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Carcinogenesis Advance Access originally published online on January 3, 2008
Carcinogenesis 2008 29(2):227-236; doi:10.1093/carcin/bgm288
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Estrogenic status modulates aryl hydrocarbon receptor—mediated hepatic gene expression and carcinogenicity

Rohit Singhal1, Kartik Shankar1,3, Thomas M. Badger2,3 and Martin J. Ronis1,3,*

1 Department of Pharmacology and Toxicology and
2 Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, AR, USA
3 Arkansas Children’s Nutrition Center, 1212 Marshall Street, Little Rock, AR 72205, USA

* To whom correspondence should be addressed. Tel: +1 501 364 2796; Fax: +1 501 364 3161; Email: ronismartinj{at}uams.edu


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
 References
 
Estrogenic status is thought to influence the cancer risk in women and has been reported to affect toxicity of carcinogenic polycyclic aromatic hydrocarbons (PAHs) in animals. The objective of this study was to examine the influence of estradiol (E2) on hepatic gene expression changes mediated by 7,12-dimethylbenz(a)anthracene (DMBA), a potent PAH. Sprague–Dawley rats were ovariectomized on postnatal day 50 and infused with E2 (5 µg/kg/day) or polyethylene glycol using osmotic pumps and 14 days later gavaged with DMBA (50 mg/kg) or sesame oil and killed 24 h thereafter. To understand the mechanism of DMBA-mediated hepatocarcinogenicity in the presence of E2, microarray analysis (Rat 230 2.0 Affymetrix-GeneChip) was performed. Two hundred and sixteen genes were downregulated; whereas, 106 genes were upregulated significantly (±1.5-fold, P < 0.05) by DMBA treatment. Hierarchical clustering revealed that the expression profile of 39 genes, regulated by DMBA, was significantly modified by E2 supplementation. Interestingly, 71 genes were uniquely modulated in the combined treatment of DMBA and E2, but not by either treatment alone. Results from chromatin immunoprecipitation assay demonstrate that in animals cotreated with E2 and DMBA, there was enhanced recruitment of estrogen receptor-{alpha} to the regulatory regions of CYP1A1 and aryl hydrocarbon receptor (AhR) genes compared with that observed in animals treated with DMBA alone. E2 supplementation leads to increased DMBA-induced CYP1A1 transcription, while the AhR gene was upregulated in the presence of E2 +DMBA only. Our data suggest that estrogenic status is (i) important in AhR regulation and can influence the effects of xenobiotics and (ii) may be an important factor in DMBA-mediated carcinogenicity.

Abbreviations: AhR, aryl hydrocarbon receptor; BW, body weight; E2, estradiol; ER, estrogen receptor; cdk, cyclin-dependent kinase; ChIP, chromatin immunoprecipitation; CYP, cytochrome P450; DMBA, 7,12-dimethylbenz(a)anthracene; Gstm, Glutathione S-transferase mu; mRNA, messenger RNA; Nqo1, reduced nicotinamide adenine dinucleotide phosphate quinone reductase-1; PAH, polycyclic aromatic hydrocarbon; PCR, polymerase chain reaction; QRTPCR, quantitative real-time PCR; TCDD, 2,4,7,8-tetracholorodibenzo-p-dioxin; XRE, xenobiotic response element


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
 References
 
Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants. The potential sources include oil slicks, automobile exhaust, smoking and barbecued food (1). Some PAHs act as ligands for the aryl hydrocarbon receptor (AhR). Activation of AhR may initiate a cascade of secondary and tertiary changes in gene expression leading to carcinogenicity, wasting syndrome, teratogenicity, hepatotoxicity, immunomodulation and enzyme induction (2,3). AhR normally remains in the cytoplasm in the native state bound with the chaperone proteins, heat shock protein 90 and immunophillin-associated protein (XAP2) that maintain the stability of the receptor. Upon ligand binding, the AhR translocates to the nucleus, heterodimerizes with AhR nuclear translocator and binds xenobiotic response elements (XREs) in the regulatory region of many genes, including the xenobiotic-metabolizing enzymes from the AhR gene battery: cytochrome P450 (CYP) 1 family (CYP1A1, CYP1A2 and CYP1B1) and reduced nicotinamide adenine dinucleotide phosphate quinone reductase-1 (Nqo1) (4). Induction of CYP1 enzymes, in general, serves as a means of maintaining the homeostasis of the chemical environment in cells. CYP1A1 catalyzes the metabolic activation of procarcinogenic PAHs to ultimate carcinogens and hence its induction might be detrimental to humans exposed to high levels of PAHs such as by cigarette smoking. Besides these CYPs, several genes involved in critical pathways, such as cell-cycle regulation, mitogen-activated protein kinase cascades and retinoblastoma protein-related functions, also appear to be effected at the transcriptional level by the direct or indirect interactions with the AhR [reviewed in (5)]. A number of studies suggest a role for AhR ligands in modulating the expression of estrogen-responsive genes (6–9). Inhibitory AhR–estrogen receptor-alpha (ER{alpha}) cross talk has been extensively studied in multiple reproductive tissues and cell lines with the emphasis given to the effect of ligand-activated AhR on estrogen-responsive genes. These studies established AhR ligands as antiestrogens acting as potential selective ER modulators. Although the precise molecular mechanisms are unclear, they may include binding of activated AhR on the XRE present in estrogen-responsive genes, such as heat shock protein 27, cathepsin D, c-fos and pS2 (10–12).

Fewer studies have been conducted to evaluate the effects of estrogens on AhR-mediated signaling, especially in liver that is generally considered a nonclassical estrogen-responsive tissue that selectively expresses ER{alpha} (13,14). However, accumulating evidence suggest that estrogens play roles in transcriptional and translational modulation of hepatic genes involved in cellular growth (15) and development (16). Metabolism of 17 β-estradiol (E2), the major endogenous estrogenic hormone, by some CYPs can result in DNA adduct formation, oxidative DNA damage and carcinogenesis (17). Epidemiological reports on smokers indicate significantly higher levels of bulky DNA adduct and higher susceptibility to lung cancer among females compared with males (18,19). In vivo studies also suggest decreased 2,4,7,8-tetracholorodibenzo-p-dioxin (TCDD)-induced hepatic DNA damage in ovariectomized rats as compared with control females, suggesting a role of estrogens in dioxin and PAH-mediated carcinogenicity (20). This might indicate that PAH-mediated toxicities are influenced by the endogenous estrogenic status and a different PAH signature in pre- and postmenopausal women.

The present studies were performed to elucidate the mechanisms and overall changes in hepatic gene expression upon exposure of female Sprague–Dawley rats to PAH in the presence and absence of estrogens. Herein, we demonstrate that hepatic gene expression by a PAH, 7,12-dimethylbenz(a)anthracene (DMBA) is modified after cotreatment with E2. Comparisons among various groups were determined, using microarray analysis, in order to identify genes affected by DMBA treatment and modulated by E2 treatment. Interestingly, we observed a unique gene expression profile with E2+ DMBA. The effects of the treatments on AhR–ER{alpha} cross talk were evaluated using chromatin immunoprecipitation (ChIP) assay which established that induction of CYP1A1 and AhR genes is transcriptionally modified by E2-activated ER{alpha}.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Supplementary material
 Funding
 References
 
Animal care and experiment design
The experiment received prior approval from the Institutional Animal Care and Use Committee at University of Arkansas for Medical Sciences. Adult female Sprague–Dawley rats, postnatal day 30, were obtained from Charles River Laboratories (Wilmington, MA) and were housed in polycarbonate cages in an environmentally controlled room with a 12 h light–dark cycle. Rats were provided with pelleted semipurified AIN-93G diet to prevent exposure to dietary phytoestrogens found in most commercially available rat food (21). Postnatal day 50 rats were ovariectomized and subcutaneously infused with E2 or polyethylene glycol (Sigma, St Louis, MO) using Alzet 2002TM mini-osmotic pumps (Alza corp., Palo Alto, CA) that were filled to release 0.5 µl/h for 14 days at 5 µg/kg/day. On postnatal day 64, rats were orally gavaged with 50 mg/kg DMBA or sesame oil (Sigma). After 24 h, rats were briefly anesthetized with Nembutal (100 mg/kg, intraperitoneal) followed by decapitation and blood, liver and uteri were collected (refer to supplementary material for schematic representation, available at Carcinogenesis Online) and kept frozen at –80°C until used for the experiments. The treatment groups were designated as no treatment (control), DMBA treatment (DMBA), E2 treatment (E2) and cotreatment of E2 and DMBA (E2 + DMBA).

E2 level determination
Serum E2 levels were determined by ultra-sensitive radioimmunoassay, using DSL-4800TM kit (Diagnostic Systems Laboratories, Webster, TX), following manufacturer’s protocol. The kit was sensitive enough to detect as low as 2.2 pg/ml of estrogen.

Total RNA isolation and microarray preparation
Total RNA was isolated from 100 mg of hepatic tissue using TRI reagent (Molecular Research Center, Cincinnati, OH) according to the manufacturer’s instruction and cleaned using RNeasy kit (Qiagen, Valencia, CA). Contaminating DNA was enzymatically degraded with RNase-free DNase (Qiagen). RNA was quantified and analyzed for integrity using RNA 6000 Nano Chip on Agilent 2100 Bioanalyzer (Agilent technologies, Palo Alto, CA). First- and second-strand cDNA synthesis, biotin-labeled cRNA synthesis, fragmentation of cRNA and hybridization reactions were performed using one cycle cDNA synthesis kit (Affymetrix, Santa Clara, CA). Briefly, 8 µg of purified RNA was used to synthesize cDNA. Labeled cRNA was synthesized from cDNA using a GeneChip IVT labeling kit (Affymetrix) according to the manufacturer’s instructions. Twenty microgram cRNA was then fragmented in a solution of 5x fragmentation buffer and RNase for 35 min. Complementary RNA was hybridized by filling up with 300 µl volume of the clarified hybridization cocktail to the Rat genome 230 2.0 array. The cRNA was hybridized for 16 h at 45°C in the hybridization oven set at 60 r.p.m. After hybridization, the cocktail from the probe array was removed. The probe array was completely washed using wash buffer and stained with the staining cocktail, using the wash and stain kit (Affymetrix) in GeneChip fluidics station 450 (Affymetrix). The probe array was scanned after the wash and staining protocols with GeneChip Scanner 3000 (Affymetrix).

For each of the 31 099 genes on the Affymetrix Rat genome 230 2.0 array, the data on induction or repression values were analyzed using GeneChip Operating Software obtained from Affymetrix.

Microarray data normalization and analysis
The intensity values of different genes (probe sets) generated by Affymetrix GeneChip Operating Software were imported into GeneSpring version 7.3X software (Silicon Genetics, Redwood City, CA) for data analysis. The data files (.CEL files) containing the probe-level intensities were processed using the robust multiarray analysis algorithm (GeneSpring) for background correction (22). Subsequently, the data were subjected to normalization by setting measurements <0.01–0.01 and by per-chip and per-gene normalization using GeneSpring normalization algorithms. The normalized data were then subjected to a series of pairwise comparisons. Comparisons were made between various treatments: DMBA versus control; E2 versus control and E2 + DMBA versus control. The resulting gene lists generated from each pairwise comparisons included only the genes that had a fold change value greater than +1.5 or lesser than –1.5 (i.e. ±1.5) and a P < 0.05. A list of all those genes that were expressed differentially was generated by combining the gene lists from individual pairwise comparisons and subjected to hierarchical clustering. Hierarchical clustering was performed using the ‘smooth correlation for distance measure’ algorithm to identify samples and genes with similar pattern of expression.

Evaluation of E2 treatment on DMBA-affected gene expression
A comparison between E2 + DMBA versus E2 was made to identify set of genes changed by DMBA in the presence of E2, but not by E2 itself. List of genes common to DMBA-induced genes modified by E2 treatment and genes induced by DMBA only by the interaction with E2, not by either treatment itself, were generated. Among the DMBA-induced genes modified by E2 treatment, those genes were identified whose expression changes ±1.5-fold by E2 + DMBA treatment as compared with DMBA alone and cluster analysis was performed using Cluster 2.1.1 and ‘Tree View’ version 1.60 software supplied by Eisen Lab, Stanford University (http://rana.lbl.gov/EisenSoftware.htm).

ChIP assay
The ChIP-ITTM Enzymatic kit (Active Motif, Carlsbad, CA) was used for ChIP assay and modified for in vivo samples. Briefly, the frozen liver tissue (200 mg) was finely minced with razor blade. The crosslinking of the proteins in the tissue was performed by incubating the minced tissue with 20 ml of 37% formalin (Sigma) solution for 10 min on a rocking shaker at room temperature. The crosslinking reaction was stopped by adding 5 ml of glycine stop-fix solution [3 ml of 10x glycine buffer; 3 ml 10x phosphate-buffered saline (provided with the kit) and 24 ml water] followed by homogenization, using a dounce homogenizer, in 2 ml of ice-cold phosphate-buffered saline supplemented with protease inhibitor and 100 mM phenylmethylsulfonyl fluoride and the homogenate was passed through 100 µM cell strainer (BD FalconTM, Bedford, MA) followed by centrifugation for 10 min at 2500 r.p.m. at 4°C and processed further as mentioned in the kit protocol. The chromatin obtained by enzymatic shearing of the nuclei was ‘precleared’ with protein G agarose beads to reduce nonspecific background followed by incubation with 2 µg of antibodies (Santa Cruz, CA): (i) anti-AhR goat polyclonal antibody (sc-8088) or (ii). anti-ER{alpha} (sc-542) on a rotator at 4°C overnight. The yield of target region DNA-possessing GCGTG was analyzed by conventional polymerase chain reaction (PCR) with the following primer (IDT, Skokie, IL) sequence: CYP1A1-XRE, F 5'-CGC CCT TGC AAA GCT TAA GAC-3' and R 5'-TCC CAG TGC TGT CAC GCT AG-3'; AhR-XRE, F 5'-GAC AGC GGA GGA GCG GGC TC-3' and R 5'-GGG TCG CTC CGG GCT GAA CC-3'.

Microarray validation by real-time PCR
Total RNA (1 µg) was reverse transcribed using iSCRIPTTM cDNA synthesis kit (Bio-Rad, Hercules, CA) following the manufacturer’s instructions. Primers (supplementary data are available at Carcinogenesis Online) were designed by Primer ExpressTM Software (Applied Biosystems, Foster City, CA). cDNA samples were amplified using 2x SYBR Green PCR Master Mix (Bio-Rad) at optimal concentrations (10 nmol/l) of primers in a total reaction volume of 25 µl under the conditions recommended by the manufacturer: (i) preincubation at 50°C for 2 min; (ii) DNA polymerase activation at 95°C for 10 min and (iii) 40 PCR cycles of 95°C for 15 s and 60°C for 1 min. Expression levels of genes were normalized to that of glyceradehyde 3-phosphate dehychogenase to control for input gene. Samples were assayed in duplicate using the ABI Prism 7000 detection system (Applied Biosystems).

Assessment of apoptosis
Terminal deoxynucleotidyl transferase-mediated deoxy-uridine triphosphate nick-end labeling assay to detect apoptotic cells was performed as described by Eason et al. (23). Terminal deoxynucleotidyl transferase-mediated deoxy-uridine triphosphate nick-end labeling-positive cells were counted from three to four randomly selected fields at x100 per slide, and two slides were evaluated for each tissue block from four rats per treatment.

Statistical analysis
Statistical analysis was performed using Sigma Stat and graphs were generated using Sigma Plot (SPSS, Chicago, IL). Relative organ weights were obtained by normalizing the organ weight with the body weight (BW) of the animal. Data (organ weights, serum E2 levels; Table I and real-time reverse transcriptase–PCR; Figure 1) were analyzed by two-way analysis of variance followed by Student–Newman post hoc test and were considered significant if P < 0.05. Differences between treatment groups in microarray data were analyzed by ‘fold changes in volcano plot’ (GeneSpring Software) and changes were considered significant at ±1.5-fold and P < 0.05.


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  		Table I. Relative organ weights and serum E2 level of rats with different treatments

 

   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Supplementary material
 Funding
 References
 
Organ weight and serum E2 levels
A decrease (P < 0.05) was observed in the BW in E2-infused rats compared with non-E2-infused groups. However, the relative weights (organ weight normalized to the BW) of liver and uterus increased (P < 0.05) by E2 infusion, while DMBA treatment had no effects on the BW, liver weight relative to body weight, uterus weight relative to body weight or E2 levels. Serum E2 levels were higher in the E2-infused groups compared with non-E2-infused groups (P < 0.05). Cotreatment with E2 and DMBA resulted in greater (P < 0.05) E2 levels compared with E2 alone (Table I).

Hierarchical clustering
Gene lists from individual comparison of treatments against control were combined as described under ‘microarray data normalization and analysis’ in Materials and Methods section. This combined gene list included 1339 genes with known biological functions. Unsupervised hierarchical clustering analysis was performed based on the treatment and gene expression type. Among the three treatments, E2 + DMBA and E2 clustered together; whereas, DMBA clustered alone, suggesting greater common effects of E2 treatment on hepatic genes in the presence and absence of DMBA than DMBA alone. Using pseudogene lines, the heat map was divided into seven subclusters: I, genes induced by E2; II, genes induced by E2 + DMBA; III, genes induced by DMBA; IV, genes repressed by DMBA; V, genes repressed by E2 + DMBA; VI, genes repressed by E2 and VII, genes induced by DMBA and repressed by E2 + DMBA (supplementary data are available on Carcinogenesis Online).

Hepatic gene expression effected by the interaction between DMBA and E2 treatments
A Venn diagram illustrates that 283 genes affected by DMBA treatment were unaffected by E2 supplementation; these genes were deemed as ‘DMBA affected genes not modulated by E2’. Thirty-nine genes were found common between genes modulated by DMBA alone (DMBA versus control) and genes altered by DMBA in the presence of E2, but not by E2 itself (E2 + DMBA versus E2); these genes were deemed as ‘DMBA-affected genes modulated by E2’, while 71 genes were found to be affected by DMBA only in the presence of E2, not by either treatment alone; these genes were deemed as ‘genes affected by DMBA only in the presence of E2’ (supplementary data are available on Carcinogenesis Online).

Among 39 ‘DMBA-affected genes modulated by E2’ genes, only 18 were modified ±1.5-fold by the cotreatment of E2 + DMBA as compared with DMBA alone (E2 + DMBA versus DMBA). Hierarchical clustering of these 18 genes revealed xenobiotic-metabolizing genes—CYP1A1 (1.5-fold) and abhydrolase domain containing 2 (Abdh2; 2.0-fold) are further stimulated whereas glutathione S-transferase mu 3 (Gstm3; –1.45-fold) and Nqo1 (–1.5-fold) are repressed by E2 supplementation. DMBA-mediated repression of cellular differentiation-associated Gas6 (2.47-fold) and apoptotic gene disulfide isomerase 3 (Pdia3; 1.58) was reversed upon E2 supplementation. DMBA-repressed transcription factors—nuclear receptor subfamily 0, group B, member 2 (Nr0b2, 1.7) was increased whereas insulin receptor substrate 3 (1.7) was further downregulated (Table II). Interestingly, a gene involved in axon regeneration, neuronal regeneration-related protein was suppressed by 11.7-fold. The role of neuronal regeneration-related protein in liver physiology is unknown (Table II; supplementary data are available on Carcinogenesis Online).


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  		Table II. Functional characterization and fold changes of DMBA-affected genes modulated by E2 supplementation

 


Figure 1
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  		Fig. 1. Pattern of change in mRNA expression level, n = 6; percent of control, upon treatment in nine selected genes, normalized with the expression level of glyceraldehyde 3-phosphate dehydrogenase (internal control), determined by real-time PCR. Both real-time PCR and microarray data are represented as percent of control. Real-time PCR were analyzed by two-way analysis of variance followed by Student–Newman post hoc test (P < 0.05). #, $ and @ represent effects of DMBA, E2 and interaction between DMBA and E2, respectively.

 
Seventy-one genes ‘affected by DMBA only in the presence of E2’ were changed by the interaction between E2 and DMBA. Out of 71 genes, 14 genes were repressed; whereas, 57 genes were induced. Xenobiotic-metabolizing phase II genes—Gstm1 and microsomal Gstm3—were downregulated, whereas oxidative stress–associated copper chaperone for superoxide dismutase was upregulated. Cellular proliferation and apoptosis-related genes—nuclear ubiquitous casein kinase and cyclin-dependent kinase (cdk) substrate and growth arrest and DNA-damage-inducible 45 alpha, ER-binding fragment-associated gene 9 and apoptosis inhibitor 5 were upregulated by the combination of E2 and DMBA as compared with either treatment alone. Genes related to signal transduction and transcription—AhR, nuclear receptors-1d1 (Nr1d1), Nr1d2, peroxisome proliferator-activated receptor alpha, hairy and enhancer of split 6 and CCAAT/enhancer binding protein gamma, were induced by the interaction of DMBA and E2, (Table III; supplementary data are available on Carcinogenesis Online).


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  		Table III. Functional categorization and fold change of genes affected only in the presence of both E2 and DMBA

 
Treatment with DMBA for 24 h time period led to significant changes in expression of 322 genes (±1.5-fold, P < 0.05) as compared with control group; 105 genes were induced and 217 genes were repressed. Upregulated genes included genes involved in xenobiotic metabolism such as phase I genes—CYP1A1, CYP1A2, epoxide hydrolase 2 (Ephx2), Nqo1 and flavin monooxygenase 1 (Fmo1) and phase II genes such as uridine glucosyltransferase 1 family a (Ugt1a), glutathione synthetase (Gss) and reductase (Gsr). Among all the genes, CYP1A1 was induced to the highest level viz. 199-fold. Genes involved in cellular proliferation for example cyclins—A2, D 1 and 2, growth arrested specific 6 (Gas6), cell division cycle 1 and 2 were found to be repressed. Proapoptotic gene-Bel-2-associated-X-protein (Bax) was induced by 1.6-fold while antiapoptotic gene- Bcl-2-modifying factor (Bmf) was repressed by 1.5-fold. E2 treatment resulted in changes of 587 genes, as compared with the control group; 471 genes were downregulated and 116 were upregulated. Treatment with DMBA in the group infused with E2 (E2 + DMBA) resulted in a significant change in the expression profiles of 925 genes: 259 were induced and 666 were repressed, determined by comparing E2 + DMBA with control group (supplementary data are available on Carcinogenesis Online).

Verification of microarray response
Quantitative real-time PCR (QRTPCR) was used to verify the fold changes in the transcript levels observed by microarrays. The pattern of expression of 20 genes was evaluated using QRTPCR, all of which displayed similar pattern of treatment-mediated changes both in microarrays and real-time PCR except CYP1B1 expression which was not found to be changed significantly by DMBA in microarray data analysis. Figure 1 displays QRTPCR verification of nine selected genes.

DMBA-dependent recruitment of E2-activated ER{alpha} is associated with enhanced CYP1A1 and AhR genes transcription
Binding of transcription factors AhR and ER{alpha} to the regulatory regions of CYP1A1 and AhR genes was determined by ChIP assay. DMBA treatment either in the presence or absence of E2 resulted in the binding of AhR to the XRE—containing enhancer region, GCGTG element, at—1116 base pairs upstream of transcription start site of CYP1A1, while recruitment of ER{alpha} was also observed to the same region but only when DMBA treatment was supplemented with E2 (Figure 2A, left panel). Similar GCGTG elements were recognized at –300 and –336 base pair upstream of transcription start site of AhR gene. Treatment with DMBA or E2 resulted in the recruitment of AhR and ER{alpha}, respectively, to the regulatory regions whereas DMBA treatment in combination with E2 resulted in the recruitment of both AhR and ER{alpha} to the same sites (Figure 2A, right panel). Change in messenger RNA (mRNA) expression profile of CYP1A1 and AhR genes was determined by QRTPCR. DMBA treatment significantly (P < 0.05) induced CYP1A1 mRNA levels, both in the presence and absence of E2; whereas, E2 supplementation further enhanced DMBA-mediated CYP1A1 induction by 1.5-fold (Figure 2C, left panel). DMBA or E2 treatment itself had no effect on the AhR mRNA expression level, whereas E2-supplemented DMBA treatment resulted in significant (P < 0.05) induction (Figure 2C, right panel). Two-way analysis of variance also suggested a significant interaction between E2 and DMBA both at CYP1A1 and AhR genes.


Figure 2
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  		Fig. 2. DMBA-dependent recruitment of E2-activated ER{alpha} enhances CYP1A1 (panel left) and AhR (panel right) genes transcription. ChIP assays were performed with chromatin prepared from rat liver subjected under treatments using anti-AhR or anti-ER{alpha} antibodies as mentioned in Materials and Methods. (A) PCRs of DNA from ChIP assay for input or immunoprecipitated fractions CYP1A1 and AhR. (B) Schematic representation of rat CYP1A1 and AhR genes promoter region. Arrows indicate the location of amplified PCR sequence. Circle represents XRE/AhR-binding site and solid block represents GC-rich region/Sp1-binding site. (C) Real-time PCR amplification of CYP1A1 and AhR genes. Differences in mRNA expression were analyzed by two-way analysis of variance followed by Student–Newman post hoc test (P < 0.05). #, $ and @ represent effects of DMBA, E2 and interaction between DMBA and E2, respectively.

 
Assessment of apoptosis
DMBA treatment resulted in a marginal increase in percent positive cells (0.091 ± 0.02) as compared with control (0.076 ± 0.04) and E2 treatment (0.070 ± 0.003). Cotreatment with E2 and DMBA resulted in 1.6-fold increase in percent positive apoptotic cell count (0.15 ± 0.03) compared with DMBA alone. Because of the interanimal variability, the differences did not reach a level of statistical significance.


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Supplementary material
 Funding
 References
 
In the present study, we used an animal model and a biomarker gene, CYP1A1, to address a critical question concerning the cancer risk of estrogenized women (premenopausal or on estrogen replacement therapy) exposed to PAHs. The present study represents the first comprehensive in vivo examination of the hepatic gene expression profile in response to a common PAH (DMBA) in the presence and absence of E2. In addition, the mechanism of increased DMBA-mediated induction of CYP1A1, an enzyme responsible for PAH–DNA adducts formation and carcinogenesis, in the presence of E2, has been investigated. Most of the earlier studies conducted at the genomic levels have used TCDD as a model AhR ligand to interpret the toxicities exhibited by the activation of this receptor. Herein, we used DMBA which is a model PAH. DMBA is preferentially metabolized by CYP1B1 that is primarily an extrahepatic enzyme. However, CYP1A1, which has high inducibility in liver, can also catalyze the formation of dihydrodiols from DMBA (24,25). Twenty-four hours of exposure to DMBA resulted in no significant changes in BW or liver weight; whereas, in studies performed using single-oral dose of TCDD (200 µg/kg), an increase in liver weight, but not the BW, was observed (26–28). However, a significant reduction in BW and increase in liver weight and uterine weight was observed by E2 infusion. Surprisingly, an increase in serum E2 level was observed in the combination of E2 and DMBA. The reason is difficult to understand but one of the possibilities might be reduced clearance of E2 from the body in the presence of DMBA either as the result of reduced metabolism (29). This could also be a dose effect and difficult to analyze in small group of animals.

Functional annotation obtained from Affymetrix Web site (http://www.affymetrix.com/index.affx) indicated that interaction between DMBA and E2 may contribute to changes in xenobiotic metabolism, immune response, cellular proliferation and apoptosis and signal transduction, which is described below.

Xenobiotic metabolism and oxidative stress
Genes encoding products associated with oxidoreductase, monooxygenase and xenobiotic metabolism were induced by DMBA, some of which have been previously characterized as members of the ‘AhR gene battery’ including CYP1A1, CYP1A2, Nqo1 and Ugt1a6 (4). Although induction of these proteins serves important roles in detoxification, the respective activities also contribute to the formation of reactive oxygen species, which can lead to cellular oxidative stress, lipid peroxidation, DNA adducts formation and initiate malignant cell transformation (30–32). Microarray and QRTPCR data suggest that induction of phase I enzyme CYP1A1 is increased whereas the detoxification enzymes, Nqo1 and Gstm3, are reduced by the interactions between E2 and DMBA as compared with DMBA itself. Similar increases in TCDD-mediated DNA adduct formation were observed in ovariectomized rats compared with intact rats (20) and female rats compared with male rats (33), suggesting estrogen-dependent enhancement of AhR ligand-mediated DNA adduct formation and reduction in detoxification. Real-time PCR also suggests an increase in CYP1B1 mRNA by DMBA in the presence and absence of E2. CYP1B1 is expressed mainly in extrahepatic tissues; however, its induction in liver cannot be negated and has been demonstrated by many laboratories, including ours (34–36). This detection may be subjected to conditions and sensitivity of real-time PCR and that is why not replicated by microarrays.

Immune response and apoptosis
The mechanism of PAH-mediated immunosuppression, though not well understood, seems to involve CYP1A1-generated, PAH-dihydrodiol-induced B-cell apoptosis and a deficit in the T-helper cell function (30,37). Our studies, though not in bone marrow, suggest DMBA-mediated downregulation of genes responsible for antigen presentation and complement activation. E2 did not have a significant affect on DMBA-modulated immune-responsive genes. Moreover, a cancer biomarker, ER-binding fragment-associated gene 9 (also known as RCAS1), an estrogen responsive gene whose product induces apoptosis in active immune cells and has been directly associated with tumor progression and invasiveness in hepatocellular, breast, prostate and renal cancer (38–40), was increased by E2 only in the presence of DMBA.

A DMBA-associated induction of proapoptotic genes (Bel-2-associated-X-protein, tumor protein p53-induced nuclear protein 1—Trp53inp1 and Bel-2-associated-X-protein-associated mitochondrial modulator of apoptosis protein—Moap1) and repression of antiapoptotic gene, Bcl-2-modifying factor, indicates a conducive environment for apoptosis that may be a direct response to DMBA-initiated apoptosis or an indirect response related to DMBA-mediated oxidative stress and does not necessarily depend on the presence of estrogen. However, an increase in antiapoptotic apoptosis inhibitor 5 (Api5) gene was observed by E2 and DMBA cotreatment only. Apoptosis inhibitor 5 has been shown to serve as a marker for tumorigenic cells, since it allows only tumor cells to survive because of inhibition of E2 promoter-binding factor-dependent apoptosis (41). This indicates that PAHs could generate both apoptotic and antiapoptotic signals in hepatic cells and the presence of estrogens could be a crucial deciding factor in which process predominates. Terminal deoxynucleotidyl transferase-mediated deoxy-uridine triphosphate nick-end labeling assay, a measure of DNA damage and apoptosis, suggests an increase, though not statistically significant, in apoptotic index by E2 and DMBA cotreatment compared with either E2 or DMBA which is consistent with our hypothesis that DMBA activation is increased by E2 supplementation.

Cellular proliferation
AhR plays a crucial role in cell-cycle regulation, since AhR null mice exhibit increased epidermal hyperplasia and adenocarcinoma of liver and lung (42). A large body of evidence suggests that AhR ligands, especially TCDD, inhibit cell proliferation and induce cell-cycle arrest. Mechanisms may include interaction of activated AhR with retinoblastoma/E2 promoter-binding factor-binding proteins that causes inhibition of some cyclins and Cdk expression leading to cell-cycle arrest at various check points (5,43). DMBA treatment repressed the expression profile of many genes associated with cell proliferation and differentiation including cyclins—A2, B2, D2 and B1. Estrogens, on the other hand, are known mitogens and contribute to the transformation of quiescent cells through cell cycle (44). This suggests an antagonizing affect of E2 on DMBA-mediated cell-cycle arrest. Cell-cycle mediators—Cdk inhibitor 1A, growth arrest and DNA-damage-inducible 45 alpha, Gas6 and nuclear ubiquitous casein kinase and cdk substrate were induced only by the cotreatment of E2 and DMBA. Overexpression of growth arrest and DNA-damage-inducible 45 alpha in conjunction with Cdk inhibitor 1A , though a homeostatic response to increased cellular stress, has been linked to poor prognosis in pancreatic cancer (45) and might serve as another indicator for increased DNA damage by cotreatment of E2 and DMBA.

Signal transduction and transcription factors
Activated AhR has been known to produce cellular changes through partnering with various transcription factors (46–48) and various coactivators and corepressors (49,50). Our data suggest that DMBA treatment alone results in reduced expression profile of number of genes related to signal transduction and transcription, including growth hormone receptor and sterol regulatory element-binding factor 1 that may provide insight into DMBA-mediated changes in cellular signaling not dependent upon estrogenic status. However, nuclear hormone receptors—Nr1d1 and Nr1d2 (also known as Rev-erb alpha and beta, respectively) were upregulated only upon cotreatment with E2 and DMBA. Amplified levels have been linked with retinoic acid receptor alpha- and beta-mediated toxicities (51), breast cancer biopsy (52) and mouse hepatocarcinoma cell lines (53).

The toxic effects of AhR ligands have been shown to be highly dependent on gender. TCDD at 100 ng/kg/day was shown to exhibit higher incidence of hepatocellular inflammatory, necrotic and degenerative changes in female rats when compared with male rats (54). On the other hand, estrogens and ER had been associated with an increased metabolic activation of PAHs in breast and lung tissues (55–57). To elucidate the molecular mechanism underlying estrogen-mediated changes in DMBA signaling, we picked two genes to be analyzed by ChIP. There is a considerable evidence for direct protein–protein interaction between ER{alpha} and AhR at the enhancer region of CYP1A1 gene. Most of the work done has been conducted in MCF-7 mammary cell lines using TCDD as AhR ligand and suggest an ER-mediated transrepression of CYP1A1 gene transcription (9,58,59). However, studies performed in liver of female Long–Evans rats (60) and HuH7, a human liver carcinoma cell line (59), suggest an E2-mediated enhancement of TCDD-induced CYP1A1 transcription. Matthews et al. also observed an increase in CYP1A1 activity as a result of TCDD-bound AhR-mediated recruitment of ER{alpha} in the copresence of E2 and TCDD as compared with TCDD alone (52). The current data are the first to show an in vivo analysis of the hepatic CYP1A1 enhancer region and suggest a recruitment of E2-activated ER{alpha} to DMBA-activated AhR on CYP1A1 XRE only in the group cotreated with E2 and DMBA, but not in DMBA alone. This E2-dependent increase in ligand-activated CYP1A1 might also explain female-specific TCDD-mediated induction of hepatic DNA adducts in Sprague–Dawley rats (33) and in the lungs of female smokers (19). As with CYP1A1, XREs were identified in the rat AhR promoter region. Once again, this is the first time to our knowledge that AhR gene has been analyzed at the regulatory region for the recruitment of AhR and ER{alpha}. DMBA-specific AhR and E2-specific ER{alpha} recruitments were observed by either treatment alone without any significant changes in AhR gene expression profile. Cotreatment of E2 and DMBA led to the corecruitment of both receptors leading to increased AhR mRNA. This suggests an auto-upregulation of AhR gene in the presence of both E2 and DMBA. The differences in the AhR–ER{alpha}-mediated increase in the CYP1A1 and AhR gene could be explained by the differences in their respective coregulators and corepressors and need further exploration. Contrary to our findings, an increase in AhR mRNA by E2 itself as well in combination with TCDD was reported in MCF-7 cells by Spink et al. (61). The differences may be due to the comparison of mammary cell lines with the whole-liver tissue, TCDD compared with DMBA, and different E2 treatment regimes.

PAHs are ubiquitously present in the environment and human exposure is nearly inevitable. Using the genome analysis tools we anticipate increased risk of carcinogenesis by exposure to PAHs under conditions that might be expected to occur in women with normal circulating levels of estrogens (premenopausal and estrogen replacement). Our speculation is supported by epidemiological studies performed by Mollerup et al. who demonstrated an increased DNA adduct levels and CYP1A1 expression in the lungs of female smokers compared with male smokers (18,19). Although the hepatocarcinogenic effects of estrogens have been attributed to stimulated growth and proliferation, increased CYP1-mediated formation of genotoxic catechol and quinone estrogen metabolites and resulting oxidative damage to DNA, proteins and lipids following exposure to PAHs may also be contributing factors (reviewed in refs 44 and 62). As demonstrated in the hypothetical model, Figure 3, our data suggest a novel scenario where auto-upregulation of AhR gene, as a result of E2 and DMBA interaction, might lead to increased phase I enzyme—associated procarcinogen activation, reduced phase-II-associated detoxification, increased proliferation and reduced apoptosis in precancerous cells. This might increase the risk of PAH-mediated carcinogenesis in the premenopausal women. Further work is required to see if the same phenomenon occurs in cancer target tissue such as the mammary gland.


Figure 3
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  		Fig. 3. A hypothetical model. Exposure to PAH would lead to CYP1A1 activation, DNA adduct formation, oxidative stress and altered signal transduction, which might result in carcinogenesis (dotted arrow). Exposure of PAH in the presence of E2 would cause autoinduction of AhR (and AhR-mediated signaling), enhanced CYP1A1 induction, quinone and free radical generation, increase in oxidative stress, DNA adduct formation, cellular proliferation and increase in altered signal (solid arrow).

 

   Supplementary material
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 Supplementary material
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Supplementary material can be found at http://carcin.oxfordjournals.org/.


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
References
 
U.S. Department of Agriculture Current Research Information System (6251-51000-005-02S).


   Acknowledgments
 
Conflict of Interest Statement: None declared.


   References
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 Introduction
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Received June 21, 2007; revised November 15, 2007; accepted December 8, 2007.


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