pyrene in Human Cell Lines - American Chemical Society

Chem. Res. Toxicol. 2007, 20, 1797–1810

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AHR- and DNA-Damage-Mediated Gene Expression Responses Induced by Benzo(a)pyrene in Human Cell Lines Sarah L. Hockley,*,† Volker M. Arlt,† Daniel Brewer,† Robert te Poele,‡ Paul Workman,‡ Ian Giddings,†,§ and David H. Phillips† Section of Molecular Carcinogenesis, The Institute of Cancer Research, Brookes Lawley Building, 15 Cotswold Road, Sutton, Surrey SM2 5NG, United Kingdom, Cancer Research UK Centre for Cancer Therapeutics, The Institute of Cancer Research, Haddow Laboratories, 15 Cotswold Road, Sutton, Surrey, SM2 5NG, United Kingdom, and Cancer Research U.K. DNA Microarray Facility, Institute of Cancer Research, 15 Cotswold Road, Sutton, Surrey, SM2 5NG, United Kingdom ReceiVed July 11, 2007

Carcinogens induce complex transcriptional responses in cells that may hold key mechanistic information. Benzo(a)pyrene (BaP) modulation of transcription may occur through the activation of the aryl hydrocarbon receptor (AHR) or through responses to DNA damage. To characterize further the expression profiles induced by BaP in HepG2 and MCF-7 cells obtained in our previous study, they were compared to those induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which activates AHR but does not bind to DNA, and anti-benzo(a)pyrene-trans-7,8-dihydrodiol-9,10-epoxide (BPDE), which binds directly to DNA but does not activate AHR. A total of 22 genes had altered expression in MCF-7 cells after both BaP and TCDD exposure, and a total of 29 genes had altered expression in HepG2 cells. In both cell lines, xenobiotic metabolism was upregulated through induction of NQO1, MGST1, and CYP1B1. A total of 78 expression changes were induced by both BaP and BPDE in MCF-7 cells, and a total of 29 expression changes were induced by both BaP and BPDE in HepG2 cells. These genes were predominantly involved in cell cycle regulation, apoptosis, and DNA repair. BaP and BPDE caused the repression of histone genes in both cell lines, suggesting that regulation of these genes is an important component of the DNA damage response. Interestingly, overlap of the BPDE and TCDD gene expression profiles was also observed. Furthermore, some genes were modulated by BaP but not by TCDD or BPDE, including induction of CRY1 and MAK, which may represent novel signaling pathways that are independent of both AHR activation and DNA damage. Promoter analysis identified candidate genes for direct transcriptional regulation by either AHR or p53. These analyses have further dissected and characterized the complex cellular response to BaP. Introduction 1

Benzo(a)pyrene (BaP) is an environmental carcinogenic polycyclic aromatic hydrocarbon (PAH) that is formed through incomplete combustion of organic materials, and common sources are tobacco smoke, automobile exhaust, and food (1, 2). BaP can be metabolically activated in mammalian cells through the formation of its reactive electrophilic metabolite, antibenzo(a)pyrene-trans-7,8-dihydrodiol-9,10-epoxide (BPDE), which covalently binds to DNA to form bulky DNA adducts (3). Although evidence suggests a direct link between BaPinduced DNA adducts and carcinogenesis (4), this DNA damage is not sufficient for complete tumor progression and other carcinogen-induced cellular responses are required, some of which may involve gene expression changes modulated by BaP. * To whom correspondence should be addressed: Section of Molecular Carcinogenesis, The Institute of Cancer Research, Brookes Lawley Building, 15 Cotswold Road, Sutton, Surrey SM2 5NG, U.K. E-mail: [email protected]. † Section of Molecular Carcinogenesis. ‡ Cancer Research UK Centre for Cancer Therapeutics. § Cancer Research U.K. DNA Microarray Facility. 1 Abbreviations: AHR, aryl hydrocarbon receptor; ARNT, AHR nuclear translocator; BaP, benzo(a)pyrene; BPDE, anti-benzo(a)pyrene-trans-7,8dihydrodiol-9,10-epoxide; DMSO, dimethyl sulphoxide; EASE, Expression Analysis Systematic Explorer; PAH, polycyclic aromatic hydrocarbon; RTqPCR, real-time quantitative polymerase chain reaction; TCDD, 2,3,7,8tetrachlorodibenzo-p-dioxin; XRE, xenobiotic response element.

Gene expression alterations induced by BaP have been reported in several studies (5–12). In one of these, we showed that exposure of MCF-7 and HepG2 cells to BaP results in a complex transcriptional response that is influenced by the time of exposure, concentration, and cell type (12). Among the cellular processes triggered in response to BaP that are likely to contribute to this complexity are the activation of the aryl hydrocarbon receptor (AHR), response to DNA damage, and oxidative stress signaling. The extent of involvement of these different factors in the BaP-induced transcription response is still largely unknown. Thus, identification of genes altered by compounds such as BaP and further characterization of their transcription response is crucial to understanding their carcinogenic mechanisms. In this present study, we have focused on investigating the roles of AHR activation and the response to DNA adduct formation in BaP-induced transcription. A number of PAHs, including BaP, can bind to and activate AHR (13, 14). The activated receptor is capable of modulating the expression of target genes through recognition of an enhancer DNA element, known as the xenobiotic response element (XRE), in their promoter regions (15). Several genes that encode phase I and II xenobiotic metabolizing enzymes are known to be upregulated by AHR, including those involved in the bioactivation of BaP to BPDE (16, 17). This transcription factor receptor is therefore an important component of the PAHinduced gene expression response, and the loss of AHR has

10.1021/tx700252n CCC: $37.00  2007 American Chemical Society Published on Web 10/19/2007

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Figure 1. Hypothetical cellular processes leading to altered gene expression induced by BaP, TCDD, and BPDE. TCDD activates AHR but does not bind to DNA, and BPDE binds directly to DNA but does not activate AHR. A comparison of TCDD- and BaP-induced expression profiles will give insight into AHR-mediated effects of the BaP response, while a comparison of BPDE- and BaP-induced expression profiles may allow for the identification of DNA-damage-specific effects of BaP.

been shown to inhibit BaP-induced carcinogenicity in mice (18). Cellular signaling responses to DNA damage also contribute to the transcriptional response of BaP. For example, the activation of the tumor suppressor protein, p53, in response to BaP-induced DNA damage leads to transcriptional modulation of genes involved in DNA repair, cell-cycle arrest, and apoptosis (19, 20), thereby protecting the cell from transformation. In this study, expression profiles induced in MCF-7 and HepG2 cells, following exposure to 2,3,7,8-tetrachlorodibenzop-dioxin (TCDD) or BPDE, have been compared to the BaPinduced expression profiles from our previous analysis (12). The proposed scheme is illustrated in Figure 1. MCF-7 and HepG2 cells are competent in metabolically activating BaP (6, 7) and express wild-type p53 (21, 22). TCDD is a powerful activator of AHR, although it is not a substrate for xenobiotic detoxification enzymes and does not form DNA adducts. BPDE is the ultimate carcinogenic metabolite of BaP and binds directly to DNA, independent of metabolic processes induced via AHR. A comparison of TCDD and BaP expression profiles will give insight into AHR-mediated effects of the BaP response, while a comparison of BPDE and BaP expression profiles may allow for the identification of DNA-damage-specific effects of BaP. BPDE and TCDD gene expression was monitored using microarrays representing 18 224 cDNA clones that were also used to obtain the BaP-induced expression profiles in our previous study (12).

Materials and Methods Cell Culture and Chemical Treatment. MCF-7 human breast carcinoma cells and HepG2 human hepatocarcinoma cells were purchased from the European Collection of Cell Cultures (ECACC, Salisbury, U.K.). The cells were grown as adherent monolayers and cultured as previously described (12). BPDE was synthesized at the Institute of Cancer Research by the method of R. G. Harvey and P. P. Fu (23), and purity was checked by thin-layer chromatography on silica, with 19:1 tetrahydrofuran/triethylamine as the eluant. TCDD was purchased from AccuStandard, New Haven, CT. For chemical exposure, T75 flasks were seeded at 0.13 × 106 MCF-7 cells/ mL or 0.27 × 106 HepG2 cells/mL in a total volume of 15 mL, and the appropriate concentrations of BPDE or TCDD dissolved in dimethyl sulphoxide (DMSO, Sigma Aldrich) were added after 48 h. DMSO alone was added to control cultures, and its volume was kept at 0.2% of the total culture medium. Cells were harvested by trypsinisation and then washed with phosphatebuffered saline (PBS). Cell Viability and DNA Adduct Measurement. Cells were exposed to BPDE (0.001–1 µM) or TCDD (1–10 nM) for up

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to 48 h in duplicate biological experiments. Control cells were treated with DMSO, and cell viability was measured as previously described (12). DNA was isolated from cells exposed to BPDE by a standard phenol–chloroform extraction method, and DNA adducts were measured for each DNA sample using the nuclease P1 enrichment version of the 32P-postlabeling method as described previously (12). Results were expressed as DNA adducts/108 nucleotides. RNA Extraction and cDNA Synthesis for Microarray Analysis. Cells were treated with BPDE (0.5 and 1 µM) for 2, 6, and 24 h or TCDD (10 nM) for 6 and 24 h. Control cells were exposed to DMSO. All incubations were performed in triplicate to give three independent replicate flasks for a particular combination of chemical, concentration, and time. Cell pellets were collected and total RNA was extracted using the Qiagen RNeasy Mini Kit protocol (RNeasy Mini Handbook, Qiagen, U.K.). After the addition of lysis buffer (RLT, Qiagen), samples were homogenized using QIAshredders (Qiagen). RNA was quantified spectrophotometrically, and integrity was determined using a 2100 Bioanalyzer (Agilent Technologies, U.K.). RNAs that had a rRNA 28S/18S ratio greater than 1.5 were used for microarray analysis. Total RNA (4 µg) was reverse-transcribed into cDNA and fluorescently labeled with Cy3 or Cy5 monoreactive dyes (Amersham Biosciences, U.K.) using the Invitrogen Indirect cDNA Labeling Kit protocol (Invitrogen, U.K.), according to the instructions of the manufacturer. After labeling, repetitive sequences within the cDNA samples were blocked with 16 µg of Human Cot-1 DNA (Invitrogen, U.K.) to prevent nonspecific sequences binding to the cDNA probes. cDNA Microarray Hybridizations. Gene expression analysis was performed using the Cancer Research U.K. DNA Microarray Facility (CRUKDMF) Human 22K Genome-Wide Array version 1.0.0. The full probe list for this array can be found at http://www.icr.ac.uk/array/array.html. The arrays were gridded onto type 7* silanized slides (GE Healthcare, U.K.), followed by cross-linking by exposure to 65K µJ UV light in a Stratalinker (Stratagene). Hybridization and washing of the slides were performed as previously described (12). The slides were then scanned on an Axon Genepix 4000A laser scanner (Axon Instruments, Sunnyvale, CA). Microarray Analysis. Image analysis using GenePix Pro version 5.1 software (Axon Instruments, Sunnyvale, CA) and data normalization within GeneSpring version 7.2 were performed as previously described (12). Within GenePix, a spot was automatically flagged as present if the signal intensity of >75% of the pixels for either the Cy3 or Cy5 channels was 1 standard deviation above the background intensity. Genes were removed from further analysis if they had not been flagged in Genepix software as present in greater than 50% of the microarrays. Within GeneSpring, a one-sample Student’s t test is calculated for Log2 transformed replicate Cy3/Cy5 ratios to test whether the mean normalized expression value for the gene is statistically different from 0. Cy3/Cy5 ratios of the three biological replicate samples were averaged, and these data were then used to identify genes modulated by at least 1.4-fold and had a one-sample two-tailed Student’s t test p value of less than 0.05. This list of significantly altered genes was used to perform hierarchical clustering within GeneSpring. Expression Analysis Systematic Explorer (EASE) software (http://david.ncifcrf.gov/ease/ease.jsp) was used to identify biological processes associated with the AHR and DNA damage transcriptional responses. The one-tailed Fisher exact probability test was used to measure over-representation of biological

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processes in the gene lists when compared to their total representation on the microarrays. Biological processes with a p value < 0.05 were considered significant. Because of the small numbers of genes being annotated by this method (range ) 14–78), biological processes that were associated with only one gene were still considered significant because they provide an indication of an effect. Real-Time Quantitative Polymerase Chain Reaction (RTqPCR). Reverse transcription PCR was used to generate cDNA for relative quantitation analysis using real-time fluorescent PCR on an ABI PRISM 7900HT Sequence Detection System (Applied Biosystems, U.K.), performed as previously described (12). To detect the modulated expression of selected target genes 20× Assays-On-Demand TM gene expression primers and probes (Applied Biosystems) were used (CYP1A1Hs00153120_m1, CYP1B1-Hs00164383_m1, NQO1Hs00168547_m1, AKR1C3-Hs00366267_m1, BAX-Hs00180269, PCNA-Hs00427214_g1,CDKN1A-Hs00355782_m1,andHIST1H3DHs00371415_m1). Relative gene expression was calculated using the comparative threshold cycle (CT) method as performed previously (12). Gene Promoter Analysis. To identify AHR- and p53-binding sites, the 10 000 bp upstream region and the first exon for each genewereobtainedfromTRASER(http://genome-www6.stanford.edu/ cgi-bin/Traser) using the Entrez Gene ID. The sequence data were based on NCBI Human Genome Build 36, version 1. These sequences were imported into Transplorer software (Biobase GmbH, Wolfenbüttel, Germany), and Dragon Promoter Finder was used to identify promoter regions based on the methodology described in ref 24. The sequences of identified potential promoter regulatory elements were exported from the software. If the regulatory region was within 5000 bp of the transcription start site, only the identified potential promoter region sequence as identified by Dragon Promoter Finder was reintroduced into the Transplorer software. Because classic promoters were not identified for CDKN1A, a known p53-regulated gene, or NQO1, a known AHR-regulated gene, the promoters of these two genes were taken from the Genomatix promoter database. Matrix searches were then performed on the promoter sequences in Transplorer. Matrices were obtained from the TRANSFAC database. AHR-binding sites were searched for using matrices M00778 (11 bases), M00235 (16 bases), and M00237 (19 bases) that are AHR-specific, and p53-binding sites were searched for using matrices M00034 (20 bases), M00272 (10 bases), and M00761 (10 bases) that are p53-specific, using a 90% cutoff for the matrix similarity score for both the core promoter and the entire matrix (25).

Results Cell Viability and DNA Adduct Analysis. Cell viability and DNA adducts were measured in MCF-7 and HepG2 cells exposed to up to 1 µM BPDE for up to 48 h (Figures 2 and 3). The viability of MCF-7 cells exposed to 1 µM BPDE for 48 h was 43%, while the viability of similarly treated HepG2 cells was 73%. In contrast, no loss of viability was observed in either cell line exposed to up to 5 µM BaP for up to 48 h (12). DNA adduct formation was concentration-dependent in both cell lines (Figure 3). Concentrations of 0.5 and 1 µM were selected for microarray experiments, because although the loss of cell viability did occur, adduct levels detected after exposure to these concentrations (up to 615 adducts/108 nucleotides in MCF-7 cells and up to 962 adducts/108 nucleotides in HepG2 cells) are similar to those observed in these cells after BaP exposure

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Figure 2. Effects of BPDE on MCF-7 and HepG2 cell viability relative to controls. The values are the mean ( range of two independent experiments.

Figure 3. DNA adduct formation measured by 32P-postlabeling in MCF-7 cells and HepG2 cells after BPDE exposure. The values are the log10 mean ( range of duplicate cell incubations; each DNA sample was analysed twice.

(12). No loss of viability was observed in cells exposed to up to 10 nM TCDD (data not shown). CYP1A1 RTqPCR. The CYP1A1 gene is highly inducible by both BaP and TCDD through activation of AHR (17); however, a clone for this gene was not present on the cDNA array used in this study. RTqPCR was therefore used to measure the expression of CYP1A1 after exposure of the cells to TCDD or BaP (Figure 4). CYP1A1 induction by BaP and TCDD was higher in HepG2 cells than in MCF-7 cells. TCDD in the range of 1–10 nM gave similar levels of modulation of CYP1A1 as BaP in the range of 1–5 µM, with the exception of 10 nM for 24 h of exposure in the HepG2 cells, where the induction was approximately 2-fold higher. BPDE-Induced Gene Expression Profiling. Microarray analysis identified a total of 1081 genes with significantly (p < 0.05) altered expression by at least 1.4-fold after BPDE exposure in MCF-7 cells and a total of 95 genes in HepG2 cells (Supplementary Table 1 in the Supporting Information). A total of 45 of the expression alterations were common to both cell


Figure 4. Expression of CYP1A1 as measured by RTqPCR in MCF-7 and HepG2 cells after the exposure to BaP or TCDD. Relative expression was calculated using the formula 2-∆∆CT, and cells treated with DMSO were used as the calibrator sample.

lines (shaded in Supplementary Table 1 in the Supporting Information), although a large number of changes were celltype specific. The common alterations included down-regulation of histone genes HIST1H 2BN, 2AC, 3D, 1C, 4B, 3E, and 2BJ. Hierarchical clustering of the data (Figure 5) reveals that 6 and 24 h of exposure to 1 µM BPDE had the greatest effect on gene expression in MCF-7 cells (Figure 5A), the same concentration that resulted in the greatest loss of viability (Figure 2). While an effect of the BPDE concentration is not so evident in HepG2 cells, it appears that gene expression effects are greater at the earlier time points of 2 and 6 h than at 24 h (Figure 5B). The time and concentration had similar effects on the transcriptional response of the cells to BPDE to that observed after BaP exposure (12), suggesting that gene expression profiles of cells exposed to genotoxic compounds are largely determined by DNA-damage-mediated responses. EASE analysis performed on the BPDE expression profiles to identify biological processes that are significantly over-represented (Fisher exact test p value < 0.05) in the gene lists when compared to their total representation on the microarrays indicated that apoptotic processes were induced in MCF-7 cells but not significantly in HepG2 cells (data not shown). The upregulation of apoptosis genes (ALOX15B, BAX, CDKN1A, CRADD, PMAIP1, PRKCE, STK17A, TNFRSF19, and FAS) in MCF-7 cells occurred after BPDE concentrations that resulted in a reduced viability of MCF-7 cells (Supplementary Table 1 in the Supporting Information and Figure 2). TCDD-Induced Gene Expression Profiling. A total of 28 genes had significantly altered expression by at least 1.4-fold in MCF-7 cells exposed to TCDD, and this number was 134 in HepG2 cells (Supplementary Table 2 in the Supporting Information). Hierarchical clustering (Figure 5) showed that time influenced TCDD-induced gene expression in HepG2 cells, with a greater effect seen after 24 h than after 6 h (Figure 5A). By a comparison, the 6 and 24 h TCDD gene expression profiles are relatively similar for MCF-7 cells (Figure 5B). The time-

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dependent effect of TCDD in HepG2 cells is also evident in CYP1A1 expression measured by RTqPCR (Figure 4). Only three of the TCDD-modulated transcripts were common to both cell lines, NQO1, MGST1, and SLC7A5 (shaded in Supplementary Table 2 in the Supporting Information), all of which were upregulated. The TCDD-induced expression profiles were largely cell-type-specific and probably represented genes that are both directly and indirectly regulated by AHR. The greater effect of TCDD on gene expression in HepG2 cells than in MCF-7 cells is consistent with the higher number of xenobiotic metabolism genes identified as induced by BaP in these cells (12) and with the higher induction of CYP1A1 gene expression (Figure 4). Comparison of BaP-, BPDE-, and TCDD-Induced Gene Expression Profiles. BaP and BPDE. A comparison of the BaP gene expression profiles obtained from our previous study (12) with the BPDE gene expression profiles obtained here revealed 78 and 29 gene expression changes induced by both compounds in MCF-7 and HepG2 cells, respectively (Figure 6 and Tables 1 and 2), the majority of which were modulated in the same direction by both compounds. A total of 10 of these altered genes were common to both cell lines (shaded in Tables 1 and 2). BaP and TCDD. Overlap between the BaP gene expression profiles (12) and those of TCDD was also evident. In MCF-7 cells, both compounds modulated the expression of 22 clones, and in HepG2 cells, this number was 29 (Figure 6 and Tables 3 and 4). All expression changes common to BaP and TCDD were changed in the same direction by both compounds. Only two of the shared modulated genes, MGST1 and NQO1, were common to both cell lines (shaded in Tables 3 and 4). BPDE and TCDD. Although the overall gene expression profiles of the genotoxin, BPDE, and the nongenotoxin, TCDD, were very different as shown by hierarchical clustering (Figure 5), some overlap between their expression profiles did occur with 19 clones modulated by both compounds in MCF-7 and 14 in HepG2 cells (Figure 6 and Supplementary Table 3 in the Supporting Information). These common alterations included a number of genes involved in xenobiotic metabolism. The AHRinducible gene CYP1B1 was, as expected, upregulated after exposure to TCDD but was also induced to a lesser extent after BPDE exposure in both cell lines, as measured by microarray analysis (Supplementary Table 3 in the Supporting Information) and by RTqPCR (Table 6). RTqPCR measurement of CYP1A1 revealed a similar response to BPDE exposure in the two cell lines (data not shown). Interestingly, BaP affected the expression of many genes not affected by BPDE or TCDD (Supplementary Table 4 in the Supporting Information), with 117 changes occurring in MCF-7 cells and 77 changes occurring in HepG2 cells. A number of genes are represented by more than one clone on the microarray, which may introduce some variation to the data because of differences between the clones. Aside from this, some of the genes modulated by BaP but not by BPDE or TCDD may represent BaP-modulated expression that is independent of AHR activation and DNA damage and may indicate novel pathways related to the mechanism of action of BaP. Only two genes CRY1 and MAK, were modulated in the same direction (upregulated) by BaP alone in both MCF-7 and HepG2 cells. Affected Biological Processes. EASE analysis was performed to identify biological processes associated with the BaP-TCDD and BaP-BPDE overlapping gene lists. The affected biological processes are summarized in Table 5. In both cell lines, genes common to BaP and BPDE exposure were linked to cell cycle



Figure 5. Hierarchical clustering of BPDE- and TCDD-exposed MCF-7 and HepG2 cells. Clustering was performed using the total list of genes significantly (p < 0.05) modulated by at least 1.4-fold by either BPDE or TCDD in (A) MCF-7 (1090 genes) and (B) HepG2 (215 genes) cells.

Figure 6. Venn diagrams to illustrate the overlap of BaP-, BPDE-, and TCDD-induced gene expression profiles in MCF-7 and HepG2 cells.

regulation, DNA repair, xenobiotic metabolism, and chromosome organization. Additionally, biological processes involved in protein metabolism were affected in MCF-7 cells by both BaP and BPDE. Expression changes common to BaP and TCDD exposure in the two cell lines were linked to a number of different biological processes, with xenobiotic metabolism genes being affected in both cell lines. Genes involved in cellular transport were also upregulated in MCF-7 cells, and genes linked to electron transport were downregulated. In HepG2 cells, changes in genes linked to oxidative stress signaling were also evident after BaP and TCDD exposure through induction of TXNRD1 and genes linked to angiogenesis, fatty acid metabolism, and transcription were downregulated. EASE analysis of the expression changes modulated by BaP only did not reveal any pathways or biological processes that were significantly over-represented in these gene lists, except for complement activation/humoral immune response processes through downregulation of three genes (APOH, C1QG, and C4A) in HepG2 cells. RTqPCR. RTqPCR was used to validate the microarray results for seven selected genes identified as AHR- or DNAdamage-induced. CDKN1A, PCNA, BAX, and HIST1H3D were all identified as DNA-damage-modulated on the microarrays in one or both of the cell lines (Table 6). RTqPCR confirmed that in the majority of cases these genes are modulated by BPDE but not by TCDD. The only exception was that of CDKN1A, which showed an altered expression in MCF-7 cells in response to both TCDD and BPDE when measured by RTqPCR. CYP1B1, NQO1, and AKR1C3 were identified as AHR-induced on the microarrays. The latter two were also identified as

downregulated by BPDE in HepG2 cells, and CYP1B1 was upregulated in MCF-7 cells by this compound. RTqPCR confirmed the induction of CYP1B1 and NQO1 but not of AKR1C3 by TCDD. CYP1B1 was identified as upregulated in HepG2 cells by RTqPCR but not on the microarrays. This result has also been observed in other studies (5, 12) and may be a result of the lower sensitivity of the microarrays to detect changes in expression when basal levels are low, as is the case for this transcript in HepG2 cells (12). RTqPCR showed the induction of CYP1B1 by BPDE in both cell lines and confirmed the repression of NQO1 but not of AKR1C3 in HepG2 cells. AHR- and p53-Binding Site Analysis. To investigate whether the AHR- or DNA-damage-mediated differentially expressed genes are potential transcriptional targets for AHR or p53, the promoter regions of the BaP–TCDD common genes were searched for AHR-binding sites (Tables 1 and 2) and the BaP–BPDE common genes were searched for p53-binding sites (Tables 3 and 4). Within the promoter regions identified for the DNA damage response genes (BaP–BPDE common genes), for both cell lines, all identified promoters contained p53-binding sites at a high frequency (between 10 and 62) and these included both down- and upregulated genes. For the genes with potential AHR-mediated expression (BaP–TCDD common genes), the majority of those with identifiable promoters, with the exception of five, contained AHR-binding sites (between 2 and 11) and included both up- and down-regulated genes.

Discussion Exposure of cells to BaP results in complex transcription responses that involve multiple cellular signaling cascades (5–12), many of which are cell-specific (12). Studies into cellular responses induced by BaP can give added insight into the basic nature of the carcinogenicity of this chemical. In this study, we have exposed MCF-7 and HepG2 cells to TCDD and BPDE and compared the resulting expression profiles to those induced by BaP in our previous study (12), to identify BaP-induced expression changes that are altered specifically via activation of AHR or in response to DNA damage. From the comparisons made in this study and in our previous study (12), it is evident that the degree of overlap between the compound-induced expression profiles of MCF-7 and HepG2 cells is surprisingly small, suggesting that the majority of transcriptional responses are cell-type-specific. A comparison of the gene expression profiles from BaP-treated A549 cells, a lung


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Table 1. Genes Significantly (p < 0.05) Modulated in MCF-7 Cells by at Least 1.4-Fold in Response to BaP and BPDEa


Chem. Res. Toxicol., Vol. 20, No. 12, 2007 1803 Table 1. Continued

a BPDE expression profiles were compared to BaP expression profiles identified in our previous study (12), identifying 78 genes as altered by both compounds in MCF-7 cells. This table contains expression ratios of these genes after incubation of MCF-7 cells with BPDE. + indicates that a p53-binding site has been found; – indicates that it was not found in the promoter; and N/A indicates that the promoter region could not be identified. 1 To identify p53-binding sites, promoter regions were identified in the 10 000 bp upstream region and the first exon of each gene. The promoter sequences were then searched for p53-binding sites by a Matrix Search using M00034, M00272, and M00761 that are p53-specific, at 90% stringency. 2 Shaded genes represent genes modulated by BaP and BPDE in both MCF7 and HepG2 cells.

adenocarcinoma cell line, with those from MCF-7 and HepG2 cells gave a similar level of overlap, confirming this interesting finding (unpublished results).

AHR-Induced Gene Expression. TCDD and BaP both bind AHR, a ligand-dependent transcription factor. Upon ligand binding, AHR translocates to the nucleus and forms a het-


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Table 2. Genes Significantly (p < 0.05) Modulated in HepG2 Cells by at Least 1.4-Fold in Response to BaP and BPDEa

a BPDE expression profiles were compared to BaP expression profiles identified in our previous study (12), identifying 29 genes as altered by both compounds in HepG2 cells. This table contains expression ratios of these genes after incubation of HepG2 cells with BPDE. + indicates that a p53-binding site has been found; – indicates that it was not found in the promoter; and N/A indicates that the promoter region could not be identified. 1 To identify p53-binding sites, promoter regions were identified in the 10 000 bp upstream region and the first exon of each gene. The promoter sequences were then searched for p53-binding sites by a Matrix Search using M00034, M00272, and M00761 that are p53-specific, at 90% stringency. 2 Shaded genes represent genes modulated by BaP and BPDE in both MCF7 and HepG2 cells.

erodimer with the AHR nuclear translocator (ARNT). This complex recognizes an enhancer DNA element, known as the xenobiotic response element (XRE), in the promoter of target genes, which results in their transcriptional activation. AHRinducible genes (17, 26, 27) include those encoding xenobiotic metabolizing enzymes, such as CYP1A1 and CYP1B1, which are involved in the bioactivation of PAHs, such as BaP. Animal knockout studies have shown that the loss of AHR results in

reduced toxicity and carcinogenicity of TCDD and BaP (18, 28). NQO1 and MGST1 were the only genes on the microarrays modulated by both BaP and TCDD in both cell lines and both encode enzymes important in the detoxification of BaP (29, 30). The AHR-inducible enzymes CYP1A1 and CYP1B1 are key enzymes in BaP bioactivation (31, 32). Consistent with this, induction of the CYP1A1 gene was detected by RTqPCR in both cell lines after exposure to both BaP and TCDD. The CYP1B1



Table 3. Genes Significantly (p < 0.05) Modulated in MCF-7 Cells by at Least 1.4-Fold in Response to BaP and TCDDa

a TCDD expression profiles were compared to BaP expression profiles identified in our previous study (12), identifying 22 genes as altered by both compounds in MCF-7 cells. This table contains expression ratios of these genes after incubation of MCF-7 cells with TCDD. + indicates that an AHR-binding site has been found; – indicates that it was not found in the promoter; and N/A indicates that the promoter region could not be identified. 1 To identify AHR-binding sites, promoter regions were identified in the 10 000 bp upstream region and the first exon of each gene. The promoter sequences were then searched for AHR-binding sites by a Matrix Search using M00778, M00235, and M00237 that are AHR-specific, at 90% stringency. 2 Shaded genes represent genes modulated by BaP and TCDD in both MCF7 and HepG2 cells.

gene was also upregulated by BaP and TCDD in both cell lines, shown by RTqPCR, but was only demonstrated by microarray analysis to be upregulated in MCF-7 cells. The lack of detection of CYP1B1 expression modulation on the microarrays for HepG2 cells is a consistent observation for this particular gene that is probably due to the very low basal levels of this transcript in HepG2 cells (12). The four AHR-regulated genes, CYP1A1, CYP1B1, NQO1, and MGST1, are likely to play important roles in the BaP cellular response and may represent biomarkers of exposure for compounds such as BaP and TCDD. Xenobiotic metabolism was the only process significantly altered in both MCF-7 and HepG2 cells via AHR-driven gene expression, confirming that AHR plays a key role in the metabolism of BaP. Karyala et al. (33) compared BaP and TCDD gene expression profiles from mouse smooth muscle cells and found that the compounds produced quite different cellular responses, with

only a small overlap of genes already known to be modulated by both compounds (e.g., Cyp1b1). Unlike BaP, TCDD is resistant to metabolism and is therefore likely to persist in cells for longer, which may explain the different cellular responses to these two compounds. Not all genes altered through activation of AHR contain an XRE. Some studies have shown that AHR can mediate gene expression by other mechanisms, such as by interaction with other proteins affecting gene transcription (34–36). In addition, some genes may be modulated through AHR activation as a result of downstream signaling in response to initial direct expression changes. Analysis of promoter sequences, identified in genes altered via AHR activation, revealed that most of them, including up- and downregulated genes, contained XRE sequences. Although the presence of these sequences may not


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Table 4. Genes Significantly (p < 0.05) Modulated in HepG2 Cells by at Least 1.4-Fold in Response to BaP and TCDDa

a TCDD expression profiles were compared to BaP expression profiles identified in our previous study (12), identifying 29 genes as altered by both compounds in HepG2 cells. This table contains expression ratios of these genes after incubation of HepG2 cells with TCDD. + indicates that a AHR-binding site has been found; – indicates that it was not found in the promoter; and N/A indicates that the promoter region could not be identified. 1 To identify AHR-binding sites, promoter regions were identified in the 10 000 bp upstream region and the first exon of each gene. The promoter sequences were then searched for AHR-binding sites by a Matrix Search using M00778, M00235, and M00237 that are AHR-specific, at 90% stringency. 2 Shaded genes represent genes modulated by BaP and TCDD in both MCF7 and HepG2 cells.

necessarily imply that these genes are AHR-regulated, they may represent direct targets for AHR-regulated transcription.

DNA-Damage-Induced Gene Expression. BPDE, the ultimate carcinogenic metabolite of BaP, forms DNA adducts (3)



Table 5. Biological Processes Affected by Both BaP and BPDE Exposure and Both BaP and TCDD Exposure in MCF-7 and HepG2 Cells

1

v, upregulated expression; V, downregulated expression.

and elicits a DNA damage response that includes induction of the p53 protein and gene regulation. A comparison of the BPDE and BaP transcription profiles identified genes linked to the cell cycle as upregulated via the DNA damage response in both cell lines, including the p53-inducible gene encoding the cell-cycle inhibitor CDKN1A (37, 38). In MCF-7 cells, two other p53regulated genes, CCNG1 and BTG2, linked to cell-cycle inhibition (39, 40), were upregulated as a result of DNA damage. CDKN1A and BTG2 both signal cells to arrest in G1 (38, 40) or G2 (37). In contrast to this observation, at the gene expression level, cells have been seen to arrest in the S phase after BaP exposure (12, 41, 42). In HepG2 cells, E2F6, a gene encoding a transcription factor involved in the repression of genes that are normally activated in the S phase (43), was upregulated. This gene may therefore play a role in the S-phase arrest in

response to BaP. Exposure to BPDE results in S-phase arrest of cells (data not shown), confirming that the effect of BaP on the cell cycle is in response to DNA damage. The gene encoding PCNA, involved in cellular proliferation and DNA repair (44), was induced in HepG2 cells by DNA damage. A p53-regulated gene, DDB2, was the only DNA repair gene (45) induced by DNA damage in MCF-7 cells. Induction of apoptotic signaling in response to DNA damage was evident in both cell lines. Thus, RPS27L, a p53-regulated gene, whose product has been shown to promote apoptosis (46) was induced by DNA damage in both cell lines, together with the pro-apoptotic functioning gene BAX. The expression of histone genes is tightly correlated to DNA synthesis (47), and their repression was seen in response to DNA damage in both cell lines. We found previously that histone expression levels correlated inversely with DNA adduct levels


Hockley et al.

Table 6. Summary of the Agreement between Microarray and RTqPCR Data of Selected AHR- and DNA-Damage-Mediated Expression Changesa

cells lacking AHR suggests an alternative pathway of Cyp1b1 induction (33). BaP Gene Expression Effects Independent of AHR and DNA Damage. Many genes induced by BaP were found to be independent of AHR and DNA damage expression signaling and may represent novel pathways in the mechanistic action of BaP. These genes included CRY1 and MAK that were similarly induced in both cell lines. Cryptochrome 1 encoded by CRY1 is a core component of the mammalian circadian clock. It is thought that, because disruption of the cell cycle can cause cancer, then so can disruption of the circadian rhythm (53, 54). Upregulation of CRY1 in response to BaP exposure may therefore represent a defense response for maintaining normal circadian rhythm. MAK encodes a serine/threonine kinase that is induced in prostate cells in response to androgen and that may be involved in prostate cancer progression (55). Upregulation of this gene may indicate a novel kinase-signaling cascade induced in response to BaP. Further investigation is required to confirm the apparent AHR independence of these two genes.

a Agreement between the microarray and RTqPCR data is 79% for both the BPDE- and TCDD-modulated genes. 1 +, altered expression. 2 -, unaltered expression.

Conclusions

in MCF-7 cells (12), strengthening the concept that histone gene repression occurs in response to genotoxic insult. The BPDEinduced histone gene expression profiles mirrored those induced by BaP, with repression at 6 and 24 h in MCF-7 cells but with only temporary repression followed by recovery at 24 h in HepG2 cells. The probable purpose of this inhibition of DNA synthesis is to allow time for DNA repair (48), and histone gene expression may therefore indicate the rate of repair of DNA damage. The delay of DNA synthesis is evident in cells exposed to BaP that arrest in the S phase of the cell cycle (12, 41, 42). Repression of histone expression has been seen in response to ionizing-radiation-induced DNA damage (49, 50) and, together with S-phase arrest, was identified in yeast cells exposed to aflatoxin B1 (51), suggesting that this response is evolutionarily conserved and important in maintaining DNA integrity after genotoxic insult. p53 plays an important role in the BaP-induced gene expression profile by modulating the transcription of a number of genes that protect cells from DNA damage. Promoter analysis of the BaP–DNA-damage-induced gene expression changes revealed the p53-binding motif to be present at a high frequency in all genes with identifiable promoters, indicating that they are potential direct targets of p53-regulated transcriptional modulation. As with the promoters found to contain XRE motifs, the presence of these potential p53-binding sequences does not establish unequivocally that these genes are p53-regulated and further experiments, such as chromatin immunoprecipitation assays, are required. Overlap of BPDE and TCDD Expression Profiles. It was interesting to observe the overlap between the gene expression profiles of the genotoxin BPDE and the nongenotoxin TCDD, including a number of genes involved in xenobiotic metabolism. BPDE has a very short half-life in aqueous solution and undergoes hydrolysis to tetrols (52), and it can not be ignored that such compounds may be influencing the gene expression response to BPDE. It is plausible that BPDE and/or its tetrol derivatives are capable of binding to AHR, although with lower affinity than that of BaP. Another possibility is that these genes are induced in an AHR-independent manner. For example, increased transcription of Cyp1b1 after BaP exposure of mouse

A comparison of gene expression profiles induced by BaP with those induced by TCDD and BPDE has defined AHR- and DNA-damage-mediated responses. AHR-regulated effects consisted primarily of xenobiotic metabolism processes that lead to bioactivation or detoxification of BaP. The DNA damage response consisted largely of p53-response genes that signal for cell-cycle arrest, DNA repair, and apoptosis. The downregulation of histone genes was also identified as a key response to DNA damage in both MCF-7 and HepG2 cells, which may be important in the inhibition of DNA synthesis to allow for repair of the damage. Work is underway to study further the role of p53 in the transcriptional response to compounds such as BaP. Acknowledgment. We thank the technical staff of the Cancer Research U.K. DNA Microarray Facility for production of the microarrays. This work was supported by S. Hockley’s Ph.D. studentship from the Institute of Cancer Research and by Cancer Research U.K. The authors (S.L.H., V.M.A., and D.H.P.) are partners of Environmental Cancer Risk, Nutrition, and Individual Susceptibility (ECNIS), a network of excellence operating within the European Union 6th Framework Program, Priority 5: “Food Quality and Safety” (contract number 513943). Supporting Information Available: Genes with significantly altered expression by at least 1.4-fold in either MCF-7 or HepG2 cells after exposure to BPDE (Supplementary Table 1), genes with significantly altered expression by at least 1.4-fold in either MCF-7 or HepG2 cells after exposure to TCDD (Supplementary Table 2), gene expression changes common to BPDE and TCDD exposure in MCF-7 and HepG2 cells (Supplementary Table 3), and genes with altered expression after BaP but not BPDE or TCDD exposure in MCF-7 and HepG2 cells (Supplementary Table 4). This material is available free of charge via the Internet at http://pubs.acs.org.

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