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.
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 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.
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 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.
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 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.
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.
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.
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 (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.
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.
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.
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 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.
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 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.
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.
Cooxidation of xenobiotics (X) during the conversion of arachidonic acid to PGH2by prostaglandinH synthase.
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.
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.
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.
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).
Catalytic cycle of cytochrome P450.
Cytochrome P450 catalyzes the following types of oxidation reactions:
hydroxylation of an aliphatic or aromatic carbon;
epoxidation of a double bond;
heteroatom (S-, N-, and I-) oxygenation and N-hydroxylation;
heteroatom (O-, S-, N-, and Si-) dealkylation;
oxidative group transfer;
cleavage of esters;
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.
Examples of reactions catalyzed by cytochrome P450: hydroxylation of aliphatic carbon.
Examples of reactions catalyzed by cytochrome P450: hydroxylation of aromatic carbon.
Examples of reactions catalyzed by cytochrome P450: epoxidation.
Examples of reactions catalyzed by cytochrome P450: heteroatom oxygenation.
Examples of reactions catalyzed by cytochrome P450: heteroatom dealkylation.
Examples of reactions catalyzed by cytochrome P450: oxidative group transfer.
Examples of reactions catalyzed by cytochrome P450: cleavage of esters.
Examples of reactions catalyzed by cytochrome P450: dehydrogenation.
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:
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.
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.
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.
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.
Table 6–2 Examples of clinically relevant substrates, inhibitors, and inducers of the major human liver microsomal P450 enzymes involved in xenobiotic biotransformation.
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.
Table 6–3 Examples of xenobiotics activated by human P450. ||Download (.pdf)
Table 6–3 Examples of xenobiotics activated by human P450.
Amino acid pyrolysis products (DiMeQx, MelQ, MelQx, Glu P-2, IQ, PhlP, Trp P-1, Trp P-2)
CYP2A6 and 2A13
NNK and bulky nitrosamines
CYP2C8, 9, 18, 19
CYP1A1 and 1B1
Benzo[a]pyrene and other polycyclic aromatic hydrocarbons
Aflatoxin B1 and G1
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.
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.