Localization, distribution, and induction of xenobiotic-metabolizing enzymes and aryl hydrocarbon hydroxylase activity within lung.

Pharraac. Ther. Vol. 47, pp. 419-445, 1990 Printed in Great Britain. All rights reserved

0163-7258/90$0.00+ 0.50 © 1990PergamonPress pie

Specialist Subject Editor: T. E. GRAM

LOCALIZATION, DISTRIBUTION, A N D I N D U C T I O N OF XENOBIOTIC-METABOLIZING ENZYMES A N D ARYL H Y D R O C A R B O N HYDROXYLASE ACTIVITY WITHIN L U N G JEFFREY BARON a n d JEFFREY M. VOIGT* Department of Pharmacology, The University of Iowa, Iowa City, Iowa 52242, U.S.A. Abstract--The metabolism of xenobiotics within lung often leads to toxicity, although certain pulmonary cells are more readily damaged than others. This differential susceptibility can result from cell-specific differences in xenobiotic activation and detoxication. The localization and distribution of xenobioticmetabolizing enzymes (cytochromes P-450, NADPH-cytochrome P-450 reductase, epoxide hydrolase, glutathione S-transferases, UDP-glucuronosyltransferases, and a sulfotransferase) and of aryl hydrocarbon (benzo[a]pyrene) hydroxylase activity determined immunohistochemically and histochemically, respectively, within lung are discussed. Findings reveal that xenobiotics can be metabolized in situ, albeit to different extents, by bronchial epithelial cells, Clara and ciliated bronchiolar epithelial cells, and type II pneumocytes and other alveolar wall cells and that enzymes and activities are not necessarily induced uniformly among these cells.

CONTENTS 1. 2. 3. 4.

Introduction Overview of Xenobiotic-Metabolizing Enzymes Intrapulmonary Localization of Xenobiotic-Metabolizing Enzymes Methods of Study 4.1. Immunohistochemical localization of xenobiotic-metabolizing enzymes within lung 4.1.1. Enzymes and antibodies 4.1.2. Tissue preparation 4.1.3. Immunoperoxidase staining 4.1.4. Immunofluorescence staining 4.1.5. Microfluorometric analysis of immunofluorescence staining 4.2. Histochemical demonstration of benzo[a]pyrene hydroxylase activity 5. Localization and Distribution of Xenobiotic-Metabolizing Enzymes and Benzo[a]pyrene Hydroxylase Activity within Lungs of Untreated Rats 5.1. Xenobiotic-metabolizing enzymes 5.2. Benzo[a]pyrene hydroxylase activity 6. Effects of 3-Methylcholanthrene and Aroclor 1254 on Xenobiotic-Metabolizing Enzymes and Benzo[a]pyrene Hydroxylase Activity within Rat Lung 6.1. Xenobiotic-metabolizing enzymes 6.2. Benzo[a]pyrene hydroxylase activity 7. Concluding Comments Acknowledgements References

1. I N T R O D U C T I O N The lung is a major portal of entry of xenobiotics into the body and, as such, is continually exposed to airborne environmental chemicals. It is also continually exposed to those xenobiotics that enter the body *Present address: Department of Pharmacology and Toxicology, Philadelphia College of Pharmacy and Science, Philadelphia, Pennsylvania 19104, U.S.A.

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through other portals and are present in the general circulation. As a consequence of this exposure, the lung represents a major target for necrosis, tumorigenesis, and other chemically-induced toxicities. Many pulmonary toxins and most chemical carcinogens, however, are relatively inert substances that must be bioactivated in order to exert their cytotoxic and/or tumorigenic actions (Miller, 1978; Boyd, 1980a,b; Wright, 1980; Miller and Miller, 1982, 1985; Boyd and Statham, 1983; Minchin and

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Boyd, 1983; Yost et al., 1989). The bioactivation of these xenobiotics usually involves their transformation into electrophilically-reactive metabolites that, presumably by attacking and binding to nucleophilic sites on DNA and other cellular macromolecules, induce genomic alterations and various toxicities (Heidelberger, 1975; Miller, 1978; Boyd, 1980a,b; Wright, 1980; Miller and Miller, 1982, 1985; Minchin and Boyd, 1983; Hecht, 1985; Yost et al., 1989). Xenobiotics that produce toxicities as a result of their transformation into reactive metabolites often selectively damage specific cells: in the lung, these chemicals most frequently injure the nonciliated bronchiolar epithelial cell, commonly referred to as the Clara cell (Reid et al., 1973; Boyd et al., 1974, 1978b; Huang et al., 1977; Boyd, 1980a,b; Forkert and Reynolds, 1982; Tong et al., 1982; Baron and Kawabata, 1983; Haschek et al., 1983; Krijgsheld et al., 1983; Minchin and Boyd, 1983; Kehrer and Kacew, 1985; Buckpitt and Franklin, 1989; Yost et al., 1989). A number of factors undoubtedly contribute to the basis for the differential susceptibilities of cells in lung as well as in other tissues to toxicities resulting from the formation of reactive metabolites. These include differences in the uptake of xenobiotics into cells, differences in intracellular levels of reduced glutathione which can form conjugates with and thereby inactivate potentially toxic electrophiles, cell-specific differences in repair processes, and differences in the abilities of cells to activate and detoxicate xenobiotics. Xenobiotic metabolism is an especially important determinant of a cell's susceptibility to toxicities produced by reactive metabolites since the intracellular balance between xenobiotic activation and detoxication processes dictates whether or not reactive metabolites will accumulate within a cell in which they could then exert their toxic actions. In view of this, it should be apparent that the identification of those sites at which xenobiotics are activated and detoxicated within lung can provide significant insight into the underlying biochemical basis for the cell-selective nature of toxicities that frequently occur as a result of the pulmonary metabolism of xenobiotics. Primarily for this reason, the in situ localizations of xenobiotic-metabolizing enzymes in lung have received considerable attention during the past decade. This review is focused on immunohistochemical findings on the intrapulmonary localizations of cytochromes P-450, NADPH-cytochrome P-450 reductase, epoxide hydrolase, glutathione S-transferases, UDP-glucuronosyltransferases, and a sulfotransferase. Emphasis is placed on our own semiquantitative immunohistochemical findings on the relative levels of xenobiotic-metabolizing enzymes in bronchial epithelial cells, Clara cells, and type II pneumocytes and on our histochemical findings on aryl hydrocarbon (benzo[a]pyrene) hydroxylase activity in lungs of untreated and xenobiotic pretreated rats. Additionally, an overview of xenobiotic-metabolizing enzymes and the roles that they play in the activation and detoxication of xenobiotics is presented together with a summary of the metabolism of xenobiotics by different types of pulmonary cells.

2. OVERVIEW OF XENOBIOTICMETABOLIZING ENZYMES Among those enzymes that catalyze the activation and detoxication of xenobiotics, the cytochromes P-450-containing monooxygenase enzyme systems play a pivotal role and have clearly received the greatest attention. These enzyme systems are associated with the endoplasmic reticulum and nuclear envelope and consist of NADPH-cytochrome P-450 reductase and different forms of cytochrome P-450 (Masters and Okita, 1980; Guengerich, 1987). The reductase mediates the NADPH-dependent reduction of cytochromes P-450 (Masters et al., 1971; Masters and Okita, 1980) which, in their ferrous state, catalyze the oxidative and reductive metabolism of a vast array of substances exhibiting diverse chemical structure (Wislocki et al., 1980; Guengerich, 1987). Despite the fact that cytochromes P-450-catalyzed reactions constitute a critically important process for inactivating and thereby limiting the durations of action of drugs and other xenobiotics, they also frequently result in the transformation of relatively inert chemicals into highly toxic, electrophilicallyreactive metabolites such as epoxides (e.g. arene oxides), carbonium and nitrenium ions, and carbonand oxygen-centered radicals (Miller, 1978; Boyd, 1980a; Wright, 1980; Miller and Miller, 1982, 1985; Cummings and Prough, 1983; Minchin and Boyd, 1983; Guengerich and Liebler, 1985; Guengerich, 1988; Yost et al., 1989). The generation of reactive metabolites from xenobiotics often precedes the appearance of necrosis, mutagenesis, carcinogenesis, and other toxicities (Miller, 1978; Boyd, 1980a; Wright, 1980; Miller and Miller, 1982, 1985; Boyd and Statham, 1983; Minchin and Boyd, 1983), and the cytochromes P-450-mediated formation of these metabolites indeed appears to be the initial step leading to the occurrence of pulmonary damage following in vivo exposure to a great many xenobiotics, including 4-ipomeanol and 3-methylfuran (Boyd et al., 1978a; Boyd, 1980a,b), naphthalene (Tong et al., 1982; Buckpitt and Franklin, 1989), butylated hydroxytoluene (Witschi et al., 1989), 3methylindole (Bray and Carlson, 1979; Hanafy and Bogan, 1980), and both polycyclic aromatic hydrocarbons and N-nitrosamines (Miller, 1978; ReznikSchfiller and Reznik, 1979; Wright, 1980; Miller and Miller, 1982, 1985; Hecht, 1985; Guengerich, 1988). Additionally, xenobiotics such as nitrofurantoin (Holtzman et al., 1981) and mitomycin C (Komiyama et al., 1979) can be transformed into reactive metabolites that are capable of damaging the lung under the influence of NADPH-cytochrome P-450 reductase alone. The different forms of cytochrome P-450 comprise a superfamily of monooxygenases, with many tissues containing a number of these enzymes which exhibit differing, yet often overlapping, substrate specificities (Guengerich, 1979, 1987, 1988; Lu and West, 1980). Also, certain cytochrome P-450 forms, NADPHcytochrome P-450 reductase, and a variety of monooxygenase activities can be induced in numerous tissues by a multitude of xenobiotics, including phenobarbital, certain other drugs, steroids, polycyclic aromatic hydrocarbons, polychlorinated

Enzymes within lung biphenyls, and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (Conney, 1967, 1982; Guengerich, 1977b, 1978, 1979, 1987, 1990; Boyd, 1980a; Lu and West, 1980; Masters and Okita, 1980; Guengerich et al., 1982a,b; Halpert, 1988). Since a metabolite generated under the influence of one form of cytochrome P-450 might be highly cytotoxic, mutagenic, and/or carcinogenic, whereas another metabolite produced from the same parent chemical under the influence of a different form of cytochrome P-450 might be biologically inactive, differences in both the catalytic activities and relative levels of the cytochrome P-450 forms that are present clearly represent important factors contributing to both tissue- and cell-specific differences in the generation and accumulation of toxic, reactive metabolites. As recently reviewed in depth by Guengerich (1990) in another article in this series, pulmonary cytochromes P-450 have been studied and characterized mostly in rabbits and, to a lesser extent, rats. In both species, the forms of cytochrome P-450 that have been detected in lung to date appear to closely resemble forms found in liver (Guengerich, 1977a, 1990; Guengerich and Mason, 1979; Slaughter et al., 1981; Guengerich et al., 1982b; Parandoosh et al., 1987; Ueng et al., 1988). Furthermore, pulmonary and hepatic microsomal NADPH-cytochrome P-450 reductases in each species are also very similar, if not identical (Buege and Aust, 1975; Guengerich, 1977a; Serabjit-Singh et al., 1979). Rabbit lung contains three cytochromes P-450, designated forms 2, 5 and 6 (Domin et al., 1986), that have been studied quite extensively. Forms 2 and 5 account for more than 90% of the total microsomal cytochrome P-450 content in lungs of untreated animals (Domin et al., 1986) and are responsible for catalyzing the activation of 4-ipomeanol (Wolf et al., 1982). Cytochrome P-450 form 6 is present at only very low levels in untreated rabbits (Domin et al., 1986), catalyzes aryl hydrocarbon hydroxylase activity more efficiently than do either forms 2 or 5 (Cheung et al., 1984), and can be dramatically *The nomenclature of the individual forms of rat cytochrome P-450 discussed in this article has been described by Guengerich (1987) and Nebert et al. (1989). In the classification of Nebert et al., cytochrome P-450 BNF-B is P450IA1, the product of the C Y P 1 A I gene as is, presumably, cytochrome P-450 MC-B which is apparently identical to cytochrome P-450 BNF-B; cytochrome P-450 BNF/ISF-G is P4501A2, the C Y P 1 A 2 gene product; cytochrome P-450 PB-B is P450IIBI, the product of the C Y P 2 B 1 gene; cytochrome P-450 PB-D is P450IIB2, the C Y P 2 B 2 gene product; and cytochrome P-450 PCN-E appears to be the product of the C Y P 3 A 2 gene. The C Y P 3 A family encodes at least 4 forms of cytochrome P-450 in rat liver: cytochrome P-450 PCNb (P450IIIA2), which appears to be identical to P-450 PCN-E and is the product of the C Y P 3 A 2 gene (Halpert, 1988); cytochrome P-450 PCNa (P450111AI), the C Y P 3 A I gene product (Halpert, 1988); cytochrome P-450 PCNc (Halpert, 1988); and a fourth protein recently described by Shimada et al. (1989). Neither cytochrome P-450 PCNc nor the protein isolated by Shimada et al. have been assigned to gene sequences. It is not known which of the proteins in the P450IIIA family other than cytochrome P-450 PCN-E are expressed in rat lung.

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induced by TCDD, polycyclic aromatic hydrocarbons, and polychlorinated biphenyls (Domin et al., 1986; Guengerich, 1990). A fourth cytochrome P-450, form 4, has been detected in lungs of TCDD pretreated rabbits but does not appear to be present in untreated animals (Dees et al., 1982). Four cytochromes P-450 have also been detected to date in rat lung. The cytochrome P-450 found at the greatest level corresponds to the major phenobarbital-inducible form in rat liver, cytochrome P-450 PB-B* (Guengerich, 1977b, 1978, 1979, 1987; Guengerich and Mason, 1979; Guengerich et al., 1982a,b; Keith et aL, 1987). This protein is quite similar to rabbit cytochrome P-450 form 2 (Guengerich et al., 1982a,b; Guengerich, 1990) and appears to be responsible for catalyzing the activation of 4-ipomeanol (Guengerich, 1977a,b). A second form resembles cytochrome P-450 BNF/MC-B, the major form induced in rat liver by fl-naphthoflavone and 3-methylcholanthrene (Guengerich, 1979, 1987; Guengerich and Mason, 1979; Guengerich et al., 1982a,b; Foster et al., 1986; Keith et al., 1987). This enzyme appears to be the ortholog of rabbit form 6 (Guengerich, 1990), catalyzes aryl hydrocarbon hydroxylase activity more efficiently than do other forms of cytochrome P-450 (Guengerich et al., 1982a), is present at extremely low levels in untreated rats (Guengerich et al., 1982b; Keith et al., 1987; Guengerich, 1990), and can be markedly induced by polycyclic aromatic hydrocarbons, polychlorinated biphenyls, and TCDD (Foster et al., 1986; Keith et al., 1987; Guengerich, 1990). A third form corresponds to the major cytochrome P-450 induced in rat liver by pregnenolone-16~t-carbonitrile, cytochrome P-450 PCN-E (Guengerich et al., 1982a,b) and is also present at very low levels (Baron et al., 1986b, 1988; Voigt et al., 1990). The involvement of this cytochrome P-450 in pulmonary xenobiotic metabolism remains to be elucidated, however. The fourth cytochrome P-450 that has been found in rat lung appears to be the ortholog of rabbit form 5, is also present at extremely low levels in untreated as well as in phenobarbital and TCDD pretreated rats, and is capable of catalyzing the N-hydroxylation of the promutagen, 2-aminofluorene (Vanderslice et al., 1987). Electrophilically-reactive and toxic epoxides are frequently generated during the cytochromes P-450mediated oxidative metabolism (i.e. epoxidation) of aromatic chemicals such as naphthalene (Buckpitt and Franklin, 1989) and benzo[a]pyrene (Miller, 1978; Gelboin, 1980; Wislocki et al., 1980; Wright, 1980; Miller and Miller, 1982, 1985; Cummings and Prough, 1983). It has also been suggested (Boyd et al., 1978a) that an epoxide of 4-ipomeanol is responsible, at least in part, for pulmonary damage that occurs following in vivo exposure to the parent furan. Once produced, epoxides can be detoxicated by spontaneous rearrangement to form phenols, by conjugation with reduced glutathione (discussed below), and by epoxide hydrolase which catalyzes their hydration to yield the corresponding trans-dihydrodiols (Oesch, 1973; Lu and Miwa, 1980). Despite the fact that trans-dihydrodiols are stable, nontoxic chemicals, certain trans-dihydrodiols generated from polycyclic aromatic hydrocarbons can be further

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monooxygenated by polycyclic aromatic hydrocarbon- and polyhalogenated hydrocarbon-inducible forms of cytochrome P-450 (i.e. rat cytochrome P-450 BNF/MC-B and rabbit cytochrome P-450 form 6) to yield t r a n s - d i h y d r o d i o l - e p o x i d e s (Sims et al., 1974; Thakker et al., 1976; Levin et al., 1977; Gelboin, 1980; Guengerich, 1988), extremely reactive electrophiles that are generally considered to represent the ultimate toxic metabolites derived from polycyclic aromatic hydrocarbons (Huberman et al., 1976; Thakker et al., 1976; Miller, 1978; Gelboin, 1980). Thus, epoxide hydrolase serves a dual role in xenobiotic metabolism: on the one hand, it mediates the inactivation of reactive and toxic epoxides, while on the other hand, it catalyzes the formation of precursors for ultimate cytotoxic, mutagenic, and carcinogenic metabolites. Epoxide hydrolase has not as yet been isolated from lung. However, preparations derived from lungs of rats (Oesch et al., 1977; Smith et al., 1978; DePierre et al., 1983; Jones et al., 1983), rabbits (Smith et al., 1978; Gill and Hammock, 1980: Devereux et al., 1985), and other laboratory species, as well as from humans (Oesch et al., 1980: Devereux et al., 1986), exhibit epoxide hydrolase activity. Moreover, the demonstration by Guengerich and his colleagues (Guengerich et al., 1979a) that an antibody raised against rat hepatic microsomal epoxide hydrolase was capable of precipitating greater than 90% of the epoxide hydrolase activity in rat lung microsomes revealed that pulmonary and hepatic epoxide hydrolases are immunochemically similar. The glutathione S-transferases also play critical roles in xenobiotic metabolism, particularly in the detoxication of epoxides and other reactive metabolites (Boyland and Chasseaud, 1969; Jakoby and Habig, 1980). The members of this family of enzymes catalyze the conjugation of the sulfhydryl group of reduced glutathione with a wide variety of electrophilic chemicals (Boyland and Chasseaud, 1969; Fjellstedt et al., 1973; Jakoby and Habig, 1980), a reaction representing the initial step leading to the formation of mercapturic acid derivatives (Boyland and Chasseaud, 1969; Habig et al., 1974; Jakoby and HaNg, 1980). The glutathione S-transferases, together with reduced glutathione which can also be conjugated with electrophiles nonenzymatically (Boyland and Chasseaud, 1969; Buckpitt and Boyd, 1980), are thus capable of inactivating electrophilically-reactive metabolites derived from a multitude of xenobiotics, including 4-ipomeanol (Boyd et al., 1978a; Boyd, 1980a,b; Buckpitt and Boyd, 1980), naphthalene (Buckpitt and Franklin, 1989), benzo[a]pyrene (Gelboin, 1980; Robertson et al., 1986), and others that are toxic to the lung, thereby preventing their covalent interaction with cellular macromolecules. Additionally, the glutathione Stransferases are able to bind, and in this manner inactivate, a large number of potentially toxic, hydrophobic substances that are not conjugated with reduced glutathione (Ketley et al., 1975; Litwack et al., 1977). Glutathione S-transferase activity towards a number of substrates has been seen in rat (Smith et al., 1978; Jones et al., 1983; Morgenstern et al., 1984: Robertson et al.. 1986), rabbit (Gram et al.,

1974; Smith et al., 1978), and human (Partridge el al., 1984) lung preparations. Furthermore, as reviewed by Guengerich (1990), a number of glutathione S-transferases have been isolated and purified to apparent homogeneity from both rat and human lung. In many instances, the protein subunit compositions and catalytic activities of the glutathione S-transferases purified from rat lung correspond to those of forms present in rat liver (Singh and Awasthi, 1984; Awasthi et al., 1985; Partridge et al., 1985; Robertson et al., 1985). Glucuronidation is another important process in xenobiotic metabolism. In this reaction, the glucuronic acid moiety of UDP-glucuronic acid is covalently linked to a variety of xenobiotics, xenobiotic metabolites, and other acceptor molecules to produce more water-soluble compounds that, in general, are more readily excreted from the body than are the parent chemicals (Dutton, 1980). This biotransformation reaction usually results in the detoxication of xenobiotics (Dutton0 1980; Kasper and Henton, 1980) and is involved in the metabolism of certain chemicals that are capable of damaging the lung; for example, naphthalene (Buckpitt and Franklin, 1989) and benzo[a]pyrene (Gelboin, 1980). It must be appreciated, however, that glucuronidation can also give rise to the formation of reactive metabolites from certain xenobiotics, notably arylamines, and in this manner can contribute to the initiation of the neoplastic process and the production of other toxicities that occur following in vitro exposure to these chemicals (Bock, 1977; Miller, 1978; Kasper and Henton, 1980). Glucuronidation occurs in numerous tissues and is catalyzed by another family of enzymes collectively named the UDP-glucuronosyltransferases (Dutton, 1980; Bock et al., 1983). Although neither these enzymes nor xenobiotic glucuronidation have been studied appreciably in lung, rat (Bock et al., 1980; Jones et al., 1983) and rabbit (Gram et al., 1974; Lucier el al., 1977) pulmonary preparations do display UDP-glucuronosyltransferase activity. Sulfation represents another major pathway for the conjugative metabolism of xenobiotics. In this reaction, members of another family of enzymes, the sulfotransferases (Jakoby et al., 1980; Sekura et al., 1981), catalyze the conjugation of 3'-pbosphoadenosine 5'-phosphosulfate with a wide spectrum of acceptor molecules possessing a hydroxyl functional group (Roy, 1960; Jakoby et al., 1980). Substrates for sulfation include reactive and toxic metabolites of naphthalene (Buckpitt and Franklin, 1989), butylated hydroxytoluene (Witschi et al., 1989), benzo[a]pyrene (Gelboin, 1980), and certain other xenobiotics that can damage the lung. Sulfate esters are generally less toxic, more water soluble, and more readily excreted in comparison to the parent chemicals from which they are formed (Jakoby et al., 1980). However, as was noted for glucuronidation, sulfation can also result in the enhanced toxicity of a number of xenobiotics (Bock, 1977; Miller, 1978; Jakoby el al., 1980). Although a sulfotransferase has been isolated and purified from bovine lung (Baranczyk-Kuzma and Szymczyk, 1987), virtually nothing is currently known about either xenobiotic sulfation or the

Enzymes within lung enzymes that catalyze this biotransformation in rat or rabbit lung.

3. I N T R A P U L M O N A R Y LOCALIZATION OF XENOBIOTIC-METABOLIZING ENZYMES The intrapulmonary metabolism of xenobiotics is unquestionably of paramount importance in the etiology of many chemically-induced pulmonary toxicities. Moreover, the balance between the activation and detoxication of xenobiotics within individual cells in this organ might very well be the principal factor that determines why certain pulmonary cells are much more highly susceptible than others to toxicities that occur as a consequence of the bioactivation of many xenobiotics. Thus, delineation of the intrapulmonary localizations and distributions of xenobiotic-metabolizing enzymes as well as of the abilities of the different types of pulmonary cells to metabolize xenobiotics is clearly a prerequisite for elucidating the basis for the cell-selective nature of lung damage that is frequently seen following in vivo exposure to many chemicals. Unfortunately, the identification of those pulmonary cells within which xenobiotics are activated and detoxicated in vivo has been severely impeded by the complexity and cellular diversity of the lung, an organ that contains at least 40 distinctive cell types (Sorokin, 1970). Despite such obstacles, the use of a variety of experimental approaches, especially during the past decade, has permitted significant advances to be made in defining the major sites at which xenobiotics are metabolized within lung. In their pioneering histochemical studies on aryl hydrocarbon hydroxylase activity, Wattenberg and Leong (1962) demonstrated that benzo[a]pyrene was hydroxylated within the alveolar wall in rat lung but could not detect the monooxygenase activity in either the bronchus or bronchiole. Their inability to detect benzo[a]pyrene's hydroxylation in the bronchus and bronchiole is difficult to reconcile with results of subsequent studies and might have been due to methodological problems in the histochemical assay. Indeed, as discussed elsewhere in this article, by modifying the procedure developed by Wattenberg and Leong, we have detected the hydroxylation of benzo[a]pyrene within rat bronchial and bronchiolar epithelial cells as well as alveolar wall cells. Furthermore, employing another histochemical assay, Grasso et al. (1971) found that aniline was hydroxylated by rat bronchial epithelial and alveolar wall cells. Thus, it can be concluded from these histochemical findings that the bronchus, bronchiole, and alveolar wall represent major sites for the in situ oxidative metabolism of xenobiotics in lung. Autoradiographic findings on the covalent binding of reactive xenobiotic metabolites within lung provided evidence for the participation of specific types of pulmonary cells in the metabolism of xenobiotics, Boyd (1977) demonstrated that 4-ipomeanol, which selectively damages Clara cells, was preferentially activated by cytochrome P-450 within Clara cells to yield reactive metabolite(s) that bound covalently to these cells in rats, mice, and hamsters and concluded JPT 473--G

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that the Clara cell was a primary site for xenobiotic monooxygenation in lung. The subsequent finding that reactive metabolites of several carcinogenic Nnitrosamines were also preferentially formed and covalently bound within rat and hamster Clara cells (Reznik-Schiiller and Lijinsky, 1979; Boyd and Reznik-Schfiller, 1984) provided additional support for this conclusion as well as for the existence of a direct relationship between cell-specific toxicity and xenobiotic activation. Despite the fact that these findings might suggest that only Clara cells are capable of activating xenobiotics, results of other autoradiographic studies revealed that xenobiotics can be activated by additional types of pulmonary cells. For instance, although 4-ipomeanol is also activated to the greatest extent by rabbit Clara cells, the formation and covalent binding of its metabolite(s) occur to considerable degrees in rabbit type II pneumocytes (Devereux et al., 1982). Furthermore, while the exposure of rats to the tritiated tobaccospecific carcinogen, 4-(N-methyl-N-nitrosamino)-l(3-pyridyl)-l-butanone, results in Clara cells being labeled predominantly, other cells in the bronchiole and alveolar wall were also seen to be labeled, as were alveolar macrophages (Belinsky et al., 1987). In addition, the cytochrome P-450-mediated metabolism of 3-methylindole, which damages both pneumocytes and bronchiolar epithelial cells in goats, results in the covalent binding of its metabolite(s) to ciliated bronchiolar epithelial cells, type I pneumocytes, and type II pneumocytes as well as to Clara cells in this species (Becker et al., 1984). Thus, while there are some inconsistencies among these autoradiographic findings, and although there could be significant species differences either in the ability of a specific pulmonary cell to metabolize xenobiotics or in the balance between xenobiotic activation and detoxication processes within a given type of pulmonary cell, these results nevertheless clearly illustrate that xenobiotics can be transformed into reactive metabolites by type II pneumocytes and other pulmonary cells in addition to Clara cells. It is evident from these findings, however, that xenobiotics are most often activated to the greatest extent by Clara cells, and this is clearly consistent with the fact that Clara cells are most readily damaged as a consequence of the bioactivation of many pulmonary toxins. Results of studies on the formation and accumulation of alkylated DNA bases and DNA adducts in carcinogen pretreated animals also demonstrated that different types of pulmonary cells can metabolize xenobiotics and, further, that there can be significant cell type-specific differences in the extent of xenobiotic activation. For instance, following the exposure of rats to 4-(N-methyl-N-nitrosamino)-l(3-pyridyl)-1 °butanone, O6-methylguanine is found in type II pneumocytes, endothelial cells, and alveolar macrophages but accumulates to a considerably greater degree in Clara cells (Belinsky et aL, 1987; Devereux et al., 1988). Levels of O6-ethylguanine are also much greater in Clara cells than in type II pneumocytes and alveolar macrophages in Nnitrosodiethylamine-treated hamsters (Fong and Rasmussen, 1987). In marked contrast, O6-methylguanine accumulates to similar extents in Clara cells, type II pneumocytes, endothelial cells, and alveolar

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macrophages in rats treated with N-nitrosodimethylamine (Belinsky et al., 1987; Devereux et al., 1988). Comparable levels of the major D N A adduct produced as the result of the activation of benzo[a]pyrene, (+)-r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10tetrahydrobenzo[a]pyrene: deoxyguanosine, have also been detected in Clara cells, type II pneumocytes, and alveolar macrophages following the administration of the polycyclic aromatic hydrocarbon to rabbits (Horton et al., 1985). Thus, in addition to demonstrating that a number of different cells are capable of metabolizing carcinogens in lung, these findings suggest that the extent to which xenobiotics are activated by different pulmonary cell types might be highly dependent on the particular xenobiotics to which the lung is exposed and this, in turn, could reflect the existence of significant differences in the intrapulmonary distributions of certain cytochrome P-450 forms as well as of other xenobiotic-metabolizing enzymes. The use of more classic biochemical approaches conclusively demonstrated that xenobiotics can be metabolized in vitro by bronchial epithelial cells, Clara cells, and type II pneumocytes, as well as by alveolar macrophages. Results of studies on cultured human bronchial epithelial cells established that these cells are capable of transforming polycyclic aromatic hydrocarbons (Harris et al., 1977b; Autrup et al., 1980) and N-nitrosamines (Harris et al., 1977a; Castonguay et al., 1983) into reactive metabolites that bind covalently to protein and DNA. Cultured rat, mouse, and hamster bronchial epithelial cells are also able to activate benzo[a]pyrene (Autrup et al., 1980). Furthermore, the cytochromes P-450-catalyzed monooxygenations of several other xenobiotics occur in both rabbit (Sabourin et al., 1988) and dog (Bond et al., 1988) bronchi. Additionally, while only limited information is currently available regarding the detoxication of xenobiotics in the bronchus, epoxide hydrolase, glutathione S-transferase, glucuronosyltransferase, and sulfotransferase activities have each been detected in this tissue (Autrup et al., 1980; Bond et al., 1988). As reviewed in depth by Devereux et al. (1989) in another article in this series, results of numerous studies on Clara cells, type II pneumocytes, and alveolar macrophages isolated from the lungs of various species have clearly shown that each of these cells is capable of metabolizing xenobiotics. In most instances, however, they do not metabolize xenobiotics equally: Clara cells usually exhibit the greatest metabolic activity, followed by type II pneumocytes and then alveolar macrophages (Jones et al., 1982, 1983; Urade et al., 1982; Devereux et al., 1985; Domin et al., 1986), although significant species differences have been detected in the patterns of distribution of certain xenobiotic monooxygenase activities among these three cell types (Urade et al., 1982; Massey et al., 1987; Belinsky et al., 1987; Devereux et al., 1989). Since comparable studies have not been conducted with ciliated epithelial cells, type I pneumocytes, or other cells present in the lung, it must be emphasized that the question of whether or not these cells also participate in the pulmonary metabolism of xenobiotics remains to be answered.

Consistent with the fact that Clara cells are usually more active in monooxygenating xenobiotics than are other pulmonary cells, cytochrome P-450 forms 2, 5 and 6 are more greatly concentrated in these cells than in type II pneumocytes and alveolar macrophages in rabbits (Aune et al., 1985; Domin et al., 1986; Devereux et al., 1989). The distribution of cytochrome P-450 form 6 among the three cells apparently differs from those of forms 2 and 5, however (Domin et al., 1986). Rabbit Clara cells also contain a higher level of NADPH-cytochrome P-450 reductase than do type II pneumocytes (Devereux and Fouts, 1981; Domin et al., 1986) which, in turn, contain a greater amount of this enzyme than do alveolar macrophages (Domin et al., 1986). Similar differences in epoxide hydrolase (Jones et al., 1983; Devereux et al., 1985, 1986), glutathione S-transferase (Jones et al., 1983), and UDP-glucuronosyltransferase (Jones et al., 1983) activities have also been detected among the three cell types, at least with the limited number of substrates that have been studied to date. Thus, while Clara cells, type II pneumocytes, and alveolar macrophages are each clearly capable of activating and detoxicating xenobiotics, these findings illustrate that xenobioticmetabolizing enzymes and activities are not distributed uniformly among them and provide a plausible explanation for why the Clara cell is the major target site for toxicities resulting from the metabolism of 4-ipomeanol and certain other xenobiotics. Immunohistochemical approaches have been employed in order to more definitively establish where xenobiotic-metabolizing enzymes are localized within lung and to provide further insight into the abilities of different pulmonary cells to both activate and detoxicate xenobiotics. It should be appreciated that knowledge of the precise localizations of enzymes within tissues is often essential for defining the metabolic capabilities of specific cells. In many instances, however, this knowledge can only be obtained through the application of exquisitely sensitive immunohistochemical staining techniques with which enzymes and other antigens can be visualized in tissues and cells. It must be emphasized, however, that despite the fact that immunohistochemistry offers a number of rather obvious advantages for investigating the localization of enzymes within a complex tissue such as the lung, its use is also associated with several limitations, while a considerable amount of caution must be exercised to ensure that valid, reproducible data are generated (Baron et al., 1986a). For instance, differences in staining for the same or closely related enzymes, as well as the inability to immunohistochemically detect a specific enzyme, in fixed tissue specimens could result from fixation-induced modifications in the threedimensional structure and/or epitopes of the protein, either of which could significantly alter the interaction between the enzyme and its antibody, especially in biologic membranes. The use of unfixed tissues or different fixatives for preparing tissue specimens, different preparations of the same antigen, different polyclonal and/or monoclonal antibodies raised to the same antigen, and different staining procedures could certainly explain, at least in part,

Enzymes within lung the basis for the discrepant immunohistochemical findings on certain enzymes that have appeared in the literature. Furthermore, the presence of significant degrees of nonspecific background staining in tissue sections can greatly hinder, if not prevent, the proper interpretation of immunohistochemical results, particularly when the intensity of staining for an antigen is extremely weak. In addition, both false-positive and false-negative results can frequently be obtained. In spite of such limitations, the application of immunohistochemistry permits the study of enzymes that are present within only either a small percentage of cells or limited regions of tissues and allows examinations of enzymes whose overall tissue and/or cellular levels may be too low to be detected by means of more conventional biochemical, biophysical, immunological, and molecular biological methodologies. The intrapulmonary localizations of NADPHcytochrome P-450 reductase and certain forms of cytochrome P-450 and glutathione S-transferase have been the subjects of several studies, whereas findings on the Iocalizations of epoxide hydrolase, glucuronosyltransferases, and sulfotransferases within lung have not as yet been described by other laboratories. The initial reports of immunohistochemical findings on xenobiotic-metabolizing enzymes within lung appeared in 1980 and concerned, in part, NADPHcytochrome P-450 reductase. Dees et al. (1980) detected immunohistochemical staining for this flavoprotein in the bronchial and bronchiolar epithelia and alveolar wall in lungs of rats and minipigs, with identical results being obtained using saline and phenobarbital pretreated animals. The specific cells that were stained for the reductase were not identified, however. Serabjit-Singh et al. (1980a) also reported that an antibody to NADPH-cytochrome P-450 reductase intensely stained Clara cells in rabbit lung but did not comment on staining of other cells. Subsequently, the reductase was immunohistochemically shown to be present in Clara cells of the bronchus and bronchiole (Plopper et al., 1987; Serabjit-Singh et al., 1988) and, at a significantly lower level, in type II pneumocytes (Serabjit-Singh et al., 1988) in rabbits. Only equivocal staining for the enzyme was seen in rabbit ciliated bronchiolar epithelial cells, however, while its presence could not be demonstrated in either type I pneumocytes or endothelial cells (Serabjit-Singh et al., 1988). In human lung, Hall et al. (1989) reported that an antibody to NADPH-cytochrome P-450 reductase stained the bronchial and bronchiolar epithelia and alveolar wall, as well as alveolar macrophages, although cells other than Clara cells and alveolar macrophages that were stained for the reductase were not identified. The localizations of several forms of cytochrome P-450 have also been investigated in lung, especially in rabbits. Results of immunohistochemical studies on cytochrome P-450 form 2 in isolated rabbit lung cells revealed that both Clara cells and type II pneumocytes contain this hemeprotein (SerabjitSingh et al., 1980a; Devereux et al., 1981). When sections prepared from fixed paraffin-embedded tissue specimens were examined, however, staining for form 2 appeared to be restricted to Clara cells (Serabjit-Singh et al., 1980a,b; Devereux et al., 1981).

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The reason for this discrepancy remains unknown. On the other hand, using a different antibody raised against cytochrome P-450 form 2 and unfixed frozen sections, Dees et al. (1982) detected the presence of this cytochrome P-450 in the bronchial and bronchiolar epithelia as well as in type I and possibly type II pneumocytes. These investigators also found that staining for form 2 was not altered in lung after rabbits were pretreated with either phenobarbital or TCDD. More recently, Plopper et al. (1987) and Serabjit-Singh et al. (1988) demonstrated that an antibody to cytochrome P-450 form 2 stained Clara cells and, to a lesser extent, ciliated epithelial cells in both the bronchus and bronchiole. Serabjit-Singh et al. (1988) also detected staining for form 2 in type II pneumocytes, and, in marked contrast to the findings of Dees et al. (1982), in capillary endothelial cells but not type I pneumocytes. The explanation for these conflicting observations is also unknown, although it must be kept in mind that different antibodies to cytochrome P-450 form 2 were utilized in the two studies; Dees and her colleagues examined staining at the light microscopic level in unfixed frozen sections whereas Serabjit-Singh and her associates employed an electron microscopic technique and fixed, embedded tissue specimens. Philpot and his associates have also examined the localization of cytochrome P-450 form 5 in rabbit lung (Serabjit-Singh et al., 1980a, 1988; Devereux et al., 1981; Plopper et al., 1987). Analogous to their findings on cytochrome P-450 form 2, they detected the presence of form 5 in Clara and ciliated cells in the bronchus and bronchiole, in type II pneumocytes, and in capillary endothelial cells but not in type I pneumocytes. Both forms of cytochrome P-450, moreover, appeared to be most greatly concentrated in Clara cells. On the other hand, differences in staining for the two forms were noted within Clara and ciliated cells in the bronchiole, especially with respect to the intensities with which antibodies to forms 2 and 5 stained agranular endoplasmic reticulum in Clara cells and Golgi membranes in both Clara and ciliated cells (Serabjit-Singh et al., 1988). The biologic significance of such differences in the intracellular localizations of different cytochrome P-450 forms remains to be determined, however. Neither cytochrome P-450 forms 4 nor 6 have been immunohistochemically detected in lungs of untreated rabbits, but both can be markedly induced by TCDD (Dees et al., 1982; Domin et al., 1986; Guengerich, 1990). In TCDD pretreated rabbits, Dees et al. (1982) found that antibodies to forms 4 and 6 stained the bronchial and bronchiolar epithelia, with staining for form 6 being more intense, although they did not identify the epithelial cells that were stained for either form of cytochrome P-450. Additionally, the antibodies to forms 4 and 6 intensely stained the endothelia of pulmonary arteries, veins, and capillaries in TCDD pretreated rabbits. Antibodies raised against the major forms of cytochrome P-450 induced in rat liver by phenobarbital (i.e. cytochrome P-450 PB-B, the ortholog of rabbit form 2) and polycyclic aromatic hydrocarbons (i.e. cytochrome P-450 BNF/MC-B, the ortholog of rabbit form 6) have been employed to investigate the localizations of immunochemically-related forms of

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cytochrome P-450 in lungs of untreated and xenobiotic pretreated rats and mice. In rat lung, Keith et al. (1987) found that a polyclonal antibody to the major phenobarbital-inducible cytochrome P-450 stained a large percentage of bronchiolar Clara cells and type II pneumocytes, whereas staining of other cells was not apparent. Neither phenobarbital nor 3-methylcholanthrene altered staining for this cytochrome P-450. Significantly different results were obtained when a polyclonal antibody to the major cytochrome P-450 induced by 3-methylcholanthrene was used (Keith et al., 1987). In untreated rats, this antibody stained only a limited number of Clara ceils and type II pneumocytes. Moreover, staining was detected in only approximately 40% of the rats examined. However, after rats were treated with 3-methylcholanthrene, markedly enhanced staining was evident in many, but not all, Clara cells and type II pneumocytes. Additionally, staining was seen in the endothelia of arteries, arterioles, veins, venules, and septal capillaries of 3-methylcholanthrene pretreated rats. In marked contrast to these findings, Foster et al. (1986) reported that a monoclonal antibody to the major /3-naphthoflavone-inducible cytochrome P-450 produced detectable staining of only bronchiolar Clara cells in both untreated and /~-naphthoflavone pretreated rats. These discrepant results could possibly be due to the fact that Keith and his colleagues used a polyclonal antibody and fixed, embedded sections whereas Foster and his associates employed a monoclonal antibody and frozen sections. Alternatively, they might reflect the existence of significant strain differences in the intrapulmonary localization and induction of the cytochrome P-450 since Sprague-Dawley rats were used by Keith et al. while Foster et al. studied Wistarderived rats. In mouse lung, Forkert et al. (1986, 1989) found that a monoclonal antibody prepared against rat hepatic microsomal cytochrome P-450 PB-B stained the bronchiolar epithelium, especially Clara cells, as well as type II pneumocytes. Staining was not apparent in other pulmonary cells, however, nor was staining altered when mice were pretreated with either phenobarbital or 3-methylcholanthrene. Despite the fact that forms of cytochrome P-450 corresponding to those induced in rat liver by polycyclic aromatic hydrocarbons have not been detected in lungs of untreated mice, they are present in 3-methylcholanthrene-treated mice (Forkert et al., 1986, 1989; Anderson et al., 1987). Using monoclonal antibodies that recognize murine cytochrome P~-450, a form corresponding to rat cytochrome P-450 BNF/MC-B, Forkert et al. (1989) detected staining in alveolar wall cells, especially type II pneumocytes, but not in other pulmonary cells in mice pretreated with 3-methylcholanthrene. On the other hand, a monoclonal

4.1.1. E n z y m e s a n d A n t i b o d i e s

*On the basis of the recommendation by Jakoby et al. (1984), the glutathione S-transferases discussed in this article are named according to the numbers assigned to their two constituent subunits. Formerly, glutathione S-transferases 1 1 and I-2 were referred to as either gutathione S-transferase B or ligandin, while glutathione S-transferases 3-3, 3-4, and 5-5 were called, respectively, glutathione S-transferases A, C, and E.

To investigate the localizations, distributions, and inductions of xenobiotic-metabolizing enzymes within rat lung, polyclonal rabbit or sheep antibodies were raised against the following enzymes that had been isolated and purified to apparent homogeneity from rat liver. NADPH-cytochrome P-450 reductase (Taira et al., 1979); cytochromes P-450 BNF-B (Guengerich et al., 1982a), MC-B (Guengerich,

antibody specific for an epitope common to both rat liver cytochrome P-450 BNF/MC-B and cytochrome P-450 BNF/ISF-G, a form corresponding to murine cytochrome P3-450, stained the capillary endothelium as well as the alveolar wall (Anderson et al., 1987; Forkert et al., 1989). However, while Anderson et al. (1987) found that this antibody stained these cells in C57BL/6 mice, a strain responsive to the inducing actions of 3-methylcholanthrene, but not in DBA/2 mice, a strain nonresponsive to 3-methylcholanthrene, Forkert et al. (1989) reported that discernible differences in staining were not apparent between 3-methylcholanthrene-treated C57BL/6 and DBA/2 mice. No explanation can be offered for these contradictory results which are particularly perplexing inasmuch as the same monoclonal antibody was used by the two groups. The intrapulmonary localizations of glutathione S-transferases have received only limited study and, moreover, have only been investigated in rats. Awasthi et al. (1984) reported that antibodies raised against rat liver glutathione S-transferases 1-2" and 3-4 and a form apparently unique to the lung produced staining throughout the bronchiolar epithelium and alveolar wall. These investigators, however, did not identify the cells that were stained by any of the antibodies. These immunohistochemical findings on the localizations of NADPH-cytochrome P-450 reductase, cytochromes P-450, and glutathione S-transferases within lung are, thus, quite consistent with the results of histochemical, autoradiographic, and biochemical studies and provide further evidence for the participation of a number of different pulmonary cells in the metabolism of xenobiotics. Our laboratory has also investigated the localizations of NADPH-cytochrome P-450 reductase and different cytochromes P-450 and glutathione S-transferases, as well as of epoxide hydrolase, three UDP-glucuronosyltransferases, and an aryl sulfotransferase, in rat lung. The remainder of this article will focus on our qualitative and semiquantitative immunohistochemical findings on the localizations and distributions of these enzymes and on our histochemical findings on aryl hydrocarbon (benzo[a]pyrene) hydroxylase activity in lungs of untreated rats and rats pretreated with 3-methylcholanthrene and Aroclor 1254, a mixture of polychlorinated biphenyls.

4. METHODS OF STUDY 4.1. IMMUNOHISTOCHEMICALLOCALIZATION OF XENOBIOTIC-METABOLIZING ENZYMES WITHIN LUNG

Enzymes within lung 1977b, 1978), PB-B (Guengerich, 1977b, 1978), and PCN-E (Guengerich et al., 1982a); epoxide hydrolase (Guengerich et al., 1979b); glutathione S-transferases 1-1 (Habig et al., 1974), 3--4 (Habig et al., 1974), and 5-5 (Fjellstedt et al., 1973); p-nitrophenol, 3~thydroxysteroid, and 17fl-hydroxysteroid UDPglucuronosyltransferases (Falany and Tephly, 1983; Kirkpatrick et al., 1984; Knapp et al., 1988; Tephly et al., 1988a,b); and aryl sulfotransferase IV (Sekura and Jakoby, 1981; Sekura et al., 1981; Duffel and Janss, 1986). Antibodies, the specificities of which have been described previously, raised to these rat liver enzymes were then utilized in immunoperoxidase and immunofluorescence staining techniques to localize antigens at the light microscopic level in sections prepared from fixed, paraffin-embedded lung specimens. In view of similarities that exist among some forms of cytochrome P-450, glutathione S-transferase, UDP-glucuronosyltransferase, and aryl sulfotransferase, the matter of cross-reactivity needs to be considered. Cytochromes P-450 BNF-B and MC-B represent the major forms of rat hepatic microsomal cytochrome P-450 induced by fl-naphthoflavone and 3-methylcholanthrene, respectively, and appear to be identical as judged by their spectral, electrophoretic, and immunologic properties (Guengerich, 1977b, 1978; Guengerich et al., 1982a). Cytochrome P-450 BNF/MC-B shares common epitopes with P-450 BNF/ISF-G, a minor constitutive form of cytochrome P-450 in rat liver (Guengerich et al., 1982a). Cytochrome P-450 PB-B, the major phenobarbitalinducible form of rat hepatic microsomal cytochrome P-450, shares common epitopes with P-450 PB-D, another minor constitutive form of rat liver cytochrome P-450 (Guengerich et al., 1982a). In rat lung, cytochrome P-450 PB-B appears to be the major constitutive form of the hemeprotein present, with cytochrome P-450 BNF/MC-B being found at much lower levels in untreated rats (Guengerich et al., 1982b; Keith eta/., 1987; Guengerich, 1990). Neither cytochromes P-450 BNF/ISF-G nor PB-D appear to be expressed in rat lung (Goldstein and Linko, 1984; Omiecinski, 1986; Keith et al., 1987). Cytochrome P-450 PCN-E is the major rat hepatic microsomal form induced by pregnenolone-16~-carbonitrile (Guengerich e t a / . , 1982a; Halpert, 1988). Although the antibody raised against cytochrome P-450 PCN-E probably recognizes more than one of the rat liver proteins that are encoded by the C Y P 3 A gene family, in Western blots of rat pulmonary microsomes, it reacts with only a single band that exhibits the same mobility as that of the major band in hepatic microsomes of pregnenolone-16~-carbonitrile pretreated rats (Voigt et a/., 1990). Glutathione S-transferases 1-1 and 1-2 share a common subunit and are therefore immunochemically similar, as are glutathione S-transferases 3-4 and 3-3 (Habig et al., 1974). For this reason, immunohistochemical results obtained using the antibodies raised against glutathione S-transferases 1-1 and 3-4 must be interpreted as being indicative of the presence of transferases 1-1 and/or 1-2 and transferases 3-3 and/or 3-4, respectively. The antibody to glutathione S-transferase 5-5 is specific for this enzyme, however (Jakoby and Habig, 1980). The

427

antibody raised against p-nitrophenol UDPglucuronosyltransferase is also specific for this enzyme, whereas the antibodies to 3~t-hydroxysteroid and 17fl-hydroxysteroid UDP-glucuronosyltransferases are each capable of cross-reacting with the heteroiogous antigen in solubilized rat liver microsomes (Knapp et al., 1988). Despite this, each of the anti-UDP-glucuronosyltransferases recognizes distinct proteins in tissue sections (Knapp et al., 1988). Finally, although four aryl sulfotransferases are found in rat liver, aryl sulfotransferase IV is antigenically distinct from aryi sulfotransferases I and II (Sekura and Jakoby, 1981; Sekura et al., 1981) as well as from aryl sulfotransferase III (Duffel et al., unpublished observations). While the antibodies to rat liver NADPH-cytochrome P-450 reductase, cytochromes P-450, epoxide hydrolase, glutathione S-transferases, UDP-glucuronosyltransferases, and aryl sulfotransferase IV are capable of cross-reacting with rat pulmonary proteins, it must be emphasized that immunohistochemical staining produced by these antibodies within rat lung may only be indicative of the presence of antigens that are immunochemically-similar, rather than identical, to the rat liver enzymes. For the sake of simplicity, however, these antigens will be referred to using the names of the hepatic enzymes. 4.1.2. Tissue Preparation Male Sprague-Dawley rats weighing 160-180g were used in these studies. For immunohistochemical analyses, rats were sacrificed by decapitation, and their lungs were immediately perfused through the pulmonary artery with ice-cold 0.9% NaCl. After cannulating the trachea, the lungs were inflated with ice-cold fixative solution consisting of 1% acetic acid in 95% ethanol. Following tracheal ligation, the lungs and heart were removed en bloc, and 1- to 2-mm-thick slices of tissue obtained from each pulmonary lobe were fixed at 4°C for 4 hr by constant shaking in fixative. We have found that fixation of lung specimens with 95% ethanol:l% acetic acid provides optimal retention of the epitopes of the proteins studied while preserving the morphologic characteristics of the tissue, whereas fixatives such as paraformaldehyde and p-benzoquinone yield much less satisfactory immunohistochemical results. The fixed tissue specimens were dehydrated, cleared, and embedded in paraffin, and 4-/~m-thick serial sections were prepared and mounted on Histostik~-coated microscope slides. NADPH-cytochrome P-450 reductase, cytochromes P-450, epoxide hydrolase, glutathione S-transferases, UDP-glucuronosyltransferases, and aryl sulfotransferase IV were then localized at the light microscopic level by means of both immunoperoxidase (avidin-biotin-peroxidase) and immunofluorescence (avidin-fluorescein isothiocyanate) staining as described below. For histochemical analyses of benzo[a]pyrene hydroxylase activity, after the trachea was cannulated, lungs were inflated with 0.9% NaC1 containing 4% gelatin and immersed in ice-cold 0,9% NaCl. After the gelatin had solidified, l- to 2-mm-thick slices of unfixed lung could be obtained without

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J. BARONand J. M. VOIGT with an epithelium consisting of Clara and ciliated cells that also rests on a basement membrane: as the diameter of the bronchiole decreases, the ratio of Clara to ciliated cells increases. The Clara cell is readily identified by its dome-shaped apex that protrudes into the lumen of the bronchiole (Fig. 1B). The alveolar wall contains two epithelial cells, type I and type II pneumocytes. Type I pneumocytes are flat cells with long, thin cytoplasmic extensions that cover a large surface area. At the light microscopic level, however, type I pneumocytes cannot be distinguished from capillary endothelial cells that are also associated with the alveolar wall. Type II pneumocytes are more cuboidal in shape, are located in niches at junctions of alveolar septa, and are characterized~ in part, by the presence of lamellar inclusion bodies in which surfactant is stored (Fig. IC). 4.1.3. Immunoperoxidase

FIG, I. Bronchial (A) and bronchiolar (B) epithelia and alveolar walls (C) in sections of rat lung stained with toluidine blue. Arrowheads in B point to Clara cells, and arrowheads" in C point to type I1 pnenmocytes. collapsing alveoli. The tissue slices were then frozen by immersion in a 2-methylbutane/dry ice slurry, and unfixed cryostat, sections 6Ftin in thickness, were prepared at - 2 0 C . The sections were mounted on coverslips coated with an adhesive solution containing 1% gelatin and 0.05% chrome alum and allowed to air dry for 2 hr at - 2 0 - C prior to use. The identification of specific cells in immunohistochemically-stained sections and in those used for histochemical studies on benzo[a]pyrene hydroxylase activity was confirmed by examining serial sections stained with toluidine blue (Fig. 1). In rat lung, the larger, conducting bronchial airways are lined with an epithelium composed of basal, ciliated, and secretory cells (Fig. IA). These cells rest on a basement membrane, but only ciliated and secretory cells extend from the basement membrane to the bronchial lumen. The smaller, bronchiolar airways are lined

Staining

Xenobiotic-metabolizing enzymes were localized at the light microscopic level in rat lung using the avidin-biotin-peroxidase (ABC) staining technique (Hsu et al., 1981). In comparison to other immunoenzymatic staining procedures, the ABC method, which makes use of the extraordinary affinity of the glycoprotein, avidin, for biotin, provides greater sensitivity for antigen detection in tissue sections together with lower nonspecific staining. Briefly, in the staining protocol used (Baron et al., 1986a; Voigt et al., 1990), sections were sequentially exposed to aqueous dimethylsulfoxide to enhance the uniform penetration of antibodies into the tissue, normal (nonimmune) serum to reduce nonspecific antibody binding, rabbit or sheep antiserum or immune IgG raised against the xenobiotic-metabolizing enzyme being studied, biotinylated antisheep or antirabbit immunoglobulins, and avidin complexed with biotinylated horseradish peroxidase. In this procedure, avidin acts as a link between the biotinylated peroxidase and the biotinylated immunoglobulins which bind to the antibody directed against and bound to the xenobiotic-metabolizing enzyme in the tissue section. The subsequent exposure of the section to 3,3'-diaminobenzidine tetrahydrochloride and H202 results in the oxidative polymerization of 3,3'diaminobenzidine to form an insoluble product that, when osmicated, can be easily seen without further staining under the light microscope as a dark, particulate deposit at the site of the antigen-antibody complex (Seligman et al., 1968). Since endogenous peroxidase activity in tissue sections can give rise to false-positive immunohistochemical results, sections were initially exposed to phenylhydrazine (Straus, 1972). We have found that phenylhydrazine is superior to methanolic hydrogen peroxide for blocking endogenous peroxidase activity in sections (Kawabata et al., 1984; Baron et al., 1986a). In order to assess the presence and degree of nonspecific staining resulting from endogenous peroxidase activity and/or the nonspecific binding of antibodies, serial sections were exposed either to equivalent dilutions or concentrations of the appropriate normal serum of IgG or to preparations from which the antibody had been removed by adsorption

Enzymes within lung with the homogeneous antigen (Taira et al., 1979; Baron et al., 1981, 1986a; Kawabata et al., 1981). Little if any immunoperoxidase staining for xenobiotic-metabolizing enzymes was evident when lung sections were exposed to normal sera or adsorbed antisera as well as when the biotinylated immunoglobulins were replaced with normal sera. 4.1.4. Irnmunofluorescence Staining The use of multiple staining methods very greatly minimizes the generation of false-positive and falsenegative immunohistochemical results. For this reason, in part, antigens immunochemically-related to rat liver xenobiotic-metabolizing enzymes were also investigated in rat lung employing an immunofluorescence staining technique utilizing avidin complexed with fluorescein isothiocyanate (Baron et al., 1988; Voigt et al., 1990). In this procedure, after the tissue section has been exposed to dimethylsulfoxide, normal serum, the antibody to the enzyme being studied, and biotinylated immunoglobulins as in the avidin-biotin-peroxidase staining protocol, it is exposed to an avidin-fluorescin isothiocyanate complex. Thus, the antibody directed against and bound to the xenobiotic-metabolizing enzyme in the tissue section is labeled with a fluorochrome. Immunofluorescence staining is then visualized by means of incident-light (epi-illumination) fluorescence microscopy (Baron et al., 1978, 1981, 1986a). Since fluorescein isothiocyanate emits a bright, apple-green fluorescence (emission maximum, 525 nm), the presence of the fluorescently-labeled antigen-antibody complex can be readily detected in the section. The apple-green fluorescence, moreover, is either not apparent or is of only minimal intensity when sections are exposed to normal sera or IgG or to adsorbed antibody preparations as well as when the biotinylated immunoglobulins are replaced with normal sera. 4.1.5. Microfluorometric Analysis o f lrnmunofluorescence Staining

Immunohistochemical staining is routinely evaluated by means of visual inspection and subjective grading of staining intensity. The proper interpretation of immunohistochemical results frequently requires much more precise analysis, however. This is especially true when staining intensity is quite weak and/or a significant degree of nonspecific staining occurs. To circumvent these and related problems associated with the visual evaluation of immunohistochemical staining and, therefore, to more accurately analyze staining for xenobiotic-metabolizing enzymes within rat lung, the intensities with which each of the antibodies employed stained bronchial epithelial cells, Clara cells, and type II pneumocytes were determined microfluorometrically after completion of immunofluorescence staining (Baron et al., 1978, 1981, 1986a, 1988; Taira et al., 1979; Kawabata et al., 1981, 1984; Voigt et al., 1990). These microfluorometric determinations permitted calculations of the relative extents to which each antibody bound to these three types of pulmonary cells. When immunohistochemical staining procedures are opti-

429

mized, as they were in these studies, the intensity of immunohistochemical staining is limited solely by the content of the antigen. Therefore, determinations of the relative extents to which an antibody binds to different cells are directly proportional to and, hence, serve as indices of the relative levels of the antigen within those cells (Baron et al., 1986a). Microfluorometric measurements of immunofluorescence staining intensities were obtained using a modified Leitz MPV-I microscope photometer system (Baron et al., 1978, 1981, 1986a; Voigt et al., 1990). Fluorescence emitted at 525 nm from within 7-#m 2 circular areas in the cytoplasm of bronchial epithelial cells, Clara cells, and type II pneumocytes was detected by a photomultiplier tube, the output of which was converted into absorbance units. The relative extent to which an antibody bound to a specific cell type was calculated by subtracting the mean intensity of fluorescence emitted from within cells in sections exposed to normal serum or IgG from that emitted from within corresponding cells in serial sections exposed to antiserum of immune IgG. Since absorbance decreases as the intensity of emitted fluorescence increases, absorbance values are subtracted from 1, and the results expressed in terms of 1 - absorbance (x 100) so that there is a positive, linear relationship between immunofluorescence staining intensity and the extent of antibody binding. 4.2. HISTOCHEMICALDEMONSTRATIONOF BENZO[A] PYRENEHYDROXYLASEACTIVITY Despite the fact that immunohistochemistry yields information that, in many cases, cannot be otherwise obtained, it does not provide any insight into the biologic functions and/or activities of antigens within the cells in which they are localized. However, biochemical correlates to immunohistochemical findings on cytochromes P-450 and NADPH-cytochrome P-450 reductase can be obtained through the use of histochemical techniques with which the intratissue localizations of certain cytochromes P-450-catalyzed monooxygenations can be investigated (Wattenberg and Leong, 1962; Grasso et al., 1971; Wattenberg, 1971). In our studies, sites for the in situ hydroxylation of benzo[a]pyrene in rat lung were identified using modifications (Baron et al., 1984, 1986b, 1988; Baron and Kawabata, 1987; Voigt et al., 1990) of the fluorescence histochemical procedure described by Wattenberg and Leong (1962) and Bresnick et al. (1977) for visualizing phenolic metabolites of benzo[a]pyrene. To accomplish this, unfixed cryostat sections were exposed to HPLC-purified benzo[a]pyrene and then incubated with a reduced pyridine nucleotide-generating system consisting of NADPH, NADH, glucose 6-phosphate, and glucose 6-phosphate dehydrogenase (Voigt et al., 1990). Sections were then mounted in alkaline glycerol and maintained at 0 ~ ° C during observation and photography. In alkaline media, phenolic benzo[a]pyrene metabolites emit a greenish-yellow fluorescence (excitation, 400nm; emission, 522 nm). This fluorescence was visualized in tissue sections by means of incident-light fluorescence microscopy using a Leitz Orthoplan microscope equipped with a Ploemopak ~- 2.1 fluorescence illuminator containing an H2 filter cube

430


(excitation, 390--490nm; RKP 510 beam-splitting mirror; and LP 515 suppression filter). The use of HPLC-purified benzo[a]pyrene together with a cold stage to minimize fluorescence fading as well as the diffusion of polar metabolites in the sections resulted in significant increases in both the sensitivity and accuracy of the histochemical assay for aryl hydrocarbon hydroxylase activity. The necessity for these modifications could explain why benzo[a]pyrene hydroxylase activity was originally not detected in the bronchial and bronchiolar epithelia (Wattenberg and Leong, 1962; Wattenberg, 1971).

5. L O C A L I Z A T I O N A N D DISTRIBUTION OF X E N O B I O T I C - M E T A B O L I Z I N G ENZYMES A N D BENZO[a]PYRENE H Y D R O X Y L A S E ACTIVITY W I T H I N LUNGS O F U N T R E A T E D RATS 5.1. XENOBIOTIC-METABOLIZ1NGENZYMES

The exposure of sections prepared from ethanol : acetic acid-fixed, paraffin-embedded lungs of untreated, male Sprague-Dawley rats to the polyclonal antibodies raised against rat liver NADPHcytochrome P-450 reductase, cytochromes P-450 BNF-B, PB-B, and PCN-E, epoxide hydrolase, glutathione S-transferases 1 1, 3-4, and 5 5, p-nitrophenol, 3~-hydroxysteroid and 17/~-hydroxysteroid UDP-glucuronosyltransferases, and aryl sulfotransferase IV resulted in immunohistochemical staining within the bronchial and bronchiolar epithelia and alveolar wall (Figs 2-4). Despite the fact that the antibody to rat liver cytochrome P-450 MC-B stained, albeit very weakly, bronchi, bronchioles, and alveolar walls in unfixed cryostat sections, little if any staining by this antibody was discernible in fixed, embedded lung specimens. Since we have not detected significant differences in staining produced by antibodies raised against cytochromes P-450 MC-B and BNF-B in extrapulmonary tissues, it is possible that the epitopes recognized by the anticytochrome P-450 MC-B might be much more labile in lung than in other tissues and, thus, might have been modified during fixation. Indeed, fixation-induced alterations in this protein's epitopes might explaifi why Keith et al. (1987) found that a comparable polyclonal antibody stained only a few Clara cells and type II pneumocytes in fixed, embedded specimens and in only 40% of the untreated rats studied. Differences in immunoperoxidase and immunofluorescence staining produced by the various antibodies used were not evident among different pulmonary lobes or between Sprague-Dawley and Holtzman rats. Moreover, specific staining was not apparent in smooth muscle, connective tissue, or endothelia of arteries, arterioles, veins, and venules in sections exposed to any of the antibodies. However, since endothelial cells of alveolar capillaries cannot be unequivocally identified at the light microscopic level, possible staining of these cells could not be evaluated. lmmunohistochemical staining for NADPH-cytochrome P-450 reductase was clearly visible in bronchial epithelial cells, both Clara and ciliated bronchiolar epithelial cells, and type II pneumocytes

in untreated rats, with much less staining being associated with type I pneumocytes and/or capillary endothelial cells (Fig. 2A). Visually, Clara cells appeared to be much more intensely stained by the antireductase than were other cells. The results of microfluorometric analyses of immunofluorescence staining intensities presented in Table 1 confirmed this observation and revealed that, while the antireductase bound equally to bronchial epithelial cells and type II pneumocytes, approximately 90% more antibody bound to Clara cells. These data therefore demonstrate that bronchial epithelial cells and type II pneumocytes in untreated rats contain similar levels of NADPH-cytochrome P-450 reductase whereas Clara cells contain almost twice as much enzyme. Interestingly, a comparable difference in NADPH-cytochrome P-450 reductase activity has been found between rabbit Clara cells and type II pneumocytes (Devereux and Fouts, 1981; Domin et al., 1986). Consistent with the fact that pulmonary microsomes of untreated rats contain only an extremely low amount of cytochrome P-450 BNF-B (Guengerich et al., 1982b; Keith et al., 1987; Guengerich, 1990), the antibody to this cytochrome P-450 produced very weak staining within lungs of untreated rats. Nonetheless, staining for cytochrome P-450 BNF-B was detectable in bronchial epithelial cells, Clara as well as ciliated bronchiolar epithelial cells, and alveolar wall cells (Fig. 2B), Furthermore, the presence of the hemeprotein was verified by microfluorometric analysis of anticytochrome P-450 BNF-B binding, the results of which disclosed that this antibody bound to a similar, extremely low, but statistically significant (p < 0.05), extent to bronchial epithelial cells, Clara ceils, and type II pneumocytes (Table 1). Thus, unlike NADPH-cytochrome P-450 reductase, cytochrome P-450 BNF-B is distributed quite uniformly among these three cell types in untreated rats. The level of pulmonary microsomal cytochrome P-450 PB-B in untreated rats is much greater than that of cytochrome P-450 BNF-B (Guengerich et al., 1982b; Keith et al., 1987; Guengerich, 1990). In agreement with this, bronchial epithelial cells, Clara and ciliated bronchiolar cells, and alveolar wall cells of untreated rats were much more intensely stained by the antibody to cytochrome P-450 PB-B (Fig. 2C) than by the anticytochrome P-450 BNF-B. Additionally, and in marked contrast to findings on cytochrome P-450 BNF-B, the microfluorometric data in Table 1 show that while the anticytochrome P-450 PB-B bound equally to bronchial epithelial cells and type II pneumocytes, approximately 50% more antibody bound to Clara cells. Identical findings were obtained when immunoperoxidase staining for cytochrome P-450 PB-B was evaluated by means of microdensitometry using a Bioquant Image Analysis System with a Video Counting and Microdensitometry (VCMTE) program (R&M Biometrics, Inc., Nashville, TN). The use of image analysis, moreover, permitted determinations of the intensities with which the anticytochrome P-450 PB-B stained ciliated bronchiolar cells and type I pneumocytes/capillary endothelial cells. Using image analysis, the extents to which the antibody bound to cells of untreated rats

Enzymes within lung

431

ID,'-.

B b,

C

D

50 pm FIG. 2. Immunoperoxidase staining produced by antibodies raised against NADPH-cytochrome P-450 reductase (A) and cytochromes P-450 BNF-B (B), PB-B (C), and PCN-E (D) within bronchial (left column) and bronchiolar (middle column) epithelia and alveolar walls (right column) in lungs of untreated rats. Arrowheads point to Clara cells and type II pneumocytes in bronchioles and alveolar walls, respectively.

were determined to be (data are given as mean integrated absorbance x 100): bronchial epithelial cells, 29.5; Clara cells, 42.7; ciliated bronchiolar cells, 20.7; type II pneumocytes, 30.2; and type ! pneumocytes/capillary endothelial cells, 23.2. Thus, in addition to establishing that cytochrome P-450 PB-B is present in ciliated cells and in cells other than type II pneumocytes that are associated with the alveolar wall, these findings conclusively demonstrate that there are marked differences in the patterns of distribution of different forms of cytochrome P-450 within rat lung. Furthermore, these results are con-

sistent with the finding that rat Clara cells exhibit greater cytochrome P-450 PB-B-catalyzed activities than do type II pneumocytes (Jones et al., 1983; Belinsky et al., 1987) as well as with the fact that rabbit Clara cells contain 2- to 3-fold more cytochrome P-450 form 2, the ortholog of rat cytochrome P-450 PB-B, than do type II pneumocytes (Domin et al., 1986). Of greater importance with respect to chemically-induced pulmonary damage, the finding that Clara cells in untreated rats contain the greatest amounts of both NADPH-cytochrome P-450 reductase and cytochrome P-450 PB-B, the form of

432


A

A

C gE

"'6

iv ~

D FIG. 3. Immunoperoxidase staining produced by antibodies raised against epoxide hydrolase (A) and glutathione S-transferases 1-I (B), 3~t (C), and 5-5 (D) within bronchial (left column) and bronchiolar (middle column) epithelia and alveolar walls (right column) in lungs of untreated rats. Arrowheads point to Clara cells and type II pneumocytes in bronchioles and alveolar walls, respectively. cytochrome P-450 that appears to be primarily responsible for catalyzing the activation of 4-ipomeanol (Guengerich, 1977a,b), is also clearly consistent with and may explain why Clara cells are so readily damaged following in vivo exposure to 4-ipomeanol. Bronchial epithelial cells, Clara and ciliated bronchiolar cells, type II pneumocytes, and other alveolar wall cells in untreated rats were also stained by the antibody raised to rat liver cytochrome P-450 PCN-E (Fig. 2D). In contrast to the results obtained using antibodies to cytochrome P-450 PB-B and NADPH-

cytochrome P-450 reductase, however, the anticytochrome P-450 PCN-E bound comparably to bronchial epithelial cells, Clara cells, and type II pneumocytes (Table 1). Thus, in untreated rats, cytochrome P-450 PCN-E, like cytochrome P-450 BNF-B, is distributed uniformly among these three cell types. These findings therefore indicate that, while cytochrome P-450 PB-B-catalyzed monooxygenations occur to the greatest extent in Clara cells, those mediated by both cytochromes P-450 PCN-E and BNF-B in untreated rats would be likely

Enzymes within lung

433

FIG. 4. Immunoperoxidase staining produced by antibodies raised against p-nitrophenol (A), 3ct-hydroxysteroid (B), and 17fl-hydroxysteroid (C) UDP-glucuronsyltransferases and aryl sulfotranferase IV (D) within bronchial (left column) and bronchiolar (middle column) epithelia and alveolar walls (right column ) m lungs of untreated rats.-Arrowheads point to Clara cells and type II pneumocytes in bronchioles and alveolar walls, respectively. to occur to equivalent extents in bronchial epithelial cells, Clara cells, and type II pneumocytes. In sections of lungs of untreated rats exposed to the antibody raised against rat liver epoxide hydrolase, staining was evident in bronchial epithelial cells, Clara and ciliated bronchiolar cells, and alveolar wall cells (Fig. 3A). Analogous to immunohistochemical findings on NADPH-cytochrome P-450 reductase and cytochrome P-450 PB-B, the antibody to epoxide hydrolase appeared to stain Clara cells most intensely, and microfluorometric analyses revealed

that, although the antiepoxide hydrolase bound equally to bronchial epithelial cells and type II pneumocytes, approximately 2-fold more antibody bound to Clara cells (Table I). Thus, in addition to containing the highest levels of cytochrome P-450 PB-B and NADPH-cytochrome P-450 reductase, Clara cells of untreated rats contain the greatest amount of epoxide hydrolase. This finding is clearly consistent with the fact that Clara cells of rats (Jones et al., 1983) and rabbits (Devereux et al., 1985) display much greater epoxide hydrolase activity than

434

J. Bakoy and J. M. VO1OT

TABtE 1. Binding of Antibodies Raised Against Rat Liver Xenobiotic-Metabolizing Pulmonary Cells of Untreated Rats

Enzymes to

Antibody binding to Antibody raised against NADPH-cytochrome P-450 reductase Cytochrome P-450 BNF-B Cytochrome P-450 PB-B Cytochrome P-450 PCN-E Epoxide hydrolase Glutathione S-transferase 1-1 Olutathione S-transferase 3~, Glutathione S-transferase 5 5 p-Nitrophenol UDPglucuronosyltransferase 3:~-Hydroxysteroid UDPglucuronosyltransferase 17,8-Hydroxysteroid UDPglucuronosyltransferase Aryl sulfotransferase IV

Bronchial epithelial cells 58.9-+2.3 9.6 ,+ 2.0 37.0+2.4 23.4 + 1.8 24.0 ,+ 1.7 42.3 4- 2.2 68.8± 1.8" 56.0 ,+ 1.4

Clara cells

Type II pneumocytes

109.1 _+2.2* 58.4_+2.3 5.8 + 1.3 4.0 + 0.8 5 5 . 1 _ + 2 . 1 " 38.6,+1.5 26.6 + 1.5 21.9 ,+ 1.2 69.9 ,+ 3.2* 22.6 _+ 1.3 55.0 4 1.9" 25.2 + 1.3t 55.1 + 1.8 21.5_+ 1.3t 61.3 ,+ 2.1 39.4 ,+ 1.55"

59.5±3.5

57.3,+2. l

51.4,+3.3

22.7 _+2,3

26.0 + 3.7

20.2 + 2.8

58.2 -+ 4.5 53.3 _+ 1.4"

57.8 _+4.7 43.5 ± 1.4

41.3 _+3.6t 46.6 + 3.6

Antibody binding values, expressed in terms of I-absorbance (x 100), represent the mean + standard error of at least 20 separate microfluorometric determinations made after completion of immunofluorescence staining using 5 sets of serial sections obtained from each of at least 5 untreated rats. *Significantlygreater (P < 0.05) than the extent of binding to the other cell types. tSignificantly lesser (P < 0.05) than the extent of binding to the other cell types. do type II pneumocytes. It was nonetheless somewhat surprising, however, inasmuch as the generation of an epoxide from 4-ipomeanol has been suggested (Boyd et al., 1978a) to be at least partially responsible for damaging Clara cells. Thus, in view of these immunohistochemical and biochemical findings, one could certainly question whether the epoxidation of 4ipomeanol is, in fact, a major route for the bioactivation of the furan in Clara cells. The antibodies raised against rat liver glutathione S-transferases 1-1 (Fig. 3B), 3-4 (Fig. 3C), and 5-5 (Fig. 3D) each stained bronchial epithelial cells, Clara as well as ciliated bronchiolar cells, and alveolar wall cells in untreated rats. Pronounced differences in the distribution of staining produced by the three antibodies were readily apparent between the bronchus and bronchiole, however. Microfluorometric findings disclosed that, although each antibody bound to the least extent to type II pneumocytes, the antibody to glutathione S-transferase I-1 bound most greatly to Clara cells, whereas the antitransferase 3-4 bound to a significantly greater extent to bronchial epithelial cells, while the antibody to transferase 5-5 bound comparably to Clara and bronchial epithelial cells (Table 1). Thus, these findings demonstrate that, as was observed for different forms of cytochrome P-450, different glutathione S-transferases are distributed in significantly different manners within rat lung. These results further indicate that the glutathione S-transferase-catalyzed conjugations of electrophilically-reactive metabolites with reduced glutathione would occur to the least degree in type II pneumocytes and are, thus, consistent with the finding that benzo[a]pyrene-4,5-oxide is much more extensively conjugated with reduced glutathione in rat Clara cells than in type II pneumocytes (Jones et al., 1983). Of course, since reduced glutathione can be conjugated with electrophiles in the absence of an enzyme, it must be stressed that the extent of glu-

tathione conjugation within a particular cell would obviously be greatly dependent on the intracellular level of this nucleophile. At the present time, however, virtually nothing is known regarding the in situ distribution of reduced glutathione within lung. Staining by the antibodies to rat liver p-nitrophenol (Fig. 4A), 3~-hydroxysteroid (Fig. 4B), and 17/~-hydroxysteroid (Fig. 4C) UDP-glucuronosyltransferases was readily detected in bronchial epithelial cells, Clara and ciliated bronchiolar epithelial cells, and alveolar wall cells of untreated rats, although the anti-3:~-hydroxysteroid UDP-glucuronosyltransferase stained these cells much less intensely than did the other two antibodies. Visually, the antibodies to p-nitrophenol and 3c~-hydroxysteroid UDP-glucuronosyltransferases each appeared to stain bronchial epithelial cells, Clara cells, and type II pneumocytes comparably, but while the anti-17/~hydroxysteroid UDP-glucuronosyltransferase also appeared to stain bronchial epithelial and Clara cells similarly, it stained type II pneumocytes somewhat less intensely. Microfluorometric determinations (Table 1) revealed that the antibodies to p-nitrophenol and 3~-hydroxysteroid UDP-glucuronosyltransferases each bound comparably to the three cell types and that while the anti-17/~-hydroxysteroid UDP-glucuronosyltransferase also bound equally to bronchial epithelial and Clara cells, approximately 30% less bound to type II pneumocytes. These findings demonstrate that antigens related to rat liver p-nitrophenol, 3~-hydroxysteroid, and 17/~-hydroxysteroid UDP-glucuronosyltransferases are present in rat lung and, further, that they are distributed much more uniformly among bronchial epithelial cells, Clara cells, and type II pneumocytes than are NADPH-cytochrome P-450 reductase, cytochrome P-450 PB-B, epoxide hydrolase, and glutathione Stransferases 1-1, 3-4, and 5 5. While these results suggest that xenobiotics would be glucuronidated to

Enzymes within lung similar extents by these cells, it is quite likely that rat lung contains other UDP-glucuronosyltransferases, and, further, that they may not be uniformly distributed among different pulmonary cells. This could certainly explain why rat Clara cells are much more active than type II pneumocytes in glucuronidating 4-methylumbelliferone (Jones et al., 1983), a reaction catalyzed most efficiently in rat liver by p-nitrophenol UDP-glucuronosyltransferase (Tephly et al., 1988a). Bronchial epithelial cells, Clara and ciliated bronchiolar epithelial cells, and type II pneumocytes as well as other cells associated with the alveolar wall in untreated rats were also stained by the antibody raised against rat liver aryl sulfotransferase IV (Fig. 4D). In this case, staining appeared to be somewhat more intense in the bronchus, and the data in Table 1 show that 15-25% more antiaryl sulfotransferase IV bound to bronchial epithelial cells than to type II pneumocytes and Clara cells. These results therefore demonstrate that rat lung contains an antigen related to rat liver aryl sulfotransferase IV and that it is present at the greatest level in bronchial epithelial cells. The question of whether rat lung also contains other aryl sulfotransferases remains to be addressed.

435

5.2. BENZO[A]PYRENEHYDROXYLASEACTIVITY When unfixed cryostat sections prepared from lungs of untreated rats were incubated with benzo[a]pyrene and reduced pyridine nucleotides, phenolic metabolites emitting a greenish-yellow fluorescence were generated from the polycyclic aromatic hydrocarbon in bronchial epithelial cells, Clara as well as ciliated bronchiolar cells, and type II pneumocytes and other ceils associated with the alveolar wall (Fig. 5A). The appearance of connective tissue in the region of the basement membrane in both the bronchus and bronchiole resulted from nonspecific autofluorescence and was also seen in sections that were incubated in the absence of benzo[a]pyrene and reduced pyridine nucleotides. Under the microscope, the brownish-yellow nonspecific autofluorescence was readily distinguished from the greenish-yellow fluorescence emitted by phenolic benzo[a]pyrene metabolites. Although some variability was evident, benzo[a]pyrene appeared to be hydroxylated to comparable extents in bronchial and bronchiolar epithelial cells and type II pneumocytes, with considerably less activity being seen in other alveolar wall cells. Little if any activity was discernible in the absence of reduced pyridine nucleotides. Furthermore, when

FIG. 5. Benzo[a]pyrene hydroxylase activity within bronchial (left column) and bronchiolar (middle column) epithelia and alveolar walls (right column) in lungs of untreated (A), 3-methylcholanthrene pretreated (B), and Aroclor 1254 pretreated (C) rats. The greenish-yellow fluorescence resulting from the generation of benzo[a]pyrene phenols was visualized in unfixed cryostat sections incubated with benzo[a]pyrene, NADPH, NADH, glucose 6-phosphate, and glucose 6-phosphate dehydrogenase (Voigt et al., 1990). The bright (brownish-yellow) fluorescence in the basement membranes of the bronchus and bronchiole is due to nonspecific autofluorescence. Arrowheads in bronchi denote the limits of the epithelium; arrowheads and asterisks in bronchioles indicate Clara cells and ciliated bronchiolar epithelial cells, respectively; and arrowheads in alveolar areas point to type II pneumocytes.

436


sections were preincubated with antibodies to NADPH-cytochrome P-450 reductase and/or the different forms of cytochrome P-450 studied, dramatic inhibition of monooxygenase activity occurred throughout the lung. These histochemical results thus correlate quite well with immunohistochemical findings on the intrapulmonary localizations of cytochromes P-450 and NADPHcytochrome P-450 reductase. Indeed, the fact that benzo[a]pyrene appeared to be monooxygenated to similar extents by bronchial and bronchiolar epithelial cells and type II pneumocytes is clearly consistent with findings on the intrapulmonary distribution of cytochrome P-450 BNF-B, the form of cytochrome P-450 which, in addition to cytochrome P-450 MC-B, is most active in catalyzing aryl hydrocarbon hydroxylase activity (Guengerich et al., 1982a).

6. EFFECTS OF 3 - M E T H Y L C H O L A N T H R E N E AND AROCLOR 1254 ON XENOBIOTICMETABOLIZING ENZYMES AND BENZO[a]PYRENE HYDROXYLASE ACTIVITY W I T H I N RAT LUNG 6.1. XENOBIOTIC-METABOLIZINGENZYMES Certain forms of cytochrome P-450 and many other xenobiotic-metabolizing enzymes can be induced in a variety of tissues, including lung, by polycyclic aromatic hydrocarbons, polychlorinated biphenyls, and a multitude of other xenobiotics (Conney, 1967, 1982; Guengerich, 1977b, 1978, 1979, 1987, 1990; Boyd, 1980a; Lu and West, 1980; Guengerich et al., 1982a,b; Devereux et al., 1989). In order to determine where within lung xenobiotic metabolism can be affected by inducers, immunohistochemical studies were conducted using tissue obtained from rats pretreated with either 3-methylcholanthrene, administered i.p. in corn oil at a dose of 40 mg/kg/day for 5 consecutive days, or Aroclor 1254, administered i.p. in corn oil at a dose of 500mg/kg once 7 days prior to animal sacrifice. Differences in the staining of lung cells by the antibodies to rat liver NADPH-cytochrome P-450 reductase cytochromes P-450 PB-B and PCN-E, epoxide hydrolase, glutathione S-transferases, UDPglucuronosyltransferases, and aryl sulfotransferase IV were not apparent among untreated rats and rats treated with corn oil, 3-methylcholanthrene, or Aroclor 1254. This finding is, in general, consistent with biochemical results and the limited number of immunohistochemical observations reported by other laboratories that were discussed previously. On the other hand, Jones et aL 0983) found that fl-naphthoflavone enhanced the glucuronidation of 4-methylumbelliferone by rat type II pneumocytes, while Bock et al. (1980) reported that Aroclor 1254 induced the pulmonary glucuronidation of 1-naphthol, a reaction catalyzed in rat liver by both p-nitrophenol and 17fl-hydroxysteroid UDP-glucuronosyltransferases (Tephly el al., 1988a). With respect to the explanation for this discrepancy, we have found that 3-methylcholanthrene and several other xenobiotics are capable of greatly inducing p-nitrophenol and 17flhydroxysteroid UDP-glucuronosyltransferase activi-

ties in rat liver, but that increases in these activities are accompanied by only very modest enhancements in staining of hepatocytes for the two enzymes. This suggests that increased enzyme expression is not the sole factor responsible for the induction of UDPglucuronosyltransferase activities. In marked contrast to findings on other xenobioticmetabolizing enzymes, both 3-methylcholanthrene and Aroclor 1254 dramatically enhanced staining by the antibodies raised against rat hepatic microsomal cytochromes P-450 BNF-B and MC-B within rat lung. This finding is consistent with reports on the induction of pulmonary cytochrome P-450 forms that correspond to rat liver cytochrome P-450 BNF/MCB by polycyclic aromatic hydrocarbons and polychlorinated biphenyls (Dees et al., 1982; Jones et al., 1982, 1983; Domin et al., 1986; Foster et al., 1986; Anderson et al., 1987; Keith et al., 1987; Devereux et al., 1989; Forkert et al., 1989; Guengerich, 1990). On the other hand, a striking difference was clearly apparent between the effects of 3-methylcholanthrene and Aroclor 1254 on staining produced by the antibodies to cytochromes P-450 BNF-B and MC-B. Since 3-methylcholanthrene and Aroclor 1254 affected staining by the two anticytochromes P-450 in identical manners, and since cytochromes P-450 BNF-B and MC-B appear to be identical (Guengerich, 1977b, 1978; Guengerich et al., 1982a), only results obtained using the antibody to cytochrome P-450 MC-B will be considered here. Despite the fact that the antibody to cytochrome P-450 MC-B produced only barely detectable staining in lungs of untreated rats (Fig. 6A), it clearly stained Clara cells and alveolar wall cells, especially type II pneumocytes, in 3-methylcholanthrene pretreated rats (Fig. 6B). In contrast, 3-methylcholanthrene did not appear to alter staining of either ciliated bronchiolar or bronchial epithelial cells by the anticytochrome P-450 MC-B. Results of microfluorometric analyses of immunofluorescence staining intensities presented in Table 2 confirmed that 3methylcholanthrene markedly induced cytochrome P-450 BNF/MC-B in Clara cells and type II pneumocytes and that it failed to affect the enzyme in the bronchial epithelium. Interestingly, 3-methylcholanthrene induced the cytochrome P-450 to similar extents in Clara cells and type II pneumocytes as evidenced by the fact that these cells contained comparable levels of the hemeprotein in 3-methylcholanthrene pretreated rats. Aroclor 1254 produced an even more striking increase in the staining of Clara cells, type II pneumocytes, and other alveolar wall cells by the antibody to cytochrome P-450 MC-B, but like 3-methylcholanthrene, did not appear to alter staining of ciliated bronchiolar epithelial cells (Fig. 6C). In marked contrast to 3-methylcholanthrene, however, Aroclor 1254 also greatly enhanced staining of the bronchial epithelium, although this was detected in only 28% of the cells. Unfortunately, at the light microscopic level and in 4-/zm-thick sections prepared from fixed, paraffin-embedded specimens, the bronchial cells that were stained by the anticytochrome P-450 MC-B could not be identified. Microfluorometric analyses revealed that, seven days after the administration of a single i.p. dose of Aroclor 1254, essentially identical

Enzymes within lung

437

FIG. 6. Immunoperoxidase staining produced the antibody raised against cytochrome P-450 MC-B within bronchial (left column) and bronchiolar (middle column) epithelia and alveolar walls (right column) in lungs of untreated (A), 3-methylcholanthrene pretreated (B), and Aroclor 1254 pretreated (C) rats.

TABLE 2. Effects of 3-Methylcholanthrene and Aroclor 1254 Pretreatments on Binding of the Antibody Raised Against Rat Liver Cytochrome P-450 MC-B to Rat Pulmonary Cells Anticytochrome P-450 MC-B binding to Pretreatment None Corn oil* 3-Methylcholanthrenet Corn oil:~ Aroclor 125~

Bronchial epithelial cells 0.8 0.9 0.8 0.3 34.7

___1.5 ___1.9 ___1.7 + 1.8 + 2.8 II¶

Clara cells

Type II pneumocytes

0.5 __+1.3 0.5 +_ 1.2 24.1 + 2.211 1.7 ___1.5 39.8 + 3.011

1.1 __+1.5 1.1 -t- 1.7 27..4 + 1.811 1.2 ___1.4 34.0 ___2.311

Antibody binding values are expressed in terms of 1 - absorbance ( x 100). *Vehicle for 3-methylcholanthrene, administered i.p. once a day for 5 consecutive days. tAdministered at a dose of 40 mg/kg/day, i.p. in corn oil, for 5 consecutive days. :~Vehicle for Aroclor 1254, administered once i.p. §Administered once at a dose of 500 mg/kg, i.p. in corn oil, and animals were sacrificed 7 days later. 11Significantly greater (P

Induction by cigarette smoke of aryl hydrocarbon hydroxylase activity in the rat kidney and lung.

Aryl hydrocarbon hydroxylase activity in the first-trimester human placenta: induction by carcinogens and chemoprotectors.

Placental aryl hydrocarbon hydroxylase activity and placental calcifications.

Human skin aryl hydrocarbon hydroxylase. Induction by coal tar.

Induction of aryl hydrocarbon hydroxylase in rodent lung by cigarette smoke: a potential short-term bioassay.

Questionable relation of aryl hydrocarbon hydroxylase to lung-cancer risk.

Activity of aryl hydrocarbon hydroxylase in psoriatic skin.

The activity of aryl hydrocarbon hydroxylase in adult human skin.

Effects of some epoxides on aryl hydrocarbon hydroxylase activity.

Induction of aryl hydrocarbon hydroxylase by 3-methylcholanthrene in liver, lung and kidney of gonadectomized and sham-operated Wistar rats.

Genetics of aryl hydrocarbon hydroxylase induction in mice: response of the lung to cigarette smoke and 3-methylcholanthrene.

Analysis of the aryl hydrocarbon hydroxylase assay.

Comparison of aryl hydrocarbon hydroxylase induction in cultured blood lymphocytes and pulmonary macrophages.

Aryl hydrocarbon hydroxylase in asbestos workers.

Aryl hydrocarbon hydroxylase inducibility and carcinoma of oral cavity.

Letter: Aryl hydrocarbon hydroxylase inducibility and laryngeal carcinomas.

Relationship of aryl hydrocarbon hydroxylase activity to benzo(a)pyrene-metabolizing activity of cells in culture.

Effect of aging and other factors on monocyte aryl hydrocarbon hydroxylase activity.

On the occurrence of cytochrome P-450 and aryl hydrocarbon hydroxylase activity in rat brain.

Strain differences in aryl hydrocarbon hydroxylase induction by 3-methylcholanthrene in rabbits.

Selections for and against cells possessing cytochrome P450IA1-dependent aryl hydrocarbon hydroxylase activity.

Aryl hydrocarbon hydroxylase in cultured lymphocytes of twins.

Effects of recombinant human interferon alpha on aryl hydrocarbon hydroxylase activity in cultured human peripheral lymphocytes.

Monocyte aryl hydrocarbon hydroxylase (AHH) activity mimics Kupffer cell and hepatocyte AHH activity in an animal model of liver disease.