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Carcinogenesis Advance Access originally published online on February 1, 2007
Carcinogenesis 2007 28(7):1606-1612; doi:10.1093/carcin/bgm013
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Increase of carcinogenic risk via enhancement of cyclooxygenase-2 expression and hydroxyestradiol accumulation in human lung cells as a result of interaction between BaP and 17-beta estradiol

Louis W. Chang1, Yun-Ching Chang1,2, Chia-Chi Ho1,3, Ming-Hsien Tsai1 and Pinpin Lin1,3,*

1 Division of Environmental Health and Occupational Medicine, National Health Research Institutes, No. 35, Keyan Road, Zhunan Town, Miaoli County 350, Taiwan, Republic of China
2 Institute of Biochemistry and Biotechnology, Chung Shan Medical University, Taichung, Taiwan
3 Institute of Medical and Molecular Toxicology, Chung Shan Medical University, Taichung, Taiwan

* To whom correspondence should be addressed. Tel: +886 37 246 166 ext. 36508; Fax: +886 37 587 406; Email: pplin{at}nhri.org.tw


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Animal studies demonstrated that females are more susceptible than males to benzo[a]pyrene (BaP)-induced toxicities, including lung carcinogenesis. Elevation of cyclooxygenase-2 (COX-2) expression has been shown to increase the risk of cancer development. BaP induces COX-2 expression, and an interaction between BaP and estrogen in relation to COX-2 expression is suspected. In the present study, 10 µM BaP alone only slightly increased COX-2 mRNA expression and 10 nM 17-beta estradiol (E2) alone slightly increased prostaglandin E2 (PGE2) secretion in human bronchial epithelial cells. However, co-treatment with BaP and E2 potentiated COX-2 mRNA expression and significantly elevated PGE2 secretion. Utilizing specific inhibitors and reporter assays, we further investigated the potentiation mechanisms of E2 on BaP-induced COX-2 expression. First, E2 activated estrogen receptor to increase PGE2 secretion, which directly increased COX-2 expression. Second, E2 potentiated BaP-induced nuclear factor-{kappa}B (NF-{kappa}B) activation, which regulates COX-2 expression. Third, although the aryl hydrocarbon receptor (AhR) did not play a role in BaP-induced COX-2 expression, the potentiation effect of E2 itself was AhR dependent. We further demonstrated that BaP induced the production of genotoxic E2 metabolites (2- and 4-hydroxyestradiols) via AhR-up-regulated cytochromes P450 1A1 and 1B1. These metabolites could directly activate NF-{kappa}B to further promote COX-2 mRNA expression in human lung epithelial cells. These findings were further supported by increased PGE2 secretion in rat lung slice cultures. Our findings that the BaP–E2 interaction enhanced COX-2 expression and hydroxyestradiol accumulation in the media of cultivated lung cells and tissues provide the needed scientific basis for higher risk of BaP-associated lung cancer in females.

Abbreviations: AhR, aryl hydrocarbon receptor; BaP, benzo[a]pyrene; COX-2, cyclooxygenase-2; CYP1A1, cytochrome P450 1A1; CYP1B1, cytochrome P450 1B1; DMF, 3',4'-dimethoxyflavone; DMSO, dimethyl sulfoxide; E2, 17-beta estradiol; ER, estrogen receptor; LHC, laboratory of human carcinogenesis MeOE2, methoxyestradiol; NF-{kappa}B, nuclear factor-{kappa}B; OHE2, hydroxyestradiol; PGE2, prostaglandin E2; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Prostaglandins, the primary metabolites of cyclooxygenase-2 (COX-2)-catalyzed oxygenation of arachidonic acid, are important immune modulators in inflammatory lung diseases. Recently, several lines of evidence have shown that chronic inflammation predisposes to cancer (1). Elevated COX-2 expression is common in a variety of tumors, including lung cancer (2–4), and COX-2 transcription is induced by cytokines and carcinogens (5–7). Increased prostaglandin E2 (PGE2) has also been reported in specimens from lung cancer patients (8). The increased COX-2 expression and PGE2 secretion seem to facilitate survival of tumor cells by inhibiting apoptosis and increasing cell proliferation, invasiveness and angiogenesis (9–11).

Exposure to polycyclic aromatic hydrocarbon-contaminated air pollutants has been associated with the occurrence of pulmonary diseases (12). Benzo[a]pyrene (BaP), found in cigarette smoke and by-products of combustion, is one of the most widely studied polycyclic aromatic hydrocarbons (13,14). Several animal studies have demonstrated that BaP induces lung tumors in a gender-dependent manner, with females more susceptible than males (15,16). However, the mechanism of the gender difference is still unclear. Other studies have demonstrated that BaP induces COX-2 expression and PGE2 secretion in a variety of cell types, including lung adenocarcinoma cells (17–19). Lu et al. (20) and Pavan et al. (21) reported that 17-beta estradiol (E2) increases PGE2 secretion in human U937-derived macrophages and amnion-derived WISH cells, respectively. Therefore, we hypothesized that the combination of BaP and E2 would enhance COX-2 expression and PGE2 secretion to promote cancer development.

The mechanisms for BaP-induced COX-2 expression or E2-induced PGE2 secretion have been investigated in different cell lines. Miller et al. (17) reported that BaP-induced COX-2 expression is aryl hydrocarbon receptor (AhR) dependent in breast cancer cells. Yan et al. (19) demonstrated that nuclear factor-{kappa}B (NF-{kappa}B) mediates BaP-induced COX-2 expression in vascular smooth muscle cells. In addition, increased PGE2 secretion in response to E2 has been suggested to be estrogen receptor (ER) dependent (20,21). The ER has two isoforms, alpha and beta; the alpha isoform is present in female reproductive tissues and is more responsive to E2 than the beta isoform (22). However, lung cells mainly express ER beta. The function of E2 on PGE2 secretion has yet to be clarified in lung cells or tissues.

It is well established that AhR mediates expression of cytochrome P450 1A1 (CYP1A1) and cytochrome P450 1B1 (CYP1B1), which catalyzes 2- and 4-hydroxylations of E2, respectively, to form 2-hydroxyestradiol (2OHE2) and 4OHE2 (23,24). Both 2OHE2 and 4OHE2 cause DNA damage, such as quinone DNA adducts and apurinic sites (25). Furthermore, 4OHE2 induces renal tumors in hamsters (26). Previously, we demonstrated that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) activates AhR, which subsequently up-regulates CYP1A1 and CYP1B1 expression and increases the accumulation of the E2 metabolites 2-methoxyestradiol (2MeOE2) and 4-methoxyestradiol (4MeOE2) (the methylated products of OHE2) in the human bronchial epithelial BEAS-2B cells (27). BaP is also an AhR agonist. Thus, it is likely that BaP can induce CYP1A1 and CYP1B1 expression to modulate E2 metabolism in BEAS-2B cells. To understand the role of E2 metabolites on the interaction between BaP and E2, we evaluated the effect of BaP on E2 metabolism and the effects of accumulated E2 metabolites on COX-2 expression in BEAS-2B cells. Our data highlight the importance of the BaP–E2 interaction on the inflammatory response in the lung.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Materials
E2, Liquid Chromatography grade ethanol, methanol, insulin, hydrocortisone-21-hemisuccinate, Krebs–Henseleit buffer, RPMI 1640 medium (phenol red free), BaP and dimethyl sulfoxide (DMSO) were purchased from Sigma Chemical Co. (St Louis, MO). 2OHE2, 4OHE2, 2MeOE2 and 4MeOE2 were from Steraloids (Newport, RI) and dissolved in methanol for storage. BaP was dissolved in DMSO, stored in aliquots and kept at –20°C until used. Internal standards 17-{alpha} ethynylestradiol and pentobarbital were obtained from TCI (Tokyo, Japan). Formic acid and ammonia formate (used as High Performance Liquid Chromatography mobile-phase modifiers) and ascorbic acid were obtained from Honeywell Riedel-de-Haën AG (Seelze, Germany). ICI182780 was purchased from Tocris Bioscience (Ellisville, MO). Bay117085 was purchased from BIOMOL International, L.P. (Plymouth Meeting, PA). 3',4'-Dimethoxyflavone (DMF) was purchased from INDOFINE Chemical Company (Hillsborough, NJ). PGE2 was purchased from Cayman Chemical Company (Ann Arbor, MI) and dissolved in ethanol for storage.

Cell culture
The human bronchial epithelial cell line BEAS-2B cells immortalized with SV40 (American Type Culture Collection, Manassas, VA) were maintained in serum-free Laboratory of Human Carcinogenesis-9 (LHC-9) medium (BioSource International, Nivelles, Belgium) in a 37°C incubator with a humidified mixture of 5% CO2 and 95% air. The medium was changed twice a week, and cells were passaged by trypsinization every week. Before treatment, cells were incubated with phenol red-free LHC-8 medium supplemented with 0.33 nM retinoic acid and 0.5 ng/ml epinephrine for 48 h. Phenol red-free medium was used because phenol red has been reported to have estrogen-like effects (28).

Cytotoxicity assay
Cytotoxicity of BaP and/or E2 was determined with dimethylthiazol-diphenyltetrazolium bromide assay. Cells (2 x 104 per well) were seeded in 96-well plates for 24 h and then incubated with vehicle (0.1% DMSO + 0.1% methanol), 10 nM E2 and/or 10 µM BaP for 24 or 72 h. Subsequently, 1 mg/ml dimethylthiazol-diphenyltetrazolium bromide was added to the medium and cells were incubated for an additional 4 h. Precipitated formazan was dissolved in 0.5 ml DMSO and the absorbance was measured at 535 nm. The data are presented as the percentage of controls. All vehicles, including 0.1% DMSO, 0.1% methanol and 0.1% ethanol (for PGE2 treatment), had no significant cytotoxicity.

Quantitative real-time reverse transcription–polymerase chain reaction assay
BEAS-2B cells (2 x 105 cells per well) were seeded into six-well dishes and then treated with 1 µM and 10 µM BaP or 10 nM E2 for 24 h and in some cases with inhibitors, including 25 nM ICI182780, 2.5 µM Bay117085 or 10 µM DMF. Treatment with neither 25 nM ICI182780 nor 2.5 µM Bay117085 had cytotoxicity (determined with dimethylthiazol-diphenyltetrazolium bromide assay). But treatment with 10 µM DMF reduced 14% of cell viability. BaP and E2 were, respectively, dissolved in DMSO and methanol. Therefore, the vehicle control for BaP and/or E2 treatment was 0.1% DMSO and/or 0.1% methanol. For OHE2 treatment, cells were treated with 10 nM 2OHE2, 10 nM 4OHE2 or the combination of equal amounts of 0.01, 0.1, 1 or 10 nM 2OHE2 and 4OHE2. Total RNA was prepared using TriReagent (Life Technologies, Rockville, MD) and the chloroform extraction method. Synthesis of cDNA was performed using Moloney Murine Leukemia virus (M-MIV) reverse transcriptase, deoxyribonucleotide triphosphate and RNase inhibitor (Promega, Madison, WI) with 1.5 µg total RNA mixed with 0.5 µg oligo-dT (Mission Biotech, Taipei, Republic of China). Quantitative polymerase chain reaction was carried out using the TaqMan® Universal polymerase chain reaction Master Mix (Perkin-Elmer Applied Biosystems, Foster City, CA) and analyzed on an ABI PRISM 7700 Sequence Detector System (Perkin-Elmer Applied Biosystems). Primers were chosen with the assistance of the computer program Primer Express (Perkin-Elmer Applied Biosystems). The primer sequences and their optimal primer concentrations of CYP1A1, CYP1B1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) have been described previously (29,30). The primers for COX-2 were 5'-GCTGGAACATGGAATTACCC-3' and 5'-ATCTGCCTGCTCTGGTCAAT-3'. The probe for COX-2 was 5'-ACCAGCAACCCTGCCAGCAA-3'. The polymerase chain reactions consisted of an initial step of 2 min at 50°C and a polymerase activation step for 15 min at 95°C, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Quantitative values were obtained from the threshold cycle (CT) number. Target gene expression level was normalized to GAPDH mRNA expression in each sample. The relative mRNA level of the target gene is Formula, Formula.

Enzyme-linked immunosorbent assay
BEAS-2B cells (5 x 104 cells per well) were seeded into 24-well dishes and then treated with 10 µM BaP and/or 10 nM E2 for 3 days, and in some cases with inhibitors (25 nM ICI182780, 2.5 µM Bay117085 or 10 µM DMF). The culture medium was replaced with 0.5 ml LHC-9 medium per well, and conditioned medium was collected 72 h later. PGE2 concentration was determined using the PGE2 enzyme-linked immunosorbent assay kit for humans and rats (Cayman Chemical Company) according to the manufacturer's instructions.

Quantification of E2 metabolites with HPLC/MS/MS
This method has been described in our previous work (27). E2 metabolites in the medium were extracted and purified with a solid-phase extraction column (Extrelut® NT 20, Merck, Darmstadt, Germany). E2 metabolites in the extracts were quantified with a triple-stage quadrupole mass spectrometer (API 3000, PE-SCIEX, Concord, Ontario, Canada) coupled to an HPLC system (PE series 200, Perkin-Elmer, Norfolk, CT). Detection limits are 0.5 pg/ml medium for 2OHE2 and 4OHE2 and 5 pg/ml medium for 2MeOE2 and 4MeOE2.

Animals
Six-week-old male Sprague-Dawley rats (150–250 g) were purchased from BioLASCO Biotechnology (Taiwan, Republic of China). The animals were housed in standard cages under a 12-h light–dark cycle and received food and water ad libitum. All procedures and experiments with animals in this study were approved by the Animal Care and Use Committee at the National Health Research Institutes, Taiwan, Republic of China.

Rat lung slice preparation and incubation
Rats were anesthetized with pentobarbital sodium (100 mg/kg) via intra-peritoneal injection. The lung was perfused with saline through the right pulmonary artery for 5 min to wash the blood away and prevent blood coagulation in small vessels. Then, the lungs were precisely excised and immediately inflated with 1.0% low-melting agarose dissolved in culture medium at 37°C. The culture medium was RPMI 1640 containing 1 µM insulin, 0.1 mM hydrocortisone-21-hemisuccinate, 5% fetal calf serum, 50 µg/ml streptomycin and 50 IU/ml penicillin. Subsequently, the rat lungs were placed in ice-cold Krebs–Henseleit buffer, pH 7.2, until completely gelled (31,32). Cylindrical tissue cores (8 mm diameter) were prepared from the lung tissues, which were cut to form 450-µm-thick lung slices using a Vitron tissue slicer (Vitron, Tucson, AZ). A total of two lung slices were floated onto the titanium mesh of a single Teflon roller insert (Vitron). Each insert was placed in a 20 ml glass scintillation culture vial containing 1.8 ml of culture medium. Culture vials were capped (each cap had a central 2 mm hole) and placed horizontally into the dynamic organ culture incubator at 37°C, 5% CO2 and 95% O2. After 2 h, the culture medium was changed and the lung slices were treated as indicated in the figure legends for 72 h. The culture medium was collected at the end of the 72 h incubation, and the secreted PGE2 concentration was determined using a PGE2 enzyme-linked immunosorbent assay kit for humans and rats.

Reporter gene assay
To assess ER activity on a canonical E2 response element, the pGL2-3ERE reporter containing three consensus estrogen-responsive elements upstream of a PRL TATA box and the firefly luciferase gene was obtained from Clontech (San Diego, CA). The pNF-{kappa}B-Luc (Stratagene, La Jolla, CA) vector contained the Photinus pyralis (firefly) luciferase reporter gene driven by a basic promoter element (TATA box) plus five repeats of the {kappa}B cis-enhancer element (TGGGGACTTTCCGC) (33). For the luciferase assay, human BEAS-2B cells (5 x 104) were seeded onto 12-well plates, cultured overnight and then incubated continuously with LHC-8 medium for 48 h. The cells were transfected with the plasmids using lipofectamine transfection reagent (Invitrogen, Grand Island, NY) for 6 h at 37°C. After 6 h of incubation, the medium was carefully removed and fresh LHC-8 medium containing various reagents was added into the wells. Cells were stimulated with DMSO, 10 µM BaP, 10 nM E2 or 10 µM BaP plus 10 nM E2 for 72 h. Cells were collected and transcriptional activity was assayed with the Luciferase Assay System (Promega) and a luminometer (Berthold Analytical Instruments, Nashua, NH). A plasmid expressing the bacterial ß-galactosidase gene was co-transfected in each experiment to serve as an internal control of transfection efficiency. The transcriptional activity of each reporter plasmid was normalized relative to ß-galactosidase activity, and the activity in cells treated with vehicle was set at 1.0 and defined as relative luciferase activity. Each experiment was repeated at least three times.

Statistical analysis
Treated and control groups were compared using the one-way analysis of variance followed by Tukey's Honestly Significant Difference test (P < 0.05 significance level). E2 metabolite levels among groups from LC/MS/MS analysis were compared using the Student's t-test (P < 0.05).


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Effect of interaction between BaP and E2 on COX-2 induction in BEAS-2B cells
Several animal studies have demonstrated that females are more sensitive to BaP than males with regard to immunotoxicity (34,35) and carcinogenicity (15,36,37). We therefore determined whether the addition of E2 affected BaP's ability to induce an inflammatory response in the human bronchial epithelial cell line BEAS-2B using COX-2 expression as a measure of inflammatory reaction. In BEAS-2B cells, COX-2 mRNA levels were increased by 10 µM BaP to 5.3-fold of control (P < 0.01) (Figure 1A). E2 (10 nM) alone failed to induce COX-2 expression (Figure 1A). However, co-treatment with 10 µM BaP and 10 nM E2 significantly enhanced BaP-induced COX-2 expression to 13.8-fold of control (P < 0.01) (Figure 1A). These data suggest that 10 nM E2 might potentiate the 10 µM BaP-induced inflammatory reaction in lung cells. Treatment with 10 µM BaP reduced cell viability to 51.9 or 64.3% of vehicle controls at 24 or 72 h (Table I). Co-treatment with 10 nM E2 slightly increased cell viability (Table I).


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  		Table I. Cytotoxicity of BaP and/or E2 in BEAS-2B cells

 


Figure 1
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  		Fig. 1. Effects of ER antagonist ICI182780 (ICI) on the interaction between BaP and E2 in BEAS-2B cells. Cells were incubated with the indicated treatments, including vehicle control (0.1% DMSO + 0.1% methanol), 10 nM E2, 10 µM BaP or 25 nM ICI182780 for 24 h (COX-2 mRNA assay) or 72 h (PGE2 assay). (A) COX-2 mRNA levels were quantified by real-time reverse transcription–polymerase chain reaction. (B) PGE2 was detected with an enzyme-linked immunosorbent assay (pg/ml). (C) ER activity was detected with a reporter assay. Cells were transfected with the pGL2-3ERE luciferase reporter. Transfected cells were incubated with the indicated treatments for 24 h before collection to measure luciferase activity. (D) Cells were treated with 0.1% ethanol, 20 pg/ml PGE2 or 40 pg/ml PGE2 for 24 h. COX-2 mRNA levels were quantified by real-time reverse transcription–polymerase chain reaction. Three samples were assayed in each experiment. Data are the mean ± SD for three independent experiments. *P < 0.05 as compared with vehicle-treated cells, {dagger}P < 0.05 as compared with 10 µM BaP-treated cells, {ddagger}P < 0.05 as compared with 10 nM E2-treated cells and §P < 0.05 as compared with 10 µM BaP + 10 nM E2-treated cells.

 
An ER antagonist prevents the potentiation of BaP-induced COX-2 expression by E2
It has been reported that BEAS-2B cells mainly express ER beta (38). Therefore, we investigated what role ER activation might play in the interaction between BaP and E2. Co-treatment with ICI182780, an ER antagonist, prevented the potentiation effect of E2 on BaP-induced COX-2 expression (P = 0.01) (Figure 1A). Furthermore, ICI182780 slightly inhibited E2-induced PGE2 secretion (P = 0.05) and strongly inhibited the interaction between BaP and E2 on PGE2 secretion (P < 0.01) (Figure 1B). It appears that the ER was not only involved in E2-induced PGE2 secretion but also in the potentiation effect of E2. Utilizing an ER reporter assay, we found, however, that BaP reduced E2-induced ER activity (P < 0.05) (Figure 1C). These data suggest that BaP did not directly enhance ER activation to potentiate E2-induced PGE2 secretion. On the other hand, treatment with 20 or 40 pg/ml PGE2 significantly increased COX-2 mRNA levels to 2.1- or 2.7-fold of controls (P < 0.05, P < 0.01) (Figure 1D), suggesting that E2-induced PGE2 might partially enhance COX-2 expression in the presence of BaP.

An I{kappa}B kinase alpha inhibitor prevents the potentiation of BaP-induced COX-2 expression by E2 in BEAS-2B cells
Several studies have demonstrated that BaP up-regulates COX-2 mRNA levels via the NF-{kappa}B signaling pathway (38,39). Therefore, we investigated the role of NF-{kappa}B activation in the potentiation effect of E2 on COX-2 expression. Co-treatment of BaP with Bay117085, an I{kappa}B kinase alpha inhibitor that prevents NF-{kappa}B activation, prevented BaP-induced COX-2 expression (P = 0.001) and the potentiation effect of E2 on BaP-induced COX-2 expression (P < 0.001) (Figure 2A). Whereas BaP alone significantly increased NF-kB reporter activity to 3.5-fold of control (P < 0.01), co-treatment with 10 nM E2 significantly enhanced BaP-induced NF-{kappa}B activity to 7.1-fold of control (P < 0.001) (Figure 2B). These data indicate that E2 potentiated BaP-induced COX-2 expression by enhancing NF-{kappa}B activation.


Figure 2
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  		Fig. 2. Effects of the I{kappa}B kinase alpha inhibitor (Bay117085) or the AhR antagonist (DMF) on the interaction between BaP and E2in BEAS-2B cells. Cells were incubated with the indicated treatments, including 10 µM BaP, 10 nM E2, 2.5 µM Bay117085 and/or 10 µM DMF for 24 h. The vehicle control for BaP and E2co-treatment was 0.1% DMSO + 0.1% methanol. The vehicle control for BaP and DMF co-treatment was 0.1% DMSO. (A) The COX-2mRNA level was quantified by real-time reverse transcription–polymerase chain reaction. (B) NF-{kappa}B activity was detected with a reporter assay. BEAS-2B cells were transfected with the pNF-{kappa}B-Luc reporter. Transfected cells were incubated with the indicated treatments for 24 h before collection to measure luciferase activity. (Cand D) BEAS-2B cells were incubated with the indicated treatments for 24 h. The mRNA levels for CYP1A1and CYP1B1(C) as well as COX-2(D) were quantified by real-time reverse transcription–polymerase chain reaction. *P<0.05 as compared with vehicle-treated cells, {dagger}P<0.05 as compared with 10 µM BaP-treated cells and §P<0.05 as compared with 10 µM BaP + 10 nM E2-treated cells.

 
An AhR antagonist prevents the potentiation of BaP-induced COX-2 expression by E2 in BEAS-2B cells
Utilizing the AhR antagonist, DMF, we next evaluated the role of AhR activation in the interaction between BaP and E2. DMF significantly prevented BaP-induced (10 µM) CYP1A1 and CYP1B1 expression (P = 0.001) (Figure 2C). Whereas DMF failed to prevent BaP-induced COX-2 expression, it prevented the potentiation effect of E2 on BaP-induced COX-2 expression (P < 0.001) (Figure 2D). These results indicate that BaP induced COX-2 expression in an AhR-independent manner but E2 potentiated BaP-induced COX-2 expression in an AhR-dependent manner.

Estrogen metabolites increase COX-2 expression in BEAS-2B cells
It has been shown that AhR ligands, such as BaP, induce both CYP1A1, which catalyzes 2-hydroxylation of E2, and CYP1B1, which catalyzes 4-hydroxylation of E2 (23). In the presence of 100 nM E2, treatment with 10 µM BaP significantly increased the accumulation of 2OHE2 or 4OHE2 in the BEAS-2B cell culture medium (Table II). However, the levels of methoxyestradiols (MeOH2) decreased following BaP treatment. Interestingly, 10 nM 2OHE2 and 4OHE2, but not the MeOH2, significantly increased COX-2 mRNA levels to 2.5-fold of control (P < 0.001, P < 0.05) (Figure 3A). Co-treatment with Bay117085 prevented COX-2 induction by 2OHE2 and 4OHE2 (P < 0.05) (Figure 3B). In addition, combined 2OHE2 and 4OHE2 treatment significantly increased COX-2 expression at the doses of 0.01–10 nM (Figure 3C). Furthermore, 2OHE2 and 4OHE2 individually increased NF-{kappa}B activity (Figure 3D). These results indicate that 2OHE2 and 4OHE2 activated NF-{kappa}B to up-regulate COX-2 expression. Thus, the elevation of OHE2 levels by BaP might contribute to the potentiation effect of E2 on COX-2 expression in lung cells.


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  		Table II. Effects of BaP on the accumulation of E2 metabolites in the media of BEAS-2B cells

 


Figure 3
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  		Fig. 3. Effects of estrogen metabolites on COX-2expression in BEAS-2B cells. (A) Cells were treated with 0.1% methanol, 10 nM OHE2or 10 nM MeOH2for 24 h. (B) Cells were treated with vehicle control (0.1% DMSO + 0.1% methanol), 10 nM 2OHE2or 10 nM 4OHE2with or without Bay117085 for 24 h. COX-2mRNA levels were quantified by real-time reverse transcription–polymerase chain reaction. (C) Cells were treated with 0.1% methanol or 0.1–10 nM combined 2OHE2and 4OHE2(equal concentrations of 2OHE2and 4OHE2) for 24 h. COX-2mRNA levels were quantified by real-time reverse transcription–polymerase chain reaction. (D) NF-{kappa}B activity was detected by a reporter assay. BEAS-2B cells were transfected with the pNF-{kappa}B-Luc reporter. Transfected cells were incubated with the indicated treatments for 24 h before collection to measure luciferase activity. Three samples were assayed in each experiment. Data are the mean ±SD for three independent experiments. *P<0.05 as compared with vehicle-treated cells and §P<0.05 as compared with 10 nM 2/4OHE2-treated cells.

 
Interaction between BaP and E2 on PGE2 secretion in rat lung slice cultures
Tissue slice cultures are an in vitro system that retains the biochemical capacity and the metabolic function of the whole organ (40,41). Recently, we have successfully induced CYP1A1 and CYP1B1 protein expression with TCDD and BaP in rat lung slice cultures (29). In our present study, we confirmed the interaction between BaP and E2 in rat lung slice cultures. It has been well established that COX-2 promotes PGE2 biosynthesis (42,43). Therefore, we confirmed that BaP and E2 together would affect PGE2 secretion in rat lung slice cultures. E2, but neither 1 nor 10 µM BaP, increased PGE2 secretion (Figure 4A). Co-treatment with 10 µM BaP, but not 1 µM BaP, enhanced 10 nM E2-induced PGE2 secretion (P < 0.05) (Figure 4A), which was prevented by ICI182780, Bay117085 or DMF (P < 0.001) (Figure 4B, C and D).


Figure 4
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  		Fig. 4. Interaction between BaP and E2on PGE2 secretion in rat lung slice cultures. (A–D) Rat lung slices were cultivated in medium containing the vehicle control (0.1% DMSO + 0.1% methanol), 1 µM BaP, 10 µM BaP, 10 nM E2, 25 nM ICI, 2.5 µM Bay117085 and/or 10 µM DMF for 72 h. The amount of PGE2 released into the culture medium was detected with an enzyme-linked immunosorbent assay. A total of 8–10 slices were examined for each condition. Each experiment was repeated at least three times. *P<0.05 as compared with vehicle-treated cells, {ddagger}P<0.05 as compared with 10 nM E2-treated cells and §P<0.05 as compared with 10 µM BaP + 10 nM E2-treated cells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
Several studies have suggested that COX-2 and other proteins in the same pathway serve as modulators of tumor growth (9,11,44). In addition, increased COX-2 expression has been observed in non-small cell lung cancers (3,4). It has been proposed that COX-2 increases angiogenesis and mediates resistance to apoptosis (44). Our present study demonstrates the potentiation effect of E2 on BaP-induced COX-2 expression in human bronchial epithelial cells. COX-2 regulates biosynthesis of prostaglandins, including PGE2. In BEAS-2B cells, PGE2 further induces COX-2 expression. A similar effect of PGE2 has also been reported in intestinal adenomas (45). Our work suggests that COX-2 and PGE2 are mutually and positively regulated in BEAS-2B cells. We thus predict that COX-2 and PGE2 stimulate each other to magnify subsequent inflammatory reactions in the lung. This interaction was confirmed by increased PGE2 secretion in rat lung slice cultures following treatment with E2 and BaP. These data suggest that the co-existence of E2 and BaP enhances inflammatory reactions—even carcinogenesis—in the lung. In the future, we plan to conduct animal studies to test whether the interaction between E2 and BaP occurs in vivo and whether it can be prevented by AhR, ER or NF-{kappa}B inhibitors.

In human bronchial epithelial cell cultures, BaP increased COX-2 expression via the NF-{kappa}B-dependent pathway. Our results suggest that E2 might potentiate BaP-induced COX-2 expression via three mechanisms (Figure 5). First, BaP exposure promotes the conversion of E2 into OHE2, which then activate NF-{kappa}B and induce COX-2 expression. Second, E2 enhances NF-{kappa}B activation by BaP. Third, E2 also increases PGE2 secretion to potentiate COX-2 induction. A combination of these pathways could significantly enhance BaP-induced COX-2 expression. However, other mechanisms remain to be explored, such as an interaction between ER and NF-{kappa}B.


Figure 5
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  		Fig. 5. A model for the potentiation effects of E2on BaP-induced COX-2mRNA accumulation.

 
Metabolic activation of estrogen to genotoxic metabolites such as OHE2 is one of the major mechanisms for estrogen-related carcinogenesis (46). In particular, 4OHE2 can be converted further into E2-3,4-quinone, which attacks DNA and leads to mutations (47,48). In addition to genotoxicity, some studies have demonstrated that OHE2, especially 4OHE2, modulate cell proliferation, apoptosis and even invasiveness (49–51). Our present study is the first to demonstrate that OHE2 induce COX-2 expression, suggesting that OHE2 might promote lung carcinogenesis via COX-2-related pathways.

Our present study demonstrated that in the presence of E2, BaP elevated the production of E2 metabolite, OHE2 (Table I). Since E2 only enhanced COX-2 expression in the presence of BaP (Figure 1A), OHE2 was believed to be the factor responsible in COX-2 induction. Indeed, addition of OHE2 to the cells showed a dose-related increase in COX-2 expression (Figure 3A). Despite our clear demonstrations on the inter-relationship between BaP–E2 interactions, OHE2 production and COX-2 elevation, our present observations were limited by two factors: first, only the OHE2 levels in the culture media were measured and the intracellular level of OHE2 remained unknown; second, we only used a relatively higher level of E2 (100 nM), in the presence of BaP, for the demonstration on the production of OHE2 but we were successful in demonstrating that a much lower level of E2 (10 nM), in the presence of BaP, was sufficient to induce an elevation of COX-2. The actual level of OHE2 produced by this low level of E2 (10 nM) needs to be addressed in future studies.

Although BaP and TCDD both are AhR agonists and increased CYP1A1 and CYP1B1 expression, they had different effects on the accumulation of E2 metabolites in BEAS-2B cells. Previously, we demonstrated that TCDD increases the accumulation of 2MeOE2 and 4MeOE2 without significantly altering OHE2 levels (27). It appears that TCDD induced OHE2 that were quickly converted to MeOH2. Methylation of OHE2 by catechol-O-methyl transferase is considered a detoxification reaction (52).

We report here the novel finding that, in addition to its own carcinogenic ability, BaP, in the presence of E2, enhanced the accumulation of another carcinogen, 4OHE2, as well as a genotoxic agent, 2OHE2. BaP-induced OHE2 failed to be further methylated, although the reason for that is still unknown. We have measured catechol-O-methyl transferase activity in purified cytosol, and it was not significantly reduced by BaP treatment (data not shown). But we did not rule out the possibility that BaP might reduce the level of S-adenosylmethionine, the methyl donor for OHE2, to decrease intracellular catechol-O-methyl transferase activity. Furthermore, OHE2, but not MeOH2, induced COX-2 expression in BEAS-2B cells. Therefore, the presence of both BaP and E2 leads to a higher carcinogenic risk than BaP alone. These results supply a rationale for the increased prevalence of BaP-induced lung tumors in females.


   Acknowledgments
 
This work was supported by a research grant, DOH95-TD-G-111-020, from the National Research Program for Genomic Medicine and Department of Health, and EO-095-PP-14 from the Division of Environmental Health and Occupational Medicine, National Health Research Institutes, Taiwan, Republic of China. The scientific content of the manuscript does not necessarily signify the views and policies of the Department of Health or the Division of Environmental Health and Occupational Medicine/National Health Research Institutes or condemn, endorse or recommend for use on the issue presented.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received September 4, 2006; revised December 18, 2006; accepted January 17, 2007.


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C.-C. Ho, Y.-C. Ling, L. W. Chang, H.-T. Tsai, M.-H. Tsai, and P. Lin
17-Beta Estradiol and Hydroxyestradiols Interact via the NF-Kappa B Pathway to Elevate Cyclooxygenase 2 Expression and Prostaglandin E2 Secretion in Human Bronchial Epithelial Cells
Toxicol. Sci., August 1, 2008; 104(2): 294 - 302.
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