ToxSci Advance Access originally published online on April 1, 2008
Toxicological Sciences 2008 104(1):67-73; doi:10.1093/toxsci/kfn058
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Pregnane X Receptor Protects HepG2 Cells from BaP-Induced DNA Damage
* College of Veterinary Medicine and Biomedical Sciences
School of Rural Public Health, Texas A&M University System Health Science Center, College Station, Texas 77843
1 To whom correspondence should be addressed at Department of Veterinary Physiology and Pharmacology, Mail Stop 4466, Texas A&M University, College Station, TX 77843. Fax: (979) 458-4485. E-mail: ytian{at}cvm.tamu.edu.
Received December 4, 2007; accepted March 14, 2008
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Pregnane X receptor (PXR) is a nuclear receptor that coordinately regulates transcriptional expression of both phase I and phase II metabolizing enzymes. PXR plays an important role in the pharmacokinetics of a broad spectrum of endogenous and xenobiotic compounds and appears to have evolved in part to protect organisms from toxic xenobiotics. Metabolism of benzo[a]pyrene (BaP), a well-established carcinogen and ubiquitous environmental contaminant, can result in either detoxification or bioactivation to its genotoxic forms. Therefore, PXR could modulate the genotoxicity of BaP by changing the balance of the metabolic pathways in favor of BaP detoxification. To examine the role of PXR in BaP genotoxicity, BaP–DNA adduct formation was measured by 32P-postlabeling in BaP-treated parental HepG2 cells and human PXR-transfected HepG2 cells. The presence of transfected PXR significantly reduced the level of adducts relative to parental cells by 50–65% (p < 0.001), demonstrating that PXR protects liver cells from genotoxicity induced by exposure to BaP. To analyze potential PXR-regulated detoxification pathways in liver cells, a panel of genes involved in phase I and phase II metabolism and excretion was surveyed with real-time quantitative reverse transcription PCR. The messenger RNA levels of CYP1A2, GSTA1, GSTA2, GSTM1, UGT1A6, and BCRP (ABCG2) were significantly higher in cells overexpressing PXR, independent of exposure to BaP. In addition, the total GST enzymatic activity, which favors the metabolic detoxification of BaP, was significantly increased by the presence of PXR (p < 0.001), independent of BaP exposure. Taken together, these results suggest that PXR plays an important role in protection against DNA damage by polycyclic aromatic hydrocarbons (PAHs) such as BaP, and that these protective effects may be through a coordinated regulation of genes involved in xenobiotic metabolism.
Key Words: benzo[a]pyrene; pregnane X receptor; DNA adducts; HepG2.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Pregnane X receptor (PXR) is a nuclear receptor with broad specificity that is activated by structurally diverse lipophilic compounds. It is expressed mainly in the liver, intestine and colon. PXR plays an important role in pharmacokinetics of a broad spectrum of endogenous and xenobiotic compounds and appears to have evolved in part to protect organisms from toxic xenobiotics (Kliewer, 2003
; Xie and Evans, 2001). PXR regulates metabolism and disposition of these compounds through coordinate regulation of the gene expression of phase I and II metabolizing enzymes and transport proteins (Maglich et al., 2002; Rosenfeld et al., 2003; Staudinger et al., 2003). PXR protects the liver against toxic insult by both endogenous and xenobiotic compounds (Sonoda et al., 2005; Staudinger et al., 2001; Xie et al., 2001).
Benzo[a]pyrene (BaP) is a common environmental contaminant found in air, water, soil, sediment and cooked foods. BaP is a well-established animal carcinogen and a probable human carcinogen; human exposure to mixtures containing BaP has been linked with an increased risk of cancer (Boffetta et al., 1997; Mastrangelo et al., 1996; Mumtaz et al., 1996; Sasco et al., 2004). A critical step in the induction of cancer by BaP is bioactivation by phase I metabolizing enzymes, most notably by members of the cytochrome P450 (CYP) superfamily (Ramesh et al., 2004). These electrophilic metabolites are capable of reacting with nucleophilic sites of DNA to form DNA adducts. The formation and persistence of carcinogen–DNA adducts have been shown to be critical events for the initiation of neoplasia in target cells. However, phase I metabolism, when coupled with phase II conjugation and subsequent elimination by membrane-bound transporters, is also an important mechanism for detoxifying BaP. The toxicity of BaP in a particular organism, tissue, or cell type is determined by the balance among these competing metabolic pathways.
In this study, the major goal was to test the hypothesis that PXR could modulate the genotoxicity of BaP. A cell culture model was developed based on the well-characterized human hepatoma cell line HepG2. HepG2 cells possess many functional xenobiotic-metabolizing enzymes and are sensitive to PAHs (Knasmuller et al., 2004). However, constitutive expression of PXR is low or undetectable in HepG2 cells. PXR expression was restored by stable transfection of human PXR (hPXR) expression plasmid. The effect of PXR on BaP-induced DNA damage was investigated by comparison of DNA adduct formation in the parental cells and the hPXR-enhanced cells. The results demonstrate that PXR decreases BaP genotoxicity in this cell model.
The second goal of the study was to test the hypothesis that PXR attenuates BaP genotoxicity by regulation of metabolizing enzymes involved in detoxification pathways. The gene expression of 20 BaP-metabolizing enzymes and five PXR-regulated transport proteins was measured by quantitative reverse transcription PCR (RT-PCR). In addition, the total GST enzymatic activity was measured. The results of the gene expression and enzyme activity experiments suggest that PXR reduces BaP genotoxicity by upregulating metabolizing enzymes that contribute to detoxification.
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MATERIALS AND METHODS |
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Materials.
The human HepG2 cell line was purchased from American Type Culture Collection (ATCC, Manassas, VA). LipofectAMINE and oligonucleotides used as PCR primers were obtained from Invitrogen (Carlsbad, CA). Dulbecco's modified Eagle's medium (DMEM) was from HyClone. Fetal bovine serum (FBS) was from Atlanta Biologicals (Lawrenceville, GA). Plasmid DNA purification kits, rifampicin (RIF), nuclease P1, spleen phosphodiesterase, micrococcal endonuclease, and potato apyrase were from Sigma (St Louis, MO). Polynucleotide kinase was from USB Corp. (Cleveland, OH). [
-32P]-adenosine triphosphate was from MP Biomedicals (Irvine, CA). BaP was obtained from Sigma and 5000x solutions were prepared in American chemical society-grade dimethyl sulfoxide (DMSO) from EMD Science (Gibbstown, NJ).
Stable transfection.
The expression vector for hPXR, p3XFlag-hPXR (Fig. 1A), was constructed as described previously (Gu et al., 2006). The PCR primers were designed based on the published hPXR sequence (Lehmann et al., 1998). HepG2 cells were maintained in DMEM supplemented with 10% FBS and 1% antibiotic/antimycotic in 5% CO2 at 37°C. For transfection, cells were seeded onto six-well plates, and when cell density reached 50% confluency, transfected with PXR expression plasmid (0.5 µg/well) by lipofection for 5 h using LipofectAMINE according to manufacturer's instructions. After the transfected cells were cultured for two days, G418 (1 mg/ml) was added to the medium for an extended selection period. The G418-resistant colonies were pooled and further selected with medium containing 400 µg/ml G418. Surviving single colonies were cloned, expanded, and tested for PXR expression and ligand responsiveness.
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Cell culture and treatment.
Parental and hPXR-enhanced cells were cultured in DMEM supplemented with 10% FBS and 1% antibiotic/antimycotic (100 units/ml penicillin G sodium, 100 µg/ml streptomycin sulfate and 0.25 µg/ml amphotericin B). Cells were pretreated with 5µM RIF for 24 h, then treated with BaP and collected after 24 h. RIF and BaP controls consisted of an equivalent volume of DMSO.
DNA adduct assay.
Cells were cultured in 150- x 25-mm culture dishes. BaP was administered to the cells at three doses (0.5, 2, and 5µM). DNA was isolated from the cells using QIAGEN 100/G Genomic-tips (Qiagen, Valencia, CA) according to the manufacturer's protocol.
DNA adducts were quantified by the nuclease P1-enhanced 32P-postlabeling assay (Phillips and Arlt, 2007) using 6 µg DNA. Multidirectional anion-exchange thin-layer chromatography (conditions described previously in Cizmas et al., 2004) was used to separate 32P-labeled DNA adducts on polyethyleneimine-cellulose sheets. Profile and radioactivity of DNA adducts from individual samples were determined by Instant Imager (PerkinElmer, Waltham, MA). Student's t-test was used to evaluate the effects of cell type and inducer treatment on adduct levels at each dose.
Real-time quantitative RT-PCR.
Cells were seeded in 12-well plates at 25% confluency and treated as described above. BaP was administered at 5µM (24 h). Total RNA was isolated from the cells using Trizol Reagent (Invitrogen) according to the manufacturer's instructions. RNA from three replicate wells was pooled. Complimentary DNA was synthesized by reverse transcription using Invitrogen's M-MLV Reverse Transcriptase according to the manufacturer's instructions. PCR primers were designed based on published sequences and purchased from Invitrogen. Real-time quantitative PCR was performed on a 7900HT Real Time PCR system from Applied Biosystems (Foster City, CA) using SYBR Green Master Mix (Applied Biosystems). Results were normalized to β-actin.
GST assay.
Cells were cultured as described above and treated with RIF (5µM, 24 h) or DMSO. The total activity of glutathione S-transferase (GST) was determined using the GST Assay Kit (Cayman Chemicals, Ann Arbor, MI) following the manufacturer's protocol. The conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) with reduced glutathione was measured spectrophotometrically (320 nm) and linear regression was performed to calculate a rate (slope). Student's t-test was used to evaluate the effects of cell type and inducer treatment on the slopes.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Establishment of Stable Transfected Cell Line
A modified HepG2 cell line, PXR-G2, was produced for this study using an hPXR expression plasmid, p3XFlag-hPXR (Fig. 1A). Stable transfected colonies were selected with G418 and expanded. The expression of transfected hPXR was confirmed by quantitative RT-PCR (Fig. 1B). PXR messenger RNA (mRNA) level was 13-fold higher in the stable transfected PXR-G2 cells relative to the parental HepG2 cells. The presence of transfected hPXR in the PXR-G2 cells increased the responsiveness of CYP3A4 to RIF (Fig. 1C). Induction of CYP3A4 in response to RIF treatment (5µM) was measured in both cell lines by real-time quantitative RT-PCR. Response to RIF over DMSO control increased from twofold to 16-fold with the presence of transfected hPXR.
Impact of PXR on BaP-induced DNA Adduct Formation in HepG2 Cells
The effect of hPXR expression on BaP-induced genotoxic effects in HepG2 and PXR-G2 cells was investigated. DNA adduct formation was measured by 32P-postlabeling in both cell lines, with or without RIF (5µM) pretreatment, at three BaP doses (0.5, 2, and 5µM). HepG2 and PXR-G2 cells exhibited qualitatively similar profiles of BaP-induced DNA adducts. The DNA adduct patterns with or without RIF in each cell line were also comparable (Fig. 2). The major spot, Spot 3, was characterized in previous 32P-postlabeling analyses (Lu et al., 1986) as BaP-diol-epoxide-N2-deoxyguanosine (BPDE-dG).
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Treatment with BaP, both with and without RIF pretreatment, resulted in formation of DNA adducts in a dose-dependent manner in both HepG2 and PXR-G2 cells (Fig. 3). 4121 ± 248 adducts per 109 nucleotides were detected in HepG2 cells, whereas 2128 ± 134 adducts per 109 nucleotides were detected in PXR-G2 cells at the highest BaP dose (Fig. 4). The presence of transfected hPXR had a statistically significant effect on adduct levels at 5µM BaP both without inducer treatment (p = 0.002) and with inducer treatment (p < 0.001). Treatment with inducer significantly (p = 0.008) decreased adduct levels in PXR-G2 cells (37%), but decreased adducts levels in HepG2 cells by only 11%. BPDE-dG (Spot 3) levels were significantly lower in PXR-G2 cells both without inducer pretreatment (p = 0.003) and with inducer (p < 0.001) (data not shown). These results indicate that PXR protects the cells from genotoxic effects of BaP.
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PXR Regulation of Genes Involved in Xenobiotic Metabolism and Elimination
The effects of BaP and PXR on expression of a panel of 25 genes were investigated by quantitative RT-PCR. The products of these selected genes, which include metabolizing enzymes and drug transporters, are involved in xenobiotic pharmacokinetics. Relative to parental HepG2 cells, stable PXR-HepG2 cells exhibited higher constitutive mRNA expression of many phase I and II xenobiotic enzymes including CYPs, GSTs, and UGTs in the absence of BaP (Table 1). Most notably, mRNA levels of CYP1A2 increased 8.8-fold, GSTM1 increased 140-fold and UGT1A6 increased 200-fold. In addition, mRNA levels of breast cancer resistance protein (BCRP), a transporter that excretes BaP-sulfate metabolites in colon cells (Ebert et al., 2005
), increased in PXR-HepG2 cells. Upregulation of these genes in cells overexpressing PXR may reduce BaP toxicity by enhancing detoxification pathways.
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Following exposure to BaP, there was an increase in mRNA levels of some phase I and II enzymes in HepG2 cells, notably CYP1A1, CYP1A2, and GSTP1 (Table 1). A similar increase in mRNA levels of phase I and II enzymes was observed in the PXR-HepG2 cells following BaP exposure, although there was a notably higher increase in GSTP1 and a lower increase in CYP1A1 (Table 1).
Effect of PXR on GST Enzymatic Activity
The total activity of GST (cytosolic and microsomal) was assessed to determine the GST enzymatic levels. The rate of CDNB conjugation, which is directly proportional to the GST activity, was measured in both cell lines, with or without RIF treatment (5µM, 24 h). The GST activity level was 8.23 ± 0.61 units per minute in HepG2 cells and 34.0 ± 2.3 units per minute in PXR-G2 cells (Fig. 5), a fourfold increase. The presence of transfected hPXR had a statistically significant effect (p < 0.001) on GST activity both without and with RIF pretreatment. Treatment with RIF had no effect on GST activity in HepG2 cells, but increased GST activity in PXR-G2 cells by 21% (p = 0.13). These results, considered with the increased mRNA levels of GSTM1, GSTA1, and GSTA2, suggest that PXR increases the enzyme levels of GSTs in liver cells through transcriptional regulation of GST gene expression.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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PXR is a broad specificity, low affinity nuclear receptor that transcriptionally regulates many genes associated with xenobiotic metabolism and clearance, and therefore performs an important function for detoxification of xenobiotics (Kliewer, 2003
). PXR is considered to be a xenosensor because of its ability to respond to many xenobiotics, including pharmaceuticals, dietary nutrients, and environmental contaminants. Consistent with this protective role, PXR is mainly expressed in the liver, intestine and colon, which are sites of first-pass elimination. Because PXR regulates a wide range of the genes expressed in the hepato-intestinal system, it may affect the pharmacokinetics of environmental contaminants. The major goal of this study was to investigate the role of PXR in the genotoxicity of BaP, a well-known carcinogen and environmental contaminant. Although PXR can be activated by many therapeutic drugs, naturally occurring chemicals and environmental contaminants, its role as a sensor of BaP and effector on BaP detoxification has not been well understood.
In the liver, PXR upregulates multiple phase I and phase II enzymes which metabolize BaP. Metabolism is important in both the detoxification of BaP and its transformation to genotoxic metabolites. For example, recent in vivo studies utilizing knockout mouse strains (Sagredo et al., 2006; Uno et al., 2001, 2004, 2006) indicate that CYP isoforms involved in phase I metabolism of BaP contribute to both the protective and potentiating pathways. In the current model of BaP bioactivation, BaP is transformed to highly electrophilic dihydrodiol-epoxides (BPDEs), which then can form DNA adducts, through a series of steps mediated by phase I enzymes. The formation of an epoxide is catalyzed by CYPs, followed by conversion to a dihydrodiol by epoxide hydrolases (EH), and further conversion by CYPs to BPDE (Ramesh et al., 2004). Reactive ortho-quinones, which are also capable of forming DNA adducts, can be produced by oxidation of BaP-dihydrodiols by aldo–keto reductases (AKR) (Burczynski et al., 1998). Phase I enzymes also contribute to detoxification by alternate reactions that form nontoxic or less toxic compounds, such as the conversion of BPDEs to tetraols by EH (Ramesh et al., 2004).
Phase I metabolism adds functional groups which allow conjugation by phase II enzymes, a step that effectively detoxifies BaP by preventing further transformation to carcinogenic metabolites, and facilitates excretion from the cell. In a discussion of the paradoxical effects of the CYP1 enzymes on BaP toxicity (Nebert et al., 2004), the authors suggest that detoxification may be enhanced in cells, such as hepatocytes and gastrointestinal epithelial cells, in which the CYP1s are tightly coupled to phase II enzymes.
To analyze the role of PXR in BaP detoxification, we created a cell culture model using HepG2 cells, which lack PXR, and PXR-transfected HepG2 cells. Levels of DNA adducts were significantly decreased by the expression of PXR and, in cells overexpressing PXR, by pretreatment with RIF. Levels of BPDE-dG, the major adduct, were significantly lower in cells overexpressing PXR. Although the other spots have not been identified, they seem to be BPDE-related adducts because they display a similar pattern to adducts detected in cells treated with pure BPDE (Li et al., 2001). These results demonstrate that PXR protects the cells from the genotoxic effects of BaP, and suggest that PXR may reduce the formation of BPDE.
In our cell culture model, CYP1A2, GSTM1, UGT1A6, GSTA1, and GSTA2 were upregulated by PXR, independent of BaP (Table 1). Consistent with the GST gene expression results, total GST activity was significantly increased by PXR, independent of BaP (Fig. 5). GSTs catalyze the formation of glutathione conjugates of primary epoxides and BPDEs; UGTs catalyze the formation of glucuronide conjugates of dihydrodiols (Ramesh et al., 2004). Following exposure to BaP, there was an increase in mRNAs of phase I and II enzymes in HepG2 cells (Table 1). A similar increase in mRNAs of phase I and II enzymes was observed in PXR-HepG2 cells following BaP exposure, although there were notably higher increases in mRNAs of CYP1A2 and GSTP1 (Table 1). As expected, the Ah receptor agonist BaP caused large increases in CYP1A1 mRNA in both HepG2 and PXR-HepG2 cells; interestingly, the increase was attenuated in PXR-transfected cells. The mechanism for this attenuation is unclear and it is conceivable that a squelching mechanism may play a role where PXR competes with the Ah receptor for the common transcriptional coregulators.
mRNA levels of membrane-bound drug transporter BCRP, which has been shown to be regulated by PXR in mice (Anapolsky et al., 2006), increased 12-fold in the hPXR-enhanced cells. BCRP actively excretes sulfate metabolites of BaP in Caco-2 cells (Ebert et al., 2005) and has been shown to transport glutathione, glucuronide and sulfate conjugates of other compounds (Adachi et al., 2005; Suzuki et al., 2003). BCRP may contribute to detoxification by removing BaP conjugates from the cell and thus decreasing bioavailability.
Our results strongly suggest a mechanism in which PXR coordinately regulates phase I and II enzymes and transporters that are relevant for BaP metabolism and disposition, leading to BaP detoxification (Fig. 6). Upregulated CYP increases initial BaP metabolism to epoxides. A higher level of GST favors rapid conjugation of the epoxides rather than further metabolism to dihydrodiols by EH, which was not affected by PXR. Higher levels of UGT increase conjugation of dihydrodiols. Dihydrodiols are converted to BPDEs by upregulated CYP, but increased GST also favors rapid conjugation of BPDEs, reducing the amount available to bond to DNA. Finally, upregulated BCRP enhances elimination of conjugated species from the cell.
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In conclusion, this research demonstrates that PXR decreases the formation of BaP-induced adducts in an hPXR-enhanced liver cell line, indicating that PXR plays an important role in protecting these cells from genotoxicity induced by exposure to PAHs. The gene expression of several BaP-metabolizing phase I and phase II enzymes and one PXR-regulated drug transporter was increased in the hPXR-enhanced cells. Enzymatic activity of GST, an important family of phase II enzymes, was significantly higher in HepG2 cells overexpressing PXR. Taken together, these data suggest that PXR may effectively decrease bioavailability of carcinogenic BaP metabolites by coordinately regulating a pathway of biphasic metabolism and excretion that reduces intracellular concentrations of BaP and procarcinogenic phase I metabolites. These findings further our understanding of factors influencing the genotoxicity of BaP and can guide the design of in vivo studies that may help improve risk assessment of PAHs.
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FUNDING |
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National Institute of Enivironmental Health Sciences grants P42 (ES04917, P30 ES09106, and ES09859); and by American Heart Association grant (0355131Y).
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