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Carcinogenesis |
Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6084
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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6–12 h), suggesting a requirement for
(±)-trans-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene
metabolism. Aldo-keto reductase (AKR) inhibitors blocked this effect,
suggesting that benzo(a)pyrene-7,8-dione (BPQ), a planar
PAH o-quinone generated by AKRs, was the downstream
inducer. BPQ was found to be a potent and rapid inducer of CYP1A1, with
an EC50 value in HepG2 cells identical to that of the
parent benzo(a)pyrene. BPQ was a more potent inducer of
CYP1A1 when compared with the 1,6-, 3,6-, and
6,12-benzo(a)pyrene-diones. Multiple PAH
o-quinones caused induction of CYP1A1, demonstrating that
this was a general property of AKR-generated PAH
o-quinones. HepG2-101L cells stably transfected with a
XRE-luciferase construct showed that BPQ activated CYP1A1
transcription via a XRE-dependent mechanism. BPQ failed to induce
CYP1A1 in AhR-deficient and AhR nuclear translocator-deficient murine
hepatoma cell lines and confirmed that induction of CYP1A1 was AhR and
AhR nuclear translocator-dependent. Electrophoretic mobility shift
assays demonstrated the specific appearance of BPQ-activated AhR in the
nucleus, and immunofluorescence studies confirmed that BPQ mediated
nuclear translocation of the AhR. Classical bifunctional inducers
elevate CYP1A1 expression via a XRE and are subsequently
converted by CYP1A1 to electrophiles that induce phase II enzymes via
an electrophilic response element/antioxidant response element. PAH
o-quinones represent a novel class of bifunctional inducer
because they are electrophiles produced by phase II enzymes that
simultaneously induce phase I enzymes via a XRE and phase II enzymes
via a electrophilic response element/antioxidant response element (see
also M. E. Burczynski et al., Cancer Res.,
59: 607–614, 1999). This study shows that the AhR provides
the only known mechanism by which genotoxic PAH o-quinones
generated in the cytosol can be targeted to the nucleus with
specificity. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A second pathway of PAH activation is catalyzed by AKRs (11
, 12) . The AKRs are monomeric cytosolic NADP(H)-dependent
oxidoreductases (34 kDa), and several human isoforms
(AKR1C1–AKR1C4) catalyze the oxidation of PAH
trans-dihydrodiols to PAH o-quinones
(12, 13)
.4
In the case of B(a)P, the proximate carcinogen
B(a)P-diol is converted by AKRs to BPQ. PAH
o-quinones are electrophilic metabolites that enter futile
redox cycles and amplify ROS multiple times (15)
. PAH
o-quinones are formed in rat hepatocytes by AKR1C9 and are
both cytotoxic and genotoxic in vitro (16
, 17)
.
These quinones form stable and depurinating adducts with DNA, and their
propensity to redox cycle could lead to oxidative damage of DNA
(e.g., formation of 8'-hydroxy-2-deoxyguanosine, strand
scission, and base propenals; Refs. (18, 19, 20)
. PAH
o-quinones are gen-erated by AKRs in the cytosolic
compartment, and how PAH o-quinones gain access to the
nucleus is presently unknown. In disposition studies in primary rat
hepatocytes, a significant amount of 20 µM
[3H]BPQ (30%) was sequestered into the cell pellet (DNA,
RNA, and protein) within 0.5 h (17)
. Treatment of
primary rat hepatocytes with these toxicological concentrations of BPQ
results in extensive strand scission of the genomic DNA. These data
suggest that BPQ generated in the cytosol, like anti-BPDE
formed in the microsomes, can reach the nucleus.
PAHs are considered bifunctional inducers: they enhance the expression
of both the CYPs and phase II (de)toxification enzymes (including the
AKRs) through two distinct mechanisms (21)
. In the first
mechanism, PAHs bind directly to a cytosolic receptor termed the AhR
that demonstrates a strict specificity for planar aromatic compounds
(22)
. Upon binding ligand, the cytosolic AhR dissociates
from heat shock protein 90 and is rapidly translocated into the
nucleus, where it forms a complex with its heterodimeric partner, ARNT.
The ligand-bound AhR/ARNT complex then robustly activates the
expression of a battery of genes containing XREs within their
regulatory regions, including CYP1A1 (for reviews, see Refs. 23
and 24
; Fig. 2
).
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The present studies sought to determine whether downstream electrophilic metabolites of PAH might also feedback stimulate the expression of the CYP1A1 gene. In HepG2 cells, B(a)P-diol robustly enhanced CYP1A1 mRNA levels with delayed kinetics, implying that a downstream metabolite of B(a)P-diol was the actual CYP inducer. Subsequent studies identified PAH o-quinones generated by human AKRs as the inducers. PAH o-quinones induce CYP1A1 via the AhR signaling pathway, demonstrating that these electrophilic metabolites represent novel bifunctional inducers that require no further metabolism to simultaneously induce both phase II (de)toxification enzymes (via an EpRE/ARE due to their electrophilic/redox-active nature) and phase I activating enzymes (via the XRE due to their restored planarity). These studies also imply that the cytosolic colocalization of AKR enzymes and the AhR provides a mechanism whereby PAH o-quinones generated in the cytosol can be targeted to the nucleus with specificity. The nuclear targeting of these redox-active o-quinones by the AhR may contribute to the spectrum of oxidative DNA damage observed after exposure of cells to PAH.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Culture
HepG2 hepatoma cells (passages 5–20) were maintained in Eagle’s MEM
supplemented with 10% heat-inactivated fetal bovine serum and 100
units/ml penicillin/streptomycin solution. Hepa1c1c7, hepa1c1c4, and
hepa1c1c12 cells (kindly provided by Dr. Oliver Hankinson, University
of California Los Angeles, Los Angeles, CA) were maintained in 
-MEM
(without nucleosides) supplemented with 10% heat-inactivated fetal
bovine serum, 100 units/ml penicillin/streptomycin, and 2.5 µg/ml
fungizone solution (Irvine Scientific, Santa Anna, CA). HepG2-101L
cells stably expressing a portion of the CYP1A1 gene
promoter (-1612/+292) that bears three consensus XREs (a kind gift
from Dr. Robert H. Tukey, University of California, San Diego, La
Jolla, CA) linked to a luciferase reporter were maintained in
DMEM (with low glucose) supplemented with 5% non-heat-inactivated FCS,
5% non-heat-inactivated NuSerum IV, and 0.4 mg/ml G418 to maintain
selection. Cells were incubated at 37°C in a humidified atmosphere
containing 5% CO2 and passaged every 5 days at a 1:20
dilution (HepG2, hepa1c1c7, hepa1c1c4, and hepa1c1c12 cell lines) or
every 7 days at a 1:4 dilution (HepG2-101L). To measure either the
induction of CYP1A1 RNA or the nuclear translocation of the AhR by
electrophoretic mobility shift assays, 48 h before treatment,
3 x 106 hepatoma cells were seeded into
10-cm dishes containing fresh media. Two days later (60–70%
confluence), cells were exposed to the various inducers. Aliquots (100
µl) of 100 x stock solutions in DMSO were added to 10
ml of fresh culture medium, and cells were incubated for the indicated
times before harvesting. For XRE-luciferase reporter gene assays,
24 h before treatment, 1 x 106 cells
were seeded into 6-well plates. One day later (70–80% confluence),
cells were treated with the various inducers 12 h before
harvesting.
RNA Isolation and Northern Analysis
Cellular RNA was isolated using the Trizol reagent. Total RNA (10 µg)
was separated by electrophoresis on 1.0% agarose/formaldehyde gels and
transferred overnight to Duralon-UV membranes (Stratagene, La Jolla,
CA). Membranes were prehybridized in hybridization buffer (30%
formamide, 10% dextran sulfate, 1 M NaCl, and 1% SDS)
with 100 µg/ml sheared salmon sperm DNA at 42°C for 2 h. After
prehybridization, membranes were hybridized to 107 dpm of
[-32P]dATP probes corresponding to either:
(a) a 1-kb EcoRI fragment of the human CYP450 1A1
3' untranslated region (phP1-450-3', ATCC 57259); (b) a 1-kb
EcoRI fragment of the murine CYP450 1A1 (pMP1-450-3', ATCC
63006); or (c) an 855-bp EcoRI fragment of human
colon AKR1C1(hcDD/DD1) cDNA (kindly provided by Dr. Paul Ciaccio and
Dr. Ken Tew, Fox Chase Cancer Center, Philadelphia, PA) that were
labeled by random priming to a specific activity of greater than
109 cpm/µg DNA. Hybridization was performed at 42°C for
16–24 h. After hybridization, blots were subjected to two
high-stringency washes with 0.1x SSC plus 1% SDS at 60°C for 45 min
and 30 min, respectively. Signal intensities were measured using the
PhosphorImager system (Molecular Dynamics), and blots were exposed to
X-ray film at -80°C overnight. For purposes of normalization, blots
were stripped and reprobed with a 780-bp
PstI/XbaI fragment of human GAPDH labeled by
random priming as described above.
Luciferase Reporter Assays
The luciferase assay was carried out using the Dual Luciferase Reporter
Assay system from Promega. At the end of the treatment period,
HepG2-101L cells were rinsed twice in ice-cold PBS and then incubated
in 1 ml of 1x passive lysis buffer for 15 min on an orbital shaker at
room temperature. Lysates were centrifuged for 30 s at
14,000 rpm in a microfuge, and aliquots of the cleared lysates were
assayed immediately or stored at -70°C. The luciferase assay was
carried out using 20 µl of cell lysate and 100 µl of Luciferase
Assay Reagent II as provided. The light emitted was measured for
10 s using a Berthold Lumat LB-9501 luminometer. To normalize for
protein present in the stable transfectant cell lysates, quantitation
of protein content was performed on 100-µl aliquots of the cell
lysate using the Bradford reagent (Biorad, Hercules, CA). All assays
were found to be in the linear range versus time and protein
concentration.
Nuclear Extract Preparation
Nuclear extracts were prepared by the method of Dignam as described in
Ausubel et al. (33)
, with minor modifications.
After harvesting, cells were resuspended in hypotonic lysis buffer [10
mM HEPES (pH 7.9) containing 1.5 mM
MgCl2, 10 mM KCl, 0.5 mM DTT, and
0.2 mM phenylmethylsulfonyl fluoride] and centrifuged at
3,000 rpm for 5 min. Pelleted cells were resuspended in hypotonic lysis
buffer and disrupted with 10 strokes in a Dounce homogenizer (>90%
lysis by trypan blue exclusion). Nuclei were pelleted at 14,000 rpm for
30 min and then resuspended in 0.5x packed nuclear volume of (low
salt) nuclear extraction buffer [20 mM HEPES (pH 7.9)
containing 1.5 mM MgCl2, 0.2 mM
EDTA, 25% glycerol, 0.02 M KCl, 0.5 mM DTT,
and 0.2 mM phenylmethylsulfonyl fluoride). After dropwise
addition of an equal volume of nuclear extraction buffer containing
high salt (0.8 M KCl), samples were mixed on a tiltboard
for 30 min at 4°C. Samples were centrifuged at 14,000 x g for 30 s, and the supernatants (nuclear
extracts) were assayed immediately for protein content using the
Bradford reagent (Biorad) and used in subsequent electrophoretic
mobility shift assays.
Electrophoretic Mobility Shift Assay
Electrophoretic mobility shift assays were performed using a
double-stranded oligonucleotide containing a consensus XRE-binding site
from the CYP1A1 promoter corresponding to -998 to -969
(5'-dTTCTCCGGTCCTTCTCACGCAACGCCTGGGCA-3'
(italic letters correspond to the core XRE sequence) of the human
CYP1A1 gene (DRE-983, as described previously in Ref. 34
).
After annealing, the probes were labeled with
[-32P]dATP using the Klenow fragment (to fill in extra
TT dinucleotide overhangs at either end of the annealed
double-stranded oligonucleotide) and then purified through Qiaquik
nucleotide removal columns (Qiagen, Valencia, CA). Nuclear extracts (5
µg of protein) were incubated in binding buffer [25 mM
HEPES (pH 7.9) containing 100 mM KCl, 6.25 mM
MgCl2, 0.5 mM EDTA, 0.5 mM DTT, 1
mM phenylmethylsulfonyl fluoride, and 10% glycerol] in
the presence of 2.5 µg of BSA, 0.2 µg of poly(dI-dC), and 50 fmol
of -32P-labeled XRE oligonucleotide (20,000 cpm) in a
total volume of 15 µl at room temperature for 30 min. The reaction
mixture was then subjected to electrophoresis through a 4.5%
polyacrylamide gel containing 1x TBE and 1% glycerol. The gels were
dried under a vacuum for autoradiography (overnight exposure) and
subsequent PhosphorImager analysis. For competition experiments,
nuclear extracts were incubated before the addition of labeled
oligonucleotide for 10 min at room temperature with an excess of
unlabeled oligonucleotides and then incubated in the binding reactions
described.
Cell Growth and Fixation for Immunofluorescence Microscopy
Hepa1c1c7 cells were detached by trypsinization and seeded into 60-mm
culture dishes containing sterile glass coverslips previously coated in
a solution of collagen (100 µg/ml) for 1 h. Cells were seeded at
30% confluence and allowed to attach overnight. The following day,
coverslips were washed with PBS at room temperature and then incubated
in 4% paraformaldehyde (pH 7.4) for 15 min. After three rinses in PBS,
coverslips were incubated in 0.05% Triton in PBS for 10 min.
Immunofluorescence Staining and Microscopy
Coverslips were blocked in 2% goat IgG in 0.05% Triton in PBS for 30
min. After aspiration, coverslips were incubated in a 1:50 dilution
of primary antibody solution (rabbit antimurine AhR) at room
temperature for 90 min. After three rinses in 1x PBS, coverslips were
incubated in a 1:1000 dilution of secondary antibody solution (goat
antirabbit IgG fluorescein conjugate) at room temperature for 90 min in
the dark. Coverslips were washed three times with 1x PBS and then
mounted onto glass slides in mounting media. Once dry, fluorescence was
observed with a Nikon Diaphot fluorescent microscope.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The delayed but robust induction of CYP1A1 via B(a)P-diol
suggested that this metabolite either (a) induced
CYP1A1 expression via a novel mechanism or (b)
required activation to a downstream metabolite capable of inducing
CYP1A1 mRNA.
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. Both 6MPA and ursodeoxycholic acid inhibited the
B(a)P-diol-dependent induction of CYP1A1 mRNA in HepG2 cells
with IC50 values that were higher but approach the
IC50 values observed for the inhibition of purified
recombinant AKR1C1 in vitro, respectively (Ref. 30 ; data not
shown). These data suggest that the AKR1C1-dependent oxidation of
B(a)P-diol to BPQ is the metabolic event responsible for the
induction of CYP1A1 in B(a)P-diol-treated HepG2 cells.
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. The ED50 for the TCDD-dependent induction of CYP1A1 mRNA in
HepG2 cells was approximately 1 nM. B(a)P and
BPQ were nearly equipotent in their ability to induce CYP1A1 mRNA
(ED50 1 µM), and both were less
efficacious than TCDD. Importantly both compounds produced a rapid
(1 h) and robust induction of CYP1A1 mRNA, suggesting that neither
inducer required metabolism to an active species. The finding that
B(a)P is approximately 1000-fold less potent than the
nonmetabolizable inducer TCDD is consistent with previous reports
comparing TCDD and PAH (35)
. anti-BPDE was
incapable of inducing CYP1A1 levels, even at the highest doses tested
(30 µM). Thus, BPQ is the only downstream
B(a)P-diol metabolite that acts as a direct inducer of
CYP1A1 mRNA in HepG2 cells.
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. In the first experiment, three extended quinones of B(a)P
derived from CYP-generated phenol metabolites were compared with the
AKR-generated ortho-quinone BPQ for their ability to induce
CYP1A1 in HepG2 cells. BPQ was a superior inducer of CYP1A1 mRNA when
compared with the 1,6-, 3,6-, and the
6,12-benzo(a)pyrene-diones. The abilities of various PAH
o-quinones generated by AKRs to induce CYP1A1 were
widely disparate (Fig. 6)
. The rank order of potency was BPQ >
BAQ > NPQ > DMBAQ. This rank order inversely follows their reactivity
with cellular thiols, e.g., glutathione (data not shown),
suggesting that increased electrophilicity of PAH o-quinones
may limit their ability to induce CYP1A1 mRNA in HepG2 cells.
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20- and 13-fold,
respectively) from the XRE-reporter (Fig. 7)
, demonstrating that BPQ mediates transcription of CYP1A1 mRNA via XREs
located in the 5' flanking region of the CYP1A1 gene.
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, it failed to induce CYP1A1 in either AhR- or ARNT-deficient hepatoma
cells. These results clearly indicate that BPQ, like TCDD and
B(a)P, activates CYP1A1 expression via an
AhR/ARNT-dependent pathway.
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. This complex was specific for the XRE, because increasing
concentrations of cold competitor XRE oligonucleotide successfully
eliminated this complex without causing loss of the lower nonspecific
band observed in all of the reactions.
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. In untreated cells, all immunofluorescence staining associated with the
AhR was present in the cytosol (Fig. 10A)
, and this staining
pattern remained unchanged after treatment with anti-BPDE
(Fig. 10C)
. However, after treatment with either BPQ (Fig. 10D)
or the classical AhR ligand TCDD (Fig. 10B)
,
a significant fraction of cells demonstrated intense AhR staining in
the nucleus, confirming the ability of BPQ to cause translocation of
AhR into the nucleus.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Because X-ray crystallographic studies show that the oxidation of the nonplanar B(a)P-diol by CYP1A1 results in the even more torsionally strained anti-BPDE (37) , it was not surprising that anti-BPDE failed to be an inducer of CYP1A1. In contrast, the oxidation of B(a)P-diol by AKRs initially results in a catechol, which then undergoes successive one-electron oxidations to form an o-quinone with carbons that are now sp2-hybridized at the 7 and 8 positions, restoring planarity to this electrophilic metabolite. Although there are no crystal structures of PAH o-quinones to date, energy minimization of B(a)P-diol, anti-BPDE, and BPQ structures using QUANTA/CHARMm verified the uniquely planar nature of BPQ (data not shown), a hallmark of AhR ligands.
These studies definitively show that PAH o-quinones utilize the classical AhR signal transduction pathway to cause the induction of CYP1A1. This was demonstrated by the ability of BPQ to (a) induce CYP1A1 expression over a rapid time course, activate XRE-dependent reporter gene expression, and cause translocation of the AhR to the nucleus; and (b) by the failure of BPQ to induce murine CYP1A1 in AhR- and ARNT-deficient murine hepatoma cells.
We have previously reported that AKR1C1 is constitutively expressed in HepG2 cells and that it oxidizes B(a)P-diol to BPQ efficiently (12 , 30) . This would suggest that the induction of CYP1A1 by B(a)P-diol should occur rapidly and not over the delayed time course reported here. However, a sufficient reservoir of "free" BPQ must first build up to act as a ligand of the AhR. Studies on [3H]B(a)P-diol metabolism in HepG2 cells reveal that there is a high level of glucuronyl- and sulfo-transferase activity present, and that free BPQ is only detected after 12–24 h.5 Thus, the time required to detect free BPQ after B(a)P-diol treatment is consistent with the time course observed for the indirect induction of CYP1A1 by B(a)P-diol.
Parent PAHs are classified as bifunctional inducers because as planar
molecules they induce CYP1A1 expression via the AhR/XRE pathway, and
after metabolism, their downstream metabolites induce phase II
(de)toxification enzymes via the ARE pathway (21)
.
However, PAH o-quinones define a novel class of bifunctional
inducer that does not require metabolism to induce gene expression via
both mechanisms. In previous studies (12
, 30)
, we
demonstrated that AKR1C1 is induced (most likely via an ARE/EpRE-type
mechanism) by its reaction product, the electrophilic and redox-active
BPQ, thereby establishing an autoregulatory loop. PAH
o-quinones produced by this pathway are therefore
bifunctional because in the present studies they were also found to
induce CYP1A1 via the AhR/XRE pathway. Thus, due to their uniquely dual
planar/electrophilic nature, PAH o-quinones possess features
that allow them to simultaneously induce both phase I and phase II gene
expression through different mechanisms without a requirement for
further metabolism. We compare the mechanism of classical bifunctional
inducers described by Prochaska et al. (21)
with the mechanism of action of the new bifunctional inducers PAH
o-quinones (Fig. 11)
. Because PAH o-quinones can differ in their electrophilicity
by several orders of magnitude, their chemical reactivity may determine
their relative abilities to activate phase I versus phase II
(de)toxification gene expression via the XRE versus the ARE
pathways, respectively.
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Our discovery that PAH o-quinones generated in the cytosol
are shuttled into the nuclear compartment via the AhR reveals the only
known mechanism by which genotoxic PAH o-quinones can be
targeted to the nucleus with specificity. Although higher
concentrations of BPQ (20 µM) can be sequestered in a
nonspecific manner into the cell pellet, significantly lower
concentrations of BPQ (100–300 nM) result in detectable
CYP1A1 induction. Thus, at low concentrations, preferential targeting
may indeed be achieved by the AhR. Because BPQ is a redox active
compound and will enter into futile cycles, this specific targeting of
low concentrations of BPQ could contribute to its genotoxic profile. In
this regard, it is well known that after ligand-dependent translocation
of the AhR to the nucleus, the AhR is rapidly degraded to limit the
duration of CYP1A1 induction (38
, 39)
. It is unclear,
however, what happens to the previously bound ligand. Local liberation
of PAH o-quinones in proximity to genomic DNA, which is
known to contain copper (40
, 41)
, will enhance redox
cycling in the presence of nuclear reducing equivalents and provides a
mechanism for the oxidative DNA damage observed with parent PAH
in vitro and in vivo (42
, 43)
. In
the future, it will be interesting to determine whether the genotoxic
effects of BPQ are attenuated in AhR-deficient cells.
In addition to the implications for the initiation phase of PAH carcinogenesis, the activation of the AhR by PAH o-quinones may also play a role in the promotion stage. The propensity of PAH o-quinones to redox cycle and amplify ROS in PAH-exposed cells may mimic the tumor-promoting effects of phorbol myristoyl acetate by generating oxidative stress, which can lead to the inappropriate activation of protein kinase C and expression of the proto-oncogenes c-fos and c-jun (44) . TCDD is also a well-established tumor promoter (45, 46, 47, 48) , and the ability of PAH o-quinones to mimic a well-characterized property of TCDD (CYP1A1 induction) may represent an epigenetic property of PAH o-quinones that contributes to the ability of PAHs to act as complete carcinogens.
In summary, these studies demonstrate that PAH o-quinones generated by human AKRs induce the PAH-activating enzyme CYP1A1 via an AhR-dependent mechanism. The ability of genotoxic PAH o-quinones to enter the nucleus and mimic the potent tumor promoter TCDD may have important implications for both the initiation and promotion phases of PAH carcinogenesis. These and other in vitro studies underscore the need to test PAH o-quinones as endogenously generated tumor-initiating/promoting metabolites of PAHs in in vivo models of chemical carcinogenesis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by NIH Grants CA39504 and
CA55711 (to T. M. P.) and a Pharmaceutical Research and
Manufacturers of America Foundation Advanced Predoctoral Fellowship (to
M. E. B.). 
2 To whom requests for reprints should be
addressed, at Department of Pharmacology, University of Pennsylvania
School of Medicine, 3620 Hamilton Walk, Philadelphia, PA 19104-6084.
Phone: (215) 898-9445; Fax: (215) 573-2236; E-mail: penning{at}pharm.med.upenn.edu
3 The abbreviations used are: PAH,
polycyclic aromatic hydrocarbon; B(a)P,
benzo(a)pyrene; AhR, aryl hydrocarbon receptor; AKR,
aldo-keto reductase; ARE/EpRE, antioxidant response
element/electrophilic response element; BPQ,
benzo(a)pyrene-7,8-dione; CYP, cytochrome P450;
B(a)P-diol,
(±)-trans-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene;
ARE, antioxidant response element; EpRE, electrophilic response
element; XRE, xenobiotic response element; anti-BPDE,
(±)-anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene;
TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; ARNT, AhR nuclear
translocator; ROS, reactive oxygen species; 6MPA, 6-medroxyprogesterone
acetate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; BAQ,
benz[a]anthracene-3,4-dione; NPQ, naphthalene-1,2-dione;
DMBAQ, dimethylbenz[a]anthracene-3,4-dione; DD,
dihydrodiol dehydrogenase
[trans-1,2-dihydrobenzene-1,2-diol dehydrogenase (EC
1.3.1.20)].
4 The nomenclature for the AKR superfamily was
proposed by Jez et al. (Ref. 14
) and adopted at the 8th
International Symposium on Enzymology and Molecular Biology of Carbonyl
Metabolism in Deadwood, South Dakota, June 29–July 3, 1996.
5 C. Yarosh and T. M. Penning, unpublished
observations.
Received 8/ 2/99. Accepted 12/ 9/99.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-hydroxysteroid-dihydrodiol dehydrogenase for trans-dihydrodiol metabolites of polycyclic aromatic hydrocarbons. J. Biol. Chem., 261: 6184-6191, 1986.-napthoquinone. Org. Synth., 2: 430-435, 1943.
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Y.-W. Kwon, S. Ueda, M. Ueno, J. Yodoi, and H. Masutani Mechanism of p53-dependent Apoptosis Induced by 3-Methylcholanthrene. INVOLVEMENT OF p53 PHOSPHORYLATION AND p38 MAPK J. Biol. Chem., January 11, 2002; 277(3): 1837 - 1844. [Abstract] [Full Text] [PDF]
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), B(a)P-diol (
),
anti-BPDE (
), or BPQ (
) in DMSO were added to fresh
media to yield the appropriate range of final concentrations in 0.1%
DMSO. After 6 h, total RNA was harvested, and 10 µg of RNA per
lane were electrophoresed, transferred to membranes, and sequentially
analyzed for CYP1A1 and GAPDH mRNA as described in "Materials and
Methods." Results for each time point were determined by
PhosphorImager analysis and are expressed as the fraction of the
maximal CYP1A1:GAPDH ratio (observed with 30 nM TCDD).
