From the Numerous foreign chemicals (xenobiotics) confront us daily; such
compounds often are lipophilic and may accumulate to toxic levels
unless they are metabolized to water-soluble products that can be
readily excreted. A cytochrome P450 enzyme(s) often catalyzes the
initial step in such detoxification pathways. Multiple forms of
cytochrome P450 exist; these enzymes are notable for their broad and
overlapping substrate specificities. Although P450s usually mediate
detoxification reactions, under some circumstances they activate their
substrates to carcinogenic, mutagenic, and/or cytotoxic products. They
also contribute to the oxidative metabolism of endogenous hormones,
fatty acids, and
cytokines(1, 2, 3, 4, 5) . Some cytochrome P450 genes are expressed constitutively, while
others (particularly those involved in xenobiotic metabolism) are
inducible. In many cases, inducers are also substrates for the induced
enzymes; therefore, P450 activities remain elevated only as needed.
Enzyme induction usually enhances detoxification; thus, under most
conditions, induction is a protective
mechanism(3, 5, 7) . Induction most often
occurs at the level of transcription(6, 7) . Table 1lists the major xenobiotic-inducible cytochrome P450s and
some of their substrates.
The inducible cytochrome P450 enzymes
represent interesting experimental systems for analyzing the mechanisms
by which small molecules enhance the transcription of specific genes.
Such knowledge can provide insights into gene regulation that are of
relatively broad interest. Here, we briefly summarize the mechanisms by
which xenobiotics induce cytochrome P450 gene transcription.
Aromatic Hydrocarbon-inducible Cytochrome P450s Polycyclic aromatic hydrocarbons, such as the carcinogens
3-methylcholanthrene (3-MC) (
The AhR's structure, deduced from its
cDNA sequence, reveals several features: 1) a basic helix-loop-helix
(bHLH) domain; 2) two regions, designated as ``PAS,'' because
of their homology with the regulatory proteins Per, Arnt, and Sim; 3) a
glutamine-rich region(14, 15) . Mutational analyses
imply that the bHLH domain is important for protein-protein
interactions and for binding to
DNA(14, 16, 17) . The PAS domains appear
necessary for protein-protein interactions and for ligand binding,
while the C-terminal half of the AhR is required for
transactivation(14, 16, 17, 18) .
The in vitro translated receptor does not bind strongly to
DNA, even in the presence of TCDD; acquisition of DNA binding
capability requires that the receptor heterodimerize with the Arnt
protein (see below)(14, 15, 16) . Additional
proteins that may participate with the AhR in the induction response
have recently been
identified(8, 19, 20, 21, 22) .
The deduced sequence of AhR reveals that it is a novel type of
ligand-activated transcription factor, distinct from those described
previously.
Several factors have facilitated the mechanistic analyses of
cytochrome P4501A1 induction: (a) uninduced activity
(background) is low and induced activity (signal) is high; (b)
induction occurs in cells in culture; (c) a high potency,
non-metabolized inducer (TCDD) is available; (d) induction
exhibits a genetic polymorphism in mice; (e) methods are
available to isolate induction-defective cells, which enables genetic
analyses. As described in more detail in subsequent sections, analyses
of the induction of other cytochrome P450 enzymes suffer from the lack
of one or more of these factors. Therefore, for other cytochrome P450
enzymes, our understanding of the induction mechanism is less complete
than it is for cytochrome P4501A1.
Phenobarbital-inducible Cytochrome P450s The induction of drug- and steroid-metabolizing enzyme
activity by phenobarbital (PB) was one of the seminal observations
leading to scientific interest in cytochrome P450. PB induces several
forms of cytochrome P450 (Table 1) as well as other
xenobiotic-metabolizing enzymes. In addition, compounds that are
structurally unrelated to PB (e.g. 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT),
certain polychlorinated biphenyls, and others) induce a similar pattern
of enzyme activities and are considered to be
``PB-like''(6, 7, 25) . In rat
liver, PB rapidly increases the rate of transcription of the CYP2B1/2
genes(6, 25) . In chick embryo liver, PB rapidly
induces CYP2H1 and CYP2H2, which are related structurally to CYP2B1/2,
by a combination of transcriptional and post-transcriptional
mechanisms(5, 6, 25) . In Bacillusmegaterium, PB induces fatty acid monooxygenases (CYP102
and CYP106) by increasing transcription of the corresponding
genes(26) . Because PB induces cytochrome P450 transcription
in bacteria, birds, and mammals, we might expect the induction
mechanism to be highly conserved; however, this is not so. For example,
in rat hepatocytes, PB induces CYP2B1/2 and CYP3A by different
mechanisms, which are distinguishable by their sensitivity to
cycloheximide (ongoing protein synthesis is required for induction of
CYP2B1/2 but not that of CYP3A(25) ). Similar differences have
also been reported in other systems(25) .
Shaw et al.(31) have identified a PB-responsive DNA region
upstream of the CYP2B1 and CYP2B2 genes. They also demonstrated that PB
responsiveness was influenced by glucocorticoids and suggested that PB
might act indirectly by causing the accumulation of an endogenous
steroid. A PB-responsive element(s) has also been localized to a region
between -5.9 and -1.1 kilobases upstream of the start site
of CYP2H1 transcription(25) . Omiecinski and colleagues (32) used transgenic mice to localize a PB-responsive DNA
regulatory element(s) within the -0.8 to -19-kilobase
region upstream of the transcription start site of the rat CYP2B2
gene(32) . The properties of these PB-responsive DNA elements
remain to be determined.
Peroxisome Proliferator-inducible Cytochrome P450s Several structurally dissimilar compounds, including fibrate
hypolipidemic drugs, phthalate ester plasticizers, and halogenated
aromatic solvents induce peroxisome proliferation in mammalian liver.
This response involves increased peroxisomal In rat liver, clofibrate rapidly increases
CYP4A1/6 gene transcription (33, 34) . Likewise,
peroxisome proliferators rapidly increase the transcription rates of
the genes for fatty acyl-CoA oxidase (ACO) and enoyl-CoA
hydratase/3-hydroxyacyl-CoA dehydrogenase, components of the
peroxisomal fatty acid
PPs induce the transcription of the cytochrome P4504A and ACO genes;
however, the induction mechanisms for the two genes may differ. For
example, studies in rat hepatocytes imply that induction of CYP4A1/6
transcription is a primary response to PPs and precedes the induction
of ACO transcription, which is a secondary response(40) .
Furthermore, mechanism-based inactivators of cytochrome P4504A1
inhibited the induction of ACO but not the induction of P4504A1. In
addition, hexadecanedioic acid, a long chain fatty acid formed by
cytochrome P4504A1, induced ACO but not P4504A1(41) . These
data suggest that induction of the cytochrome P4504A1 enzyme and the
resultant generation of long chain fatty acids are required for the
subsequent induction of peroxisomal
Steroid-inducible Cytochrome P450s In the early 1970s, Selye (42) observed that the
``catatoxic'' steroid, pregnenolone-16
The cytochrome P450 superfamily consists of over 150 P450
genes present in a variety of organisms(46) . It has been
proposed that the genes evolved during ``animal-plant
warfare'' as a mechanism by which animals protected themselves
from dietary and environmental toxicants (47) . Induction is
likely to be advantageous from an evolutionary standpoint, allowing
enhanced detoxification following exposure to xenobiotics. On the other
hand, it is unclear whether the regulatory pathways evolved
specifically to deal with dietary and environmental xenobiotics or
whether they evolved from regulatory pathways already present in the
cell for other reasons. For example, cytochrome P450 gene regulation
may have evolved as a mechanism to maintain metabolic homeostasis with
respect to endogenous lipophilic substrates (e.g. steroids,
fatty acids, etc.). If true, this scenario would predict the existence
of endogenous ligands that regulate cytochrome P450 transcription.
Identification of such ligands would generate a better understanding of
the biochemical pathways by which cells recognize and respond to
chemical stimuli.
INTRODUCTION
Aromatic Hydrocarbon-inducible Cytochrome P450s
Phenobarbital-inducible Cytochrome P450s
Peroxisome Proliferator-inducible Cytochrome P450s
Steroid-inducible Cytochrome P450s
Conclusion
FOOTNOTES
REFERENCES
)and benzo(a)pyrene,
are prototypical inducers of several P450s, most notably P4501A1/1A2
and
1B1(6, 7, 8, 9, 10, 11) .
Nuclear ``run-on'' experiments have revealed that induction
primarily reflects an increase in the rate of transcription of these
genes(8, 9, 10, 11) , although
posttranscriptional effects on P4501A2/1B1 have been
reported(3, 12) . Our understanding of cytochrome P450
induction by aromatic hydrocarbons is based largely upon analysis of
P4501A1 gene transcription.
The Ah Receptor
The environmental contaminant
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is an unusually
potent inducer of P4501A1 (up to 30,000 times more potent than 3-MC (13) ). Poland et al.(13) showed that mouse
liver contained a protein that bound TCDD saturably, reversibly, and
with high affinity and, thus, had the ligand binding properties of a
receptor. The protein has been designated as the ``Ah
receptor'' because it binds other aromatic hydrocarbons (such as
3-MC and benzo(a)pyrene) in addition to TCDD. Biochemical and
genetic evidence implicates the Ah receptor in the induction of CYP1A1
transcription. For example, structure-activity studies reveal a
correlation between receptor binding affinity and potency as an
inducer. Furthermore, genetic studies in inbred mouse strains reveal
quantitative differences in responsiveness to TCDD, and the same
genetic locus governs both TCDD binding and enzyme
induction(13) .The Arnt Protein
Hankinson (15) identified
a cDNA that complements the defect in cells that fail to respond to
TCDD, even though their AhR is normal. Hankinson (15) designated the corresponding protein as ``Arnt''
(for Ah receptor nuclear translocator). Its deduced amino acid sequence
reveals that Arnt has an organization that is similar to AhR: 1) a bHLH
domain; 2) two PAS homologies; 3) a glutamine-rich region. Mutational
studies implicate the bHLH region in DNA binding and heterodimerization
with AhR; the C-terminal region provides a transactivation
function(15, 18, 23, 24) . The Arnt
protein does not bind TCDD and does not bind to DNA in the absence of
liganded AhR(15) . The data imply that Arnt heterodimerizes
with liganded AhR to generate an active, enhancer binding transcription
factor.Activation of Transcription
Recombinant DNA and
transfection experiments revealed the presence of a TCDD-inducible,
AhR-dependent, and Arnt-dependent transcriptional enhancer and a
transcriptional promoter upstream of the CYP1A1
gene(8, 15) . The enhancer participates in inducible,
AhR-dependent, and Arnt-dependent protein-DNA interactions, as expected
for the binding of the AhRArnt heterodimer to DNA. The
heterodimer recognizes a specific nucleotide sequence (which has been
designated as a xenobiotic-responsive element or a dioxin-responsive
element); furthermore, AhR
Arnt binds within the major DNA groove
and bends the DNA in vitro near the site of the protein-DNA
interaction(8) .
Chromatin Structure
Studies in intact cells reveal
that the inactive enhancer appears relatively inaccessible to
DNA-binding proteins in vivo. Exposure to TCDD leads to the
rapid occupation of multiple binding sites for
AhRArnt(8, 15) . The data suggest that
AhR
Arnt activates transcription via a mechanism that does not
require other enhancer-binding proteins. In uninduced cells,
constitutively expressed, general transcription factors fail to bind to
the CYP1A1 promoter. However, in the presence of TCDD, rapid occupation
of protein-binding sites on the promoter is observed(8) . Thus,
the binding of AhR
Arnt to the enhancer facilitates the binding of
transcription factors to the promoter. The inactive enhancer/promoter
region assumes a nucleosomal configuration with a nucleosome
specifically positioned at the promoter. The nucleosomal organization
provides an explanation for the inaccessibility of the CYP1A1
regulatory region in uninduced cells. The loss of nucleosomes at the
promoter plausibly accounts for the increase in CYP1A1 transcription
that occurs in induced cells(8) .
Future Research
CYP1A1 induction constitutes an
interesting system for analyzing the mechanism by which bHLH
transcription factors alter chromatin structure and enhance gene
expression; the results of such analysis may be of relatively broad
interest. Molecular genetic techniques are being used to study the
functional organization of AhR and Arnt and to identify and
characterize additional proteins that may participate in the induction
response. In addition, CYP1A1 induction is a prototype for the analysis
of the mechanism by which bHLH transcription factors alter chromatin
structure and enhance gene expression. CYP1A1 induction provides a
model for other TCDD-inducible cytochrome P450 genes, like CYP1A2 and
CYP1B1. Induction of these genes is AhR-dependent, largely
transcriptional, and, at least in the case of the CYP1A2 gene, it
involves dioxin-responsive transcriptional
enhancers(7, 9, 10, 11, 12) .
A Phenobarbital Receptor?
The structural diversity among
PB-like inducers is difficult to reconcile with the existence of a
specific receptor for these compounds. Studies using radiolabeled PB as
a ligand have failed to detect a specific PB-binding protein. A potent
inducer, 1,4-bis[2-(3,5-dichloropyridyloxy)] benzene,
appeared potentially useful for ligand binding studies; however, it is
active in mice, but not in guinea pigs or rats. Therefore,
1,4-bis[2-(3,5-dichloropyridyloxy)] benzene probably does not
act by a mechanism identical to that of PB (7, 25) .Activation of Transcription
In B. megaterium, the upstream regulatory region of the
barbiturate-inducible CYP102 (BM-3) gene contains a 17-base pair
element (designated as the ``Barbie Box''), which regulates
the response to PB(26, 27, 28) . Transfection
experiments in primary rat hepatocytes demonstrated that a Barbie Box
sequence could confer PB inducibility upon a heterologous gene; a
mutant consensus sequence was inactive(29) . Consensus Barbie
Box sequences have been identified upstream of the bacterial CYP102 and
CYP106 (BM-1) genes, the rat CYP2B1/2 and CYP3A2 genes, and other
PB-responsive genes(27, 28) . The consensus sequence
bound proteins from both bacteria and rat liver nuclei in a
PB-dependent manner in vitro(26, 27, 28, 29) . Notably, PB
treatment reduced the binding of the bacterial proteins to DNA but
increased the binding of the mammalian proteins. These findings imply
the existence of a PB-dependent protein(s) that binds to DNA near the
promoter region of PB-responsive cytochrome P450
genes(26, 27, 28, 29, 30) .
Whether the protein(s) is expressed constitutively or is induced by PB
is unresolved, and its function remains to be determined.Future Research
We understand relatively little
about the mechanism by which PB and PB-like chemicals induce cytochrome
P450 gene expression. The lack of convenient PB-responsive cell culture
systems, as well as the lack of genetic polymorphisms in PB
responsiveness, have hindered mechanistic analyses. Recently developed
primary or continuous cell lines that respond to PB have been
reported(25) , and their utility in mechanistic analyses of PB
action is being exploited(25, 29, 31) .
Identification of PB-responsive elements by transfection provides an
avenue for purification and cloning of the cognate transactivating
factors. The lack of structural similarity among PB-like inducers is
difficult to reconcile with the concept of a specific ``PB
receptor.'' It seems more likely that PB-like compounds act
indirectly to induce cytochrome P450 gene transcription. Thus, the
question of PB responsiveness remains an intriguing experimental issue
for the future.
-oxidation of fatty
acids, as well as induction of microsomal lauric acid
-hydroxylase activity, which is catalyzed by cytochrome P4504A1.
The induced enzymes may play a role in the metabolism of long chain
fatty acids(33) .
-oxidation
pathway(35, 36) .
Peroxisome Proliferator-activated Receptor
The
transcriptional nature of the induction response led to the hypothesis
that peroxisome proliferators (PPs) act via a nuclear protein related
to steroid hormone receptors. Cloning and functional analyses of
``orphan'' receptor cDNAs led to the identification of a
``peroxisome proliferator-activated receptor'' (PPAR) cDNA,
which mediates the biological response to
PPs(35, 36) . Certain androgens and fatty acids can
also activate rat liver PPAR(37, 38) . Direct binding
of ligands to the PPAR has not been demonstrated; furthermore, the lack
of structural similarity among PPs suggests that they might act
indirectly to induce gene transcription.Activation of Transcription
Tugwood et
al.(39) identified a DNA domain upstream of the ACO gene
that conferred PP responsiveness upon a reporter gene; electrophoretic
mobility shift experiments suggested that the PPAR binds to its cognate
response element in vitro. Johnson and co-workers (34) identified a functional PP-responsive element upstream of
the rabbit cytochrome P4504A6 gene; they subsequently demonstrated its
specific interaction with an activated PPARRXR heterodimer. These
observations imply that the PPAR acts via a mechanism analogous to that
described for the steroid/thyroid/retinoid family of nuclear receptors.
-oxidation enzymes.
Future Research
The lack of structural similarity
among PPs raises the question as to the identity of the ligand(s) that
actually binds to the PPAR. Identification of such ligands might
provide new insights into the contributions of the PPAR, peroxisomal
enzymes, and cytochrome P4504A enzymes to lipid metabolism. In some
cases, PPAR-dependent and other pathways may converge to generate novel
patterns of gene regulation. For example, the PPAR can form
heterodimers with another member(s) of the nuclear receptor family,
permitting the target gene to respond to a more diverse set of chemical
signals(35, 36) . Future studies in this area may
provide new insights into the mechanisms by which PPs produce their
biological effects.
-carbonitrile
(PCN), induced hepatic xenobiotic-metabolizing enzyme activity.
Subsequent studies demonstrated that PCN induced a novel form of
cytochrome P450(7) . More recently, others have reported that
PCN or high doses of dexamethasone or anti-glucocorticoids induce
additional forms of cytochrome P450 (Table 1). The PCN-inducible
enzymes, designated as the cytochrome P4503A family, metabolize a
variety of substrates, including testosterone and numerous
drugs(6, 7) .
Mechanism of Induction
Exposure of rats either to PCN or
to high doses of dexamethasone leads to an increased rate of hepatic
cytochrome P4503A gene transcription(6, 7) . Guzelian
and co-workers (43) have identified a
dexamethasone/PCN-responsive DNA element between -220 and
-56 upstream of the CYP3A1 gene. However, the exact cis-acting
element(s) responsible for the induction response remains to be
determined. Studies with a variety of steroid hormone analogs indicate
that induction of cytochrome P4503A, 2B1/2, and 2C6 occurs in response
to dexamethasone and glucocorticoid receptor antagonists and thus does
not involve a known steroid
receptor(7, 43, 44) .Future Research
The recent development of cell
culture systems in which the CYP3A gene(s) responds to steroids,
phenobarbital, and other inducers should facilitate future analyses of
the induction mechanism(45) . The discovery of more potent
inducers and the development of a photoaffinity ligand might permit the
identification of the receptor that mediates induction. Identification
of PCN-responsive elements by transfection provides an avenue for
purification and cloning of the cognate transactivating factor(s).
Isolation of induction-defective cells would allow the application of
genetic techniques to the study of this interesting system.
-carbonitrile; AhR, aromatic
hydrocarbon receptor; Arnt, AhR nuclear translocator.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.