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Chem. Res. Toxicol. 2008, 21, 70–83
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
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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
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* 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/
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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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Chem. Res. Toxicol., Vol. 21, No. 1, 2008
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
Guengerich
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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Chem. Res. Toxicol., Vol. 21, No. 1, 2008 79
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).
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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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