26510318-Bio-Transformation-of-Xenobiotics

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153

Figure 6-17. Glutathione-dependent dehalogenation of 1,2-dihaloethane to ethylene.

A. Nucleophilic attack on carbon. B. Nucleophilic attack on halide.

form heterodimers). The different molecular forms of ADH are divided into four major classes. Class I contains ADH1, ADH2, and ADH3, which can be considered isozymes. ADH1 contains either two alpha subunits or one alpha subunit plus a beta or gamma subunit. ADH2 contains either two beta subunits (which could be 1,2, or 3) or a beta subunit plus a gamma subunit (which could be1 or 2). ADH3 contains two gamma subunits (which could be 1 or 2). ADH2 enzymes that differ in the type of subunits are

known as allelozymes, as are ADH3 enzymes that differ in the type of subunit. Accordingly, ADH2*1 is an allelozyme composed of1 units; ADH2*2 is an allelozyme composed of 2 subunits, and ADH2*3 is an allelozyme composed of 3 subunits.

Class II contains ADH4, which is made up of two subunits (named pi because this form of ADH is not inhibited by pyrazole). Class III contains ADH5, which is made up of two subunits (for which reason it is also known as chi-ADH). Class IV contains ADH6 (originally named ADH7, as mentioned above), which is made up of two subunits designated or (Agarwal and Goedde, 1992; Jörnvall and Höög, 1995; Edenberg, 2000).

The class I ADH isozymes ( -ADH, -ADH, and -ADH) are responsible for the oxidation of ethanol and other small aliphatic alcohols, and they are strongly inhibited by pyrazole and its 4-alkyl derivatives (e.g., 4-methylpyrazole). High levels of class I ADH isozymes are expressed in liver and adrenals, with lower levels in kidney, lung, blood vessels [in the case of ADH2 (i.e.,-ADH)] and other tissues, but not brain. Class II ADH ( -ADH) is primarily expressed in liver (with lower levels in stomach), where it preferentially oxidizes larger aliphatic and aromatic alcohols. Class II ADH differs from the class I ADH in that it plays little or no role in ethanol and methanol oxidation, and it is not inhibited by pyrazole. Long-chain alcohols (pentanol and larger) and aromatic alcohols (such as cinnamyl alcohol) are preferred substrates for class III ADH ( -ADH). Like class II ADH, class III ADH is not inhibited by pyrazole. However, in contrast to class II ADH, which is largely confined to the liver, class III ADH is ubiquitous, being present in virtually all tissues (including brain), where it appears to play an important role in detoxifying formaldehyde. In fact, class III ADH and formaldehyde dehydrogenase are identical enzymes (Koivusalo et al., 1989). Class IV ADH ( - or -ADH) is a low affinity (high Km), high capacity ADH (high Vmax), and is the most active of the medium-chain ADHs in oxidizing retinol. It is the major ADH expressed in human stomach and other areas of the gastrointestinal tract (esophagus, gingiva, mouth, and tongue). In contrast the other ADHs, class IV ADH is not expressed in adult human liver. Inasmuch as class IV ADH is expressed in the upper gastrointestinal tract, where chronic alcohol consumption leads to cancer development, there is growing interest in the role of class IV ADH in the conversion of ethanol to acetaldehyde (a suspected upper GI tract carcinogen or cocarcinogen) and in its role in the metabolism of retinol (a vitamin required for epithelial cell growth and differentiation), which might be inhibited by alcohol consumption (Seitz and Oneta, 1998).

The class I isozymes of ADH differ in their capacity to oxidize ethanol. Even the allelozymes, which differ in a single amino acid, differ markedly in the affinity (Km) and/or capacity (Vmax) for oxidizing ethanol to acetaldehyde. The homodimer 2 2 and heterodimers containing at least one 2 subunit (i.e., the ADH2*2

Figure 6-18. Dehydrochlorination of the pesticide DDT to DDE, a glutathione-dependent reaction.

154	UNIT 2 DISPOSITION OF TOXICANTS

Figure 6-19. Oxidation of alcohols to aldehydes and carboxylic acids by alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH).

allelozymes) are especially active in oxidizing ethanol at physiologic pH. ADH2*2 is known as atypical ADH and is responsible for the unusually rapid conversion of ethanol to acetaldehyde in 90 percent of the Pacific Rim Asian population (Japanese, Chinese, Korean). The atypical ADH is expressed to a much lesser degree in Caucasians ( 5 percent of Americans, 8 percent of English,12 percent of Germans, and 20 percent of Swiss), African Americans ( 10 percent), Native Americans (0 percent) and Asian Indians (0 percent) (Agarwal and Goedde, 1992). The three ADH2 alleles, ADH2*1 ( 1-ADH), ADH2*2 ( 2-ADH), and ADH2*3 ( 3-ADH), are mainly expressed in Caucasians (up to 95 percent), Pacific Rim Asians ( 90 percent) and Africans/African Americans ( 24 percent), respectively. These population differences in ADH2 allelozyme expression contribute to ethnic differences in alcohol consumption and toxicity, as discussed in the next section, “Aldehyde Dehydrogenase.”

Unlike the allelic variants of ADH2, the allelic variants of ADH3 do not differ markedly in their ability to oxidize ethanol. However, as in the case of the ADH2 allelozymes, the expression of the ADH3 allelozymes also varies from one ethnic group to the next. The two allelozymes of ADH3, namely ADH3*1 ( 1-ADH) and ADH3*2 ( 2-ADH) are respectively expressed 50:50 in Caucasians, but 90:10 in Pacific Rim Asians (Li, 2000).

The various class I ADHs in liver oxidize ethanol with a Km of 50 M to 4 mM. For comparison, legal intoxication in the United States is defined as a blood alcohol level of 0.1%, which corresponds to 22 mM (Edenberg, 2000). Therefore, during intoxication, the hepatic metabolism of ADH becomes saturated, and the kinetics of ethanol disappearance conform to zero-order kinetics, meaning that a constant amount of ethanol is metabolized per unit time. When the concentration of ethanol falls within the range of Km, the kinetics of ethanol disappearance conform to a first-order process, meaning that a constant percentage of the remaining ethanol is metabolized per unit time.

Compared with hepatic ADH, gastric ADH has a lower affinity (higher Km) but higher capacity (larger Vmax) for oxidizing ethanol, the former being dominated by the class I ADHs, the latter by class IV ADH. Although ethanol is largely biotransformed by hepatic ADH, gastric ADH nevertheless can limit the systemic bioavailability of alcohol. This first-pass elimination of alcohol by gastric ADH can be significant, depending on the manner in which the alcohol is consumed; large doses over a short time produce high ethanol concentrations in the stomach, which compensate for the low affinity (high Km) of gastric ADH. Young women have lower gastric ADH activity than do men, and gastric ADH activity tends to be lower in alcoholics (Frezza et al., 1990). Some alcoholic women have no detectable gastric ADH, and blood levels of ethanol after oral consumption of alcohol are the same as those

that are obtained after intravenous administration. Gastric ADH activity decreases during fasting, which is one reason why alcohol is more intoxicating when it is consumed on an empty stomach. Several commonly used drugs (cimetidine, ranitidine, aspirin) are noncompetitive inhibitors of gastric ADH. Under certain circumstances these drugs increase the systemic availability of alcohol, although the effect is too small to have serious medical, social, or legal consequences (Levitt, 1993). About 30 percent of Asians appear to be genetically deficient in class IV ADH, the main gastric ADH. In addition to biotransforming ethanol and retinol, class IV ADH also detoxifies the dietary carcinogen nitrobenzaldehyde. It has been suggested that a lack of class IV ADH in Japanese subjects may impair their ability to detoxify nitrobenzaldehyde and may possibly be linked to the high rate of gastric cancer observed in the Japanese population (Seitz and Oneta, 1998).

Alcohols can be oxidized to aldehydes by non-ADH enzymes in microsomes and peroxisomes, although these are quantitatively less important than ADH for ethanol oxidation (Lieber, 1999). The microsomal ethanol oxidizing system (formerly known as MEOS) is the cytochrome P450 enzyme CYP2E1. The corresponding peroxisomal enzyme is catalase. The oxidation of ethanol to acetaldehyde by these three enzyme systems is shown in Fig. 6-20.

Aldehyde Dehydrogenase Aldehyde dehydrogenase (ALDH) oxidizes aldehydes to carboxylic acids with NAD+ as the cofactor. Several ALDH enzymes are involved in the oxidation of xenobiotic aldehydes (Goedde and Agarwal, 1992). The enzymes also have esterase activity (Yoshida et al., 1998). Formaldehyde dehydrogenase, which specifically oxidizes formaldehyde complexed with glutathione, is not a member of the ALDH family but is a class III ADH (Koivusalo et al., 1989). Twelve ALDH genes (known as ALDH1 to 10, SSDH, and MMSDH) have been identified in humans, and a correspondingly large number of ALDH genes appear to be present in other mammalian species. The name, tissue distribution, subcellular location, and major substrate for the 12 human ALDHs are summarized in Table 6-2. The ALDHs differ in their primary amino acid sequences. They may also differ in the quaternary structure. For example, ALDH3 appears to be a dimer of two 85-kDa subunits, whereas ALDH1 and ALDH2 appear to be homotetramers of 54-kDa subunits (Goedde and Agarwal, 1992). In contrast to ALDH1 and ALDH2, which specifically reduce NAD , ALDH3 reduces both NAD and NADP due to its low affinity for these cofactors.

As shown in Fig. 6-20, ALDH2 is a mitochondrial enzymes that, by virtue of its high affinity, is primarily responsible for oxidizing simple aldehydes, such as acetaldehyde (Km for acetaldehyde 5 M at pH 7.4). A genetic polymorphism for ALDH2 has been documented in humans. A high percentage (45 to 53 percent)

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Figure 6-20. Oxidation of ethanol to acetaldehyde by ethanol dehydrogenase (ADH), cytochrome P450 (CYP2E1), and catalase.

Note the oxidation of ethanol to acetic acid involves multiple organelles.

of Japanese, Chinese, Koreans, Taiwanese, and Vietnamese populations are deficient in ALDH2 activity due to a point mutation (Glu487 n Lys487). This inactive allelic variant of ALDH2 is known as ALDH2*2, to distinguish it from the active, wild-type enzyme, ALDH2*1. This same population (i.e., Pacific Rim Asians) also has a high incidence of the atypical form of ADH (i.e., ADH2*2), which means that they rapidly convert ethanol to acetaldehyde but only slowly convert acetaldehyde to acetic acid. (They also have a relatively high prevalence of a deficiency of class IV ADH activ-

ity, which impairs gastric metabolism of ethanol.) As a result, many Asian subjects experience a flushing syndrome after consuming alcohol due to a rapid buildup of acetaldehyde, which triggers the dilation of facial blood vessels through the release of catecholamines. Native Americans also experience a flushing syndrome after consuming alcohol, apparently because they express another allelic variant of ALDH2 and/or because acetaldehyde oxidation in blood erythrocytes is impaired in these individuals, possibly due to the expression of a variant form of ALDH1. The functional ge-

Table 6-2

Properties of the Human Aldehyde Dehydrogenases (ALDHs)

ENZYME		SUBCELLULAR
(ABBREVIATED SYMBOL)	TISSUE	DISTRIBUTION	MAJOR SUBSTRATE
ALDH1	Liver, stomach, etc.	Cytosol	Retinal
ALDH2	Liver, stomach, etc.	Mitochondria	Acetaldehyde
ALDH3	Stomach, lung, etc.	Cytosol	Fatty and aromatic aldehydes
ALDH4	Liver, kidney	Mitochondria	Glutamate, -semialdehyde
ALDH5	Testis, liver	Mitochondria	Propionaldehyde
ALDH6	Salivary gland, stomach, kidney	Cytosol	Aliphatic aldehyde, retinal
ALDH7	Kidney, lung	Microsomes	Aliphatic and aromatic aldehydes
ALDH8	Parotid	Microsomes	Unknown
ABHD	Liver, kidney, muscle	Cytosol	Amine aldehyde
FALDH	Liver, heart, muscle	Microsomes	Fatty and aromatic aldehydes
SSDH	Brain, liver, heart	Mitochondria	Succinic semialdehyde
MMSDH	Kidney, liver, heart	Mitochondria	Methylmalonate semialdehyde

KEY: ABHD, 4-aminobutyraldehyde dehydrogenase; FALDH, fatty aldehyde dehydrogenase; SSDH, succinic dehydrogenase; MMSDH, methylmalonate semialdehyde dehydrogenase.

SOURCE: Yoshida et al., 1998.

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netic variants of ADH that rapidly convert ethanol to acetaldehyde (i.e., ADH2*2), and the genetic variants of ALDH that slowly detoxify acetaldehyde both protect against heavy drinking and alcoholism. Inhibition of ALDH by disulfiram (Antabuse) causes an accumulation of acetaldehyde in alcoholics. The nauseating effect of acetaldehyde serves to deter continued ethanol consumption (Goedde and Agarwal, 1992). However, it is important to note that a predisposition toward alcoholism is not simply determined by factors that affect the pharmacokinetics of ethanol and its metabolites. Studies in humans and rodents implicate serotonin 1b receptor, dopamine D2 receptor, tryptophan hydroxylase, and neuropeptide Y as candidate targets of genetic susceptibility in the pharmacodynamic actions of ethanol (Li, 2000). Monoamine oxidase may also be a risk modifier for alcoholism, as discussed under “Monoamine Oxidase,” further on.

Genetic deficiencies in other ALDHs impair the metabolism of other aldehydes, which is the underlying basis of certain diseases. For example, ALDH4 deficiency disturbs proline metabolism and causes type II hyperprolinemia, symptoms of which include mental retardation and convulsions. A deficiency of ALDH10, which detoxifies fatty aldehydes, disturbs the metabolism of membrane lipids. This is the underlying basis of SjörgenLarson syndrome, symptoms of which include ichthyosis, neurologic problems, and oligophrenia (Yoshida et al., 1998).

The toxicologic consequences of an inherited (i.e., genetic) or acquired (e.g., drug-induced) deficiency of ALDH illustrate that aldehydes are more cytotoxic than the corresponding alcohol. This is especially true of allyl alcohol (CH2 PCHCH2OH), which is converted by ADH to the highly hepatotoxic aldehyde acrolein (CH2 PCHCHO). The oxidation of ethanol by ADH and ALDH leads to the formation of acetic acid, which is rapidly oxidized to carbon dioxide and water. However, in certain cases, alcohols are converted to toxic carboxylic acids, as in the case of methanol and ethylene glycol, which are converted via aldehyde intermediates to formic acid and oxalic acid, respectively. Formic and oxalic acids are considerably more toxic than acetic acid. For this reason, methanol and ethylene glycol poisoning are commonly treated with ethanol, which competitively inhibits the oxidation of methanol and ethylene glycol by ADH and ALDH. The potent inhibitor of ADH 4-methylpyrazole (fomepizole) is also used to treat methanol and ethylene glycol poisoning.

The reduction of aldehydes and ketones to primary and secondary alcohols by carbonyl reductases has already been discussed (see “Carbonyl Reduction,” above). In contrast to ADH and ALDH, carbonyl reductases typically use NADPH as the source of reducing equivalents. Aldehydes can also be oxidized by aldehyde oxidase and xanthine oxidase, which are discussed below under “Molybdenum Hydroxylases.”

Dihydrodiol Dehydrogenase In addition to several hydroxysteroid dehydrogenases and aldose reductases, the aldo-keto reductase (AKR) superfamily includes several forms of dihydrodiol dehydrogenase (Penning, 1997). As previously mentioned in the section on carbonyl reductase, dihydrodiol dehydrogenases are monomeric ( 34 kDa), cytosolic, NADP(H)-requiring oxidoreductases that oxidize the trans-dihydrodiols of various polycyclic aromatic hydrocarbons to the corresponding ortho-quinones, as shown in Fig. 6-6 (Burczynski and Penning, 2000). The conversion of such dihydrodiols to ortho-quinones has toxicological implications, which were discussed earlier in the section on “Quinone Reduction.”

Molybdenum Hydroxylases (Molybdozymes) Two major molybdenum hydroxylases or molybdozymes participate in the biotransformation of xenobiotics: aldehyde oxidase and xanthine dehydrogenase/xanthine oxidase (XD/XO) (Rettie and Fisher, 1999). Sulfite oxidase is third molybdozyme; it is not described here except to say that sulfite oxidase, as the name implies, oxidizes sulfite, an irritating air pollutant, to sulfate, which is relatively innocuous. All three molybdozymes are flavoprotein enzymes consisting of two identical 150-kDa subunits, each of which contains FAD, molybdenum, in the form of a pterin molybdenum cofactor {[MoVI (PS) (PO)]2 } and two iron-sulfur (Fe2S2) centers (known as FeSI and FeSII). The catalytic cycle involves an interaction between the molybdenum center with a reducing substrate, which results in the reduction of the molybdenum cofactor. After this, reducing equivalents are transferred intramolecularly to the flavin and iron-sulfur centers, with reoxidation occurring via the flavin moiety by molecular oxygen (in the case of aldehyde oxidase and xanthine oxidase) or NAD (in the case of xanthine dehydrogenase). During substrate oxidation, aldehyde oxidase and xanthine oxidase are reduced and then reoxidized by molecular oxygen; hence, they function as true oxidases. The oxygen incorporated into the xenobiotic is derived from water rather than oxygen, which distinguishes the oxidases from oxygenases. The overall reaction is as follows:

H2O

RH 2H , 2e ROH

Additional details of the catalytic cycle are described below, under “Xanthine Dehydrogenase–Xanthine Oxidase.” Xanthine oxidase and aldehyde oxidase catalyze the oxidation of electrondeficient sp2-hybridized (i.e., double-bonded) carbon atoms found, more often than not, in nitrogen heterocycles, such as purines, pyrimidines, pteridines, and iminium ions. This contrasts with oxidation by cytochrome P450, which generally catalyzes the oxidation of carbon atoms with a high electron density. For this reason, xenobiotics that are good substrates for molybdozymes tend to be poor substrates for cytochrome P450, and vice versa (Rettie and Fisher, 1999). In nitrogen heterocycles, the carbon atom with lowest electron density is adjacent to the nitrogen atom, for which reason xanthine oxidase and aldehyde oxidase tend to hydroxylate the-carbon atom to form a hydroximine that rapidly tautomerizes to the corresponding -aminoketone. As the name implies, aldehyde oxidase can convert certain aldehydes to the corresponding carboxylic acid—a property that is also shared by xanthine oxidase, albeit with lower activity. Certain aromatic aldehydes, such as tamoxifen aldehyde and benzaldehyde, are good substrates for aldehyde oxidase and xanthine oxidase, whereas aliphatic aldehydes tend to be poor substrates. Consequently, aldehyde oxidase and xanthine oxidase contribute negligibly to the metabolism of acetaldehyde. Some reactions catalyzed by aldehyde oxidase and xanthine oxidase are shown in Fig. 6-21. Under certain conditions, both enzymes can also catalyze the reduction of xenobiotics containing one or more of the following functional groups: azo, nitro, N-oxide, nitrosamine, hydroxamic acid, oxime, sulfoxide, and epoxide. This is discussed further below, in the section entitled “Aldehyde Oxidase.”

Xanthine Dehydrogenase – Xanthine Oxidase Xanthine dehydrogenase (XD) and xanthine oxidase (XO) are two forms of the same enzyme that differ in the electron acceptor used in the final step of catalysis. In the case of XD, the final electron acceptor is

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157

Figure 6-21. Examples of reactions catalyzed by the molybdozymes, xanthine oxidase, and aldehyde oxidase.

NAD (dehydrogenase activity), whereas in the case of XO the final electron acceptor is oxygen (oxidase activity). XD is converted to XO by oxidation of cysteine residues (Cys993 and Cys1326 of the human enzyme) and/or proteolytic cleavage. The conversion of XD to XO by cysteine oxidation appears to be reversible. Under normal physiologic conditions, XD is the predominant form of the enzyme found in vivo. However, during tissue processing, the dehydrogenase form tends to be converted to the oxidase form, hence, most in vitro studies are conducted with XO or a combination of XO and XD. The induction (up-regulation) of XD and/or the con-

version of XD to XO in vivo is thought to play an important role in ischemia-reperfusion injury, lipopolysaccharide (LPS)-mediated tissue injury, and alcohol-induced hepatotoxicity. During ischemia, XO levels increase because hypoxia induces XD/XO gene transcription and because XD is converted to XO. During reperfusion, XO contributes to oxidative stress and lipid peroxidation because the oxidase activity of XO involves the reduction of molecular oxygen, which can lead to the formation of reactive oxygen species of the type shown in Fig. 6-13. Similarly, treatment with LPS, a bacterial endotoxin that triggers an acute inflammatory response, in-

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creases XO activity both by inducing XD/XD transcription and by converting XD to XO. The associated increased in oxidative stress has been implicated in LPS-induced cytotoxicity. Ethanol facilitates the conversion of XD to XO, and the conversion of ethanol to acetaldehyde provides a substrate and, hence, a source of electrons for the reduction of oxygen.

Typical reactions catalyzed by XD/XO are shown in Fig. 6-21. XD/XO contributes significantly to the first-pass elimination of several purine derivatives (e.g., 6-mercaptopurine and 2,6- dithiopurine) and limits the therapeutic effects of these cancer chemotherapeutic agents. On the other hand, certain prodrugs are activated by xanthine oxidase. For example, the antiviral prodrugs 6-deoxyacyclovir and 2 -fluoroarabino-dideoxypurine, which are relatively well absorbed after oral dosing, are oxidized by xanthine oxidase to their respective active forms, acyclovir and 2 -fluo- roarabino-dideoxyinosine, which are otherwise poorly absorbed (see Fig. 6-21). Furthermore, XD/XO has been implicated in the bioactivation of mitomycin C and related antineoplastic agents, although this bioactivation reaction is also catalyzed by DTdiaphorase (see section entitled “Quinone Reduction,” below).

XD/XO catalyzes an important physiologic reaction, namely the sequential oxidation of hypoxanthine to xanthine and uric acid, as shown in Fig. 6-21 (Rajagopalan, 1980). By competing with hypoxanthine and xanthine for oxidation by XD/XO, allopurinol inhibits the formation of uric acid, making allopurinol a useful drug in the treatment of gout (a complication of hyperuricemia). Allopurinol can also be used to evaluate the contribution of XD/XO to xenobiotic biotransformation in vivo. Like allopurinol, hydroxylated coumarin derivatives, such as umbelliferone (7-hydroxy-

coumarin) and esculetin (7,8-dihydroxycoumarin), are potent inhibitors of XD/XO.

Monomethylated xanthines are preferentially oxidized to the corresponding uric acid derivatives by XD/XO. In contrast, dimethylated and trimethylated xanthines, such as theophylline (1,3- dimethylxanthine) and caffeine (1,3,7-trimethylxanthine), are oxidized to the corresponding uric acid derivatives primarily by cytochrome P450. Through two sequential N-demethylation reactions, cytochrome P450 converts caffeine to 1-methylxanthine, which is converted by XD/XO to 1-methyluric acid. The urinary ratio of 1-methylxanthine to 1-methyluric acid provides an in vivo marker of XD/XO activity.

The mechanism of catalysis of xanthine oxidase has been reviewed by Bray et al. (1996). The pteridin molybdenum cofactor {[MoVI (PS) (PO)]2+} contains both a sulfido and oxo ligand. It was once thought that the oxo group was transferred and incorporated into the substrate, such as the C8-position of xanthine. More recent studies suggest that the oxo group does not participate directly in substrate oxidation. Instead, catalysis appears to involve the transient formation of a Mo–C bond between the molybdenum metal center and the C8 position of xanthine, as illustrated in Fig. 6-22. The formation of this Mo-C intermediate is preceded by deprotonation of xanthine (at the C8 position), which reduces the sulfido ligand (MoPS) to Mo–SH. Deprotonation of xanthine produces a carbanion that reacts with molybdenum to form the Mo–C intermediate, which reacts with water to produce a three-ringed intermediate involving Mo, oxygen and the C8 position of xanthine, which rearranges to yield the hydroxylated substrate (uric acid). The catalytic cycle is completed by the transfer of electrons to

Figure 6-22. Catalytic cycle of xanthine dehydrogenase (XD) and xanthine oxidase (XO).

XD and XO are two forms of the same molybdozyme. During the final step of the catalytic cycle, the enzyme is reoxidized by transferring electrons to NAD (in the case of XD) or oxygen (in the case of XO). The same mechanism of catalysis likely applies to aldehyde oxidase, although there is no dehydrogenase form of this molybdozyme.

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NAD (dehydrogenase activity) or oxygen (oxidase activity). A similar mechanism of catalysis likely holds true for aldehyde oxidase.

In humans, XD/XO is a cytosolic enzyme that is widely distributed throughout the body, with the highest levels in heart, brain, liver, skeletal muscle, pancreas, small intestine, colon, and placenta. In humans, XD/XO appears to encoded by a single gene. Although the sequences of two enzymes have been reported in the literature, the first report is now known to describe the sequence of human aldehyde oxidase. A complete deficiency of XD/XO (which may also involve a deficiency of aldehyde oxidase) gives rise to the rare genetic disorder known as xanthinuria.

Aldehyde Oxidase Aldehyde oxidase is the second of two molybdozymes that play an important role in xenobiotic biotransformation, the other being xanthine oxidase (discussed in the preceding section). Whereas xanthine oxidase exists in two forms, a dehydrogenase form (XD) that relays electrons to NAD and an oxidase form (XO) that relays electrons to molecular oxygen, aldehyde oxidase exists only in the oxidase form, apparently because it lacks an NAD binding site (Terao et al., 1998). Another significant difference between these two molybdozymes is that high levels of xanthine oxidase appear to be widely distributed throughout the body, whereas high levels of aldehyde oxidase are found in the liver, with considerably less activity in other tissues, at least in humans. Aside from these differences, many of the features of xanthine oxidase apply to aldehyde oxidase, including subcellular location (cytosol), enzyme structure, and cofactor composition, mechanism of catalysis, preference for oxidizing carbon atoms adjacent to the nitrogen atoms in nitrogen heterocycles, and its preference for oxidizing aromatic aldehydes over aliphatic aldehydes. Furthermore, aldehyde oxidase also transfers electrons to molecular oxygen, which can generate reactive oxygen species and lead to oxidative stress and lipid peroxidation. Therefore, the pathophysiologic features described for xanthine oxidase may similarly apply to aldehyde oxidase, especially in the case of ethanol-induced liver damage.

As shown in Fig. 6-21, aldehyde oxidase can oxidize a number of substituted pyrroles, pyridines, pyrimidines, purines, pteridines, and iminium ions by a mechanism that is presumably similar to that described for xanthine oxidase in the previous section. Aldehyde oxidase can oxidize aldehydes to their corresponding carboxylic acids, but the enzyme shows a marked preference for aromatic aldehydes (e.g., benzaldehyde, tamoxifen aldehyde). Consequently, aldehyde oxidase contributes negligibly to the oxidation of aliphatic aldehydes, such as acetaldehyde. Rodrigues (1994) found that, in a bank of human liver samples, aldehyde oxidase activity toward N1-methylnicotinamide varied more than 40-fold, whereas activity toward 6-methylpurine varied less than 3-fold. Although this suggests that human liver cytosol contains two or more forms of aldehyde oxidase, subsequent Southern blot analysis has provided evidence for only a single copy of the aldehyde oxidase gene in humans (Terao et al., 1998). On the other hand, two aldehyde oxidase genes have been identified in mice.

A number of physiologically important aldehydes are substrates for aldehyde oxidase, including homovanillyl aldehyde (formed from dopamine), 5-hydroxy-3-indoleacetaldehyde (formed from serotonin), and retinal, which is converted by aldehyde oxidase to retinoic acid, an important regulator of cell growth, differentiation, and morphogenesis. The catabolism of catecholamines by monoamine oxidase produces dihydromandelalde-

hyde, which is oxidized by aldehyde oxidase to dihydromandelic acid. Therefore, aldehyde oxidase plays an important role in the catabolism of biogenic amines and catecholamines. In humans, the gene for aldehyde oxidase has been mapped to chromosome 2q22q33, placing it near a genetic marker that cosegregates with the recessive familial form of amyotrophic lateral sclerosis. In mouse brain, aldehyde oxidase is localized in the choroid plexus and motor neurons, which lends further support to the proposal that aldehyde oxidase is a candidate gene for this particular motor neuron disease (Bendotti et al., 1997). Furthermore, combined deficiency of molybdoproteins, which affects aldehyde oxidase and XD/XO, leads to an impairment in the development of the central nervous system and is accompanied by severe neurologic symptoms.

In general, xenobiotics that are good substrates for aldehyde oxidase are poor substrates for cytochrome P450, and vice versa (Rettie and Fisher, 1999). Naphthalene (with no nitrogen atoms) is oxidized by cytochrome P450 but not by aldehyde oxidase, whereas the opposite is true of pteridine (1,3,5,8-tetraazanaphthalene), which contains four nitrogen atoms. The intermediate structure, quinazolone (1,3-diazanaphthalene) is a substrate for both enzymes. This complementarity in substrate specificity reflects the opposing preference of the two enzymes for oxidizing carbon atoms; cytochrome P450 prefers to oxidize carbon atoms with high electron density, whereas aldehyde oxidase (and XD/XO) prefers to oxidize carbon atoms with low electron density. The substrate specificity of aldehyde oxidase differs among mammalian species, with substrate size being the main differentiating factor. The active site of human aldehyde oxidase accommodates much smaller substrates than rabbit or guinea pig aldehyde oxidase. Substituents on a substrate that increase electronegativity tend to enhance Vmax, whereas substituents that increase lipophilicity tend to increase affinity (decrease Km). Another interesting species difference is that dogs possess little or no aldehyde oxidase activity. However, aldehyde oxidase in human liver has proven to be rather unstable, which complicates an in vitro assessment of species differences in aldehyde oxidase activity (Rodrigues, 1994; Rettie and Fisher, 1999). A further complication is the observation of species differences in the relative roles of aldehyde oxidase and XD/XO in xenobiotic biotransformation. For example, the 6-oxidation of antiviral deoxyguanine prodrugs is catalyzed exclusively in rats by XD/XO, but by aldehyde oxidase in humans (Rettie and Fisher, 1999).

Aldehyde oxidase is the second of two enzymes involved in the formation of cotinine, a major metabolite of nicotine excreted in the urine of cigarette smokers. The initial step in this reaction is the formation of a double bond (CPN) in the pyrrole ring, which produces nicotine 1 ,5 -iminium ion. Like nicotine, several other drugs are oxidized either sequentially or concomitantly by cytochrome P450 and aldehyde oxidase, including quinidine, azapetine, cyclophosphamide, carbazeran, and prolintane. Other drugs that are oxidized by aldehyde oxidase include bromonidine (an2-adrenoceptor agonist), O6-benzylguanine (a cancer chemotherapeutic agent), quinine (an antimalarial), pyrazinamide (a tuberculostatic agent), methotrexate (an antineoplastic and immunosuppressive agent) and famciclovir (an antiviral prodrug that is converted by aldehyde oxidase to penciclovir). Several pyrimidine derivatives are oxidized by aldehyde oxidase, including 5-ethyl- 2(1H)-pyrimidone, which is converted by aldehyde oxidase to 5- ethynyluracil. Like Sorivudine, 5-ethynyluracil is a metabolismdependent (suicide) inactivator of dihydropyrimidine dehydrogenase (see Fig. 6-14).

160	UNIT 2 DISPOSITION OF TOXICANTS

Menadione, hydralazine, methadone and proadifen are inhibitors of aldehyde oxidase. Menadione is a potent inhibitor of aldehyde oxidase (Ki 0.1 M) and can be used together with allopurinol to discriminate between aldehyde oxidaseand xanthine oxidase-catalyzed reactions. Hydralazine has been used to assess the role of aldehyde oxidase in human drug metabolism in vivo. The ability of proadifen to inhibit aldehyde oxidase is noteworthy because this methadone analog, commonly known as SKF 525A, is widely used as a cytochrome P450 inhibitor.

Under certain conditions, aldehyde oxidase and xanthine oxidase can also catalyze the reduction of xenobiotics, including azo-reduction (e.g., 4-dimethylaminoazobenzene), nitro-reduction (e.g., 1-nitropyrene), N-oxide reduction (e.g., S-(-)-nicotine-1 -N- oxide), nitrosamine reduction (e.g., N-nitrosodiphenylamine), hydroxamic acid reduction (e.g., N-hydroxy-2-acetylaminofluorene), sulfoxide reduction [e.g., sulindac (see Fig. 6-12)] and epoxide reduction [e.g., benzo(9a)pyrene 4,5-oxide]. Oximes (CPNOH) can also be reduced by aldehyde oxidase to the corresponding ketimines (CPNH), which may react nonenzymatically with water to produce the corresponding ketone or aldehyde (CPO) and ammonia. An analogous reaction allows aldehyde oxidase to catalyze the reductive ring-opening of Zonisamide and 1,2-benzisoxazole, which results in formation of an oxo-containing metabolite and ammonia (Sugihara et al., 1996). Xenobiotic reduction by aldehyde oxidase requires anaerobic conditions or the presence of a reducing substrate, such as N1-methylnicotinamide, 2-hydroxypyrimidine or benzaldehyde. These “cosubstrates” reduce the enzyme, which in turn catalyzes azo-reduction, nitro-reduction, etc., by relaying electrons to xenobiotics (rather than molecular oxygen). These unusual requirements make it difficult to assess the degree to which aldehyde oxidase functions as a reductive enzyme in vivo.

Monoamine Oxidase, Diamine Oxidase, and Polyamine Oxidase Monoamine oxidase (MAO), diamine oxidase (DAO), and polyamine oxidase (PAO) are all involved in the oxidative deamination of primary, secondary, and tertiary amines (Weyler et al., 1992; Benedetti and Dostert, 1994). Substrates for these enzymes include several naturally occurring amines, such as the monoamine serotonin (5-hydroxytryptamine), the diamines putrescine and histamine, and monoacetylated derivatives of the polyamines spermine and spermidine. A number of xenobiotics are substrates for these enzymes, particularly MAO. Oxidative deamination of a primary amine produces ammonia and an aldehyde, whereas oxidative deamination of a secondary amine produces a primary amine and an aldehyde. [The products of the former reaction (i.e., an aldehyde and ammonia) are those produced during the reductive biotransformation of certain oximes by aldehyde oxidase, as described in the preceding section on aldehyde oxidase]. The aldehydes formed by MAO are usually oxidized further by other enzymes to the corresponding carboxylic acids, although in some cases they are reduced to alcohols. Examples of reactions catalyzed by MAO, DAO, and PAO are shown in Fig. 6-23. Monoamine oxidase is located throughout the brain and is present in the liver, kidney, intestine, and blood platelets in the outer membrane of mitochondria. Its substrates include milacemide (Fig. 6-23), a dealkylated metabolite of propranolol (Fig. 6-23), primaquine, haloperidol, doxylamine, -phenylethylamine, tyramine, catecholamines (dopamine, norepinephrine, epinephrine), tryptophan derivatives (tryptamine, serotonin), and tryptophan analogs known as triptans, which include the antimigraine drugs sumatriptan, zolmitriptan, and rizatriptan.

Figure 6-23. Examples of reactions catalyzed by monoamine oxidase (MAO), diamine oxidase (DAO), and polyamine oxidase (PAO).

Note that pheneizine is a metabolism-dependent (mechanism-based) inhibitor of MAO-A and MAO-B.

There are two forms of monoamine oxidase, called MAO-A and MAO-B. MAO-A preferentially oxidizes serotonin (5-hydroxy- tryptamine), norepinephrine, and the dealkylated metabolite of propranolol. It is preferentially inhibited by clorgyline, whereas MAO-B preferentially oxidizes -phenylethylamine and benzylamine and is preferentially inhibited by l-deprenyl (selegiline). Species differences in the substrate specificity of MAO have been documented. For example, dopamine is oxidized by MAO-B in humans but by MAO-A in rats and by both enzymes in several other mammalian species. Most tissues contain both forms of the enzyme, each encoded by a distinct gene, although some tissues express only one MAO. In humans, for example, only MAO-A is expressed in the placenta, whereas only MAO-B is expressed in blood

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platelets and lymphocytes. The distribution of MAO in the brain shows little species variation, with the highest concentration of MAO-A in the locus ceruleus and the highest concentration of MAO-B in the raphe nuclei. MAO-A is expressed predominantly in catecholaminergic neurons, whereas MAO-B is expressed largely in serotonergic and histaminergic neurons and glial cells. The distribution of MAO throughout the brain does not always parallel that of its substrates. For example, serotonin is preferentially oxidized by MAO-A, but MAO-A is not found in serotonergic neurons.

MAO-A and -B are encoded by two distinct genes, both localized on the X-chromosome (Xp11.23) and both comprising 15 exons with an identical intron-exon organization, which suggests they are derived from a common ancestral gene (Shih et al., 1999). The amino acid sequence of MAO-A (Mr 59.7 kDa) is 70 percent identical to that of MAO-B (Mr 58.0 kDa). The deletion of both MAO-A and -B gives rise to Norrie disease, an X-linked recessive neurologic disorder characterized by blindness, hearing loss, and mental retardation (Shih et al., 1999). Selective loss of MAO-A (due a point mutation) gives rise to abnormal aggressiveness, whereas alterations in MAO-B have been implicated in Parkinson’s disease (discussed later in this section).

The mechanism of catalysis by monoamine oxidase is illustrated below:

RCH2NH2 FAD n RCHPNH FADH2

RCHPNH H2O n RCHO NH3

FADH2 O2 n FAD H2O2

The substrate is oxidized by the enzyme, which itself is reduced (FAD n FADH2). The oxygen incorporated into the substrate is derived from water, not molecular oxygen; hence the enzyme functions as a true oxidase. The catalytic cycle is completed by reoxidation of the reduced enzyme (FADH2 n FAD) by oxygen, which generates hydrogen peroxide (which may be a cause of oxidative stress). The initial step in the catalytic cycle appears to be abstraction of hydrogen from the -carbon adjacent to the nitrogen atom; hence, the oxidative deamination of xenobiotics by MAO is generally blocked by substitution of the -carbon. For example, amphetamine and other phenylethylamine derivatives carrying a methyl group on the -carbon atom are not oxidized well by MAO. (Amphetamines can undergo oxidative deamination, but the reaction is catalyzed by cytochrome P450.) The abstraction of hydrogen from the -carbon adjacent to the nitrogen atom can occur stereospecifically; therefore, only one enantiomer of an-substituted compound may be oxidized by MAO. For example, whereas MAO-B catalyzes the oxidative deamination of both R- and S- -phenylethylamine, only the R-enantiomer is a substrate for MAO-A. The oxidative deamination of the dealkylated metabolite of propranolol is catalyzed stereoselectively by MAO-A, although in this case the preferred substrate is the S-enantiomer (which has the same absolute configuration as the R-enantiomer of-phenylethylamine) (Benedetti and Dostert, 1994).

Clorgyline and l-deprenyl (selegiline) are metabolismdependent inhibitors (i.e., mechanism-based or suicide inactivators) of MAO-A and MAO-B, respectively. Both enzymes are irreversibly inhibited by phenelzine, a hydrazine that can be oxidized either by abstraction of hydrogen from the -carbon atom, which leads to oxidative deamination with formation of benzaldehyde and benzoic acid, or by abstraction of hydrogen from the terminal ni-

trogen atom, which leads to formation of phenylethyldiazene and covalent modification of the enzyme, as shown in Fig. 6-23.

Monoamine oxidase has received considerable attention for its role in the activation of MPTP (1-methyl-4-phenyl-1,2,5,6- tetrahydropyridine) to a neurotoxin that causes symptoms characteristic of Parkinson’s disease in humans and monkeys but not rodents (Gerlach et al., 1991). In 1983, Parkinsonism was observed in young individuals who, in attempting to synthesize and use a narcotic drug related to meperidine (demerol), instead synthesized and self-administered MPTP, which causes selective destruction of dopaminergic neurons in the substantia nigra. MPTP crosses the blood–brain barrier, where is it oxidized by MAO in the astrocytes (a type of glial cell) to 1-methyl-4-phenyl-2,3-dihydropyridine (MPDP ), which in turn autooxidizes to the neurotoxic metabolite, 1-methyl-4-phenylpyridine MPP , as shown in Fig. 6-24. Because it is transported by the dopamine transporter, MPP concentrates in dopaminergic neurons, where it impairs mitochondrial respiration. The neurotoxic effects of MPTP can be blocked with pargyline (an inhibitor of both MAO-A and MAO-B) and by l-deprenyl (a selective inhibitor of MAO-B) but not by clorgyline (a selective inhibitor of MAO-A). This suggests that the activation of MPTP to its neurotoxic metabolite is catalyzed predominantly by MAO-B. This interpretation is consistent with the recent finding that MAO-B knockout mice (i.e., transgenic mice that lack MAO-B) do not sustain damage to the dopaminergic terminals of nigrostriatal neurons after MPTP treatment (Shih et al., 1999).

Genetic and environmental factors both appear to play important roles in the etiology of Parkinson’s disease. Apart from MPTP, parkinsongenic neurotoxins to which humans are exposed have not been identified unequivocally, hence, the environmental factors that cause Parkinson’s disease remain to be identified. It is interesting that the bipyridyl herbicide paraquat is similar in structure to the toxic metabolite of MPTP, as shown in Fig. 6-24. Some epidemiologic studies have shown a positive correlation between herbicide exposure and the incidence of Parkinsonism in some but not all rural communities. Haloperidol can also be converted to a potentially neurotoxic pyridinium metabolite (Subramanyam et al., 1991).

Figure 6-24. Activation of MPTP (1-methyl-4-phenyl-1,2,5,6-tetrahy- dropyridine) to the neurotoxic metabolite, MPP (1-methyl-4-phenylpyri- dine), by monoamine oxidase B.

The toxic pyridinium metabolite, MPP , is structurally similar to the herbicide paraquat. MPDP , 1-methyl-4-phenyl-2,3-dihydropyridine.

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MAO-B may be among the genetic factors that affect susceptibility to Parkinson’s disease. MAO-B activity in the human brain increases with aging, perhaps due to a proliferation of glial cells. It has been proposed that increased oxidation of dopamine by MAO-B in the elderly may lead to a loss of dopaminergic neurons in the substantia nigra, which underlies Parkinson’s disease. Such damage may be caused by the oxidative stress associated with the oxidative deamination of dopamine by MAO-B. In support of this proposal, it has been found that patients with Parkinson’s disease have elevated MAO-B activity in the substantia nigra, and the MAO-B inhibitor l-deprenyl (selegiline) delays the progression of symptoms (Sano et al., 1997). Furthermore, there are allelic variants of MAO-B, some of which (such as allele 1 and allele B4) appear to be associated with an increased risk of developing Parkinson’s disease (Shih et al., 1999). No such association has been found between Parkinson’s disease and MAO-A gene polymorphisms. Recently, cigarette smoking, which carries a number of health risks, has been shown nevertheless to provide some protection against Parkinson’s disease (Gorell et al., 1999). Although the mechanism of protection remains to be determined, it is interesting to note that cigarette smokers are known to have decreased levels of MAO-B (and MAO-A) (Shih et al., 1999), the degree of which is proportional to cigarette usage (i.e., it is dose-related) (Whitfield et al., 2000).

MAO-A knockout mice have elevated brain levels of serotonin and a distinct behavioral syndrome, including enhanced aggression in adult males. The enhanced aggressive behavior exhibited by MAO-A knockout mice is consistent with the abnormal aggressive behavior in individuals who lack MAO-A activity due to a point mutation in the MAO-A gene (Shih et al., 1999). Other polymorphisms in the MAO-A gene appear to be risk modifiers for alcoholism among Euro-Americans and Han Chinese (Shih et al., 1999). MAO-B may also be a factor in alcoholism, inasmuch as alcoholics (especially male type 2 alcoholics) tend to have lower MAO activity in platelets, which contain only MAO-B. However, MAO-B activity is not lower in alcoholics when cigarette smoking status is taken into account, which suggests that MAO-B activity tends to be lower in alcoholics because smoking and alcohol and dependence are strongly associated with each other (Whitfield et al., 2000).

Although not present in mitochondria, PAO resembles MAO in its cofactor requirement and basic mechanism of action. Both enzymes use oxygen as an electron acceptor, which results in the production of hydrogen peroxide. The MAO inhibitor pargyline also inhibits PAO. The anticonvulsant milacemide is one of the few xenobiotic substrates for PAO, although it is also a substrate for MAO (Fig. 6-23). By converting milacemide to glycine (via glycinamide), MAO plays an important role in anticonvulsant therapy with milacemide (Benedetti and Dostert, 1994).

Diamine oxidase is a cytosolic, copper-containing pyridoxal phosphate-dependent enzyme present in liver, kidney, intestine, and placenta. Its preferred substrates include histamine and simple alkyl diamines with a chain length of four (putrescine) or five (cadaverine) carbon atoms. Diamines with carbon chains longer than nine are not substrates for DAO, although they can be oxidized by MAO. DAO or a similar enzyme is present in cardiovascular tissue and appears to be responsible for the cardiotoxic effects of allylamine, which is converted by oxidative deamination to acrolein. Although histamine is a substrate for DAO, there is little or no DAO in brain (nor is there a receptor-mediated uptake system for histamine, in contrast to other neurotransmitters). For this reason, the major path-

way of histamine metabolism in the brain is by methylation (see “Methylation,” below).

Aromatization The conversion of MPTP to MPP (Fig. 6-24) is an example of a reaction involving the introduction of multiple double bonds to achieve some semblance of aromaticity (in this case, formation of a pyridinium ion). Aromatization of xenobiotics is an unusual reaction, but some examples have been documented. A mitochondrial enzyme in guinea pig and rabbit liver can oxidize several cyclohexane derivatives to the corresponding aromatic hydrocarbon, as shown in Fig. 6-25 for the aromatization of cyclohexane carboxylic acid (hexahydrobenzoic acid) to benzoic acid. Mitochondria from rat liver are less active, and those from cat, mouse, dog, monkey, and human are completely inactive. The reaction requires magnesium, coenzyme A, oxygen, and ATP. The first step appears to be the formation of hexahydrobenzoyl-CoA, which is then dehydrogenated to the aromatic product. Glycine stimulates the reaction, probably by removing benzoic acid through conjugation to form hippuric acid (a phase II reaction). The conversion of androgens to estrogens involves aromatization of the A- ring of the steroid nucleus. This reaction is catalyzed by CYP19, one of the cytochrome P450 enzymes involved in steroidogenesis.

Peroxidase-Dependent Cooxidation The oxidative biotransformation of xenobiotics generally requires the reduced pyridine nucleotide cofactors NADPH and NADH. An exception is xenobiotic biotransformation by peroxidases, which couple the reduction of hydrogen peroxide and lipid hydroperoxides to the oxidation of other substrates—a process known as cooxidation (Eling et al., 1990). Several different peroxidases catalyze the biotransformation of xenobiotics, and these enzymes occur in a variety of tissues and cell types. For example, kidney medulla, platelets, vascular endothelial cells, the GI tract, brain, lung, and urinary bladder epithelium contain prostaglandin H synthase (PHS); mammary gland epithelium contains lactoperoxidase; and leukocytes contain myeloperoxidase. PHS is one of the most extensively studied peroxidase involved in the xenobiotic biotransformation. This enzyme possesses two catalytic activities: a cyclooxygenase that converts arachidonic acid to the cyclic endoperoxide-hydroperoxide PGG2 (which involves the addition of two molecules of oxygen to each molecule of arachidonic acid) and a peroxidase that converts the hydroperoxide to the corresponding alcohol PGH2 (which can be accompanied by the oxidation of xenobiotics). The conversion of arachidonic acid to PGH2, which is subsequently converted to a variety of eicosanoids (prostaglandins, thromboxane, and prostacyclin), is shown in Fig. 6-26. PHS and other peroxidases play an important role in the activation of xenobiotics to toxic or tumorigenic metabolites, particularly in extrahepatic tissues that contain low levels of cytochrome P450 (Eling et al., 1990).

Figure 6-25. Aromatization of cyclohexane carboxylic acid, a reaction catalyzed by rabbit and guinea pig liver mitochondria.