Cytochrome P450 and Chemical Toxicology - Chemical Research in

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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, and many important papers on the roles of P450s in chemical toxicology have appeared in Chemical Research in 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 structures of several of the major human P450s are now in hand. The animal P450s are still important in the context of metabolism and safety testing. Many well-defined examples exist for roles of P450s in decreasing the adverse effects of drugs through biotransformation, and an equally interesting field of investigation is the bioactivation of chemicals, including drugs. Unresolved problems include the characterization of the minor “orphan” P450s, ligand cooperativity and kinetic complexity of several P450s, the prediction of metabolism, the overall contribution of bioactivation to drug idiosyncratic problems, the extrapolation of animal test results to humans in drug development, and the contribution of genetic variation in human P450s to cancer incidence. Contents 1. Introduction and Background 1.1. Current Knowledge about P450s 2. Roles of P450s in Reducing Toxicity 3. Bioactivation by P450s 3.1. Aflatoxin B1 3.2. Ethyl Carbamate 3.3. Coupling of Norharman and Aniline 3.4. Troglitazone 3.5. Other Bioactivation Reactions 3.6. Mechanism-Based Activation 3.7. P450s and Oxidative Damage 4. Current and Future Issues 4.1. Functions of “Orphan” P450s 4.1.1. Analysis of Suspects 4.1.2. Transgenic Animal Models 4.1.3. Library Screening 4.1.4. Untargeted Metabolomic Strategies in Vitro 4.1.5. Untargeted in Vitro Strategies with Isotope Editing 4.2. Ligand Cooperativity 4.3. Predictions of Metabolism 4.4. Overlaps of Detoxication and Bioactivation 4.5. Roles of P450s in Idiosyncratic Drug Toxicity 4.6. Predicting Human Toxicity 4.7. Understanding P450 Gene Polymorphisms and Disease 5. Conclusion

1. Introduction and Background 70 70 72 73 73 74 74 74 75 75 76 77 77 77 77 77 77 77 77 77 78 78 78 78 79

* To whom correspondence should be addressed. Tel: 615-322-2261. Fax: 615-322-3141. E-mail: [email protected].

Cytochrome P450 (P450) research can be traced back to in vitro studies on the metabolism of steroids, drugs, and carcinogens in the 1940s (1). Some of the major developments were the spectral observation of P450 (2), photochemical action studies implicating P450 as the oxidase in the electron transport system (3), the separation (4) and subsequent purification of P450 (5, 6), and several studies implicating multiple P450 enzymes (7, 8). Other early seminal studies include the extensive biochemical and biophysical work with the bacterial P450 101A1 (P450cam) (9) and the first complete nucleotide sequence of a P450 (10). Studies on the chemistry of oxygen activation developed, and one of the key studies underpinning our current models was evidence for a stepwise process involving C–H bond breaking (11). During the past 20 years, we have seen a major shift of emphasis to human P450s, which had seemed almost impossible in the early research. The knowledge about the human P450s has had important ramifications in understanding the metabolism of drugs. In comparison to the situation ∼25 years ago, far fewer drugs fail in development due to pharmacokinetic problems in humans, because of the reiterative approach of chemical synthesis, target screening, and in vitro metabolism studies in place in pharmaceutical companies (Figure 1). However, less progress has been made in accurately predicting human toxicity problems with drugs and the challenge remains considerable (13, 14). In retrospect, one of the driving forces for the study of P450s has been the quest for information to better understand and predict the metabolism and toxicity of drugs and other chemicals [e.g., thalidomide (15–17)]. 1.1. Current Knowledge about P450s. This section will focus on important developments that have occurred over the past 20 years, that is, since this journal began. One is certainly the completion of the human genome project, which set the number of human P450 (“CYP”) genes at 57 (Table 1) (and the number of pseudogenes at 58) (http://drnelson.utmem.edu/

10.1021/tx700079z CCC: $40.75  2008 American Chemical Society Published on Web 12/06/2007

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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 Major Substrate Class (18, 19) sterols

xenobiotics

fatty acids

eicosanoids

vitamins

1B1 7A1 7B1 8B1 11A1 11B1 11B2 17A1 19A1 21A2 27A1 39A1 46A1 51A1

1A1 1A2 2A6 2A13 2B6 2C8 2C9 2C18 2C19 2D6 2E1 2F1 3A4 3A5 3A7

2J2 4A11 4B1 4F12

4F2 4F3 4F8 5A1 8A1

2R1 24A1 26A1 26B1 26C1 27B1

unknown 2A7 2S1 2U1 2W1 3A43 4A22 4F11 4F22 4V2 4×1 4Z1 20A1 27C1

Figure 2. Basic P450 catalytic cycle (37).

CytochromeP450.html), thus putting speculation about this number to rest. However, some uncertainties exist about the expression of some of the genes at the mRNA and particularly the protein levels (e.g., P450 4A22). Twenty years ago, the biochemical purification of several of the major human liver P450s was achieved (20–22). The development of recombinant DNA technology was well underway, and heterologous expression was done in low-yield systems. Some breakthroughs in the early 1990s led to successful high-level bacterial expression (23–25), which was critical for crystallography work. In the past few years, the number of crystal structrures of human P450s has increased rapidly, and today, high-resolution structures are available for human P450s 1A2 (26), 2A6 (27), 2C8 (28), 2C9 (29, 30), 2D6 (31), and 3A4 (32–34), the major P450s involved in drug metabolism. These structures have replaced less accurate homology models based mainly on bacterial P450s (P450s 101A1 and 102A1) and also serve as reasonable templates for other closely related subfamily P450 members. One general observation with these and other animal and bacterial P450 structures is that most undergo major conformational changes upon ligand binding (35), and ligand-free structures are of limited use in understanding the functions of these proteins. However, with some of the P450s having large active sites, the positions of ligands in the crystal structures still leave many questions open about the interactions (32, 34, 36).

The generally accepted catalytic cycle for P450 reactions is shown in Figure 2. However, the point should be made that this is a simplified version and that the system is dynamic, and the steps do not necessarily proceed in a linear order around the cycle. For instance, substrate can be bound and released at other steps along the cycle (38, 39). Also, most of the oxygenated intermediates (or all?) have the potential to dismute, generating reactive oxygen species (vide infra), and the coupling efficiencies of most P450 systems in vitro are low. The concepts developed by Groves regarding a formal perferryl oxygen intermediate and a stepwise oxygenation mechanism (Figure 3) have been proved useful in rationalizing most oxidation reactions (37, 41), with provision for one-electron oxidation (37, 40, 41). In the past decade, several alternate mechanisms have been proposed, and these remain controversial. One oftendiscussed mechanism is that proposed by Newcomb and involving FeO2+ or FeO2H2+ instead of FeO3+ as the oxidant (42, 43). This mechanism had initial impetus from a chemical proposal for the third step in the P450 19A1 aromatase reaction (44, 45), although an alternate FeO3+ reaction has recently been suggested to be more tenable, based on density functional theory calculations (46). The proposals for concerted mechanisms have been based largely on results obtained with lack of rearrangement of radical clocks (47), and the interpretation is controversial (37, 48, 49). Another proposal is the “two-spin state” system of Shaik and co-workers, with the FeO3+ complex providing

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Figure 4. Contributions of enzymes to the metabolism of marketed drugs. The results are from a study of Pfizer drugs (57), and similar percentages have been reported by others in other pharmaceutical companies (58). (A) Fraction of reactions on drugs catalyzed by various human enzymes. FMO, flavin-containing monoxygenase; NAT, Nacetyltransferase; and MAO, monoamine oxidase. (B) Fractions of P450 oxidations on drugs catalyzed by individual P450 enzymes. The segment labeled 3A4 (+3A5) is mainly due to P450 3A4, with some controversy about exactly how much is contributed by other subfamily 3A P450s. Reprinted with permission from ref 57. Copyright 2004 American Society for Pharmacology and Experimental Therapeutics.

Figure 3. General mechanism for P450 oxidation reactions involving a perferryl oxygen intermediate and odd-electron chemistry (see Figure 2) (40).

both high- and low-spin populations for discrete reaction pathways and multiple products (50, 51). This mechanism has intellectual attraction in explaining many of the intricacies of P450 reactions, although the evidence is all theoretical. Rate-limiting steps in P450 reactions (Figure 2) probably vary considerably, depending upon the reaction and the in vitro experimental setting. Reduction (first electron) (52–54), C–H bond breaking (38), and a step following product formation (conformational change?) (55) have been identified in some cases, and the rate of transfer of the second electron has been proposed 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), which is fortuitous in simplifying the task of assigning new reactions to individual P450s (57). One issue in drug development is bioavailability, and a common initial study is usually “microsomal stability” to predict if most of a drug will be eliminated too rapidly in a “first-pass” effect (59). Another issue is side effects due to the inherent pharmacology of the parent drug. Drug doses are adjusted so that 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 by another drug, toxicity may develop, particularly if drug accumulation occurs upon multiple doses. Drug–drug interactions are recognized to be a major cause of adverse drug reactions. These phenomena can often be understood and in many cases predicted in the context of individual human P450 enzymes. One well-documented example is terfenadine, the first marketed nonsedating antihistamine (60) (Figure 5). Normally, terfenadine is 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). In individuals who used drugs that inhibit P450 3A4 (e.g., ketoconazole and erythromycin), terfenadine accumulated in the plasma and cardiac tissue. Dietary constituents (e.g., grapefruit) can also inhibit P450s, although not to the extent to present serious danger with terfenadine (61). Terfenadine itself is an antagonist of the human ether-a-go-go (hERG) receptor and causes torsade de pointes (and arrhythmia), invoked in a number of deaths (62). Following the deaths, the U.S. Food and Drug Administration (FDA)1 first introduced a contraindication labeling for use of azoles and erythromycin with terfenadine and subsequently withdrew terfenadine from the market. Fexofenadine (Allegra) does not have this liability and has replaced terfenadine on the market, along with other antihistamines such as loratadine. Another example of toxicity of a parent drug is the anticoagulant warfarin, which has a relatively narrow therapeutic window (i.e., little variation in dose between being effective and being toxic in different individuals). Too low a level of warfarin can yield clotting, and too high a level can give rise to hemorrhaging. The “effective dose” can be adjusted in individuals, and this dose has been shown to be influenced by polymorphisms that affect catalytic activity in the P450 2C9 coding region (63). Thus, the *2 (Arg144 Cys change) and *3 (Ile359 Leu change) alleles both lower the dose needed for maintenance dosing (and conversely raise the risk of an individual hemorrhaging at a fixed dose). The risk of hemorrhaging is particularly high if the patient is ill or changing medications. However, the P450 2C9 polymorphisms have been estimated to account collectively for only ∼10% of the total variation in warfarin doses among patients (64). Similar cases involve some environmental toxicants and carcinogens, although generally the parent compounds generate little if any pharmacological activity of their own. However, one issue is metabolism (to innocuous products) that will prevent distribution to tissues in which bioactivation may occur, thus preventing toxicity. For instance, metabolism in the liver can prevent distribution of polycyclic hydrocarbons to the lung and other target tissues (65). Although P450 1A1 is often considered dangerous because it activates polycyclic hydrocarbons, deletion 1

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

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Figure 5. Role of P450 3A4 in terfenadine toxicity. Terfenadine is rapidly converted to two products by P450 3A4, one involving hydroxylation of 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) to the 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 by drugs such as erythromycin or ketoconazole, terfenadine begins to accumulate in the plasma and tissues. The high affinity of terfenadine for the hERG receptor can lead to arrhythmias and deaths.

of the gene rendered mice more sensitive to benzo[a]pyrene toxicity (66). The point was made (67) that P450 1 family induction is often erroneously viewed by pharmaceutical companies as a liability in drug development; a more accurate statement is that this potential issue has been considered a problem by regulatory agencies, for example, the FDA (68, 69), and safety assessment departments have necessarily had to be defensive in some cases. The successful drug omeprazole provides an outstanding example of a compound that does induce P450 1 family enzymes (at least in individuals deficient in P450 2C19, which oxidizes the drug) but has not proven to be a problem. For further discussion of the issue, see a recent review by Ma and Lu (70).

3. Bioactivation by P450s The concept of metabolic conversion of chemicals to reactive products that covalently bind macromolecules can be attributed to the late James and Elizabeth Miller (71), and P450s are major players in this paradigm. The list of chemicals known to be activated is considerable, and the reader is referred to lists of drugs (72, 73), toxicants, and carcinogens (74–76). As with drugs, a subset of the human P450s appear to be responsible for most of the cases, although the list changes from drug metabolism (Figure 4B). P450s 2C8, 2C9, and 2D6 contribute little 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 role of P450 2E1 in acetaminophen toxicity is well-established (77). The concept of bioactivation reactions is not new to P450 science (72, 78). Twenty-five years ago, the knowledge of which individual P450s catalyzed the activation of particular compounds was very meager, and the knowledge of the human P450s was almost nil (79). By 1991, a fairly extensive compilation of the human P450s involved in activation of carcinogens and protoxicants was available (80). Several examples of bioactivation by P450s from the past 20 years will be presented as examples of the range of the P450s and, despite the apparent diversity, the point that common chemical mechanisms can be invoked.

Most of our understanding of bioactivation reactions is in the context of the generation of electrophilic products that become covalently bound to proteins and DNA. With drugs, there are examples in which a drug metabolite may have more intrinsic 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 can cause mutations, as can be demonstrated experimentally in various ways (83, 84). In the somatic mutation theory (85), these would go on to produce cancer. This field has developed in terms of both basic and human studies (76, 86, 87). The formation of protein adducts with electrophiles has a long history, even preceding DNA work (71). There is considerable correlative evidence linking protein modification with drug toxicity in experimental systems, going back to the classic work of Gillette and Brodie (88). Today, protein adduct formation is even used in some screening paradigms in pharmaceutical development (89). Unfortunately, it is not possible to introduce a defined protein adduct into a biological system and produce a direct toxic effect, in the way that DNA studies can be done. The reader is referred elsewhere to recent reviews on the significance of protein adducts (14, 89, 90). 3.1. Aflatoxin B1. Although a mechanism involving the 8,9epoxide had been proposed for some time (91), the evidence was indirect. The chemical synthesis of the epoxide (92) was a critical advance and ultimately led to a battery of studies that have provided considerable insight (Figure 6) (93). The exo- and endo-8,9-epoxides are both produced, in varying ratios, in chemical synthesis (92, 96) and by individual P450 enzymes (96, 97). The exo isomer is g10 (3)-fold more mutagenic and genotoxic than the endo isomer (97, 98). An important measurement was the half-life of the exo-epoxide, which is 1 s at neutral pH and room temperature (99). The synthesis and this kinetic study led to other experiments that established 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 reaction involves several interesting features including DNA-catalyzed acid hydrolysis, base intercalation, and a very rapid SN2 attack of the N7 atom of guanine on the exo-epoxide (95, 98, 103). The endo-epoxide can effectively be considered a detoxication

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Figure 6. Major pathways involved in human aflatoxin B1 metabolism (94). The indicated parameters are either measured second-order rate constants or 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.

product (97, 98). Direct reaction of the epoxide with proteins is possible, although a more likely route involves reaction with the dialdehyde formed by base-catalyzed rearrangement of the dihydrodiol (94, 99). Collectively, the body of knowledge allows a scheme to be developed (Figure 6) with second-order rate constants for chemical reactions and kcat/Km parameters for the major human enzymes involved in these processes. This framework allows for the development of logical paradigms to be used for chemoprevention methods, for example, use of compounds such as oltipraz to inhibit and induce particular enzymes (104). 3.2. Ethyl Carbamate. This compound, also known as urethane, is a commodity chemical and has been shown to be carcinogenic in rodents (105). A pathway of bioactivation proposed by the Millers (106) involved desaturation to vinyl carbamate (Figure 7). P450 2E1 has been shown to have a role in the bioactivation of many low Mr chemicals, including halogenated hydrocarbons and vinyl monomers (107). The Millers and their associates had been able to show that vinyl carbamate was more tumorigenic than ethyl carbamate (108) but had been unable to demonstrate the desaturation process. A careful search, utilizing selective extraction and GC-MS, revealed trace formation of vinyl carbamate formed from ethyl carbamate (107). Furthermore, vinyl carbamate was also converted to a reactive product that reacted with adenosine (to form 1,N (6)-ethenoadenosine) and was presumed to be vinyl carbamate epoxide (107). The steadystate levels of vinyl carbamate and its epoxide are consistent with the pathway shown in Figure 7, and a slow desaturation of ethyl carbamate by P450 2E1 followed by epoxidation at a rate 103-fold faster (107). The epoxide has been synthesized using dimethyldioxirane (t1/2 of 10 min in H2O) and has been shown to be mutagenic and tumorigenic (109). This level of stability would be expected to allow considerable migration, in

that the t1/2 is 600-fold longer than that of aflatoxin B1 8,9epoxide (vide supra). 3.3. Coupling of Norharman and Aniline. Norharman is found in cigarette smoke and pyrolyzed food and was discovered to be a “comutagen” in the 1970s (110). That is, the addition of norharman to an “S9” or other P450-based system used in bacterial mutagenesis tests was found to enhance the mutagenicity of aniline and some other simple arylamines. One possible explanation is heterotropic activation of a P450, a phenomenon observed with some P450s (18, 111) (vide infra). However, an alternate explanation appears to be the case. Norharman and aniline react to form a new heterocyclic compound, 9-(4′-aminophenyl)-9H-pyrido[3,4-b]indole, which subsequently undergoes N-oxygenation to a hydroxylamine that is acetylated and then reacts with DNA (Figure 8A) (112, 113). P450 1 family and P450 3A4 enzymes are involved in these processes (113, 114). No direct work has been done on the catalytic mechanism of coupling, but a tenable mechanism is presented in Figure 8B (41, 113). 3.4. Troglitazone. Troglitazone was the first of the thiazolidinedione “glitazone” drugs, developed as a peroxisome proliferator-activated receptor γ-agonist and used to treat diabetes. After introduction on the market, the drug was withdrawn in 2000 due to what was considered an unacceptably high incidence of hepatotoxicity (73, 115, 116). Subsequent in vitro work was used to establish the course of biochemical transformation shown in Figure 9. Troglitazone is highly bound to albumin and metabolized primarily in the liver. The catalysts have been implicated as P450s 3A4 and, to a lesser extent, P450 2C8 (117, 118). Both the thiazolidinedione and the chromane ring systems can be activated, as judged by the analysis of GSH conjugates (Figure 7). The P450 reactions are readily rationalized in the context of known chemistry (37, 41).

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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).

Newer glitazone drugs have been developed to meet the needs of diabetics, for example, rosiglitazone and pioglitazone. These compounds contain the thiazolidinedione ring and show covalent binding 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 hepatoxicity (119). Interestingly, in cell-based systems, P450 inhibition did not protect against the in vitro parameters presumed related to toxicity (120). For further discussion of the relationship of covalent binding and toxicity of drugs see, refs 14 and 89. 3.5. Other Bioactivation Reactions. The current literature contains many P450 reactions leading to bioactivation, and the majority have probably been published in the course of the last 20 years. A comprehensive list is not presented here, but a number of reviews provide a wealth of information (37, 41, 72, 73, 89, 121, 122). The formation of reactive products has become an issue in the pharmaceutical industry, and efforts are being made to identify drug toxicity in preclinical screens. The goals, issues, and technology are discussed elsewhere (14, 89).

3.6. Mechanism-Based Activation. One feature of P450 reactions that occurs with considerable frequency is mechanismbased inactivation by drugs and other chemicals. This process was recognized early, although not well-understood (123). Today, we realize that many classes of compounds can cause such inactivation (e.g., olefins, acetylenes, and cyclopropylamines), and in many cases, the mechanisms are well-understood at the chemical level (124). In most cases, an intermediate in the oxidative pathway reacts with either the heme or the apoprotein. Probably less common are amines and a few other compounds (e.g., methylene dioxyphenyls) that yield products that bind tightly (but not covalently) to the heme iron (“metabolic inhibitory complexes”) and are recognized by the spectral changes that they produce (125, 126). There are still some categories of compounds that often act as mechanistic-based inactivators but for which the chemistry underlying the process is not so obvious (e.g., piperazines) (127). Inhibition and even destruction of P450s do not in themselves produce toxicity, unless one is dealing with a P450 involved in a critical physiological pathway (e.g., steroidogenesis). In this

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

regard, the level of rat liver P450 can be lowered ∼75% by 1-aminobenztriazole without any apparent adverse effects (128). With some drugs, the production of heme adducts can trigger porphyrias because the adducts disrupt porphyrin synthesis (129). The most relevant issue with mechanism-based inhibition by drugs is drug–drug interactions arising because inhibition of a P450 will attenuate metabolism and lead to higher plasma and tissue levels of that drug. For instance, several HIV-1 protease inhibitors (e.g., ritonavir) are potent P450 3A4 inhibitors and block the metabolism of drugs (that are P450 3A4 substrates) used concurrently (130, 131). If a drug shows mechanism-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 effect of consumption of grapefruit juice on drug metabolism, which is related to mechanism-based inactivation of intestinal P450 3A4 by bergamottin (132). Does finding mechanism-based inactivation of a P450 indicate a tendency for the production of reactive products that would attack other proteins and result in toxicity? The answer is not necessarily. Some compounds do both, but in principle, the two processes are clearly distinct (133). For instance, consider the cases of 1-aminobenztriazole and bergamottin presented above. Also, some drugs are still developed on the basis of mechanismbased inactivation, even for P450 19A1 (134). 3.7. P450s and Oxidative Damage. P450s can catalyze oneelectron reductions (135) or produce oxygenation products that are unstable and reduce molecular oxygen (e.g., catechol estrogens) (136). Conceivably, one-electron oxidation products could undergo radical propagation reactions with oxygen, but the list of documented stable one-electron oxidation products

is sparse (137). Early studies in the P450 field demonstrated poor 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 involvement in the generation of oxidative damage, invoking P450s in the 1A, 2A, 2B, 2E, 3A, and 4A subfamilies (141–144). However, closer inspection indicates that almost all of the literature is based on in vitro systems, mainly either microsomes or cultured cells. Many of the biomarkers used as parameters of oxidative damage in in vivo work have not been well-validated. The production of isoprostanes has been shown to be the most accurate measure available for assessing oxidative damage (145) and can be utilized in vivo, even in human studies. In a recent study, rats were treated in classical regimens known to induce particular P450s, and parameters of oxidative damage were measured. Liver microsomes showed a variety of changes (in NADPH oxidation, H2O2 formation, and thiobarbiturate-reactive product formation), but liver and plasma isoprostanes were found only to be substantially elevated in association with a barbiturate type response (treatment with phenobarbital or Aroclor 1254). The lack of change in isoprostanes upon treatment with β-naphthoflavone, isoniazid, pregnenolone 16R-carbonitrile, or clofibrate, which induce P450s in the 1A, 2E, 3A, and 4A subfamilies, respectively, was notable (146). Thus, P450s in the 2B subfamily (or others that might be induced by barbiturates, e.g., 2C6) are associated with oxidative damage, but the others are not, in in vivo settings.

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4. Current and Future Issues Although P450 can be considered a relatively mature research field in many ways, many questions and challenges still exist, and the area holds many opportunities for dedicated young scientists. The following list is not intended to be comprehensive and is oriented toward some issues relevant to toxicology. 4.1. Functions of “Orphan” P450s. The term “orphans” is used to designate relatively recently identified human (and other) P450s for which little information is available, using the terminology originally applied to the steroid hormone receptor family (147). This term can be used for about one-fourth of the 57 human P450 genes (19, 148). The Human Genome Project has provided important knowledge about gene locations and genetic variants of recently discovered P450s. Orphan P450s are likely not to be major contributors to the metabolism of drugs but may have roles in the activation of carcinogens and protoxicants. For instance, the orphan P450 2W1 has been shown to be expressed only in tumor tissue (149) and also to activate a variety of chemical carcinogens (150). In a broad sense, the identification of functions of newly identified proteins is one of the major problems in biology. Several approaches can be used to define the functions of orphan P450s in humans and animal models (19), and some of these have been employed already. 4.1.1. Analysis of Suspects. If related P450s (e.g., same P450 family) catalyze reactions with a class of chemicals, then these can be used in trial assays. For instance, when P450 27C1 was purified, it was tested with vitamin D compounds because P450 27A1 and 27B1 catalyze such reactions (151) (however, no activity was observed). 4.1.2. Transgenic Animal Models. One option is to delete what is expected to be the orthologous gene from a mouse and then interrogate the animals for differences. In some cases, this can be done with a “metabolic” approach. Thus, P450 2R1 was characterized as a retinoic acid hydroxylase (152). Alternatively, a human enzyme could be overexpressed in mice and the animals could be examined (in a metabolomic approach) to look for differences. 4.1.3. Library Screening. In this approach, components of a selected set of perhaps 50–300 chemicals, representing a broad spectrum of chemical classes, are catalyzed for interaction with a purified P450. If even weak activity is found with a representative, for example, an androgen, then further studies are done with more class representatives. 4.1.4. Untargeted Metabolomic Strategies in Vitro. The purified P450 is incubated in a cofactor-fortified system with an extract of the tissue in which the P450 is expressed. Changes in the composition of the extract are interrogated for changes using LC-MS, using principal component analysis or other approaches to compare the extract before and after the incubation (19). 4.1.5. Untargeted in Vitro Strategies with Isotope Editing. This approach is similar to the former one, except that an incorporated cofactor (O2) is partially tagged with an isotope (e.g., 18O) and the extract is examined for an isotopic signature with an expected 18O (16):O ratio). Software employing this approach has been developed and used with model systems (153). 4.2. Ligand Cooperativity. A full treatise on this issue is far beyond the scope of this review. In brief, some of the P450s exhibit rather aberrant catalytic behavior (18, 111, 154–156). Ligand cooperativity is of two types: (i) homotropic cooperativity, in which sigmoidal plots of reaction velocity vs substrate are seen, and (ii) heterotropic cooperativity, in which the

addition of a compound to the enzyme stimulates the oxidation of a substrate (the enhancing compound may also be a substrate itself) (18). Work with animal models suggests that this phenomenon (at least heterotropic cooperativity) can occur in vivo (157, 158). With regard to homotropic cooperativity, the patterns are often modest (in terms of apparent Hill plot parameters, e.g., n ) 1.3–1.5), and care is required in analysis. However, in a recent study in this laboratory, we have observed an apparent n value of ∼6 for the 1-hydroxylation of pyrene by rabbit P450 1A2 (2). A number of hypotheses have been proposed, including classical allosteric effects keyed to binding at a remote site, multiple occupancy of the active site, multiple protein conformers that are selected by binding to ligands, and mixtures of the above (18, 111, 159). Unfortunately, much of the literature in this area is based on simplistic steady-state kinetic analyses, and the recently elucidated crystal structures (of P450s 2C8 and 3A4) have not been able to resolve the issues (32, 34, 36). Our own work in this area has shown the involvement of slow protein–ligand changes that occur during the time scale of substrate oxidation (39, 160). That is, conformational changes can still be occurring while the enzyme is oxidizing substrates, and effectively, a mixture (or perhaps more properly a continuum) of enzyme forms exists in the course of the reaction. The results for substrate binding kinetics (39, 160) also suggest that the substrate (or inhibitor) for P450 3A4 first interacts with a peripheral site on the enzyme and then somewhat slowly moves toward the heme iron. Whether or not this putative peripheral site is that occupied by progesterone (32) or testosterone (161) in some X-ray crystal structures is unclear. Recent results with rabbit P450 1A2 also indicate multiple steps associated with ligand binding, and fluorescence evidence for the formation of pyrene excimers (dimers) in the active site has been obtained,2 reminiscent of a steady-state study with P450 3A4 (162). 4.3. Predictions of Metabolism. P450s are involved in ∼75% of all drug metabolism (Figure 4A). The ability to predict sites and rates of oxidation of new substrates would greatly facilitate drug development, as well as considerations of potential carcinogens and toxicants. Efforts toward this goal have been made. Possible approaches can involve either comparisons of existing databases of biotransformation data, docking and electronic information about P450s and substrates, or a mixture of the two. Some of the efforts have been made within pharmaceutical organizations and some by smaller private organizations. One unresolved issue is that available crystal structures have deficiencies in providing all of the details that are desired. In the absence of such information, the energy of different substrate bonds is not a reliable guide to prediction (163). Better drug design predictions can be made within series of closely related compounds, while de novo estimates (in unrelated series) are still far more difficult and remain a challenge for the pharmaceutical industry. Although computational tools are on the market, these have generally not yet found major success in the pharmaceutical industry (in drug metabolism or safety assessment), 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 is evident; few of the structures of ligand-bound P450s correctly predict regioselectivity of oxidation (29). For instance, 5,6benzoflavone 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 from the heme iron (26, 97, 155, 164). 4.4. Overlaps of Detoxication and Bioactivation. A simple view of P450s is that some do good things and some do bad things, and appropriate induction and inhibition have the potential to produce a more favorable situation. However, the situation is usually much more complex. Mention has already been made of the significance of metabolism in individual tissues and the resulting balance of detoxication and activation (65, 67). An even more complicating situation occurs when an individual P450 can both activate and detoxicate the same molecules, for example, P450 1A1 with benzo[a]pyrene (165) or P450 3A4 with aflatoxin B1 (97, 166). Some insight can be gained with detailed studies of the enzymology, but a more appropriate understanding of the overall situation requires (i) a more complex system (at least cellular and possibly in vivo), (ii) distribution analysis (pharmacokinetic) of the chemical and its products in various tissues (“target” and nontarget), (iii) analysis of the systems at varying doses (concentrations), and (iv) knowledge of the distribution of the P450 in various tissues (and cells). Of course, analysis in complex systems is more difficult if multiple P450s are involved in any of the reactions. These problems require many measurements and also methods of pharmacokinetic modeling/fitting. 4.5. Roles of P450s in Idiosyncratic Drug Toxicity. Idiosyncratic drug reactions are defined as those highly individualized in occurrence, and their pharmacological basis is unknown. In practice they occur in ∼1/10 (3) to 1/10 (4) individuals and have been very hard to predict with animal models and even in clinical trials. Unfortunately, some of these problems are not identified before new drugs are introduced to the market. Although the statement is often made that these responses are dose-independent, this is not really established, and many do not accept this premise. Two phenomena often discussed in relation to idiosyncrasies are bioactivation (to yield covalent binding) and hypersensitivity/ allergic responses. P450s certainly play a role in bioactivation and covalent binding, and covalent binding and autoimmune antibodies accompany some idiosyncratic drug reactions (167, 168), but exactly how these events fit together and what is causal are still rather unclear. We do not have a good estimate of the contribution of P450s to idiosyncratic reactions, and this question warrants further study. The problems are extremely complex. With both tienilic acid (168) and dihydralazine (167), a small set of the patients develop hepatitis and also have circulating antibodies that recognize a P450 involved in bioactivation, P450 2C9 in the case of tienilic acid and P450 1A2 in the case of dihydralazine. Drug adducts are formed with the P450 (in in vitro experiments). Questions still exist as to how the P450s are processed to generate antibodies (169). The antibodies recognize unmodified P450s (but do not inhibit oxidations in vivo). Not all patients with antibodies develop hepatitis. Also, efforts to produce animal models have produced antibodies and hepatotoxicity but not together (170). Thus, we are left with questions about causality (171) and are still very limited in the availability of animal models for predicting idiosyncratic events (172). 4.6. Predicting Human Toxicity. The problem of predicting toxicity has already been mentioned. The scope of the problem is considerable (12, 14) and goes far beyond P450 issues. One of the challenges is the difficulty of extrapolating from animal toxicity data to humans. Some improvement in this area may come with more knowledge about P450s, perhaps to the extent

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that drug metabolism extrapolations to humans may help improve predictions in this area. One classic example of the problems is acetaminophen. We know that deletion of P450 2e1 in mice nearly eliminates hepatoxicity, and simultaneous deletion of P450 1a2 is even more effective (77, 173). Presumably the load of reactive products is decreased, although apparently the effect on the load has not been reported. A real issue is that the biological events following the adduction process are still rather vague, even in animal models. Although the involvement of mouse P450s 2e1 and 1a2 in toxicity is quite convincing (77, 173, 174), extrapolations to human P450s are not direct (175). Thus, we still have many questions, and it is hoped that major progress will occur in this area. 4.7. Understanding P450 Gene Polymorphisms and Disease. Today, extensive information about the major polymorphisms in many of the human P450s (http://www.cypalleles. ki.se) is available. There has been considerable discussion of the potential use of this information in developing personalized medicine, facilitating drug development, and identifying cancer risk. However, the reality is far from this. Today, the FDA does not prescribe genotyping for any drug (some assays are approved, but none are required). There are several issues involved, including costs, genotype/phenotype correspondence, the limited contribution of genotype as compared to environmental influence with P450 3A4, and the lack of a strong case history for the use of genotyping in prediction of idiosyncrasies to date. Thus, the challenges are many, but ultimately, sound approaches that include functional analysis are most likely to be successful. The relationships between P450 genotypes and cancer are even more difficult to establish than with drugs, and many of the reports on associations of P450 polymorphisms with genotypes have not held up in meta analysis (176–179). Cancer risk estimates associated with individual single nucleotide polymorphisms must be considered spurious at best. An example in point is the relationship of P450 2D6 with lung cancer. In 1983, poor metabolism (phenotype, based on in vivo debrisoquine metabolism) was reported to be strongly associated with less lung cancer in cigarette smokers (180). One reason for such a relationship could be the activation of procarcinogens in tobacco smoke by P450 2D6. However, the expression of P450 2D6 in lung is relatively low, and a number of searches have not identified any carcinogens that are preferentially activated by P450 2D6 (181, 182) even in studies with cigarette smoke condensate extracts (183). Further epidemiological studies have yielded mixed answers on the relationship of the poor metabolizer phenotype and also P450 2D6 genotypes with lung cancer (184), and the relationship is weak at best in meta analysis (185). Another related study involves the relationship between P450 1A1 induction and lung cancer, first reported in 1973 (186, 187). This relationship is still unclear (188) and cannot be understood in the context of present knowledge about P450 1A1 (189, 190) or the Ah receptor (191). One possible lead is P450 1B1, in that this enzyme has high activity toward polycyclic aromatic hydrocarbons such as benzo[a]pyrene (192, 193) and has been shown 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 with clinical trials, for which the problems have already been mentioned. In large clinical trials, there are often thousands of individuals being administered a single, well-defined drug under controlled 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, the record of exposure to chemicals is sparse at best, there is limited evidence that a single chemical causes the cancer, the time between the initial exposure and the outcome (cancer) is decades, and the disease is usually heterogeneous. In the future, studies in this area will probably need to involve larger studies of more homogeneously exposed individuals, including those exposed to high concentrations of single cancer suspects (e.g., vinyl monomers).

5. Conclusion P450 research developed largely because of its potential to explain the metabolism and toxicity of drugs and carcinogens. Today, we are at a position where the biochemical understanding of these systems is rich but still not complete. The field has been highly successful in the context of providing better predictions about human drug metabolism. However, many challenges still remain in further developing and applying our knowledge of P450s to unresolved problems in chemical toxicity. With the challenges come many opportunities awaiting dedicated researchers who have vision in the P450 field. Acknowledgment. Work in this area in my laboratory is supported in part by U.S. Public Health Service Grants R37 CA090426 and P30 ES000267. Thanks are extended to K. Stark for comments on a draft version and to K. Trisler for assistance in preparation of the manuscript. Congratulations and thanks are in order to all who have played a role in the first 20 years of Chemical Research in Toxicology.

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