Influence of xenobiotics on drug metabolism and its sensitive detection

Exp. Pathol. 1990; 39: 187-194 Gustav Fischer Verlag lena

Friedrich Schiller University Jena, Institute of Pharmacology and Toxicology, Jena, GDR

Influence of xenobiotics on drug metabolism and its sensitive detection By D. MULLER With 6 figures

Address for correspondence: Doz. Dr. D. MULLER, Institut fUr Pharmakologie und Toxikologie, Friedrich-Schiller-Universitat Jena, LobderstraBe 1, DDR -6900 Jena

Key words: xenobiotics; biotransfonnation: drug metabolism; cytochrome P-4S0; induction; inhibition; ethoxycoumarin O-deethylation; thoxyresorufin O-deethylation; pentoxyresorufin O-depentylation; ethylmorphine Ndemethylation; caffeine elimination

Summary Biotransformation of drugs and other xenobiotics in the liver plays an important role in toxication and detoxication processes, i.e. changes in biotransformation activity have significant biological consequences. For that reason the determination of biotransformation activity is useful. The rate of some biotransformation reactions can be markedly changed by very small doses of xenobiotics. These changes may be used for the sensitive detection of the exposure to xenobiotics. Among biotransformation reactions cytochrome P-450-dependent monooxygenation is very important. It can be induced and inhibited by xenobiotics. Induction ofP-450 forms can be detected by various methods, e.g. by using model reactions. Ethoxycoumarin O-deethylation, ethoxyresorufin O-deethylation, pentoxyresorufin O-depentylation and ethylmorphine N-demethylation are catalyzed at different P-450 forms, and their determination is described in detail.

General aspects of drug metabolism One of the major functions of the liver is the metabolism (biotransformation) of drugs and other xenobiotics. It is useful to determine biotransformation activity in the liver as a tool of detecting effects of xenobiotics on this organ for at least 2 reasons: Biotransformation is very important for detoxication and toxication ofxenobiotics, i.e. changes of this process following exposure to xenobiotics have significant biological consequences. The rate of some biotransformation reactions or the concentration of the corresponding enzymes can be markedly changed by very small doses ofxenobiotics. Even if these changes per se are of minor biological significance, they can be used for the sensitive detection of the exposure to xenobiotics, e.g. toxic substances. Thus drug-metabolizing enzymes can be used for biomonitoring (e.g. detection of water and air pollution). A good many biotransformation enzymes and reactions are known. These enzymes mostly have a relatively low substrate specificity, i.e. the big number of xenobiotics is metabolized by a relatively low number of enzymes. Table 1 shows some possible biotransformation reactions. The elimination rate of many drugs is determined by their metabolization in the liver. Metabolites are Exp. Pathol. 39 (1990) 3-4

187

Table 1. Examples of drug-metabolizing reactions in the liver. Phase I reactions (1) oxidations C-oxygenation epoxidation oxidative N-, 0-, S-dealkylation N-, S-oxidation alcohol and aldehyde oxidation

(3) Hydrolyses (2) Reductions ester hydrolysis carbonyl reduction hydrolysis of C-N epoxide reduction nitroreduction bond azoreduction glycoside hydrolysis reductive dehalogenahydrolytic dehalogetion nation

Phase II reactions glucuronidation sulphatation conjugation with glutathione (-+ formation of mercapturic acid)

methylation acylation

usually more polar and cannot be reabsorbed in the renal tubules. Table 2 demonstrates the influence of biotransformation on elimination half life time of barbiturates with different lipid solubility. The higher the lipophily, the slower will be the elimination without metabolism and the more important is the metabolization for the elimination (e.g. hexobarbital). Very often xenobiotics loose their biological effectiveness following metabolization.

Table 2. Relations between lipid solubility of some drugs and their theoretical and real elimination half life time (according to REMMER 1966). Drug

Barbital Phenobarbital Pentobarbital Glutethimide Hexobarbital

Solubility in chloroform (% extracted from water)

Half-life time (days) theoretical (without metaboliz. )

real (without metaboliz. )

40 96 100 100 100

2-3 10-20 60-150 60-150 60-150

2-3 3-6 0.5-3 0.3-0.6 0.2

Renal excretion (% unchanged)

90 40 0 0 0

Reactive metabolites, however, can be also formed. Usually more than one reaction is involved in the metabolism ofaxenobiotic. If a toxic metabolite is formed, it may be detoxified by other biotransformation reactions. If detoxication is not sufficient, the reactive metabolite may bind to macromolecules in the liver cell. The consequences are cell damage, mutagenic effects and/or initiation of carcinogenesis. Thus the balance between toxication and detoxication reactions is very important for the effects of xenobiotics. This is demostrated in fig. 1 for the metabolism of acetaminophen (paracetamol). About 95 % of the drug are metabolized to non-toxic glucuronides and sulphates. About 5 % are metabolized at cytochrome P-450 to a reactive metabolite which can bind to macromolecules or cause the formation of oxygen radicals. Cell damage can be caused by both mechanisms. These toxic effects can be prevented if the detoxication of this metabolite by the glutathione system is sufficient.

188

Exp. Pathol. 39 (1990) 3-4

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Moonooxygenation at cytochrome P458 (P458) One of the most important biotransformation reactions, also for toxication, is the P-4S0dependent monooxygenation in the endoplasmic reticulum of the liver. P-4S0 is a hemoprotein which together with the NADPH-P-4S0 reductase and phospholipids catalyzes the following reaction: R-H+NADPH+H+

---P450

R - OH + H2 0 + NADP

(R - H = substrate, R - OH = hydroxylated metabolite). N- and O-dealkylations (s . below) are spontaneous reactions after the P-4S0-dependent cx-C hydroxylation of a side chain of a molecule . For the more detailed mechanism of P-4S0-dependent monooxygenation cf. ESTABROOK (1984) . Since changes of the activity of the P-4S0-dependent monooxygenase system following exposure to xenobiotics are of particular biological relevance and can be also used for a very sensitive detection of the exposure to many xenobiotics, only this system will be considered in the following sections.

Induction of P-45O-dependent monooxygenation and its detection P-4S0 can be induced by many xenobiotics, depending on the different chemical P-4S0 forms, which differ, e.g ., in substrate specificity. In the rat liver at least 26 P-4S0 forms have been cloned and sequenced so far (GONZALES 1989). Polycyclic aromatic hydrocarbons (PAH) such as 3methylcholanthrene and ~-naphthoflavone (~-NF) induce 2 P-4S0 forms (P-4S0 IAI and IA2) (cf. GONZALEZ 1989). These P-4S0 forms metabolize benzo(a)pyrene , 2-acetylaminoflurene, aflatoxin Bl to form reactive metabolites. Therefore both enzymes of this P-4S0 family could playa very important role in chemically induced cancer. Phenobarbital induces P-4S0 forms (e.g. in the P-450 II family) which are involved in the metabolism of many drugs. Ethanol induces P-450 lIE 1 which is Exp . Pathol. 39 (1990) 3-4

189

responsible for the formation of toxic metabolites from N-nitrosodimethylamine, halothane and other substances (cf. GONZALEZ 1989). These few examples show that induction of P-4S0 forms may be associated with increased formation of toxic metabolites. Therefore a sensitive detection of the' exposure to certain inducers is necessary. Only relatively simple methods are to be demonstrated, wich can be used in many laboratories. Model reactions which show a high induction factor and which are catalyzed by one or few P-4S0 forms can be used for this purpose. For the determination of the following 3 P-4S0-dependent reactions only a few milligrams of liver tissue are necessary, which can be obtained from the rat even by fine needle aspiration biopsy, if the same animal must be investigated several times (FOUTS 1980). 7-Ethoxycoumarin 0deethylation is catalyzed preferentially by PAR-induced P-450 forms, and to a smaller extent also by phenobarbital-induced forms (GUENGERICH et al. 1982). Because of well-detectable induction even by small doses of the inducers and the strong fluorescence of the metabolite 7-hydroxycoumarin this reaction is useful as a screening method for the detection of an induction. Fig. 2 shows the induction of this reaction by ~-NF and phenobarbital. 7-Ethoxyresorufin O-deethylation is a reaction which is specifically dependent on P-4S0 forms which are induced by PAR (GUENGERICH et al. 1982). The induction factor is very high, and even very low ~-NF doses alter this reaction rate (fig. 3). 7-Pentoxyresorufin O-depentylation is a very sensitive parameter for the detection of the induction of the phenobarbital type (LUBET et al. 1985). The induction factor following phenobarbital administration is considerably higher than with other reactions (fig. 4), thus this reaction is a very sensitive parameter for the detection of a phenobarbital-type induction. It is, however, also induced to a smaller extent by ~-NF (fig. 4), i.e., it is not specific for the induction by phenobarbital. A fourth reaction, which can be used for detecting induction, is ethylmorphine Ndemethylation. In contrast to the above-mentioned 3 reactions more liver tissue is needed for the determination, and this reaction depends on more P-450 forms (constitutive, phenobarbital- and pregnenolone-16cx-carbonitrile/dexamethasone-induced forms (GUENGERICH et al. 1982). An advantage of this reaction is the lack of induction by PAR (fig. S). A detailed description of these 4 reactions is given in the appendix.

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Fig. 2. 7-Ethoxycoumarin O-deethylation in liver 9,000 g supernatant from 33-day-old male rats (own investigations). C: control rats, PB: rats treated with phenobarbital (3 x 60 mg/kg), ~NF: rats treated with ~-naphthoflavone (l x 0.2 or SO mg/kg).

Fig.3. 7-Ethoxyresorufin O-deethylation in liver 9,000 g supernatant from 33-day-old male rats (own investigations). c: control rats, PB: rats treated with phenobarbital (3 x 60 mg/kg), treated with ~-naphthoflavone (l x 0.2 or 50 mg/kg). 190

Exp. Pathol. 39 (1990) 3-4

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Fig. 5. Ethylmorphine N-demethylation in liver 9,000 g supernatant from 33-day-old male rats (own investigations). C: control rats, PB : rats treated with phenobarbital (3 X 60 mglkg) , ~NF: rats treated with ~-naphthoflavone (1 X 50 mg/kg), PCN : rats treated with pregnenolone 16cxcarbonitrile (3 X 20 mg/kg).

There are a good many of other substrates whose metabolization can be used for the characterization of P-450 forms. Steroids (e.g. testosterone) and warfarin are hydroxylated stereospecifically at different positions by different P-450 forms (cf. ASTROM and DEPIERRE 1986). By using only one substrate and measuring the pattern of metabolites one can get information on the activity of different P-450 forms. Induction can be also detected in vivo, if the elimination of a drug is measured which depends mainly on the metabolization at P-450. Caffeine is demethylated preferentially by P-450 forms induced by PAH (ALDRIDGE et al. 1977). Fig. 6 demonstrates that even small doses of~-NF shorten caffeine half-life time in rats considerably . The sensitivity is similar to that of ethoxycoumarin and ethoxyresorufin O-deethylation in vitro . If the methyl groups of caffeine are labelled (l3C , 14C) you need not determine caffeine in serum or saliva, only the exhaled labelled CO 2 is measured . A further non-invasive method is the determination of antipyrine metabolites in the urine, whose pattern is differently influenced by inducers of the phenobarbital and PAH type (BOTTCHER et al. 1982). The induction of P-450 forms can be also detected by methods other than the measurement of model reactions. P-450 forms can be easily characterized by immunochemical methods (cf. ASTROM and DEPIERRE 1986) , if you have antibodies against the different P-450 forms. In the future these antibodies will be commercially available, i.e. , many laboratories can use these immunochemical techniques. Under these conditions the measurement of model reactions becomes less important.

IBItihitioR of P45O-c1ependent IBODooxygenation ad its detection Xenobiotics are able to inhibit the P-450-dependent metabolism of other xenobiotics. This inhibition is mostly reversible and is caused by the binding of the substance to the heme site or the substrate binding site of P-4S0 . Reversal of inhibition can occur by metabolism of the inhibitor or Exp. Pathol. 39 (1990) 3-4

191

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after diffusion of the inhibitor away from the enzyme. Among the drugs potent inhibitors of this type are antifungal imidazoles, HTreceptor antagonists and quinoline drugs (MURRAY 1987). This type of inhibition may be easily detected by addition of the potential inhibitor in different concentrations to the incubation medium which is used for the determination of the above-mentioned biotransformation reactions (e.g. ethoxycoumarin O-deethylation or ethylmorphine N-demethylation). With the help of the Michaelis-Menton kinetics inhibition type and IC so (inhibitor concentration which causes 50 % inhibition) can be obtained. The binding of xenobiotics can be also detected directly by optical difference spectroscopy, since following binding spectral changes of P-450 occur: type I spectral change after binding to the apoprotein, type II spectral change after binding to the heme (SCHENKMAN et al. 1967). Apart from reversible inhibition of monooxygenation there are more complex mechanisms of inhibition. A large number of therapeutic agents undergo metabolic activation by P-450 to inhibitory products (cf. MURRAY 1987). These metabolic products may generate relatively stable complexes with P-450 so that P-450 is sequestered in a functionally inactive state. Examples for this type of inhibition are macrolide antibiotics, amphetamines and hydrazines (cf. MURRAY 1987). Alternately the metabolite may inactivate P-450 via heme or apoprotein modification (autocatalytic inactivation or suicide processing). The classical substance that modifies the heme is allylisopropylacetamide, but also some anesthetic agents have similar effects. The P-450 apoprotein can be modified by coumarin derivatives, disulfiram, spironolactone and chloramphenicol (cf. MURRAY 1987). The inhibitory action can be also detected by the above-mentioned model reactions either after in vivo treatment with the inhibitor or in vitro addition of the inhibitor to the incubation medium which is used for the determination of the monooxygenase reactions. So far total cytochrome P-450 concentration was not mentioned at all concerning the detection of induction or inhibition. The changes of total P-450 concentration are little in comparison to those of the model reactions. Thus this parameter need not be determined in the first step of screening, even if the determination is very simple. 192

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Appendix (Description of methods) Preparation of liver 9,000g supernatant 30- to 60-day-old male rats (good inducibility of monooxygenases !) are sacrificed, and the liver is removed, weighed and rinced in ice-cold 0.9 % NaCI solution. All further procedures are carried out at 4°C. The liver is homogenized in 0.1 M sodium phosphate buffer (l g liver + 2ml buffer). The homogenate is centrifuged at 9,000g for 20min. The 9,000g supernatant is used as P-450 source for the determination of the below-mentioned model reactions. Besides it is necessary to determine protein content of the supernatant by common methods. If the supernatant is not used within a few hours (storage at 4 0c) it must be stored in a freezer (- 80°C or below) to avoid loss of activity. Determination of7-ethoxycoumarin O-deethylation (according to Amo 1978, with modifications) 200nmoles of 7-ethoxycoumarin (dissolved in 0.1 ml methanol) are added to a glass tube, and then methanol is completely evaporated. After evaporation the following substances are added: O.lml O.IM sodium phosphate buffer, pH 7.4, 0.1 ml glucose-6-phosphate (2.5 [lmoles, dissolved in buffer), O.lml MgCb (lO[lmoles, dissolved in water), O.lml diluted 9,000g supernatant (dilution with buffer I: IO for controls rats, 1 :20 for PAH-induced rats). Start of the reaction with: 0.1 ml NADPH (0.25 [lmoles, dissolved in buffer). Final incubation volume 0.5 m!. After incubation at 37°C for IOmin the reaction is stopped by addition of 0.5 ml of 0.31 M trichloroacetic acid. Then 4 ml of 0.05 M NaOH are added and after centrifugation at 6,000rev/min for IOmin the main metabolite 7-hydroxycoumarin is fluorimetrically determined in the clear supernatant (excitation at 375nm, emission at 454nm). The blanks have the same composition, but trichloroacetic acid is added before NADPH. The standards contain different amounts of 7-hydroxycoumarin; NADPH, glucose-6-phosphate, MgCl 2 and ethoxycoumarin can be omitted, if these compounds are also omitted in the blanks of the standards. Determination of 7-ethoxyresorufin O-deethylation (according to POHL and FOUTS 1980, with modifications) 1 nmole of 7-ethoxyresorufin (dissolved in 0.1 ml methanol) is added to a glass tube, and then methanol is completely evaporated. The composition of the medium is the same as for 7ethoxycoumarin O-deethylation (see above). The dilution of 9,000 g supernatant is generally 1: 10, after maximal induction by PAH 1: 40. After incubation at 37°C for 5 min the reaction is stopped by addition of 1 ml ice-cold methanol. After centrifugation at 6,000rev/min for 5 min the metabolite resorufin is fluorimetrically determined in the clear supernatant (excitation 540nm, emission 585nm). The blanks have the same composition, but methanol is added before NADPH. The standards contain different amounts of resorufin in buffer without ethoxyresorufin, NADPH, supernatant, MgCl 2 and glucose-6-phosphate. Determination of 7-pentoxyresorufin O-depentylation This reaction is determined in the same way as ethoxyresorufin O-deethylation, since the same metabolite (resorufin) is formed. There are only a few differences: addition of 5 nmoles pentoxyresorufin, dilution of the 9,000g supernatant generally I: 10, after maximal induction by phenobarbital 1: 20, incubation time 10 min. Determination of ethylmorphine N-demethylation The following substances are added to a glass tube: O.Sml O.SM sodium phosphate buffer, pH 7.4, O.OSml NADP (H) (0.3 [lmoles, dissolved in 0.1 M sodium phosphate buffer, pH 7.4), 13

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0.05 ml glucose-6-phosphate (5 !1moles, dissolved in 0.1 M buffer), 0.05ml MgCl 2 (6!1moles, dissolved in H 20), 0.05 ml semicarbacide (lO!1moles, dissolved in 0.1 M buffer), 0.2ml diluted 9,OOOg supernatant (dilution 1:4 with O.IM buffer). Start of the reaction with: 0.1 ml ethylmorphine (6!1moles, dissolved in 0.1 M buffer). After incubation at 37°C for lOmin the reaction is stopped by addition of 0.1 ml 75 % trichloroacetic acid. After centrifugation at 6,000rev/min for lOmin the formaldehyde formed from ethylmorphine is determined in the clear supernatant by using the method of NASH (1953), modified by KLEEBERG and KLINGER (1982). 0.5ml NASH reagent are added to 0.5ml supernatant, and after incubation at 37°C for 40min the samples are cooled with tap water, and the absorbance is measured at 412nm. The blanks have the same composition, ethylmorphine is omitted. The standards contain different formaldehyde concentrations instead of ethylmorphine. NASH reagent contains 30g ammonia acetate, O.4ml acetylacetone, 0.6ml 99% acetic acid and H 20 ad 100ml.

References AITIO, A.: A simple and sensitive assay of 7-ethoxycoumarin deethylation. Anal. Biochem. 1978; 85: 488-491. ALDRIDGE, A., PERSONS, W.-D., NEIME, A. H.: Stimulation of caffeine metabolism in the rat by 3-MC. Life Sciences 1977; 21: 967-974. ASTROM, A., DEPIERRE, J. W.: Rat liver microsomal cytochrome P-450 purification, characterization, multiplicity and induction. Biochim. Biophys. Acta 1986; 853: 1-27. BOTTCHER, J., BASSMANN, H., SCHlJPPEL, R.: Modification of hydroxylation and conjugation pattern of antipyrine by PB and 3-MC in the rat. In: Microsomes, drug oxidations, and drug toxicity (eds.: R. SATO and R. KATO). Japan Scientific Societies Press, Tokyo 1982, pp. 441-442. ESTABROOK, R. W.: Cytochrome P-450 and oxygenation reactions: a status report. In: Drug metabolism and toxicity (eds.: J. R. MITCHEL and M. G. HORNING). Raven Press, New York 1984, pp. 1-20. FOUTS, J. R.: Assays of mixed function oxidase activity of small samples of liver. In: Fine needle aspiration biopsy of the rat liver (ed. G. ZBINDEN). Pergamon Press, Oxford 1980, pp. 33-37. GONZALES, F. J.: The molecular biology of cytochrome P-450 s. Pharmacol. Rev. 1989; 40: 243-288. GUENGERICH, F. P., DANNAN, G. A., WRIGHT, S. T., MARTIN, W. Y., KAMINSKY, L. S.: Purification and characterization of liver microsomal cytochromes P-4S0: Electrophoretic, spectral, catalytic and immunological porperties and inducibility of eight isozymes isolated from rats treated with phenobarbital or ~-naphtho­ flavone. Biochemistry 1982; 21: 6019-6013. KLEEBERG, U., KLINGER, W.: Sensitive formaldehyde determination with NASH's reagent and a 'tryptophan reaction'. J. Pharmacol. Meth. 1982; 8: 19-31. LUBET, R. A., MAYER, R. T., CAMERON, 1. W., NIMS, R. W., BURKE, M. D., WOLFF, TH., GUENGERICH, F. P.: A rapid and sensitive assay for measuring induction of cytochrome(s) P-450 by phenobarbital and other xenobiotics in the rat. Arch. Biochem. Biophys. 1985; 238: 43-48. MURRAY, M.: Mechanisms of the inhibition of cytochrome P-450-mediated drug oxidation by therapeutic agents. Drug Metab. Rev. 1987; 18: 55-81. NASH, T.: The colorimetric estimation of formaldehyde by means of the Hantzsch reaction. Biochem. J. 1953; 55: 416-421. NEBERT, D. W., JENSEN, N. M.: The Ah locus: genetic regulation of the metabolism of carcinogens, drugs, and other environmental chemicals by cytochrome P-4S0-mediated monooxygenases. Crit. Rev. Biochem. 1979; 6: 401-437. POHL, R. J., FOUTS, J. R.: A rapid method for assaying the metabolism of 7-ethoxyresorufin by microsomal subcellular fractions. Anal. Biochem. 1980; 107: 150-155. REMMER, H.: Die Entgiftung von Pharmaka. Dtsch. med. Wochenschr. 1966; 91: 289-296. Schenkman, J. B., Remmer, H., Estabrook, R. W.: Spectral studies of drug interaction with hepatic microsomal cytochrome. Molec. Pharmacol. 1967; 3: 1\3-123. (Accepted January 17, 1990)

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