Chapter 6. Biotransformation of Xenobiotics

• Biotransformation is the metabolic conversion of endogenous and xenobiotic chemicals to more water-soluble compounds.
• Xenobiotic biotransformation is accomplished by a limited number of enzymes with broad substrate specificities.
• Phase I reactions involve hydrolysis, reduction, and oxidation. These reactions expose or introduce a functional group (—OH, —NH2, —SH, or —COOH), and usually result in only a small increase in hydrophilicity.
• Phase II biotransformation reactions include glucuronidation, sulfonation (more commonly called sulfation), acetylation, methylation, and conjugation with glutathione (mercapturic acid synthesis), which usually result in increased hydrophilicity and elimination.

Biotransformation is the metabolic conversion of endogenous and xenobiotic chemicals to more water-soluble compounds. Generally, the physical properties of a xenobiotic are changed from those favoring absorption (lipophilicity) to those favoring excretion in urine or feces (hydrophilicity). An exception to this general rule is the elimination of volatile compounds by exhalation.

Chemical modification of a xenobiotic by biotransformation may alter its biological effects. Some drugs undergo biotransformation to active metabolites that exert their pharmacodynamic or toxic effect. In most cases, however, biotransformation terminates the pharmacologic effects of a drug and lessens the toxicity of xenobiotics. Enzymes catalyzing biotransformation reactions often determine the intensity and duration of action of drugs and play a key role in chemical toxicity and chemical tumorigenesis.

Basic Properties of Xenobiotic Biotransforming Enzymes

Xenobiotic biotransformation is accomplished by a limited number of enzymes with broad substrate specificities. The synthesis of some of these enzymes is triggered by the xenobiotic (by the process of enzyme induction), but in most cases the enzymes are expressed constitutively (i.e., they are synthesized in the absence of a discernible external stimulus). Although the synthesis of steroid hormones is catalyzed by cytochrome P450 enzymes in steroidogenic tissues, this family of enzymes in the liver converts steroid hormones into water-soluble metabolites to be excreted.

The structure (i.e., amino acid sequence) of a given biotransforming enzyme may differ among individuals, which can give rise to differences in rates of xenobiotic biotransformation. The study of the causes, prevalence, and impact of heritable differences in xenobiotic biotransforming enzymes is known as pharmacogenetics.

Biotransformation versus Metabolism

The terms biotransformation and metabolism are often used synonymously, particularly when applied to drugs. The term metabolism is often used to describe the total fate of a xenobiotic, which includes absorption, distribution, biotransformation, and elimination. However, metabolism is commonly used to mean biotransformation, which is understandable from the standpoint that the products of xenobiotic biotransformation are known as metabolites. Furthermore, individuals with a genetic enzyme deficiency resulting in impaired xenobiotic biotransformation are described as poor metabolizers rather than poor biotransformers.

Stereochemical Aspects of Biotransformation

Stereochemical properties influence the interaction between a xenobiotic and its biotransforming enzyme. Many xenobiotics, especially drugs, contain one or more chiral centers and can exist in two mirror-image stereoisomers or enantiomers. The biotransformation of some chiral xenobiotics occurs stereoselectively, which means that one enantiomer (stereoisomer) is biotransformed faster than its antipode.

Categories of Xenobiotic Biotransforming Enzymes

The reactions catalyzed by xenobiotic biotransforming enzymes are generally divided into four categories: (1) hydrolysis, (2) reduction, (3) oxidation, and (4) conjugation (Table 6–1). Hydrolysis, reduction, and oxidation reactions expose or introduce a functional group (—OH, —NH2, —SH, or —COOH), and usually result in only a small increase in hydrophilicity. Conjugation biotransformation reactions include glucuronidation, sulfonation (more commonly called sulfation), acetylation, methylation, conjugation with glutathione (mercapturic acid synthesis), and conjugation with amino acids (such as glycine, taurine, and glutamic acid). Most result in a large increase in xenobiotic hydrophilicity; hence, they greatly promote the excretion of foreign chemicals.

Distribution of Xenobiotic Biotransforming Enzymes

Xenobiotic biotransforming enzymes are widely distributed throughout the body and are present in several subcellular compartments. In vertebrates, the liver is the richest source of enzymes catalyzing biotransformation reactions. These enzymes are also located in the skin, lung, nasal mucosa, kidney, eye, gastrointestinal tract, as well as numerous other tissues. Intestinal microflora play an important role in the biotransformation of certain xenobiotics. Biotransformation enzymes are located primarily in the endoplasmic reticulum (microsomes) or the soluble fraction of the cytoplasm (cytosol), with lesser amounts in mitochondria, nuclei, and lysosomes (see Table 6–1).

Hydrolysis

Carboxylesterases, Cholinesterases, and Paraoxonase

The hydrolysis of carboxylic acid esters, amides, and thioesters is largely catalyzed by carboxylesterases and by two cholinesterases: true acetylcholinesterase in erythrocyte membranes and pseudocholinesterase, which is also known as butyrylcholinesterase and is located in serum. Phosphoric acid esters are hydrolyzed by paraoxonase, a serum enzyme also known as aryldialkylphosphatase. Phosphoric acid anhydrides are hydrolyzed by a related organophosphatase.

Carboxylesterases in serum and tissues and serum cholinesterase collectively determine the duration and site of action of certain drugs. In general, enzymatic hydrolysis of amides occurs more slowly than that of esters. The hydrolysis of xenobiotic esters and amides in humans is largely catalyzed by just two carboxylesterases called hCE1 and hCE2.

Carboxylesterases are glycoproteins that are present in serum and most tissues. Carboxylesterases hydrolyze numerous endogenous lipid compounds and generate pharmacologically active metabolites from several ester or amide prodrugs. In addition, carboxylesterases may convert xenobiotics to toxic and tumorigenic metabolites.

Cholinesterases play an important role in limiting the toxicity of organophosphates, which inhibit acetylcholinesterase and thus the termination of acetylcholine action. Factors that decrease esterase activity potentiate the toxic effects of organophosphates, whereas factors that increase serine esterase activity have a protective effect.

Paraoxonases, calcium-dependent enzymes containing a critical sulfhydryl group, catalyze the hydrolysis of a broad range of organic compounds, including lactones. Thus, “lactonase” is a more encompassing name for this group of enzymes.

Prodrugs and Alkaline Phosphatase

Many prodrugs are designed to be hydrolyzed by hydrolytic enzymes such as carboxylesterases, cholinesterases, and alkaline phosphatase. Thus, these enzymes may be used to activate prodrugs in vivo and thereby generate potent anticancer agents in highly selected target sites, releasing the drug in the vicinity of the tumor cells.

Peptidases

Numerous human peptides and several recombinant peptide hormones, growth factors, cytokines, soluble receptors, and monoclonal antibodies are used therapeutically. These peptides are hydrolyzed in the blood and tissues by a variety of peptidases, which cleave the amide linkage between adjacent amino acids.

Epoxide Hydrolase

Epoxide hydrolase catalyzes the trans-addition of water to alkene epoxides and arene oxides, and is present in virtually all tissues. It plays an important role in detoxifying electrophilic epoxides that might otherwise bind to proteins and nucleic acids and cause cellular toxicity and genetic mutations. There are five distinct forms of epoxide hydrolase in mammals: microsomal epoxide hydrolase (mEH), soluble epoxide hydrolase (sEH), cholesterol epoxide hydrolase, LTA4 hydrolase, and hepoxilin hydrolase. The latter three enzymes appear to hydrolyze endogenous epoxides exclusively and have virtually no capacity to detoxify xenobiotic oxides.

In contrast to the high degree of substrate specificity displayed by the cholesterol, LTA4, and hepoxilin epoxide hydrolases, the mEH and sEH hydrolyze many alkene epoxides and arene oxides. Generally, these two forms of epoxide hydrolases and cytochrome P450 enzymes, which are often responsible for producing the toxic epoxides, have a similar cellular localization that presumably ensures the rapid detoxication of alkene epoxides and arene oxides generated during the oxidative biotransformation of xenobiotics.

Epoxide hydrolase is one of the several inducible enzymes in liver microsomes. Induction of epoxide hydrolase is invariably associated with the induction of cytochrome P450.

Reduction

Certain metals and xenobiotics containing an aldehyde, ketone, disulfide, sulfoxide, quinone, N-oxide, alkene, azo, or nitro group are often reduced in vivo. The reaction may proceed enzymatically or nonenzymatically by interaction with reducing agents, such as the reduced forms of glutathione, FAD, FMN, and NADP. Likewise, enzymes, such as alcohol dehydrogenase (ADH), aldehyde oxidase, and cytochrome P450, can catalyze both reductive and oxidative reactions depending on the substrate and conditions.

Azo- and Nitro-Reduction

Azo- and nitro-reduction are catalyzed by intestinal microflora and under certain conditions (i.e., low oxygen tension), by two liver enzymes: cytochrome P450 and NADPH-quinone oxidoreductase (also known as DT-diaphorase). The reactions require NADPH and are inhibited by oxygen. The anaerobic environment of the lower gastrointestinal tract is well suited for azo- and nitro-reduction.

Carbonyl Reduction

The reduction of certain aldehydes to primary alcohols and of ketones to secondary alcohols is catalyzed by NAD(P)H-dependent reductases belonging to one of the two superfamilies, the aldo-keto reductases (AKRs) and the short-chain dehydrogenases/reductases (SDRs). AKRs are members of a superfamily of cytosolic enzymes that reduce both xenobiotic and endobiotic compounds. SDR carbonyl reductases are monomeric enzymes, present in blood and the cytosolic fraction of various tissues. Hepatic carbonyl reductase activity is present mainly in the cytosolic fraction, with a different carbonyl reductase present in the microsomes.

Disulfide Reduction

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 can reduce sulfoxides, which were formed by cytochrome P450. Under reduced oxygen tension, the NADPH-dependent reduction of N-oxides in liver microsomes may be catalyzed by cytochrome P450 or NADPH–cytochrome P450 reductase.

Quinone Reduction

Quinones can be reduced to hydroquinones by two cytosolic flavoproteins, NQO1 and NQO2, without oxygen consumption. NADPH-quinone oxidoreductase-1 (DT-diaphorase) and NADPH-quinone oxidoreductase-2 have different substrate specificities. The two-electron reduction of quinones also can be catalyzed by carbonyl reductase. This pathway of quinone reduction is essentially nontoxic and is not associated with oxidative stress.

The second pathway of quinone reduction catalyzed by microsomal NADPH–cytochrome P450 reductase results in the formation of a semiquinone free radical by a one-electron reduction of the quinone. 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.

The properties of the hydroquinone determine whether, during the metabolism of quinine-containing xenobiotics, NQO functions as a protective antioxidant or a prooxidant activator leading to the formation of reactive oxygen species and reactive semiquinone free radicals.

Dehalogenation

There are three major mechanisms for removing halogens (F, Cl, Br, and I) from aliphatic xenobiotics: (1) reductive dehalogenation involves replacement of a halogen with hydrogen; (2) oxidative dehalogenation replaces a halogen and hydrogen on the same carbon atom with oxygen; and (3) double dehalogenation involves the elimination of two halogens on adjacent carbon atoms to form a carbon–carbon double bond. A variation of this third mechanism is dehydrohalogenation, in which a halogen and hydrogen on adjacent carbon atoms are eliminated to form a carbon–carbon double bond.

Oxidation

Alcohol Dehydrogenase

ADH is a cytosolic enzyme present in several tissues including the liver, which has the highest levels, the kidney, the lung, and the gastric mucosa. There are five major classes of ADH. The class I ADH isozymes (α-ADH, β-ADH, and γ-ADH) are responsible for the oxidation of ethanol and other small aliphatic alcohols. Class II ADH (π-ADH) is primarily expressed in liver where it preferentially oxidizes larger aliphatic and aromatic alcohols. Long-chain alcohols (pentanol and larger) and aromatic alcohols are preferred substrates for class III ADH (χ-ADH). Class IV ADH (σ- or μ-ADH), which is not expressed in liver, is the most active of the medium-chain ADHs in oxidizing retinol. Class V ADH has no subunit designation.

Aldehyde Dehydrogenase

Aldehyde dehydrogenase (ALDH) oxidizes aldehydes to carboxylic acids with NAD+ as the cofactor. The enzymes also have esterase activity. The 19 identified ALDHs differ in their primary amino acid sequences and in the quaternary structure. In contrast to ALDH1A1 and ALDH2, which specifically reduce NAD+, ALDH3A1 reduces both NAD+ and NADP+.

As shown in Figure 6–1, ALDH2 is a mitochondrial enzyme that, by virtue of its high affinity, is primarily responsible for oxidizing simple aldehydes, such as acetaldehyde. Genetic deficiencies in other ALDHs impair the metabolism of other aldehydes.

Dihydrodiol Dehydrogenase

The AKR superfamily includes several forms of dihydrodiol dehydrogenases, which are cytosolic, NADPH-requiring oxidoreductases that oxidize various polycyclic aromatic hydrocarbons to potentially toxic metabolites.

Molybdenum Hydroxylases

Two major molybdenum hydroxylases or molybdozymes participate in the biotransformation of xenobiotics: aldehyde oxidase and xanthine oxidoreductase (also known as xanthine oxidase [XO]). Sulfite oxidase, a third molybdozyme, oxidizes sulfite, an irritating air pollutant, to sulfate, which is relatively innocuous. All three molybdozymes are flavoprotein enzymes. During substrate oxidation, aldehyde oxidase and XO are reduced and then reoxidized by molecular oxygen. The oxygen incorporated into the xenobiotic is derived from water rather than oxygen, which distinguishes the oxidases from oxygenases. Xenobiotics that are good substrates for molybdozymes tend to be poor substrates for cytochrome P450, and vice versa.

Xanthine Oxidoreductase

Xanthine dehydrogenase (XD) and 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 NAD+, whereas in the case of XO the final electron acceptor is oxygen. XD is converted to XO by oxidation of cysteine residues and/or proteolytic cleavage. The conversion of XD to XO in vivo may be important in ischemia–reperfusion injury, lipopolysaccharide-mediated tissue injury, and alcohol-induced hepatotoxicity. XO contributes to oxidative stress and lipid peroxidation because the oxidase activity of XO involves reduction of molecular oxygen, which can lead to the formation of reactive oxygen species.

Allopurinol and other xanthine oxidoreductase inhibitors are being evaluated for the treatment of various types of ischemia–reperfusion and vascular injury that appear to be mediated, at least in part, by xanthine oxidoreductase.

Aldehyde Oxidase

The molybdozyme aldehyde oxidase exists only in the oxidase form. Cytosolic aldehyde oxidase transfers electrons to molecular oxygen, which can generate reactive oxygen species and lead to lipid peroxidation. Aldehyde oxidase plays an important role in the catabolism of biogenic amines and catecholamines.

Monoamine Oxidase

Monoamine oxidases (MAO) are involved in the oxidative deamination of primary, secondary, and tertiary amines, including serotonin and a number of xenobiotics. 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 aldehydes formed by MAO are usually oxidized further by other enzymes to the corresponding carboxylic acids. MAO is located throughout the brain and in the outer membrane of mitochondria of the liver, kidney, intestine, and blood platelets.

The substrate is oxidized by MAO, which itself is reduced using FAD. The oxygen incorporated into the substrate is derived from water, not molecular oxygen. The catalytic cycle is completed by reoxidation of the reduced enzyme (FADH2 → FAD) by oxygen, which generates hydrogen peroxide.

Semicarbazide-sensitive amine oxidase (SSAO) is a copper-containing enzyme that catalyzes fundamentally the same reaction as MAO. It can be distinguished from MAO by its sensitivity to inhibitors and presence in plasma and various cell surfaces, whereas MAO is found in mitochondria.

Peroxidase-Dependent Cooxidation

Oxidative bio-transformation of xenobiotics by peroxidases couples the reduction of hydrogen peroxide and lipid hydroperoxides to the oxidation of other substrates via a process known as cooxidation. An important peroxidase is prostaglandin H synthetase (PHS), which possesses two catalytic activities: a cyclooxygenase that converts arachidonic acid to prostaglandins and a peroxidase that converts the hydroperoxide to the corresponding alcohol PGH2. PSH has two forms (PSH1 and PSH2) that are better known as two forms of cyclooxygenase, namely, COX1 and COX2. PSH peroxidases are important in the activation of xenobiotics to toxic or tumorigenic metabolites, particularly in extrahepatic tissues that contain low levels of cytochrome P450. Oxidation of xenobiotics by peroxidases involves direct transfer of the peroxide oxygen to the xenobiotic, as shown in Figure 6–2 for the conversion of substrate X to product XO.

Xenobiotics that serve as electron donors, such as amines and phenols, can also be oxidized to free radicals during the reduction of a hydroperoxide by peroxidases. 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 two-electron process), two molecules of xenobiotic can be oxidized (each by a one-electron process). Many of the metabolites produced are reactive electrophiles that can cause tissue damage.

PSH2 may play at least two distinct roles in tumor formation: it may convert certain xenobiotics to DNA-reactive metabolites and initiate tumor formation, and it may promote subsequent tumor growth, perhaps through formation of growth-promoting eicosanoids.

PHS is unique among peroxidases because it can both generate hydroperoxides and catalyze peroxidase-dependent reactions, as shown in Figure 6–2. Xenobiotic biotransformation by PHS is controlled by the availability of arachidonic acid, whereas conversion by other peroxidases is controlled by the availability of hydroperoxide substrates.

Flavin Monooxygenases

Liver, kidney, intestine, brain, and lung contain one or more FAD-containing monooxygenases (FMO) that oxidize the nucleophilic nitrogen, sulfur, and phosphorus heteroatom of various xenobiotics. The mammalian FMO gene family comprises five microsomal enzymes that require NADPH and O2, and many of the reactions catalyzed by FMO can also be catalyzed by cytochrome P450.

The mechanism of catalysis by FMO is depicted in Figure 6–3. 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 relatively stable peroxide. During the oxygenation of xenobiotics, the flavin peroxide oxygen is transferred to the substrate (depicted as X → XO in Figure 6–3). The final step in the catalytic cycle involves restoration of FAD to its oxidized state and release of NADP+. This final step is rate-limiting, and it occurs after substrate oxygenation.

Cytochrome P450

The cytochrome P450 (CYP) system ranks first in terms of catalytic versatility and the sheer number of xenobiotics it detoxifies or activates. The highest concentration of CYP enzymes involved in xenobiotic biotransformation is found in hepatic endoplasmic reticulum (microsomes), but CYP enzymes are present in virtually all tissues. All CYP enzymes are heme-containing proteins that catalyze the monooxygenation of one atom of oxygen into a substrate, and the other oxygen atom is reduced to water with reducing equivalents derived from NADPH.

During catalysis, CYP does not interact directly with NADPH or NADH. In the endoplasmic reticulum, electrons are relayed from NADPH to cytochrome P450 via a flavoprotein called NADPH–cytochrome P450 reductase. In mitochondria, electrons are transferred from NADPH to CYP via ferredoxin and ferredoxin reductase.

There are notable exceptions to the principle that cytochrome P450 requires a second enzyme (i.e., a flavoprotein) for catalytic activity. One exception applies to thromboxane A synthase (CYP5A1) and prostaglandin I2 synthase (prostacyclin synthase or CYP8A1), which are involved in the conversion of arachidonic acid to eicosanoids. In both cases, cytochrome P450 functions as an isomerase and catalyzes a rearrangement of the oxygen atoms introduced into arachidonic acid by cyclooxygenase. The second exception involves two CYP enzymes expressed in the bacterium Bacillus megaterium. These CYP enzymes are considerably larger than most CYP enzymes because the P450 moiety and oxidoreductase flavoprotein are expressed in a single protein encoded by a single gene.

Cytochrome P450 and NADPH–cytochrome P450 reductase are embedded in the phospholipid bilayer of the endoplasmic reticulum, which facilitates their interaction. As shown in Figure 6–4, the first part of the catalytic cycle involves the activation of oxygen, and the final part involves substrate oxidation, which entails the abstraction of a hydrogen atom or an electron from the substrate followed by oxygen rebound (radical recombination). Following the binding of substrate to the CYP enzyme, the heme iron is reduced from the ferric (Fe3+) to the ferrous (Fe2+) state by the addition of a single electron from NADPH–cytochrome P450 reductase. Release of the oxidized substrate returns cytochrome P450 to its initial state. If the catalytic cycle is interrupted, oxygen is released as superoxide anion (O2-) or hydrogen peroxide (H2O2).

Cytochrome P450 catalyzes the following types of oxidation reactions:

1. hydroxylation of an aliphatic or aromatic carbon;

2. epoxidation of a double bond;

3. heteroatom (S-, N-, and I-) oxygenation and N-hydroxylation;

4. heteroatom (O-, S-, N-, and Si-) dealkylation;

5. oxidative group transfer;

6. cleavage of esters;

7. dehydrogenation.

Liver microsomes from all mammalian species contain numerous P450 enzymes, each with the potential to catalyze the various reactions shown in Figures 6–5, 6–6, 6–7, 6–8, 6–9, 6–10, 6–11, and 6–12. In general, CYP enzymes are classified into subfamilies based on amino acid sequence identity.

The function and regulation of CYP1A1, CYP1A2, CYP1B1, CYP2E1, CYP2R1, CYP2S1, CYP2U1, and CYYP2W1 are highly conserved among mammalian species and these proteins have the same names in all mammalian species. In most other cases, the CYP enzymes are named in a species-specific manner. The levels and activity of each CYP enzyme vary from one individual to the next, due to environmental and/or genetic factors. Decreased CYP enzyme activity can result from (1) a genetic mutation that either blocks the synthesis of a CYP enzyme or leads to the synthesis of a catalytically compromised, inactive, or unstable enzyme, which gives rise to the poor and intermediate metabolizer genotypes, (2) exposure to an environmental factor (such as an infectious disease or an inflammatory process) that suppresses CYP enzyme expression, or (3) exposure to a xenobiotic that inhibits or inactivates a preexisting CYP enzyme. By inhibiting cytochrome P450, one drug can impair the biotransformation of another, which may lead to an exaggerated pharmacologic or toxicologic response to the second drug. Increased CYP enzyme activity can result from (1) gene duplication leading to over-expression of a CYP enzyme; (2) exposure to drugs and other xenobiotics that induce the synthesis of cytochrome P450; or (3) stimulation of preexisting enzyme by a xenobiotic.

Induction of cytochrome P450 by xenobiotics increases CYP enzyme activity. By inducing cytochrome P450, one drug can stimulate the metabolism of a second drug and thereby decrease or ameliorate its therapeutic effect. Allelic variants, which arise by point mutations in the wild-type gene, are another source of interindividual variation in CYP activity. Environmental factors known to affect CYP levels include medications, foods, social habits (e.g., alcohol consumption and cigarette smoking), and disease status (diabetes, inflammation, viral and bacterial infection, hyperthyroidism, and hypothyroidism). When environmental factors influence CYP enzyme levels, considerable variation may be observed during repeated measures of xenobiotic biotransformation (e.g., drug metabolism) in the same individual. Due to their broad substrate specificity, it is possible that two or more CYP enzymes can contribute to the metabolism of a single compound.

The pharmacologic or toxic effects of certain drugs are exaggerated in a significant percentage of the population due to a heritable deficiency in a CYP enzyme. Inasmuch as the biotransformation of a xenobiotic in humans is frequently dominated by a single CYP enzyme, the considerable effort in identifying which CYP enzyme or enzymes are involved in eliminating the drug is known as reaction phenotyping or enzyme mapping. Four approaches to reaction phenotyping are as follows:

1. Correlation analysis involves measuring the rate of xenobiotic metabolism by several samples of human liver microsomes and correlating reaction rates with the variation in the level or activity of the individual P450 enzymes in the same microsomal samples.

2. Chemical inhibition evaluates the effects of known CYP enzyme inhibitors on the metabolism of a xenobiotic by human liver microsomes. Inhibitors of cytochrome CYP must be used cautiously because most of them can inhibit more than one CYP enzyme.

3. Antibody inhibition determines the effects of inhibitory antibodies against selected CYP enzymes on the biotransformation of a xenobiotic by human liver microsomes. This method alone can potentially establish which human CYP enzyme is responsible for biotransforming a xenobiotic.

4. Biotransformation by purified or recombinant human CYP enzymes establishes whether a particular CYP enzyme can or cannot biotransform a xenobiotic, but it does not address whether that CYP enzyme contributes substantially to reactions catalyzed by human liver microsomes.

Examples of substrates, inhibitors, and inducers for each CYP enzyme in human liver microsomes are given in Table 6–2. Because reaction phenotyping in vitro is not always carried out with toxicologically relevant substrate concentrations, the CYP enzyme that appears responsible for biotransforming the drug in vitro may not be the CYP enzyme responsible for biotransforming the drug in vivo.

Activation of Xenobiotics by Cytochrome P450

The role of human CYP enzymes in the activation of procarcinogens and protoxicants and some cytochrome P450-dependent reactions are summarized in Table 6–3. Many of the chemicals listed in Table 6–3 are also detoxified by cytochrome P450 by conversion to less toxic metabolites. In some cases, the same CYP enzyme catalyzes both activation and detoxication reactions. For example, CYP3A4 activates aflatoxin B1 to the hepatotoxic and tumorigenic 8,9-epoxide, but it also detoxifies aflatoxin B1 by 3-hydroxylation to aflatoxin Q1. Complex factors determine the balance between xenobiotic activation and detoxication.

Inhibition of Cytochrome P450

In addition to predicting the likelihood of some individuals being poor metabolizers due to a genetic deficiency in P450 expression, information on which human CYP enzyme metabolizes a drug can help predict or explain drug interactions. Inhibitory drug interactions generally fall into two categories: direct and metabolism-dependent inhibition. Direct inhibition can be subdivided into two types. The first involves competition between two drugs that are metabolized by the same CYP enzyme. The second is also competitive in nature, but the inhibitor is not a substrate for the affected CYP enzyme. Metabolism-dependent inhibition occurs when cytochrome P450 converts a xenobiotic to a metabolite that is a more potent inhibitor, either reversible or irreversible, than the parent compound.

Induction of Cytochrome P450

Inducers of cytochrome P450 increase the rate of xenobiotic biotransformation. Some of the CYP enzymes in human liver microsomes are inducible (Table 6–2). As an underlying cause of serious adverse effects, P450 induction lowers blood levels, which compromises the therapeutic goal of drug therapy but does not cause an exaggerated response to the drug.

Although induction of cytochrome P450 may increase the activation of procarcinogens to DNA-reactive metabolites, there is little evidence from either human epidemiologic studies or animal experimentation that P450 induction enhances the incidence or multiplicity of tumors caused by known chemical carcinogens. In fact, most evidence points to a protective role of enzyme induction against chemical-induced neoplasia. Cytochrome P450 induction can cause pharmacokinetic tolerance whereby larger drug doses must be administered to achieve therapeutic blood levels due to increased drug biotransformation.

P450 Knockout Mice

Transgenic mice that lack one or more CYP enzymes may be used to evaluate the role of specific CYP enzymes in xenobiotic activation. Studies in knockout mice are relevant to humans because their counterpart can be found in those individuals who lack certain CYP enzymes or other xenobiotic biotransforming enzymes. Experiments in knockout mice underscore how genetic polymorphisms in the human population are risk modifiers for the development of chemically induced disease.

Conjugation reactions include glucuronidation, sulfonation (more commonly called sulfation), acetylation, methylation, conjugation with glutathione (mercapturic acid synthesis), and conjugation with amino acids (such as glycine, taurine, and glutamic acid). The cosubstrates for these reactions, which are shown in Figure 6–13, react with functional groups that are either present on the xenobiotic or are introduced or exposed during oxidation, reduction, or hydrolysis. With the exception of methylation and acetylation, conjugations result in a large increase in xenobiotic hydrophilicity, which greatly facilitates excretion of foreign chemicals. Glucuronidation, sulfation, acetylation, and methylation involve reactions with activated or “high-energy” cosubstrates, whereas conjugation with amino acids or glutathione involves reactions with activated xenobiotics. Except for the glucuronosyltransferases, most conjugation enzymes are mainly located in the cytosol (Table 6–1).

Glucuronidation

Glucuronidation requires the cosubstrate uridine diphosphate-glucuronic acid (UDP-glucuronic acid), and the reaction is catalyzed by UDP-glucuronosyltransferases (UGTs). Examples of xenobiotics that are glucuronidated are shown in Figure 6–14. The site of glucuronidation is generally an electron-rich nucleophilic heteroatom (O, N, or S) as found in aliphatic alcohols and phenols, carboxylic acids, primary and secondary aromatic and aliphatic amines, and free sulfhydryl groups. Endogenous substrates for glucuronidation include bilirubin, steroid hormones, and thyroid hormones.

Glucuronide conjugates of xenobiotics and endogenous compounds are polar, water-soluble metabolites. Whether glucuronides are excreted from the body in bile or urine depends on the size of the aglycone (parent compound or unconjugated metabolite). The carboxylic acid moiety of glucuronic acid, which is ionized at physiologic pH, promotes excretion because (1) it increases the aqueous solubility of the xenobiotic and (2) it is recognized by the biliary and renal organic anion transport systems, which enables glucuronides to be secreted into urine and bile. Glucuronides of xenobiotics are substrates for β-glucuronidase present in the intestinal microflora. The intestinal enzyme can release the aglycone, which undergoes enterohepatic circulation delaying elimination of the xenobiotic.

Cofactor availability can limit the rate of glucuronidation of drugs that are administered in high doses and are conjugated extensively, such as aspirin and acetaminophen.

UGTs expressed in rat liver microsomes belong to two gene families, UGT1 and UGT2, each containing several subfamilies with many similar members. Members of gene family 2 are composed of six exons that are not shared between members with the exception of UGT2A2 and UGT2A1. In contrast, members of family 1 are formed from a single gene with multiple copies of the first exon, each of which can be connected in cassette fashion with a common set of exons.

Sulfonation

Many xenobiotics and endogenous substrates undergo sulfonation. Sulfate conjugation is catalyzed by sulfotransferases, a multigene family of enzymes that generally produces a highly water-soluble sulfuric acid ester. The cosubstrate for the reaction is 3′-phosphoadenosine-5′-phosphosulfate (PAPS; see Figure 6–13).

Sulfate conjugation involves the transfer of sulfonate, not sulfate (i.e., SO3-, not SO4-) from PAPS to the xenobiotic. (The commonly used terms sulfation and sulfate conjugation are used here, even though sulfonation and sulfonate conjugation are more appropriate descriptors.) Table 6–4 lists examples of xenobiotics and endogenous compounds that are sulfonated without prior biotransformation by oxidation enzymes. An even greater number of xenobiotics are sulfated after a hydroxyl group is exposed or introduced during oxidative or hydrolytic biotransformation.

Sulfate conjugates of xenobiotics are excreted mainly in urine. Sulfatases present in the endoplasmic reticulum and lysosomes primarily hydrolyze sulfates of endogenous compounds. Some sulfate conjugates are substrates for further biotransformation.

PAPS is synthesized from inorganic sulfate (SO42-) and ATP in a two-step reaction. The major source of sulfate required for the synthesis of PAPS appears to be derived from cysteine through a complex oxidation sequence. The low cellular concentration of PAPS (˜75 μM versus ˜350 μM UDP-glucuronic acid and ˜10 mM glutathione) limits the capacity for xenobiotic sulfonation.

Multiple sulfotransferases have been identified in all mammalian species examined. There are two major enzyme classes: membrane-bound enzymes are found in the Golgi apparatus and soluble enzymes are located in the cytoplasm. Sulfotransferases are arranged into gene families (SULT1 to SULT5) that share at least 45 percent amino acid sequence identity, and are further subdivided into several subfamilies. Each family appears to work on a specific functional group (i.e., phenols, alcohols, and amines).

In general, sulfonation is an effective means of decreasing the pharmacologic and toxicologic activity of xenobiotics. However, as shown in Figure 6–15, sulfonation has a role in the activation of aromatic amines, methyl-substituted polycyclic aromatic hydrocarbons, and safrole to tumorigenic metabolites.

Methylation

Methylation, a minor pathway of biotransformation, generally decreases the water solubility of xenobiotics and masks functional groups that might otherwise be conjugated by other enzymes. Methylation can also lead to increased toxicity. The cosubstrate for methylation is S-adenosylmethionine (SAM) (Figure 6–13). The methyl group bound to the sulfonium ion in SAM is transferred to xenobiotics and endogenous substrates by nucleophilic attack from an electron-rich heteroatom (O,N, or S) leaving S-adenosylhomocysteine. Examples of xenobiotics and endogenous substrates that undergo O-, N-, or S-methylation are shown in Figure 6–16.

The O-methylation of phenols and catechols is catalyzed by two different enzymes known as phenol O-methyltransferase (POMT) in microsomes and catechol-O-methyltransferase (COMT) in cytosol and microsomes. In rats and humans, COMT is encoded by a single gene with two different promoters and transcription initiation sites. Transcription at one site produces a cytosolic form of COMT, whereas transcription from the other site produces a membrane-bound form by adding a 50-amino acid segment that targets COMT to the endoplasmic reticulum. Substrates for COMT include several catecholamine neurotransmitters and catechol drugs, such as L-DOPA and methyldopa.

Several N-methyltransferases have been described in humans and other mammals. Phenylethanolamine N-methyltransferase catalyzes the N-methylation of the neurotransmitter norepinephrine to form epinephrine in the adrenal medulla and in certain regions of the brain, and is of minimal significance in xenobiotic biotransformation. However, histamine and nicotine N-methyltransferases expressed in liver, intestine, and/or kidney do methylate xenobiotics.

S-Methylation is an important pathway in the biotransformation of sulfhydryl-containing xenobiotics. In humans, S-methylation is catalyzed by thiopurine methyltransferase in cytosol and thiol methyltransferase in microsomes.

Acetylation

N-Acetylation is a major route of biotransformation for xenobiotics containing an aromatic amine (R—NH2) or a hydrazine group (R—NH—NH2), which are converted to aromatic amides (R—NH—COCH3) and hydrazides (R—NH—NH—COCH3), respectively. N-Acetylation masks an amine with a nonionizable group, so that many N-acetylated metabolites are less water soluble than the parent compound. Nevertheless, N-acetylation of certain xenobiotics, such as isoniazid, facilitates their urinary excretion.

Xenobiotic N-acetylation catalyzed by cytosolic N-acetyltransferases requires the cosubstrate acetyl-coenzyme A (acetyl-CoA; Figure 6–13). The two-step reaction involves: (1) transfer of the acetyl group from acetyl-CoA to an active site cysteine residue within the enzyme with release of coenzyme A and (2) subsequent transfer of the acetyl group from the acylated enzyme to the amino group of the substrate with regeneration of the enzyme.

NAT1 and NAT2, the two acetyltransferases existing in humans, are 79 to 95 percent identical in amino acid sequence with an active site cysteine residue in the N-terminal region. Although encoded by genes on the same chromosome, NAT1 is expressed in most tissues of the body, whereas NAT2 is mainly expressed only in liver and intestine. Most (but not all) of the tissues that express NAT1 also appear to express low levels of NAT2, at least at the level of mRNA. NAT1 and NAT2 also have different but overlapping substrate specificities. Examples of drugs that are N-acetylated by NAT1 and NAT2 are shown in Figure 6–17.

Genetic polymorphisms for N-acetylation have been documented in humans, hamsters, rabbits, and mice. Polymorphisms in NAT2 have a number of pharmacologic and toxicologic consequences: slow NAT2 acetylators are predisposed to drug toxicities, including excessive hypotension from hydralazine, peripheral neuropathy from isoniazid and dapsone, systemic lupus erythematosus from hydralazine and procainamide, and the toxic effects of coadministration of the anticonvulsant phenytoin with isoniazid.

The N-acetyltransferases detoxify aromatic amines by converting them to the corresponding amides that are less likely to be activated to DNA-reactive metabolites. However, N-acetyltransferases can activate aromatic amines if they are first N-hydroxylated by cytochrome P450. The acetoxy esters of N-hydroxyaromatic amines, like the corresponding sulfonate esters (Figure 6–15), can break down to form highly reactive nitrenium and carbonium ions that bind to DNA. Whether fast acetylators are protected from or predisposed to the cancer-causing effects of aromatic amines depends on the nature of the aromatic amine and other risk modifiers.

Amino Acid Conjugation

Two principal pathways by which xenobiotics are conjugated with amino acids are illustrated in Figure 6–18. The first involves conjugation of xenobiotics containing a carboxylic acid group with the amino group of amino acids such as glycine, glutamine, and taurine (see Figure 6–13). After activation of the xenobiotic by conjugation with CoA, the acyl-CoA thioether reacts with the amino group of an amino acid to form an amide linkage. The second pathway involves conjugation of xenobiotics containing an aromatic hydroxylamine with the carboxylic acid group of such amino acids as serine and proline. This pathway involves activation of an amino acid by aminoacyl-tRNA synthetase, which reacts with an aromatic hydroxylamine to form a reactive N-ester.

Substrates for amino acid conjugation are restricted to certain aliphatic, aromatic, heteroaromatic, cinnamic, and arylacetic acids. The ability of xenobiotics to undergo amino acid conjugation depends on steric hindrance around the carboxylic acid group, and by substituents on the aromatic ring or aliphatic side chain. Amino acid conjugates of xenobiotics are eliminated primarily in urine. The acceptor amino acid used for conjugation is both species- and xenobiotic-dependent.

Amino acid conjugation of N-hydroxy aromatic amines (hydroxylamines) is an activation reaction producing N-esters that can degrade to form electrophilic nitrenium and carbonium ions. Conjugation of hydroxylamines with amino acids is catalyzed by cytosolic aminoacyl-tRNA synthetases and requires ATP (Figure 6–18).

Glutathione Conjugation

Conjugation of xenobiotics with glutathione includes an enormous array of electrophilic xenobiotics, or xenobiotics that can be biotransformed to electrophiles. The tripeptide glutathione comprises of glycine, cysteine, and glutamic acid (Figure 6–13). Glutathione conjugates are thioethers, which form by nu cleophilic attack of glutathione thiolate anion (GS-) with an electrophilic carbon, oxygen, nitrogen, or sulfur atom in the xenobiotic. This conjugation reaction is catalyzed by a family of glutathione S-transferases that are present in most tissues, where they are localized in the cytoplasm (>95 percent) and endoplasmic reticulum (<5 percent).

Substrates for glutathione S-transferase are commonly hydrophobic, contain an electrophilic atom, and react nonenzymatically with glutathione at some measurable rate. The mechanism by which glutathione S-transferase increases the rate of glutathione conjugation involves deprotonation of GSH to GS-. The concentration of glutathione in liver is extremely high (˜5 to 10 mM); hence, the nonenzymatic conjugation of certain xenobiotics with glutathione can be significant. However, some xenobiotics are conjugated with glutathione stereoselectively, indicating that the reaction is largely catalyzed by glutathione S-transferase. Like glutathione, the glutathione S-transferases are themselves abundant cellular components, accounting for up to 10 percent of the total cellular protein. These enzymes bind, store, and/or transport a number of compounds that are not substrates for glutathione conjugation. The cytoplasmic protein formerly known as ligandin, which binds heme, bilirubin, steroids, azo-dyes, polycyclic aromatic hydrocarbons, and thyroid hormones, is an alpha-class GST.

As shown in Figure 6–19, substrates for glutathione conjugation can be divided into two groups: those sufficiently electrophilic to be conjugated directly and those that must first be biotransformed to an electrophilic metabolite prior to conjugation. The conjugation reactions themselves can be divided into two types: displacement reactions, in which glutathione displaces an electron-withdrawing group, and additionreactions, in which glutathione is added to an activated double bond or strained ring system.

The displacement of an electron-withdrawing group by glutathione typically occurs when the substrate contains halide, sulfate, sulfonate, phosphate, or a nitro group (i.e., good leaving groups) attached to an allylic or benzylic carbon atom.

The addition of glutathione to a carbon–carbon double bond is also facilitated by the presence of a nearby electron-withdrawing group; hence, substrates for this reaction typically contain a double bond attached to —CN, —CHO, —COOR, or —COR.

Glutathione can also conjugate xenobiotics with an electrophilic heteroatom (O, N, and S). In each of the examples shown in Figure 6–20, the initial conjugate formed between glutathione and the heteroatom is cleaved by a second molecule of glutathione to form oxidized glutathione (GSSG). The initial reactions are catalyzed by glutathione S-transferase, whereas the second reaction (which leads to GSSG formation) generally occurs nonenzymatically.

Glutathione conjugates formed in the liver can be effluxed into bile and blood, and they can be converted to mercapturic acids in the kidney and excreted in urine. As shown in Figure 6–21, the conversion of glutathione conjugates to mercapturic acids involves the sequential cleavage of glutamic acid and glycine from the glutathione moiety, followed by N-acetylation of the resulting cysteine conjugate.

Glutathione S-transferases are dimers composed of identical subunits, although some forms are heterodimers. Each subunit contains 199 to 244 amino acids and one catalytic site. Numerous subunits have been cloned and sequenced and differ in substrate specificity, tissue location, and cellular location. Conjugation with glutathione represents an important detoxication reaction because electrophiles are potentially toxic species that can bind to critical nucleophiles, such as proteins and nucleic acids, causing cellular damage and genetic mutations. Glutathione is also a cofactor for glutathione peroxidase, which is important in protecting cells against lipid and hemoglobin peroxidation.

In some cases, conjugation with glutathione enhances the toxicity of a xenobiotic. Glutathione conjugates of various compounds can activate xenobiotics to become toxic by releasing a toxic metabolite, being inherently toxic itself, or being degraded to a toxic metabolite.