Current Topics in Medicinal Chemistry 2004, 4, 1767-1788
1767
Cytochromes P450 in the Bioactivation of Chemicals
Costas Ioannides* and David F.V. Lewis
Molecular Toxicology Group, School of Biomedical and Molecular Sciences, University of Surrey, Guildford, Surrey,
GU2 7XH, UK
Abstract: The initial view that the cytochrome P450 enzyme system functions simply in the deactivation of xenobiotics is
anachronistic on the face of mounting evidence that this system can also transform many innocuous chemicals to toxic
products. However, not all xenobiotic-metabolising cytochrome P450 subfamilies show the same propensity in the
bioactivation of chemicals. For example, the CYP2C, 2B and 2D subfamilies play virtually no role in the bioactivation of
toxic and carcinogenic chemicals, whereas the CYP1A, 1B and 2E subfamilies are responsible for the bioactivation of the
majority of xenobiotics. Electronic and molecular structural features of organic chemicals appear to predispose them to
either bioactivation by one cytochrome P450 enzyme or deactivation by another. Consequently, the fate of a chemical in
the body is largely dependent on the cytochrome P450 profile at the time of exposure. Any factor that modulates the
enzymes involved in the metabolism of a certain chemical will also influence its toxicity and carcinogenicity. For
example, many chemical carcinogens bioactivated by CYP1, on repeated administration, selectively induce this family,
thus exacerbating their carcinogenicity. CYP1 induction potency by chemicals appears to be determined by a combination
of their molecular shape and electron activation. The function of cytochromes P450 in the bioactivation of chemicals is
currently being exploited to design systems that can be used clinically to facilitate the metabolic conversion of prodrugs to
their biologically-active metabolites in cells that poorly express them, such as tumour cells, in the so-called gene-directed
prodrug therapy.
INTRODUCTION
The human body is continuously bombarded by an
immense diversity of xenobiotics, both natural and
anthropogenic, which it cannot exploit in any way to its
advantage, and so they are undesirable. By far the vast
majority of xenobiotics that find their way into the human
body are naturally-occurring, largely phytochemicals,
although the relatively few man-made chemicals have
received most attention by toxicologists, since the focus of
regulation worldwide is largely confined to these. The body
recognises the xenobiotics as foreign, potentially detrimental
to its survival, and its immediate response is to protect itself.
In order for this objective to be achieved, the body has
developed, as a first line of defence, a number of transporter
systems, such as the P-glycoprotein, which prevent their
absorption through the gastrointestinal tract by facilitating
their efflux from the enterocytes into the lumen [1-3]. For
chemicals which, at least partly, overcome this obstacle and
enter the body, the route of elimination is through excretion.
However, as most of these chemicals are largely lipophilic,
an essential requirement for them to traverse the body’s
lipoid membranes, they are very difficult to eliminate
through excretion in the bile and urine. Consequently, in
order to facilitate their excretion, the body has to
metabolically convert them to readily excretable hydrophilic
entities. The body has, therefore, developed a number of
enzyme systems that ensure these lipophilic chemicals are
rendered hydrophilic through metabolism. Moreover, such
*Address correspondence to this author at Molecular Toxicology Group,
School of Biomedical and Life Sciences, University of Surrey, Guildford,
Surrey, GU2 7XH, UK; Tel: 01483 689709; Fax: 01483 576978; E-mail:
[email protected]
1568-0266/04 $45.00+.00
metabolism, in most cases, results in the abolition of their
biological activity, as these metabolites, in contrast to the
parent compounds, are unable to reach their site of action
and/or fail to interact with the appropriate receptors.
A number of enzyme systems, capable of metabolising
xenobiotics have been identified and extensively investigated
[4]. Undoubtedly, the most important enzyme system is the
cytochrome P450-dependent mixed-function oxidases, a
ubiquitous system of haem-thiolate enzymes encountered in
almost every human organ, but with the highest
concentration in the liver, which consequently functions as
the centre of xenobiotic metabolism, being capable of
catalysing both oxidation and reduction pathways [5].
Although this review will concentrate on the role of
cytochrome P450 in the metabolism of exogenous chemicals,
it should be emphasised that this enzyme system is also a
crucial catalyst of the metabolism, both biosynthesis and
degradation, of endogenous substrates including steroid and
other hormones, eicosanoids and certain vitamins [6].
CHARACTERISTICS OF THE CYTOCHROME P450
SYSTEM
A principal attribute of the cytochrome P450 system is
the unprecedented broad substrate specificity it displays,
explaining its pivotal role in xenobiotic metabolism. It
catalyses efficiently the metabolism of chemicals varying
enormously in their size and shape. It metabolises small
molecular weight compounds such as the solvent methanol
(MW=42) as well as large molecular weight compounds
such as the immunosuppressant cyclosporin (MW=1203); it
effects the metabolism of planar molecules such as benzo[a]
pyrene, a carcinogenic polycyclic aromatic hydrocarbon, as
© 2004 Bentham Science Publishers Ltd.
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Current Topics in Medicinal Chemistry, 2004, Vol. 4, No. 16
well as of globular molecules such as the anticonvulsant
phenobarbitone. Naturally, no single enzyme can
accommodate in its substrate-binding site such a diversity of
substrates, and thus it is not surprising that cytochrome P450
is not a single enzyme, but a superfamily comprising a
number of enzymes, each with a different substrate
specificity. This cytochrome P450 superfamily is divided
into a number of families, which in turn are subdivided into
subfamilies, each of which may consist of one or more
enzymes (isoforms). Classification of cytochrome P450
enzymes within families and subfamilies is carried out
strictly on the basis of primary sequence homology, with no
consideration of their function in xenobiotic metabolism.
Cytochrome P450 enzymes belonging to the same family
share at least a 40% structural homology whereas homology
between enzymes within a subfamily is at least 55 %. For
example, CYP3A4 enzyme denotes a cytochrome P450
protein belonging to family 3, subfamily A and is protein 4.
The families responsible for the metabolism of xenobiotics
are CYP1 to CYP3, and to a much lesser extent CYP4,
whereas cytochrome P450 enzymes belonging to other
families are concerned with the metabolism of endogenous
substrates. It is believed that over 90% of drug oxidations in
humans are catalysed by the first three families [7].
BIOACTIVATION OF XENOBIOTICS
For a long time, it was widely believed that the exclusive
outcome of cytochrome P450-mediated metabolism was an
increase in a chemical’s polarity, leading to its enhanced
excretion. In the 70s, however, it was realised that for the
majority of chemicals, toxicity is mediated by metabolites
and not by the parent compounds. Studies with 2acetylaminofluorene revealed that, for this compound to
express its hepatocarcinogenicity, metabolism through Nhydroxylation was essential, and this reaction was catalysed
by the cytochrome P450 system, later shown to be the CYP1
family. Thus, paradoxically, the same system that for such a
long time had been associated with the deactivation and
elimination of chemicals, could metabolise chemicals to
electrophilic intermediates that could promote carcinogenicity and other forms of toxicity through irreversible
interaction with cellular macromolecules, by engaging into
covalent bonding. This process by which an innocuous
chemical could be metabolically converted to a reactive
metabolite, potentially deleterious to the cell is referred to as
‘bioactivation’ or ‘metabolic activation’. For most chemicals,
their toxicity is inextricably linked to their metabolism which
generates electrophiles such as epoxides, carbonium ions and
nitrenium ions. Any factor that influences their metabolism,
quantitatively and/or qualitatively, will also modify their
toxicity.
Although cytochromes P450 frequently catalyse the first
step in the bioactivation of most chemicals, other enzyme
systems, such as the sulphotransferases and acetylases, are
also essential in the generation of the ultimate toxic species,
the entity that interacts with the cellular macromolecules. As
already discussed, the carcinogen 2-acetylaminofluorene
undergoes cytochrome P450-mediated N-hydroxylation to
yield the hydroxylamine; this further esterifies to form the
sulphatoxy or acetoxy esters which break down spontaneously
Ioannides and Lewis
to release the nitrenium ion, believed to be the ultimate
genotoxic intermediate that interacts with DNA. Clearly, the
cytochrome P450 enzymes act in concert with other
xenobiotic-metabolising systems to produce eventually the
reactive intermediates. If the reaction leading to a reactive
intermediate of a chemical cannot be catalysed by the body’s
enzyme systems, then this chemical will not effect toxicity.
For example, 4-aminobiphenyl is a human carcinogen whose
activation proceeds through a cytochrome P450-catalysed Nhydroxylation to form a genotoxic hydroxylamine. The
hydroxylamine of its isomer, 2-aminobiphenyl, is also
genotoxic but, because neither cytochromes P450 nor any
other enzyme system can effect the N-hydroxylation, this
compound is devoid of carcinogenic activity [8].
The generated reactive intermediates may interact with
DNA, as already described for 2-acetylaminofluorene, to
form adducts which, if they escape the repair mechanisms of
the cell, may be fixed and passed to the progeny, thus giving
rise to mutations ‘Fig. (1)’. The reactive intermediates of
chemicals may also interact covalently with proteins,
disturbing physiological homeostasis, leading to cell death. It
is now recognised that reactive intermediates may also
function as haptens, conferring on proteins antigenic
potential and eliciting immunotoxicity [9, 10]. Drugs, such
as the antimalarial amodiaquine, are metabolically converted
to metabolites that bind covalently to proteins to generate
neoantigens resulting in the production of autoantibodies.
Amodiaquine is oxidised to the electrophile, amodiaquine pquinoneimine, and this electrophile metabolite binds
covalently to proteins to produce neoantigens. Subsequent
exposure to this drug may provoke an autoimmune response
leading to hepatitis. Paracetamol (acetaminophen) is also
oxidised to a quinoneimine which, following interaction with
proteins, gives rise to cell death.
Reactive intermediates can induce toxicity and
carcinogenicity through an additional mechanism, involving
interaction with molecular oxygen to yield short-lived highly
reactive oxygen species, such as the superoxide anion which,
in the presence of traces of iron salts, can be transformed to
the highly reactive hydroxyl radical (OH·), a powerful
oxidant. Indeed, reactive oxygen species are currently being
implicated in the aetiology and progression of a number of
major chronic diseases including atherosclerosis, cancer,
cardiovascular disease, diabetes, rheumatoid arthritis,
reperfusion injury and ischaemia [11-14]. Reactive oxygen
species (ROS) can elicit cellular damage similar to that
resulting from the covalent interaction of the reactive species
of chemicals with cellular components; they oxidise DNA to
induce mutations, oxidise lipids to form lipid peroxides
which appear to play an important role in the promotion and
progression stages of chemical carcinogenesis, and also
oxidise proteins [15]. For example quinones, oxidation
products of aromatic hydrocarbons, can undergo oneelectron reductions to form the semiquinone radical, which
may directly attack DNA. It causes DNA damage also
indirectly, through reactive oxygen species produced as a
consequence of their interaction with molecular oxygen [16].
.
The most reactive oxygen species is the hydroxyl radical
(OH ). It possesses an unpaired electron and so tends to
form bonds with other species in order for the unpaired
Cytochromes P450 in the Bioactivation of Chemicals
Current Topics in Medicinal Chemistry, 2004, Vol. 4, No. 16
1769
Fig. (1) Bioactivation of chemicals.
electron to become paired and thus stable. Other biologically
relevant reactive oxygen species are the superoxide anion,
•
also a radical (O–
2 ), and hydrogen peroxide (H2O2). In the
•
presence of iron, the OH can be generated from either O–
2
(Haber-Weiss reaction) or H2O2 (Fenton reaction).
.
Cytochrome P450 enzymes can also function as
generators of deleterious reactive oxygen species when
NADPH is oxidised by undergoing futile cycling in the
absence of substrate metabolism [17]. The superoxide anion
is released which, by the action of superoxide dismutase,
may be converted to hydrogen peroxide that yields the
hydroxyl radical in the presence of iron.
FATE OF REACTIVE INTERMEDIATES
Liver is the principal site of the bioactivation of
chemicals since most xenobiotic-metabolising enzyme
systems are expressed in this tissue at high concentrations.
Extrahepatic tissues contain a more restricted number of
enzyme systems, generally being present at much lower
concentrations compared with the liver. Therefore, it would
be logical to expect that the liver would also be the major
site of chemically-induced tumours. In reality, however, the
breast, lung and colon are far more frequent sites of
tumorigenesis, despite their limited metabolic competence.
This raises the possibility that reactive intermediates
produced in the liver may be exported systemically to other
tissues where they can exert their deleterious effects. The
breast is a frequent site of tumorigenesis despite the fact that
its capacity to metabolise and bioactivate chemicals through
oxidation appears to be minimal [18]. It has been proposed
that heterocyclic carcinogenic amines, such as PhIP (2amino-1-methyl-6-phenylimidazo[4,5-b]pyridine), are Noxidised in the liver and, in the form of either the
hydroxylamine or following esterification to the
acetoxyester, are then transported to extrahepatic tissues
such as the colon to yield DNA adducts [19]. Extrahepatic
tissues appear capable of catalysing the esterification of
hydroxylamines but poor in catalysing the initial oxidation of
the parent heterocyclic amine as a result of the very low
levels of cytochrome P450 [20]. Polycyclic aromatic
hydrocarbons, such as benzo[a]pyrene, are potent pulmonary
carcinogens. When the liver from rats pretreated with
benzo[a]pyrene were transplanted in untreated animals, the
extent of DNA binding in the lung, liver and kidney was the
same in both, those animals exposed directly to the
carcinogen and those who received the liver transplants,
implying that the liver was the major source of the of the
reactive intermediates that interacted with DNA, not only for
the liver, but also for the lung and kidney [21]. It appears
that reactive intermediates may be sufficiently stable to
traverse membranes to reach tissues distant to the generating
tissue. In the case of aromatic amines, the reactive
hydroxylamine is stabilised by forming a glucuronide that is
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Current Topics in Medicinal Chemistry, 2004, Vol. 4, No. 16
transported to the bladder where it is released to exert its
carcinogenic effect.
DETOXICATION OF REACTIVE INTERMEDIATES
The living organism is endowed with a number of
defensive mechanisms that protect its DNA from genotoxic
attack by chemical reactive intermediates and reactive
oxygen species. The most prominent enzyme system in the
defence against reactive intermediates is the glutathione Stransferases, a superfamily of largely cytosolic proteins
expressed in most tissues [22]. These enzymes catalyse the
conjugation of the tripeptide glutathione, a nucleophile
present intracellularly at high levels, with electrophilic
reactive intermediates, forming a bond between the sulphur
of the cysteine moiety of glutathione and the electrophile.
The generated conjugate is further processed enzymically
and excreted in the urine or bile as the N-acetylcysteine
conjugate (mercapturate).
A number of enzyme systems protect the cell against
oxidative damage by effectively detoxifying these reactive
oxygen species, and prevent a state of ‘oxidative stress’,
where the cell is unable to cope with the generation of
reactive oxygen species. Such a state may ensue as a result
of overproduction of reactive oxygen species and/or a
decreased ability to deactivate them. Hydrogen peroxide is
broken down enzymically by glutathione peroxidase, an
enzyme present in the cytosol and mitochondria, and
catalase, a peroxisomal enzyme ‘Fig. (2)’. Superoxide
dismutase, an enzyme localised in the mitochondria and
cytosol, is an effective defence against the superoxide anion,
and NAD(P)H-quinone reductase (DT-diaphorase), an
enzyme found primarily in the cytosol, protects against
quinone-derived oxygen radicals by converting the quinone
to the hydroquinone through a two-electron reduction. An
increasing number of chemicals are known to function as
antioxidants, scavenging oxygen radicals, and in this way
afford protection against oxidative damage. Such
antioxidants include endogenous chemicals, e.g. uric acid,
melatonin etc., and numerous phytochemicals that are
ingested with food in substantial amounts, e.g. polyphenols.
Glutathione
peroxidase
Catalase
2H2O + O2
Superoxide dismutase
H2 O2 + O2
O2 + O2 + 2H
O
O
OH
DT-diaphorase
DT-diaphoras e
BALANCE OF ACTIVATION/DEACTIVATION
Carcinogens and other chemical toxins are metabolised
simultaneously through a number of routes, most of which
result in the generation in biologically inactive products, and
are accordingly regarded as deactivation pathways. In most
cases it is only a single, usually minor pathway, that is
associated with the production of genotoxic intermediates
and the cellular defensive mechanisms can deal effectively
with the low levels of reactive intermediates that are
produced. If an animal species, however, favours the
activation of a carcinogen then it would be vulnerable to its
carcinogenicity whereas, if its metabolism proceeds
primarily through the deactivation pathways, then it will
display resistance. Species variations in chemical
carcinogenesis usually reflect differences in the metabolic
pathways and the balance of activation/deactivation. Many
examples have been documented where various animal
species displayed a markedly different response to the same
chemical. For example, the guinea pig is resistant to the
carcinogenicity of aromatic amines such as 2-acetylaminofluorene [23] whereas the cynomolgus monkey is
refractive to the carcinogenicity of the heterocyclic amine 2amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) [24],
and these have been attributed to genetic differences in the
metabolism of these carcinogens.
To illustrate the pathways of activation/deactivation that
chemical carcinogens are simultaneously subjected to,
the food carcinogen IQ (2-amino-3-methylimidazo-[4,5f]quinoline), another heterocyclic amine, is used as an
example ‘Fig. (3)’. The activation pathway involves Nhydroxylation, followed by esterification of the
hydroxylamine with sulphate and acetate to generate the
sulphatoxy and acetoxy esters respectively, which break
down spontaneously to yield the nitrenium ion, the presumed
ultimate carcinogen. Ring hydroxylation and conjugation of
the parent compound or the ring-hydroxylated metabolite are
strictly deactivation pathways. The ring-hydroxylated
metabolite is eventually excreted in conjugated form.
Any factor that disturbs the delicate balance between
activation and deactivation will alter the response to the
toxicity of a chemical. Such situations may arise when:
•
GSSG + 2H2O
2GSH + H2O2
2H2O2
Ioannides and Lewis
O
O
Quinone
Semiquinone radical
OH
Hydroquinone
Fig. (2). Enzyme systems protecting against reactive oxygen
species.
GSH, reduced glutathione.
The deactivation pathways are saturated or impaired,
so that more of the metabolism is directed towards the
activation pathway. For example, the hepatotoxicity
associated with paracetamol overdose is due to the
fact that its principal deactivation pathways, through
conjugation with sulphate and glucuronic acid, are
saturated because the levels of the essential cofactors
3′-phosphoadenosine-5′-phosphosulphate (PAPS) and
uridine diphosphate glucuronic acid (UDPGA), the
activated forms of sulphate and glucuronic acid
respectively, are depleted. As a consequence, the
bioactivation step through cytochrome P450 oxidation
to produce a reactive quinoneimine (Nacetylbenzoquinoneimine), which at normal doses is a
minor metabolic step, assumes greater importance so
that the levels of the hepatotoxic intermediate rise. As
the quinoneimine is normally detoxicated by
glutathione conjugation, the increased demand for
Cytochromes P450 in the Bioactivation of Chemicals
Current Topics in Medicinal Chemistry, 2004, Vol. 4, No. 16
1771
N+H
N
N
CH3
N
Nitrenium ion
H+
H
N
OSO3
H
OCOCH3
N
N
OH
NH
N
N
N
CH3
N
N
CH3
N
N-Sulphatoxy IQ
H
N-Acetoxy IQ
OH
O
C
NH2
CH3
N
N
N
N
CH3
N
N-Acetyl IQ
NH2
CH3
H
N
N
N
N
N
N-Hydroxy IQ N-glucouronide
IQ
SO3
GlcA
N
H
N
N
N
CH3
N
CH3
N
N-Hydroxy IQ
N
N
CH3
N
GlcA
N
CH3
N
CH3
N
N
IQ N-Sulphamate
OH
N
5-Hydroxy IQ
IQ N-Glucuronide
5-O-conjugation with sulphate
or glucuronic acid
Fig. (3). Metabolic activation and deactivation of IQ.
IQ, 2-amino-3-methylimidazo-[4,5-f]quinoline.
glutathione leads to its eventual depletion, so that the
body defences are unable to cope with the higher
levels and the body succumbs to its toxicity.
Similarly, genetic deficiencies in conjugating systems
may lead to increased sensitivity to the toxicity of
chemicals that rely heavily on these enzymes for their
deactivation. For example, in humans, glucuronyl
transferase activity may be totally lacking in
individuals with the Crigler-Najjar syndrome type I, a
severe and fatal disease. These patients, being unable
to eliminate bilirubin through glucuronidation,
develop jaundice [25]. Such patients may be sensitive
to drugs whose principal pathway of metabolism is
through glucuronidation. A milder condition is
Gilbert’s syndrome where the patient experiences
only intermittent jaundice [25]. These patients display
low glucuronidation capacity, and when taking
paracetamol they excrete less in the form of
glucuronides, with more of the metabolism being
directed towards oxidation forming the hepatotoxic Nacetylbenzoquinoneimine [26]. The anticancer drug
iminotecan provokes severe toxicity in patients with
Gilbert’s syndrome as a result of suppressed
glucuronidation [27]. Moreover, intake of drugs metabolised by glucuronidation may induce jaundice, as
the drug competes with bilirubin for glucuronidation
[28]. Clearly, genetic polymorphisms in xenobioticmetabolising enzymes, including cytochromes P450,
glutathione S-transferases and N-acetylases may
influence significantly the biological outcome
following exposure to toxic compounds [29, 30].
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•
•
Current Topics in Medicinal Chemistry, 2004, Vol. 4, No. 16
Ioannides and Lewis
The detoxication of the electrophilic reactive
intermediates is suppressed and as a result these
accumulate and the toxic effect is exacerbated.
Bromobenzene, a toxic compound, is metabolically
converted to an epoxide that mediates its toxicity ‘Fig.
(4)’. The epoxide may rearrange to bromophenols or
be deactivated by hydration, catalysed by epoxide
hydrolase, or by conjugation with glutathione,
catalysed by the glutathione S-transferases. However,
in situations where the levels of glutathione are
limiting, such as when rats have been starved, the
toxicity of bromobenzene rises dramatically ‘Fig. (5)’.
characterises this disease. The ketone, acetone, is an
established potent inducer of CYP2E1. Induction of
cytochrome P450 enzymes has also been associated to
higher chemical carcinogenicity, and this will be
discussed in more detail (vide infra).
•
Many of the xenobiotic-metabolising enzyme
systems, and especially the cytochromes P450, may
be up-regulated as a result of previous exposure to
chemicals, both natural and synthetic [31], or the
presence of disease [32]. If the cytochrome P450
enzyme(s) that functions as the major catalyst of the
bioactivation of a chemical is induced, then increased
production of reactive intermediates will lead to
enhanced toxicity. One of the cytochrome P450
enzymes catalysing the bioactivation of paracetamol
to the reactive quinoneimine (vide supra), namely
CYP2E1, is elevated as a result of alcohol exposure,
and consequently chronic alcoholics are characterised
by high levels of activity. As a result they are
vulnerable to the hepatotoxicity of paracetamol [33,
34]. Increased levels of CYP2E1 have been observed
in patients with insulin-dependent diabetes as well as
in animal models of the disease [32, 35]. In this
case, the up-regulation of this enzyme appears to
be a consequence of the hyperketonaemia that
Increased deactivation of reactive intermediates
primarily through enhanced enzymic conjugation with
glutathione. During the last decade a number of
dietary phytochemicals have been identified, capable
of antagonising the carcinogenicity of chemicals in
animal models. Increased detoxication of chemical
reactive intermediates, as a result of enhanced
glutathione S-transferase activity, is believed to
contribute significantly to their anticarcinogenic
activity. For examples indoles and isothiocyanates,
both components of cruciferous vegetable, are potent
inducers of glutathione conjugation [36, 37].
CYTOCHROME
P450
ENZYMES
IN
THE
BIOACTIVATION OF CHEMICAL CARCINOGENS
AND OTHER TOXINS
Although most xenobiotic-metabolising enzyme systems
may participate in chemical bioactivation, unquestionably
the cytochromes P450 are by far the most prominent. The
unprecedented versatility of the cytochrome P450 system in
the metabolism of both exogenous and endogenous
substrates is the characteristic that sets it aside from other
enzyme systems There are striking differences, however, in
the role of individual cytochrome P450 enzymes in the
metabolic activation of chemicals, with some isoforms
making virtually no contribution. For example, in the case of
Br
Bromobenzene
Br
Br
Covalent
binding to proteins
OH
3-Bromophenol
Br
Heptatotoxicity
O
Bromobenzene 3,4-oxide
GSH
OH
GSH
Br
Br
Br
4-Bromophenol
H
HO
H
OH
Bromobenzene 3,4-dihydrodiol
Fig. (4). Metabolic activation and deactivation of bromobenzene.
GSH, reduced glutathione.
OH
SG
SG
OH
Cytochromes P450 in the Bioactivation of Chemicals
Current Topics in Medicinal Chemistry, 2004, Vol. 4, No. 16
1773
Fig. (5). Effect of starvation on bromobenzene toxicity.
SGPT, serum glutamate-pyruvate transaminase. Data from [222].
2-naphthylamine, the activation process entails an initial Nhydroxylation that appears to be catalysed by a single
cytochrome P450 enzyme, namely CYP1A2, whereas a
number of enzymes catalyse the ring-hydroxylation reactions
which are deactivation pathways ‘Fig. (6)’ [38]. Similarly, in
coumarin metabolism, the activation pathway involving
arene oxidation to form the 3,4-epoxide is catalysed by
CYP1A enzymes whereas detoxification through 7hydroxylation is brought about by CYP2A6 [39].
Of the various xenobiotic-metabolising enzyme systems,
cytochromes P450 are the most sensitive to chemical
exposure that may lead to induction or inhibition of their
expression [40], and may have clinical and toxicological
consequences, leading to drug-drug [41], herbal-drug [42]
and diet-drug [31] interactions. Finally, cytochrome P450
induction can impact chemical carcinogenicity, and this will
be discussed in more detail (vide infra).
CYP1 FAMILY
One of the first cytochrome P450 families to be identified
and subsequently purified, CYP1 is consequently one of the
most extensively studied, originally called cytochrome P448
because of the absorption maximum of the CO complex with
the reduced cytochrome, in contrast to the other families, is
at 448 rather that at 450 nm. It is a small family comprising
two subfamilies, 1A and 1B. The former subfamily contains
two proteins whereas the latter contains a single protein.
CYP1A2 is primarily expressed in the liver, but the hepatic
levels of CYP1A1 and CYP1B1 are very low. In contrast,
the latter two proteins are present in appreciable amounts in
extrahepatic tissues [43, 44]. However, a CYP1A2-like
protein and activity have been reported to be present in the
human lung and other extrahepatic tissues [45-47].
In both human and rodent liver, CYP1 is not a major
cytochrome P450 family, comprising less than 5 % of the
cytochrome P450 content in the liver of rats and about 10 %
in human liver. Higher expression appears to occur in the
liver of cattle, deer and in some monkey strains compared
with the rat [48-50] as exemplified by the O-deethylation of
ethoxyresorufin, the most frequently employed marker for
CYP1 activity. CYP1 is probably the most conserved family
within the phylogenetic tree so that the human CYP1
proteins share extensive structural similarity and display
similar specificity to the orthologous rodent proteins,
alluding to an important role for this family in a vital
biological function [51]. In studies employing purified
cytochrome P450 proteins in reconstituted systems, CYP1A2
catalysed the oxidation of uroporphyrinogen to uroporphyrin
[52] and this isoform appears to be involved in chemicallyinduced uroporphyria, a condition associated with high
hepatic levels and urinary excretion of uroporphyrin and
other porphyrins. The involvement of CYP1A2 expression
was confirmed in Cyp1a2 knockout mice where no increase
in uroporphyrin was observed following treatment with
agents that induce uroporphyria in wild-type animals [53].
Recent studies have provided experimental evidence that
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Current Topics in Medicinal Chemistry, 2004, Vol. 4, No. 16
Ioannides and Lewis
H
N
OSO3
OH
NH2
1-hydroxy-2-naphthylamine
Sulphos transfer ase
NH2
2-naphthylamine
NH2
NHOH
H+
CYP1A2
NH2
N-sulphate-2-naphthylamine
CYP1A1, CYP1A2
CYP2B, CYP2C11
N-hydroxy-2-naphthylamine
CYP1A1
CYP1A2
O-acetyl-transferase
Nitrenium ion
H
N
OCOCH3
HO
6-hydroxy-2-naphthylamine
N-acetoxy-2-naphthylamine
Fig. (6). Role of cytochrome P450 enzymes in the activation and deactivation of 2-naphthylamine
CYP1A2 may be also involved in human porphyria cutanea
tarda, a rare condition that is also associated with elevated
uroporphyrin levels [54]. A role has been ascribed for this
isoform in the microsomal catabolism and elimination of
bilirubin [55, 56]. Consequently, induction of CYP1A2
would facilitate bilirubin elimination in conditions
characterised with hyperbilirubinaemia, such as the genetic
condition Crigler-Najjar syndrome type I. In Gunn rats, an
animal model of the disease, plasma levels of bilirubin fell
following treatment with the CYP1A2 inducer indole-3carbinol, a phytochemical present in cruciferous vegetables
[57]. CYP1A2 knockout mice, however, do not display
hyperbilirubinaemia [58], suggesting that, at least in this
species, the CYP1A2-mediated metabolism of bilirubin is
not critical to the elimination of the toxin, and glucuronide
conjugation is the prevailing detoxication pathway. Furthermore, the fact that the levels of CYP1 are downregulated in
the adult animal implies an important role in foetal and
neonatal development [59], which remains to be defined.
CYP1, however, is probably the most inducible family,
being induced by planar compounds in the liver and
extrahepatic tissues of animals and humans [60, 61].
Treatment with the polycyclic aromatic hydrocarbon, 3methylcholanthrene, elevates the expression of this family in
rats so that it comprises as much as 80 % of the total hepatic
cytochrome P450 content [62]. Induction is not confined to
the liver, but pulmonary and placental activities were also
induced as a result of smoking and following accidental
exposure to polychlorinated biphenyls in both animals and
humans [47, 63-67]. Intake of cruciferous vegetables,
presumably as a result of their indole content, as well as
numerous other dietary anutrients, including compounds
formed by the cooking process, such as polycyclic aromatic
hydrocarbons and heterocyclic amines, have been shown to
upregulate CYP1 activity in the liver and other tissues [31,
68, 69]. In humans, CYP1 expression can also be
upregulated, in both the liver and other tissues, by exposure
to therapeutic doses of drugs such as omeprazole and by
smoking [70, 71].
The substrates of the CYP1 family are essentially
lipophilic planar molecules, composed of fused aromatic
rings and characterised by a small depth and a large
area/depth2 ratio, which presumably facilitates their
interaction with DNA [72, 73]. 4-Aminobiphenyl is such a
planar molecule and is readily N-hydroxylated by CYP1A
but its isomer, 2-aminobiphenyl, being non-planar by virtue
of the amino group being at the ortho-position is not
similarly N-hydroxylated by CYP1A, and consequently lacks
carcinogenic activity [8]. However, the width of the
molecule may also be critical as molecules characterised by a
large length may be denied access to the substrate-binding
site [74]. Computer modelling studies revealed that the
molecular dimensions of the active of the CYP1A1 isoform
are characterised by a depth of 3.261 Å, hence the
requirement of planarity, and a width of 8.321Å, so that
molecules having a larger width, even if planar, are excluded
e.g. benzo(e)pyrene and 4-acetylaminoflluorene [74].
CYP1 is undoubtedly the dominant cytochrome P450
family in the bioactivation of chemicals, including many
major and ubiquitous classes of carcinogens to which
humans are unavoidably exposed, such as air pollutants and
dietary contaminants. Although the initial experimental
studies on the role of CYP1 enzymes in the bioactivation of
chemical carcinogens were conducted using animal tissues
and isolated proteins, subsequent work using human tissues
and proteins confirmed the observations in animals. They are
believed to be responsible for the activation of more than 90
% of known carcinogenic chemicals [75]. Most ubiquitous
environmental and dietary carcinogens to which humans are
frequently exposed are molecularly planar in nature, and
therefore favoured substrates of the CYP1 family. The CYP1
Cytochromes P450 in the Bioactivation of Chemicals
family, through arene oxidation and N-oxidation activates
polycyclic aromatic hydrocarbons, aromatic amines,
aflatoxins, aminoazobenzenes and heterocyclic amines
[60,76-79]. Generally, CYP1A1 is more effective in
catalysing arene oxidation and CYP1A2 N-oxidation [60].
However, CYP1B1 is also an excellent catalyst of the arene
oxidation of polycyclic aromatic hydrocarbons, and can
catalyse both of the cytochrome P450-mediated oxidations
that are necessary to produce the ultimate carcinogenic
species, the dihydrodiol epoxides [78]. In fact, of the three
members of the CYP1 family, CYP1B1 was the most
efficient in converting benzo[a]pyrene to its 7,8-diol,
benzo[c]phenanthrene to its 3,4-dihydrodiol, the precursors
of the ultimate carcinogens, dibenz[a]anthracene to the 3,4oxide, the proximate carcinogen, and dibenzo[a,l ]pyrene to
DNA-binding adducts (77, 80-82]. Its importance in the
bioactivation of polycyclic aromatic hydrocarbons can be
seen by the observation that lymphoma incidence following
exposure to 7,12-dimethylbenz[a]anthracene was much
lower in CYP1B1-null mice compared with the wild-type
mice [83]. All three members of the CYP1 family have also
been shown to be effective in the activation of the tobaccospecific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1butanone (NNK), a metabolite of nicotine [84, 85]. Both
CYP1A proteins, especially CYP1A1, are involved in the
hydroperoxide-dependent oxidation of catechol oestrogens
including the carcinogenic synthetic stilbene, diethylstilboestrol, to the genotoxic quinones [86]. CYP1B1 also
hydroxylates 17β-oestradiol, primarily at the 4-position and
appears to be the principal catalyst of this hydroxylation
pathway, whereas CYP1A1 metabolises the steroid less
efficiently, at different positions [87]. In subsequent studies,
CYP1A2 was shown to be an important catalyst of the 2- and
4-hydroxylations of oestradiol and oestrone [88]. It has been
suggested that the 4-hydroxyoestradiol can directly interact
covalently with DNA, or be a precursor for such a genotoxic
metabolite, probably a quinone or a semiquinone, which may
also act as a generator of genotoxic free radicals and may be
involved in the aetiology of breast cancer. In the hamster
kidney tumour model, the 4-hydroxyoestrogen, but not the 2isomer, induced tumours [89-91]. In the liver, the 4hydroxyoestradiol is a minor metabolite but considerable
activity has been detected in extrahepatic tissues [91]. High
activity is encountered in human tumours, including breast
tumours, where higher activity is found in the cancerous
tissue than in adjacent normal tissue, implying a role in the
pathogenesis of hormonal cancers [92]. Interestingly,
CYP1B1 appears to be over-expressed in many human
tumours [93] which is in contrast to other cytochrome P450
proteins whose levels are usually depressed in tumours [32].
As a result of their tissue localisation, CYP1A2 is more
active in the liver whereas CYP1B1 and CYP1A1 are more
active in extrahepatic tissues such as the lungs. Because of
its prominent role in the activation of chemical carcinogens,
high levels of CYP1 are considered undesirable [94].
A number of studies have linked CYP1 activity to human
cancer incidence. High levels of CYP1A in human peripheral
lymphocytes have been directly correlated with susceptibility
to lung cancer in smokers [95]. A very significant correlation
has been reported between pulmonary levels of CYP1A1 and
DNA adducts levels with the tobacco-derived polycyclic
Current Topics in Medicinal Chemistry, 2004, Vol. 4, No. 16
1775
aromatic hydrocarbons [96]. The belief was that humans
displaying high levels of CYP1 as a result of smoking were
more effective in metabolically converting tobacco smoke
polycyclic aromatic hydrocarbons to their genotoxic
metabolites. Clear positive relationships have been
established between lung cancer and CYP1A1 in the lung of
smokers [97, 98]. What is of interest is that, among smokers,
those who develop tumours display high CYP1A1 activity
[99]. It is noteworthy that the bioactivation of polycyclic
aromatic hydrocarbons by CYP1A appears to occur in the
same part of the airways and in the same cell types in which
peripheral carcinomas are observed [98]. In more recent
studies, a positive correlation was established between
aromatic/hydrophobic DNA adduct levels and CYP1A1
determined immunohistochemically [100].
CYP2 FAMILY
This is the largest mammalian cytochrome P450 family,
comprising a number of distinct subfamilies which, unlike
the CYP1 subfamilies, exhibit markedly different substrate
specificity, and are under different regulatory control.
CYP2A Subfamily
This subfamily is composed of three members in humans,
namely CYP2A6, 2A7 and 2A13 which comprise only about
1 % of the total hepatic cytochrome P450. Three proteins are
also expressed in the rat, CYP2A1, CYP2A2 and CYP2A3.
The CYP2A subfamily is also expressed in extrahepatic
tissues such as testes, Leydig cells and the olfactory mucosa
[101, 102]. At least at the mRNA level, CYP2A13 is
expressed primarily in extrahepatic tissues, particularly the
nasal mucosa, with only low levels in the liver [103]. A
characteristic of CYP2A is that the rodent proteins exhibit
different substrate specificity when compared with the
human orthologues. The rat protein CYP2A1 effectively
hydroxylates steroids at the 7α-position but is unable to
hydroxylate the anticoagulant drug coumarin at the 7position, whereas the human CYP2A6 readily hydroxylates
coumarin but is not a steroid hydroxylator [104]. The cattle
and cervine livers resemble the human protein in that they
display high coumarin 7-hydroxylase activity [49, 50]. Rat
CYP2A2, structurally similar to CYP2A1, hydroxylates
testosterone principally at the 15α-position [105]. The last
member of this subfamily in the rat, CYP2A3, appears to be
expressed in the lung and olfactory mucosa, but not in the
liver or other tissues [106, 107].
The role of CYP2A proteins in the bioactivation of
environmental chemical carcinogens is not extensive. They
appear to make a significant contribution to the human
hepatic metabolic activation of N-nitrosamines, including a
number of tobacco nitrosamines, and of 1,3-butadiene
[85,108-110]; however, in most cases the principal catalyst
of the bioactivation of these compounds is CYP2E1. In the
case of tobacco nitrosamines, however, in studies utilising
Salmonella strains engineered to express human cytochromes
P450, CYP2A6 was the most efficient of the isoforms
studied, followed by CYP2E1, in catalysing the bioactivation
of N-alkylnitrosamines, such as dimethylnitrosamine, and of
cyclic nitrosamines, such as N-nitrosopiperidine [85]. On the
whole, it appears that human CYP2A6 is the major catalyst
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Current Topics in Medicinal Chemistry, 2004, Vol. 4, No. 16
of the activation of alkylnitrosamines having a bulky chain,
whereas CYP2E1 is active in the bioactivation of short-chain
alkylnitrosamines [111]. A role has also been ascribed for
the CYP2A subfamily in the activation of the fungal
contaminant aflatoxin B1 [112]; furthermore CYP2A6 may
make a small contribution to the activation, through Nhydroxylation, of the carcinogen 4,4-methylenebis(2chloroaniline) (MOCA) [113]. Rat CYP2A3 and human
CYP2A6, which are present in olfactory mucosa, can
bioactivate the nasal toxin 2,6-dibenzonitrile and the
carcinogen hexamethylphosphoramide [107]. CYP2A6
contributes to the 4-hydroxylation of the anticancer drug
cyclophosphamide leading to the formation of the
biologically active acrolein and phosphoramide mustard;
similarly it catalyses the 4-hydroxylation of ifosfamide
[114]. Human CYP2A13 displays high activity in the
bioactivation of the nitrosamine NNK [103].
In rats, CYP2A1 is modestly induced by chemicals such
as phenobarbital and β-naphthoflavone which, however, are
much more potent inducers of the CYP1A and CYP2B
subfamilies respectively [115]. In contrast, the same
inducing agents downregulate the hepatic levels of CYP2A2.
The anticancer drug cisplatin has also been shown to
stimulate the expression of CYP2A1 in the liver of rats
[114]. CYP2A3 also appears to be inducible [106]. There is
no evidence that the human CYP2A proteins are inducible,
however. Marked interindividual variation in CYP2A6
expression in human liver has been observed [116].
CYP2B Subfamily
In humans, CYP2B is a minor subfamily comprising only
a single enzyme (CYP2B6) and constitutes less than 2% of
the total hepatic cytochrome P450 content, but is also
expressed in extrahepatic tissues [117]. In rats the CYP2B
subfamily comprises at least six proteins, CYP2B1 and
CYP2B2 being the most extensively studied. CYP2B
displays a very broad substrate specificity metabolising a
wide variety of chemicals [118]. CYP2B1 and CYP2B2
appear to have identical substrate specificity, but the former
displays higher catalytic activity.
The CYP2B subfamily is highly inducible, especially by
phenobarbitone, the first identified potent inducer of the
cytochrome P450 family. Indeed, high inducibility, at least in
animals, is brought about by numerous structurally diverse
chemicals, including pesticides like DDT, and environmental
contaminants such as structurally non-planar polyhalogenated
biphenyls, and is one of its major features [118].
Its bioactivation reactions include metabolic transformation of cyclophosphamide to its pharmacologically active
metabolites (vide supra), and of cocaine in the rat [119-121].
Although it is involved in the metabolism of many chemical
carcinogens, CYP2B appears to direct the metabolism
primarily towards the formation of inactive metabolites, so
that a deactivating role has been ascribed to this subfamily
[60]. For example, CYP2B proteins can metabolise effectively
aromatic amines and amides, but they are unable to carry out
N-hydroxylation, the activation pathway, and only catalyse
ring-oxidations that lead to biologically inactive metabolites
[38, 122]. However, they participate in the bioactivation of a
Ioannides and Lewis
number of long-chain nitrosamines to genotoxic metabolites
[123] and of 4,4(bis)methylenechloroaniline [124]. The
human CYP2B6 protein has also been shown to contribute to
the activation of aflatoxin B1 and NNK [125].
CYP2C Subfamily
This is a large subfamily, composed of at least four
enzymes in humans (CYP2C8, CYP2C9, CYP2C18 and
CYP2C19) responsible for the metabolism of many major
drugs and constitutes as much as 20% of the total human
hepatic P450. The CYP2C isoforms are modestly induced by
drugs like phenobarbitone and dexamethasone in the liver of
animals such as rat and rabbit, and experimental evidence
has been presented that induction of this subfamily occurs
also in humans exposed to rifampicin [126]. In rats, chronic
alcohol intake has been shown to enhance the expression of
CY2C7, involved in the 4-hydroxylation of retinoids, in the
liver and colon [127].
The human CYP2C subfamily appears to play a very
minor role in the bioactivation of chemical carcinogens
[128]. In rats, however, members of the CYP2C family are
efficient in converting aflatoxin B1 to the reactive 8,9epoxide [129] whereas human CYP2C enzymes can
bioactivate polycyclic aromatic hydrocarbons, but much less
effectively compared with CYP1A1, and thus may contribute
to their hepatic bioactivation [130, 131]. The human CYP2C
subfamily is responsible for the metabolic conversion of the
diuretic drug tienilic acid to yield a protein-interacting
intermediate, believed to be a sulphoxide, which can
function as a hapten eliciting immunotoxicity [10].
CYP2D Subfamily
In humans, CYP2D6, the only member of the CYP2D
subfamily, is the first recognised polymorphically-expresed
cytochrome P450 enzyme and is a principal catalyst of the
metabolism of currently used major drugs [132]. In rat six
isoforms are expressed all of which metabolise drugs such as
bufurarol [133]. However, in deer and cattle this subfamily
appears to be poorly expressed [49, 50]. CYP2D proteins
appear to be strictly constitutive and no inducer has so far
been identified. No major role in the bioactivation of
chemicals is associated with this subfamily. However, it has
been implicated in the bioactivation of the anticancer drug
tamoxifen and of the carcinogen NNK [134].
CYP2E Subfamily
In both humans and animals this subfamily consists of a
single enzyme (CYP2E1), the only exception being the
rabbit where two proteins appear to be expressed. It is
expressed in both hepatic and extrahepatic tissues [117, 135].
The CYP2E subfamily, in common with the CYP1A, is one
of the most conserved subfamilies in animal species, and the
orthologues share the same substrate specificity. A possible
endogenous role is in the metabolism and elimination of
acetone [136], and the increase in activity seen in conditions
of hyperketonaemia e.g. insulin-dependent diabetes and
starvation, may be viewed as an adaptive response to
eliminate ketone bodies [32].
Cytochromes P450 in the Bioactivation of Chemicals
It is very active in the metabolism of small molecular
weight compounds such as short-chain alcohols and organic
solvents like ether and chloroform. CYP2E1 substrates are
characterised by a small molecular diameter of <6.5 Å [137].
CYP2E plays a dominant role in the metabolism of small
molecular weight carcinogenic compounds such as
azoxymethane and to a lesser extent of its metabolite
methylazoxymethanol, acrylonitrile, benzene, 1,3-butadiene,
nitrosamines like dimethylnitrosamine and nitrosopyrrolidine, and halogenated hydrocarbons such as carbon
tetrachloride and vinyl chloride [85, 113, 138]. Indeed,
benzene and carbon tetrachloride failed to induce toxicity in
CYP2E1-knockout mice whereas severe toxicity was seen in
the wild-type animals [139, 140]. Moreover, it converts
paracetamol to the hepatotoxic quinoneimine, and the
anaesthetic halothane to the trifluoroacetyl intermediate,
which binds covalently to hepatic proteins, generating
neoantigens that are responsible for the hepatitis associated
with this drug [10]. A major and toxicologically important
characteristic of the CYP2E subfamily is its high propensity
to generate reactive oxygen species [141]. In the rat,
CYP2E1 was the most active in comparison with other
cytochrome P450 enzymes in producing hydrogen peroxide
[142]. Reactive oxygen species are not only genotoxic (vide
supra), but can also stimulate cellular proliferation and
dysplastic growth, thus accelerating the promotional stage of
carcinogenesis. CYP2E may therefore facilitate carcinogenesis by two distinct mechanisms, namely the oxidative
activation of chemicals and by the generation of reactive
oxygen species.
CYP2E1 is an inducible enzyme, its levels being elevated
following exposure to small molecular weight xenobiotics
such as acetone, alcohol, pyrazole, imidazole, isoniazid, and
in humans it is elevated in chronic alcoholics and following
isoniazid intake [143, 144]. CYP2E1 is stimulated by alcohol
exposure and consequently chronic alcoholics are
characterised by high levels of activity, and consequently are
vulnerable to the toxicity of drugs such as paracetamol (vide
supra).
CYP2F Subfamily
This subfamily, which appears to contain a single gene, is
poorly expressed in the human liver but is present in the
lung. It metabolises the lung toxin 3-methylindole (skatole)
[145]. In mouse lung it is localised in Clara cells and can
catalyse the oxidation of benzo[a]pyrene to the 7,8-epoxide,
as well as epoxidation of the pulmonary toxin naphthalene
[146].
CYP2G Subfamily
CYP2G1 is a protein expressed only in the olfactory
mucosa of animals. The murine protein has been shown
recently to catalyse the bioactivation of the drug paracetamol
and of the herbicide 2,6-dichlorobenzonitrile [147, 148].
CYP2J Subfamily
Expressed primarily in extrahepatic tissues [5, 117], this
is an inducible subfamily [149]; no role in the bioactivation
of chemical carcinogens has so far being ascribed to it.
Current Topics in Medicinal Chemistry, 2004, Vol. 4, No. 16
1777
CYP3 FAMILY
CYP3 is the most abundant family in human liver,
comprising a single subfamily, the principal protein being
CYP3A4, which is particularly adept in catalysing Ndealkylation reactions, and among cytochrome P450
enzymes it is the most active contributor to drug metabolism
[75, 150]. It is also present at high concentrations in the
intestine [151, 152] where it is responsible for the first-pass
(presystemic) metabolism of many drugs, such as the
immunosuppressant cyclosporin. Not surprisingly, it is
involved in many clinically relevant drug interactions [150].
There are two additional proteins within this family;
CYP3A5, which has similar substrate specificity to CYP3A4
but lower metabolic rates, and is the major CYP3A protein in
human lung. The third member of this family is CYP3A7,
which is expressed at very high levels in the foetus, where it
constitutes the major cytochrome P450 enzyme comprising
some 50 % of the cytochrome P450 content in the liver, but
disappears shortly before birth, possibly being replaced by
CYP3A4 [153, 154].
It is an inducible family both in rats and humans and, in
the latter, drugs like barbiturates, dexamethasone, erythromycin, phenobarbitone, rifampicin and troleandomycin were
shown to elevate its expression in the liver and/or intestine
[155, 156]. An interesting observation is that in the rabbit,
CYP3A is highly expressed in the aorta but is downregulated
by treatment with rifampicin [135].
CYP3A substrate specificity is very broad, its substratebinding site being sufficiently large to accommodate high
molecular weight compounds like the macrolide antibiotics
but also sufficiently versatile to contribute to the metabolism
of small size molecules such as paracetamol [157]. A
number of carcinogenic chemicals have been shown to be
bioactivated by the CYP3A subfamily, the most important
being aflatoxin B1 and dihydrodiols of polycyclic aromatic
hydrocarbons in whose metabolism other cytochrome P450
proteins also participate (vide supra) [112, 128, 158, 159].
Members of the CYP3A family have also been shown to
activate pyrrolizidine alkaloids such as senecionine [160],
and to contribute to the bioactivation of the heterocyclic
amine 2-amino-3-methylimidazo-[4,5-f]quinoline (IQ) [158]
and 6-aminochrysene [161]. The human CYP3A subfamily
also catalyses the N-demethylation of cocaine leading to the
generation of hepatotoxic metabolites [162, 163].
CYP4 FAMILY
Three subfamilies have been identified in humans and
rats, CYP4A, CYP4B and CYP4F, being particularly
expressed in the kidney and to a lesser extent in the liver.
CYP4A is poorly expressed in the liver of cattle and deer
[49, 50]. The catalytic activity of these towards xenobiotics
has not been clearly defined, and their predominant role is
primarily in the metabolism of fatty acids and eicosanoids.
An interesting aspect of this family is its induction by
peroxisomal proliferators, which are epigenetic carcinogens
that increase the number and size of peroxisomes and cause
cellular proliferation. In the liver, CYP4A activity is
enhanced by fibrate hypolipidaemic drugs such as clofibrate
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Current Topics in Medicinal Chemistry, 2004, Vol. 4, No. 16
Ioannides and Lewis
and ciprofibrate, and anti-inflammatories such as benoxaprofen and ibuprofen [164, 165]. CYP4B has been shown to
be inducible by phenobarbital, at least in the rabbit [166].
In the rabbit, a protein expressed principally in the lung
and other extrahepatic tissues, CYP4B1, activates a number
of aromatic amines, such as 2-aminofluorene and 2aminoanthracene, through N-hydroxylation, and the
pulmonary toxin ipomeanol [167, 168], but the rat and
human orthologues display very low activity [169].
Clearly, the role of cytochrome P450 proteins in
chemical carcinogenesis, as well as their induction by other
chemicals are isoform-specific, and these characteristics are
summarised in table 1.
Table 1.
CYTOCHROME P450 INDUCTION AND CHEMICAL
CARCINOGENESIS
The pathways of carcinogen metabolism are determined
by the enzyme profile at the time of exposure as well as the
tissue concentrations of the carcinogen since, at high tissue
concentrations, more than one form of cytochrome P450 may
be involved, as low affinity forms may also start contributing
significantly to metabolism. As many chemical carcinogens
can themselves alter the profile of xenobiotic-metabolising
enzyme systems, and especially of cytochromes P450 by
inducing some isoforms at the expense of others, further
doses of the carcinogen may be metabolised by a totally
different complement of enzymes.
Xenobiotic-metabolising Cytochromes P450 and their Role in the Bioactivation of Chemical Carcinogens.
Cytochro
me P450
subfamily
Human
proteins
Rat proteins
Role in
chemical
carcinogenesis
Major classes of
activated carcinogens
Inducibility
Principal characteristics
CYP1A
1A1, 1A2
1A1, 1A2
Very extensive
PAH, AA, HA, MC,
NNK, AAB
Very high
Highly conserved; 1A1 largely
extrahepatic, 1A2 hepatic
CYP1B
1B1
1B1
Very extensive
PAH, AA, HA, MC,
NNK, AAB, Oestrogens
Very high
Over-expressed in human tumours.
CYP2A
2A6, 2A7,
2A13
2A1, 2A2, 2A3
Moderate
NA, OP
Moderate
Marked species differences in
substrate specificity.
CYP2B
2B6
2B1, 2B2, 2B3,
2B12, 2B15, 2B16
Moderate
NA, OP
High
Catalyses the bioactivation of
many chemical carcinogens.
CYP2C
2C8, 2C9,
2C18,
2C19
2C6, 2C7, 2C11,
2C12, 2C13,
2C22, 2C23, 2C24
Minor
PAH
Moderate
Polymorphically expressed.
Important role in drug metabolism.
CYP2D
2D6
2D1, 2D2, 2D3,
2D4, 2D5, 2D18
Poor
NNK
Not inducible
Polymorphically expressed
CYP2E
2E1
2E1
Extensive
NA, HH
High
Propensity to generate reactive
oxygen species
CYP2F
2F1
Not evaluated
-
Not evaluated
Principally expressed in the lung
Not evaluated
-
Not evaluated
Principally expressed in the
olfactory mucosa
Not evaluated
-
Not evaluated
Principally expressed in
extrahepatic tissues.
CYP2G
2G1
CYP2J
CYP3A
3A4, 3A5,
3A7
3A1, 3A2, 3A9,
3A18, 3A23
Moderate
PAH, MC, PA
High
Most abundant cytochrome P450
enzyme in liver and intestine. Most
important in drug metabolism.
CYP4A
4A9, 4A11
4A1, 4A2, 4A3,
4A8
None
-
High
Primarily involved in fatty acid
metabolism. Localised primarily in
the kidney.
CYP4B
4B1
Minor
AA
Not evaluated
Expressed only in extrahepatic
tissues
PAH, Polycyclic aromatic hydrocarbons; AA, Aromatic amines; HA, Heterocyclic amines; MC, Mycotoxins; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; AAB,
Aminoazobenzenes; OP, Oxazophosphorines; HH, Halogenated hydrocarbons; PA, Pyrrolizidine alkaloids.
Cytochromes P450 in the Bioactivation of Chemicals
Although the mechanisms of cytochrome P450 induction
are complex and there are clear differences between the
various systems of regulation, it is common for the inducing
agent to displace a bound endogenous ligand from the
nuclear receptor protein complex with heat shock protein
(HSP). The associated conformational change in the
receptor, now containing the bound inducer, leads to
dimerisation of the ligand-receptor complex and its
translocation from the cytosol to the nucleus. Activation of
the relevant cytochrome P450 gene occurs following binding
of the ligand-bound receptor (either as a homodimer or
heterodimer) to a palindromic response element in the
regulatory region of the gene [170-172]. For example
induction of the CYP1 genes by chemicals such as the
polycyclic aromatic hydrocarbons occurs through binding to
the aromatic hydrocarbon (Ah) receptor, a soluble protein.
Since the liver is by far the major site of xenobiotic
metabolism, hepatic cytochrome P450 profile is an important
determinant of carcinogenic activity, particularly for orally
taken chemicals. In carcinogenicity studies, the evaluated
chemical is administered, not only at high doses, but also
daily. Such a treatment, after a single or few doses of the
chemical, may lead to selective induction of the bioactivating
enzymes, thereby exaggerating the production of reactive
intermediates, overwhelming the detoxication processes, and
markedly increasing the likelihood of a positive carcinogenic
effect. Indeed, in many cases carcinogenic activity within a
class of compounds, may be related to the propensity to
induce the bioactivating cytochrome P450 protein,
particularly in the case of CYP1-mediated bioactivation. The
association of elevated CYP1 levels to chemical carcinogenesis is supported by extensive experimental studies [94,
173, 174]. For example, DBA/2 mice, a strain refractive to
CYP1 induction, were less susceptible to the carcinogenicity
of benzo[a]pyrene compared with the C57/BL strain, where
CYP1 activity is readily inducible [173]. In another animal
study, of ten generations of rats exposed to 3´-methyl-4dimethylaminoazobenzene, a CYP1-activated carcinogen,
the 4th to 8 th generations were resistant to the carcinogenicity
of this chemical compared to the other generations; the
resistant generations also displayed poor CYP1 inducibility
and, consequently, their ability to bioactivate this carcinogen
was diminished [175, 176]. These studies support the
premise that the ability of a chemical to stimulate its own
activation pathways may be a critical factor in determining
its carcinogenic activity, and help explain the markedly
different carcinogenic activity between structurally-related
chemicals, even isomers [177]. For example, between
isomers, carcinogenic potency could be related to the ability
to upregulate CYP1A. Benzo[a]pyrene, 4-aminobiphenyl and
2-naphthylamine, all established carcinogens, were far more
potent inducers of hepatic CYP1A than their noncarcinogenic isomers benzo[e]pyrene, 2-aminobiphenyl and
1-naphthylamine [178. 179]. In recent studies [180], it was
reported that the carcinogenic activity of polycyclic aromatic
hydrocarbons was related to their ability to induce CYP1A1
and CYP1B1 activity through interaction with the Ah
receptor, confirming the previous studies [179]. In order to
further define this relationship, extensive studies were
undertaken to correlate CYP1A induction, binding to the Ah
receptor that regulates CYP1 expression, and carcinogenic
Current Topics in Medicinal Chemistry, 2004, Vol. 4, No. 16
1779
activity among isomers or structurally related compounds.
Such studies revealed that the treatment of animals with the
carcinogenic 2-acetylaminofluorene resulted in the induction
of both CYP1A isoforms, whereas the 4-isomer increased the
levels of only CYP1A2; in concordance with these findings,
the 2-isomer binds to the Ah receptor markedly more avidly
than the 4-isomer [181]. In studies involving six
azobenzenes, a correlation was established between binding
to the Ah receptor and CYP1A induction on the one hand,
and carcinogenic activity on the other hand [182]. A similar
picture emerged when isomeric diaminotoluenes or
diaminonaphthalenes and related compounds were used as
models [183, 184]. In addition to CYP1 induction, the Ah
receptor mediates many other activities including protein
kinase C, whose activation culminates in accelerated DNA
replication, dedifferentiation and cellular proliferation,
critical stages in the carcinogenesis process [185, 186]. It
may be, therefore, that an increase in the promotion stages of
carcinogenesis, mediated by the Ah receptor, is also an
important aspect, in addition to CYP1 induction. It is
noteworthy that Ah receptor knockout mice were resistant to
the carcinogenicity of benzo[a]pyrene [187].
It is thus reasonable to expect that high CYP1 levels may
predispose to increased carcinogenicity when exposure to a
CYP1-mediated carcinogen occurs. As a paradigm, cigarette
smoking is a documented CYP1A2 inducer in human liver
[188], and consequently the carcinogenicity of heterocyclic
amines, which are activated by CYP1A2, may be enhanced,
and this may help to provide a rationale for the positive
relationship between smoking and liver cancer [189]. In
recent studies conducted in rats exposed to cigarette smoke,
the hepatocarcinogenicity of MeIQx, when given during the
initiation stage, was related to the induced CYP1A2 levels
[190].
The role of induction in chemical carcinogenesis is not
confined to the CYP1 family. Treatment of rats with alcohol,
a CYP2E1 inducer, exacerbated the carcinogenicity of
azoxymethane by facilitating its metabolism to the reactive
methyldiazonium ion [191]. The bioactivation of
azoxymethane is catalysed primarily by CYP2E1 [192].
Clearly, induction of cytochromes P450 is an important
phenomenon that may predispose humans to the
carcinogenicity of chemicals. Although cytochrome P450
induction is seen readily in animals, it is much less common
in humans, and this may be one of the reasons why humans
survive in an environment littered with toxic and
carcinogenic pollutants. Such a scenario of elevated
cytochrome P450 activity most probably occurs in long-term
carcinogenicity studies, where animals are chronically
exposed to the compound undergoing testing, and thus may
not always be applicable to the human situation where
exposure may be occasional or intermittent. It has been
frequently been pointed out that too many chemicals come
out as positive in long-term carcinogenicity studies in
rodents, where excessive doses, equivalent to the maximum
tolerated dose, are employed [193]. At high doses tissue
damage and inflammation may occur triggering cell division,
a critical event in the carcinogenesis process [194]. An
additional mechanism that may contribute to the high rate of
positives is cytochrome P450 induction. It is unlikely that
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Current Topics in Medicinal Chemistry, 2004, Vol. 4, No. 16
lower doses and/or intermittent intake that occurs in humans
will result in cytochrome P450 induction.
IN SILICO EVALUATION OF CHEMICAL TOXICITY
BASED ON CYP1 CATALYSIS
We have developed a technique, known a COMPACT,
which is able to evaluate quantitatively the likely degree of
CYP1 induction for a given chemical based on a
combination of two elements of its electronic and molecular
structure [195]. Specifically, this involves a calculation of
molecular planarity (area/depth2 ratio) and electronic
interaction energy (∆E) using molecular mechanics and
molecular orbital procedures, respectively [195-198]. The
two parameters can be combined in a single descriptor
variable, termed the COMPACT radius, CR, which is given
by the following expression:
CR =
(a/d2 − 15) 2 + ( ∆E − 7) 2
where a/d2 is the ratio of molecular area to the square of
depth, and ∆E is given by the difference between frontier
orbital energies, ELUMO and EHOMO.
Although this parameter exhibits a good overall
correlation with CYP1 induction potential, this can be
improved by combining CR with molecular rectangularity
(length/ width ratio) and log P, where P is the octanol-water
partition coefficient [199]. For example, the following
equation was generated for a series of 12 structurally diverse
chemicals [94] exhibiting a range of CYP1 induction
potential activity:
log CYP1 induction = 0.23 log P - 0.40 l/w - 0.15 CR + 2.67
(±0.04)
(±0.17)
(±0.03)
n = 12; s = 0.209; R = 0.99; F = 98.6
where n is the number of compounds, s is the standard error;
R is the correlation coefficient and F is the variance ratio
[200]. However, for a smaller group of polyaromatic
hydrocarbons binding to the Ah receptor, it is found that a
very good correlation exists between area/depth2 alone and log EC50 as follows:- log EC50 = 0.44 a/d2 + 2.62 n = 7; s = 0.230; R = 0.98; F = 112.6
(±0.03)
where EC50 is the effective concentration (nM) for
displacement of 3-methylcholanthrene from the Ah receptor.
QUANTITATIVE STRUCTURE-ACTIVITY RELATIONSHIPS FOR P450-MEDIATED TOXICITY
It has been established that structure-activity
relationships in the field of P450-mediated toxicity and
metabolism of xenobiotics tend to show the importance of
frontier orbital energies [199, 200-204]. A possible rationale
for this finding could relate to the mechanism by which the
P450 substrate may react with an active form of oxygen,
with an electrophilic oxygen species [FeO]3+ representing the
most likely intermediate [205]. Consequently, one might
expect that the energy of the highest occupied molecular
Ioannides and Lewis
orbital (E HOMO), or the ionisation energy, would indicate the
degree of substrate interaction with an electrophilic
intermediate. In fact, this appears to be the case for several
series of different classes of P450 substrates [206].
For these examples, the rate of cytochrome P450mediated metabolism (or clearance in some cases) can be
correlated with expressions involving either the EHOMO or its
equivalent ionisation energy (where IE = -EHOMO) and these
terms may be combined with other descriptors in the overall
QSAR equation [207]. However, under some circumstances
the lowest unoccupied molecular orbital energy (ELUMO)
appears in correlations with P450-mediated toxicity or
metabolic rate and, furthermore, the difference between
ELUMO and EHOMO (i.e. ∆E) can sometimes be important. For
example, the carcinogenicity of symmetric dialkylnitrosamines correlates closely with ∆E [204] as does the rate of
CYP2E1-mediated metabolism of alkylbenzenes [208]. The
involvement of these different frontier orbital energies points
to the importance of electron transfer interactions either
between the substrate and cytochrome P450 active site
region or between the active species and the cellular target,
such as DNA in the case of carcinogenicity or mutagenicity.
It is known, for example, that the reactive dihydrodiol
epoxides of carcinogenic polycyclic aromatic hydrocarbons,
such as benzo(a)pyrene, are able to intercalate with DNA
base pairs prior to covalent adduct formation and, therefore,
the initial interaction is almost certainly going to be a
frontier orbital-controlled process [201].
In addition to their employment in QSAR expressions,
molecular and electronic structural descriptors such as
area/depth2 and ∆E, for example, can be utilized in the
differentiation of CYP1 substrates and inducers from those
of other isoforms. Although it is found that the combination
of area/depth2 and ∆E provides a relatively good
discrimination of CYP1-selective compounds, which may be
used predictively [209-211], it has been found necessary to
consider additional factors for distinguishing compound
selectivity towards CYP2E and CYP3A enzymes [210, 211].
In fact, it can be shown that a total of 5 structural descriptors
are required ‘Fig. (7)’ for differentiating satisfactorily (R =
0.92) between 48 substrates of 8 human P450s [212, 213].
However, when one considers the structures of definite
human carcinogens, it is apparent that these are either directacting electrophilic compounds (such as nitrogen mustards)
or are metabolically activated by CYP1 (e.g. 2naphthylamine) and CYP2E1 (e.g. benzene and vinyl
chloride). Consequently, such agents are readily identified
via a combination of structural alert and cytochrome P450
selectivity [212, 213].
CONCLUSIONS
Most carcinogenic compounds are chemically inert,
precluding any interactions with cellular macromolecules. In
the liver primarily, but also in other tissues, however, these
chemicals are metabolically transformed to highly reactive
intermediates capable of irreversible interactions with
cellular components with deleterious consequences. It is
apparent that toxicity is not simply a consequence of the
intrinsic molecular structure of the chemical, but is also
determined by the nature of the enzymes present at the time
Cytochromes P450 in the Bioactivation of Chemicals
Current Topics in Medicinal Chemistry, 2004, Vol. 4, No. 16
1781
Fig. (7). A decision tree approach to determining cytochrome P450 specificity.
Collision diameter is defined as the distance between the centres of two adjacent molecules at their closest approach. It is approximately
equivalent to the diameter of the solvent-accessible (Connolly) surface.
of exposure. Clearly for a chemical to provoke toxicity at
least two prerequisites must be fulfilled: (a) the chemical
must be, or must have the propensity to be metabolically
converted to a reactive intermediate(s), and (b) the living
organism, at the time of exposure, must possess the
necessary enzyme(s) required for the activation of the
chemical. The enzyme systems that metabolise toxic
chemicals, both their bioactivation and deactivation, are
regulated genetically [30, 214, 215] but are also modulated
by environmental factors such as diet [38] and previous
exposure to chemicals [40, 41] and by the presence of
disease [32, 216]. The fate of a chemical in the body will be
determined largely by the complement of enzymes involved
on the bioactivation/deactivation of the chemical, especially
cytochromes P450, at the time of exposure. The overall
contribution of an individual cytochrome P450 enzyme in
the bioactivation of chemicals will depend not only on the
catalytic activity of the isoform, but also on its tissue levels
as well as the tissue concentration of the chemical. At high
tissue concentrations of the chemical, more than one isoform
of cytochrome P450 may be involved, as low affinity
isoforms may also contribute significantly to metabolism.
Toxicologists refer to chemicals as been toxic or non-toxic,
while in the strictest sense the vast majority of chemicals are
innocuous, and it is the living organism that renders them
toxic through metabolism. As we are becoming more
competent in phenotyping humans for xenobioticmetabolising activity, and thus their precise metabolic
competence, we should be in a position to identify
individuals vulnerable to the toxicity of certain classes of
chemical toxin. It may well become more appropriate to talk
of ‘toxicophilic’ and ‘toxicophobic’ individuals rather than
of toxic and non-toxic chemicals, depending on their
propensity to bioactivate or deactivate chemicals.
Increasingly, the potential utilisation of cytochrome P450
enzymes in commercial applications, such as in the
bioactivation of prodrugs and in bioremediation to facilitate
1782
Current Topics in Medicinal Chemistry, 2004, Vol. 4, No. 16
the breakdown of environmental pollutants, is being
explored seriously with positive outcomes [217, 218].
Systems are currently being designed, exploiting the
propensity of cytochrome P450 enzymes to produce reactive
intermediates. This approach allows for the metabolic
transformation of prodrugs to their biologically-active
metabolites in cells that poorly express the necessary
enzymic apparatus, in the so-called gene-directed prodrug
therapy (GDEPT). The objective is to elevate the
concentration of the active form of the drugs within tumour
cells, in the case of anticancer prodrugs that necessitate
metabolic activation, with minimal toxicity to normal cells.
Usually cancer drugs such as oxazaphosphorines are given at
high doses to ensure that sufficient drug, in its active form,
reaches the tumour. Bioactivation of the drug occurs in the
liver and the active metabolites transferred to the tumours,
which may be distal to the liver. However, as the active
metabolites are also distributed to other tissues, such
treatment is associated with major side effects that limit their
use. Delivery of the drug and the enzyme directly to the
tumour would markedly lower the required doses of the
drugs and thus reduce systemic toxicity. The relevant
enzyme systems, therefore, such as cytochrome P450, are
transferred to the target cells, so that activation of the drug
occurs in situ to form the therapeutically relevant metabolite
[219]. This approach has been particularly applied to
tumours to facilitate activation of the oxazophosphorines
cyclophosphamide and ifosfamide within the tumour cell,
whose capacity to activate these prodrugs is poor, thus
minimising adverse effect to the normal cells, and in this
way suppress the serious side effects that are associated with
cancer chemotherapy and enhance the therapeutic benefit. A
better outcome is achieved if the reductase is also supplied to
the cell simultaneously with the cytochrome P450 enzyme,
so that generation of the cytotoxic effect is increased since
metabolism does not have to rely on the normally low levels
of the reductase present in tumour cells [220].
Cyclophosphamide is activated by an initial 4-hydroxylation,
catalysed by CYP2B6, which eventually leads to the
generation of the phosphoramide mustard, the therapeutic
metabolite [121]. The major difficulty encountered at present
is the efficiency of gene transfer. It has become apparent,
however, that even with poor transfection, active metabolites
and/or their precursors can diffuse into neighbouring cells
where they can exert their cytotoxicity; this is referred to as
the ‘bystander’ effect and to some extent offsets the problem
of low gene transfer. The first clinical trials in patients with
advanced, inoperable pancreatic cancer, where ifosfamide
was used as the chemotherapeutic drug and CYP2B1 was
supplied to the cancer cells, are promising [221].
Ioannides and Lewis
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