classical inducers phenobarbital and 3-methylcholanthrene and by the newly characterized inducers trans-stilbene oxide and 2-acetylaminofluorene. A number ...
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Bock, K. W., Bock-Hennig, B. S., Lilienblum, W. & Volp, R. (1981) Chem.-Biol. Interact. 36, 167-177 Bock, K. W., Lilienblum, W., Pfeil, H. & Eriksson, L. C. (1982) Cancer Res. 42, 3747-3752 Bock, K. W., Burchell, B., Dutton, G. J., Hanninen, O., Mulder, G. J., Owens, I. S., Siest, G. & Tephly, T. R. (1983) Biochem. Pharmacol. 32, 953-955 Bock, K. W., Bock-Hennig, B. S., Fischer, G., Lilienblum, W. & Ullrich, D. (1984) in Biochemical Basis of Chemical Carcinogenesis: Workshop Conference, Hoechst, 13th. in the press Burchell, B. (1982) Biochem. J . 201, 653456 Conney, A. H., Miller, E. C. & Miller, J. (1956) Cancer Res. 16, 450-459 Dutton, G. J. (1980) Glucuronidation of Drugs and Other Compounds, CRC Press., Boca Raton, FL Dutton, G. J. & Leakey, J. E. A. (1981) Progr. Drug Res. 25, 189273 Eriksson, L. C., Astrom, A., DePierre, J. W. &Bock, K. W. (1981) Biochem. SOC.Trans. 9, 271P
Fischer, G., Ullrich, D., Katz, N., Bock, K. W. & Schauer, A. (1983) Virchows Arch. 42, 193-200 Inscoe, J. K. & Axelrod, J. (1960) J . Pharmaco/. Exp. Ther. 129, 128-1 3 1 Lilienblum, W., Walli, A. K. & Bock, K. W. (1982) Biochem. Pharmacol. 31, 907-913 Lueders, K. K., Dyer, H. M., Thompson, E.B. & Kuff, E. L. (1970) Cancer Res. 30,274-279 Matern, H., Matern, S . & Gerok, W. (1982) J. Eiol. Chem. 257, 7422-7429 Owens, I. S. (1977) J. Biol. Chem. 252, 2827-2833 Pfeil, H. & Bock, K. W. (1983) Eur. J. Biochem. 131, 619423 Quinn, G. P., Axelrod, J. & Brodie, B. B. (1958) Biochem. Pharmacol. 1, 152-159 Tsuda, H., Lee, G. & Farber, E. (1980) Cancer Res. 40,1157-1164 Ullrich, D. & Bock, K. W. (1983) Bwchem. Pharmacol. in the press Watkins, J. B., Gregus, Z., Thompson, T. N. & Klaassen, C. D. (1982) Toxicol. Appl. Pharmacol. 64, 4 3 9 4 6 Wishart, G. J. (1978~)Biochem. J . 174, 671472 Wishart, G. J. (19786) Biochem. J . 174, 4855489
Induction of xenobiotic-metabolizing enzymes by trans-stilbene oxide and 2-acetylaminofluorene: observations on enzyme induction by drugs JOSEPH W. DEPIERRE, JANERIC SEIDEGARD, RALF MORGENSTERN, L E N N A R T BALK, JOHAN MEIJER and ANDERS ASTROM Department of Biochemistry, Arrhenius Laboratory, University of Stockholm, S-106 91 Stockholm, Sweden During the past few years our laboratory has been interested in the induction of xenobiotic-metabolizing enzymes by, among other substances, trans-stilbene oxide and 2-acetylaminofluorene (Seidegird et al., 1979; Guthenberg et al., 1980; Lind et al., 1980; Suzuki et al., 1980; Astrom & DePierre, 1981; Carlberg et al., 1981;DePierre et al., 1981 ; Seidegird et al., 1981 ; Astrom & DePierre, 1982; Meijer et al., 1982; Seidegdrd & DePierre, 1982a,b; Astrom et al., 1983; Meijer & DePierre, 1983). These studies have led to certain insights about enzyme induction by drugs which we would like to share with you. In particular, it is time to dispel certain myths about this process which have been around for a long time. Phenobarbital and 3-methylcholanthrene are representative inducers of drug-metabolizing enzymes . . . or are they? Table 1 presents the patterns of induction by the so-called classical inducers phenobarbital and 3-methylcholanthrene and by the newly characterized inducers trans-stilbene oxide and 2-acetylaminofluorene. A number of characteristics of these patterns should be noted. In the first place transstilbene oxide has been found to induce the same isoenzyme of cytochrome P-450 as is induced by phenobarbital, whereas 2-acetylaminofluorene induces a form of this cytochrome which is clearly distinct from those induced both by phenobarbital and 3-methylcholanthrene. This seems to be a rather general phenomenon: many xenobiotics induce the same isozymes of cytochrome P-450 as do phenobarbital or 3-methylcholanthrene, whereas many other xenobiotics induce distinct isoenzymes. In addition, many xenobiotics induce more than one isoenzyme of cytochrome P-450. An important task for the immediate future will be to compare the distinct isoenzymes induced by different xenobiotics to determine which of them are the same and thereby classify them into different groups. Table 1 also shows that 3-methylcholanthrene induces the phase-I P-450 system to a much greater extent than the phase-I1 enzymes microsomal epoxide hydrolase and
cytosolic glutathione transferases; phenobarbital induces both phase-I and -11 reactions to approximately the same extent; whereas both trans-stilbene oxide and 2-acetylaminofluorene induce microsomal epoxide hydrolase and cytosolic glutathione transferases to a much greater extent than they induce the total amount of cytochrome P-450.Of course, many more detailed patterns of induction will be discernible when the induction of different isoenzymes and of other enzymes such as UDP-glucuronosyltransferase, sulphotransferase and DT-diaphorase (NADCP)H :oxidoreductase) are also taken into account. trans-Stilbene oxide, for instance, has been found to induce all the major isoenzymes of cytosolic glutathione transferase in rat liver (Guthenberg et al., 1980) as well as to be a powerful inducer of cytosolic DT-diaphorase (Lind et al., 1980) (see also below). It is, of course, of great importance to characterize the pattern of induction of drug-metabolizing enzymes by different xenobiotics in such detail. Since reactive intermediates produced by the cytochrome P-450 system are often the direct cause of the toxic and genotoxic effects of different xenobiotics, the differential induction of phase-I and -11 activities may have a profound influence on the toxicity and genotoxicity of the inducer itself and of other xenobiotics as well (synergistic effects). In addition, the metabolism of endogenous compounds may be strongly affected by such differential induction (Meijer & DePierre, 1983). There are also distinct differences in the induction of drug-metabolizing enzymes in extrahepatic tissues by phenobarbital, 3-methylcholanthrene and trans-stilbene oxide (DePierre et al., 1984). For instance, 3-methylcholanthrene induces cytosolic glutathione transferase activity significantly only in the liver, and phenobarbital induces this activity in the intestine as well, whereas trans-stilbene oxide induces cytosolic glutathione transferase activity in the liver, kidney and adrenal gland. Such differences may again have a great influence on the relative susceptibility of different organs to toxic and genotoxic effects. In view of these and other considerations, it does not seem fruitful to continue to consider phenobarbital and 3methylcholanthrene as representative inducers with which other inducers should be compared. It is also apparent from Table 1 that neither phenobarbital, 3-methylcholanthrene, trans-stilbene oxide nor 21984
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Table 1. Pattern of induction of hepatic drug-metabolizing enzymes by four different xenobiotics All of these experiments were performed with male Sprague-Dawley rats, except the attempted induction of cytosolic epoxide hydrolase, which was performed with mice. For further details, see DePierre et al. (1981). Abbreviation: PB, phenobarbital. Inducer A
r
Parameter Specific microsomal content of cytochrome P-450 Cytwhrome P-450-catalysed reactions
PB Induced
Isoenzyme of cytochrome P-450 induced Microsomal epoxide hydrolase
PB-type
Cytosolic epoxide hydrolase Cytosolic glutathione transferase activity Microsomal glutathione transferase
No change Induced 100-200% No change
300400%
Many induced 300-400%
Induced 100-200%
3-Methylcholanthrene Induced 3OWOO%
Selective 10-20-fold induction of benzo[a]pyrene mono-oxygenase Cytochrome P-448 No change or small (30%) induction No change Induced 40-60% No change
acetylaminofluorene induces cytosolic epoxide hydrolase activity in mouse liver or microsomal glutathione transferase in rat liver. (Attempts to achieve induction of cytosolic epoxide hydrolase are generally performed with mouse liver, since the rat demonstrates a much lower hepatic activity of this enzyme than do other rodents. It would be interesting to know why.) We have tried to achieve induction of these two activities with a large number of different xenobiotics to date, and only succeeded in observing a slight increase in mouse liver microsomal glutathione transferase after treatment with antioxidants (Morgenstern & Dock, 1982) and an approx. 2-fold induction of cytosolic epoxide hydrolase activity in the same tissue by the plasticizer and peroxisome-proliferating substance phthalate. Hammock & Ota (1983) have also observed induction of cytosolic epoxide hydrolase by drugs that cause peroxisome proliferation. These observations raise the question as to whether the cytosolic epoxide hydrolase and microsomal glutathione transferase are intimately involved in xenobiotic metabolism, or whether they have other endogenous functions.
The process of induction of drug-metabolizing enzymes by xenobiotics is substrate induction . . . or is it? It has long been loosely assumed that the induction of drug-metabolizing enzymes is substrate induction, i.e. that xenobiotics induce the enzymes by which they are metabolized. This assumption seems reasonable in light of the fact that substrate induction is a common phenomenon in bacteria and that such induction would serve a useful purpose, allowing the animal to detoxify and excrete the xenobiotic to which it is chronically exposed more rapidly. However, a number of observations have now been made, both in our laboratory and in others, that lead us to question the general correctness of this assumption. For instance, we have found that 2-acetylaminofluorene is a powerful inducer of microsomal epoxide hydrolase (Table l), but there is no evidence to date that epoxide metabolites that could serve as substrate for this enzyme can be produced from 2-acetylaminofluorene. In addition, benzil and benzoin, metabolites of trans-stilbene oxide in mammalian cells, are also potent inducers of microsomal epoxide hydrolase and cytosolic glutathione transferase activity in VOl. 12
\
trans-Stilbene oxide Induced 120%
Many induced 100-200%
2-Acetylaminofluorene Induced 45-50% Selective 4-5-fold induction of 2-acetylaminofluorene metabolism
PB-type
New form
Induced 620%
Induced 660%
No change Induced 200-300% No change
No change Induced 100- 1 50% No change
Table 2. Effect of treating rats with trans-stilbene oxide on hepatic enzymes only indirectly involved in drug metabolism (see Carlberg et al., 1981 ;Seidegird & DePierre. 1982a.b) Enzyme induced ‘Rationale’ Glucose-6-phosphate Rate-limiting step in the production of dehydrogenase cytoplasmic NADPH, which is utilized by NADPH-cytochrome P-450 reductase UDP-glucose Final step in the synthesis of UDPdehydrogenase glucuronic acid, cofactor for the UDP-glucuronosyltransferases
Glutathione reductase Maintains glutathione in the reduced state necessary to achieve both enzymic and non-enzymic conjugation with reactive intermediates of xenobiotic metabolism
rat liver (Seidegird et al., 1981), but there is no reason to believe that either of these substances are substrates for either of these enzymes. Finally, T C D D (2,3,7,8-tetrachlorodibenzo-p-dioxin) is a powerful inducer of cytochrome P-450,but does not seem to be metabolized by this system. Thus the question as to whether induction of drugmetabolizing enzymes is substrate induction remains, especially for the phase-I1 enzymes. Of course, it is not impossible that phase-I and -11 enzymes are induced by different compounds even when a single xenobiotic is administered, i.e. by the xenobiotic itself and by a metabolite, or by two different metabolites. There might also be some type of ‘coupling’ between the genetic control of the phase-I and -11 systems.
The induction of drug-metabolizing enzymes is spec@ . . . or is it? Many investigators still believe that the induction of drug-metabolizing enzymes is relatively specific, i.e. other enzyme systems are not affected by this process. Recent findings indicate that this is clearly not the case. As shown in Table 2, trans-stilbene oxide induces a number of enzymes only indirectly involved in the process of drug
BIOCHEMICAL SOCIETY TRANSACTIONS
60 metabolism. In addition, various inducers affect the liver weight, the number of hepatocytes, the cellular content of endoplasmic reticulum, and the phospholipid composition of this organelle (DePierre et al., 1981). Many inducers also increase the rates of synthesis of DNA, RNA, protein and haem (DePierre et al., 1981). Of course, in all cases a rationale for these effects can be suggested (see Table 2). Indeed, since all the different metabolic processes in a cell are more or less interdependent, it was somewhat naive of us to believe that xenobiotics could induce drug-metabolizing enzymes without having profound effects on other aspects of cellular metabolism as well. These studies were supported by grant no.] RO 1 CA 2626-1-04 from the National Cancer Institute, Department of Health, Education and Welfare, U.S.A., and by grants from the Swedish Natural Science Research Council, the Swedish Medical Research Council. and National Swedish Environmental Protection Board. Astrom, A. & DePierre, J. W. (1981) Biochim. Biophys. Acra 673, 225-233 Astrom, A. & DePierre, J. W. (1982) Carcinogenesis 3, 711-713 Astrom, A., Meijer, J. & DePierre, J. W. (1983) Cancer Res. 43, 342-348 Carlberg, I., DePierre, J. W. & Mannervik, B. (1981) Biochim. Biophys. Acra 677, 140-145
DePierre, J. W., Seidegird, J., Morgenstem, R., Balk, L., Meijer, J. & Astrom, A. (1981) in Mitochondria and Microsomes (Lee, C. P., Schatz, G . & Dallner, G., eds.), pp. 585-610, AddisonWesley, Boston DePierre, J. W., SeidegArd, J., Morgenstem, R., Balk, L., Meijer, J., Astrom, A., Norelius, 1. & Emster, L. (1984) Xenobiotica in the press Guthenberg, C., Morgenstern, R., DePierre, J. W. & Mannervik, B. (1980) Biochem. Biophys. Acta 631, 1-10 Hammock, B. D. & Ota, K. (1983) Toxicol. Appl. Pharmacol. in the press Lind, C., Hojeberg, B., Seidegird, J., DePierre, J. W. & Ernster, L. (1980) FEBS Letr. 116, 289-292 Meijer, J. & DePierre, J. W. (1983) J . Steroid Biochem. 18,425435 Meijer, J., Astrom, A., DePierre, J. W., Guengerich, F. P. & Ernster, L. (1982) Biochem. Pharmacol. 31, 3907-3916 Morgenstern, R. & Dock, L. (1982) Acra Chem. Scand. Ser. B 36, 255-256 SeidegBrd, J. & DePierre, J. W. (1982~)Chem.-Biol. Interact. 40, 15-25 Seidegird, J. & DePierre, J. W. (19826) Biochern. Pharmacol. 31, 1717-1 721 Seidegird, J., Morgenstern, R., DePierre, J. W. & Emster, L. (1979) Biochim. Biophys. Acta 586, 10-21 SeidegBrd, J., DePierre, J. W., Morgenstern, R.,Pilotti, A. & Ernster, L. (1981) Biochem. Biophys. Acta 672, 65-78 Suzuki, Y., DePierre, J. W. & Emster, L. (1980) Biochim. Biophys. Acra 601, 532-543
Dynamics of the localization of drug metabolizing enzymes in tissues and cells C. R. WOLF,* A. BUCHMANN,? T. FRIEDBERG,$ E. MOLL,$ W. D. KUHLMANN,? H. W. KUNZt and F. OESCHS *Imperial Cancer Research Fund, Medical Oncology Unit, Western General Hospital, Edinburgh EH4 ZXU, U.K.; tlnstitute of Biochemistry, German Cancer Research Centre, Heidelberg 6900, Federal Republic of Germany ;$Institute of Pharmacology, University of Mainz, Mainz 6500, Federal Republic of Germany Introduction Over the last few years many advances have been made in the identification of cytochrome P-450 isoenzymes and GSTs. There are now many studies that demonstrate that isoenzymes of both of these groups of proteins play very different roles in both the activation of foreign compounds to toxic and carcinogenic products as well as their deactivation. In this respect it is extremely important to understand the factors that regulate the concentrations of particular isoenzymes within the liver and other tissues. It has been known for many years that many enzymes are distributed unevenly in the hepatocyte population of the liver lobes. The distribution of drug metabolizing enzymes is potentially very important as a cell which contains a high concentration of P-450 may contain very low concentrations of the GSTs, making that cell particularly susceptible to certain chemicals activated by the P-450 system. There are now reports on the localization of cytochrome P-450 components in the liver, and other tissues, that demonstrate cell specific localizations (Serabjit-Singh et al., 1979; Dees et al., 1982;Baron & Kawabata, 1983). In this study we have isolated and characterized four different cytochrome P-450 Abbreviations used: ARO, Arochlor 1254; Clo, Clofibrate; DEN, diethylnitrosamine; DMN, dimethylnitrosamine; EH, epoxide hydrolase ; ELISA, enzyme-linked immunosorbent assay; GST, glutathione S-transferase; IgG, immunoglobulin G ; Iso, isosafrole; 3-MC, 3-methylcholanthrene; 8-NF, P-naphthaflavone; P-450, cytochrome P-450; PCN, pregnenolone carbonitrile; SDS, sodium dodecyl sulphate; TSO, trans-stilbene oxide.
isoenzymes(two from PB-treated rat liver and two from rats treated with 3-MC), two major forms of the GSTs and hepatic microsomal EH in order to investigate their regulation relative to each other. Several experimental approaches have been taken. These are the immunochemical quantification of isoenzyme concentration in hepatic microsomal samples from animals treated with different inducing agents, the..immunochemicallocalization of these isoenzymes in serial sections of rat liver, the effect of inducing agents on these levels and the regulation of these isoenzymes in preneoplastic foci during experimental hepatocarcinogenesis. Materials and methods Cytochrome P-450 isoenzymes (PB,, PB2, MC, and MC,), GST Band C and EH were purified from the livers of male Sprague-Dawley rats (180-2OOg) using methods previously described (Bentley & Oesch, 1975; Friedberg et al., 1983; Wolf & Oesch, 1983).Antibodies to these proteins were raised in rabbits using reported procedures (Wolf & Oesch, 1983). Immunohistochemical localizations were carried out on either paraffin sections or frozen sections fixed with benzoquinnone. A double-antibody procedure was used; the second antibody was labelled with horseradish peroxidase. Peroxidase activity was visualized by incubation of the sections with 3,3'-diaminobenzidine and H202. Male or female Sprague-Dawley rats (150-200g) were used for all enzyme quantification and localization studies. Certain groups of animals were treated with PB, 3-MC, Iso, Clo, /?-NF, TSO, ARO or PCN using conventional induction schedules for maximal induction of the cytochrome P-450 isoenzymes. Preneoplastic lesions were induced in the livers of 70g female rats by using DEN (50 or 100 p.p.m.) in the drinking water for 10 days or by continuous treatment with either DEN (10mg/kg) or DMN (3mg/kg) by stomach tube 5 days a week for up to 22 weeks. Carcinogen treatment was stopped 14 days before preparation of the tissue samples. 1984
