated with 1,2-dihydro-1-hydroxy-2Scysteinylnaphthalene or the N-acetyl cysteine congener. Further support that phenols may not arise directly from precursor epoxides in vivo has come from studies with bromobenzene. Lertratanangkoon and coworkers (1987) have demonstrated the formation of 3- and 4-bromophenol following administration of cysteine adducts derived from bromobenzene-3,4-epoxide. In fact, these authors have concluded that generation of aromatic phenols via sulfur conjugated metabolites may be the major route of formation of these metabolites in vivo. The importance of the origin of phenolic metabolites arises from the studies showing that various naphthoquinones, which are metabolic derivatives of the phenols may be important in the covalent binding and cytotoxicity of naphthalene and 2-methylnaphthalene. This will be discussed in further detail later. 4.3. METABOLICCONVERSIONOF 1-NAPHTHOLAND 1,2-DIHYDROXY-1,2-DIHYDRONAPHTHALENE 1-Naphthol is a good substrate for the glucuronyl transferases (Bock et al., 1976) and, depending upon the dose and the species, the glucuronide conjugate is a major urinary metabolite after administration of either naphthalene (12% of the urinary radioactivity, Chen and Dorough, 1979) or 1-naphthol (31% of the administered dose, Chern and Dauterman, 1983). 1-Naphthol ultimately may be metabolized to 1,4-naphthoquinone via a naphthoxy free radical mechanism (Fluck et al., 1984), by an NADPH-supported reaction in rat liver microsomes (d'Arcy Doherty and Cohen, 1984) in which the enzymatically-formed metabolite may be envisaged as the 1,4-dihydroxynaphthalene which then undergoes autoxidation to the 1,4-quinone, by tyrosinase (d'Arcy Doherty et al., 1986a) or by horseradish peroxidase (d'Arcy Doherty et al., 1986b). 1,2-Naphthoquinone also has been postulated as an in vitro metabolite of 1-naphthol (d'Arcy Doherty et al., 1985; Miller et al., 1986). However, possibly because of the chemical reactivity of the compound, attempts to measure its formation directly in rat liver microsomes were unsuccessful (d'Arcy Doherty et al., 1984; Fluck et al., 1984). Several lines of evidence favor the possibility that naphthoquinones are proximate toxic and covalently bound metabolites from naphthalene. Early work on cataract formation suggested the involvement of naphthoquinones (Rees and Pirie, 1967). Subsequent studies with benzene have attributed toxicity and irreversible binding of reactive metabolites to metabolites arising from phenol and catechol rather than directly from benzene oxide (reviewed by Kalf, 1987). The further metabolism of phenolic naphthalene metabolites, possibly to naphthoquinones, was invoked in the studies of Hesse and Mezger (1979) in order to explain some observations on the irreversible (covalent) binding of radioactivity

Relationship of naphthalene and 2-methylnaphthalene

401

to microsomal protein. At low concentrations of naphthalene, covalent binding lagged behind the formation of 1-naphthol and there was a good correlation between the rate of l-naphthol generation and the appearance of covalently bound metabolites. In addition, inhibition of sulfation or glucuronidation in isolated hepatocyte suspensions markedly enhanced the covalent binding of radioactivity from radiolabelled naphthalene (Schwarz et al., 1980). Using ethylene diamine to selectively react with 1,2-naphthoquinone, d'Arcy Doherty and Cohen (1984) have shown that the covalent binding from 1-naphthol occurs primarily via 1,4-naphthoquinone. Thus, while there is ample experimental evidence to suggest that conversion of naphthalene to 1-naphthol and subsequent conversion of the naphthol catalyzed by a variety of enzymes can result in the formation of reactive metabolites, the role of these species in bronchiolar necrosis or covalent binding in vivo is far less certain. Administration of 1-naphthol does not result in bronchiolar necrosis (O'Brien et al., 1985; Buckpitt et al., 1985) nor in glutathione depletion (in contrast, marked and concentration dependent decreases in glutathione content of isolated hepatocytes are observed during incubations with 1-naphthol). Furthermore, the levels of covalently bound metabolites in lung, liver and kidney in vivo are comparable after administration of radiolabelled naphthalene or naphthol. The results of these studies, therefore, argue against the involvement of l-naphthol as a primary mediator of either covalent binding or toxicity of naphthalene in vivo. Additionally, there is some in vitro data to suggest that 1-naphthol may not be an obligate precursor to the formation of covalently bound metabolites. The rate of metabolism of naphthalene to 1-naphthol in mouse lung microsomal incubations is nearly three times that catalyzed by liver microsomes, yet the rate of formation of covalently bound metabolites is similar. The lack of difference in the rate of reactive metabolite formation, in light of the differences in the rate of generation of 1-naphthol from naphthalene, was not due to differences in the rate of conversion of 1-naphthol to covalently bound species (Buckpitt et al., 1985). Further, radiolabel from 3H-naphthalene oxide is bound covalently to microsomal protein and this conversion does not appear to require catalysis by the P-450 monooxygenase system (Buckpitt, unpublished). Further work will have to concentrate on the chemical identification of bound residues from both in vivo and in vitro sources to completely resolve these issues. 4.4. 2-METHYLNAPHTHALENE METABOLISM

In contrast to those experiments involving naphthalene, pretreatments with piperonyl butoxide, SKF 525-A (Griffin et al., 1982) or cobalt protoporphyrin (Buckpitt et al., 1984b) failed to protect mice against a pulmonary toxic dose of 2-methylnaphthalene. The inducing agents, phenobarbital and 3-methylcholanthrene, did provide some defense against 2-methylnaphthalene-induced pulmonary damage, possibly by altering the disposition of the parent hydrocarbon in the lung (Griffin et al., 1982). Depletion of glutathione by pretreatment with diethylmaleate enhanced the lethality of 2-methylnaphthalene in mice (Griffin et al., 1982) and guinea-pigs (Teshima et al., 1983) without noticeable effects on the murine pulmonary lesion. Thus, in contrast to naphthalene where there were clear indications for the involvement of cytochrome P-450 and reduced glutathione in modulating the bronchiolar necrosis, such relationships were far from straightforward with 2-methylnaphthalene. Moreover, as discussed later, radioactivity from [14C-ring]-2-methylnaphthalene was bound covalently to tissue macromolecules but an association between reactive metabolite binding and bronchiolar injury was obscure (Griffin et al., 1982, 1983). The dependence of reactive 2-methylnaphthalene metabolite formation on metabolism by the P-450's was verified in vitro by showing the covalent binding to be NADPH-dependent, inhibited by piperonyl butoxide, SKF 525-A or a N2 atmosphere. Covalent binding also was decreased substantially by inclusion of reduced glutathione in the incubation mixtures (Buckpitt et al., 1986). 2-Methylnaphthalene is metabolized to 3 isomeric dihydrodiols (3,4-, 5,6- and 7,8-dihydrodiols) both in vivo and in vitro (Breger et al., 1981; Melancon et al., 1982; Breger et al., 1983) presumably through intermediate epoxides. In addition, there are a number of products resulting from side chain oxygenation (i.e. 2-naphthoic acid

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and the glycine conjugate) which are quantitatively significant metabolites in the rat (Melancon et al., 1982) and guinea pig (Teshima et al., 1983) but not the trout (Melancon et al., 1982). In vitro studies have suggested that isozymes of P-450 control the regioselectivity of 2-methylnaphthalene epoxidation. However, interpretation of these studies was complicated by the finding that ratios of dihydrodiol isomers were found to be dependent upon the nature of the buffer used in the incubation (Melancon et al., 1985). Nevertheless, comparative studies to examine target vs nontarget tissue metabolism of 2-methylnaphthalene failed to reveal significant differences in dihydrodiol ratio (Griffin et al., 1982, 1983) in microsomal incubations of mouse lung vs liver. Approximately 10% of a dose of 2-methylnaphthalene is excreted as a cysteine conjugate (7-S-cysteinyl-2-methylnaphthalene) in the guinea pig. The implication, namely that glutathione conjugation occurs with the 7,8-epoxy-2-methylnaphthalene, is consistent with the slight depletion of glutathione noted in guinea pig liver after oral administration of 2-methylnaphthalene. There may be species differences in glutathione conjugation with 2-methylnaphthalene epoxides since cysteine/mercapturic acid metabolites have not been reported in rat or mouse urine and because little if any depletion of glutathione was observed in tissues from these species (Grimes and Young, 1956; Griffin et al., 1982). In preliminary studies using labelled glutathione and/or labelled 2-methylnaphthalene in microsomal incubations, we have been unable to find evidence for a glutathione conjugate. Ongoing studies are comparing metabolic profiles for 2-methylnaphthalene in dissected tracheobronchiolar tissue vs hepatocytes in hopes of discerning metabolic differences in these two preparations without possible interference from the large number of nontarget cells that are represented in microsomal incubations (Pang, personal communication). Renal necrosis and possible metabolic mechanisms responsible for the injury have been discussed above with naphthalene. A proximal tubular lesion has been reported in mice with 2-methylnaphthalene (Griffin et al., 1983) but potential mechanisms have not been explored. 2-Methylnaphthalene was only slowly metabolized by mouse renal microsomal preparations using either arachidonic acid or NADPH as cofactors. Substantially more effort will be needed to determine which metabolic processes (if any) are involved in the renal toxicity. 4.5. SITE OF GENERATION AND RELATIONSHIP OF REACTIVE METABOLITE BINDING TO CYTOTOXICITY

A close interrelationship between reactive metabolite binding and toxicity, such as that noted for the lung toxic furan, 4-ipomeanol, has not been demonstrated with either naphthalene or 2-methylnaphthalene (Griffin et al., 1981; Warren et al., 1982; Buckpitt and Warren, 1983). At all doses studied, covalent binding levels in liver and kidney were as high if not higher than in the most sensitive tissue, the lung (Fig. 3). Although there was o3 ..J w

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Relationship of naphthalene and 2-methylnaphthalene

403

not a close association between the target tissue for injury and organoselective reactive metabolite binding, pretreatments that resulted in a change in naphthalene-induced injury to the bronchiolar epithelium resulted in a parallel shift in pulmonary covalent binding levels. In contrast, a similar interrelationship between reactive metabolite binding in the lung and the extent/severity of 2-methylnaphthalene-induced bronchiolar injury has not been established. Marked glutathione depletion was observed after naphthalene administration in lung, liver and kidney; reduced glutathione levels in all three tissues fell to less than 20% of control at high doses (400mg/kg). By comparison, administration of 4-ipomeanol at twice the LDs0 resulted in only a 30% depletion of pulmonary glutathione. The lack of tissue specificity for binding of reactive metabolites and the dissimilarity of the data obtained with naphthalene and 4-ipomeanol suggested that there might be multiple reactive metabolites which might be sufficiently stable to be formed in one cell or tissue and deplete glutathione in another. There is considerable experimental support for the view that reactive naphthalene metabolite binding in a particular tissue is not a good measure of reactive metabolite formation in situ within that tissue. Selective depletion of hepatic and renal but not pulmonary glutathione levels by pretreatment with buthionine sulfoximine in mice results in marked increases in covalent binding of reactive metabolites in lung as well as liver and kidney. In addition, there is little correlation between the rate of formation of covalently bound metabolites by microsomal enzymes with the levels of covalently bound metabolites in vivo. Renal microsomal activation of naphthalene occurs very slowly yet in vivo covalent binding levels in the kidney are as high as in the liver or lungs (Fig. 4) (Buckpitt and Warren, 1983; Buckpitt et al., 1986). These data suggest that either liver or lungs are responsible for the formation of metabolites that become covalently bound in the kidney or that bound metabolites in the kidney are generated from metabolites of naphthalene and not the parent hydrocarbon. More direct support for the idea that reactive naphthalene metabolites are semistable and can efflux from cells/tissues comes from studies showing that approximately a third of the total naphthalene oxide formed in isolated hepatocytes can be trapped with labelled glutathione extracellularly (Richieri and Buckpitt, 1987). Surprisingly, the fraction of naphthalene oxide effluxing from the cells vs the total amount of epoxide formed did not change with increasing concentrations of substrate. This suggests that the efflux of naphthalene oxide was not dependent upon substantial depletion of intracellular glutathione stores. In addition to the efflux of naphthalene oxide, metabolites capable of becoming bound covalently to proteins in the extracellular medium also were observed in isolated hepatocyte incubations. In the case of covalent binding, the relative intracellular and extracellular levels of bound metabolite were concentration dependent. At low substrate concentrations the binding was predominantly extracellular whereas at concentrations above 0.25 mM intracellular covalent binding was highest. These data are consistent with the idea that at high substrate concentration where glutathione in the cells is depleted, intracellular proteins are the primary targets for reactive metabolites. Further demonstration that naphthalene oxide is capable of diffusing across intact cellular membranes comes from studies showing that addition of the epoxide to

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404

A.R. BUCKPITTand R. B. FRANKLIN

hepatocyte suspensions results in a concentration dependent depletion of intracellular glutathione (Buonarati et al., 1987). Recent studies indicate that not only is the liver a potential source of circulating reactive metabolites but the lung may be as well. Incubation of airway pieces blunt dissected from mice in the presence of bovine serum albumin (BSA) results in the covalent binding of large amounts of reactive metabolite to protein (Suverkropp et al., 1988). Likewise, high amounts of covalently bound radioactivity are observed in BSA containing media in the isolated perfused mouse lung system during the infusion of ~4C-naphthalene (Kanekal, personal communication). The toxicologic significance of circulating naphthalene oxide is uncertain. Naphthalene oxide is capable of depleting pulmonary glutathione when administered in bolus doses intravenously (Richieri and Buckpitt, 1988) and thus, it is possible that circulating epoxide exacerbates the effects of reactive metabolites produced in the lung by depleting glutathione. However, very high doses of naphthalene oxide were required to produce significant depletion of pulmonary glutathione and thus, the significance of this finding is still unclear. Naphthalene oxide is cytotoxic to isolated hepatocytes (Buonarati, personal communication) and produces capillary endothelial and bronchiolar epithelial cell injury when infused in an isolated perfused lung system. Thus, the potential for circulating naphthalene epoxide to produce cytotoxicity directly exists but the question of whether the levels that are in the blood are capable of playing a role in naphthalene-induced lung injury needs additional study. Although there were no obvious distinctions between hydrocarbon inducible and noninducible strains of mice (C57BL/6J and DBA/2J) in the response of the bronchiolar epithelium to i.p. 2-methylnaphthalene, there were differences in the levels of covalently bound reactive metabolites and glutathione depletion in the two strains. In C57B1/6J mice, pulmonary and renal glutathione levels were not different from control after administration of 400mg/kg 2-methylnaphthalene and hepatic thiol levels were depleted moderately only at the 3 and 6 hr time points (Griffin et al., 1982). In contrast, at the same dose, hepatic and pulmonary levels of reduced thiol were decreased to less than 50% of control 4-6 hr after 2-methylnaphthalene in the DBA strain. Differences also were noted in the extent of in vivo covalent binding of chemically reactive metabolites of 2-methylnaphthalene to lung, liver and kidney macromolecules in the two mouse strains. Not only was the extent of in vivo covalent binding greater in the C57BL/6J mice (at a toxic dose of 400 mg/kg), but the organ distribution of covalently bound metabolites was different. In both strains, pretreatments which markedly altered the covalent binding of reactive naphthalene metabolites and naphthalene-induced bronchiolar injury (i.e. piperonyl butoxide and diethylmaleate) failed to affect either the toxicity or the covalent binding of reactive 2-methylnaphthalene metabolites. Clearly, the relationship between the formation of reactive metabolites, depletion of tissue free sulfhydryls and pulmonary toxicity of 2-methylnaphthalene is far more equivocal than that noted with naphthalene. There are several possibilities which must be considered with regard to a role of chemically reactive metabolites in 2-methylnaphthalene-induced Clara cell necrosis. Like naphthalene, reactive metabolites from 2-methylnaphthalene may be semistable. Thus, levels of covalently bound metabolite in one tissue may not necessarily reflect the rate of activation of compound within that tissue. The lung is an organ with multiple cell types and measurements of in viro covalent binding necessarily measure bound metabolite in nontarget as well as target cells. Perhaps autoradiography studies would clarify this point by showing that labelling in Clara cells is far more abundant than binding in nontarget cell types. As is the case with naphthalene there are several unstable metabolites arising during the biotransformation of 2-methylnaphthalene which could become bound covalently to tissue macromolecules. Several of these could be responsible for the gross levels of binding assayed in vivo but only one may be toxicologically relevant. Lastly, the methods employed in the covalent binding assay are incapable of distinguishing between binding to critical and non-critical macromolecules. Thus, differences in macromolecular

Relationship of naphthalene and 2-methylnaphthalene

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targets may account for the target organ selectivity by both naphthalene and 2-methylnaphthalene. Additional studies will be needed to resolve these issues. 5. FUTURE DIRECTIONS Naphthalene has been a popular substrate for both metabolic and toxicologic investigations for more than 100 years. The ocular toxicity of the compound was recognized 80 years ago and the elegant studies of van Heyningen, Pirie and Rees (van Heyningen and Pirie, 1967; Rees and Pirie, 1967) related this pathology to the formation of 1,2-dihydroxynaphthalene (or 1,2-naphthoquinone). The pulmonary damage by naphthalene and 2-methylnaphthalene was reported a little more than a decade ago and while some information is available regarding the mechanisms, the relationship of a specific metabolite to cytotoxic insult to the Clara cell is by no means established. Perhaps two of the most important issues raised by this work are related to the formation and toxicologic role of reactive metabolites that appear to circulate and the suitability of the mouse as an animal model for the human in studies of pulmonary toxic agents. The issue of circulating reactive metabolites and their influence on cytotoxic and/or carcinogenic events occurring in other tissues or cells has been recognized and studied by a number of investigators (for appropriate discussions see Guengerich and Liebler, 1985). These questions are particularly important when considering xenobiotic metabolism in human lung. Normally very low or nondetectable levels of P-450 related metabolism have been reported in studies using human lung samples (Jakobsen et al., 1982) and thus contributions made by the liver to steady state levels of cytotoxic or carcinogenic agents in the lung could play a major role in pulmonary toxicology. The finding that vinyl chloride produces neoplastic transformation in endothelial cells rather than in hepatocytes where its rate of biotransformation is highest suggests some role for reactive metabolites formed in hepatocytes. The data demonstrating the efflux of reactive vinyl chloride metabolites from isolated hepatocyte suspensions would certainly support this conclusion (Ottenwalder et al., 1983). Sensitivity of hepatic endothelial cells could then be based on a lack of detoxication or repair capabilities. Similar arguments can be put forth for benzo(a)pyrene diol epoxide with the level of BPDE-DNA adducts in vivo (Stowers and Anderson, 1984). This also is consistent with studies examining the effect of pretreatment of mice with agents that are cytotoxic to Clara cells (4-ipomeanol, naphthalene or 2-methylnaphthalene) on the overall levels of bound benzo(a)pyrene metabolite in the lung (Griffin and Franklin, 1982). If the pulmonary Clara cell is primarily responsible for the binding of benzo(a)pyrene metabolites in the lung, a decrease in covalent binding levels in toxicant treated animals would have been expected. However, there were not significant differences between treated and control animals, a finding consistent with the formation and circulation of epoxides from other tissues. Additional questions regarding the mechanism for formation and exit of reactive metabolites from intact cells remain. The possibility that these metabolites are formed on the plasma membrane has been raised by recent studies localizing the P-450 monooxygenases on the exterior of the cell (Serabjit-Singh et al., 1988). Finally, the precise role of these metabolites in pulmonary toxicity has not been defined. We hope to be able to establish conditions for short term incubations of bronchiolar explants which are appropriate to maintenance of the tissue in a morphologically and biochemically viable condition. With these techniques we can precisely control the levels of naphthalene oxide and other potential precursor reactive metabolites to determine their role in pulmonary injury. A further issue that needs to be resolved is what the levels of circulating epoxide are in the in vivo situation. The fact that both naphthalene and 2-methylnaphthalene are highly selective for mouse lung makes them excellent models for further development of techniques for examining potential toxicity of agents that injure the lung in vitro in species ranging from the mouse to the human. As mentioned in the introduction to this review, much of the etiology of human lung disease is poorly understood. In part this is due to the difficulty of epidemiologic studies in various human populations against a high background level

406

A.R. BucKPlrr and R. B. Fl~ANI~LXN

of pulmonary disease caused by cigarette use. Development of the methodology for evaluating the potential pulmonary toxicity of environmental agents is, therefore, a critical link to our further understanding of the etiologic factors in lung disease. Further work is needed with both naphthalene and 2-methylnaphthalene to understand which metabolite(s) are essential to the pulmonary and renal toxicity. The work of O'Brien et al. (1985) has indicated that the extent of depletion of non-protein sulfhydryls and the rate (and extent) of naphthalene metabolism differed in the mouse and the rat. In vitro studies with lung microsomal preparations have supported this conclusion and have shown marked differences in the stereochemistry of epoxide formation between the mouse and rat (Buckpitt et al., 1987). These in vitro differences in microsomal activation are surely due to the composition of P-450 isozymes in mouse vs rat. What is not certain, however, is whether the epoxides are the toxic species, whether the enantiomers differ in toxic potency or whether the selective toxicity is potentially due to enantioselectivity of the glutathione transferases or epoxide hydrolases. Possibly isozyme composition of murine pulmonary glutathione transferases is altered by multiple doses of naphthalene and this plays a central role in the induction of tolerance to naphthalene. The recent discovery of a renal lesion from naphthalene, combined with the data showing that renal microsomal preparations lack the ability to rapidly metabolize the parent hydrocarbon, suggests that the underlying biochemical process resulting in this lesion is different from that which appears to be active in the lung. Glutathione conjugates were long thought to be the products of detoxication reactions. However, recent studies with hexachlorobutadiene, bromobenzene and several other compounds have demonstrated the further activation of these adducts in the kidney, leading to renal necrosis (Wolf et al., 1984; Monks and Lau, 1987; Anders and Lash, 1987). The question of involvement of secondary metabolites such as naphthoquinone in both Clara and proximal tubular cell necrosis needs further resolution. Naphthol is cytotoxic to isolated hepatocyte suspensions and naphthoquinones have been implicated in the loss of cell viability (Smith et al., 1982; d'Arcy Doherty et al., 1984, 1987). However, there is growing evidence that the aromatic phenols are not derived solely through spontaneous rearrangement of epoxides and thus, the relevance of this pathway to in vivo toxicity is debatable. In addition, direct administration of 1-naphthol failed to produce a pulmonary lesion in mice (Buckpitt et al., 1985; O'Brien et al., 1985). Similarly, with 2-methylnaphthalene, further metabolism of naphthols and dihydrodiols is a distinct, but as yet unexplored, possibility. The extensive work of Orrenius and coworkers (Thor et al., 1982; DiMonte et al., 1984) on menadione (2-methyl-l,2-naphthoquinone) has shown this quinone to be a potent cytotoxic agent in isolated hepatocyte suspensions. Again, it is possible that conversion of 2-methylnaphthalene to this quinone is critical to the lung lesion. While there is some evidence that the covalent binding of reactive metabolites to critical macromolecules is central to the toxic response, particularly with naphthalene, the alternative possibility that reactive oxygen species, possibly generated by cyclic reduction and oxidation of quinones, must be considered. In discussing the ocular toxicity of 1,2-naphthoquinone, Rees and Pirie (1967) addressed the possibility of hydrogen peroxide formation. While the authors do not pursue the possibilities of toxicity associated with hydrogen peroxide generation, recent events in, for example, the field of ischemia, might cause one to evaluate not only the ocular toxicity of hydrogen peroxide, but also the pulmonary toxicity; after all, is not glutathione actively involved in the detoxication of hydrogen peroxide? Finally, at this time, a definitive relationship between covalent binding of naphthalene and 2-methylnaphthalene metabolites and toxicity is obscure. One of the major reasons for this is the failure of methodology used to assess covalent binding to distinguish between specific and non-specific sites of interaction. The application of recent developments in this methodology (Sun and Dent, 1984; Hoffman et al., 1985; Bryant et al., 1987; Roberts et al., 1987) and the further improvement of these methods are likely to yield significant new insights regarding the role of covalent binding in naphthalene and 2-methylnaphthalene induced toxicity.

Relationship of naphthalene and 2-methylnaphthalene

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Acknowledgements--Work from the authors' laboratories has been supported by grants from the Toxic

Substances Research and Teaching Program, NIEHS 03365, 04311 and 04699. The authors would like to acknowledge Michael Buonarati for his careful reading of the manuscript.

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