26510318-Bio-Transformation-of-Xenobiotics

Figure 6-26. Cooxidation of xenobiotics (X) during the conversion of arachidonic acid to PGH2 by prostaglandin H synthase.

In certain cases, the oxidation of xenobiotics by peroxidases involves direct transfer of the peroxide oxygen to the xenobiotic, as shown in Fig. 6-26 for the conversion of substrate X to product XO. An example of this type of reaction is the PHS-catalyzed epoxidation of benzo[a]pyrene 7,8-dihydrodiol to the corresponding 9,10-epoxide (see Fig. 6-6). Although PHS can catalyze the final step (i.e., 9,10-epoxidation) in the formation of this tumorigenic metabolite of benzo[a]pyrene, it cannot catalyze the initial step (i.e., 7,8-epoxidation), which is catalyzed by cytochrome P450. The 9,10-epoxidation of benzo[a]pyrene 7,8-dihydrodiol can also be catalyzed by 15-lipoxygenase, which is present at high concentrations in human pulmonary epithelial cells, and by peroxyl radicals formed during lipid peroxidation in skin. Cytochrome P450 and peroxyl radicals formed by PHS, lipoxygenase, and/or lipid peroxidation may all play a role in activating benzo[a]pyrene to metabolites that cause lung and skin tumors. These same enzymes

Figure 6-27. Activation of aflatoxin B1 by cytochrome P450, leading to liver tumor formation, and by peroxidases, leading to renal papilla neoplasia.

Fig. 6-26, xenobiotics that can serve as electron donors, such as amines and phenols, can be oxidized to free radicals during the reduction of a hydroperoxide. In this case, the hydroperoxide is still converted to the corresponding alcohol, but the peroxide oxygen is reduced to water instead of being incorporated into the xenobiotic. For each molecule of hydroperoxide reduced (which is a twoelectron process), two molecules of xenobiotic can be oxidized (each by a one-electron process). Important classes of compounds that undergo one-electron oxidation reactions by peroxidase include aromatic amines, phenols, hydroquinones, and polycyclic hydrocarbons. Many of the metabolites produced are reactive electrophiles. For example, polycyclic aromatic hydrocarbons, phenols, and hydroquinones are oxidized to electrophilic quinones. Acetaminophen is similarly converted to a quinoneimine, namely N-acetyl-benzoquinoneimine, a cytotoxic electrophile that binds to cellular proteins, as shown in Fig. 6-28. The formation of this toxic metabolite by cytochrome P450 causes centrilobular necrosis of the liver. However, acetaminophen can also damage the kidney medulla, which contains low levels of cytochrome P450 but relatively high levels of PHS; hence, PHS may play a significant role in the nephrotoxicity of acetaminophen. The two-electron oxidation of acetaminophen to N-acetyl-benzoquinoneimine by PHS likely involves the formation of a one-electron oxidation product, namely N-acetyl-benzosemiquinoneimine radical. Formation of this semiquinoneimine radical by PHS likely contributes to the

nephrotoxicity of acetaminophen and related compounds, such as phenacetin and 4-aminophenol.

Like the kidney medulla, urinary bladder epithelium also contains low levels of cytochrome P450 but relatively high levels of PHS. Just as PHS in kidney medulla can activate aflatoxin and acetaminophen to nephrotoxic metabolites, so PHS in urinary bladder epithelium can activate certain aromatic amines —such as benzidine, 4-aminobiphenyl, and 2-aminonaphthalene—to DNA-reac- tive metabolites that cause bladder cancer in certain species, including humans and dogs. PHS can convert aromatic amines to reactive radicals, which can undergo nitrogen–nitrogen or nitro- gen–carbon coupling reactions, or they can undergo a second oneelectron oxidation to reactive diimines. Binding of these reactive metabolites to DNA is presumed to be the underlying mechanism by which several aromatic amines cause bladder cancer in humans and dogs. In some cases the one-electron oxidation of an amine leads to N-dealkylation. For example, PHS catalyzes the N-demethylation of aminopyrine, although in vivo this reaction is mainly catalyzed by cytochrome P450. In contrast to cytochrome P450, PHS does not catalyze the N-hydroxylation of aromatic amines.

Many of the aromatic amines known or suspected of causing bladder cancer in humans have been shown to cause bladder tumors in dogs. In rats, however, aromatic amines cause liver tumors by a process that involves N-hydroxylation by cytochrome P450,

followed by conjugation with acetate or sulfate, as shown in Fig. 6-9. This species difference has complicated an assessment of the role of PHS in aromatic amine-induced bladder cancer, because such experiments must be carried out in dogs. However, another class of compounds, the 5-nitrofurans, such as N-[4-(5-nitro-2- furyl)-2-thiazole]formamide (FANFT) and its deformylated analog 2-amino-4-(5-nitro-2-furyl)thiazole (ANFT), are substrates for PHS and are potent bladder tumorigens in rats. The tumorigenicity of FANFT is thought to involve deformylation to ANFT, which is oxidized to DNA-reactive metabolites by PHS. The ability of FANFT to cause bladder tumors in rats is blocked by the PHS cyclooxygenase inhibitor aspirin, which suggests that PHS plays an important role in the metabolic activation and tumorigenicity of this nitrofuran. Unexpectedly, combined treatment of rats with FANFT and aspirin causes forestomach tumors, which are not observed when either compound is administered alone. This example underscores the complexity of chemically induced tumor formation. A further complication in interpreting this result is the recent finding that there are two forms of cyclooxygenase: COX- 1, which is constitutively expressed in several tissues, and COX- 2, which is inducible by growth factors and mediators of inflammation. Increased expression of the latter isozyme has been documented in a number of tumors, including human colorectal, gastric, esophageal, pulmonary, and pancreatic carcinomas (Gupta and DuBois, 1998; Molina et al., 1999). Aspirin and other NSAIDs block the formation of colon cancer in experimental animals, and there is epidemiological evidence that chronic NSAID usage decreases the incidence of colorectal cancer in humans (Gupta and DuBois, 1998). The incidence of intestinal neoplasms in Apc 716 knockout mice is dramatically suppressed by crossing these transgenic animals with COX-2 knockout mice (Oshima et al., 1996). From these few examples it is apparent that cyclooxygenase may play at least two distinct roles in tumor formation—it may convert certain xenobiotics to DNA-reactive metabolites (and thereby initiate tumor formation), and it may somehow promote subsequent tumor growth, perhaps through formation of growth-promoting eicosanoids.

Many phenolic compounds can serve as reducing substrates for PHS peroxidase. The phenoxyl radicals produced by oneelectron oxidation reactions can undergo a variety of reactions, including binding to critical nucleophiles, such as protein and DNA; reduction by antioxidants such as glutathione; and self-coupling. The reactions of phenoxyl radicals are analogous to those of the nitrogen-centered free radicals produced during the one-electron oxidation of aromatic amines by PHS. Peroxidases appear to play an important role in the bone marrow suppression produced by chronic exposure to benzene. Liver cytochrome P450 converts benzene to phenol, which in turn is oxidized to hydroquinone, which can be converted to DNA-reactive metabolites by PHS in bone marrow and by myeloperoxidase in bone marrow leukocytes. The myelosuppressive effect of benzene can be blocked by the PHS inhibitor indomethacin, which suggests an important role for perox- idase-dependent activation in the myelotoxicity of benzene. The formation of phenol and hydroquinone in the liver is also important for myelosuppression by benzene. However, such bone marrow suppression cannot be achieved simply by administering phenol or hydroquinone to mice, although it can be achieved by coadministering hydroquinone with phenol. Phenol stimulates the PHS-dependent activation of hydroquinone. Therefore, bone marrow suppression by benzene involves the cytochrome P450– dependent oxidation of benzene to phenol and hydroquinone in the

liver, followed by phenol-enhanced peroxidative oxidation of hydroquinone to reactive intermediates that bind to protein and DNA in the bone marrow (Fig. 6-29). It is noteworthy that the cytochrome P450 enzyme responsible for hydroxylating benzene has been identified as CYP2E1 (see section on cytochrome P450, below). Although CYP2E1was first identified in liver, this same enzyme has been identified in bone marrow, where it can presumably convert benzene to phenol and possibly hydroquinone (Bernauer et al., 2000). The importance of CYP2E1 in the metabolic activation of benzene was recently confirmed by the demonstration that CYP2E1 knockout mice (null mice) are relatively resistant to the myelosuppressive effects of benzene (Gonzalez and Kimura, 1999; Buters et al., 1999).

The ability of phenol to enhance the peroxidative metabolism of hydroquinone is analogous to the interaction between the phenolic antioxidants butylated hydroxytoluene (BHT), and butylated hydroxyanisole (BHA). In mice, the pulmonary toxicity of BHT, which is a relatively poor substrate for PHS, is enhanced by BHA, which is a relatively good substrate for PHS. The mechanism by which BHA enhances the pulmonary toxicity of BHT appears to involve the peroxidase-dependent conversion of BHA to a phenoxyl radical that interacts with BHT, converting it to a phenoxyl radical (by one-electron oxidation) or a quinone methide (by two-electron oxidation), as shown in Fig. 6-30. Formation of the toxic quinone methide of BHT can also be catalyzed by cytochrome P450, which is largely responsible for activating BHT in the absence of BHA.

Several reducing substrates—such as phenylbutazone, retinoic acid, 3-methylindole, sulfite, and bisulfite—are oxidized by PHS to carbonor sulfur-centered free radicals that can trap oxygen to form a peroxyl radical, as shown in Fig. 6-31 for phenylbutazone. The peroxyl radical can oxidize xenobiotics in a peroxidative manner. For example, the peroxyl radical of phenylbutazone can convert benzo[a]pyrene 7,8-dihydrodiol to the corresponding 9,10-epoxide.

PHS is unique among peroxidases because it can both generate hydroperoxides and catalyze peroxidase-dependent reactions, as shown in Fig. 6-26. Xenobiotic biotransformation by PHS is

Figure 6-29. Role of cytochrome P450 and peroxidases in the activation of benzene to myelotoxic metabolites.

ABBREVIATION: PHS, prostaglandin H synthase.

controlled by the availability of arachidonic acid. The biotransformation of xenobiotics by other peroxidases is controlled by the availability of hydroperoxide substrates. Hydrogen peroxide is a normal product of cellular respiration, and lipid peroxides can form during lipid peroxidation. The levels of these peroxides and their availability for peroxidase reactions depends on the efficiency of hydroperoxide scavenging by glutathione peroxidase and catalase.

Uetrecht and others have implicated myeloperoxidase in the formation of reactive metabolites of drugs that cause agranulocytosis, including clozapine, aminopyrine, vesnarinone, propylthiouracil, dapsone, sulfonamides, procainamide, amodiaquine, and ticlopidine (references to this large body of work can be found in Liu and Uetrecht, 2000). Activation of these drugs involves their oxidation by HOCl, which is the principal oxidant produced by myeloperoxidase (in the presence of hydrogen peroxide and chloride ion) in activated neutrophils and monocytes. In the case of ticlopidine, myeloperoxidase converts the thiophene ring of this antiplatelet drug to a thiophene-S-chloride, a reactive metabolite that rearranges to 2-chloroticlopidine (minor) and dehydroticlopidine (major), or reacts with glutathione, as shown in Fig. 6-32. When catalyzed by activated neutrophils, ticlopidine oxidation is inhibited by low concentrations of azide and catalase. When catalyzed by purified myeloperoxidase, ticlopidine oxidation requires hydrogen peroxide and chloride, although all components of this purified system can be replaced with HOCl. It is not known

whether drugs that cause agranulocytosis are activated in the bone marrow by neutrophils or their precursors that contain myeloperoxidase or are activated in neutrophils in the general circulation. In the latter case, agranulocytosis would presumably involve an immune response triggered by neoantigens formed in neutrophils by the covalent modification of cellular component by one or more of the reactive metabolites formed by myeloperoxidase.

Flavin Monooxygenases Liver, kidney, and lung contain one or more FAD-containing monooxygenases (FMO) that oxidize the nucleophilic nitrogen, sulfur, and phosphorus heteroatom of a variety of xenobiotics (Ziegler, 1993; Lawton et al., 1994; Cashman, 1995; Rettie and Fisher, 1999; Cashman, 1999). The mammalian FMO gene family comprises five enzymes (designated FMO1 to FMO5) that contain about 550 amino acid residues each and are 50 to 58 percent identical in amino acid sequence across species lines. Each FMO enzyme contains a highly conserved glycine-rich region (residues 4 to 32) that binds 1 mole of FAD (noncovalently) near the active site, which is adjacent to a second highly conserved glycine-rich region (residues 186 to 213) that binds NADPH.

Like cytochrome P450, the FMOs are microsomal enzymes that require NADPH and O2, and many of the reactions catalyzed by FMO can also be catalyzed by cytochrome P450. Several in vitro techniques have been developed to distinguish reactions catalyzed by FMO from those catalyzed by cytochrome P450. In con-

trast to cytochrome P450, FMO is heat-labile and can be inactivated in the absence of NADPH by warming microsomes to 50°C for 1 min. By comparison, cytochrome P450 can be inactivated with nonionic detergent, such as 1% Emulgen 911, which has a minimal effect on FMO activity. The pH optimum for FMOcatalyzed reactions (pH 8 to 10) tends to be higher than that for most (but not all) P450 reactions (pH 7 to 8). Antibodies raised against purified P450 enzymes can be used not only to establish the role of cytochrome P450 in a microsomal reaction but also to identify which particular P450 enzyme catalyzes the reaction. In contrast, antibodies raised against purified FMO do not inhibit the enzyme. The use of chemical inhibitors to ascertain the relative contribution of FMO and cytochrome P450 to microsomal reactions is often complicated by a lack of specificity. For example, cimetidine and SKF 525A, which are well-recognized cytochrome P450 inhibitors, are both substrates for FMO. Conversely, the FMO inhibitor methimazole is known to inhibit several of the P450 enzymes in human liver microsomes (namely CYP2B6, CYP2C9, and CYP3A4). The situation is further complicated by the observation that the various forms of FMO differ in their thermal stability and sensitivity to detergents and other chemical modulators (examples of which are described later in this section).

FMO catalyzes the oxidation of nucleophilic tertiary amines to N-oxides, secondary amines to hydroxylamines and nitrones, and primary amines to hydroxylamines and oximes. Amphetamine,

benzydamine, chlorpromazine, clozapine, guanethidine, imipramine, methamphetamine, olanzapine, and tamoxifen are examples of nitrogen-containing drugs that are N-oxygenated by FMO (and by cytochrome P450 in most cases). FMO also oxidizes several sulfur-containing xenobiotics (such as thiols, thioethers, thiones, and thiocarbamates) and phosphines to S- and P-oxides, respectively. Cimetidine and sulindac sulfide are examples of sulfur-con- taining drugs that are converted to sulfoxides by FMO. (Fig. 6-12 shows how sulindac is reduced to sulindac sulfide, only to be oxidized by FMO back to the parent drug in what is often called a futile cycle.) Hydrazines, iodides, selenides, and boron-containing compounds are also substrates for FMO. Examples of FMOcatalyzed reactions are shown in Fig. 6-33A and B.

In general, the metabolites produced by FMO are the products of a chemical reaction between a xenobiotic and a peracid or peroxide, which is consistent with the mechanism of catalysis of FMO (discussed later in this section). The reactions catalyzed by FMO are generally detoxication reactions, although there are exceptions to this rule, described below (this section). Inasmuch as FMO attacks nucleophilic heteroatoms, it might be assumed that substrates for FMO could be predicted simply from their pKa values (i.e., from a measure of their basicity). Although there is some truth to this—for example, xenobiotics containing an sp3- hybridized nitrogen atom with a pKa of 5 to 10 are generally good substrates for FMO—predictions of substrate specificity based on

pKa alone are not very reliable, presumably because steric effects influence access of substrates to the FMO active site (Rettie and Fisher, 1999).

With few exceptions, FMO acts as an electrophilic oxygenating catalyst, which distinguishes it from most other flavoprotein oxidases and monooxygenases (which will be discussed further in the section on cytochrome P450). The mechanism of catalysis by FMO is depicted in Fig. 6-34. After the FAD moiety is reduced to FADH2 by NADPH, the oxidized cofactor, NADP , remains bound to the enzyme. FADH2 then binds oxygen to produce a peroxide (i.e., the 4a-hydroperoxyflavin of FAD). The peroxide is relatively stable, probably because the active site of FMO comprises nonnucleophilic, lipophilic amino acid residues. During the oxygenation of xenobiotics, the 4a-hydroperoxyflavin is converted to 4a-

hydroxyflavin with transfer of the flavin peroxide oxygen to the substrate (depicted as X XO in Fig. 6-34). From this latter step, it is understandable why the metabolites produced by FMO are generally the products of a chemical reaction between a xenobiotic and a peroxide or peracid. The final step in the catalytic cycle involves dehydration of 4a-hydroxyflavin (which restores FAD to its resting, oxidized state) and release of NADP . This final step is important because it is rate-limiting, and it occurs after substrate oxygenation. Consequently, this step determines the upper limit of the rate of substrate oxidation. Therefore all good substrates for FMO are converted to products at the same maximum rate (i.e., Vmax is determined by the final step in the catalytic cycle). Binding of NADP to FMO during catalysis is important because it prevents the reduction of oxygen to H2O2. In the absence of bound

NADP , FMO would function as an NADPH-oxidase that would consume NADPH and cause oxidative stress through excessive production of H2O2.

The oxygenation of substrates by FMO does not lead to inactivation of the enzyme, even though some of the products are strong electrophiles capable of binding covalently to critical and noncritical nucleophiles such as protein and glutathione, respectively. The products of the oxygenation reactions catalyzed by FMO and/or the oxygenation of the same substrates by cytochrome P450 can inactivate cytochrome P450. For example, the FMOdependent S-oxygenation of spironolactone thiol (which is formed by the deacetylation of spironolactone by carboxylesterases, as

shown in Fig. 6-2) leads to the formation of an electrophilic sulfenic acid (R – SH R – SOH), which inactivates cytochrome P450 and binds covalently to other proteins.

In humans, FMO plays an important role in the biotransformation of several drugs (e.g., benzydamine, cimetidine, clozapine, guanethidine, methimazole, olanzapine, sulindac sulfide, tamoxifen and various dimethylaminoalkyl phenothiazine derivatives such as chlorpromazine and imipramine), xenobiotics (e.g., cocaine, methamphetamine, nicotine, tyramine), and endogenous substrates (e.g., trimethylamine, cysteamine). The major flavin monooxygenase in human liver microsomes, FMO3, is predominantly if not solely responsible for converting (S)-nicotine to

Figure 6-33. (continued)

(S)-nicotine N-1 -oxide (which is one of the reactions shown in Fig. 6-33A). The reaction proceeds stereospecifically; only the trans isomer is produced by FMO3, and this is the only isomer of (S)-nicotine N-1 -oxide excreted in the urine of cigarette smokers or individuals wearing a nicotine patch. Therefore, the urinary excretion of trans-(S)-nicotine N-1 -oxide can be used as an in vivo probe of FMO3 activity in humans. FMO3 is also the principal enzyme involved in the S-oxygenation of cimetidine, an H2 antagonist widely used in the treatment of gastric ulcers and other acidrelated disorders (this reaction is shown in Fig. 6-33B ). Cimetidine is stereoselectively sulfoxidated by FMO3 to an 84:16 mixture of ( ) and ( ) enantiomers, which closely matches the 75:25 enantiomeric composition of cimetidine S-oxide in human urine. There-

fore, the urinary excretion of cimetidine S-oxide, like that of (S)-nicotine N-1 -oxide, is an in vivo indicator of FMO3 activity in humans.

Sulindac is a sulfoxide that exists in two stereochemical forms (as do most sulfoxides), and a racemic mixture of R- and S-sulindac is used therapeutically as a NSAID. As shown in Fig. 6-12, the sulfoxide group in sulindac is reduced to the corresponding sulfide (which is achiral), which is then oxidized back to sulindac (a process often described as futile cycling). In human liver, the sulfoxidation of sulindac sulfide is catalyzed by FMO3 with little or no contribution from cytochrome P450. At low substrate concentrations (30 M), FMO3 converts sulindac sulfide to R- and S-sulindac in an 87:13 ratio (Hamman et al., 2000). Consequently,

Figure 6-34. Catalytic cycle of flavin monooxygenase (FMO).

X and XO are the xenobiotic substrate and oxygenated product, respectively. The 4a-hydroperoxyflavin and 4a-hydroxyflavin of FAD are depicted as FADHOOH and FADHOH, respectively.

although sulindac is administered as a racemic mixture (i.e., a 1:1 mixture of R- and S-enantiomers), the reduction of this drug to the corresponding sulfide and its preferential sulfoxidation by FMO3 to R-sulindac results in stereoselective enrichment of R-sulindac in serum and urine.

In the case of sulindac sulfide, stereoselective sulfoxidation occurs not only with human FMO3 (the major FMO in human liver) but also with porcine FMO1 (the major form expressed in pig liver) and rabbit FMO2 (the major form expressed in rabbit lung) (Hamman et al., 2000). However, this conformity is the exception rather than the rule. For example, in contrast to the stereoselective oxygenation of (S)-nicotine and cimetidine by human FMO3 (see above), FMO1 (which is the major FMO expressed in pig, rat, and rabbit liver) converts (S)-nicotine to a 1:1 mixture of cis- and trans-(S)-nicotine N-1 -oxide and similarly converts cimetidine to a 1:1 mixture of ( ) and ( ) cimetidine S-oxide, respectively. Therefore, statements concerning the role of FMO in the disposition of xenobiotics in humans may not apply to other species, or vice versa.

Several sulfur-containing xenobiotics are oxygenated by FMO to electrophilic reactive intermediates. Such xenobiotics include various thiols, thioamides, 2-mercaptoimidazoles, thiocarbamates, and thiocarbamides. The electrophilic metabolites of these xenobiotics do not inactivate FMO, but they can covalently modify and inactivate neighboring proteins, including cytochrome P450. Some of these same xenobiotics are substrates for cytochrome P450, and their oxygenation to electrophilic metabolites leads to inactivation of cytochrome P450, a process known variously as metab- olism-dependent inhibition, mechanism-based inhibition and suicide inactivation. 2-Mercaptoimidazoles undergo sequential S-oxygenation reactions by FMO, first to sulfenic acids and then to sulfinic acids (R–SH R–SOH R–SO2H). These electrophilic metabolites, like the sulfenic acid metabolite produced from spironolactone thiol (see above), bind to critical nucleophiles (such as proteins) or interact with glutathione to form disulfides. The thiocarbamate functionality present in numerous agricultural

chemicals is converted by FMO to S-oxides (sulfoxides), which can be further oxygenated to sulfones. These reactions involve S-oxygenation adjacent to a ketone, which produces strong electrophilic acylating agents, which may be responsible for the toxicity of many thiocarbamate herbicides and fungicides. The hepatotoxicity of thiobenzamide is dependent on S-oxidation by FMO and/or cytochrome P450. As shown in Fig. 6-33B, the S-oxidation of thiobenzamide can lead to the formation of an oxathiirane (a three-membered ring of carbon, sulfur, and oxygen) that can bind covalently to protein (which leads to hepatocellular necrosis) or rearrange to benzamide, a reaction known as oxidative group transfer.

Endogenous FMO substrates include cysteamine, which is oxidized to the disulfide, cystamine, and trimethylamine (TMA), which is converted to TMA N-oxide (Fig. 6-33B). By converting cysteamine to cystamine, FMO may serve to produce a low- molecular-weight disulfide-exchange agent, which may participate in the formation of disulfide bridges during peptide synthesis or the renaturation of proteins. By converting TMA to TMA N-oxide, FMO converts a malodorous and volatile dietary product of choline, lecithin, and carnitine catabolism to an inoffensive metabolite. TMA smells of rotting fish, and people who are genetically deficient in FMO3 suffer from trimethylaminuria or fish-odor syndrome, which is caused by the excretion of TMA in urine, sweat, and breath (Ayesh and Smith, 1992). The underlying genetic basis of trimethylaminuria is a mutation (Pro153 Leu153) in exon 4 of the FMO3 gene (Dolphin et al., 1997). Although this mutation (and hence trimethylaminuria) occurs only rarely, it is now known to be just one of several mutations that decrease or eliminate FMO3 activity; these other mutations include missense mutations (Met66 Ile66, Met82 Thr82, Arg492 is Trp492) and the nonsense mutation Glu305 X305 (Cashman et al., 2000). As might be expected, trimethylaminuria is associated with an impairment of nicotine N-oxidation and other pathways of drug biotransformation that are primarily catalyzed by FMO3 (Rettie and Fisher, 1999).

Humans and other mammals express five different flavin monooxygenases (FMO1, FMO2, FMO3, FMO4, and FMO5) in a speciesand tissue-specific manner, as shown in Table 6-3 (adapted from Cashman, 1995). For example, the major FMO expressed in human and mouse liver microsomes is FMO3, whereas FMO1 is the major FMO expressed in rat, rabbit, and pig liver. (Although FMO3 is the major FMO in adult human liver, the major FMO in fetal human liver is FMO1.) In humans, high levels of FMO1 are expressed in the kidney, and low levels of FMO2 are expressed in the lung. However, lung microsomes from other species, particularly rabbit, mouse, and monkey, contain high levels of FMO2. The uncharacteristically low levels of FMO2 in human lung are due to a mutation (a C T transition at codon 472) in the major human FMO2 allele, which results in the synthesis of a nonfunctional, truncated protein (one lacking the last 64–amino acid residues from the C-terminus) (Dolphin et al., 1998). FMO4 appears to be expressed at low levels in the brain of several mammalian species, where it might terminate the action of several centrally active drugs and other xenobiotics.

The various forms of FMO are distinct gene products with different physical properties and substrate specificities. For example, FMO2 N-oxygenates n-octylamine, whereas such long aliphatic primary amines are not substrates for FMO1, although they stimulate its activity toward other substrates (in some cases causing a change in stereospecificity). Conversely, short-chain tertiary amines, such as chlorpromazine and imipramine, are sub-

strates for FMO1 but not FMO2. Certain substrates are oxygenated stereospecifically by one FMO enzyme but not another. For example, FMO2 and FMO3 convert (S)-nicotine exclusively to trans-(S)-nicotine N-1 -oxide, whereas the N-oxides of (S)-nico- tine produced by FMO1 are a 1:1 mixture of cis and trans isomers. FMO2 is heat-stable under conditions that completely inactivate FMO1, and FMO2 is resistant to anionic detergents that inactivate FMO1. Low concentrations of bile salts, such as cholate, stimulate FMO activity in rat and mouse liver microsomes but inhibit FMO activity in rabbit and pig liver.

The FMO enzymes expressed in liver microsomes are not under the same regulatory control as cytochrome P450. In rats, the expression of FMO1 is repressed rather than induced by treatment with phenobarbital or 3-methylcholanthrene (although some studies point to of a modest [ 3-fold] induction of rat FMO1 by 3-methylcholanthrene). Indole-3-carbinol, which induces the same P450 enzymes as 3-methylcholanthrene, causes a marked decrease in FMO activity in rat liver and intestine. A similar decrease in FMO3 activity occurs in human volunteers following the consumption of large amounts of Brussels sprouts, which contain high levels of indole-3-carbinol and related indoles. The decrease in FMO3 activity may result from direct inhibition of FMO3 by in- dole-3-carbinol and its derivatives rather than from an actual decrease in enzyme levels (Cashman et al., 1999). The levels of FMO3 and, to a lesser extent, of FMO1 in mouse liver microsomes are sexually differentiated (female male) due to suppression of expression by testosterone. The opposite is true of FMO1 levels in rat liver microsomes, the expression of which is positively regulated by testosterone and negatively regulated by estradiol. In pregnant rabbits, lung FMO2 is positively regulated by progesterone and/or corticosteroids.

Species differences in the relative expression of FMO and cytochrome P450 appear to determine species differences in the toxicity of the pyrrolizidine alkaloids, senecionine, retrorsine, and monocrotaline. These compounds are detoxified by FMO, which catalyzes the formation of tertiary amine N-oxides, but they are activated by cytochrome P450, which oxidizes these alkaloids to

pyrroles that generate toxic electrophiles through the loss of substituents on the pyrrolizidine nucleus (details of which appear in the section on cytochrome P450). Rats have a high pyrrole-forming cytochrome P450 activity and a low N-oxide forming FMO activity, whereas the opposite is true of guinea pigs. This likely explains why pyrrolizidine alkaloids are highly toxic to rats but not to guinea pigs. Many of the reactions catalyzed by FMO are also catalyzed by cytochrome P450, but differences in the oxidation of pyrrolizidine alkaloids by FMO and cytochrome P450 illustrate that this is not always the case.

Cytochrome P450 Among the phase I biotransforming enzymes, the cytochrome P450 system ranks first in terms of catalytic versatility and the sheer number of xenobiotics it detoxifies or activates to reactive intermediates (Guengerich, 1987; Waterman and Johnson, 1991). The highest concentration of P450 enzymes involved in xenobiotic biotransformation is found in liver endoplasmic reticulum (microsomes), but P450 enzymes are present in virtually all tissues. The liver microsomal P450 enzymes play a very important role in determining the intensity and duration of action of drugs, and they also play a key role in the detoxication of xenobiotics. P450 enzymes in liver and extrahepatic tissues play important roles in the activation of xenobiotics to toxic and/or tumorigenic metabolites. Microsomal and mitochondrial P450 enzymes play key roles in the biosynthesis or catabolism of steroid hormones, bile acids, fat-soluble vitamins, fatty acids, and eicosanoids, which underscores the catalytic versatility of cytochrome P450.

All P450 enzymes are heme-containing proteins. The heme iron in cytochrome P450 is usually in the ferric (Fe3 ) state. When reduced to the ferrous (Fe2 ) state, cytochrome P450 can bind ligands such as O2 and carbon monoxide (CO). The complex between ferrous cytochrome P450 and CO absorbs light maximally at 450 nm, from which cytochrome P450 derives its name. The absorbance maximum of the CO complex differs slightly among different P450 enzymes and ranges from 447 to 452 nm. All other hemoproteins that bind CO absorb light maximally at 420 nm. The unusual ab-