[CANCER RESEARCH 48, 2946-2954, June 1, 1988]
Perspectivesin CancerResearch
Roles of Cytochrome P-450 Enzymes in Chemical Carcinogenesis and Cancer
Chemotherapy1
F. Peter Guengerich2
Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232
Studies involving the metabolism of chemical carcinogens
were important in the discovery and initial characterization of
the cytochrome P-450 enzyme system. The Millers and their
associates identified NADPH-dependent microsomal enzymes
involved in the biotransformation of azo dyes (1, 2). These
reactions, reduction and mixed-function oxidation, were also
found to be important in the processing of many other carcin
ogens, as well as drugs and steroids. Subsequent work led to
the identification of the hemoprotein P-4503 as the terminal
oxidase involved in such microsomal mixed-function oxidations
(3,4). Other early studies revealed that administration of many
different chemicals to animals could alter the metabolism of
carcinogens (5-7). As we know now, many forms of P-450 exist
and the expression of these enzymes is influenced by the com
pounds which are given to the animals. Administration of the
carcinogenic polycyclic hydrocarbon 3-methylcholanthrene to
rats and analysis of the liver microsomes provided some of the
early evidence that multiple forms of P-450 exist (8, 9). Since
that time, a great deal of effort has been concentrated upon
understanding the biochemistry of these P-450 enzymes, in
terms of how they catalyze reactions and how their expression
is regulated. Although much of the focus regarding P-450
proteins has involved their contributions to the metabolism of
steroids and drugs, considerable effort is still directed towards
understanding the roles of P-450s in carcinogen metabolism.
The characteristics of P-450 enzymes will not be reviewed at
length here.4 The microsomal enzymes are found in most tissues
but are concentrated in liver. All have characteristic ferrouscarbon monoxide complex Soret peaks near 450 nm, have
montimene molecular weights of about 50,000, and accept
electrons from the flavoprotein NADPH-P-450
reducÃ-ase.
Nearly 20 different P-450 proteins have been isolated from rat
liver and all appear to be distinct gene products. Levels of the
individual proteins are altered by administration of or exposure
to a wide variety of chemicals, many of which are carcinogens
themselves (including tumor initiators such as the polycyclic
hydrocarbons and tumor promoters such as phénobarbital).
Mitochondrial P-450s accept electrons from ferridoxins and
seem to be primarily involved in anabolic steroid metabolism,
although some evidence for roles in carcinogen oxidation has
been presented (14).
Several reviews and monographs deal with various aspects of
the catalytic mechanisms and regulation of the P-450 enzymes
Received 12/28/87; revised 2/10/88; accepted 2/25/88.
1Research in the author's laboratory has been supported in part by USPHS
Grants ES 00267, ES 01590, ES 02205, ÇA30907, and ÇA44353.
1 Burroughs-Wellcome Scholar in Toxicology, 1983-1988.
3 The abbreviation used is: P-450, cytochrome P-450.
* Individual P-450 enzymes are denoted by designations in the original litera
ture or else equated to preparations used in the author's laboratory when possible,
for purposes of simplification; for reference see Refs. 10-12. Recently a tentative
nomenclature for the P-450 genes has been published (13). The term "isozymes"
is not used here. Although this term has been used extensively in the past, it
technically refers to situations in which a single reaction is catalyzed by several
proteins. Much of the attention is now focused on highly selective reactions of
individual I' 45()s and the term is often not very appropriate.
and the reader is directed to these (15-18). This "Perspectives"
article will deal with the current state of information regarding
the metabolism of carcinogens and questions related to the
general importance of such processes. The pathways of P-450mediated oxidation of carcinogens have been reviewed recently
by Kadi uba r and H am nions (19), and the entire subject of
metabolism in the activation of chemical carcinogens is treated
in a comprehensive series (20) and in a number of other review
articles (21-31). Another area discussed here is the biotransformation of cancer chemotherapeutic agents by P-450 enzymes.
Although metabolism may be of considerable importance in
influencing the efficacy and side effects of these drugs, the
current knowledge is rather limited.
Catalytic Specificity of P-450 Enzymes
What Do We Know about Which Carcinogens Are Activated and
Detoxicated by Specific P-450 Enzymes?
The literature contains many studies regarding the catalytic
specificities of the P-450 enzymes of rats, rabbits, and other
experimental animal species, as well as human P 450s. This
"Perspectives" article is not intended to serve as a compilation
of this work. More comprehensive reviews are found elsewhere
(10,11,19, 32). Table 1 includes some carcinogens which have
been studied and representative literature references. Nearly all
classes of carcinogens have been examined to at least some
degree, and a few have been studied in great detail. Invariably,
however, our knowledge is far from complete.
What are some of the ways in which the catalytic specificity
of individual P-450 enzymes can be discerned? With animal
models, some initial references can be made by comparing the
in vitro rates of a particular catalytic activity using animals
treated with different inducing agents (10). In some cases sex
differences in metabolism can be exploited. In this regard, one
can also use a series of microsomal preparations and compare
the rates of a catalytic activity under consideration with those
of a "marker" activity which has been assigned to a particular
P-450 enzyme; this approach can be used either with animal
models (47) or with humans (48). One way of elucidating
catalytic specificity is to purify the individual P-450 enzymes
and directly measure catalytic activities in reconstituted sys
tems. This approach gives a generally appropriate qualitative
picture of microsomal activity (49) although the unexplained
low activity of some P-450s (50) and possible enzyme interac
tions (51) may complicate interpretations. A second way to
address the problem involves insertion of cloned genes into
eukaryotic expression vectors and measurement of catalytic
activity (52).
One problem associated with the above studies is the esti
mation of the fraction of total activity catalyzed by a particular
form of P-450. The major approach involves titration of a
catalytic activity in microsomal samples with selective inhibi
tors. These inhibitors can be classified into three groups: Group
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CYTOCHROME P-450 AND CANCER
1, competitive inhibitors (e.g., see Ref. 53); Group 2, noncom
petitive and mechanism-based inhibitors (54); and Group 3,
antibodies (55). When families of P-450 enzymes very closely
related in primary structure are under consideration, the estab
lishment of specificity of these reagents can be difficult, even
when monoclonal antibodies are used.
Some other problems also arise. Several P-450s may all
catalyze a single reaction but with different A",,,values (44, 56).
Another problem is that the use of different end points may
generate varying pictures of catalytic specificity. For example,
the activation of the carcinogen aflatoxin H, to the putative
electrophilic 2,3-epoxide can be considered. Different patterns
of specificity have been seen with purified enzymes (and even
in rat liver microsomes) depending upon whether covalent DNA
binding (34), Salmonella typhimurium reversion (35, 36), or
bacterial SOS-linked response (37) is used to monitor the
reaction. Finally, caution should be exercised when attempts
are made to extrapolate catalytic specificity across species on
the basis of similarity in primary amino acid sequence. Notable
differences exist, for example, in the warfarin 6- and 8-hydroxylase activities of rat P-450tfNFB and its mouse orthologue
Pi-450 (57), the 7-ethoxycoumarin O-deethylase activity of rat
P-450pB-Band rabbit P-450LM-2(58, 59), and the 17/3-estradiol2-hydroxylase and progesterone 21-hydroxylase activities of
rabbit P-450 1 and human P-450MP (60).
As mentioned above, several forms of P-450 are found in the
liver of each animal species, and a number of lines of evidence
suggest that 10-20 P-450 proteins can exist in rat, rabbit, or
human liver. The P-450 enzymes often display catalytic speci
ficity. The current thought is that the chemistry involved is very
similar with all of the enzymes but that specificity is determined
by structural features in the substrate binding site. Specificity
can be demonstrated at several levels (10): (a) different P-450s
vary in their rates of catalysis of a single reaction involving a
single substrate; (b) several P-450s can metabolize a single
substrate but regioselectively catalyze reactions at different sites
of the molecule; (c) enantiomeric or diastereomeric pairs of a
single substrate can be transformed at different rates by each
P-450; (d) in a pro-chiral substrate, a P-450 can selectively use
one group (namely, hydrogen) as opposed to the other. Catalytic
specificity can, then, have important consequences, since some
oxidations of a single compound can render it more electro
philic (and capable of reacting with macromolecules) while
other oxidations can render it less biologically active and aid in
elimination from the body. Further, the rates of reaction are a
function of the amount of each P-450 form present, which can
be regulated by environmental and genetic factors as well as
sexual dimorphism and development.
Evidence That Differences in P-450 Composition Can Influence
Susceptibility to Cancer
Considerable evidence has now been accumulated in support
of the view that changes in P-450 composition can affect in
vitro and in vivo metabolism of drugs in both animal models
and humans. The evidence is less clear in the case of carcino
gens, which cannot be dealt with so easily in humans. However,
information regarding the general hypothesis does exist and
can be considered under three different headings.
Tumor Initiation in Experimental Animal Systems. Indirect
evidence for P-450 roles is provided by experiments in which
in vitro mutagenesis and DNA adduct formation are affected
by changes in the P-450 composition. Many examples exist;
the changes in P-450 composition can be brought about by
genetic means (61) or by enzyme induction or reconstitution of
purified enzymes (33).
Studies dealing with in vivo tumor initiation are more limited
and are largely restricted to the murine model of Neben and
his associates. Although increases in P-450 enzyme activity
occur in the liver and extrahepatic tissues of genetically respon
sive mice (21), only extrahepatic tissues show enhanced tumor
formation, and these experiments have been restricted to polycyclic hydrocarbons, which induce the P-450 enzyme under
consideration (1V450) in the genetically responsive animals.
Examples of cancers produced in these mice by polycyclic
hydrocarbons include lung tumors [3-methylcholanthrene], leu
kemias [benzo(a)pyrene, 3-methylcholanthrene],
s.c. tumors
[benzo(<z)pyrene, 3-methylcholanthrene] (62), brain tumors [3-
Table 1 Activation of procarcinogens by rat P-450 emymes"
Rat P-450 enzymes involved in
bioactivation4
Procarcinogen
AaC(2-amino-9//-pyrido[2,3-e]indole)
2-Acetylaminofluorene
Aflatoxin H,
2-Aminoanthracene
o-AminoazotoIuene
4-Aminobiphenyl
2-Aminofluorene
Benzo(a)pyrene
4,4' (Bis)mcthyk'm- chloroaniline (MOCA)
1,2,3,4-Dibenzanthracene
7,12-Dimethylbenz(a)anthracene
F„P-450,SF<¡
P-450r,.„P-45ÕW.B,P-450,SF-G
P-450uT.A,P-45ÕW.GP-450PB-B,
P-450irr.i
P-45ÕW., P-45IW.., P-450BFJG
P-45(Wo
-BM
P-450flNF.B,P-450,sF-o
P-45ÕWB, P-45<W.
P-45<W., P-450,SFJG
P-450PB-8,P-45<W „,
P-450.S«;
P-45ÕW.S
P-450J
A1,,V-1>inid h ylnitn >sam inu
Representative
references
33
33
34-37
33
38
39
38, 40, 41
31,42
39
33
33
43,44
Glu-P-l(2-amino-6-methyldipyrido[l,2-a:3',2'-«/]imidazole)
P-450.S«;
31,33,45
Glu-P-2(2-aminodipyrido[l,2-a:3',2'-«/]imidazole)
P-450„NF..,
P-4501SFJG
31
P-45<W„ P-450.SF*
IQ(2-amino-3-methylimidazo[4,5-/]quinoline)
45
P-45<W.B, P-450ISFJC
MeAaC(2-amino-3-methyl-9A/-pyrido[2,3-*lindole)
33
P-45<W., P-450.SF-G
MeIQ(2-amino-3,4-dimethylimidazo[4,S-/]quinoline)
33
P-45<W., P-450.SF-C
MeIQx(2-amino-3,8-dimethylimidazo[4,5-/]quinoline)
33
P-45IW.
3-Methylcholanthrene
33
P-45<W„,P-450.SF.G
JV-Methyl-4-aminoazobenzene
34,46
P-450BP-G
2-Naphthylamine
41
P-45ÕW.,,,
P-450.SF-G
Trp-P-1(3-amino-1,4-dimethyl-5//-pyrido|4,3-o]indole)
33
,sF<;,P-450aNF..
Trp-P-2(3-amino-1 -methy-5//-pyrido[4,3-*|indole)
31,33,45
" See also Ref. 19. The point was made in the text but should be reemphasized here that these are enzymes which have been shown to activate the compounds but
are not necessarily the most important catalysts of oxidation. In some cases only a limited number of P-450 enzymes have been studied, sometimes the highly
inducible enzymes may not be relevant to bioactivation in uninduced animals, and sometimes other enzymes such as prostaglandin synthase or flavin-containing
monooxygenase may be more important.
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CYTOCHROME P-450 AND CANCER
methylcholanthrene] (63), and lymphomas and lymphosarcomas [7,12-dimethylbenz(a)anthracene]
(64). However, caveats
must be made because the Ah locus is a pleiotropic response
and involves changes in a number of different enzymes poten
tially catalyzing the biotransformation of these compounds.
Further, most of the polycyclic hydrocarbons were used in
single-dose complete carcinogen bioassays, and the possibility
must be considered that the effects might not be only on
initiation.
While some reservations exist concerning experiments in
volving genetic manipulation of P-450 enzymes, studies in
which the effects of enzyme inducers on carcinogenesis have
been examined are probably even more complex. For instance,
Kouri et al. (65) reported that 2,3,7,8-tetrachlorodibenzo-pdioxin was an effective cocarcinogen in enhancing 3-methylcholanthrene-induced s.c. rodent tumors. Subsequent studies
by Poland et al. (66) indicate that the dioxin is a very potent
tumor promoter and, in view of the long retention of the
compound, this effect cannot be easily isolated. Studies on
enzyme induction by phénobarbitalare also complicated by the
knowledge that this compound and some other barbiturates can
be tumor promoters. Thus any experiments designed to show
roles of enzymes in tumor initiation must be carefully set up to
show that the enzyme inducers are truly acting as cocarcinogens
and not as promoters (some barbiturates and hydantoins are
inducers but not promoters). Usually the effect of enzyme
induction is to decrease tumor incidence, such as with the
reduction of aflatoxin Bi-induced (67, 68) or dimethylnitrosarnine induced liver tumors (69-71) by phénobarbitaland reduc
tion of aromatic amine-induced liver tumors by 3-methylcholanthrene (72, 73). When results of this type are obtained, one
must also consider the effects of the inducer on other enzymes
such as those involved in conjugation and detoxication.
Tumor Promotion in Experimental Animal Systems. Promo
tion has been addressed far less than initiation in terms of
influences due to metabolism of carcinogens. In this article, the
term promoter is used in an operational sense. That is, pro
moters are chemicals which increase the number of tumors
when administered after initiating carcinogens at doses which
by themselves do not produce tumors (and are distinguished
from cocarcinogens, which must be administered very close to
the time of initiation). With this loose functional definition,
mechanisms are not specified, and epigenetic and genetic pos
sibilities can be considered. Many of the classic promoting
agents probably do not require biotransformation to exert their
effects; for instance, phorbol esters may exert their actions by
binding to protein kinase C (74). The characteristics and mech
anism of action of individual promoting agents do vary, how
ever; for instance, the phorbol ester 12-O-tetradecanoylphorbol13-acetate differs from 7,12-dimethylbenz(a)anthracene
in a
number of aspects of its promoting properties (75, 76). Many
chemicals are effective as complete carcinogens as well as being
distinct initiators or promoters. Thus, these complete carcino
gens should possess both initiating and promoting properties.
Of interest here is the possibility that formation of electrophilic metabolites of carcinogens can play a role in their tumorpromoting activities. The concept is not new, although it should
again be emphasized that for some model promoting agents
no metabolism is required. Evidence has not been generated
directly in P-450-related systems, but the literature contains
at least two relevant cases. The first is 7-bromomethylbenz(a)anthracene, which is nearly as potent as the most active
phorbol esters in promoting 7,12-dimethylbenz(a)anthraceneinitiated mouse skin carcinomas and papillomas (77). The
reactivity of the molecule appears to be important, for the
hydroxymethyl analogue was relatively weak as either an initi
ator or a promoter.
Another example comes from the work of Boberg et al. (78)
with l'-hydroxysafrole and rat liver tumors. The compound
acts as a complete carcinogen in rat and mouse liver; its initi
ating activity in rat liver is relatively weak but the compound is
a potent promoter of diethylnitrosamine-induced
rat liver tu
mors. Both the initiating and promoting activities of l'-hy
droxysafrole are blocked by feeding pentachlorophenol, which
inhibits the sulfation of the compound to give a reactive electrophile.
The mechanisms by which these electrophilic promoters exert
their effects are unknown. One possibility is toxicity, perhaps
through reaction with specific proteins, to cause a proliferative
response in clones of initiated cells. Alternatively, further mu
tations in some cells of these proliferating clones may occur
(78). The cases mentioned above do not involve P-450 reactions,
but the production of similar electrophiles by P-450s certainly
occurs and the hypothesis should be considered that P-450
enzymes can contribute to tumor promotion as well as initia
tion.
Studies in Humans. In humans, the evidence for association
of cancer risk with P-450 composition is less well developed
than in experimental animals. Although the concept seems
intuitively obvious and is the focus for many detailed investi
gations, there are only two major lines of evidence for the
existence of a relationship and both have associated caveats.
Kellerman et al. (79, 80) grouped individuals into a trimodai
distribution on the basis of their lymphocyte aryl hydrocarbon
hydroxylase inducibility (in culture) and reported that smokers
in the high inducibility group were more prone to develop lung
tumors. These observations were originally very exciting but
technical problems associated with the system have made these
experiments difficult to repeat (i.e., see Ref. 81). More recently,
Kouri et al. (82) have reported a relatively good correlation
between the basal level of lymphocyte aryl hydrocarbon hydrox
ylase activity and the incidence of lung cancer in smokers.
However, this observation does not allow one to discern
whether the high enzymatic activity is a cause or an effect of
lung cancer, as the authors admit. Jaiswal et al. (83) found that
the increased level of Pi-450 mRNA in lymphocytes was well
correlated with aryl hydrocarbon hydroxylase inducibility and
Karki et al. (84) showed a correlation between levels of basal
lymphocyte and lung aryl hydrocarbon hydroxylase activity.
The work of De Flora et al. (85), on the other hand, suggests
that smoking directs pulmonary metabolism more towards de
toxication than towards bioactivation. The entire body of infor
mation still does not permit definitive conclusions to be drawn
about the relationship of the lymphocyte enzyme activity to
cancer risk.
The other major line of investigation deals with correlations
involving rates of drug oxidation in humans, particularly with
regard to polymorphisms. Idle et al. (86) reported a positive
relationship between the in vivo rate of debrisoquine 4-hydroxylation and the incidence of liver cancer in Nigerians and
suggested an etiological role involving aflatoxin BI. However,
work in the author's laboratory (87) and that of Davies (88)
argues against a role for the debrisoquine 4-hydroxylase in
aflatoxin BI activation. Further, no information concerning the
actual exposure of the Nigerians to aflatoxins was presented or
is available.
A more intriguing study involves the observation by Ayesh et
al. (89) that the debrisoquine slow metabolism phenotype could
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CYTOCHROME P-450 AND CANCER
be associated with decreased incidence of lung bronchiogenic
carcinomas in smokers (and rapid hydroxylation associated
with increased tumor incidence). No evidence exists for a met
abolic basis, and, of course, the etiological factors in tobacco
smoke remain undefined. Although the results of Ayesh et al.
(89) appear intriguing, a report by Drakoulis et al. (90) indicates
that the same findings were not obtained in a similar study
involving a German population (i.e., no relationship was found).
More recently Harris et al. (91) have found some phenotypic
evidence for a positive relationship similar to that of Ayesh et
al. (89) when only lung small cell tumors are considered. If a
relationship does exist, its basis remains undefined and is not
necessarily metabolic. An abstract by Ritter et al. (92) also
suggests a positive relationship between rapid debrisoquine 4hydroxylation and carcinoma of the larynx and pharynx.
The final case involves recent work by Kaisary et al. (93)
relating hydroxylation polymorphisms to bladder cancer. Pa
tients presenting with bladder cancer were classified histologically as having nonaggressive (Stages I and II) or aggressive
(Stage III) bladder cancer. Aggressive bladder cancer showed
an association with rapid debrisoquine 4-hydroxylation. This
association was not seen when nonaggressive bladder cancer
cases were examined. The particular population studied appar
ently did not have occupational exposure to known bladder
carcinogens and the influences of ethanol consumption and
smoking were not synergistic with the extent of debrisoquine
4-hydroxylation; thus, the work does not provide evidence that
the P-450 debrisoquine 4-hydroxylase (P-450DB) is activating a
bladder carcinogen, although this possibility certainly still ex
ists. Patients with nonaggressive bladder cancer had a weaker
but significant association with rapid polymorphic 4-hydroxyl
ation of 5-mephenytoin, and this relationship was independent
of a significant interaction between smoking and ethanol intake.
While other relationships might be postulated on the basis
of some in vitro and in vivo knowledge (i.e., increased tumors
in alcoholics due to /V,/V-dimethylnitrosaniine oxidation, etc.),
definitive evidence is lacking. A difficulty exists in extending
studies of this type in that, even if further evidence is obtained
for a role of metabolic activation, identification of the chemicals
involved is not trivial. If there is no epidemiological reason to
suspect a particular compound or mixture, then the problem is
very difficult. Even if a crude mixture such as tobacco smoke is
implicated the problem is difficult because the active principle
is unknown. However, there are approaches that could be used
to address such problems. For instance, the P-450 enzymes
involved in activating crude cigarette smoke condensâtes to
mutagenic forms could, in principle, be identified. The same
phenotyping approach could be applied to some of the foods
encountered in regions of China where the local tumor inci
dence is high (94).
Current and Future Problems
The information discussed above supports the view that
differences in P-450 enzymes may contribute to variations in
the metabolism of carcinogens and to risk. What are some of
the current problems that remain unsolved and in what direc
tions will new research go?
1. Most of the Animal Models Remain Poorly Characterized
in Terms of Which Specific Forms of P-450 Are Involved in
Individual Biotransformation Reactions Related to Tumor For
mation. In every animal species, only some of the individual P450 enzymes have been examined for their roles in any partic
ular reaction. Of course, in some cases, immunochemical or
other approaches may provide strong positive evidence for a
primary role of an individual enzyme and extensive further
studies may not be justified.
The bulk of the studies to date have dealt with rats. Even in
that species, the experimental deficiencies are manifold. Many
of the more extensive studies were done with a limited number
of individual P-450s; since then, the number of isolated forms
of rat P-450 has grown. Some of these, such as lanosterol 14«demethylase (95) and aromatase (96), have important roles in
the metabolism of endogenous chemicals and their roles in
carcinogen biotransformation should be evaluated. Further,
some of the preparations previously considered to be homoge
neous have been subsequently found to be more complex [e.g.,
P-450pcN-E (10, 97)]. This situation also causes problems with
antibody and nucleic acid probes in establishing specificity.
Another point to be made is that many of the studies with
rat (and other animal) P-450 enzymes are focused upon those
forms which are induced dramatically. In practice these P-450s
may not be encountered frequently, particularly under some of
the conditions of chronic bioassays. Little attention has been
given to understanding enzymology in the same strains used
for bioassays with a particular chemical under consideration.
Further, many protocols use either young animals or animals
exposed to complex promotional schemes, and little informa
tion linking enzymology to the specific conditions used during
carcinogen exposure has been obtained. As an example, a
popular model is single-dose administration of chemicals to
preweanling male mice without further promotion (e.g., Ref.
78); suffice it to say that we have a very vague idea of the
complement of P-450s present to activate the carcinogens in
such animals. Any changes in P-450 composition which occur
during partial hepatectomy have likewise not been character
ized.
2. What Do Extrahepatic P-450s Really Mean? We recognize
that P-450s are most concentrated in the liver but are also
found at lower levels in nearly every other tissue in the body,
chiefly lung and kidney but also all others with the apparent
exceptions of striated muscle and erythrocytes. In some tissues
the P-450s are highly localized in particular regions or cell
types so that local concentrations may approach those found in
liver (98). One view is that these tissue-specific differences in
P-450 can be major determinants in extrahepatic carcinogenesis, since these P-450s can activate chemicals in close proximity
to targets. On the other hand, evidence clearly exists that some
of the ultimate carcinogens can migrate throughout the body
(28, 99).
The importance of the extrahepatic P-450s, like the liver P450s, probably varies with the particular enzyme and the chem
ical under consideration. The extrahepatic P-450s may be quite
important with compounds that are converted to very highly
reactive species or with chemicals that have the organ under
consideration as a port of entry. Examples of situations in
which this latter aspect may be important include polycyclic
hydrocarbons on skin, airborne carcinogens in the nasal cavity
and pulmonary tract, and carcinogenic foodstuffs in the gas
trointestinal tract. On the other hand, hepatic P-450s may
predominate in influencing the effects of chemical carcinogens
the ingestion of which takes them through the liver and the
ultimate reactive forms of which are more stable (100). Exam
ples here include aery Ionitrile (101), pyrrol i/.idine alkaloids (22,
102), and 4-aminobiphenyl (103). Discernment of the impor
tance of the extrahepatic P-450s will require detailed investi
gations in each particular case under consideration, and the
situation may often vary with the particular P-450 form, the
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CYTOCHROME P-450 AND CANCER
chemical, and the route of administration. It should also be
pointed out that in extrahepatic tissues high levels of flavincontaining monooxygenase (104, 105) and prostaglandin synthase (106-108) can be found; often the enzymes form the same
products that are encountered in P-450 reactions and caution
should be exercised in the assignments of roles of the various
enzymes.
3. At This Time We Know Far Less about the Roles of the
Various Human P-450 Proteins in Carcinogen Metabolism Than
We Do for the Animal Models. In recent years there has been
an explosion of interest in the enzymology of the human enzyme
systems. Several of the P-450 enzymes have now been purified
from human liver, and reconstitution and immunochemical
techniques have been used to make some in vitro assessments
of roles of individual P-450s in the metabolism of certain drugs
and carcinogens (11). One of the problems encountered in the
work with P-450s arising from multigene families is that anti
bodies are often not as selective as one would hope and may
not distinguish among closely related proteins. It is possible, at
least in principle, to develop monoclonal antibodies that rec
ognize individual proteins and inhibit catalytic activity. This
approach is not trivial, however, and must be accompanied by
protein chemistry studies in the case of relatively complex
systems. Another approach involves the expression of defined
genes in chimeric systems. To date this approach has been
applied to some of the rat and rabbit P-450s (109-111) and
more recently to human P-450Mp,5 but the systems have not
been used to look at carcinogens. It should be remembered that
such expression systems, like reconstitution experiments, do
not provide direct quantitative information on the relative
extent to which a single enzyme contributes to a particular
reaction.
Other approaches can be used in in vivo situations. Restric
tion fragment length polymorphism analysis of peripheral blood
cell DN A can be used to identify the molecular basis of individ
ual deficiencies in P-450s and to develop further diagnostic
assays; these approaches can be extended to individuals with
cancer. Another approach involves the use of selective inhibitors
of different P-450s. For instance, quinidine inhibits P-450DBmediated reactions (112) and sulfaphenazole inhibits tolbutamide methyl hydroxylation (which is mediated by a protein
closely related to but apparently distinct from P-450Mp) (113,
114). These two inhibitors have A"¡
values of about 10~7 M in
vitro, and both can be used ethically in in vivo settings with
humans. While it is easier to envision experiments involving
metabolism of drugs than of carcinogens, there are some pos
sibilities with the use of industrial chemicals of low carcinogenic
potential for which finite exposure limits exist and for which
metabolism via various routes can be estimated in a sensitive
manner (i.e., CH2C12, acrylonitrile, vinyl chloride) (115, 116).
4. Which Enzyme Reactions Are Most Relevant to Chemical
Carcinogenesis? While dealing with the question of which P450 enzymes are involved in individual reactions, we also need
to give careful evaluation to which reactions we should be most
concerned about. Many of the most pertinent questions in
chemical carcinogenesis relate to mixtures of compounds, such
as those encountered in chemical waste dumps, tobacco smoke,
etc. Although presenting mixtures of chemicals to enzymes may
not be particularly satisfying from the point of view of an
enzymologist, perhaps it might be more relevant in terms of
dealing with practical problems. In considering which reactions
to assay, we generally draw on knowledge from pathways de5W. R. Brian, D. R. Umbenhauer,
unpublished results.
R. S. Lloyd, and F. P. Guengerich,
rived in experimental animal systems and select particular
reactions related to bioactivation and detoxication. However,
in some cases our insight is not very complete and caution
needs to be exercised. One alternative is measurement of an
end point such as in vitro formation of a specific DNA adduci.
However, care must also be exercised here, for in very few cases
has unambiguous evidence for the importance of an individual
DNA adduct been obtained. Another alternative is to use mutagenesis or a linked SOS repair response as an end point (33).
This avenue has a certain appeal, although it suffers in that the
precise chemistry remains undefined. However, this approach
might be advantageous in some situations.
Another strategy involves placing individual proteins into
cells and then examining the effect of a single chemical on the
transformation (of the cells) to a malignant phenotype. Adding
a P-450 protein can be done in one of two ways. The purified
protein can be inserted using "microinjection" procedures,
either using liposomes (117) or polyethylene glycol and erythrocyte ghosts (118, 119). The incorporation of the protein into
the endoplasmic reticulum and the expression of functional
activity must be monitored, of course. Another approach is to
insert a cloned chimeric gene consisting of a P-450 comple
mentary DNA downstream from a promoter and examine
expression of this gene (16, 52). In principle, the cells should
become transformed under the appropriate conditions when
exposed to a particular procarcinogen if the inserted gene codes
for a P-450 important for bioactivation of that compound. A
variation on this theme might involve examination of the effects
of "anti-sense" oligonucleotides that block translation by hy
bridizing to segments of the individual P-450 mRNAs already
expressed constitutively in cells. A further extension of the
approach would involve expression of individual P-450 proteins
in transgenic rodents. Although these experiments are still
technically difficult, the approach could yield information about
the contribution of individual gene products to the induction of
tumors by individual chemicals. Further, organ-specific expres
sion could be achieved with the use of promoters specific to
tissues in which the appropriate trans-acting factors are gener
ated.
Although this overall approach would be useful, it still suffers
from the deficiency that the results of each individual expres
sion, even if positive, cannot be interpreted in absolute terms.
Thus, a full appreciation of the roles of the various P-450
enzymes to the tumor system under consideration would require
comparative studies with several inserted genes.
5. How Adequate Are Our Animal Models for Prediction of
Events in Humans? Most bioassays done on chemicals involve
a few select strains of rats and mice. Some other species are
also used, most often when tumors are produced specific to an
organ in that species (e.g., dog bladder in the case of 2-naphthylamine). The general tendency is to utilize models (and
doses) where the tumorigenicity of a compound can be dem
onstrated relative to a background level. In many cases such
systems involve relatively complex promotional schemes (120)
or the administration of test chemicals under physiological
conditions such that a sort of "natural promotion" occurs [e.g.,
injection of compounds into newborn or weanling mice (78,
99)]. If we hypothesize that P-450s and other enzymes have
major roles in the activation of chemicals to ultimate carcino
gens, then we must ask what the similarity of the enzymes
encountered in those model situations is to those of interest in
human populations at potential risk. If we do not feel that
appropriate models exist then we need to develop better ones,
with some consideration given to metabolism. On the other
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CYTOCHROME P-450 AND CANCER
hand, if the prevalent animal models continue to be accepted
for bioassay work, then more effort should be directed towards
understanding the enzymes encountered there (see Paragraph
2).
The involvement of P-450s in promotion was mentioned
above. This aspect has not received much thought outside of
the areas of some polycyclic hydrocarbons and estragóles and
should be considered further, particularly in regard to the
prevailing animal models.
6. The Metabolism of Cancer Chemotherapeutic Agents Is an
Area That Deserves More Consideration. The general consid
erations here differ from those encountered with most sub
strates; toxicity is desired with these compounds and the prob
lem is to make them selective for certain cells (121, 122). One
can, with some insight, exploit several properties of the tumor
cells. For instance, they tend to have lowered levels of P-450s,
or at least the major P-450s found in normal liver (123). Tumors
(at least solid tumors) also tend to have low oxygen tension
(124). These and other properties can be used to design more
effective drugs which could be inactivated by normal cells but
not by tumor cells. To date the research on the identification
of specific P-450s in the metabolism of cancer chemotherapeutic agents has been limited to a few instances such as cyclophosphamide (125, 126), procarbazine (127), and l-(2-chloroethyl)-3-(cyclohexyl)-l-nitrosourea
and l-(2-chloroethyl)-3(fra/Js-4-methylcyclohexyl)-l-nitrosourea(128).
The knowledge
of human enzymes involved in the metabolism of these and
other chemotherapeutic agents is miniscule and much more
could be done. Further, only limited information is available
concerning the role of P-450 enzymes in the multiple drug
resistance phenomenon encountered in tumor cells (129, 130).
Most cancer chemotherapeutic agents produce a number of side
effects. The rates of metabolism of these drugs are also known
to vary considerably among different individuals (131). Thus, a
better understanding of the enzyme systems that process these
compounds would be of great use in designing therapies that
will avoid unwanted toxicities. Also, in some cases the drugs
are metabolized too rapidly and either enhanced toxicity or lack
of therapeutic effectiveness can result.
7. As We Accumulate More and More Information about
Details of the Enzymology and Chemistry Related to the Metab
olism of Each Chemical, How Can We Keep This Knowledge in
Perspective? This final point is perhaps more philosophical.
Already we have amassed much information but not in the case
of any compound are our data complete. If we do all of the
experiments suggested in the above sections, will we have so
much to deal with that we will be unable to put our knowledge
into any sort of perspective?
The problem has not really been that we have collected too
much data, for the situation is clearly the opposite. However,
it can be said that a large fraction of experiments have not been
designed to put in vitro investigations into the context of in vivo
animal work. More emphasis is needed on systems in which
tumors have been shown to be produced. Thus, perhaps some
of the emphasis should be shifted away from metabolism in
rabbits (where the enzymology has been excellent but tumors
are rarely studied) to mice (where the enzymology has not been
highly developed but many bioassays have been done).
Increasing attention should be paid to the experimental hu
man studies, for in the last analysis this is the only system in
which tumors are of much concern to us per se. In vitro exper
iments establishing the roles of individual enzymes in the
metabolism of carcinogens are a first priority. These can be
complemented, in the case of the chemotherapeutic agents, by
in vivo studies; e.g., if an enzyme such as P-450MP is known to
metabolize mephenytoin (132) and a role in, for example,
procarbazine metabolism is assigned on the basis of in vitro
experiments, will individuals (undergoing therapy and high
doses of procarbazine) who are deficient in the ability to metab
olize mephenytoin also clear procarbazine at decreased rates?
Epidemiológica! links must also be strengthened through phenotyping studies. One of the most dramatic relationships between
a P-450-linked activity and a specific tumor lies in the report
of Ayesh et al. (89), which has been discussed above; however,
some controversy exists concerning the reproducibility of the
basic observation (90, 91), and to date there is no evidence that
a true metabolic relationship is an etiological factor. The rela
tionships between drug oxidation phenotype and bladder cancer
incidence reported by Kaisary et al. (93) also need further
development.
In summary, the entire field of relating cancer susceptibility
to alterations in P-450-related catalytic activities is still full of
promise. It would appear that many of the necessary research
techniques have now been developed, enabling serious and
comprehensive studies to be carried out, preferably with a focus
on a few select chemicals at first. The circumstantial evidence
that the enzymatic differences are important is strong. More
decisive experiments done in animal models and humans should
be forthcoming soon.
Acknowledgments
Thanks are extended to Drs. Fred F. Kadlubar and Lawrence J.
Marnett and several members of my research laboratory (Drs. Lisa A.
Peterson, Ghazi A. Dannan, and William R. Brian and Joan L. Cmarik)
for their comments on the article. I also thank Doris Harris for her
help in preparation of the manuscript. This "Perspective" is dedicated
to two eminent cancer research scientists who have been taken from us
in the past 2 years. Lubomir S. Hnilica, my former colleague and
Stallimeli Professor of Cancer Research at Vanderbilt University, was
an expert in the area of eliminatili research and my good friend; his
insight and sense of humor are still missed. Elizabeth C. Miller, of the
McArdle Laboratory for Cancer Research at the University of Wiscon
sin, was truly a pioneer in the field of chemical carcinogenesis. I was
always impressed by her high scientific standards, her enthusiasm, and
the encouragement she gave to me and others.
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Roles of Cytochrome P-450 Enzymes in Chemical
Carcinogenesis and Cancer Chemotherapy
F. Peter Guengerich
Cancer Res 1988;48:2946-2954.
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