Cytochrome P450-Catalyzed Oxidation of Halobenzene Derivatives

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JUNE 1997 VOLUME 10, NUMBER 6 © Copyright 1997 by the American Chemical Society

Perspective Cytochrome P450-Catalyzed Oxidation of Halobenzene Derivatives Ivonne M. C. M. Rietjens,*,† Cathaline den Besten,‡ Robert P. Hanzlik,§ and Peter J. van Bladeren| Department of Biochemistry, Agricultural University, Dreijenlaan 3, 6703 HA Wageningen, The Netherlands, Department of Drug Safety, Solvay Duphar B.V., P.O. Box 900, 1381 CP Weesp, The Netherlands, Department of Medicinal Chemistry, University of Kansas, Lawrence, Kansas 66045, and Department of Toxicology, Agricultural University, Tuinlaan 5, 6703 HE Wageningen, and TNO Nutrition and Food Research Institution, Zeist, The Netherlands Received June 26, 1996

Introduction One of the major goals in the field of toxicology is to elucidate the mechanisms by which xenobiotics produce detrimental biological responses in organisms. A large number of xenobiotics are as such biologically inert, and many of their toxicological effects are mediated through the formation of ‘active’ metabolites. Knowledge on the different pathways by which a chemical can be metabolized into reactive products increases our understanding of the relationship between the chemical structure of xenobiotics and their effects on living systems. Moreover, such knowledge will be helpful in identifying chemical groupings in a given molecule, which predispose that molecule to a sequence of processes that might eventually lead to toxicity. Halogenated benzenes, considered relatively harmless because of their chemical stability, are thought to be bioactivated to products ultimately responsible for the observed toxic effects. Various cytochromes P450 play a * Address correspondence to this author: phone, 31-317-482868; fax, 31-317-484801; e-mail, [email protected]. † Agricultural University, Dreijenlaan. ‡ Solvay Duphar B.V. § University of Kansas. | Agricultural University, Tuinlaan, and TNO Nutrition and Food Research Institution.

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pivotal role in this metabolic activation. The present paper summarizes recent in vitro and in vivo studies on halogenated benzene derivatives with the aim of providing an updated view on the cytochrome P450-catalyzed oxidation of halobenzenes. Studies attempting to explain the mechanism of toxicity of halogenated aromatic hydrocarbons have mainly focused on the model compounds bromobenzene (1-4) and hexachlorobenzene (5-8). In recent years, the routes for metabolic activation of these compounds have been found to be more complicated than previously thought. The classical view is oxidative attack by cytochromes P450 resulting in the formation of an electrophilic intermediate, most commonly depicted as an epoxide, which covalently interacts with tissue macromolecules. The spontaneous isomerization of epoxides to phenols, the conversion of epoxides to dihydrodiols as catalyzed by epoxide hydrolases, and the conjugation of epoxides with glutathione, either spontaneously or enzymatically via the glutathione S-transferases, are alternative pathways that compete with the reaction of epoxides with tissue macromolecules (9, 10). As such, these pathways are detoxifying. However, secondary P450-catalyzed oxidation of phenols to hydroquinones, followed by their enzymatic and/or uncatalyzed oxidation to benzoquinones, represents an additional possibility for bioactivation © 1997 American Chemical Society

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Figure 1. Potential harmful metabolites (A-C) of a halobenzene structure (X ) halogen(s)).

of benzene derivatives, as quinones may elicit oxidative stress through redox cycling and may also act as electrophiles and arylate macromolecules (11-15). Enzymemediated conjugation of phenols and hydroquinones with glucuronic acid or sulfate may prevent the formation of reactive benzoquinones; alternatively, once formed, benzoquinones may be reduced to their corresponding hydroquinones, by means of various electron-transport enzymes. Finally, conjugation of quinones with glutathione can lead to the formation of products which can be excreted from the liver and are targeted to the kidney, where they may elicit a toxic response through mechanisms that are still largely unknown (16). Thus, as summarized in Figure 1, multiple pathways in the generation of (proximate) toxic metabolites can be envisaged departing from a simple halobenzene structure: (A) epoxides as primary metabolites, (B) benzoquinones as secondary metabolites, and (C) benzoquinonederived glutathione conjugates.

Alkylating Properties of Epoxides and Quinones Epoxide intermediates are notoriously harmful species in the bioactivation of many unsaturated chemicals (17, 18). For example, (diol) epoxides are the ultimate reactive species initiating polycyclic aromatic hydrocarboninduced carcinogenesis, and the epoxide of vinyl chloride is thought to be responsible for the vinyl chloride-induced liver cancer. The many studies on bromobenzeneinduced hepatotoxicity suggest a crucial role in this process for its 3,4-epoxide derivative, which covalently interacts with cellular macromolecules (1, 4). However, based on more recent experimental data, it seems likely that the alkylation by quinone metabolites is quantitatively at least as important as alkylation by epoxide metabolites of halogenated benzenes. This conclusion follows mainly from the following observations. First, upon incubations of radiolabeled chlorinated benzenes with rat liver microsomes, conversion-dependent covalent binding to protein was observed for all chlorinated benzenes tested, which amounted to about

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10-30% of the total metabolites formed, depending on the incubation conditions (cytochrome P450 concentration, substrate concentration, or incubation time). Not surprisingly, in the presence of the nucleophilic scavenger glutathione, this covalent binding was inhibited by 8090% and the formation of water-soluble conjugates was observed (5-7, 11, 15). This emphasizes the electrophilic nature of the reactive species. However, both arene oxides and benzoquinones are electrophilic species and thus capable of reacting with sulfhydryl groups. The observation that in microsomal incubations of halogenated benzenes, the presence of a reducing agent like ascorbic acid diminished covalent protein binding considerably (5-7, 11, 15, 19) is strongly in favor of the benzoquinone as the protein-binding species. Moreover, if epoxides were to be the species responsible for the major amount of covalent protein binding observed, the addition of 1,1,1-trichloropropene oxide (TCPO),1 a known epoxide hydrolase inhibitor which increases the life span of epoxides by inhibition of their detoxication, should have increased covalent binding. In contrast, both total oxidation and covalent binding were unaffected (15) or slightly decreased (5). A second line of evidence in favor of benzoquinones as the main alkylating species formed upon P450-mediated oxidation of chlorinated benzenes is found in the strong correlation between the rate of covalent binding and the rate of secondary metabolism of phenol metabolites to the corresponding hydroquinones, both demonstrating an inital lag phase in contrast to the initial burst of primary oxidation (15). Third, recent attempts to isolate and characterize bromobenzene-derived protein adducts from rat liver demonstrated that bromobenzene epoxide-derived proteinsulfur adducts account for less than ca. 1% of total protein adducts (20, 21). The S-alkylation of protein nucleophiles in vivo by bromobenzene-derived benzoquinone metabolites was 10-15 times more extensive (21), indicating that quinone metabolites derived from bromobenzene may have greater alkylating properties or are at least more effective in alkylation than the primary epoxide intermediates. An intriguing, if indirect, conclusion from these observations is that epoxide metabolites derived from halobenzenes appear to be rather weak alkylating agents, or they do not have as much opportunity to exert their electrophilic character as might be expected. This, then, sheds an interesting light on the role of epoxide intermediates in the toxicity of halogenated benzenes. The widely accepted view on bromobenzene-induced hepatotoxicity presumes a role for the 3,4-epoxide as the ultimate reactive species (1, 3, 4). This view is mainly based on indirect evidence such as the observation of a good correlation between, on the one hand, urinary excretion of the p-bromophenylmercapturic acid (formation of which involves the intermediacy of the 3,4-epoxide) and, on the other hand, depletion of cellular glutathione resulting in subsequent covalent binding of bromobenzene derivatives to protein. However, conjugation of bromobenzene 3,4-epoxide with glutathione has been shown to require enzymatic catalysis (3, 22), suggesting that in contrast to quinones this species has low spon1Abbreviations: TCPO, 1,1,1-trichloropropene oxide; CumOOH, cumene hydroperoxide; Fe(III)TDFPPCl-mCPBA ) meso-tetrakis(2,6difluorophenyl)porphinatoiron(III) chloride m-chloroperoxybenzoic acid; HOX, hypohalous acid.

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Figure 2. Reaction pathway of the cytochrome P450-catalyzed aromatic hydroxylation of (A) a chlorinated site para with respect to a substituent with an “acidic” proton in pentachlorophenol and (B) a fluorinated site para with respect to a substituent with an “acidic” proton in pentafluoroaniline.

taneous alkylating properties toward (protein-) sulfur nucleophiles. At this point, however, it is of importance to stress that the relationship of the extent to which a molecule undergoes covalent binding to tissue macromolecules is not necessarily a good prediction of its toxicity. Thus, the greater S-alkylating properties of quinone as compared to epoxide metabolites of bromobenzene need not necessarily imply a greater role for quinones in the toxic mechanism of the halobenzenes. Trans-Stilbene (23) and m-hydroxyacetanilide (24) become covalently bound to liver tissue but do not elicit hepatotoxicity. Monks et al. (4), comparing the toxicity and covalent binding of bromobenzene and 4-bromophenol in vivo, concluded that hepatotoxicity was more likely to be mediated by bromobenzene 3,4-epoxide, with 4-bromophenol giving rise to alkylating, but nontoxic (probably quinone), metabolites. However, depletion of glutathione by quinone metabolites might enhance the chances for covalent binding of the epoxide. Clearly, the total amount of covalent binding of metabolites does not necessarily predict the toxicity of a compound (25, 26), but the interplay of the various reactive metabolites deserves further attention.

Mechanism of Benzoquinone Formation The apparent importance of benzoquinone metabolites in the metabolic activation of chlorinated benzenes to alkylating, potentially harmful metabolites warrants a closer look at the mechanism of formation of these reactive products from the primary phenol metabolites. In the past it has been suggested that oxidation of arenes at a halogenated position, as in the case of pentachlorophenol, proceeds by a mechanism analogous to arene oxidation at a nonhalogenated position resulting in a hydroxylated product (7, 27). For example, tetrachlorohydroquinone has been detected as a metabolite of pentachlorophenol both in vitro (7) and in vivo (28). Taking the cytochrome P450 reaction as a net twoelectron transfer, the halogen in this reaction is formally lost as a halogen cation. However, the nature of the halogen ion lost has never been unambiguously demonstrated. Interestingly, support for the formation of electrophilic halogen could not be obtained by trapping experiments with 2,4- or 2,6-dimethylphenol added to a microsomal incubation with pentachlorophenol or hexachlorobenzene,2 although this method has been success2Van

Ommen, unpublished results.

fully applied to demonstrate the formation of chlorine cations in studies on the dechlorination of carbon tetrachloride (29). Direct formation of tetrachlorobenzoquinone upon oxidation of pentachlorophenol accompanied by loss of a halogen anion thus seems a much more likely alternative pathway (8) (Figure 2A). Exclusive evidence for the nature of the primary oxidation products of halophenols (i.e., either hydroquinones or benzoquinones) can not be obtained under standard conditions with molecular oxygen and NADPH as the electron donor for the cytochrome P450-mediated oxidation, since (i) the direct formation of benzoquinones may be masked by their swift covalent binding to proteins due to their high reactivity (11) and (ii) the presence of reducing agents like NADPH makes it impossible to probe the cytochrome P450-dependent oxidation independently, since any benzoquinone formed may subsequently be directly reduced to the corresponding hydroquinone, either by NAD(P)H alone or through NAD(P)Hcytochrome reductase and/or cytochrome P450 (30-32). These problems were circumvented in recent studies on the cytochrome P450-catalyzed conversion of parafluorinated anilines by using alternative oxygen donors. The direct formation of benzoquinonimines with the concomitant loss of fluoride anions was observed (33, 34). In the presence of reducing equivalents like NAD(P)H, the benzoquinonimine is reduced to give the corresponding aminophenol (Figure 2B). In analogy to these studies, anaerobic incubations with rat liver microsomes were performed using fluorophenols as model substrates and cumene hydroperoxide (CumOOH) as the oxygen donor (32). These experiments demonstrated that the mechanism of benzoquinone formation is dependent on the substitution pattern of the halophenol precursor. As depicted in Figure 3A, cytochrome P450-mediated C4-oxidation of a halogenated phenol unsubstituted at C4 proceeds via the formation of the hydroquinone as the primary product. This hydroquinone may subsequently be oxidized to its benzoquinone derivative by a cytochrome P450-mediated reaction (11), by as yet unidentified enzymes (11), or through autoxidation followed by disproportionation of the semiquinone radicals formed. However, the cytochrome P450-catalyzed oxidation of a C4-halogenated phenol (Figure 3B) was demonstrated to result in a primary product that could be reduced to the hydroquinone in the presence of NAD(P)H (32). Based on these considerations, it is proposed that the cytochrome P450catalyzed oxidation of a para-halogenated phenol proceeds by the direct formation of a benzoquinone metabo-

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Figure 3. Reaction pathway of the cytochrome P450-catalyzed aromatic hydroxylation of (A) a nonhalogenated C4 position in a halophenol and (B) a halogenated C4 position in a halophenol (32-36).

Figure 4. Oxidative dehalogenation of hexahalogenated benzenes. The order of reduction, dehalogenation, and product release is not well established and can be, as indicated, either dehalogenation and product release followed by reduction of the halophenoxenium cation (pathway I) or dehalogenation followed by reduction and product release (pathway II). The additional two electrons required may be provided to the enzyme-bound intermediate by the NADPH/NADPH-cytochrome reductase/cytochrome P450 pathway (30, 44). The halophenoxenium cation is an additional resonance form of a reaction product able to dissociate from the enzyme before being reduced (pathway I) or, perhaps more likely, an additional resonance form of an intermediate that remains bound to the active site and is reduced before being released (pathway II).

lite without the obligatory intermediacy of the hydroquinones. In a recent study, Ohe et al. (35) even demonstrated the formation of benzoquinone as the metabolite resulting from conversion of C4-halogenated phenols by a chemical cytochrome P450 model system, i.e., the meso-tetrakis(2,6-difluorophenyl)porphinatoiron(III) chloride m-chloroperoxybenzoic acid (Fe(III)TDFPPCl-mCPBA) system. The ease of halogen elimination in this cytochrome P450 model system (35), and also in a microsomal cytochrome P450-catalyzed oxidative dehalogenation (36), decreases in the order F > Cl > Br > I, as well as with the introduction of additional electron-withdrawing halogen substituents in the aromatic ring (35, 36). This observation has implications in the field of drug and agrochemical development, because incorporation of a fluorine

substituent in a drug or agrochemical has been suggested as a means of blocking biodegradation or bioactivation of the compound (37-43). Interestingly, for fully halogenated benzenes, a mechanism of cytochrome P450-catalyzed oxidative dehalogenation has been proposed that is comparable to that suggested for the phenols (see Figure 4). The reaction pathway may proceed through the formation of a halophenoxenium cation intermediate in the oxidative dehalogenation to pentahalogenated phenols (44). This mechanism includes formation of a halophenoxenium cation with a (partial) positive charge on a halogen substituent para or ortho with respect to the position of oxidative attack and subsequent elimination of the halogen from the molecule as a halogen anion. The halophenoxenium cation intermediate might be formed either directly or

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Figure 5. Proposed mechanism for cytochrome P450-catalyzed oxidative dehalogenation of 4-iodoanisole (30). The reaction is suggested to proceed either (pathway I) through a cation intermediate similar to that suggested for halophenols, haloanilines, and halobenzenes (Figures 2-4) or (pathway II) through anion elimination following or accompanying the two-electron reduction step, eliminating the role for a cation intermediate.

Figure 6. Reaction pathway for cytochrome P450-catalyzed monooxygenation of halogenated benzenes, taking into account (i) the direct formation of phenolic metabolites and (ii) the formation of the quinone type of metabolite as the primary metabolite in the case of oxidative dehalogenation. The role of keto intermediates is also taken into account, since these are already recognized as possible alternative intermediates in the route leading to formation of phenolic products as well as in the route leading to NIHshifted phenolic metabolites (48-53).

through an epoxide intermediate, and its actual status in the reaction cycle, i.e., an independent non-enzymebound transient intermediate (Figure 4, pathway I), an enzyme-bound reaction intermediate that becomes reduced to the phenol before being released (Figure 4, pathway II), or only an additional resonance form along the reaction pathway, still needs to be established.

Recently, Rizk and Hanzlik (30) demonstrated the oxidative deiodination of 4-iodoanisole to 4-hydroxyanisole to occur through similar intermediates. The oxidation was not accompanied by a detectable formation of iodinating intermediates, i.e., the iodine was not eliminated as a cation. Rather, the iodine was apparently lost as an anion, requiring two additional reduction equiva-

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lents for formation of the 4-hydroxyanisole metabolite. These electrons were suggested to originate from NADPH donated through NADPH-cytochrome P450 reductase and could be donated to the reactive primary intermediate metabolite either before or after removal of the halogen as an anion (Figure 5). Clearly this pathway for oxidative deiodination provides an alternative for the aromatic dehalogenation of iodinated benzene derivatives through halogen oxidation and formation of iodosyl compounds (8, 45, 46). This latter mechanism, proceeding by oxygenation of a halogen substituent, followed by hypohalous acid (HOX) elimination and phenol formation as a result of H2O addition, was already excluded in the literature for the elimination of fluorine and chlorine substituents on the basis of the observation that no 18O from 18O-labeled H2O was incorporated into the phenol formed from hexachlorobenzene (6, 8), as well as on the basis of the observation that in oxidative dehalogenation of pentafluorochlorobenzene a fluorine was eliminated more easily than a chlorine substituent (44). Based on the observations summarized in Figures 2-5, it becomes tempting to suggest that the proposed pathway represents a general mechanism for the cytochrome P450-dependent oxidative dehalogenation of substrates, which possess either a substituent with an acidic proton or a mesomeric electron-donating substituent ortho or para with respect to the position to be oxidized.

Role of Epoxides in the Pathway to Phenolic Metabolites The reaction schedules as presented in Figures 2-5 also pose a question related to the role of epoxides as intermediates in the pathway leading to formation of phenolic metabolites. Epoxides, once formed, can rearrange to give phenols (9, 10, 47), but this does not necessarily imply that all phenolic metabolites are formed through an epoxide precursor. Thus, the formation of meta-hydroxylated metabolites from a halobenzene can not proceed through epoxide intermediates, since the chemically synthesized halobenzene 2,3-oxide and -3,4oxide give rise to exclusive formation of respectively the o- and p-halophenol (47). More recent studies, e.g., on the hydroxylation of deuterated bromobenzenes (48) and chlorobenzenes (49), provide evidence for pathways leading to formation of phenolic metabolites from halogenated benzene derivatives without the intermediacy of epoxide and/or cyclohexadienone intermediates. Furthermore, on the basis of the absence of any deuterium loss upon conversion of deuterated [3,5-2H2]-4-iodoanisole to 2-methoxy-5-iodophenol, Rizk and Hanzlik (30) also concluded that this aromatic hydroxylation must occur almost entirely by direct aromatic hydroxylation without the formation of an epoxide or cyclohexadienone intermediate. The regioselectivity of the aromatic hydroxylation of a series of fluorobenzenes could be predicted on the basis of the calculated reactive π-electron density in the fluorobenzenes (50), indicating that the site of attack by the electrophilic ferryl-oxo group of the cytochrome P450 on the π-electrons of the fluorobenzene is also the site of hydroxylation. This observation, together with the absence of formation of NIH-shifted phenolic metabolites from the fluorobenzenes, eliminates an obligatory role for epoxides in the formation of phenolic metabolites from the fluorobenzenes. Thus, when the binding site or substrate substituents do not impose sterical hindrance leading to a selective orientation of the substrate in the

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active site, the initial attack on the arene is governed by the reactive π-electron density of the substrate. The collapse of the complex thus formed may lead to epoxides or cyclohexadienones, but it may also lead directly to phenols and, as indicated in this review, directly to benzoquinones. Thus, the view on the oxidation of halobenzenes can be updated to the scheme in Figure 6. For oxidative dehalogenation at halogenated sites, the metabolite initially formed can be a reactive benzoquinone for halophenols, a benzoquinonimine for haloanilines, or a halophenoxenium cation for halobenzenes. These intermediates require an additional two-electron reduction before the hydroxylated metabolite and a halogen anion can be formed. Since benzoquinones and benzoquinonimines are highly reactive and covalently interact with cellular macromolecules, this pathway reflects a novel route of possible significance to the toxicity in vivo.

Acknowledgment. This research was supported in part by NIH Grant GM21784 to R.P.H.

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