Regulation of the CYP1A1 gene by 2,3,7,8-tetrachlorodibenzo-p-dioxin but not by beta-naphthoflavone or 3-methylcholanthrene is altered in hepatitis C virus replicon-expressing cells.

Abstract

Exposure to hepatitis C virus (HCV) can lead to the development of cirrhosis and hepatocellular carcinoma. To examine the effects of long-term HCV infection on hepatic cytochrome P450 1A1 (CYP1A1) expression and function, we used a human hepatoma cell line expressing the HCV subgenomic replicon (Huh.8) to evaluate CYP1A1 induction by the aryl hydrocarbon receptor (AhR) ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In this study, we demonstrate that the induction of CYP1A1 expression in Huh.8 cells by TCDD but not by beta-naphthoflavone or 3-methylcholanthrene was significantly diminished. TCDD exposure of Huh.8 cells resulted in greater than 55% suppression of CYP1A1 transcription compared with the parent cell line Huh7, whereas protein levels and enzyme activities were further diminished. Suppression of CYP1A1 mRNA expression in TCDD-treated Huh.8 cells was partially reversed after pretreatment with the antioxidants N-acetylcysteine and nordihydroguaiaretic acid, suggesting a role for oxidative stress. Induced CYP1A1 message, protein, and enzyme activity were partially restored in an Huh7 cell line expressing the HCV replicon containing a deletion in the nonstructural protein NS5A. Furthermore, adenoviral expression of NS5A in Huh7 partially suppressed TCDD-induced CYP1A1 protein and enzyme activity, implicating this protein in the mechanism of suppression. These findings demonstrate that TCDD-mediated AhR signaling is impaired in hepatocytes in which HCV is present and that NS5A alone or in the presence of other nonstructural proteins of the subgenomic replicon is in part responsible.

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Regulation of the CYP1A1 Gene by 2,3,7,8-Tetrachlorodibenzo-p-
dioxin, but not β-Naphthoflavone or 3-Methylcholanthrene, is
Altered in Hepatitis C Virus Replicon-Expressing Cells
Garret R. Anderson, Aliya Hasan, Hao Yin, Ishtiaq Qadri, and Linda C. Quattrochi
From the Department of MedicineDepartment of PediatricsSchool of Medicine, and Department of
Pharmaceutical Sciences, School of PharmacyUniversity of Colorado at Denver and Health
Sciences Center, Denver, CO 80262
Abstract
Exposure to hepatitis C virus (HCV) can lead to the development of cirrhosis and hepatocellular
carcinoma. To examine the effects of chronic HCV infection on hepatic cytochrome P4501A1
(CYP1A1) expression and function, we used a human hepatoma cell line expressing the HCV
subgenomic replicon (Huh.8) to evaluate CYP1A1 induction by the aryl hydrocarbon receptor (AhR)
ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In this study, we demonstrate that the induction
of CYP1A1 expression in Huh.8 cells by TCDD, but not by β-naphthoflavone or 3-
methylcholanthrene, was significantly diminished. TCDD exposure of Huh.8 cells resulted in greater
than 55% suppression of CYP1A1 transcription compared to the parent cell line Huh7, while protein
levels and enzyme activities were further diminished. Suppression of CYP1A1 mRNA expression in
TCDD-treated Huh.8 cells was partially reversed following pretreatment with the antioxidants N-
acetylcysteine and nordihydroguaiaretic acid, suggesting a role for oxidative stress. Induced
CYP1A1 message, protein, and enzyme activity were partially restored in a Huh7 cell line expressing
the HCV replicon containing a deletion in the nonstructural protein, NS5A. Furthermore, adenoviral
expression of NS5A in Huh7 partially suppressed TCDD-induced CYP1A1 protein and enzyme
activity, implicating this protein in the mechanism of suppression. These findings demonstrate that
TCDD-mediated AhR signaling is impaired in hepatocytes in which HCV is present, and that NS5A
alone or in the presence of other nonstructural proteins of the subgenomic replicon is in part
responsible.
Abbreviations
AhR, aryl hydrocarbon receptor; Arnt, AhR nuclear translocator; βNF, β-naphthoflavone; CYP,
cytochrome P450; HCV, hepatitis C virus; MC, 3-methylcholanthrene; NAC, N-acetylcysteine;
NDGA, nordihydroguaiaretic acid; NS, nonstructural; PAHs, polycyclic aromatic hydrocarbons;
ROS, reactive oxygen species; TCDD, 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin; UGT, UDP-
glucuronosyltransferases
The cytochrome P450s (CYP), a multigene family of heme-containing proteins, are responsible
for the metabolism of numerous xenobiotics, including therapeutic drugs, environmental
chemicals, and dietary constituents, as well as endogenous substrates, such as steroids and bile
Correspondence address: Linda C. Quattrochi, Ph.D., Department of Medicine, University of Colorado at Denver and Health Sciences,
Center 4200 E. 9th Avenue, Denver, CO 80262, U.S.A., Tel.: (303) 315-3522, Fax: (303) 315-7180, E-mail:
Linda.Quattrochi@UCHSC.edu.
This work was supported by National Institutes of Health grant GM54477. Quantitative RT-PCR was performed by the University of
Colorado Cancer Center PCR Core Facility.
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Published in final edited form as:
Mol Pharmacol. 2006 September ; 70(3): 1062–1070.
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acids. Members of the CYP1 family include CYP1A1, 1A2, and 1B1. CYP1A1 is a highly
inducible enzyme that plays a critical role in the bioactivation of certain chemicals, such as
benzo[a]pyrene, to reactive intermediates associated with mutagenesis and carcinogenesis
(Gonzalez and Gelboin, 1991). Induction is mediated by the aryl hydrocarbon receptor (AhR),
a ligand-activated transcription factor that plays a pivotal role in mediating the biological
actions of a number of highly toxic chemicals including polychlorinated-dibenzo-p-dioxins (of
which 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; dioxin) is a prototype ligand),
polychlorinated-dibenzofurans, coplanar biphenyls, and polycyclic aromatic hydrocarbons
(PAHs), such as benzo[a]pyrene and 3-methylcholanthrene (MC) (Burbach et al., 1992). Upon
ligand binding, the cytosolic AhR migrates to the nucleus where it forms a dimer with Arnt
(AhR nuclear translocator), which then binds to specific DNA recognition sites, referred to as
Ah-response elements (AhRE, XRE, or DRE) located in the 5′-flanking region of target genes
(reviewed in Denison and Whitlock, 1995). TCDD, a widespread environmental pollutant
produced as the result of combustion and industrial processes, is one of the most potent agonists
of the AhR, and thus a powerful inducer of CYP1A1 activity.
In humans, infections from influenza, adenovirus, Herpes simplex, and HIV are associated
with decreases in drug biotransformation and clearance (Renton, 2004). Only a few studies
have reported the effects of hepatitis infection on drug metabolism, including impairment of
hepatic drug clearance in HCV patients, as measured by metabolism of the probe drug,
antipyrine (Ali et al., 1995; Jorquera et al., 2001). Studies of CYP function have shown that
CYP2A6 activity was depressed by hepatitis A (Pasanen et al., 1997) and induced by hepatitis
B and C (Kirby et al., 1996); while Becquemont et al. (2002) found CYP3A4 and CYP2D6
activities to be significantly lower in HCV patients than healthy volunteers. Recently, it was
shown that changes in mRNA expression of specific CYPs were linked to progression of HCV-
associated hepatocellular carcinoma (Tsunedomi et al., 2005).
HCV is a positive-stranded RNA virus that belongs to the Flaviviridae family. Chronic
infection can lead to cirrhosis and hepatocellular carcinoma (Alter, 1995). Many factors are
associated with the development of HCV-related liver damage, including exposure to such
environmental agents as cigarette smoke and alcohol. However, the molecular mechanisms
leading to cell injury are unclear. Chronic infection leads to cellular oxidative stress,
characterized by increases in cellular levels of reactive oxygen species (ROS), suggesting that
ROS may be involved in producing the damage seen in chronic HCV infection (DeMaria et
al., 1996). The development of subviral systems, consisting of stable high level expression of
HCV subgenomic replicons, for the study of replication of the viral RNA in cultured cells
(Lohmann et al., 1999; Blight et al., 2000) has facilitated studies on HCV replication and protein
function. The subgenomic replicon is composed of six nonstructural proteins (NS) that perform
various cellular functions. The NS5A protein plays a critical role in viral replication (Blight et
al., 2000), and participates in numerous cellular functions, including activation of cellular
transcription factors via oxidative stress (Gong et al., 2001) and activation of the ER stress
pathways (Waris et al., 2002).
To our knowledge, the relationship between HCV infection and the AhR signaling pathway
has not been reported. However, recent studies have investigated the role of the AhR in viral
replication. Exposure of cultured cells to AhR ligands increases the replication of human
immunodeficiency virus type 1(HIV) (Yao et al., 1995; Gollapudi et al., 1996; Ohata et al.,
2003) and human cytomegalovirus (Murayama T et al., 2002). The proposed mechanisms for
enhanced HIV replication include TCDD-dependent generation of thiol-sensitive reactive
oxygen intermediates (Yao et al., 1995), activation of NF-kB and production of TNFα
(Gollapudi et al., 1996), and increased gene expression through AhR binding to a putative XRE
(Yao et al., 1995; Ohata et al., 2003). Adult T-cell leukemia cell lines have elevated expression
of AhR and CYP1A1, suggesting a link between increased AhR expression and ATL
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leukemogenesis (Hayashibara et al., 2003). Thus, the potential of infectious agents to alter AhR
signal transduction pathways, including CYP1A1 expression and function, could lead to
increases in disease progression. Indeed, smoking, known to induce CYP1A1 and hepatic
CYP1A2, was recently shown to increase the severity of hepatic lesions in patients with chronic
hepatitis C (Pessione et al., 2001; Hezode et al., 2003).
In this study we investigated the effect of HCV on the AhR signaling pathway by examining
the induction of CYP1A1 by TCDD and other AhR ligands. We demonstrate that transcriptional
activation of the human CYP1A1 gene by TCDD, but not β-napthoflavone (βNF) or MC, is
dramatically suppressed in Huh7 cells expressing the HCV subgenomic replicon. These
findings will likely provide valuable insights into mechanisms of dioxin toxicity and the
interactions of non-inflammatory components of infectious agents on xenobiotic metabolism.
Materials and Methods
Chemicals and Reagents
Reagents were obtained as follows: TCDD from Chemsyn Science Laboratories (Lenexa, KS).
βNF, MC, and N-acetylcysteine (NAC), nordihydroguaiaretic acid (NDGA) from Sigma
Chemical Company (St. Louis, MO); [α-32P]dCTP, [γ-32P]ATP, and poly[d(I-C)] from GE
Healthcare (Piscataway, NJ); 2′ 7′- dichlorofluorescein diacetate from Alexis Biochemicals
(San Diego, CA).
Cell Culture
The cell lines used in this study, Huh7, Huh.8 and Ava.1, were provided by Dr. Charles Rice
(Rockefeller University, New York) and are described in (Blight et al., 2000). In brief, the Huh.
8 cell line contains an HCV derived expression vector stably integrated into a Huh7
background. The expression vector includes the HCV proteins, NS2, NS3, NS4A, NS4B,
NS5A and NS5B, linked to the antibiotic selection marker G-418. The Ava.1 cell line is similar
to Huh.8, but with a 47 amino acid deletion within the NS5A region, rendering this
nonstructural protein nonfunctional. All cell lines were maintained as a monolayer using
Dulbecco's Modified Eagle Medium (DMEM) (Invitrogen, Carlsbad, CA) and heat-inactivated
10% fetal bovine serum from Hyclone (Logan, UT). G-418 (geneticin)(Invitrogen) was added
to a final concentration of 800 μg/ml to medium used for culturing Huh.8 and Ava.1 cell lines.
For treatments, cells were exposed to TCDD, βNF, or MC at concentrations indicated in figure
legends or vehicle (0.1% DMSO). NDGA was dissolved in DMSO, and NAC was dissolved
in DMEM plus 25 mM Hepes and adjusted to pH 7.1 immediately before treatments.
RNA Isolation, Northern Blot Analyses, and Quantitative Real-time RT-PCR
Total RNA was extracted from near confluent cells using the RNeasy Kit (Qiagen, Valencia,
CA). Northern blot analysis was performed by electrophoresis of total RNA (10 μg) through
a 1% agarose-2.2 M formaldehyde gel, followed by blotting onto a Hybond nylon membrane
(GE Healthcare). CYP1A1 and β-actin cDNA probes were labelled with [α-32P]dCTP using
the Random Prime Kit (Invitrogen), and hybridized to the blots as previously described
(Quattrochi et al., 1985). Images were quantified by phosphorimaging in the STORM 840
Phosphorimager (GE Healthcare) and using Image Quant software from Molecular Dynamics.
For quantitative real-time RT-PCR, total RNA was treated with DNase I prior to analysis. Real-
time RT-PCR was performed using the ABI Prism 7700 sequence detector (Applied
Biosystems, Foster City, CA) with the following primers: CYP1A1 forward primer, 5′
GAATTCAGCGTGCCACTGG-3′, reverse primer, 5′-GGCATGCTTCATGGTTAGCC-3′,
and TaqMan probe, 5′-CGTGAAGGTGGACATGACCCCCAT-3′, giving a product of 69 bp;
AhR forward primer, 5′-CCTCCTTCTTGCCCTTCACC-3′, reverse primer, 5′-
GGATTTGACTTGATTCCTTCAGCT-3′, and TaqMan probe, 5′-
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CCGGTGCAGAAAACAGTAAAGCCAATCC-3′, giving a product of 75 bp; ARNT forward
primer 5′-TAGTGCCCTGGCTCGAAAA-3′, reverse primer, 5′-
CCGCAAGGACTTCATGTGAGA-3′, and TaqMan probe, 5′-
CAGACAAGCTAACCATCTTACGCATGGCA-3′, giving a product of 73 bp. Each real-time
RT-PCR reaction was performed in duplicate, and normalized to the ribosomal RNA levels in
the same sample.
Transient Transfections and Luciferase Activity Assays
Cells were plated at a density of 125,000 cells/well in 12-well plates. Transfections were
performed using Fugene 6 Transfection Reagent following manufacturer’s protocol (Roche
Applied Science, Indianapolis, IN). Five hours after transfection, DMEM containing 10% FBS
was added to each well and cultures incubated overnight. The culture media was removed after
incubation for 24 h with the transfection reagent-DNA complexes, and the cells were then
treated for 24 h with xenobiotics dissolved in DMSO. Control cells received media containing
0.1% DMSO. Following treatment, cells were rinsed with phosphate buffered saline (PBS) and
luciferase assays were performed using the Dual-Luciferase Reporter Assay System (Promega,
Madison, WI). Luciferase activity of cellular lysates was quantified with a Packard LumiCount
luminometer. Firefly luciferase activity was determined from three independent transfections,
and normalized against Renilla luciferase activities of the pRL-null control vector obtained
from the same culture.
Western Blot Analyses
Whole cell lysates were prepared by sonication of cell pellets in 250 mM sucrose/10 mM Tris
(pH 7.5) buffer. Protein concentrations were determined using the Bradford microassay from
BioRad (Hercules, CA). Western blots were prepared by electrophoresis of whole cell lysates
or nuclear extracts through 4–20% PAGE gels, and transfered to PVDF membranes (MSI,
Westboro, MA) overnight at 4°C. Blots were probed with antibody raised against human
CYP1A1 (Santa Cruz Biotechnology, Santa Cruz, CA) or AhR and Arnt antibodies (obtained
from Dr. Christopher A. Bradfield, University of Wisconsin) and subsequently with anti-actin
antibody (Oncogene Research Products, San Diego, CA) to normalize the amount of protein
loaded in each lane. As a positive control, each blot was run with one lane containing
recombinant CYP1A1 (BD Gentest, Woburn, MA). Detection was performed using
SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology, Rockford, IL),
and visualized by UVP BioImaging Systems camera and LabWorks acquisition software
(Upland, CA). Quantifications were performed using NIH Image v1.63 software.
Isolation of Nuclear Proteins and Electrophoretic Mobility Shift Assay (EMSA)
Nuclear protein fractions were isolated from near confluent cells as described by Denison et
al. (1988). Cells were treated with TCDD or βNF for 24 h prior to extraction of nuclear proteins.
Oligonucleotides were supplied by Operon Biotechnologies (Huntsville, AL). For the
preparation of the CYP1A1-XRE DNA probe, the oligonucleotides 5′-
CCGGCTCGCGTGAGAAGCG-3′ and 5′-CGCTTCTCACGCGAGCCGG-3′ were annealed
together overnight at 37°C. The probe was labeled with 32P at the 5′ ends using T4
polynucleotide kinase (Invitrogen) and [γ-32P]ATP. Labeled probes were purified through
TE-10 columns (BD Biosciences Clontech, Palo Alto, CA). For EMSA, 4 μg of nuclear extract
was incubated in a DNA binding buffer containing radiolabelled probe, 10 mM Tris (pH 8.0),
75 mM NaCl, 1 mM EDTA (pH 8.0), 1 mM DTT, 2 μg poly[d(I-C)], and 10% glycerol. Protein-
DNA complexes were separated on 6% PAGE 1X TBE (89 mM Tris, pH 8.3, 89 mM boric
acid, 2 mM EDTA) gels. Images were quantified by phosphorimaging in the STORM 840
Phosphorimager.
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P450-Glo Assay
Detection of CYP1A1 activity was performed using a P450-Glo CYP1A1 Assay Kit
(Promega). The assay was performed using cultured cells according to kit specifications. Cells
were plated on 96-well plates at 30,000 cells/well. The following day, cells were exposed to
TCDD for 24 h. The next day, cells were rinsed one time with PBS and medium containing 50
μM of the CYP1A1 specific substrate, luciferin 6′ chloroethyl ether (luciferin-CEE), was
added, and cells incubated at 37 °C for 3 h. Following the incubation, the reaction was
terminated by adding 50 μL of luciferase detection reagent, and luciferase activity quantified
with a Packard LumiCount luminometer. For each treatment group, one set of wells was
assayed in the absence of substrate and values obtained subtracted from substrate wells.
Determination of ROS Production
ROS production in Huh7 and Huh.8 cells was measured spectrofluorometrically using the cell
permeable 2′,7′-dichlorofluorescein-diacetate (DCF-DA) probe. DCF-DA is converted to its
fluorescent product dichlorofluorescein by ROS. Cells were plated on 96-well black plates
(Nunc, Roskilde, Denmark) at 25,000 cells/well (approximately 80% confluency). The
following day, media was changed to serum-free, phenol red-free DMEM, DCF-DA added to
a final concentration of 5 μM, and cells incubated for 30 min. At the end of the incubation
period, cells were rinsed one time with PBS and media containing xenobiotics was added.
Fluorescence readings were taken immediately and after various times of treatment from 15
min to 24 h using a SpectraMax Gemini EM fluorometer and SOFTmax PRO software
(Molecular Devices, Sunnyvale, CA).
Recombinant Adenovirus Expression of NS5A
The construction of the recombinant adenovirus expressing NS5A is described in (Qadri et al.,
2004). Huh7 cells were infected with a recombinant adenovirus expressing NS5A (Ad-NS5A)
or expressing green fluorescent protein (Ad-GFP), as a control, at a concentration of
approximately 2 x 103 adenovirus particles per cell for 24 h prior to the addition of TCDD.
The following day, media was changed and 10 nM TCDD was added to culture plates for an
additional 24 h. Cells were subsequently harvested for RNA and whole cell protein extracts.
Expression of Ad-GFP in Huh7 cells was detected using conventional fluorescence microscopy
as an index of efficiency of infection.
Statistics
Statistics were performed using InStat Instant Statistics (Prism 4 GraphPad Software, San
Diego, CA). Statistical differences between values were determined by a one-way ANOVA,
followed by either Bonferroni or Dunnett multiple comparisons post hoc tests or Student t test.
A p < 0.05 is considered statistically significant.
Results
Differential Effects of AhR Ligands on CYP1A1 Gene Transcription in HCV Replicon-
expressing Cells
To examine whether the AhR pathway is affected by the presence of the HCV genomic
replicon, we treated the parent cell line (Huh7) and the cells containing the HCV subgenomic
replicon (Huh.8) with TCDD, βNF, or MC, and assayed for the expression of CYP1A1 message.
We found, as expected, that exposure of Huh7 cells to all three AhR ligands resulted in
substantial induction of CYP1A1 mRNA (Fig. 1A). In contrast, exposure of Huh.8 cells to
TCDD resulted in a significant reduction in CYP1A1 mRNA as compared to Huh7 cells. Data
from Northern blots were confirmed by quantitative real-time RT-PCR data (Fig. 1B). The
expression in Huh.8 cells of TCDD-induced CYP1A1 was diminished by 55% when compared
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