26510318-Bio-Transformation-of-Xenobiotics

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can be inhibited by certain epoxides, such as 1,1,1-trichloropropene oxide and cyclohexene oxide, and certain drugs, such as valpromide (the amide analog of valproic acid) and progabide, a-aminobutyric acid (GABA) agonist. These latter two drugs potentiate the neurotoxicity of carbamazepine by inhibiting epoxide hydrolase, leading to increased plasma levels of carbamazepine 10,11-epoxide and presumably the more toxic 2,3-epoxide (Kroetz et al., 1993). Several genetic polymorphisms have been identified in the coding region and the 5 region (i.e., the regulatory region) of the gene encoding human mEH (Daly, 1999). Two variants involve substitutions at amino acid 113 (Tyr His) or amino acid 139 (His Arg), which are encoded by exons 3 and 4, respectively. Although these allelic variant forms of mEH have near normal enzymatic activity (at least 65 percent of normal), they appear to be less stable than the wild-type enzyme. The possibility that these amino acid substitutions might predispose individuals to the adverse effects of antiepileptic drugs has been examined, but no such association was found (Daly, 1999).

The mechanism of catalysis by epoxide hydrolase is similar to that of carboxylesterase, in that the catalytic site comprises three amino acid residues that form a catalytic triad. In mEH, Asp226 functions as the nucleophile, His431 the base, and both Glu376 and Glu404 as the acid, as shown in Fig. 6-7. (In sEH, the corresponding residues are Asp333, His503, and Asp495.) The attack of the nucleophile Asp226 on the carbon of the oxirane ring initiates enzymatic activity, leading to the formation of an -hydroxyester- enzyme intermediate, with the negative charge developing on the oxygen atom stabilized by a putative oxyanion hole. The His431 residue (which is activated by Glu376 and Glu404) activates a water molecule by abstracting a proton (H ). The activated (nucleophilic) water then attacks the C atom of Asp226, resulting in the hydrolysis of the ester bond in the acyl-enzyme intermediate, which restores the active enzyme and results in formation of a vicinal diol with a trans-configuration (Armstrong, 1999). The second step, namely cleavage of the ester bond in the acyl-enzyme intermediate, resembles the cleavage of the ester or amide bond in substrates for serine esterases and proteases.

Although epoxide hydrolase and carboxylesterase both have a catalytic triad comprising a nucleophilic, basic, and acidic amino acid residue, there are striking differences in their catalytic machinery, which accounts for the fact that carboxylesterases primarily hydrolyze esters and amides whereas epoxide hydrolases primarily hydrolyze epoxides and oxides. In the triad, both enzymes have histidine as the base and either glutamate or aspartate as the acid, but they differ in the type of amino acids for the nucleophile. Even during catalysis, there is a major difference. In carboxylesterases, the same carbonyl carbon atom of the substrate is attacked initially by the nucleophile Ser203 to form -hydroxyester- enzyme ester that is subsequently attacked by the activated water to release the alcohol product. In contrast, two different atoms in epoxide hydrolase are targets of nucleophilic attacks. First the less hindered carbon atom of the oxirane ring is attacked by the nucleophile Asp226 to form a covalently bound ester, and next this ester is hydrolyzed by an activated water that attacks the C atom of the Asp226 residue, as illustrated in Fig. 6-7. Therefore, in carboxylesterase, the oxygen introduced into the product is derived from the activated water molecule. In contrast, in epoxide hydrolase, the oxygen introduced into the product is derived from the nucleophile Asp226 (Fig. 6-7).

Carboxylesterases and epoxide hydrolases exhibit no primary sequence identity, but they share surprising similarities in the topol-

Figure 6-7. Catalytic cycle of microsomal carboxylesterase (left) and microsomal epoxide hydrolase (right), two A/B-hydrolase fold enzymes.

ogy of the structure and sequential arrangement of the catalytic triad. Both are members of the / -hydrolase fold enzymes, a superfamily of proteins that includes lipases, esterases and haloalkane dehydrogenases (Beetham et al., 1995; Armstrong, 1999). Functionally, proteins in this superfamily all catalyze hydrolytic reactions; structurally, they all contain a similar core segment that comprises eight -sheets connected by -helices. They all have a catalytic triad, and the arrangement of the amino acid residues in the triad (i.e., the order of the nucleophile, the acid and the base in the primary sequence) is the mirror image of the arrangement in other hydrolytic enzymes such as trypsin. All three active-site

144	UNIT 2 DISPOSITION OF TOXICANTS

residues are located on loops that are the best conserved structural features in the fold, which likely provides catalysis with certain flexibility to hydrolyze numerous structurally distinct substrates.

Reduction

Certain metals (e.g., pentavalent arsenic) and xenobiotics containing an aldehyde, ketone, disulfide, sulfoxide, quinone, N-oxide, alkene, azo, or nitro group are often reduced in vivo, although it is sometimes difficult to ascertain whether the reaction proceeds enzymatically or nonenzymatically by interaction with reducing agents [such as the reduced forms of glutathione, FAD, FMN and NAD(P)]. Some of these functional groups can be either reduced or oxidized. For example, aldehydes (RCHO) can be reduced to an alcohol (RCH2OH) or oxidized to a carboxylic acid (RCOOH), whereas sulfoxides (R1SOR2) can be reduced to a sulfide (R1SR2) or oxidized to a sulfone (R1SO2R2). In the case of halogenated hydrocarbons, such as halothane, dehalogenation can proceed by an oxidative or reductive pathway, both of which are catalyzed by the same enzyme (namely cytochrome P450). In some cases, such as azo-reduction, nitro-reduction, and the reduction of some alkenes (e.g., cinnamic acid, C6H5CH PCHCOOH), the reaction is largely catalyzed by intestinal microflora. Many of the reduction reactions described below (including azo-, nitro-, sulfoxide, and N-oxide reduction) can be catalyzed by aldehyde oxidase, but this does not appear to be the major enzyme responsible for any of the various reductive pathways of xenobiotic biotransformation.

Azoand Nitro-Reduction Prontosil and chloramphenicol are examples of drugs that undergo azoand nitro-reduction, respectively, as shown in Fig. 6-8 (Herwick, 1980). Reduction of prontosil is of historical interest. Treatment of streptococcal and pneumococcal infections with prontosil marked the beginning of specific antibacterial chemotherapy. Subsequently, it was discovered that the active drug was not prontosil but its metabolite, sul-

fanilamide (para-aminobenzene sulfonamide), a product of azoreduction. During azo-reduction, the nitrogen–nitrogen double bond is sequentially reduced and cleaved to produce two primary amines, a reaction requiring four reducing equivalents. Nitroreduction requires six reducing equivalents, which are consumed in three sequential reactions, as shown in Fig. 6-8 for the conversion of nitrobenzene to aniline.

Azoand nitro-reduction are catalyzed by intestinal microflora and by two liver enzymes: cytochrome P450 and NAD(P)H- quinone oxidoreductase (a cytosolic flavoprotein, also known as DT-diaphorase). Under certain circumstances, a third liver enzyme, aldehyde oxidase, may also catalyze azoand nitro-reduction reactions. The reactions require NAD(P)H and are inhibited by oxygen. The anaerobic environment of the lower gastrointestinal tract is well suited for azoand nitro-reduction, which is why intestinal microflora contribute significantly to these reactions. Most of the reactions catalyzed by cytochrome P450 involve oxidation of xenobiotics. Azoand nitro-reduction are examples in which, under conditions of low oxygen tension, cytochrome P450 can catalyze the reduction of xenobiotics.

Nitro-reduction by intestinal microflora is thought to play an important role in the toxicity of several nitroaromatic compounds, including 2,6-dinitrotoluene, which is hepatotumorigenic to male rats. The role of nitro-reduction in the metabolic activation of 2,6- dinitrotoluene is shown in Fig. 6-9 (Long and Rickert, 1982; Mirsalis and Butterworth, 1982). The biotransformation of 2,6- dinitrotoluene begins in the liver, where it is oxidized by cytochrome P450 and conjugated with glucuronic acid. This glucuronide is excreted in bile and undergoes biotransformation by intestinal microflora. One or more of the nitro groups are reduced to amines by nitroreductase, and the glucuronide is hydrolyzed by-glucuronidase. The deconjugated metabolites are absorbed and transported to the liver, where the newly formed amine group is N-hydroxylated by cytochrome P450 and conjugated with acetate or sulfate. These conjugates form good leaving groups, which ren-

Figure 6-8. Examples of drugs that undergo azo reduction (prontosil) and nitro reduction (chloramphenicol

and nitrobenzene).

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Figure 6-9. Role of nitro reduction by intestinal microflora in the activation of the rat liver tumorigen, 2,6-dinitrotoluene.

der the nitrogen highly susceptible to nucleophilic attack from proteins and DNA; this ostensibly leads to mutations and the formation of liver tumors. The complexity of the metabolic scheme shown in Fig. 6-9 underscores an important principle, namely that the activation of some chemical tumorigens to DNA-reactive metabolites involves several different biotransforming enzymes and may take place in more than one tissue. Consequently, the ability of 2,6-dinitrotoluene to bind to DNA and cause mutations is not revealed in most of the short-term assays for assessing the genotoxic potential of chemical agents. These in vitro assays for genotoxicity do not make allowance for biotransformation by intestinal microflora or, in some cases, the phase II (conjugating) enzymes.

Nitro-reduction by intestinal microflora also plays an important role in the biotransformation of musk xylene (1,3,5-trinitro-2- tbutyl-4,6-dimethylbenzene). Reduction of one or both of the nitro groups is required for musk xylene to induce (as well as markedly inhibit) liver microsomal cytochrome P450 (namely CYP2B) in rodents (Lehman-McKeeman et al., 1999).

Carbonyl Reduction The reduction of certain aldehydes to primary alcohols and of ketones to secondary alcohols is catalyzed by alcohol dehydrogenase and by a family of carbonyl reductases (Weiner and Flynn, 1989). Carbonyl reductases are monomeric, NADPH-dependent enzymes present in blood and the cytosolic fraction of the liver, kidney, brain, and other tissues. The major circulating metabolite of the antipsychotic drug haloperidol is a secondary alcohol formed by carbonyl reductases in the blood and liver, as shown in Fig. 6-10 (Inaba and Kovacs, 1989). Other xeno-

biotics that are reduced by carbonyl reductases include pentoxifylline (see Fig. 6-1), acetohexamide, daunorubicin, ethacrynic acid, warfarin, menadione, and 4-nitroacetophenone. The reduction of ketones to secondary alcohols by carbonyl reductases may proceed with a high degree of stereoselectivity, as in the case of pentoxifylline (Fig. 6-1) (Lillibridge et al., 1996). Prostaglandins are possibly physiologic substrates for carbonyl reductases, and they most certainly are substrates for a class of related enzymes known as prostaglandin dehydrogenases (discussed later in this section).

In liver, carbonyl reductase activity is present mainly in the cytosolic fraction, but a different carbonyl reductase is present in the microsomal fraction. These enzymes differ in the degree to which they stereoselectively reduce ketones to secondary alcohols. For example, keto-reduction of pentoxifylline produces two enantiomeric secondary alcohols: one with the R-configuration (which is known as lisofylline) and one with the S-configuration, as shown in Fig. 6-1. Reduction of pentoxifylline by cytosolic carbonyl reductase results in the stereospecific formation ( 95 percent) of the optical antipode of lisofylline, whereas the same reaction catalyzed by microsomal carbonyl reductase produces both lisofylline and its optical antipode in a ratio of about 1 to 5 (Lillibridge et al., 1996).

In rat liver cytosol, the reduction of quinones is primarily catalyzed by DT-diaphorase (see “Quinone Reduction,” below), whereas in human liver cytosol, quinone reduction is catalyzed by both DT-diaphorase and carbonyl reductases. Human liver cytosol appears to contain more than one carbonyl reductase. The activity of lowand high-affinity carbonyl reductase activity in human liver cytosol varies about tenfold among individuals (Wong et al., 1993).

146	UNIT 2 DISPOSITION OF TOXICANTS

Figure 6-10. Reduction of xenobiotics by carbonyl reductase (A) and alcohol dehydrogenase (B).

The number of carbonyl reductases involved in xenobiotic biotransformation is difficult to assess.

Structurally, carbonyl reductases belong to the short-chain dehydrogenase/reductase (SDR) superfamily (which includes certain hydroxysteroid dehydrogenases and prostaglandin dehydrogenases), although certain aldehyde reductases belong to the aldoketo reductase (AKR) superfamily (which include other hydroxysteroid dehydrogenases and aldose reductases) (Penning, 1997). This latter superfamily of enzymes includes several forms of dihydrodiol dehydrogenase, which are monomeric ( 34 kDa), cytosolic NADP(H)-requiring oxidoreductases that oxidize the trans- dihydrodiols of various polycyclic aromatic hydrocarbons to the corresponding ortho-quinones, which is depicted in Fig. 6-6 (Burczynski and Penning, 2000).

In certain cases, the reduction of aldehydes to alcohols can be catalyzed by alcohol dehydrogenase, as shown in Fig. 6-10 for the conversion of the sedative-hypnotic chloral hydrate to trichloroethanol. Alcohol dehydrogenase typically converts alcohols to aldehydes. In the case of chloral hydrate, the reverse reaction is favored by the presence of the trichloromethyl group, which is a strong electron-withdrawing group.

Disulfide Reduction Some disulfides are reduced and cleaved to their sulfhydryl components, as shown in Fig. 6-11 for the alcohol deterrent disulfiram (Antabuse). As shown in Fig. 6-11, disulfide reduction by glutathione is a three-step process, the last step of which is catalyzed by glutathione reductase. The first steps can be catalyzed by glutathione S-transferase, or they can occur nonenzymatically.

Sulfoxide and N-Oxide Reduction Thioredoxin-dependent enzymes in liver and kidney cytosol have been reported to reduce sulfoxides, which themselves may be formed by cytochrome P450 or flavin monooxygenases (Anders et al., 1981). It has been suggested that recycling through these counteracting enzyme systems may prolong the half-life of certain xenobiotics. Sulindac is a sulfoxide that undergoes reduction to a sulfide, which is excreted in bile and reabsorbed from the intestine (Ratnayake et al., 1981). This enterohepatic cycling prolongs its duration of action, such that this

nonsteroidal anti-inflammatory drug (NSAID) need only be taken twice daily. Diethyldithiocarbamate methyl ester, a metabolite of disulfiram, is oxidized to a sulfine, which is reduced to the parent methyl ester by glutathione. In the latter reaction, two molecules of glutathione (GSH) are oxidized with reduction of the sulfine oxygen to water (Madan et al., 1994), as shown below:

R1R2CPS OO 2 GSH R1R2CPS GSSG H2O

Just as sulfoxide reduction can reverse the effect of sulfoxidation, so the reduction of N-oxides can reverse the N-oxygenation of amines, which is catalyzed by flavin monooxygenases and cytochrome P450. Under reduced oxygen tension, reduction of the N-oxides of imipramine, tiaramide, indicine, and N,N-dimethyl- aniline can be catalyzed by mitochondrial and/or microsomal enzymes in the presence of NADH or NADPH (Sugiura and Kato, 1977). The NADPH-dependent reduction of N-oxides in liver mi-

Figure 6-11. Biotransformation of disulfiram by disulfide reduction (A) and the general mechanism of glutathione-dependent disulfide reduction of xenobiotics (B).

ABBREVIATIONS: GSH, glutathione; XSSX, xenobiotic disulfide; GSSG, reduced glutathione. The last reaction in (B) is catalyzed by glutathione reductase.

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crosomes appears to be catalyzed by cytochrome P450 (Sugiura et al., 1976), although in some cases NADPH-cytochrome P450 reductase may play an important role.

As a class, N-oxides are not inherently toxic compounds. However, certain aromatic and aliphatic N-oxides have been exploited as bioreductive drugs (also known as DNA-affinic drugs) for the treatment of certain cancers and infectious diseases (Wardman et al., 1995). In these cases, N-oxides have been used as prodrugs that are converted to cytotoxic or DNA-binding drugs under hypoxic conditions. The fact that N-oxides of certain drugs are converted to toxic metabolites under hypoxic conditions is the basis for their selective toxicity to certain solid tumors (namely those that are hypoxic and hence resistant to radiotherapy) and anaerobic bacteria. For example, tirapazamine (SR 4233) is a benzotriazine di-N-oxide that is preferentially toxic to hypoxic cells, such as those present in solid tumors, apparently due to its rapid activation by one-electron reduction of the N-oxide to an oxidizing nitroxide radical, as shown in Fig. 6-12 (Walton et al., 1992). This reaction is catalyzed by cytochrome P450 and NADPH-cytochrome P450 reductase (Saunders et al., 2000). Two-electron reduction of the di-N-oxide, SR 4233, produces a mono-N-oxide, SR 4317, which undergoes a second N-oxide reduction to SR 4330. Like SR 4233, the antibacterial agent quindoxin is a di-N-oxide whose cy-

totoxicity is dependent on reductive activation, which is favored by anaerobic conditions.

Bioreductive alkylating agents, which include such drugs as mitomycins, anthracyclines, and aziridinylbenzoquinones, represent another class of anticancer agents that require activation by reduction. However, for this class of agents, bioactivation also involves a two-electron reduction reaction, which is largely catalyzed by DT-diaphorase, described in the next section.

Quinone Reduction Quinones can be reduced to hydroquinones by NAD(P)H-quinone oxidoreductase, a cytosolic flavoprotein also known as DT-diaphorase (Ernster, 1987; Riley and Workman, 1992). An example of this reaction is shown in Fig. 6-13. Formation of the relatively stable hydroquinone involves a two-electron reduction of the quinone with stoichiometric oxidation of NAD[P]H without oxygen consumption. The two-electron reduction of quinones also can be catalyzed by carbonyl reductase, especially in humans. Although there are exceptions (see below, this section), this pathway of quinone reduction is essentially nontoxic; that is, it is not associated with oxidative stress, unlike the oneelectron reduction of quinones by NADPH-cytochrome P450 reductase (Fig. 6-13). In addition to quinones, substrates for DTdiaphorase include a variety of potentially toxic compounds, in-

Figure 6-12. Examples of sulfoxide and N-oxide reduction.

Note that tirapazamine (3-amino-1,2,4-benzotriazine-1,4-dioxide or SR4233) is a representative of a class of agents that are activated by reduction, which may be clinically useful in the treatment of certain tumors.

148	UNIT 2 DISPOSITION OF TOXICANTS

Figure 6-13. Two-electron reduction of menadione to a hydroquinone, and production of reactive oxygen species during its one-electron reduction to a semiquinone radical.

cluding quinone epoxides, quinoneimines, azo dyes, and C-nitroso derivatives of arylamines.

The second pathway of quinone reduction is catalyzed by NADPH-cytochrome P450 reductase (a microsomal flavoprotein) and results in the formation of a semiquinone free radical by a one-electron reduction of the quinone. Semiquinones are readily autooxidizable, which leads to nonstoichiometric oxidation of NADPH and oxygen consumption. The oxidative stress associated with autooxidation of a semiquinone free radical, which produces superoxide anion, hydrogen peroxide, and other active oxygen species, can be extremely cytotoxic, as illustrated in Fig. 6-13 for menadione. Oxidative stress appears to be an important component to the mechanism of toxicity of several xenobiotics that either contain a quinone or can be biotransformed to a quinone (Anders, 1985). The production of superoxide anion radicals and oxidative stress are responsible, at least in part, for the cardiotoxic effects of doxorubicin (adriamycin) and daunorubicin (daunomycin), the pulmonary toxicity of paraquat and nitrofurantoin, and the neurotoxic effects of 6-hydroxydopamine. Oxidative stress also plays an important role in the destruction of pancreatic beta cells by alloxan and dialuric acid. Tissues low in superoxide dismutase activity, such as the heart, are especially susceptible to the oxidative stress associated with the redox cycling of quinones. This accounts, at least in part, for the cardiotoxic effects of adriamycin and related anticancer agents.

DT-diaphorase levels are often elevated in tumor cells, which has implications for cancer chemotherapy with agents that are biotransformed by DT-diaphorase (Riley and Workman, 1992). Some cancer chemotherapeutic agents are inactivated by DT-diaphorase, such as SR 4233 (Fig. 6-12), whereas others are activated to cytotoxic metabolites, such as mitomycins, anthracyclines, and aziridinylbenzoquinones. These so-called bioreductive alkylating agents are reduced by DT-diaphorase to generate reactive intermediates that undergo nucleophilic additions with DNA, resulting in single-strand DNA breaks. The reason such drugs are preferentially toxic to tumor cells is that tumor cells, especially those in solid tumors, are hypoxic, and hypoxia induces the synthesis of DT-diaphorase [by a mechanism that involves the activator protein 1 (AP-1) and nuclear factor- B (NF- B) response elements in the 5 -promoter region of the DT-diaphorase gene]. Therefore, tumor cells often express high levels of DT-diaphorase, which predisposes them to the toxic effects of mitomycin C and related indolequinones. Interestingly, mitomycin C also up-regulates the expression of DT-diaphorase, which may enable this anticancer drug to stimulate its own metabolic activation in tumor cells (Yao et al., 1997). Induction of DT-diaphorase by mitomycin C, like that by hypoxia, involves transcriptional activation of the AP-1 and NF- B response elements in the 5 -promoter region of the DT-diaphorase gene. However, whereas activation of the DTdiaphorase AP-1 response element by mitomycin C involves both

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149

Jun and Fos, its activation by hypoxia involves only jun family dimers (Yao et al., 1997). Other factors that regulate the expression of DT-diaphorase are described below (this section).

It is now apparent that the structure of the hydroquinones produced by DT-diaphorase determines whether the two-electron reduction of quinones results in xenobiotic detoxication or activation. Hydroquinones formed by two-electron reduction of unsubstituted or methyl-substituted 1,4-naphthoquinones (such as menadione) or the corresponding quinone epoxides are relatively stable to autooxidation, whereas the methoxyl, glutathionyl, and hydroxyl derivatives of these compounds undergo autooxidation with production of semiquinones and reactive oxygen species. The ability of glutathionyl derivatives to undergo redox cycling indicates that conjugation with glutathione does not prevent quinones from serving as substrates for DT-diaphorase. The glutathione conjugates of quinones can also be reduced to hydroquinones by carbonyl reductases, which actually have a binding site for glutathione. In human carbonyl reductase, this binding site is Cys227, which is involved in binding both substrate and glutathione (Tinguely and Wermuth, 1999). Although oxidative stress is an important mechanism by which quinones cause cellular damage (through the intermediacy of semiquinone radicals and the generation of reactive oxygen species), it should be noted that quinones are Michael acceptors, and cellular damage can occur through direct alkylation of critical cellular proteins and/or DNA (reviewed in Bolton et al., 2000).

DT-diaphorase is a dimer of two equal subunits (Mr 27 kDa) each containing FAD. Mouse, rat, and human appear to possess two, three, and four forms of DT-diaphorase, respectively (Riley and Workman, 1992). The human enzymes are encoded by four distinct gene loci (DIA 1 through DIA 4). The fourth gene locus encodes the form of DT-diaphorase known as NADPH-quinone ox- idoreductase-1 (NQO1), which accounts for the majority of DTdiaphorase activity in most human tissues. This enzyme is inducible (see below, this section), and is the ortholog of rat NQO1. A second, noninducible form of DT-diaphorase, NQO2, is polymorphically expressed in humans (Jaiswal et al., 1990).

DT-diaphorase is inducible up to tenfold by two classes of agents, which have been categorized as bifunctional and monofunctional inducers (Prochaska and Talalay, 1992). The bifunctional agents include compounds like -naphthoflavone, benzo[a]pyrene, 3-methylcholanthrene and 2,3,7,8-tetrachloro- dibenzo-p-dioxin (TCDD or dioxin), which induce both phase I enzymes (such as the cytochrome P450 enzyme known as CYP1A1) and phase II enzymes (such as glutathione S-transferase and uridine diphosphate [UDP]-glucuronosyltransferase). These agents signal through two distinct mechanisms, one involving the XRE (xenobiotic-response element) and one involving the ARE (antioxidant response element), which is also known as the EpRE (electrophilic response element). (Response elements are short sequences of DNA, often located upstream in the 5 -region of a gene, that bind the transcription factors controlling gene expression.) Some enzymes, such as CYP1A1, are largely regulated by the XRE, whereas others, such as glutathione S-transferase, are largely regulated by ARE. Some enzymes, such as DT-diaphorase, are regulated by both.

In order to activate the XRE and induce the synthesis of CYP1A1, the so-called bifunctional agents must first bind to a receptor protein called the Ah receptor, which is discussed later on under “Induction of Cytochrome P450.” Unlike the bifunctional

agents, the monofunctional agents do not bind to the Ah receptor and, therefore, do not induce CYP1A1. However, like the bifunctional agents, the monofunctional agents can induce the synthesis of those phase II enzymes that are regulated by the ARE (which includes DT-diaphorase).

The monofunctional agents can be subdivided into two chemical classes: those that cause oxidative stress through redox cycling (e.g., the quinone, menadione, and the phenolic antioxidants tert- butylhydroquinone and 3,5-di-tert-butylcatechol) and those that cause oxidative stress by depleting glutathione (e.g., fumarates, maleates, acrylates, isothiocyanates, and other Michael acceptors that react with glutathione).

As previously mentioned, agents that signal through the XRE do so by binding to the Ah receptor. Agents that signal through the ARE do so by an incompletely characterized mechanism. AREs generally contain two AP1/AP1-like elements arranged as inverse or direct repeats with a 3- to 8-base-pair interval and followed by a “GC” box. Several nuclear factors—including Jun, Fos, Fra, and Nrf2 families—bind the ARE and activate transcription of DTdiaphorase and certain phase II enzymes (Favreau and Pickett, 1991; Rushmore et al., 1991; Hayes and Pullford, 1995; Radjendirane and Jaiswal, 1999). The flavonoid -naphthoflavone, the polycyclic aromatic hydrocarbon benzo[a]pyrene, and the polyhalogenated aromatic hydrocarbon TCDD all induce DTdiaphorase by both mechanisms; the parent compound binds to the Ah receptor and is responsible for inducing CYP1A1, as well as DT-diaphorase, via the XRE, whereas electrophilic and/or redox active metabolites of -naphthoflavone, benzo[a]pyrene, and TCDD are responsible for inducing glutathione S-transferase, as well as DT-diaphorase, via the ARE (Radjendirane and Jaiswal, 1999). The situation with benzo[a]pyrene is quite intriguing. This polycyclic aromatic hydrocarbon binds directly to the Ah receptor, which binds to the XRE and induces the synthesis of CYP1A1, which in turn converts benzo[a]pyrene to electrophilic metabolites (such as arene oxides and diolepoxides) and redox active metabolites (such as catechols), as shown in Fig. 6-6. These electrophilic and redox active metabolites then induce enzymes that are regulated by the ARE. However, the catechol metabolites of benzo[a]pyrene are further converted by dihydrodiol dehydrogenase to ortho-quinones (Fig. 6-6), and are thereby converted back into planar, hydrophobic compounds that are highly effective ligands for the Ah receptor (Burczynski and Penning, 2000). This may be toxicologically important, because the Ah receptor may translocate ortho-quinone metabolites of benzo[a]pyrene into the nucleus, where they might damage DNA (Bolton et al., 2000).

Among the monofunctional agents that apparently induce DTdiaphorase via ARE is sulforaphane, an ingredient of broccoli that may be responsible for the anticarcinogenic effects of this cruciferous vegetable (Zhang et al., 1992). As mentioned above (this section), hypoxia and the anticancer agent mitomycin C are also inducers of DT-diaphorase, which has implications for cancer chemotherapy.

Dihydropyrimidine Dehydrogenase In 1993, fifteen Japanese patients died as a result of an interaction between two oral medications, Sorivudine, a new antiviral drug for herpes zoster, and Tegafur, a prodrug that is converted in the liver to the anticancer agent 5-fluorouracil. The deaths occurred within 40 days of the Japanese government’s approval of Sorivudine for clinical use. The mechanism of the lethal interaction between Sorivudine and Tega-

150	UNIT 2 DISPOSITION OF TOXICANTS

fur is illustrated in Fig. 6-14 and involves inhibition dihydropyrimidine dehydrogenase, an NADPH-requiring homodimeric protein (Mr 210 kDa) containing FMN/FAD, and an iron-sulfur cluster in each subunit. The enzyme is located mainly in liver cytosol, where it catalyzes the reduction of 5-fluorouracil and related pyrimidines. Sorivudine is converted in part by gut flora to (E)-5-(2-bromovinyl) uracil (BVU), which lacks antiviral activity but is converted by dihydropyrimidine dehydrogenase to a metabolite that binds covalently to the enzyme. The irreversible inactivation (also known as suicidal inactivation) of dihydropyrimidine dehydrogenase by Sorivudine causes a marked inhibition of 5-fluorouracil metabolism, which increases blood levels of 5-flu- orouracil to toxic and, in some cases, lethal levels (Ogura et al., 1998; Kanamitsu et al., 2000).

Severe 5-fluorouracil toxicity has also been documented in rare individuals who are genetically deficient in dihydropyrimidine dehydrogenase. The incidence and underlying mechanism of this genetic polymorphism remain to be determined, although its implication is clear: the dosage of 5-fluoroacil should be decreased in individuals devoid of dihydropyrimidine dehydrogenase or substantially lacking in it. Dosage adjustment may also be necessary for heterozygotes (i.e., individuals with a single copy of the active gene) compared with individuals who are homozygous for the wildtype (active) gene (Diasio et al., 1998).

Dehalogenation There are three major mechanisms for removing halogens (F, Cl, Br, and I) from aliphatic xenobiotics (Anders, 1985). The first, known as reductive dehalogenation, involves replacement of a halogen with hydrogen, as shown below:

X

X

X

X

X

2H

X

C

C

X

C

C

H

HX

X

H

X

H

Pentahaloethane

Tetrahaloethane

In the second mechanism, known as oxidative dehalogenation, a halogen and hydrogen on the same carbon atom are replaced with oxygen. Depending on the structure of the haloalkane, oxidative dehalogenation leads to the formation of an acylhalide or aldehyde, as shown below:

X

X

X

X

X

[O]

X

C

C

X

C

C

O

HX

X

H

X

Pentahaloethane

Tetrahaloacetylhalide

Figure 6-14. Reduction of 5-fluorouracil by dihydropyrimidine dehydrogenase and its inhibition (suicide inactivation) by Sorivudine.

Note: Inhibition of dihydropyrimidine dehydrogenase is the mechanism of fatal interactions between Sorivudine and the 5-fluorouracil prodrug Tegafur.

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151

X

H

X

H

X

[O]

X

C

C

X

C

C

O

HX

X

H

X

Tetrahaloethane

Trihaloacetaldehyde

A third mechanism of dehalogenation involves the elimination of two halogens on adjacent carbon atoms to form a carbon– carbon double bond, as shown below:

		X			X					X	X
X								X	2H	C	C
		C			C
									2HX
		X			H					X	H
Pentahaloethane										Trihaloethylene

A variation on this third mechanism is dehydrohalogenation, in which a halogen and hydrogen on adjacent carbon atoms are eliminated to form a carbon–carbon double bond.

Reductive and oxidative dehalogenation are both catalyzed by cytochrome P450. (The ability of cytochrome P450 to catalyze both reductive and oxidative reactions is explained later, under “Cytochrome P450.”) Dehalogenation reactions leading to double bond formation are catalyzed by cytochrome P450 and glutathione S-transferase. These reactions play an important role in the biotransformation and metabolic activation of several halogenated alkanes, as the following examples illustrate.

The hepatotoxicity of carbon tetrachloride (CCl4) and several related halogenated alkanes is dependent on their biotransformation by reductive dehalogenation. The first step in reductive dehalogenation is a one-electron reduction catalyzed by cytochrome P450, which produces a potentially toxic, carbon-centered radical and inorganic halide. In the case of CCl4, reductive dechlorination produces a trichloromethyl radical (•CCl3), which initiates lipid peroxidation and produces a variety of other metabolites, as shown in Fig. 6-15. Halothane can also be converted by reductive dehalogenation to a carbon-centered radical, as shown in Fig. 6-16. The mechanism is identical to that described for carbon tetrachloride, although in the case of halothane the radical is generated through loss of bromine, which is a better leaving group than chlorine. Figure 6-16 also shows that halothane can undergo oxidative dehalogenation, which involves oxygen insertion at the C – H bond to generate an unstable halohydrin (CF3COHClBr) that decomposes to a reactive acylhalide (CF3COCl), which can bind to cellular proteins (particularly to amine groups) or further decompose to trifluoroacetic acid (CF3COOH).

Both the oxidative and reductive pathways of halothane metabolism generate reactive intermediates capable of binding to proteins and other cellular macromolecules. The relative importance of these two pathways to halothane-induced hepatotoxicity appears to be species-dependent. In rats, halothane-induced hepatotoxicity is promoted by those conditions favoring the reductive dehalogenation of halothane, such as moderate hypoxia (10 to 14% oxygen) plus treatment with the cytochrome P450 inducers phenobarbital and pregnenolone-16 -carbonitrile. In contrast to the situation in rats, halothane-induced hepatotoxicity in guinea pigs is largely the result of oxidative dehalogenation of halothane (Lunam et al., 1989). In guinea pigs, halothane hepatotoxicity is not enhanced by moderate hypoxia and is diminished by the use of deuterated halothane, which impedes the oxidative dehalogenation of

halothane because the P450-dependent insertion of oxygen into a carbon–deuterium bond is energetically less favorable (and therefore slower) than inserting oxygen into a carbon–hydrogen bond.

Halothane hepatitis in humans is a rare but severe form of liver necrosis associated with repeated exposure to this volatile anesthetic. In humans as in guinea pigs, halothane hepatotoxicity appears to result from the oxidative dehalogenation of halothane, as shown in Fig. 6-16. Serum samples from patients suffering from halothane hepatitis contain antibodies directed against neoantigens formed by the trifluoroacetylation of proteins. These antibodies have been used to identify which specific proteins in the endoplasmic reticulum are targets for trifluoroacetylation during the oxidative dehalogenation of halothane (Pohl et al., 1989).

The concept that halothane is activated by cytochrome P450 to trifluoroacetylhalide, which binds covalently to proteins and elicits an immune response, has been extended to other volatile anesthetics, such as enflurane, methoxyflurane, and isoflurane. In other words, these halogenated aliphatic hydrocarbons, like halothane, may be converted to acylhalides that form immunogens by bind-

Figure 6-15. Reductive dehalogenation of carbon tetrachloride to a trichloromethyl free radical that initiates lipid peroxidation.

ABBREVIATIONS: RH, unsaturated lipid; R•, lipid dienyl radical; GSH, reduced glutathione; GSSG, oxidized glutathione.

152	UNIT 2 DISPOSITION OF TOXICANTS

Figure 6-16. Activation of halothane by reductive and oxidative dehalogenation and their role in liver toxicity in rats and humans.

ing covalently to proteins. In addition to accounting for rare instances of enflurane hepatitis, this mechanism of hepatotoxicity can also account for reports of a cross-sensitization between enflurane and halothane, in which enflurane causes liver damage in patients previously exposed to halothane.

One of the metabolites generated from the reductive dehalogenation of halothane is 2-chloro-1,1-difluoroethylene (Fig. 6-16). The formation of this metabolite involves the loss of two halogens from adjacent carbon atoms with formation of a carbon–carbon double bond. This type of dehalogenation reaction can also be catalyzed by glutathione S-transferases. Glutathione initiates the reaction with a nucleophilic attack either on the electrophilic carbon to which the halogen is attached (mechanism A) or on the halide itself (mechanism B), as shown in Fig. 6-17 for the dehalogenation of 1,2-dihaloethane to ethylene. The insecticide DDT is detoxified by dehydrochlorination to DDE by DDT-dehydrochlorinase, as shown in Fig. 6-18. The activity of this glutathione-dependent reaction correlates well with resistance to DDT in houseflies.

Oxidation

Alcohol, Aldehyde, Ketone Oxidation-Reduction Systems Alcohols, aldehydes, and ketones are oxidized or reduced by a number of enzymes, including alcohol dehydrogenase, aldehyde dehydrogenase, carbonyl reductase, dihydrodiol dehydrogenase, and the molybdenum-containing enzymes, aldehyde oxidase and xanthine dehydrogenase/oxidase. For example, simple alcohols (such as methanol and ethanol) are oxidized to aldehydes (namely formalde-

hyde and acetaldehyde) by alcohol dehydrogenase. These aldehydes are further oxidized to carboxylic acids (formic acid and acetic acid) by aldehyde dehydrogenase, as shown in Fig. 6-19. NAD is the preferred cofactor for both alcohol and aldehyde dehydrogenase.

Alcohol Dehydrogenase Alcohol dehydrogenase (ADH) is a zinc-containing, cytosolic enzyme present in several tissues including the liver, which has the highest levels, the kidney, the lung, and the gastric mucosa (Agarwal and Goedde, 1992). Human ADH is a dimeric protein consisting of two 40-kDa subunits. The subunits ( , , , , , and sixth subunit known as or ) are encoded by six different gene loci (ADH1 through ADH6). [Although the human ADH subunits , , , , and are consistently reported to be encoded by genes ADH1 to ADH5, respectively, the sixth subunit ( or ) was originally reported to be encoded by gene ADH7. In this review, I have followed the recommendation of Jörnvall and Höög (1995) that the gene encoding the sixth subunit ( or ) be designated ADH6.] Updates on ADH nomenclature can be found on the Internet (http://www.gene.ucl.ac.uk./nomenclature/ADH. shtml).

There are three allelic variants of the beta subunit ( 1, 2, and3, which differ by a single amino acid) and two allelic variants of the gamma subunit ( 1 and 2, which differ by two amino acids). Consequently, the human ADH enzymes comprise nine subunits, all of which can combine as homodimers. In addition, the , , and subunits (and their allelic variants) can form heterodimers with each other (but not with the other subunits, none of which