Cytochrome P450 and Chemical Toxicology

F. Peter Guengerich*

Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt UniVersity School of Medicine,638 Robinson Research Building, 23rd and Pierce AVenues, NashVille, Tennessee 37232-0146

ReceiVed March 12, 2007

The field of cytochrome P450 (P450) research has developed considerably over the past 20 years, andmany important papers on the roles of P450s in chemical toxicology have appeared in Chemical Researchin Toxicology. Today, our basic understanding of many of the human P450s is relatively well-established,in terms of the details of the individual genes, sequences, and basic catalytic mechanisms. Crystal structuresof several of the major human P450s are now in hand. The animal P450s are still important in the contextof metabolism and safety testing. Many well-defined examples exist for roles of P450s in decreasing theadverse effects of drugs through biotransformation, and an equally interesting field of investigation isthe bioactivation of chemicals, including drugs. Unresolved problems include the characterization of theminor “orphan” P450s, ligand cooperativity and kinetic complexity of several P450s, the prediction ofmetabolism, the overall contribution of bioactivation to drug idiosyncratic problems, the extrapolation ofanimal test results to humans in drug development, and the contribution of genetic variation in humanP450s to cancer incidence.

Contents1. Introduction and Background 70

1.1. Current Knowledge about P450s 70

2. Roles of P450s in Reducing Toxicity 72

3. Bioactivation by P450s 73

3.1. Aflatoxin B1 73

3.2. Ethyl Carbamate 74

3.3. Coupling of Norharman and Aniline 74

3.4. Troglitazone 74

3.5. Other Bioactivation Reactions 75

3.6. Mechanism-Based Activation 75

3.7. P450s and Oxidative Damage 76

4. Current and Future Issues 77

4.1. Functions of “Orphan” P450s 77

4.1.1. Analysis of Suspects 77

4.1.2. Transgenic Animal Models 77

4.1.3. Library Screening 77

4.1.4. Untargeted Metabolomic Strategies inVitro

77

4.1.5. Untargeted in Vitro Strategies withIsotope Editing

77

4.2. Ligand Cooperativity 77

4.3. Predictions of Metabolism 77

4.4. Overlaps of Detoxication and Bioactivation 78

4.5. Roles of P450s in Idiosyncratic DrugToxicity

78

4.6. Predicting Human Toxicity 78

4.7. Understanding P450 Gene Polymorphismsand Disease

78

5. Conclusion 79

1. Introduction and Background

Cytochrome P450 (P450) research can be traced back to invitro studies on the metabolism of steroids, drugs, and carcino-gens in the 1940s (1). Some of the major developments werethe spectral observation of P450 (2), photochemical actionstudies implicating P450 as the oxidase in the electron transportsystem (3), the separation (4) and subsequent purification ofP450 (5, 6), and several studies implicating multiple P450enzymes (7, 8). Other early seminal studies include the extensivebiochemical and biophysical work with the bacterial P450101A1 (P450cam) (9) and the first complete nucleotide sequenceof a P450 (10). Studies on the chemistry of oxygen activationdeveloped, and one of the key studies underpinning our currentmodels was evidence for a stepwise process involving C–H bondbreaking (11).

During the past 20 years, we have seen a major shift ofemphasis to human P450s, which had seemed almost impossiblein the early research. The knowledge about the human P450shas had important ramifications in understanding the metabolismof drugs. In comparison to the situation ∼25 years ago, far fewerdrugs fail in development due to pharmacokinetic problems inhumans, because of the reiterative approach of chemicalsynthesis, target screening, and in vitro metabolism studies inplace in pharmaceutical companies (Figure 1). However, lessprogress has been made in accurately predicting human toxicityproblems with drugs and the challenge remains considerable(13, 14). In retrospect, one of the driving forces for the studyof P450s has been the quest for information to better understandand predict the metabolism and toxicity of drugs and otherchemicals [e.g., thalidomide (15–17)].

1.1. Current Knowledge about P450s. This section willfocus on important developments that have occurred over thepast 20 years, that is, since this journal began. One is certainlythe completion of the human genome project, which set thenumber of human P450 (“CYP”) genes at 57 (Table 1) (and thenumber of pseudogenes at 58) (http://drnelson.utmem.edu/

* To whom correspondence should be addressed. Tel: 615-322-2261.Fax: 615-322-3141. E-mail: f.guengerich@vanderbilt.edu.

Chem. Res. Toxicol. 2008, 21, 70–8370

10.1021/tx700079z CCC: $40.75 2008 American Chemical SocietyPublished on Web 12/06/2007

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CytochromeP450.html), thus putting speculation about thisnumber to rest. However, some uncertainties exist about theexpression of some of the genes at the mRNA and particularlythe protein levels (e.g., P450 4A22).

Twenty years ago, the biochemical purification of several ofthe major human liver P450s was achieved (20–22). Thedevelopment of recombinant DNA technology was well under-way, and heterologous expression was done in low-yieldsystems. Some breakthroughs in the early 1990s led to successfulhigh-level bacterial expression (23–25), which was critical forcrystallography work. In the past few years, the number ofcrystal structrures of human P450s has increased rapidly, andtoday, high-resolution structures are available for human P450s1A2 (26), 2A6 (27), 2C8 (28), 2C9 (29, 30), 2D6 (31), and3A4 (32–34), the major P450s involved in drug metabolism.These structures have replaced less accurate homology modelsbased mainly on bacterial P450s (P450s 101A1 and 102A1)and also serve as reasonable templates for other closely relatedsubfamily P450 members. One general observation with theseand other animal and bacterial P450 structures is that mostundergo major conformational changes upon ligand binding (35),and ligand-free structures are of limited use in understandingthe functions of these proteins. However, with some of the P450shaving large active sites, the positions of ligands in the crystalstructures still leave many questions open about the interactions(32, 34, 36).

The generally accepted catalytic cycle for P450 reactions isshown in Figure 2. However, the point should be made thatthis is a simplified version and that the system is dynamic, andthe steps do not necessarily proceed in a linear order aroundthe cycle. For instance, substrate can be bound and released atother steps along the cycle (38, 39). Also, most of theoxygenated intermediates (or all?) have the potential to dismute,generating reactive oxygen species (vide infra), and the couplingefficiencies of most P450 systems in vitro are low. The conceptsdeveloped by Groves regarding a formal perferryl oxygenintermediate and a stepwise oxygenation mechanism (Figure3) have been proved useful in rationalizing most oxidationreactions (37, 41), with provision for one-electron oxidation(37, 40, 41). In the past decade, several alternate mechanismshave been proposed, and these remain controversial. One often-discussed mechanism is that proposed by Newcomb andinvolving FeO2

+ or FeO2H2+ instead of FeO3+ as the oxidant(42, 43). This mechanism had initial impetus from a chemicalproposal for the third step in the P450 19A1 aromatase reaction(44, 45), although an alternate FeO3+ reaction has recently beensuggested to be more tenable, based on density functional theorycalculations (46). The proposals for concerted mechanisms havebeen based largely on results obtained with lack of rearrange-ment of radical clocks (47), and the interpretation is controversial(37, 48, 49). Another proposal is the “two-spin state” systemof Shaik and co-workers, with the FeO3+ complex providing

Figure 1. Reasons for the termination of drug candidates during development, based upon surveys of the pharmaceutical industry (ca. 2000) (12).See also Table 13 of ref 14.

Table 1. Classification of Human P450s Based on MajorSubstrate Class (18, 19)

sterols xenobiotics fatty acids eicosanoids vitamins unknown

1B1 1A1 2J2 4F2 2R1 2A77A1 1A2 4A11 4F3 24A1 2S17B1 2A6 4B1 4F8 26A1 2U18B1 2A13 4F12 5A1 26B1 2W111A1 2B6 8A1 26C1 3A4311B1 2C8 27B1 4A2211B2 2C9 4F1117A1 2C18 4F2219A1 2C19 4V221A2 2D6 4 × 127A1 2E1 4Z139A1 2F1 20A146A1 3A4 27C151A1 3A5

3A7Figure 2. Basic P450 catalytic cycle (37).

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both high- and low-spin populations for discrete reactionpathways and multiple products (50, 51). This mechanism hasintellectual attraction in explaining many of the intricacies ofP450 reactions, although the evidence is all theoretical.

Rate-limiting steps in P450 reactions (Figure 2) probably varyconsiderably, depending upon the reaction and the in vitroexperimental setting. Reduction (first electron) (52–54), C–Hbond breaking (38), and a step following product formation(conformational change?) (55) have been identified in somecases, and the rate of transfer of the second electron has beenproposed in some cases (56).

2. Roles of P450s in Reducing Toxicity

P450s are the major enzymes involved in drug metabolism,accounting for ∼75% (Figure 4A). Of the 57 human P450s,five are involved in ∼95% of the reactions (Figure 4B), whichis fortuitous in simplifying the task of assigning new reactionsto individual P450s (57).

One issue in drug development is bioavailability, and acommon initial study is usually “microsomal stability” to predictif most of a drug will be eliminated too rapidly in a “first-pass”effect (59). Another issue is side effects due to the inherentpharmacology of the parent drug. Drug doses are adjusted sothat most people will clear the drug at a reasonable rate.However, if an individual has an inherent (e.g., genetic)deficiency of a particular P450 or that P450 is inhibited byanother drug, toxicity may develop, particularly if drug ac-cumulation occurs upon multiple doses. Drug–drug interactionsare recognized to be a major cause of adverse drug reactions.

These phenomena can often be understood and in many casespredicted in the context of individual human P450 enzymes.One well-documented example is terfenadine, the first marketednonsedating antihistamine (60) (Figure 5). Normally, terfenadineis oxidized very rapidly by P450 3A4, and the major metabolite(fexofenadine) is responsible for the pharmacological activity

(a tert-butyl methyl group is oxidized to a carboxylic acid). Inindividuals who used drugs that inhibit P450 3A4 (e.g.,ketoconazole and erythromycin), terfenadine accumulated in theplasma and cardiac tissue. Dietary constituents (e.g., grapefruit)can also inhibit P450s, although not to the extent to presentserious danger with terfenadine (61). Terfenadine itself is anantagonist of the human ether-a-go-go (hERG) receptor andcauses torsade de pointes (and arrhythmia), invoked in a numberof deaths (62). Following the deaths, the U.S. Food and DrugAdministration (FDA)1 first introduced a contraindication label-ing for use of azoles and erythromycin with terfenadine andsubsequently withdrew terfenadine from the market. Fexofena-dine (Allegra) does not have this liability and has replacedterfenadine on the market, along with other antihistamines suchas loratadine.

Another example of toxicity of a parent drug is the antico-agulant warfarin, which has a relatively narrow therapeuticwindow (i.e., little variation in dose between being effectiveand being toxic in different individuals). Too low a level ofwarfarin can yield clotting, and too high a level can give riseto hemorrhaging. The “effective dose” can be adjusted inindividuals, and this dose has been shown to be influenced bypolymorphisms that affect catalytic activity in the P450 2C9coding region (63). Thus, the *2 (Arg144 Cys change) and *3(Ile359 Leu change) alleles both lower the dose needed formaintenance dosing (and conversely raise the risk of anindividual hemorrhaging at a fixed dose). The risk of hemor-rhaging is particularly high if the patient is ill or changingmedications. However, the P450 2C9 polymorphisms have beenestimated to account collectively for only ∼10% of the totalvariation in warfarin doses among patients (64).

Similar cases involve some environmental toxicants andcarcinogens, although generally the parent compounds generatelittle if any pharmacological activity of their own. However,one issue is metabolism (to innocuous products) that will preventdistribution to tissues in which bioactivation may occur, thuspreventing toxicity. For instance, metabolism in the liver canprevent distribution of polycyclic hydrocarbons to the lung andother target tissues (65). Although P450 1A1 is often considereddangerous because it activates polycyclic hydrocarbons, deletion

1 Abbreviation: FDA, U.S. Food and Drug Administration.

Figure 3. General mechanism for P450 oxidation reactions involvinga perferryl oxygen intermediate and odd-electron chemistry (see Figure2) (40).

Figure 4. Contributions of enzymes to the metabolism of marketeddrugs. The results are from a study of Pfizer drugs (57), and similarpercentages have been reported by others in other pharmaceuticalcompanies (58). (A) Fraction of reactions on drugs catalyzed by varioushuman enzymes. FMO, flavin-containing monoxygenase; NAT, N-acetyltransferase; and MAO, monoamine oxidase. (B) Fractions of P450oxidations on drugs catalyzed by individual P450 enzymes. The segmentlabeled 3A4 (+3A5) is mainly due to P450 3A4, with some controversyabout exactly how much is contributed by other subfamily 3A P450s.Reprinted with permission from ref 57. Copyright 2004 AmericanSociety for Pharmacology and Experimental Therapeutics.

72 Chem. Res. Toxicol., Vol. 21, No. 1, 2008 Guengerich

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of the gene rendered mice more sensitive to benzo[a]pyrenetoxicity (66). The point was made (67) that P450 1 familyinduction is often erroneously viewed by pharmaceuticalcompanies as a liability in drug development; a more accuratestatement is that this potential issue has been considered aproblem by regulatory agencies, for example, the FDA (68, 69),and safety assessment departments have necessarily had to bedefensive in some cases. The successful drug omeprazoleprovides an outstanding example of a compound that doesinduce P450 1 family enzymes (at least in individuals deficientin P450 2C19, which oxidizes the drug) but has not proven tobe a problem. For further discussion of the issue, see a recentreview by Ma and Lu (70).

3. Bioactivation by P450s

The concept of metabolic conversion of chemicals to reactiveproducts that covalently bind macromolecules can be attributedto the late James and Elizabeth Miller (71), and P450s are majorplayers in this paradigm. The list of chemicals known to beactivated is considerable, and the reader is referred to lists ofdrugs (72, 73), toxicants, and carcinogens (74–76). As withdrugs, a subset of the human P450s appear to be responsiblefor most of the cases, although the list changes from drugmetabolism (Figure 4B). P450s 2C8, 2C9, and 2D6 contributelittle to carcinogen activation, while P450s 1A1, 1B1, 2A6,2A13, 2E1, and possibly 2W1 do (as well as 1A2 and 3A4).No attempts to produce estimates such as Figure 1(for drugs)with carcinogens have been made. The “drug-metabolizing”P450s can convert some drugs to toxic products; that is, the roleof P450 2E1 in acetaminophen toxicity is well-established (77).

The concept of bioactivation reactions is not new to P450science (72, 78). Twenty-five years ago, the knowledge of whichindividual P450s catalyzed the activation of particular com-pounds was very meager, and the knowledge of the humanP450s was almost nil (79). By 1991, a fairly extensivecompilation of the human P450s involved in activation ofcarcinogens and protoxicants was available (80). Severalexamples of bioactivation by P450s from the past 20 years willbe presented as examples of the range of the P450s and, despitethe apparent diversity, the point that common chemical mech-anisms can be invoked.

Most of our understanding of bioactivation reactions is inthe context of the generation of electrophilic products thatbecome covalently bound to proteins and DNA. With drugs,there are examples in which a drug metabolite may have moreintrinsic activity with a receptor than the parent compound (81),but obvious examples related to toxicity are not available (82).A strong case exists that binding of electrophiles to DNA cancause mutations, as can be demonstrated experimentally invarious ways (83, 84). In the somatic mutation theory (85), thesewould go on to produce cancer. This field has developed interms of both basic and human studies (76, 86, 87). Theformation of protein adducts with electrophiles has a longhistory, even preceding DNA work (71). There is considerablecorrelative evidence linking protein modification with drugtoxicity in experimental systems, going back to the classic workof Gillette and Brodie (88). Today, protein adduct formation iseven used in some screening paradigms in pharmaceuticaldevelopment (89). Unfortunately, it is not possible to introducea defined protein adduct into a biological system and producea direct toxic effect, in the way that DNA studies can be done.The reader is referred elsewhere to recent reviews on thesignificance of protein adducts (14, 89, 90).

3.1. Aflatoxin B1. Although a mechanism involving the 8,9-epoxide had been proposed for some time (91), the evidencewas indirect. The chemical synthesis of the epoxide (92) was acritical advance and ultimately led to a battery of studies thathave provided considerable insight (Figure 6) (93).

The exo- and endo-8,9-epoxides are both produced, in varyingratios, in chemical synthesis (92, 96) and by individual P450enzymes (96, 97). The exo isomer is g10 (3)-fold moremutagenic and genotoxic than the endo isomer (97, 98). Animportant measurement was the half-life of the exo-epoxide,which is 1 s at neutral pH and room temperature (99). Thesynthesis and this kinetic study led to other experiments thatestablished the role of P450 3A4 in exo-epoxide formation (97)and the reactions with epoxide hydrolase, aldoketo reductase,albumin, and DNA (93–95, 100–102). The DNA reactioninvolves several interesting features including DNA-catalyzedacid hydrolysis, base intercalation, and a very rapid SN2 attackof the N7 atom of guanine on the exo-epoxide (95, 98, 103).The endo-epoxide can effectively be considered a detoxication

Figure 5. Role of P450 3A4 in terfenadine toxicity. Terfenadine is rapidly converted to two products by P450 3A4, one involving hydroxylationof a methyl of the tert-butyl group and the other leading to scission of the molecule (60). The primary alcohol is rapidly oxidized (two steps) tothe carboxylic acid, fexofenadine. In most individuals, terfenadine is oxidized rapidly, effectively acting as a pro-drug for the production of fexofenadine.Both terfenadine and fexofenadine have inherent antagonist activity of the target H1 receptor (antihistamine activity). If P450 3A4 is inhibited bydrugs such as erythromycin or ketoconazole, terfenadine begins to accumulate in the plasma and tissues. The high affinity of terfenadine for thehERG receptor can lead to arrhythmias and deaths.

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product (97, 98). Direct reaction of the epoxide with proteinsis possible, although a more likely route involves reaction withthe dialdehyde formed by base-catalyzed rearrangement of thedihydrodiol (94, 99).

Collectively, the body of knowledge allows a scheme to bedeveloped (Figure 6) with second-order rate constants forchemical reactions and kcat/Km parameters for the major humanenzymes involved in these processes. This framework allowsfor the development of logical paradigms to be used forchemoprevention methods, for example, use of compounds suchas oltipraz to inhibit and induce particular enzymes (104).

3.2. Ethyl Carbamate. This compound, also known asurethane, is a commodity chemical and has been shown to becarcinogenic in rodents (105). A pathway of bioactivationproposed by the Millers (106) involved desaturation to vinylcarbamate (Figure 7).

P450 2E1 has been shown to have a role in the bioactivationof many low Mr chemicals, including halogenated hydrocarbonsand vinyl monomers (107). The Millers and their associates hadbeen able to show that vinyl carbamate was more tumorigenicthan ethyl carbamate (108) but had been unable to demonstratethe desaturation process. A careful search, utilizing selectiveextraction and GC-MS, revealed trace formation of vinylcarbamate formed from ethyl carbamate (107). Furthermore,vinyl carbamate was also converted to a reactive product thatreacted with adenosine (to form 1,N (6)-ethenoadenosine) andwas presumed to be vinyl carbamate epoxide (107). The steady-state levels of vinyl carbamate and its epoxide are consistentwith the pathway shown in Figure 7, and a slow desaturationof ethyl carbamate by P450 2E1 followed by epoxidation at arate 103-fold faster (107). The epoxide has been synthesizedusing dimethyldioxirane (t1/2 of 10 min in H2O) and has beenshown to be mutagenic and tumorigenic (109). This level ofstability would be expected to allow considerable migration, in

that the t1/2 is 600-fold longer than that of aflatoxin B1 8,9-epoxide (vide supra).

3.3. Coupling of Norharman and Aniline. Norharman isfound in cigarette smoke and pyrolyzed food and was discoveredto be a “comutagen” in the 1970s (110). That is, the additionof norharman to an “S9” or other P450-based system used inbacterial mutagenesis tests was found to enhance the mutage-nicity of aniline and some other simple arylamines. One possibleexplanation is heterotropic activation of a P450, a phenomenonobserved with some P450s (18, 111) (vide infra). However, analternate explanation appears to be the case.

Norharman and aniline react to form a new heterocycliccompound, 9-(4′-aminophenyl)-9H-pyrido[3,4-b]indole, whichsubsequently undergoes N-oxygenation to a hydroxylamine thatis acetylated and then reacts with DNA (Figure 8A) (112, 113).P450 1 family and P450 3A4 enzymes are involved in theseprocesses (113, 114). No direct work has been done on thecatalytic mechanism of coupling, but a tenable mechanism ispresented in Figure 8B (41, 113).

3.4. Troglitazone. Troglitazone was the first of the thiazo-lidinedione “glitazone” drugs, developed as a peroxisomeproliferator-activated receptor γ-agonist and used to treatdiabetes. After introduction on the market, the drug waswithdrawn in 2000 due to what was considered an unacceptablyhigh incidence of hepatotoxicity (73, 115, 116).

Subsequent in vitro work was used to establish the course ofbiochemical transformation shown in Figure 9. Troglitazone ishighly bound to albumin and metabolized primarily in the liver.The catalysts have been implicated as P450s 3A4 and, to a lesserextent, P450 2C8 (117, 118). Both the thiazolidinedione andthe chromane ring systems can be activated, as judged by theanalysis of GSH conjugates (Figure 7). The P450 reactions arereadily rationalized in the context of known chemistry (37, 41).

Figure 6. Major pathways involved in human aflatoxin B1 metabolism (94). The indicated parameters are either measured second-order rate constantsor kcat/Km values for the major human enzyme system involved [or kcat/Kd in the case of DNA (95)]. AFAR, aflatoxin B1 aldehyde reductase; AKR,aldo-keto reductase; GST, GSH transferase; and BSA, bovine serum albumin.

74 Chem. Res. Toxicol., Vol. 21, No. 1, 2008 Guengerich

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Newer glitazone drugs have been developed to meet the needsof diabetics, for example, rosiglitazone and pioglitazone. Thesecompounds contain the thiazolidinedione ring and show covalentbinding to protein in in vitro and in vivo assays (73). However,the doses of these drugs are lower than troglitazone and,accordingly, reduce the extent of covalent binding and hepa-toxicity (119). Interestingly, in cell-based systems, P450 inhibi-tion did not protect against the in vitro parameters presumedrelated to toxicity (120). For further discussion of the relation-ship of covalent binding and toxicity of drugs see, refs 14 and89.

3.5. Other Bioactivation Reactions. The current literaturecontains many P450 reactions leading to bioactivation, and themajority have probably been published in the course of the last 20years. A comprehensive list is not presented here, but a number ofreviewsprovideawealthof information(37,41,72,73,89,121,122).The formation of reactive products has become an issue in thepharmaceutical industry, and efforts are being made to identify drugtoxicity in preclinical screens. The goals, issues, and technologyare discussed elsewhere (14, 89).

3.6. Mechanism-Based Activation. One feature of P450reactions that occurs with considerable frequency is mechanism-based inactivation by drugs and other chemicals. This processwas recognized early, although not well-understood (123).Today, we realize that many classes of compounds can causesuch inactivation (e.g., olefins, acetylenes, and cyclopropyl-amines), and in many cases, the mechanisms are well-understoodat the chemical level (124). In most cases, an intermediate inthe oxidative pathway reacts with either the heme or theapoprotein. Probably less common are amines and a few othercompounds (e.g., methylene dioxyphenyls) that yield productsthat bind tightly (but not covalently) to the heme iron (“meta-bolic inhibitory complexes”) and are recognized by the spectralchanges that they produce (125, 126). There are still somecategories of compounds that often act as mechanistic-basedinactivators but for which the chemistry underlying the processis not so obvious (e.g., piperazines) (127).

Inhibition and even destruction of P450s do not in themselvesproduce toxicity, unless one is dealing with a P450 involved ina critical physiological pathway (e.g., steroidogenesis). In this

Figure 7. Activation of ethyl carbamate by P450 2E1 (107).

Figure 8. (A) Enhancement of the mutagenicity of aniline by norharman via fusion and subsequent hydroxylation. (B) A proposed mechanism (41).

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regard, the level of rat liver P450 can be lowered ∼75% by1-aminobenztriazole without any apparent adverse effects (128).With some drugs, the production of heme adducts can triggerporphyrias because the adducts disrupt porphyrin synthesis(129). The most relevant issue with mechanism-based inhibitionby drugs is drug–drug interactions arising because inhibitionof a P450 will attenuate metabolism and lead to higher plasmaand tissue levels of that drug. For instance, several HIV-1protease inhibitors (e.g., ritonavir) are potent P450 3A4 inhibi-tors and block the metabolism of drugs (that are P450 3A4substrates) used concurrently (130, 131). If a drug showsmechanism-based inactivation of a P450, unanticipated drug–drug interactions may result, or the pharmacokinetics of the drug(the inhibitor) can vary with time. Another example is the effectof consumption of grapefruit juice on drug metabolism, whichis related to mechanism-based inactivation of intestinal P4503A4 by bergamottin (132).

Does finding mechanism-based inactivation of a P450 indicatea tendency for the production of reactive products that wouldattack other proteins and result in toxicity? The answer is notnecessarily. Some compounds do both, but in principle, the twoprocesses are clearly distinct (133). For instance, consider thecases of 1-aminobenztriazole and bergamottin presented above.Also, some drugs are still developed on the basis of mechanism-based inactivation, even for P450 19A1 (134).

3.7. P450s and Oxidative Damage. P450s can catalyze one-electron reductions (135) or produce oxygenation products thatare unstable and reduce molecular oxygen (e.g., catecholestrogens) (136). Conceivably, one-electron oxidation productscould undergo radical propagation reactions with oxygen, butthe list of documented stable one-electron oxidation products

is sparse (137). Early studies in the P450 field demonstratedpoor coupling of NADPH oxidation with substrate oxygenation,and the production of O2

-· and H2O2 was documented (138, 139).In the absence of catalase, the H2O2 can destroy heme (140).

The literature is replete with discussions of P450 involvementin the generation of oxidative damage, invoking P450s in the1A, 2A, 2B, 2E, 3A, and 4A subfamilies (141–144). However,closer inspection indicates that almost all of the literature isbased on in vitro systems, mainly either microsomes or culturedcells.

Many of the biomarkers used as parameters of oxidativedamage in in vivo work have not been well-validated. Theproduction of isoprostanes has been shown to be the mostaccurate measure available for assessing oxidative damage (145)and can be utilized in vivo, even in human studies. In a recentstudy, rats were treated in classical regimens known to induceparticular P450s, and parameters of oxidative damage weremeasured. Liver microsomes showed a variety of changes (inNADPH oxidation, H2O2 formation, and thiobarbiturate-reactiveproduct formation), but liver and plasma isoprostanes were foundonly to be substantially elevated in association with a barbituratetype response (treatment with phenobarbital or Aroclor 1254).The lack of change in isoprostanes upon treatment with�-naphthoflavone, isoniazid, pregnenolone 16R-carbonitrile, orclofibrate, which induce P450s in the 1A, 2E, 3A, and 4Asubfamilies, respectively, was notable (146). Thus, P450s in the2B subfamily (or others that might be induced by barbiturates,e.g., 2C6) are associated with oxidative damage, but the othersare not, in in vivo settings.

Figure 9. Activation of troglitazone by P450 3A4 (117, 118).

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4. Current and Future Issues

Although P450 can be considered a relatively mature researchfield in many ways, many questions and challenges still exist,and the area holds many opportunities for dedicated youngscientists. The following list is not intended to be comprehensiveand is oriented toward some issues relevant to toxicology.

4.1. Functions of “Orphan” P450s. The term “orphans” isused to designate relatively recently identified human (and other)P450s for which little information is available, using theterminology originally applied to the steroid hormone receptorfamily (147). This term can be used for about one-fourth of the57 human P450 genes (19, 148). The Human Genome Projecthas provided important knowledge about gene locations andgenetic variants of recently discovered P450s. Orphan P450sare likely not to be major contributors to the metabolism ofdrugs but may have roles in the activation of carcinogens andprotoxicants. For instance, the orphan P450 2W1 has beenshown to be expressed only in tumor tissue (149) and also toactivate a variety of chemical carcinogens (150).

In a broad sense, the identification of functions of newlyidentified proteins is one of the major problems in biology.Several approaches can be used to define the functions of orphanP450s in humans and animal models (19), and some of thesehave been employed already.

4.1.1. Analysis of Suspects. If related P450s (e.g., same P450family) catalyze reactions with a class of chemicals, then thesecan be used in trial assays. For instance, when P450 27C1 waspurified, it was tested with vitamin D compounds because P45027A1 and 27B1 catalyze such reactions (151) (however, noactivity was observed).

4.1.2. Transgenic Animal Models. One option is to deletewhat is expected to be the orthologous gene from a mouse andthen interrogate the animals for differences. In some cases, thiscan be done with a “metabolic” approach. Thus, P450 2R1 wascharacterized as a retinoic acid hydroxylase (152). Alternatively,a human enzyme could be overexpressed in mice and theanimals could be examined (in a metabolomic approach) to lookfor differences.

4.1.3. Library Screening. In this approach, components ofa selected set of perhaps 50–300 chemicals, representing a broadspectrum of chemical classes, are catalyzed for interaction witha purified P450. If even weak activity is found with arepresentative, for example, an androgen, then further studiesare done with more class representatives.

4.1.4. Untargeted Metabolomic Strategies in Vitro. Thepurified P450 is incubated in a cofactor-fortified system withan extract of the tissue in which the P450 is expressed. Changesin the composition of the extract are interrogated for changesusing LC-MS, using principal component analysis or otherapproaches to compare the extract before and after the incubation(19).

4.1.5. Untargeted in Vitro Strategies with IsotopeEditing. This approach is similar to the former one, except thatan incorporated cofactor (O2) is partially tagged with an isotope(e.g., 18O) and the extract is examined for an isotopic signaturewith an expected 18O (16):O ratio). Software employing thisapproach has been developed and used with model systems(153).

4.2. Ligand Cooperativity. A full treatise on this issue isfar beyond the scope of this review. In brief, some of the P450sexhibit rather aberrant catalytic behavior (18, 111, 154–156).Ligand cooperativity is of two types: (i) homotropic cooperat-ivity, in which sigmoidal plots of reaction velocity vs substrateare seen, and (ii) heterotropic cooperativity, in which the

addition of a compound to the enzyme stimulates the oxidationof a substrate (the enhancing compound may also be a substrateitself) (18). Work with animal models suggests that thisphenomenon (at least heterotropic cooperativity) can occur invivo (157, 158). With regard to homotropic cooperativity, thepatterns are often modest (in terms of apparent Hill plotparameters, e.g., n ) 1.3–1.5), and care is required in analysis.However, in a recent study in this laboratory, we have observedan apparent n value of ∼6 for the 1-hydroxylation of pyreneby rabbit P450 1A2 (2).

A number of hypotheses have been proposed, includingclassical allosteric effects keyed to binding at a remote site,multiple occupancy of the active site, multiple protein conform-ers that are selected by binding to ligands, and mixtures of theabove (18, 111, 159). Unfortunately, much of the literature inthis area is based on simplistic steady-state kinetic analyses,and the recently elucidated crystal structures (of P450s 2C8 and3A4) have not been able to resolve the issues (32, 34, 36).

Our own work in this area has shown the involvement ofslow protein–ligand changes that occur during the time scaleof substrate oxidation (39, 160). That is, conformational changescan still be occurring while the enzyme is oxidizing substrates,and effectively, a mixture (or perhaps more properly a con-tinuum) of enzyme forms exists in the course of the reaction.The results for substrate binding kinetics (39, 160) also suggestthat the substrate (or inhibitor) for P450 3A4 first interacts witha peripheral site on the enzyme and then somewhat slowlymoves toward the heme iron. Whether or not this putativeperipheral site is that occupied by progesterone (32) or test-osterone (161) in some X-ray crystal structures is unclear.Recent results with rabbit P450 1A2 also indicate multiple stepsassociated with ligand binding, and fluorescence evidence forthe formation of pyrene excimers (dimers) in the active site hasbeen obtained,2 reminiscent of a steady-state study with P4503A4 (162).

4.3. Predictions of Metabolism. P450s are involved in∼75% of all drug metabolism (Figure 4A). The ability to predictsites and rates of oxidation of new substrates would greatlyfacilitate drug development, as well as considerations ofpotential carcinogens and toxicants. Efforts toward this goal havebeen made. Possible approaches can involve either comparisonsof existing databases of biotransformation data, docking andelectronic information about P450s and substrates, or a mixtureof the two. Some of the efforts have been made withinpharmaceutical organizations and some by smaller privateorganizations.

One unresolved issue is that available crystal structures havedeficiencies in providing all of the details that are desired. Inthe absence of such information, the energy of different substratebonds is not a reliable guide to prediction (163). Better drugdesign predictions can be made within series of closely relatedcompounds, while de novo estimates (in unrelated series) arestill far more difficult and remain a challenge for the pharma-ceutical industry. Although computational tools are on themarket, these have generally not yet found major success inthe pharmaceutical industry (in drug metabolism or safetyassessment), and in the short term, empirical (experimental)approaches will continue to be dominant. The problem of using“rational” systems with the available P450 crystal structures isevident; few of the structures of ligand-bound P450s correctlypredict regioselectivity of oxidation (29). For instance, 5,6-benzoflavone is slowly oxidized to the 5,6-epoxide by P450 1A2,

2 Isin, E. M., Sohl, C. D., Marsch, G. A., and Guengerich, F. P.Manuscript in preparation.

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but the structure has the oxidized alkene bond furthest fromthe heme iron (26, 97, 155, 164).

4.4. Overlaps of Detoxication and Bioactivation. A simpleview of P450s is that some do good things and some do badthings, and appropriate induction and inhibition have thepotential to produce a more favorable situation. However, thesituation is usually much more complex. Mention has alreadybeen made of the significance of metabolism in individual tissuesand the resulting balance of detoxication and activation (65, 67).An even more complicating situation occurs when an individualP450 can both activate and detoxicate the same molecules, forexample, P450 1A1 with benzo[a]pyrene (165) or P450 3A4with aflatoxin B1 (97, 166).

Some insight can be gained with detailed studies of theenzymology, but a more appropriate understanding of the overallsituation requires (i) a more complex system (at least cellularand possibly in vivo), (ii) distribution analysis (pharmacokinetic)of the chemical and its products in various tissues (“target” andnontarget), (iii) analysis of the systems at varying doses(concentrations), and (iv) knowledge of the distribution of theP450 in various tissues (and cells). Of course, analysis incomplex systems is more difficult if multiple P450s are involvedin any of the reactions. These problems require many measure-ments and also methods of pharmacokinetic modeling/fitting.

4.5. Roles of P450s in Idiosyncratic Drug Toxicity. Idio-syncratic drug reactions are defined as those highly individual-ized in occurrence, and their pharmacological basis is unknown.In practice they occur in ∼1/10 (3) to 1/10 (4) individuals andhave been very hard to predict with animal models and even inclinical trials. Unfortunately, some of these problems are notidentified before new drugs are introduced to the market.Although the statement is often made that these responses aredose-independent, this is not really established, and many donot accept this premise.

Two phenomena often discussed in relation to idiosyncrasiesare bioactivation (to yield covalent binding) and hypersensitivity/allergic responses. P450s certainly play a role in bioactiva-tion and covalent binding, and covalent binding and autoimmuneantibodies accompany some idiosyncratic drug reactions(167, 168), but exactly how these events fit together and whatis causal are still rather unclear. We do not have a good estimateof the contribution of P450s to idiosyncratic reactions, and thisquestion warrants further study. The problems are extremelycomplex. With both tienilic acid (168) and dihydralazine (167),a small set of the patients develop hepatitis and also havecirculating antibodies that recognize a P450 involved in bio-activation, P450 2C9 in the case of tienilic acid and P450 1A2in the case of dihydralazine. Drug adducts are formed with theP450 (in in vitro experiments). Questions still exist as to howthe P450s are processed to generate antibodies (169). Theantibodies recognize unmodified P450s (but do not inhibitoxidations in vivo). Not all patients with antibodies develophepatitis. Also, efforts to produce animal models have producedantibodies and hepatotoxicity but not together (170). Thus, weare left with questions about causality (171) and are still verylimited in the availability of animal models for predictingidiosyncratic events (172).

4.6. Predicting Human Toxicity. The problem of predictingtoxicity has already been mentioned. The scope of the problemis considerable (12, 14) and goes far beyond P450 issues. Oneof the challenges is the difficulty of extrapolating from animaltoxicity data to humans. Some improvement in this area maycome with more knowledge about P450s, perhaps to the extent

that drug metabolism extrapolations to humans may helpimprove predictions in this area.

One classic example of the problems is acetaminophen. Weknow that deletion of P450 2e1 in mice nearly eliminateshepatoxicity, and simultaneous deletion of P450 1a2 is evenmore effective (77, 173). Presumably the load of reactiveproducts is decreased, although apparently the effect on the loadhas not been reported. A real issue is that the biological eventsfollowing the adduction process are still rather vague, even inanimal models. Although the involvement of mouse P450s 2e1and 1a2 in toxicity is quite convincing (77, 173, 174), extrapola-tions to human P450s are not direct (175). Thus, we still havemany questions, and it is hoped that major progress will occurin this area.

4.7. Understanding P450 Gene Polymorphisms andDisease. Today, extensive information about the major polymor-phisms in many of the human P450s (http://www.cypalleles.ki.se) is available. There has been considerable discussion ofthe potential use of this information in developing personalizedmedicine, facilitating drug development, and identifying cancerrisk. However, the reality is far from this. Today, the FDA doesnot prescribe genotyping for any drug (some assays areapproved, but none are required). There are several issuesinvolved, including costs, genotype/phenotype correspondence,the limited contribution of genotype as compared to environ-mental influence with P450 3A4, and the lack of a strong casehistory for the use of genotyping in prediction of idiosyncrasiesto date. Thus, the challenges are many, but ultimately, soundapproaches that include functional analysis are most likely tobe successful. The relationships between P450 genotypes andcancer are even more difficult to establish than with drugs, andmany of the reports on associations of P450 polymorphismswith genotypes have not held up in meta analysis (176–179).Cancer risk estimates associated with individual single nucle-otide polymorphisms must be considered spurious at best.

An example in point is the relationship of P450 2D6 withlung cancer. In 1983, poor metabolism (phenotype, based onin vivo debrisoquine metabolism) was reported to be stronglyassociated with less lung cancer in cigarette smokers (180). Onereason for such a relationship could be the activation ofprocarcinogens in tobacco smoke by P450 2D6. However, theexpression of P450 2D6 in lung is relatively low, and a numberof searches have not identified any carcinogens that arepreferentially activated by P450 2D6 (181, 182) even in studieswith cigarette smoke condensate extracts (183). Further epide-miological studies have yielded mixed answers on the relation-ship of the poor metabolizer phenotype and also P450 2D6genotypes with lung cancer (184), and the relationship is weakat best in meta analysis (185).

Another related study involves the relationship between P4501A1 induction and lung cancer, first reported in 1973 (186, 187).This relationship is still unclear (188) and cannot be understoodin the context of present knowledge about P450 1A1 (189, 190)or the Ah receptor (191). One possible lead is P450 1B1, inthat this enzyme has high activity toward polycyclic aromatichydrocarbons such as benzo[a]pyrene (192, 193) and has beenshown to exhibit the trimodal distribution of inducibility (194)first reported by Shaw and Kellerman in 1973 (186, 187).

The difficulty in associating cancer risks with P450 activities(in humans) is not surprising if one compares the situation withclinical trials, for which the problems have already beenmentioned. In large clinical trials, there are often thousands ofindividuals being administered a single, well-defined drug undercontrolled conditions, and a relatively simple outcome may be

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measured (e.g., blood pressure) after a short time. However,with cancers, there are usually relatively small numbers, therecord of exposure to chemicals is sparse at best, there is limitedevidence that a single chemical causes the cancer, the timebetween the initial exposure and the outcome (cancer) isdecades, and the disease is usually heterogeneous. In the future,studies in this area will probably need to involve larger studiesof more homogeneously exposed individuals, including thoseexposed to high concentrations of single cancer suspects (e.g.,vinyl monomers).

5. Conclusion

P450 research developed largely because of its potential toexplain the metabolism and toxicity of drugs and carcinogens.Today, we are at a position where the biochemical understandingof these systems is rich but still not complete. The field hasbeen highly successful in the context of providing betterpredictions about human drug metabolism. However, manychallenges still remain in further developing and applying ourknowledge of P450s to unresolved problems in chemicaltoxicity. With the challenges come many opportunities awaitingdedicated researchers who have vision in the P450 field.

Acknowledgment. Work in this area in my laboratory issupported in part by U.S. Public Health Service Grants R37CA090426 and P30 ES000267. Thanks are extended to K. Starkfor comments on a draft version and to K. Trisler for assistancein preparation of the manuscript. Congratulations and thanksare in order to all who have played a role in the first 20 yearsof Chemical Research in Toxicology.

References

(1) Mueller, G. C., and Miller, J. A. (1948) The metabolism of4-dimethylaminoazobenzene by rat liver homogenates. J. Biol. Chem.176, 535–544.

(2) Omura, T., and Sato, R. (1962) A new cytochrome in livermicrosomes. J. Biol. Chem. 237, 1375–1376.

(3) Estabrook, R. W., Cooper, D. Y., and Rosenthal, O. (1963) The lightreversible carbon monoxide inhibition of the steroid C21-hydroxylasesystem of the adrenal cortex. Biochem. Z. 338, 741–755.

(4) Lu, A. Y. H., and Coon, M. J. (1968) Role of hemoprotein P-450 infatty acid ω-hydroxylation in a soluble enzyme system from livermicrosomes. J. Biol. Chem. 243, 1331–1332.

(5) Imai, Y., and Sato, R. (1974) A gel-electrophoretically homogeneouspreparation of cytochrome P-450 from liver microsomes of phe-nobarbital-pretreated rabbits. Biochem. Biophys. Res. Commun. 60,8–14.

(6) Haugen, D. A., van der Hoeven, T. A., and Coon, M. J. (1975)Purified liver microsomal cytochrome P-450: separation and char-acterization of multiple forms. J. Biol. Chem. 250, 3567–3570.

(7) Alveres, A. P., Schilling, G., Levin, W., and Kuntzman, R. (1967)Studies on the induction of CO-binding pigments in liver microsomesby phenobarbital and 3-methylcholanthrene. Biochem. Biophys. Res.Commun. 29, 521–526.

(8) Sladek, N. E., and Mannering, G. J. (1969) Induction of drugmetabolism. II. Qualitative differences in the microsomal N-de-methylating systems stimulated by polycyclic hydrocarbons and byphenobarbital. Mol. Pharmacol. 5, 186–199.

(9) Katagiri, M., Ganguli, B. N., and Gunsalus, I. C. (1968) A solublecytochrome P450 functional in methylene hydroxylation. J. Biol.Chem. 243, 3543–3546.

(10) Mizukami, Y., Sogawa, K., Suwa, Y., Muramatsu, M., and Fujii-Kuriyama, Y. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 3958–3962.

(11) Groves, J. T., McClusky, G. A., White, R. E., and Coon, M. J. (1978)Aliphatic hydroxylation by highly purified liver microsomal cyto-chrome P-450: Evidence for a carbon radical intermediate. Biochem.Biophys. Res. Commun. 81, 154–160.

(12) Kola, I., and Landis, J. (2004) Can the pharmaceutical industry reduceattrition rates. Nat. ReV. Drug DiscoVery 3, 711–715.

(13) Stevens, J. L. (2006) Future of toxicology—Mechanisms of toxicityand drug safety: Where do we go from here. Chem. Res. Toxicol.19, 1393–1401.

(14) Guengerich, F. P., and MacDonald, J. S. (2007) Applying mechanismsof chemical toxicity to predict drug safety. Chem. Res. Toxicol. 20,344–369.

(15) Speirs, A. L. (1962) Thalidomide and congenital abnormalities. Lancet1, 303–305.

(16) Gordon, G. B., Spielberg, S. P., Blake, D. A., and Balasubramanian,V. (1981) Thalidomide teratogenesis: Evidence for a toxic arene oxidemetabolite. Proc. Natl. Acad. Sci. U.S.A. 78, 2545–2548.

(17) Lu, J., Helsby, N., Palmer, B. D., Tingle, M., Baguley, B. C., Kestell,P., and Ching, L. M. (2004) Metabolism of thalidomide in livermicrosomes of mice, rabbits, and humans. J. Pharmacol. Exp. Ther.310, 571–577.

(18) Guengerich, F. P. (2005) Human cytochrome P450 enzymes. InCytochrome P450: Structure Mechanism, and Biochemistry, 3rd ed.(Ortiz de Montellano, P. R., Ed.) pp 377–530, Kluwer Academic/Plenum Press, New York.

(19) Guengerich, F. P., Wu, Z.-L., and Bartleson, C. J. (2005) Functionof human cytochrome P450s: Characterization of the remainingorphans. Biochem. Biophys. Res. Commun. 338, 465–469.

(20) Distlerath, L. M., Reilly, P. E. B., Martin, M. V., Davis, G. G.,Wilkinson, G. R., and Guengerich, F. P. (1985) Purification andcharacterization of the human liver cytochromes P-450 involved indebrisoquine 4-hydroxylation and phenacetin O-deethylation, twoprototypes for genetic polymorphism in oxidative drug metabolism.J. Biol. Chem. 260, 9057–9067.

(21) Shimada, T., Misono, K. S., and Guengerich, F. P. (1986) Humanliver microsomal cytochrome P-450 mephenytoin 4-hydroxylase, aprototype of genetic polymorphism in oxidative drug metabolism.Purification and characterization of two similar forms involved inthe reaction. J. Biol. Chem. 261, 909–921.

(22) Guengerich, F. P., Martin, M. V., Beaune, P. H., Kremers, P., Wolff,T., and Waxman, D. J. (1986) Characterization of rat and humanliver microsomal cytochrome P-450 forms involved in nifedipineoxidation, a prototype for genetic polymorphism in oxidative drugmetabolism. J. Biol. Chem. 261, 5051–5060.

(23) Barnes, H. J., Arlotto, M. P., and Waterman, M. R. (1991) Expressionand enzymatic activity of recombinant cytochrome P450 17R-hydroxylase in Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 88,5597–5601.

(24) Larson, J. R., Coon, M. J., and Porter, T. D. (1991) Alcohol-induciblecytochrome P-450IIE1 lacking the hydrophobic NH2-terminal seg-ment retains catalytic activity and is membrane-bound when ex-pressed in Escherichia coli. J. Biol. Chem. 266, 7321–7324.

(25) Li, Y. C., and Chiang, J. Y. L. (1991) The expression of a catalyticallyactive cholesterol 7R-hydroxylase cytochrome P-450 in Escherichiacoli. J. Biol. Chem. 266, 19186–19191.

(26) Sansen, S., Yano, J. K., Reynald, R. L., Schoch, G., Griffin, K., Stout,C. D., and Johnson, E. F. (2007) Adaptations for the oxidation ofpolycyclic aromatic hydrocarbons exhibited by the structure of humanP450 1A2. J. Biol. Chem. 282, 14348–14355.

(27) Yano, J. K., Hsu, M. H., Griffin, K. J., Stout, C. D., and Johnson,E. F. (2005) Structures of human microsomal cytochrome P450 2A6complexed with coumarin and methoxsalen. Nat. Struct. Mol. Biol.12, 822–823.

(28) Schoch, G. A., Yano, J. K., Wester, M. R., Griffin, K. J., Stout, C. D.,and Johnson, E. F. (2004) Structure of human microsomal cytochromeP450 2C8. Evidence for a peripheral fatty acid binding site. J. Biol.Chem. 279, 9497–9503.

(29) Williams, P. A., Cosme, J., Ward, A., Angove, H. C., MatakVinkovic, D., and Jhoti, H. (2003) Crystal structure of humancytochrome P450 2C9 with bound warfarin. Nature 424, 464–468.

(30) Wester, M. R., Yano, J. K., Schoch, G. A., Yang, C., Griffin, K. J.,Stout, C. D., and Johnson, E. F. (2004) The structure of humancytochrome P450 2C9 complexed with flurbiprofen at 2.0-Å resolu-tion. J. Biol. Chem. 279, 35630–35637.

(31) Rowland, P., Blaney, F. E., Smyth, M. G., Jones, J. J., Leydon, V. R.,Oxbrow, A. K., Lewis, C. J., Tennant, M. G., Modi, S., Eggleston,D. S., Chenery, R. J., and Bridges, A. M. (2006) Crystal structure ofhuman cytochrome P450 2D6. J. Biol. Chem. 281, 7614–7622.

(32) Williams, P. A., Cosme, J., Vinkovic, D. M., Ward, A., Angove,H. C., Day, P. J., Vonrhein, C., Tickle, I. J., and Jhoti, H. (2004)Crystal structures of human cytochrome P450 3A4 bound tometyrapone and progesterone. Science 305, 683–686.

(33) Yano, J. K., Wester, M. R., Schoch, G. A., Griffin, K. J., Stout, C. D.,and Johnson, E. F. (2004) The structure of human microsomalcytochrome P450 3A4 determined by X-ray crystallography to 2.05Å resolution. J. Biol. Chem. 279, 38091–38094.

(34) Ekroos, M., and Sjögren, T. (2006) Structural basis for ligandpromiscuity in cytochrome P450 3A4. Proc. Natl. Acad. Sci. U.S.A.103, 13682–13687.

(35) Poulos, T. L., and Johnson, E. F. (2005) Structures of cytochromeP450 enzymes. In Cytochrome P450: Structure, Mechanism, and

PerspectiVe Chem. Res. Toxicol., Vol. 21, No. 1, 2008 79

Dow

nloa

ded

by U

NIV

OF

NO

RT

H C

AR

OL

INA

on

Aug

ust 3

1, 2

009

| http

://pu

bs.a

cs.o

rg

Pub

licat

ion

Dat

e (W

eb):

Dec

embe

r 6,

200

7 | d

oi: 1

0.10

21/tx

7000

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Biochemistry, 3rd ed. (Ortiz de Montellano, P. R., Ed.) pp 87–114,Kluwer Academic/Plenum Publishers, New York.

(36) Guengerich, F. P. (2006) A malleable catalyst dominates themetabolism of drugs. Proc. Natl. Acad. Sci. U.S.A. 103, 13565–13566.

(37) Ortiz de Montellano, P. R., and De Voss, J. J. (2005) Substrateoxidation by cytochrome P450 enzymes. In Cytochrome P450:Structure, Mechanism, and Biochemistry, 3rd ed. (Ortiz de Montel-lano, P. R., Ed.) pp 183–245, Kluwer Academic/Plenum Publishers,New York.

(38) Yun, C.-H., Kim, K.-H., Calcutt, M. W., and Guengerich, F. P. (2005)Kinetic analysis of oxidation of coumarins by human cytochromeP450 2A6. J. Biol. Chem. 280, 12279–12291.

(39) Isin, E. M., and Guengerich, F. P. (2006) Kinetics and thermodynam-ics of ligand binding by cytochrome P450 3A4. J. Biol. Chem. 281,9127–9136.

(40) Guengerich, F. P., and Macdonald, T. L. (1984) Chemical mecha-nisms of catalysis by cytochromes P-450: A unified view. Acc. Chem.Res. 17, 9–16.

(41) Guengerich, F. P. (2001) Common and uncommon cytochrome P450reactions related to metabolism and chemical toxicity. Chem. Res.Toxicol. 14, 611–650.

(42) Newcomb, M., Le Tadic-Biadatti, M. H., Chestney, D. L., Roberts,E. S., and Hollenberg, P. F. (1995) A nonsynchronous concertedmechanism for cytochrome P-450 catalyzed hydroxylation. J. Am.Chem. Soc. 117, 12085–12091.

(43) Chandrasena, R. E., Vatsis, K. P., Coon, M. J., Hollenberg, P. F.,and Newcomb, M. (2004) Hydroxylation by the hydroperoxy-ironspecies in cytochrome P450 enzymes. J. Am. Chem. Soc. 126, 115–126.

(44) Akhtar, M., Calder, M. R., Corina, D. L., and Wright, J. N. (1982)Mechanistic studies on C-19 demethylation in oestrogen biosynthesis.Biochem. J. 201, 569–580.

(45) Cole, P. A., and Robinson, C. H. (1988) A peroxide model reactionfor placental aromatase. J. Am. Chem. Soc. 110, 1284–1285.

(46) Hackett, J. C., Brueggemeier, R. W., and Hadad, C. M. (2005) Thefinal catalytic step of cytochrome P450 aromatase: A densityfunctional theory study. J. Am. Chem. Soc. 127, 5224–5237.

(47) Newcomb, M., Shen, R., Choi, S., Toy, P. H., Hollenberg, P. F.,Vaz, A. D. N., and Coon, M. J. (2000) Cytochrome P450-catalyzedhydroxylation of mechanistic probes that distinguish between radicalsand cations. Evidence for cationic but not for radical intermediates.J. Am. Chem. Soc. 122, 2677–2686.

(48) Auclair, K., Hu, Z., Little, D. M., Ortiz de Montellano, P. R., andGroves, J. T. (2002) Revisiting the mechanism of P450 enzymes withthe radical clocks norcarane and spiro[2,5]octane. J. Am. Chem. Soc.124, 6020–6027.

(49) Ortiz de Montellano, P. R., and De Voss, J. J. (2002) Oxidizingspecies in the mechanism of cytochrome P450. Nat. Prod. Rep. 19,477–493.

(50) Harris, N., Cohen, S., Filatov, M., Ogliaro, F., and Shaik, S. (2000)Two-state reactivity in the rebound step of alkane hydroxylation bycytochrome P-450: Origins of free radicals with finite lifetimes.Angew. Chem., Int. Ed. 39, 2003–2007.

(51) Shaik, S., Kumar, D., de Visser, S. P., Altun, A., and Thiel, W. (2005)Theoretical perspective on the structure and mechanism of cyto-chrome P450 enzymes. Chem. ReV. 105, 2279–2328.

(52) Diehl, H., Schädelin, J., and Ullrich, V. (1970) Studies on the kineticsof cytochrome P-450 reduction in rat liver microsomes. Z. Physiol.Chem. 351, 1359–1371.

(53) Backes, W. L., Sligar, S. G., and Schenkman, J. B. (1982) Kineticsof hepatic cytochrome P-450 reduction: Correlation with spin stateof the ferric heme. Biochemistry 21, 1324–1330.

(54) Guengerich, F. P., and Johnson, W. W. (1997) Kinetics of ferriccytochrome P450 reduction by NADPH-cytochrome P450 reductase:Rapid reduction in absence of substrate and variations amongcytochrome P450 systems. Biochemistry 36, 14741–14750.

(55) Bell-Parikh, L. C., and Guengerich, F. P. (1999) Kinetics ofcytochrome P450 2E1-catalyzed oxidation of ethanol to acetic acidvia acetaldehyde. J. Biol. Chem. 274, 23833–23840.

(56) Zhang, H., Gruenke, L., Arscott, D., Shen, A., Kasper, C., Harris,D. L., Glavanovich, M., Johnson, R., and Waskell, L. (2003)Determination of the rate of reduction of oxyferrous cytochrome P4502B4 by 5-deazariboflavin adenine dinucleotide T491V cytochromeP450 reductase. Biochemistry 42, 11594–11603.

(57) Williams, J. A., Hyland, R., Jones, B. C., Smith, D. A., Hurst, S.,Goosen, T. C., Peterkin, V., Koup, J. R., and Ball, S. E. (2004) Drug-drug interactions for UDP-glucuronosyltransferase substrates: Apharmacokinetic explanation for typically observed low exposureAUCi/AUC ratios. Drug Metab. Dispos. 32, 1201–1208.

(58) Wienkers, L. C., and Heath, T. G. (2005) Predicting in vivo druginteractions from in vitro drug discovery data. Nat. ReV. DrugDiscoVery 4, 825–833.

(59) Williams, J. A., Bauman, J., Cai, H., Conlon, K., Hansel, S., Hurst,S., Sadagopan, N., Tugnait, M., Zhang, L., and Sahi, J. (2005) InVitro ADME phenotyping in drug discovery: Current challenges andfuture solutions. Curr. Opin. Drug DiscoVery DeV. 8, 78–88.

(60) Yun, C.-H., Okerholm, R. A., and Guengerich, F. P. (1993) Oxidationof the antihistaminic drug terfenadine in human liver microsomes:role of cytochrome P450 3A(4) in N-dealkylation and C-hydroxy-lation. Drug Metab. Dispos. 21, 403–409.

(61) Rau, S. E., Bend, J. R., Arnold, J. M. O., Tran, L. T., Spence, J. D.,and Bailey, D. G. (1997) Grapefruit juice-terfenadine single-doseinteraction: Magnitude, mechanism, and relevance. Clin. Pharmacol.Ther. 61, 401–409.

(62) Kivistö, K. T., Neuvonen, P. J., and Klotz, U. (1994) Inhibition ofterfenadine metabolism: Pharmacokinetic and pharmacodynamicconsequences. Clin. Pharmacokinet. 27, 1–5.

(63) Higashi, M. K., Veenstra, D. L., Kondo, L. M., Wittkowsky, A. K.,Srinouanprachanh, S. L., Farin, F. M., and Rettie, A. E. (2002)Association between CYP2C9 genetic variants and anticoagulation-related outcomes during warfarin therapy. J. Am. Med. Assoc. 287,1690–1698.

(64) Marsh, S., and McLeod, H. L. (2006) Pharmacogenomics: Frombedside to clinical practice. Hum. Mol. Genet. 15 (Spec. No. 1), R89–R93.

(65) Nebert, D. W. (1989) The Ah locus: Genetic differences in toxicity,cancer, mutation, and birth defects. CRC Crit. ReV. Toxicol. 20, 153–174.

(66) Uno, S., Dalton, T. P., Derkenne, S., Curran, C. P., Miller, M. L.,Shertzer, H. G., and Nebert, D. W. (2004) Oral exposure tobenzo[a]pyrene in the mouse: Detoxication by inducible cytochromeP450 is more important than metabolic activation. Mol. Pharmacol.65, 1225–1237.

(67) Nebert, D. W., Dalton, T. P., Okey, A. B., and Gonzalez, F. J. (2004)Role of aryl hydrocarbon receptor-mediated induction of the CYP1enzymes in environmental toxicity and cancer. J. Biol. Chem. 279,23847–23850.

(68) Diaz, D., Fabre, I., Daujat, M., Saint Aubert, B., Bories, P., Michel,H., and Maurel, P. (1990) Omeprazole is an aryl hydrocarbon-likeinducer of human hepatic cytochrome P450. Gastroenterology 99,737–747.

(69) Ioannides, C., and Parke, D. V. (1993) Induction of cytochrome P4501as an indicator of potential chemical carcinogenesis. Drug Metab.ReV. 25, 485–501.

(70) Ma, Q., and Lu, A. Y. H. (2007) CYP1A induction and human riskassessment: An evolving tale of in Vitro and in ViVo studies. DrugMetab. Dispos. 35, 1009–1016.

(71) Miller, E. C., and Miller, J. A. (1947) The presence and significanceof bound amino azodyes in the livers of rats fed p-dimethylamino-azobenzene. Cancer Res. 7, 468–480.

(72) Nelson, S. D. (1982) Metabolic activation and drug toxicity. J. Med.Chem. 25, 753–765.

(73) Kalgutkar, A. S., Gardner, I., Obach, R. S., Shaffer, C. L., Callegari,E., Henne, K. R., Mutlib, A. E., Dalvie, D. K., Lee, J. S., Nakai, Y.,O’Donnell, J. P., Boer, J., and Harriman, S. P. (2005) A compre-hensive listing of bioactivation pathways of organic functional groups.Curr. Drug Metab. 6, 161–225.

(74) Guengerich, F. P., and Liebler, D. C. (1985) Enzymatic activationof chemicals to toxic metabolites. CRC Crit. ReV. Toxicol. 14, 259–307.

(75) Dipple, A., Michejda, C. J., and Weisburger, E. K. (1985) Metabolismof chemical carcinogens. Pharmacol. Ther. 27, 265–296.

(76) Searle, C. E., Ed. (1984) Chemical Carcinogens, Vols. 1 and 2,American Chemical Society, Washington, DC.

(77) Lee, S. S. T., Buters, J. T. M., Pineau, T., Fernandez-Salguero, P.,and Gonzalez, F. J. (1996) Role of CYP2E1 in the hepatotoxicity ofacetaminophen. J. Biol. Chem. 271, 12063–12067.

(78) Mitchell, J. R., Jollow, D. J., Potter, W. Z., Davis, D. C., Gillette,J. R., and Brodie, B. B. (1973) Acetaminophen-induced hepaticnecrosis. I. Role of drug metabolism. J. Pharmacol. Exp. Ther. 187,185–194.

(79) Distlerath, L. M., and Guengerich, F. P. (1987) Enzymology of humanliver cytochromes P-450. In Mammalian Cytochromes P-450, Volume1 (Guengerich, F. P., Ed.) pp 133–198, CRC Press, Boca Raton, FL.

(80) Guengerich, F. P., and Shimada, T. (1991) Oxidation of toxic andcarcinogenic chemicals by human cytochrome P-450 enzymes. Chem.Res. Toxicol. 4, 391–407.

(81) Stearns, R. A., Chakravarty, P. K., Chen, R., and Chiu, S. H. L.(1995) Biotransformation of losartan to its active carboxylic acidmetabolite in human liver microsomes: Role of cytochrome P4502Cand 3A subfamily members. Drug Metab. Dispos. 23, 207–215.

(82) Liebler, D. C., and Guengerich, F. P. (2005) Elucidating mechanismsof drug-induced toxicity. Nat. ReV. Drug DiscoVery 4, 410–420.

(83) Ames, B. N., Durston, W. E., Yamasaki, E., and Lee, F. D. (1973)Carcinogens are mutagens: A simple test system combining liver

80 Chem. Res. Toxicol., Vol. 21, No. 1, 2008 Guengerich

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homogenates for activation and bacteria for detection. Proc. Natl.Acad. Sci. U.S.A. 70, 2281–2285.

(84) Loechler, E. L., Green, C. L., and Essigmann, J. M. (1984) In ViVomutagenesis by O6-methylguanine built into a unique site in a viralgenome. Proc. Natl. Acad. Sci. U.S.A. 81, 6271–6275.

(85) Bauer, K. H. (1928) Mutationstheorie der Geschwulstenstehung,Springer, Berlin.

(86) Pfeifer, G. P., and Denissenko, M. F. (1998) Formation and repairof DNA lesions in the p53 gene: Relation to cancer mutations.EnViron. Mol. Mutagen. 31, 197–205.

(87) Toraason, J., Albertini, R., Bavard, S., Bigbee, W., Blair, A., Boffetta,P., Bonassi, S., Chanock, S., Christiani, D., Eastmond, D., Hanash,S., Henry, C., Kadlubar, F., Mirer, F., Nebert, D., Rapport, S., Rest,K., Rothman, N., Ruder, A., Savage, R., Schulte, P., Siemiatycki, J.,Shields, P., Smith, M., Tolbert, P., Vermeulen, R., Vineis, P.,Wacholder, S., Ward, E., Waters, M., and Weston, A. (2004)Applying new biotechnologies to the study of occupationsl cancer—Aworkshop summary. EnViron. Health Perspect. 112, 413–416.

(88) Jollow, D. J., Mitchell, J. R., Potter, W. Z., Davis, D. C., Gillette,J. R., and Brodie, B. B. (1973) Acetaminophen-induced hepaticnecrosis. II. Role of covalent binding in ViVo. J. Pharmacol. Exp.Ther. 187, 195–202.

(89) Evans, D. C., Watt, A. P., Nicoll-Griffith, D. A., and Baillie, T. A.(2004) Drug–protein adducts: An industry perspective on minimizingthe potential for drug bioactivation in drug discovery and develop-ment. Chem. Res. Toxicol. 17, 3–16.

(90) Walgren, J. L., Mitchell, M. D., and Thompson, D. C. (2005) Roleof metabolism in drug-induced idiosyncratic hepatotoxicity. Crit. ReV.Toxicol 35, 325–361.

(91) Essigmann, J. M., Croy, R. G., Nadzan, A. M., Busby, W. F., Jr.,Reinhold, V. N., Buc̈hi, G., and Wogan, G. N. (1977) Structuralidentification of the major DNA adduct formed by aflatoxin B1inVitro. Proc. Natl. Acad. Sci. U.S.A. 74, 1870–1874.

(92) Baertschi, S. W., Raney, K. D., Stone, M. P., and Harris, T. M. (1988)Preparation of the 8,9-epoxide of the mycotoxin aflatoxin B1: Theultimate carcinogenic species. J. Am. Chem. Soc. 110, 7929–7931.

(93) Guengerich, F. P., and Johnson, W. W. (1999) Kinetics of hydrolysisand reaction of aflatoxin B1exo-8,9-epoxide and relevance to toxicityand detoxication. Drug Metab. ReV. 31, 141–158.

(94) Guengerich, F. P., Arneson, K. O., Williams, K. M., Deng, Z., andHarris, T. M. (2002) Reaction of aflatoxin B1 oxidation products withlysine. Chem. Res. Toxicol. 15, 780–792.

(95) Johnson, W. W., and Guengerich, F. P. (1997) Reaction of aflatoxinB1exo-8,9-epoxide with DNA: Kinetic analysis of covalent bindingand DNA-induced hydrolysis. Proc. Natl. Acad. Sci. U.S.A. 94, 6121–6125.

(96) Raney, K. D., Coles, B., Guengerich, F. P., and Harris, T. M. (1992)The endo 8,9-epoxide of aflatoxin B1: A new metabolite. Chem. Res.Toxicol. 5, 333–335.

(97) Ueng, Y.-F., Shimada, T., Yamazaki, H., and Guengerich, F. P. (1995)Oxidation of aflatoxin B1 by bacterial recombinant human cytochromeP450 enzymes. Chem. Res. Toxicol. 8, 218–225.

(98) Iyer, R., Coles, B., Raney, K. D., Thier, R., Guengerich, F. P., andHarris, T. M. (1994) DNA adduction by the potent carcinogenaflatoxin B1: Mechanistic studies. J. Am. Chem. Soc. 116, 1603–1609.

(99) Johnson, W. W., Harris, T. M., and Guengerich, F. P. (1996) Kineticsand mechanism of hydrolysis of aflatoxin B1exo-8,9-oxide andrearrangement of the dihydrodiol. J. Am. Chem. Soc. 118, 8213–8220.

(100) Johnson, W. W., Ueng, Y.-F., Mannervik, B., Widersten, M., Hayes,J. D., Sherratt, P. J., Ketterer, B., and Guengerich, F. P. (1997)Conjugation of highly reactive aflatoxin B1 8,9-exo-epoxide catalyzedby rat and human glutathione transferases: Estimation of kineticparameters. Biochemistry 36, 3056–3060.

(101) Johnson, W. W., Ueng, Y.-F., Yamazaki, H., Shimada, T., andGuengerich, F. P. (1997) Role of microsomal epoxide hydrolase inthe hydrolysis of aflatoxin B1 8,9-epoxide. Chem. Res. Toxicol. 10,672–676.

(102) Guengerich, F. P., Cai, H., McMahon, M., Hayes, J. D., Sutter, T. R.,Groopman, J. D., Deng, Z., and Harris, T. M. (2001) Reduction ofaflatoxin B1 dialdehyde by rat and human aldo-keto reductases. Chem.Res. Toxicol. 14, 727–737.

(103) Raney, K. D., Gopalakrishnan, S., Byrd, S., Stone, M. P., and Harris,T. M. (1990) Alteration of the aflatoxin cyclopentenone ring to aδ-lactone reduces interacalation with DNA and decreases formationof guanine N7 adducts by aflatoxin epoxides. Chem. Res. Toxicol.3, 254–261.

(104) Kensler, T. W., Groopman, J. D., Sutter, T. R., Curphey, T. J., andRoebuck, B. D. (1999) Development of cancer chemopreventativeagents: Oltipratz as a paradigm. Chem. Res. Toxicol. 12, 113–126.

(105) Nettleship, A., Henshaw, P., and Meyer, H. (1943) Induction ofpulmonary tumors in mice with ethyl carbamate (urethane). J. Natl.Cancer Inst. 4, 309–319.

(106) Dahl, G. A., Miller, J. A., and Miller, E. C. (1978) Vinyl carbamateas a promutagen and a more carcinogenic analog of ethyl carbamate.Cancer Res. 38, 3793–3804.

(107) Guengerich, F. P., and Kim, D.-H. (1991) Enzymatic oxidation ofethyl carbamate to vinyl carbamate and its role as an intermediate inthe formation of 1,N (6)-ethenoadenosine. Chem. Res. Toxicol. 4,413–421.

(108) Dahl, G. A., Miller, E. C., and Miller, J. A. (1980) Comparativecarcinogenicities and mutagenicities of vinyl carbamate, ethyl car-bamate, and ethyl N-hydroxycarbamate. Cancer Res. 40, 1194–1203.

(109) Park, K. K., Liem, A., Stewart, B. C., and Miller, J. A. (1993) Vinylcarbamate epoxide, a major strong electrophilic, mutagenic andcarcinogenic metabolite of vinyl carbamate and ethyl carbamate(urethane). Carcinogenesis 14, 441–450.

(110) Levitt, R. C., Legraverend, C., Nebert, D. W., and Pelkonen, O. (1977)Effects of harman and norharman on the mutagenicity and bindingto DNA of benzo[a]pyrene metabolites in Vitro and on arylhydrocarbon hydroxylase induction in cell culture. Biochem. Biophys.Res. Commun. 79, 1167–1175.

(111) Atkins, W. M. (2006) Current views on the fundamental mechanismsof cytochrome P450 allosterism. Exp. Opin. Drug Metab. Toxicol.2, 573–579.

(112) Totsuka, Y., Hada, N., Matsumoto, K., Kawahara, N., Murakami,Y., Yokoyama, Y., Sugimura, T., and Wakabayashi, K. (1998)Structural determination of a mutagenic aminophenylnorharmanproduced by the co-mutagen norharman with aniline. Carcinogenesis19, 1995–2000.

(113) Oda, Y., Totsuka, Y., Wakabayashi, K., Guengerich, F. P., andShimada, T. (2006) Activation of aminophenylnorharman, aminom-ethylnorharman, and amnophenylharman to genotoxic metabolitesby human N,O-Acetyltransferases and cytochrome P450 enzymesexpressed in Salmonella typhimurium umu tester strains. Mutat. Res.21, 410–416.

(114) Nishigaki, R., Totsuka, Y., Takamura-Enya, T., Sugimura, T., andWakabayashi, K. (2004) Identification of cytochrome P-450s involvedin the formation of APNH from norharman with aniline. Mutat. Res.562, 19–25.

(115) Gitlin, N., Julie, N. L., Spurr, C. L., Lim, K. N., and Juarbe, H. M.(1998) Two cases of severe clinical and histologic hepatotoxicityassociated with troglitazone. Ann. Intern. Med. 129, 36–38.

(116) Isley, W. L., and Oki, J. C. (2001) Hepatotoxicity of thiazolidinedi-ones. Diabetes, Obes. Metab. 3, 389–392.

(117) Yamazaki, H., Shibata, A., Suzuki, M., Nakajima, M., Shimada, N.,Guengerich, F. P., and Yokoi, T. (1999) Oxidation of troglitazoneto a quinone-type metabolite catalyzed by cytochrome P450 2C8 and3A4 in human liver microsomes. Drug Metab. Dispos. 27, 1260–1266.

(118) Kassahun, K., Pearson, P. G., Tang, W., McIntosh, I., Leung, K.,Elmore, C., Dean, D., Wang, R., Doss, G., and Baillie, T. A. (2001)Studies on the metabolism of troglitazone to reactive intermediatesin Vitro and in ViVo. Evidence for novel biotransformation pathwaysinvolving quinone methide formation and thiazolidinedione ringscission. Chem. Res. Toxicol. 14, 62–70.

(119) Alvarez-Sanchez, R., Montavon, F., Hartung, T., and Pahler, A.(2006) Thiazolidinedione bioactivation: A comparison of the bioac-tivation potentials of troglitazone, rosiglitazone, and pioglitazoneusing stable isotope-labeled analogues and liquid chromatographytandem mass spectrometry. Chem. Res. Toxicol. 19, 1106–1116.

(120) Masubuchi, Y. (2006) Metabolic and non-metabolic factors determin-ing troglitazone hepatotoxicity: A review. Drug Metab. Pharma-cokinet. 21, 347–356.

(121) Kalgutkar, A. S., and Soglia, J. R. (2005) Minimising the potentialfor metabolic activation in drug discovery. Exp. Opin. Drug Metab.Toxicol. 1, 91–141.

(122) Isin, E. M., and Guengerich, F. P. (2007) Complex reactions catalyzedby cytchrome P450 enzymes. Biochim. Biophys. Acta 1770, 314–329.

(123) De, Matteis, F. (1971) Loss of haem in rat liver caused by theporphyrogenic agent 2-allyl-2-isopropylacetamide. Biochem. J. 124,767–777.

(124) Correia, M. A., and Ortiz de Montellano, P. R. (2005) Inhibitionand degradation of cytochrome P450 enzymes, In Cytochrome P450:Structure, Mechanism, and Biochemistry, 3rd ed. (Ortiz de Montel-lano, P. R., Ed.) pp 247–322, Kluwer Academic/Plenum Press, NewYork.

(125) Werringloer, J., and Estabrook, R. W. (1973) Evidence for aninhibitory product-cytochrome P-450 complex generated duringbenzphetamine metabolism by liver microsomes. Life Sci. 13, 1319–1330.

PerspectiVe Chem. Res. Toxicol., Vol. 21, No. 1, 2008 81

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| http

://pu

bs.a

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rg

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licat

ion

Dat

e (W

eb):

Dec

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r 6,

200

7 | d

oi: 1

0.10

21/tx

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79z

(126) Paulsen-Sörman, U. B., Jönsson, K. H., and Lindeke, B. G. A. (1984)Cytochrome P-455 nm complex formation in the metabolism ofphenylalkylamines. 8. Stereoselectivity in metabolic intermediarycomplex formation with a series of chiral 2-substituted 1-phenyl-2-aminoethanes. J. Med. Chem. 27, 342–346.

(127) Palamanda, J. R., Casciano, C. N., Norton, L. A., Clement, R. P.,Favreau, L. V., Lin, C. C., and Nomeir, A. A. (2001) Mechanism-based inactivation of CYP2D6 by 5-fluoro-2-[4-[(2-phenyl-1H-imidazol-5-yl)methyl]-1-piperazinyl]pyrimidine. Drug Metab. Dispos.29, 863–867.

(128) Meschter, C. L., Mico, B. A., Mortillo, M., Feldman, D., Garland,W. A., Riley, J. A., and Kaufman, L. S. (1994) A 13-week toxicologicand pathologic evaluation of prolonged cytochromes P450 inhibitionby 1-aminobenzotriazole in male rats. Fundam. Appl. Toxicol. 22,369–381.

(129) Tephly, T. R., Gibbs, A. H., and De Matteis, F. (1979) Studies onthe mechanism of experimental porphyria produced by 3,5-diethoxy-carbonyl-1,4-dihydrocollidine: Role of a porphyrin-like inhibitor ofprotohaem ferro-lyase. Biochem. J. 180, 241–244.

(130) Bailey, D. G., Edgar, B., Spence, J. D., Munzo, C., and Arnold,J. M. O. (1990) Felodipine and nifedipine interactions with grapefruitjuice. Clin. Pharmacol. Ther. 47, 180.

(131) Ernest, C. S., 2nd, Hall, S. D., and Jones, D. R. (2005) Mechanism-based inactivation of CYP3A by HIV protease inhibitors. J. Phar-macol. Exp. Ther. 312, 583–591.

(132) Bailey, D. G., Dresser, G. K., and Bend, J. R. (2003) Bergamottin,lime juice, and red wine as inhibitors of cytochrome P450 3A4activity: Comparison with grapefruit juice. Clin. Pharmacol. Ther.73, 529–537.

(133) Silverman, R. B. (1988) Mechanism-Based Enzyme InactiVation:Chemistry& Enzymology, CRC Press, Boca Raton, FL.

(134) Miller, W. R., Anderson, T. J., Evans, D. B., Krause, A., Hampton,G., and Dixon, J. M. (2003) An integrated view of aromatase and itsinhibition. J. Steroid Biochem. Mol. Biol. 86, 413–421.

(135) Harrelson, W. G., Jr., and Mason, R. P. (1982) Microsomal reductionof gentian violet. Evidence for cytochrome P-450-catalyzed freeradical formation. Mol. Pharmacol. 22, 239–242.

(136) Yager, J. D., and Liehr, J. G. (1996) Molecular mechanisms ofestrogen carcinogenesis. Annu. ReV. Pharmacol. Toxicol. 36, 203–232.

(137) Sato, H., and Guengerich, F. P. (2000) Oxidation of 1,2,4,5-tetramethoxybenzene to a cation radical by cytochrome P450. J. Am.Chem. Soc. 122, 8099–8100.

(138) Gillette, J. R., Brodie, B. B., and La Du, B. N. (1957) The oxidationof drugs by liver microsomes: on the role of TPNH and oxygen.J. Pharmacol. Exp. Ther. 119, 532–540.

(139) Nordblom, G. D., and Coon, M. J. (1977) Hydrogen peroxideformation and stoichiometry of hydroxylation reactions catalyzed byhighly purified liver microsomal cytochrome P-450. Arch. Biochem.Biophys. 180, 343–347.

(140) Guengerich, F. P. (1978) Destruction of heme and hemoproteinsmediated by liver microsomal reduced nicotinamide adenine dinucle-otide phosphate-cytochrome P-450 reductase. Biochemistry 17, 3633–3639.

(141) Caro, A. A., and Cederbaum, A. I. (2004) Oxidative stress, toxicology,and pharmacology of CYP2E1. Annu. ReV. Pharmacol. Toxicol 44,27–42.

(142) Zangar, R. C., Davydov, D. R., and Verma, S. (2004) Mechanismsthat regulate production of reactive oxygen species by cytochromeP450. Toxicol. Appl. Pharmacol. 199, 316–331.

(143) Robertson, G., Leclercq, I., and Farrell, G. C. (2001) Nonalcoholicsteatosis and steatohepatitis. II. Cytochrome P-450 enzymes andoxidative stress. Am. J. Physiol. Gastrointest. LiVer Physiol. 281,G1135–1139.

(144) Liu, H., Bigler, S. A., Henegar, J. R., and Baliga, R. (2002)Cytochrome P450 2B1 mediates oxidant injury in puromycin-inducednephrotic syndrome. Kidney Int. 62, 868–876.

(145) Kadiiska, M. B., Gladen, B. C., Baird, D. D., Graham, L. B., Parker,C. E., Ames, B. N., Basu, S., Fitzgerald, G. A., Lawson, J. A.,Marnett, L. J., Morrow, J. D., Murray, D. M., Plastaras, J., Roberts,L. J., 2nd, Rokach, J., Shigenaga, M. K., Sun, J., Walter, P. B., Tomer,K. B., Barrett, J. C., and Mason, R. P. (2005) Biomarkers of oxidativestress study III. Effects of the nonsteroidal anti-inflammatory agentsindomethacin and meclofenamic acid on measurements of oxidativeproducts of lipids in CCl4 poisoning. Free Radical Biol. Med. 38,711–718.

(146) Dostalek, M., Brooks, J., Milne, G. L., Moore, M. M., Sharma, A.,Morrow, J. D., and Guengerich, F. P. (2007) In ViVo oxidative damageassociated with cytochrome P450 induction in rats, Abstracts, 15thInt. Conf. Cytochrome P450, June 17–21, Bled, Slovenia.

(147) Mangelsdorf, D. J., and Evans, R. M. (1995) The RXR heterodimersand orphan receptors. Cell 83, 841–850.

(148) Stark, K., and Guengerich, F. P. (2007) The orphan human cyto-chrome P450: What is left to learn? Drug Metab ReV., in press.

(149) Karlgren, M., Gomez, A., Stark, K., Svard, J., Rodriguez-Antona,C., Oliw, E., Bernal, M. L., Ramon y Cajal, S., Johansson, I., andIngelman-Sundberg, M. (2006) Tumor-specific expression of thenovel cytochrome P450 enzyme, CYP2W1. Biochem. Biophys. Res.Commun. 341, 451–458.

(150) Wu, Z.-L., Sohl, C. D., Shimada, T., and Guengerich, F. P. (2006)Recombinant enzymes over-expressed in bacteria show broadcatalytic specificity of human cytochrome P450 2W1 and limitedactivity of human cytochrome P450 2S1. Mol. Pharmacol. 69, 2007–2014.

(151) Wu, Z.-L., Bartleson, C. J., Ham, A.-J. L., and Guengerich, F. P.(2006) Heterologous expression, purification, and properties of humancytochrome P450 27C1. Arch. Biochem. Biophys. 445, 138–146.

(152) Cheng, J. B., Motola, D. L., Mangelsdorf, D. J., and Russell, D. W.(2003) De-orphanization of cytochrome P450 2R1: A microsomalvitamin D 25-hydroxylase. J. Biol. Chem. 278, 38084–38093.

(153) Sanchez-Ponce, R., and Guengerich, F. P. (2007) Untargeted analysisof mass spectrometry data for elucidation of function of new enzymes.Anal. Chem. 79, 3355–3362.

(154) Shou, M., Grogan, J., Mancewicz, J. A., Krausz, K. W., Gonzalez,F. J., Gelboin, H. V., and Korzekwa, K. R. (1994) Activation ofCYP3A4: Evidence for the simultaneous binding of two substratesin a cytochrome P450 active site. Biochemistry 33, 6450–6455.

(155) Ueng, Y.-F., Kuwabara, T., Chun, Y.-J., and Guengerich, F. P. (1997)Cooperativity in oxidations catalyzed by cytochrome P450 3A4.Biochemistry 36, 370–381.

(156) Harlow, G. R., and Halpert, J. R. (1998) Analysis of humancytochrome P450 3A4 cooperativity: Construction and characteriza-tion of a site-directed mutant that displays hyperbolic steroidhydroxylation kinetics. Proc. Natl. Acad. Sci. U.S.A. 95, 6636–6641.

(157) Lasker, J. M., Huang, M.-T., and Conney, A. H. (1982) In ViVoactivation of zoxazolamine metabolism by flavone. Science 216,1419–1421.

(158) Tang, W., and Stearns, R. A. (2001) Heterotropic cooperativity ofcytochrome P450 3A4 and potential drug-drug interactions. Curr.Drug Metab 2, 185–198.

(159) Atkins, W. M. (2005) Non-Michaelis-Menten kinetics in cytochromeP450-catalyzed reactions. Annu. ReV. Pharmacol. Toxicol. 45, 291–310.

(160) Isin, E. M., and Guengerich, F. P. (2007) Multiple sequential stepsinvolved in the binding of inhibitors to cytochrome P450 3A4. J. Biol.Chem. 282, 6863–6874.

(161) He, Y.-A., Gajiwala, K. S., Wu, M., Parge, H., Burke, B., Lee, C. A.,and Wester, M. R. (2006) The crystal structure of human CYP3A4in complex with testosterone, Abstracts, 16th International Sympo-sium on Microsomes and Drug Oxidations (MDO 2006), August 10–14, Budapest, Hungary.

(162) Dabrowski, M. J., Schrag, M. L., Wienkers, L. C., and Atkins, W. M.(2002) Pyrene-pyrene complexes at the active site of cytochromeP450 3A4: evidence for a multiple substrate binding site. J. Am.Chem. Soc. 124, 11866–11867.

(163) Krauser, J. A., and Guengerich, F. P. (2005) Cytochrome P450 3A4-catalyzed testosterone 6�-hydroxylation: Stereochemistry, kineticdeuterium isotope effects, and rate-limiting steps. J. Biol. Chem. 280,19496–19506.

(164) Hosea, N. A., Miller, G. P., and Guengerich, F. P. (2000) Elucidationof distinct binding sites for cytochrome P450 3A4. Biochemistry 39,5929–5939.

(165) Conney, A. H. (1982) Induction of microsomal enzymes by foreignchemicals and carcinogenesis by polycyclic aromatic hydrocarbons:G. H. A. Clowes Memorial Lecture. Cancer Res. 42, 4875–4917.

(166) Raney, K. D., Shimada, T., Kim, D.-H., Groopman, J. D., Harris,T. M., and Guengerich, F. P. (1992) Oxidation of aflatoxins andsterigmatocystin by human liver microsomes: Significance of aflatoxinQ1 as a detoxication product of aflatoxin B1. Chem. Res. Toxicol. 5,202–210.

(167) Bourdi, M., Larrey, D., Nataf, J., Berunau, J., Pessayre, D., Iwasaki,M., Guengerich, F. P., and Beaune, P. H. (1990) A new anti-liverendoplasmic reticulum antibody directed against human cytochromeP-450 IA2: A specific marker of dihydralazine-induced hepatitis.J. Clin. InVest. 85, 1967–1973.

(168) Beaune, P., Dansette, P. M., Mansuy, D., Kiffel, L., Finck, M., Amar,C., Leroux, J. P., and Homberg, J. C. (1987) Human anti-endoplasmicreticulum autoantibodies appearing in a drug-induced hepatitis aredirected against a human liver cytochrome P-450 that hydroxylatesthe drug. Proc. Natl. Acad. Sci. U.S.A. 84, 551–555.

(169) Loeper, J., Descatoire, V., Maurice, M., Beaune, P., Belghiti, J.,Houssin, D., Ballet, F., Feldman, G., Guengerich, F. P., and Pessayre,D. (1993) Cytochromes P-450 in human hepatocyte plasma mem-brane: Recognition by several autoantibodies. Gastroenterology 104,203–216.

82 Chem. Res. Toxicol., Vol. 21, No. 1, 2008 Guengerich

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200

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(170) Beaune, P., Pessayre, D., Dansette, P., Mansuy, D., and Manns, M.(1994) Autoantibodies against cytochromes P450: Role in humandiseases. AdV. Pharmacol. 30, 199–245.

(171) Dansette, P. M., Bonierbale, E., Minoletti, C., Beaune, P. H., Pessayre,D., and Mansuy, D. (1998) Drug-induced immunotoxicity. Eur. J.Drug Metab. Pharmacokinet. 23, 443–451.

(172) Shenton, J. M., Chen, J., and Uetrecht, J. P. (2004) Animal modelsof idiosyncratic drug reactions. Chem.-Biol. Interact. 150, 53–70.

(173) Cheung, C., Yu, A. M., Ward, J. M., Krausz, K. W., Akiyama, T. E.,Feigenbaum, L., and Gonzalez, F. J. (2005) The cyp2e1-humanizedtransgenic mouse: Role of cyp2e1 in acetaminophen hepatotoxicity.Drug Metab. Dispos. 33, 449–457.

(174) Zaher, H., Buters, J. T., Ward, J. M., Bruno, M. K., Stern, S. T.,Cohen, S. D., and Gonzalez, F. J. (1998) Protection againstacetaminophen toxicity in cyp1a2 and cyp2e1 double-null mice.Toxicol. Appl. Pharmacol. 152, 193–199.

(175) Jaeschke, H., and Bajt, M. L. (2006) Intracellular signaling mecha-nisms of acetaminophen-induced liver cell death. Toxicol. Sci. 89,31–41.

(176) d’Errico, A., Taioli, E., Chen, X., and Vineis, P. (1996) Geneticmetabolic polymorphisms and the risk of cancer: A review of theliterature. BioMarkers 1, 149–173.

(177) Christensen, P. M., Gøtzsche, P. C., and Brøsen, K. (1997) Thesparteine/debrisoquine (CYP2D6) oxidation polymorphism and therisk of lung cancer: A meta-analysis. Eur. J. Clin. Pharmacol. 51,389–393.

(178) Ye, Z., and Parry, J. M. (2002) The CYP17 MspA1 polymorphismand breast cancer risk: A meta-analysis. Mutagenesis 17, 119–126.

(179) Masson, L. F., Sharp, L., Cotton, S. C., and Little, J. (2005)Cytochrome P-450 1A1 gene polymorphisms and risk of breastcancer: A HuGE review. Am. J. Epidemiol. 161, 901–915.

(180) Ayesh, R., Idle, J. R., Ritchie, J. C., Crothers, M. J., and Hetzel,M. R. (1984) Metabolic oxidation phenotypes as markers forsusceptibility to lung cancer. Nature 312, 169–170.

(181) Wolff, T., Distlerath, L. M., Worthington, M. T., Groopman, J. D.,Hammons, G. J., Kadlubar, F. F., Prough, R. A., Martin, M. V., andGuengerich, F. P. (1985) Substrate specificity of human livercytochrome P-450 debrisoquine 4-hydroxylase probed using immu-nochemical inhibition and chemical modeling. Cancer Res. 45, 2116–2122.

(182) Crespi, C. L., Penman, B. W., Gelboin, H. V., and Gonzalez, F. J.(1991) A tobacco smoke-derived nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, is activated by multiple human cytochromeP450s including the polymorphic human cytochrome P4502D6.Carcinogenesis 12, 1197–1201.

(183) Shimada, T., and Guengerich, F. P. (1991) Activation of amino-R-carboline, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, and acopper phthalocyanine cellulose extract of cigarette smoke condensate

by cytochrome P-450 enzymes in rat and human liver microsomes.Cancer Res. 51, 5284–5291.

(184) Roots, I., Drakoulis, N., and Brockmöller, J. (1993) Still an openquestion: Does active CYP2D6 predispose to lung cancer? InProceedings, 8th International Conference on Cytochrome P450:Biochemistry, Biophysics, and Molecular Biology (Lechner, M. C.,Ed.) p 159, John Libbey Eurotext, Chichester, United Kingdom.

(185) Rostami-Hodjegan, A., Lennard, M. S., Woods, H. F., and Tucker,G. T. (1998) Meta-analysis of studies of the CYP2D6 polymorphismin relation to lung cancer and Parkinson’s disease. Pharmacogenetics8, 227–238.

(186) Kellerman, G., Luyten-Kellerman, M., and Shaw, C. R. (1973)Genetic variation of aryl hydrocarbon hydroxylase in human lym-phocytes. Am. J. Hum. Genet. 25, 327–331.

(187) Kellerman, G., Shaw, C. R., and Luyten-Kellerman, M. (1973) Arylhydrocarbon hydroxylase inducibility and bronchogenic carcinoma.New Engl. J. Med. 298, 934–937.

(188) Kouri, R. E., McKinney, C. E., Slomiany, D. J., Snodgrass, D. R.,Wray, N. P., and McLemore, T. L. (1982) Positive correlationbetween high aryl hydrocarbon hydroxylase activity and primary lungcancer as analyzed in cryopreserved lymphocytes. Cancer Res. 42,5030–5037.

(189) Zhang, Z.-Y., Fasco, M. J., Huang, L., Guengerich, F. P., andKaminsky, L. S. (1996) Characterization of purified recombinanthuman CYP 1A1-Ile462 and Val462: Assessment of a role for the rareallele in carcinogenesis. Cancer Res. 56, 3926–3933.

(190) Persson, I., Johansson, I., and Ingelman-Sundberg, M. (1997) In Vitrokinetics of two human CYP1A1 variant enzymes suggested to beassociated with interindividual differences in cancer susceptibility.Biochem. Biophys. Res. Commun. 231, 227–230.

(191) Kawajiri, K., Watanabe, J., Eguchi, H., Nakachi, K., Kiyohara, C.,and Hayashi, S. (1995) Polymorphisms of human Ah receptor geneare not involved in lung cancer. Pharmacogenetics 5, 151–158.

(192) Shimada, T., Hayes, C. L., Yamazaki, H., Amin, S., Hecht, S. S.,Guengerich, F. P., and Sutter, T. R. (1996) Activation of chemicallydiverse procarcinogens by human cytochrome P450 1B1. Cancer Res.56, 2979–2984.

(193) Shimada, T., Gillam, E. M. J., Oda, Y., Tsumura, F., Sutter, T. R.,Guengerich, F. P., and Inoue, K. (1999) Metabolism of benzo[a]py-rene to trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene by recom-binant human cytochrome P450 1B1 and purified liver epoxidehydrolase. Chem. Res. Toxicol. 12, 623–629.

(194) Toide, K., Yamazaki, H., Nagashima, R., Itoh, K., Iwano, S.,Takahashi, Y., Watanabe, S., and Kamataki, T. (2003) Aryl hydro-carbon hydroxylase represents CYP1B1, and not CYP1A1, in humanfreshly isolated white cells: Trimodal distribution of Japanesepopulation according to induction of CYP1B1 mRNA by environ-mental dioxins. Cancer Epidemiol. Biomarkers PreV. 12, 219–222.

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