The mechanism of cytochrome P450-catalysed drug oxidations

11 The mechanism of cytochrome P450-catalysed drug oxidations V. Ullrich (Department of Physiological Chemistry, University of the Saarland, Homburg-Saar, German Federal Republic) INTRODUCTION Most compounds involved in the metabolism of cells, organs and organisms are hydrophilic in nature, possessing polar groups capable of undergoing a variety of metabolic reactions. Lipophilic molecules, like steroids or the fat-soluble vitamins, are in the minority. These latter compounds consist, in substantial part, of aromatic or alicyclic rings and/or aliphatic side-chains. These hydrocarbon groups are metabolically rather inert and in fact only one group of enzymes, the mono -oxygenases, can attack aromatic or aliphatic C - H bonds. Monooxygenases use molecular oxygen and two electrons from an external donor to introduce an oxygen atom into organic substrates according to the equation (Mason, 1957): RH + Oz + DHz -+ ROH + D + H10 where RH represents substrate and DH z represents reduced electron donor. Such enzymes play an important role in the formation and transformation of steroids, bile acids, amino acids or vitamins (Ullrich, 1972). Micro-organisms using lipophilic compounds as the sole carbon source rely on the presence ofmono-oxygenases for the initial oxidation of the substrate to alcohols, which can be further oxidised with concomitant production of chemical energy. In the examples discussed so far, the products of mono-oxygenation are further metabolised. In contrast, there are a number of mono-oxygenases which have as their only function the conversion of lipophilic compounds to more hydrophilic and, hence, excretable forms. This is known to occur with steroids and a large number of lipophilic compounds which are foreign to the organism and enter the body with food, by respiration or absorption through the skin. Examples of such compounds include food additives, natural plant constituents such as alkaloids or terpenes, industrial organic chemicals and an increasing number of drugs. The mono-oxygenase system responsible for the oxidation of these compounds may 201 G. C. K. Roberts (ed.), Drug Action at the Molecular Level © Institute of Biology Endowment Trust Fund 1977 V. Ullrich 202 be regarded as a detoxifying system, since without it an accumulation of these compounds in hydrophobic parts of the body would lead to disturbances of physiological functions. The designation 'detoxifying system' should, however, be avoided, since today we know that the same system can also potentiate the toxicity of compounds, depending on their chemical structure. Since other features of this drug mono -oxygenase system are also rather unusual, its nature and mechanism of action have elicited considerable interest. It is the purpose of this contribution to summarise the current results and ideas on the mechanism and to relate them to pharmacological, toxicological and even medical problems involved in drug metabolism. THE OCCURRENCE AND THE STRUCTURE OF THE DRUG MONO-OXYGENASE SYSTEM According to an earlier hypothesis by Brodie, Gillette and LaDu (I958), only terrestrial animals were thought to require the enzymic outfit for the oxidation of lipophilic compounds, since they are preferentially exposed to these chemicals. Since then corresponding activities have also been detected in fish (pedersen, Hershberger and Juchau, 1974), and in view of the contamination of surface water it is not surprising that even marine species contain activities. Table 11.1 lists a number of species from the North Sea in which mono-oxygenase activities could be detected. As a test the O-dealkylation of the flu orogenic substrate 7 -ethoxycoumarin was used (Ullrich and Weber, 1972). From the organs tested only the hepatopancreas and the small intestine showed O-dealkylation activity. In higher animals and in man the liver contains the highest drug mono-oxygenase activity. Small intestine (Wattenberg, Leong and Strand, 1962; Lehrmann, Ullrich and Rummel, 1973) contains considerably less, but when calculated on the basis Table 11.1 0- Dealkylation activity for 7 -ethoxycoumarin in organ homogenates of various marine species Species Organ Myaxacephalus scarp ius liver intestine liver hepatopancreas intestine hepatopancreas hepatopancreas hepatopancreas Zaarces viviparus Carcinus maenas Eupagurus bernhardus Buccinium undatum Ciana intestinalis Specific activity (nmol/mg protein) 0.21 0.60 0.08 0.04 0.02 0.06 0.11 0.014 After preparation the organs were immediately homogenised and the mixture was passed through a gauze cloth. The values represent averages of 3 -4 preparations. The Mechanism of Cytochrome P450-Catalysed Drug Oxidations 203 of the single cell, the activity of the intestinal mucosal cell is comparable to that of the hepatocyte. Lung (Wattenberg and Leong, 1965) and skin (Alvares et ai., 1973) are also active, but heart and brain are not. This is demonstrated in Table 11.2 for the different organs of mice by measuring the O-dealkylation activity in total homogenates. It is evident that only those organs which are considered by the pharmacologist as the 'ports of entry' of foreign compounds into the organism contain activity. Table 11.2 Organ distribution of 7-ethoxycoumarin O-dealkylation activity in the mouse Organ Liver Small intestine Skin Lung Kidney Brain Heart Activity per organ (nmol min-I) 147 15 7 0.7 0.1 o o % of total activity 86.5 8.5 4 1 0.1 o o The activity was determined fluorometrically in organ homogenates as described by Ullrich and Weber (1972), except for small intestine and skin, which were incubated in substrate solution for 10 min. By surface reflectance fluorimetry it was established that the reaction was linear with time. The umbelliferone formed was eluted from the tissue by ether extraction. The intracellular localisation has invariably been in the endoplasmic reticulum membranes. These membranes contain a cytochrome which, from its unusual carbon monoxide complex, has been termed cytochrome P450 (Omura and Sato, 1964). The same cytochrome had been identified as the oxygen and substrate binding component from steroid hydroxylases (Estabrook, Cooper and Rosenthal, 1963) and it is now established that it also has the same function in the drug mono-oxygenase system. The transfer of the electrons is mediated by an FADand FMN -containing NADPH-dependent reductase (Iyanagi and Mason, 1973), and by recombination of these two components the drug mono-oxygenase activity could be reconstituted (Lu, Junk and Coon, 1969; Strobel et al., 1970). One of the most interesting aspects of cytochrome P450 is its broad substrate specificity. In view of the almost endless number of lipophilic chemicals, the question arose whether all compounds are metabolised by a simple cytochrome P450 or whether a variety of specific enzymes exist. There was already evidence from various research groups that pretreatment of animals with drugs which could act as inducers of the mono-oxygenase system leads to changes in the liver microsomal fractions with respect to activities towards, several substrates as well as in the product patterns (Conney et ai., 1969; Frommer et ai., 1972; Kuntzman, 1969). 204 V. Ullrich Table 11.3 Effect of inhibitors on the O-dealkylation activity for 7 -ethoxycoumarin in rat liver microsomes after various pretreatments % Inhibition by Pretreatment Specific Metyrapone activity (2 X 10- 5 M) Naphthoflavone Tetrahydrofuran (l0-2M) (2 X 1O- 5 M) None (controls) d 0.7 ± 0.2 52 ± 10 10 ± 5 15 ± 6 None (controls) 0.4 ± 0.2 5± 3 12 ± 5 48 ± 20 Phenobarbital (3 d) 2.2 ± 0.6 72 ± 12 7±5 4± 2 3,4-Benzpyrene (2 d) 5.4 ± 2.0 2± 5 90 ± 6 2± 5 Ethanol (20 d) 0.9 ± 0.2 7± 3 10 ± 5 79 ± 10 <;> Data are taken from Ullrich et af. (1975). The specific activity is expressed as nmol (mg protein)-' min -'. For the substrate 7 -ethoxycoumarin, we could show that after various pretreatments the activity could be inhibited differently by the three compounds metyrapone, naphthoflavone and tetrahydrofuran (Ullrich, Weber and Wollenberg, 1975) (Table 11.3). The observed differences in the pattern of inhibition do not accord with the presence of only one species of cytochrome P450 fractions in liver microsomes and revealed that even up to seven species may be present (Welton and Aust, 1974; Haugen, van der Hoeven and Coon, 1975). According to our results, 7 -ethoxycoumarin is a substrate for at least three forms of cytochrome P450. Thus we would suggest that the different forms have an overlapping substrate specificity but show a preference for certain groups of compounds. Thus the 3,4-benzpyrene-induced cytochrome P450 (or P448) shows greater activity towards aromatic compounds, phenobarbital-induced cytochrome P450 towards aliphatic compounds (Ullrich, 1968; Frommer, Ullrich and Staudinger, 1970) and ethanol-induced cytochrome P450 possibly for more hydrophilic compounds such as ethers or alcohols. The use of inhibitors provides a convenient test for the presence of the multiple forms. If one applies these inhibitors to the study of the drug mono-oxygenase activity in human liver biopsies the results given in Table 11.4 are obtained. Although it is not established and even unlikely that the cytochrome P450 species in humans are identical with those in rats, the different patterns of inhibition in the various patients indicate that many cytochrome P450 species must also exist in humans. There is not yet a clear-cut answer with regard to the biological signi- 205 The Mechanism of Cytochrome P450-Catalysed Drug Oxidations Table 11.4 0- Dealkylation activities for 7 -ethoxycoumarin in human liver needle biopsies Patient (sex) S., T. (m) L., E. (f) A., E. (m) Z., A. (f) B., E. (f) R., R. (f) M., M. (m) R., G. (m) B., H. (m) S., K. (f) C., P. (m) C., K. (m) Z., J. (m) F., K. (m) S., K. (m) F., H. (m) K., A. (m) B., J. (m) W., A. (m) Age Specific activity 32 47 55 63 25 21 30 38 70 47 57 20 29 41 40 63 66 0.10 0.12 0.12 0.00 0.10 0.02 0.15 0.35 0.04 0.14 0.30 0.13 0.04 0.05 0.04 0.02 0.03 0.02 Metyrapone % Inhibition by Tetrahydrofuran Naphthoflavone 27 37 33 52 38 42 16 10 32 22 0 27 16 28 33 30 48 20 10 22 15 0 39 5 25 100 51 41 70 34 28 52 35 67 44 80 10 50 25 33 0 31 0 46 84 22 27 95 17 19 33 72 0 70 The activity is expressed as nmol umbelliferone formed per mg of protein per minute in a 500 g supernatant. The fluorometric assay was performed in a microcuvette of 0.1 ml volume (Ullrich and Weber, unpUblished). ficance of the multiple forms, but as a general rule one can assume that higher affinities for a substrate can only be achieved by increasing the substrate specificity of an enzyme. Since the efficacy of cytochrome P450 in metabolising foreign compounds is directly related to its affinity, it is apparent that a multiplicity of this cytochrome represents an evolutionary advantage. The existence of various forms of cytochrome P450 explains the different patterns of metabolites in microsomes from different origins or the multiphaSic kinetic behaviour, but it does not appear to affect the mechanism of the system, which seems to be identical for all cytochromes P450. THE MECHANISM OF CYTOCHROME P450 CATALYSIS The Substrate Binding Reaction In general, enzyme catalysis starts with the formation of an enzyme -substrate complex. This was also established for cytochrome P450 by spectroscopic studies on the interaction of substrates with the oxidised cytochrome. Additions of lipophilic compounds result in spectral changes which are characterised by a shift of the 206 V. Ullrich Soret absorption band around 420 nm to a new absorption at about 390 nm (Remmer et at., 1966) The dissociation constant characterising this spectral change corresponded to the Km of the mono-oxygenation reaction (Schenkman, Remmer and Estabrook, 1967), in accordance with the classical concept of enzymesubstrate complex formation. Using the soluble cytochrome P450 from camphoroxidising bacteria as a model, it was found that the oxidised cytochrome with the 420 nm Soret band is a low-spin complex, whereas the 390 nm absorbing species is also ferric cytochrome P450 but in a high "spin state (Peisach and Blumber, 1970). This was also confirmed in microsomes but only about 30 per cent of the cytochrome P450 reacted with the substrate cyclohexane (Waterman, Ullrich and Estabrook, 1973), as a consequence of the heterogeneity of cytochrome P450. Recent work on model complexes has further characterised the enzymesubstrate complex as a five -coordinated complex, whereas the 420 nm species is a hexa-coordinated complex (Koch et al., 1975). The ligand in the fifth position is probably a sulphide group from a cysteinylresidue in the apoprotein, whereas the sixth ligand in the substrate-free cytochrome may be a water molecule (Griffin and Peterson, 1975). The mercaptide ligand would be unique for a haemoprotein and could explain many of the unusual spectral properties of cytochrome P450. In view of this coordination chemistry, the substrate binding reaction can be formulated as shown in Fig. 11.1 Cytochrome P 450 (low spin) Enzyme - Substrate Complex (high spin) Figure 11.1 Scheme for substrate binding to cytochrome P450 The Reduction of the Enzyme -Substrate Complex Oxygen can bind only to the ferrous cytochrome P450, and, hence, the reduction of the ferric cytochrome P450 - substrate complex is a very important stage in the reaction sequence. The enzymatic reduction of cytochrome P450 can be monitored by the formation of its carbon monoxide complex at 450 nm (Gigon, Gram and Gillette, 1969). These reduction kinetics are biphasic and consist of a fast phase, which corresponds to the reduction of the enzyme -substrate complex, and a slow phase representing the reduction of free cytochrome (Diehl, Schiidelin and Ullrich 1970). In the absence of carbon monoxide (which shifts the equilibrium towards the reduced form) there seems to be hardly any electron transfer to the free cytochrome, in agreement with the very low redox potential- in the The Mechanism of Cytochrome P450-Catalysed Drug Oxidations 207 100 NADH - dep. 0- dealkylation 80 >~ ~u « ~ 60 NADPH -dep. 0- dealkylalion 40 20 4 8 12 16 mg Immunoglobulin mg Microsomal Protein Figure 11.2 Effect of anti-(NADPH-cytochrome P4S0 reductase) antibody on the NADH- and NADPH-dependent O-dealkylation of 7 -ethoxycoumarin in rat liver microsomes. Microsomes from phenobarbital pretreated rats were used. The antibody preparation was kindly donated by Dr Omura and prepared according to Morimoto et al. (1976) NADPH'- dep. 0- dealkylation 80 60 ~ :~ u NADH - dep. 0- dealkylation « 40 ~ 20 2 3 4 5 mg Immunoglobulin mg Microsomal Protein Figure 11.3 Effect of anti-(NADH-cytochrome b s reductase) antibody on the NADH- and NADPH-dependent O-dealkylation of 7 -ethoxycoumarin in rat liver microsomes. The antibody preparation was kindly donated by Dr Omura region of380mV - forlow-spin cytochrome P450 (Waterman and Mason, 1970). The high-spin enzyme -substrate complex has a considerably higher potential (Wilson, Tsibris and Gunsalus, 1973), which could explain why NADPH-oxidation is greatly enhanced in the presence of substrates. The first-order rate constant for the reduction of the enzyme - cyclohexane complex has been calculated as 1.1 S-1 by computer simulation of the reduction kinetics (Diehl et al., 1970). This 208 V. Ullrich 100 x NADPH - dep. 80 ::- 60 <{ 40 ~u o -dealkylation NADH - dep. o!! o - dealkylation 20 2 3 4 mg Immunoglobulin mg Microsomal Protein Figure 11.4 Effect of anti-(cytochrome b s ) antibody on the NADH- and NADPH-dependent O-dealkylation of 7 -ethoxycoumarin in rat liver microsomes. The antibody preparation was kindly donated by Dr Omura and was prepared according to Mannering, Kuwahara and Omura (1974) is supported by the finding that increasing the electron flow to the cytochrome by providing a second pathway involving NADH increases the hydroxylation rate (Staudt, Lichtenberger and Ullrich, 1974). The electron transport chain from NADH does not involve the NADPH-cytochrome P450 reductase but rather NADH -cytochrome bs reductase and cytochrome b s . This could be established by the use of antibodies, as shown in Figs. 11.2 -11.4. The coordination chemistry of the reduced cytochrome is less well known, since electron paramagnetic resonance (EPR) is not applicable to ferrous complexes. Since the mercaptide anion is expected to complex less well to ferrous iron than to ferric iron, the reduced enzyme -substrate complex may possibly contain a sulphydryl group at the fifth coordination position. (Fig. 11.5). ~ L "J\ 5-··. Fell (?) }I H-C / "- R3 R2 /: /' Reduced ES - Complex (high spin) Figure 11.5 Scheme for the reduction of the cytochrome P450 - substrate complex The Mechanism a/Cytochrome P450-Catalysed Drug Oxidations 209 The Oxygen Activation Process A coordinatively unsaturated ferrous cytochrome can be expected to add oxygen in a very rapid reaction to yield an oxy complex. This has been found with cytochrome P450 from camphor-grown bacteria (Gunsalus, 1970; Ishimura, Ullrich and Peterson, 1971) but not yet with microsomal cytochrome P450, although a 440 nm band in a difference spectrum has been attributed to an oxy complex by Estabrook and associates (Estabrook et at., 1971). The spectral and magnetic properties of the oxy complex are almost identical with those of oxy-haemoglobin and oxy-myoglobin. Certainly, the bound oxygen molecule in these species cannot be regarded as sufficiently activated to hydroxylate C - H bonds. The actual activation process occurs by transfer of a second electron to the oxy complex. Details of this reaction are not yet known and only indirect evidence has been accumulated for the structure of the active oxygen. From its chemical reactivity an oxenoid structure is very likely (Ullrich and Staudinger, 1968). This term characterises a complex which can transfer an oxygen atom with strongly electrophilic properties to an acceptor molecule. Since peracids are known to exhibit very similar properties to those of the enzymic active oxygen of monooxygenases, a peroxide structure was discussed (Ullrich and Staudinger, 1968). On the other hand the 0 - 0 bond may be liable enough to undergoproton-catalysed splitting with formation of water and a ferric oxygen atom complex, in which higher oxidation states of iron may participate in the stabilisation of the oxygen atom (Fig. 11.6). FellI _ ~ [Fe0 Q. _ QI 2- 2t te/ ~ [Fe-O]3+ tOW Oxy- Oxenoid Complex Complex Figure 11.6 Scheme for the oxygen activation reaction of cytochrome P450 Recent approaches towards the elucidation of the structure of active oxygen have been based on the use of oxidising agents as substitutes for molecular oxygen and NADPH. Thus it was found that cytochrome P450 can catalyse hydroxylations by using cumene hydroperoxide as a source of active oxygen (Rahimtula and O'Brien, 1974). Unfortunately, this experiment does not allow discrimination between the two postulated structures. Certain steroid hydroxylations in liver microsomes can be mediated in the presence of periodate (Hrycay et at., 1975), which indeed favours the [FeOp+ structure, but it is not clear whether periodate oxidises the iron to the pentavalent state or whether periodate serves as an oxygen atom donor for the ferric cytochrome P450. Drug hydroxylations are not catalysed by this compound 01. Ullrich, unpublished). The latter mechanism V. Ullrich 210 has been proposed for a mono-oxygenation that we observed with iodosobenzene (lichtenberger, Nastainczyk and Ullrich, 1976). In the presence of this compound cytochrome P450 could catalyse the hydroxylation of cyclohexane and the 0dealkylation of 7 -ethoxycoumarin. These reactions showed similar affinity differences for the various cytochrome P450 species to those of the oxygen - and NADPH-dependent reaction, so the active site of the cytochrome must be involved. The simplest explanation for the action of iodosobenzene would be that it is able to transfer its oxygen atom to the substrate via 9ytochrome P450. The Oxygen Atom Transfer Reaction In contrast to the high substrate and product specificities of some cytochrome P P 450- dependent steroid hydroxylases, the mono- oxygenation of drugs usually leads to several products. Generally it can be concluded that the product pattern formed is the result of the attack by an electrophilic oxygen species on the substrate. Hydroxylating model systems such as trifluoroperacetic acid lead to a product pattern very similar to that of the enzymatic reactions (Ullrich et al., 1967; Frommer and Ullrich, 1971), indicating that with small substrate molecules steric factors do not greatly influence the product distribution. This situation changes if larger molecules are used as substrates. In this case the pattern of products is even altered when microsomes induced by different pretreatments are used (Conney et al., 1969). The last step in the reaction sequence which completes the cycle can be formulated as shown in Fig.1I.7. ::/--/--..,./ .. -H-O-C ... RI / 'R R3 2 Oxenoid Complex Cytochrome P 450 (low spin) Figure 11.7 Scheme for the oxygen atom transfer from the oxenoid complex of cytochrome P450 to the substrate THE FORMAnON OF TOXIC METABOLITES Alcohols and phenols are the most common products of the mono-oxygenation reaction. Very often, however, the oxygen atom transfer leads to unstable intermediates which can undergo subsequent reactions. Examples are the de alkylation of alkyl groups at heteroatoms, which are believed to proceed via hydroxylations at the 0: - carbon atom. Phenols may often, if not always, be products of rearrangements of arene oxide intermediates (Jerina et al., 1968) and arene oxides may be The Mechanism a/Cytochrome P450-Catalysed Drug Oxidations 211 isolated only if the rearomatisation energy is low enough. Since arene oxides are rather reactive electrophiles, further reactions with water by formation of dihydrodiols or with glutathione to form conjugates may occur. Under certain conditions even reactions with thiol groups of proteins or amino groups of nucleic acids resulting in covalent binding of the metabolites may take place (Waterfall and Sims, 1973). Such a chemical alteration of nucleic acids may lead to mutagenesis or carcinogenesis. The effects of covalent binding to proteins are more difficult to evaluate and pathological reactions are difficult to predict. CONCLUSIONS The mechanism of cytochrome P450 seems to be gradually unravelling, but still a number of questions remain open. Part of these remaining problems may be interesting only to chemists, who wonder about the unique coordination chem· istry of this cytochrome and the electronic structure of the oxenoid complex. The biochemists are still struggling with the isolation of the membrane -bound components and are trying to reconstitute the system. This has become considerably more complicated by the known existence of mUltiple forms of cyto· chrome P450. Also the mode of regulation of the biosynthesis of cytochrome P450 and the reductase is far from being understood. Most relevant, however, seems to be the toxification reactions which arise from the unspecificity of the system. The number of chemicals we are exposed to is increasing every day. Some of them may be reactive per se and can be easily identified. Many of the others seem harmless at first sight, but may be metabolised to reactive intermediates in the body, perhaps with resultant irreversible damage. Since the majority of malignant tumour growth may be traced back to environmental factors, it seems worthwhile to elucidate the mechanism of action of this cytochrome completely as a step towards understanding the relationship between the structure of a compound and its potential toxicity. REFERENCES Alvares, A. P., Leigh, S., Kappas, A., Levin, W. and Canney, A. H. (1973). Drug Metab. Disposition, 1,386 Brodie, B. B., Gillette, J. R. and LaDu, B. N. (1958).Ann. Rev. Biochem., 27,427 Canney, A. H., Levin, W., Jacobson, M., Kuntzman, R., Cooper, D. Y. and Rosenthal, O. (1969). Microsomes and Drug Oxidations (ed. Gillette, Conney, Cosmides, Estabrook, Fouts and Mannering), Academic Press, New York, p. 279 Diehl, H., Schiidelin, J. and Ullrich, V. (1970). 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