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Vol. 292, Issue 3, 1008-1014, March 2000
Department of Pathology, College of Medicine, University of California-Irvine, Irvine, California
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Abstract |
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 Top
 Abstract
 Introduction
Experimental Procedures
Results
Discussion
References
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Recent studies from this laboratory have demonstrated that L-tryptophan, after oxidation either by UV-irradiation or ozone, induces aryl hydrocarbon receptor (AhR) activation and binding of the liganded AhR complex to its specific DNA recognition site, thereby initiating transcription of the cytochrome P-450 1a1 (Cyp1a1) gene with concomitant increase of CYP1A1 protein and 7-ethoxyresorufin O-deethylase activity in wild-type mouse hepatoma cells, Hepa lclc7 (Hepa-1), in culture. Temporary inhibition of protein synthesis by cycloheximide resulted in superinduction of oxidized tryptophan-inducible CYP1A1 mRNA, protein, and 7-ethoxyresorufin O-deethylase activity in Hepa-1 cells. In the present communication, the results obtained by immunoblot analyses with monoclonal CYP1A1/1A2 antibody (NIH 1-7-1) demonstrate that both UV- or ozone-oxidized tryptophan also induce CYP1A2 protein in Hepa-1 cells. CYP1A2 mRNA, detected by reverse transcription-polymerase chain reaction, was markedly induced in the UV- or ozone-oxidized tryptophan-treated cells. Temporary inhibition of protein synthesis by cycloheximide further induced oxidized tryptophan-inducible CYP1A2 mRNA as well as the protein in Hepa-1 cells. This is the first report demonstrating the induction of CYP1A2 mRNA and protein in Hepa-1 cells.
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Introduction |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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Cytochrome
P-450 (CYP) is a superfamily of hemoproteins that are able to
metabolize a large number of endogenous and exogenous compounds through
oxidative, reductive, and peroxidative mechanisms. According to the
current estimates, the P-450 gene family comprises 74 families, 14 of
which exist in all mammals examined to date (Nelson et al., 1996
). In
mammals, the CYP
family 1 consists of three
isozymes, CYP1A1, 1A2, and 1B1 (Nelson et al., 1996). Although no
endogenous substrate for CYP1A1 has been found to date, this isozyme
actively metabolizes benzo[a]pyrene and several other polycyclic
aromatic hydrocarbons (PAHs) (Guengerich and Shimada, 1991). In
contrast, CYP1A2 exhibits a high level of catalytic activity toward
2-acetylaminofluorene, acetanilide, aflotoxin B1,
4-aminobiphenyl, and other arylamines (McManus et al., 1984; Kadlubar
and Hammons, 1987; Faletto et al., 1988). Many of these PAHs and
arylamine substrates are metabolized by CYP1A1 and 1A2 to carcinogenic
and toxic intermediates. Therefore, the differences in individual risk
of carcinogenesis or toxicity might be correlated with the level of
expression of either of these genes in a particular tissue.
In general, CYP1A1 is not expressed in normal adult animal tissues but
can be induced severalfold in response to several stimuli such as PAHs
or halogenated aromatic hydrocarbons (for review, see Gonzalez, 1989),
hyperoxia (Okamoto et al., 1993; Khatsenko et al., 1997), or oxidized
tryptophan (Sindhu et al., 1996b, Sindhu and Kikkawa, 1999). CYP1A2, in
contrast, is known to be constitutively expressed in animals and is
inducible in the liver after treatment by a variety of exogenous
substances, including PAHs, ingestion of charbroiled meat, certain
unknown components of cruciferous vegetables, dietary heterocyclic
amines, and certain drugs (for review, see Guengerich and Shimada,
1991; Wrighton and Stevens, 1992 and references therein). In the aryl
hydrocarbon (Ah)-responsive mouse liver, the levels of constitutively
expressed CYP1A2 mRNA levels appear to be at least as high as maximally
induced CYP1A1 mRNA levels (Gonzalez et al., 1984). However, many
liver-derived cell lines are known to rapidly lose the expression of
CYP1A2. Therefore, in contrast to CYP1A1, the detailed molecular
mechanism(s) underlying the expression of CYP1A2 are not very well defined.
Mouse hepatoma-derived Hepa lclc7 (Hepa-1) cell line has been
considered as an excellent model for CYP1A1 studies and the wild-type
and variant Hepa-1 cells have been used by numerous laboratories to
investigate the molecular and biochemical mechanisms of CYP1A1
induction. Hepa-1 cells are known to be inducible for CYP1A1 and retain
the same high level of inducible CYP1A1 activity during several months
in culture (for review, see Hankinson, 1994). Additionally, no
detectable heterogeneity in CYP1A1 activity has been observed among
subclones of this line (Hankinson, 1994). Furthermore, mutants of the
Hepa-1 cell line that are defective in induction of CYP1A1 have been
isolated and characterized (Hankinson, 1994). However, CYP1A2 in this
cell line was either thought not to be expressed or, if expressed at
all, it was thought to be below the levels of detection.
For the past several years, this laboratory has been investigating the
molecular and biochemical mechanisms of CYP1A1 induction by oxidized
tryptophan in wild-type mouse Hepa-1 cells, in culture. As a part of
this project, we have recently demonstrated that L-tryptophan, after oxidation by either ozone (OT) or
UV-irradiation (UVT), induces aryl hydrocarbon receptor (AhR)
activation and binding of the liganded AhR complex to its specific DNA
recognition site, thereby inducing transcription of the
Cyp1a1 gene with concomitant increase of CYP1A1 protein and
7-ethoxyresorufin O-deethylase (EROD) activity in wild-type
Hepa-1 cells (Sindhu et al., 1996b, 1999). The induction of CYP1A1 by
UV-oxidized tryptophan was effectively inhibited by administration of a
15-mer antisense phosphorothioate oligonucleotide that was targeted to
the Cyp1a1 gene (Sindhu et al., 1996a). More recently, we
have found that temporary inhibition of protein synthesis by
cycloheximide causes superinduction of UVT- or OT-inducible CYP1A1
mRNA, protein and EROD activity in wild-type Hepa-1 cells (Sindhu and
Kikkawa, 1999). The present investigation was undertaken as a corollary
to this project. The results obtained in the present study demonstrate
that administration of OT or UVT to wild-type Hepa-1 cells causes
induction of CYP1A2 mRNA and the protein. Temporary inhibition of
protein synthesis by cycloheximide was found to result in the
superinduction of CYP1A2 mRNA as well as the protein.
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Experimental Procedures |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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Materials. Cycloheximide, actinomycin D, and NADPH were obtained from Sigma Chemical Co. (St. Louis, MO). L-Tryptophan was purchased from Aldrich Chemical Co. (Milwaukee, WI) and was of highest purity commercially available. 7-Ethoxyresorufin and resorufin were purchased from Pierce (Rockford, IL). 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) was obtained from Midwest Research Institute, Kansas City, MO. Monoclonal CYP1A1/1A2 antibody (NIH 1-7-1) was generously provided by Dr. S. S. Park of National Cancer Institute, Frederick, MD. All other chemicals were of highest purity commercially available.
Oxidation of Tryptophan.
O3 was
generated by passing medical grade oxygen as detailed in Yan et al.
(1999). Ozonation of tryptophan was carried out by bubbling a stream of
O3 (196 mg/m3, 100 ppm)
through a fine capillary into 500 ml of 2 mM aqueous solution of
tryptophan for 24 h. Glass-distilled water, used routinely for the
experiments, also was ozonized along with tryptophan and used as one of
the controls.
). The oxidized solutions were protected
from light and stored in the refrigerator.
To confirm the oxidation of tryptophan, unoxidized tryptophan, UVT, or
OT were analyzed by reversed phase HPLC (Perkin-Elmer Series 410)
equipped with a Zorbax C18 analytical column
(4.6 × 250 mm, 5 µm) and an LC 235 diode array detector as
described in Sindhu et al. (1996b, 1999) and Yan et al. (1999).
Cells and Growth Conditions.
Wild-type Hepa-1 cells,
generous gifts from Dr. Oliver Hankinson of UCLA, were propagated in
ribonucleoside-free-minimum essential medium (Life Technologies,
Paisley, Scotland) containing 5% heat inactivated fetal bovine serum
(Gemini Biological Products, Calabasas, CA) and 1%
antibiotic-antimycotic (Life Technologies) with
25-cm2 canted neck tissue culture flasks (Becton
Dickinson, Mountain View, CA) as described previously (Sindhu et al.,
1996b, 1999; Yan et al., 1999). When the cells were ~95% confluent,
these were incubated with OT or UVT for a total of 4 h at 37°C
as described in the text. The cells were treated with cycloheximide as
indicated during the final minutes of oxidized tryptophan incubation
period. At the end of treatment period, the medium containing oxidized tryptophan and cycloheximide was taken out and the cells were washed
twice with 10 ml each of sterile PBS. After adding 10 ml of fresh
medium, actinomycin D (1 µg/ml) was added and the cells were
incubated at 37°C for 3 more hours. The cells were then washed twice
with ice-cold PBS and scraped. The harvested cell suspension was
centrifuged at 1500g for 10 min and the cell pellet was
resuspended in 250 µl of sonication buffer (150 mM Tris-HCl, pH 8.0, containing 7.5% sucrose). The cell suspensions were sonicated on ice
with a cell disrupter (Heat Systems-Ultrasonics, Inc., Plainview, NY) at 50% power and an output setting of three with two periods of 10 s, interrupted by one interval of the same duration on ice to
avoid overheating.
.
Total protein content of the sonicated cells was measured with the
Bio-Rad protein assay kit with bovine plasma 
-globulin as the standard.
SDS-Polyacrylamide Gel Electrophoresis (PAGE) and
Immunoblotting.
The disrupted cell extracts (10 µg of protein)
were boiled with an equal volume of SDS-sample solubilization buffer
(4% SDS, 10% 
-mercaptoethanol, 20% glycerol, 125 mM Tris-HCl, pH
6.8) for 5 min. The samples were allowed to cool to room temperature and proteins were resolved by denaturing SDS-PAGE on discontinuous polyacrylamide (10%) slab gels. The proteins were then
electrophoretically transferred to nitrocellulose membranes (Bio-Rad,
Richmond, CA). The filters were blocked with SuperBlock blocking buffer
(Pierce) containing 5% nonfat milk, washed thoroughly with
Tris-buffered saline containing 0.1% Tween 20 and probed with
monoclonal anti-CYP 1A1/1A2 (1-7-1). The antigen-antibody complexes
were detected with a horseradish peroxidase-conjugated anti-mouse IgG
(Amersham, Arlington Heights, IL) with the enhanced chemiluminescence
detection system (Amersham).
Isolation of RNA and Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Hepa-1 cells, grown as described earlier, were incubated with OT or UVT (100 µM) for a total of 2 h in the presence or absence of cycloheximide. Cycloheximide (5 µg/ml medium) was included during the final 1 h of oxidized tryptophan treatment period. At the end of the incubation period, total RNA was isolated with ultraspec total RNA isolation reagent (Biotecx Laboratories, Inc., Houston, TX) according to the manufacturer's instructions except that the aqueous solution containing RNA was treated three times with phenol/chloroform/isoamyl alcohol (25:24:1) and then precipitated with isopropanol. The RNA pellet was washed two times with 75% ethanol and gently dried under house vacuum in a desiccator. Finally, the RNA was dissolved in 100 µl of nuclease-free H2O and quantified.
The isolated RNA solution (5 µg each) was diluted with 10.15 µl of nuclease-free H2O to a total volume of 11.15 µl and 1 µl (5 pmol) of poly (dT)15 (Promega, Madison, WI) was then added. The mixture was kept in boiling water for 5 min and then cooled on ice for 5 min. After adding 4 µl of reverse transcriptase buffer (250 mM Tris-HCl, pH 8.3, 375 mM KCl, 15 mM MgCl2; Life Technologies), 1 µl of 10 mM deoxynucleoside-5'-triphosphate mixture (Promega), 2 µl of 0.1 M dithiothreitol (Life Technologies), 0.25 µl (10 U) of RNAsin (Promega), and 0.6 µl of Moloney murine leukemia virus reverse transcriptase (120 U), these were incubated at 37°C for 1 h. After the cDNA synthesis, reverse transcriptase was inactivated by incubation at 75°C for 10 min. Ribonuclease H (1 µl, 1.5 U; Promega) was then added and incubated for 20 min at 37°C. The single-stranded cDNA thus obtained was used for PCR amplification. The primers for PCR amplification of CYP1A2 were determined with the computer program HYBsimulator (Advanced Gene Computing Technologies, Irvine, CA) as described (Mitsuhashi, 1996). The sequence of the
sense and the antisense primers for CYP1A2 were as follows:
5'-CTACCGATACACATCCTTTGTCCCC-3', and 5'-GATGCCTCGACAATCTTCACTTGGA-3'. -Actin was amplified as a control. The sequence of sense and the
antisense primers for -actin has been described (Sindhu et al.,
1996b). cDNA (6 µl) was mixed with 3 µl of 10× PCR buffer (Promega), 2.4 µl of 25 mM MgCl2 (Promega), 1.5 µl of each sense and antisense primers (0.1 mg/ml), 2.4 µl of 10 mM
deoxynucleoside-5'-triphosphate mixture (Promega), and 0.2 µl
of Taq polymerase (Fisher, Pittsburgh, PA) in a final
volume of 30 µl and was amplified in a programmable thermal
controller (MJ Research, Cambridge, MA) with 30 cycles by denaturation
at 94°C for 1.5 min, annealing at 55°C for 1.5 min, and extension
at 72°C for 4 min.
Statistical Analysis. Statistical analysis of the data was performed with StatView II software. Results are expressed as means ± S.D. Statistical significance of the difference between the control and experimental group was determined by one-way ANOVA and Dunnett's test.
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Results |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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Induction of EROD Activity by OT and UVT and Its Superinduction by Cycloheximide. Wild-type Hepa-1 cells were incubated with OT or UVT (100 µM) and TCDD (2 nM) for 4 h in the presence or absence of cycloheximide as described in the text. Cycloheximide (5-µg/ml medium) was added during the final 2 h of oxidized tryptophan treatment. The cells were washed twice and incubated for an additional 3 h in fresh medium containing actinomycin D (1-µg/ml medium). The cells were then washed twice, harvested, and EROD activity was determined as described above. Both OT and UVT caused a significant induction of EROD activity compared with the untreated controls (Table 1). Treatment with cycloheximide caused a superinduction of both OT- and UVT-inducible EROD activity. Similar results were obtained with TCDD (Table 1).
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Superinduction of CYP1A2 Protein.
Immunoblot analysis of the
cell extracts (10 µg of protein) was carried out with monoclonal
CYP1A1/1A2 antibody. This antibody is known to cross-react with both
CYP1A1 and CYP1A2. The assay was carried out several times with similar
results and one representative result is presented in Fig.
1. The results show that no CYP1A2 protein (lower band) could be detected in the control (lane 1) or
cycloheximide-treated samples (lane 2). However, OT treatment caused an
induction of CYP1A2 protein (lanes 3 and 4) that was further induced by
treatment with cycloheximide (lanes 7 and 8). Similarly, UVT caused a
significant induction of CYP1A2 protein (lanes 5 and 6) that was
further induced by cycloheximide treatment (lanes 9 and 10).
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Superinduction of Oxidized Tryptophan-Inducible CYP1A2 mRNA
Expression by Cycloheximide.
Results obtained with RT-PCR
amplification of CYP1A2-specific cDNA (Fig.
4, upper panel) show that no CYP1A2 cDNA
could be detected in the unoxidized tryptophan-treated controls (lane
1) or cycloheximide-treated samples (lane 2). However, with the cells that were incubated with UVT for a total of 2 h at the final
concentration of 100 µM, an expected 440-base pair (bp)-long fragment
of CYP1A2 cDNA was readily detected (lane 3). Furthermore, to the cells to which cycloheximide was added during the final 1 h of UVT
treatment, CYP1A2 mRNA was further induced (lane 4). Similarly, CYP1A2
cDNA could be detected in cells that were incubated with OT alone (lane 5) and was further induced when cycloheximide was added after treatment
of the cells with OT (lane 6). -Actin-specific cDNA of 338 bp was
detected in all the groups (Fig. 4, bottom), namely, control (lane 1),
cycloheximide (lane 2), and UVT (lane 3), UVT plus cycloheximide (lane
4), OT (lane 5), and OT plus cycloheximide (lane 6) RT-PCR
amplifications were carried out at least three times with similar
results. Similar results were obtained with TCDD (data not shown).
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Restriction Digestion of CYP1A2 cDNA with AvaII.
An analysis
of the restriction enzyme site map of CYP1A2 cDNA revealed an AvaII
restriction site at 1485 bp resulting in two fragments of 306 and 134 bp, respectively. PCR-amplified CYP1A2 cDNA was incubated with and
without AvaII (20 U) overnight at 37°C in a total volume
of 30 µl and 15 µl of the reaction mixtures was separated by
agarose (1%) gel electrophoresis and visualized by ethidium bromide
staining. The results reported in Fig. 5
show that incubation of the CYP1A2 cDNA with AvaII resulted
in its cleavage confirming its identity.
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Sequencing of CYP1A2 cDNA. The PCR amplified CYP1A2 cDNA was purified with QIAquick PCR purification kit (Qiagen, Chatsworth, CA) and sequenced. DNA sequencing was performed on an ABI PRISM 377 DNA Sequencer with protocols recommended by the manufacturer with dRhodamine chemistry. Standard runs were performed on 36-cm well-to-read gels. An analysis of the observed cDNA sequence showed 100% homology with the predicted CYP1A2 sequence, thus confirming that the PCR-amplified cDNA was indeed that of CYP1A2.
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Discussion |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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In recent years, the role of AhR in the induction of CYP1A1 has
been extensively studied. Induction of CYP1A1 seems to be regulated
exclusively at the transcriptional level (for review, see Hankinson,
1995). The unliganded AhR, found in the cytosol by conventional
subcellular fractionation, has a molecular weight of ~280 kDa and is
comprised of AhR monomer, two molecules of the 90-kDa heat shock
protein, and possibly other proteins (Hankinson, 1995). After ligand
binding, AhR interacts with aryl hydrocarbon receptor nuclear
translocator and the heterodimer of AhR and aryl hydrocarbon receptor
nuclear translocator constitutes a transcription factor, referred to as
the transformed AhR complex, which stimulates the synthesis of
CYP1A1 protein and several other proteins involved in xenobiotic
metabolism (Hankinson, 1995). Activation of transcription occurs
through interaction of the transformed AhR complex with several copies
of short sequences, termed xenobiotic-(dioxin) responsive elements,
located within the 5'-flanking region of the Cyp1a1 gene.
The regulation of Cyp1a2 gene expression, in contrast, is
not yet very well understood partly because of lack of stable cell line(s) that respond to the induction of this gene by PAHs or halogenated aromatic hydrocarbons. In addition, although the Hepa-1 cell line has been successfully used as a model system for delineating the mechanism(s) of CYP1A1 induction by numerous laboratories, no
serious attempt seems to have been made to date to investigate the
presence of CYP1A2 in these cells. The induction characteristics of
CYP1A1 and 1A2 mRNAs in Ah-responsive and Ah-nonresponsive mouse
strains have been shown to be similar (Tukey and Nebert, 1984),
suggesting that these isozymes may share a common mechanism of
induction. Therefore, although the induction of Cyp1a2 gene appears to require the AhR, there is very little similarity between the
rodent CYP1A1 and CYP1A2 5'-flanking regions, where the regulatory elements are generally thought to reside (Gonzalez et al., 1985). After
treatment with PAHs or TCDD, substantial increase in the rate of
Cyp1a2 gene transcription was observed in the livers of the
mouse (Okino et al., 1992) and rat (Pasco et al., 1993), although post-transcriptional regulation also has been reported to contribute to
the induced CYP1A2 mRNA levels (Silver and Krauter, 1988). However,
some laboratories also have reported either no increase (Pasco et al.,
1988) or only a small increase (Silver and Krauter, 1988) in the rate
of Cyp1a2 gene transcription and the reason for this
discrepancy is currently unknown. Recently, Quattrochi et al. (1994)
suggested that human CYP1A2 may be regulated through two mechanisms:
AhR specific- and promoter-specific elements.
Although the expression of CYP1A2 was initially described only in the
liver, recent studies have suggested that this isozyme is more widely
spread than originally thought. CYP1A2 expression has been described in
the brain (Farin and Omiecinski, 1993) and in cultured umbilical vein
endothelium (Farin et al., 1994). There is also evidence that CYP1A2
mRNA is present in human duodenum following treatment with omeprazole
(McDonnell et al., 1992). Pineau et al. (1995) generated a
transgenic mouse line that lacks expression of CYP1A2 and found that
mice homozygous for a targeted mutation in the Cyp1a2 gene
were nonviable. Lethality occurred shortly after birth with symptoms of
severe respiratory distress, suggesting that CYP1A2 is critical for
neonatal survival. However, Liang et al. (1996) developed a
CYP1A2-deficient mouse line by homologous recombination in embryonic
stem cells that is completely viable and fertile.
In adult mammals, the primary sites of constitutive expression of
CYP1A2 have been shown to be the liver (Sakuma et al., 1998) and
olfactory epithelia (Ding et al., 1992). Suggestions have been made
that the hepatic enzyme may be involved in an alternate disposal
pathway of bilirubin (Kapitulnik and Gonzalez, 1993). With solution
hybridization technology, Raval et al. (1991) determined the
steady-state level of control in rat hepatic CYP1A2 mRNA to be six
molecules/cell, which was increased to >30-fold on i.p. administration
of 3-methylcholanthrene (MC). CYP1A1 mRNA, in contrast, was
increased from <3 to 68 molecules by MC treatment (Raval et al.,
1991). However, in liver-derived cell lines such as mouse Hepa-1, rat
H4IIE, and human HepG2, the levels of induced CYP1A2 mRNA were
found to be either negligible or below the levels of detection as
judged by Northern blot analysis (Jaiswal et al., 1985; Xu and
Bresnick, 1990; Chung and Bresnick, 1994). However, with RT-PCR, HepG2
cells have recently been shown to constitutively express CYP1A2, which
was induced by MC treatment (Chung and Bresnick, 1994).
In the present investigation, immunoblot analyses of Hepa-1 cell-free
extracts with monoclonal antibody (NIH 1-7-1), which is known to
cross-react with CYP1A1 and 1A2, showed that both CYP1A1 and 1A2
protein were induced by OT or UVT as well as by TCDD (Figs. 1 and 2).
Further induction of both these proteins was observed in the cells that
had been incubated with cycloheximide during the final 2 h of the
inducer treatment. It should be pointed out that in earlier
communication, we reported the presence of only CYP1A1 protein after
treatment of Hepa-1 cells with oxidized tryptophan (Sindhu et al.,
1996b). The discrepancy in these results is because in the earlier
investigation after incubating the membrane with chemiluminescent
substrate, we routinely exposed the membrane to the film for 1 to 2 min
that, on development, yielded an intense CYP1A1 protein band. In
addition, we were working on the generally held notion that CYP1A2 is
either absent, or if present at all, it was thought to be below the
levels of detection in liver-derived cell lines (Jaiswal et al., 1985;
Xu and Bresnick, 1990). Because CYP1A1 and 1A2 proteins are
barely separable on 10% acrylamide gels, longer exposure of the
membrane to the film yielded only one intense band. This is confirmed
by the presence of only one very intense band when MC-induced mouse
liver microsomes were used as the standard (Sindhu et al., 1996b).
During the course of our investigations on the superinduction of
oxidized tryptophan-inducible CYP1A1 by cycloheximide, immunoblotting
showed a very intense CYP1A1 protein band. The induction of CYP1A1
protein was so huge that we began controlling the time of the exposure
of the membrane to the film at 10-s intervals. It was then that the
presence of CYP1A2 protein was consistently observed. Therefore, in
Sindhu et al. (1996b), the intense CYP1A1 protein band is comprised of both CYP1A1 and CYP1A2 proteins.
The primers for PCR amplification of CYP1A2-specific cDNA were
determined by a computer program, HYBsimulator, as described (Mitsuhashi, 1996), and the selected sequences were the most specific ones with minimum chance of cross-hybridization against CYP1A1 or other
known P-450 genes. Furthermore, the selected upstream sense primer is
located on exon 4, whereas the downstream antisense primer was chosen
from the junction of exon 6 and 7 to prevent amplification of genomic
DNA, if any, that might contaminate the RNA preparations. RT-PCR
resulted in the amplification of an expected 440-bp CYP1A2 cDNA (Fig.
4, top) and 338-bp -actin cDNA (bottom), respectively. Treatment
with OT or UVT caused an induction of CYP1A2 mRNA, whereas -actin
was not affected by oxidized tryptophan treatment. Restriction
digestion of PCR-amplified CYP1A2 cDNA by AvaII yielded two
expected fragments of 306 and 134 bp, respectively (Fig. 5). Finally,
the PCR-amplified CYP1A2 cDNA was purified with QIAquick PCR
purification kit (Qiagen) and sequenced. The observed CYP1A2 cDNA
sequence had 100% homology with the reported sequence for mouse CYP1A2 cDNA.
Treatment of Hepa-1 cells with cycloheximide during the final 2 h
of inducer treatment resulted in further induction of both CYP1A2 mRNA
as well as the protein. Similar results were obtained with CYP1A1
(Sindhu and Kikkawa, 1999) suggesting that the induction and
superinduction of these two isozymes occurs through a similar mechanism. These results are different from those reported by Teifeld
et al. (1989) who showed that although Cyp1a2 gene was transcribed in hepatocyte primary cultures, treatments that strongly superinduced CYP1A1 transcription rate had a negligible
effect on Cyp1a2 gene transcription rate.
Nemoto and Sakurai (1993) have reported that constitutive as well as
MC-inducible CYP1A2 expression was dependent on cell density in adult
mouse hepatocytes and was higher in cells cultivated at lower density.
However, OT or UVT at concentrations used in the present investigation
had no effect on the cell density, thus eliminating the possibility of
the effect of cell density on its expression. These results are similar
to those reported by Chung and Bresnick (1994) who observed that
induction of CYP1A2 in HepG2 cells by MC also was not effected by cell number.
In conclusion, the present investigation represents the first report demonstrating that both CYP1A2 mRNA and protein can be induced by oxidized tryptophan in Hepa-1 cells. These studies should be appropriate for investigating the details of the CYP1A2 induction mechanism in these cells.
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Acknowledgments |
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We thank Dr. Oliver Hankinson of UCLA for generously providing the wild-type Hepa lclc7 cells and for critically reading the manuscript. Generous gift of CYP1A1/1A2 monoclonal antibody by Dr. S. S. Park is gratefully acknowledged. We also thank Armen Minasian, Armond Aghakhani, and Rongzi Yan for their help in some of the experiments. The assistance of Pamela Sutherland, Marlene Elizarraraz, and Ronald Niece of the Biotechnology Resource Facility at the University of California-Irvine in DNA sequencing also is appreciated.
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Footnotes |
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Accepted for publication November 18, 1999.
Received for publication July 7, 1999.
1 This work was supported by Grant HL-50450 from the National Heart, Lung and Blood Institute of the National Institutes of Health, U.S. Public Health Service.
Send reprint requests to: Ram K. Sindhu, Ph.D., Department of Pathology, College of Medicine, University of California-Irvine, Irvine, CA 92697-4800. E-mail: rksindhu{at}uci.edu
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Abbreviations |
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CYP, cytochrome P-450; PAH, polycyclic aromatic hydrocarbon; Ah, aryl hydrocarbon; OT, ozone-oxidized tryptophan; UVT, UV-oxidized tryptophan; AhR, aryl hydrocarbon receptor; EROD, 7-ethoxyresorufin O-deethylase; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; PAGE, polyacrylamide gel electrophoresis; RT-PCR, reverse transcription-polymerase chain reaction; bp, base pair; MC, 3-methylcholanthrene.
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References |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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/) null mutant mice develop normally but show deficient drug metabolism.
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-DNA/EcoRI + HindIII (0.75 µg)
marker (Promega). Lane 2 had undigested CYP1A2 cDNA. Lane 3 had CYP1A2
cDNA that had been digested with AvaII as described in
the text.