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Background: The arylhydrocarbon receptor (AhR) mediates the adverse effects of environmental chemicals. Benzyl butyl phthalate (BBP) with the most estrogenic potency among Phthalate may have effect on human granulosa cells (hGC), but whether BBP can activate AhR in hGC is unaddressed.
Objective: We investigated the signaling pathway of AhR on human granulosa cell mediated by BBP. In present study, we examined the effect of BBP on Cytochrome P450 (CYP) 1A1, 1B1 in human granulosa-lutein cells.
Materials: Human GL cells cell line HO23 and Human GL cells collected from women undergoing in vitro fertilization were cultured in medium containing BBP 10-6 M. AhR expression were assayed after incubation for 24h,. AhR antagonist and SiRNA were investigated to confirm the effect of AhR. We studied the downstream of AhR, CYP1A1, CYP1B1 and reporter assay were determined. The AhR translocation from cytoplasm into nucleus were imaged by the Live Cell imaging. ChIP measured the enhancement of CYP1B1 instead of CYP1A1 in the nuclear portion. Reporter assay revealed the same stimulation effect of BBP in CYP1B1.
The diesters of 1,2-benzenedicarboxylic acid (phthalates), are man-made chemicals with wide spectrum of industrial applications such as plasticizers in flexible polyvinyl chloride products, as industrial solvents, and as chemical stabilizer in cosmetics, food packing, and medical care instruments. The widespread use of phthalates contributes to distribute and exist in the environment omnipresently. There are several potential contamination routes to humen include oral, dermal, inhalation. Because phthalates are also widely used in medicial care and consumer products, the odds is high for non-occupational human exposure. Species at the top of the food chain will be at risk because of bioaccumulation and biomagniï¬cation of such substances. Benzyl butyl phthalate (BBP) have been detected in the humen samples in the U.S. at median values 12.7 μg/l in adult human urine samples.1 It is suspected that inï¬‚uence on sex steroid-controlled in the reproductive organs is of great importance.
Previous reports have suggested phthalates resulting in an impairment of steroidogenesis of ovarian granulosa cells from rats.2, 3, 4 However, limited data reported the effect of phthalates on steroid production in human granulosa cells.5 Among the phthalates, mono-(2-ethylhexyl) phthalate (MEHP) and di-(2-ethylhexyl) phthalate (DEHP) are most studied in the granulosa steroidogenesis. Otherwise, BBP has been reported to influence the breast cancer cell line angiogenesis.6 In the recombinant yeast screen assay, BBP was the most active estrogenic phthalate among those tested.7 Previous report also showed that BBP could enter the cytoplasm, bioaccumulate, and exert mitogenic effects,8 and have the highest toxicity of embryonic development.9
Phthalates binds to the aryl hydrocarbon receptor (AhR) and induces down stream effector, Cytochrome P450 (CYP)1A1, CYP1B1, and the biological consequence in the hGC were investigated in the present study.10 The down stream of AhR ligand binding CYP1B1 high expression in the hen ovary is associated with ovarian cancer, primarily localized to the granulosa layer.11 The AhR is an universal ligand-activated member of the basic helix-loop-helix/Per-ARNT-Sim family of transcription factors.12 Increasing evidences show that the recognized role of the AhR mediate the toxic effects of various environmental disrupter, such as polycyclic aromatic hydrocarbons (PAHs).13, 14, 15, 16
In addition to its role in response to toxic exposure, AhR have effects in the female reproductive system.17 AhR knock out (AhRKO) mice have reduced fertility, increased abortion rate, and small litters.18 The reduced fertility in AhRKO mice was suspected partly due to ovarian defects. AhRKO follicles were reported to decreased granulosa cell proliferation and reduced expression of cell cycle regulators.19 Recent study demonstrated that phthalate reduced estrogen production by inhibion of aromatase activity on the transcript level in hGC.5 Collectively, these data suggest that AhR can mediate biological effect in the ovarian granulosa cells.
The aim of the this study was to evaluate the effect of BBP on AhR, and the cascade effect of AhR, such as cell biology and CYP activation were determined.
Materials and Methods
Cell lines, plasmid and chemicals.
The immortalized human ovarian granulosa cell line HO23 were obtained from Dr. Hsien-An Pan (Department of Obstetrics and Gynecology, National Cheng Kung University) and maintained in DMEM/F12 with 10% fetal bovine serum (Sigma) and 1% penicillin/streptomycin (Invitrogen). Cells were incubated in a humidified atmosphere with 5% CO2 at 37â„ƒ. HO23 is established by transfection with primary cells obtained from in vitro fertilization patients with SV40 genome, Ha-ras oncogene, and the p53 gene.20 Primary hGC are obtained from those patents receiving IVF procedure at the Department of Obstetrics and Gynecology, Kaohsiung Medical University Hospital. The recruiting criteria includes those with tubal disease, male infertility or idiopathic infertility. Long ovulation protocol were given following our previous protocol. The granulosa cells were cultured in DMEM/F12, supplemented with 5% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM L-glutamine. A pGL2-basic expression vector (Invitrogen), was used as a null expression vector for lentiviral infection. pCYP1A1 was constructed by inserting CYP1A1 promoter into pGL2-basic expression vector. pCYP1B1 was constructed by inserting CYP1B1 promoter into pGL2-basic expression vector. pEGFP-C1-AhR, a kind gift of Dr. Hsin-yu Lee (Department of Life Science, National Taiwan University), was cloned the AhR gene into pGFP-C1 (CLONTECH). BBP and 3',4'-dimethoxyflavone (3',4'-DMF) were purchased from Sigma.
Quantitative reverse transcription-PCR.
The mRNA levels of AhR, ARNT (aryl hydrocarbon nuclear translocator), CYP1A1, CYP1B1 and β-actin were analyzed by quantitative PCR. Total RNA of cells was extracted using the TRIZOL reagent (Invitrogen), followed by synthesis of the first-strand complementary cDNA using reverse transcriptase-polymerase chain reaction (RT-PCR) kit and primer oligo-dT25 (Promega). The first-strand cDNA was amplificated by PCR with specific primers for the AhR (forward, 5′-TAC TCT GCC GCC CAA ACT GG-3′ and reverse 5′-GCT CTG CAA CCT CCG ATT CC-3′), ARNT (forward, 5′-GCC AAA CCA TTC AGA CTG TGG-3′ and reverse 5′-GGC TCG AAA ACC AGA CAA GCT-3′), CYP1A1 (forward, 5′- ACG GTT TCT CAC CCC TGA TG-3′ and reverse 5′-GTA GAC AGA GTC TAG GCC TCA-3′), CYP1B1 (forward, 5′-AGT TCT CCG GGT TAG GCC AC-3′ and reverse 5′-ACT CGA GTG CAG GCA GAA TTG-3′) and β-actin (forward, 5′-CTC GCT GTC CAC CTT CCA-3′ and reverse 5′-GCT GTC ACC TTC ACC GTT C-3′). Amplified products were loaded on to 1% agarose gels and stained by ethidium bromide.
Two µg of the pCYP1A1 or pCYP1B1 were transfected to HO23 cell in plain medium using TransIT®-LT1 Transfection Reagent (Mirus) as to manufacturer's recommendations. In each transfection mixture 6 ng of pGL4 vector (Promega) was included as an internal control for transfection efficiency. Luciferase activity was tested by using Dual-Luciferase® Reporter 1000 Assay System (Promega).
Small interfering RNA (siRNA) targeting AhR (siRNA1-936507, siRNA1-936508), and CYP1B1 (siRNA1-944901, siRNA2-944902) was designed and prepared by Sigma. Transfection was carried out using TransIT®-LT1 Transfection Reagent. HO23 were transfected with AhR or CYP1B1 siRNA with a final concentration of 5 nmol/L.
Chromatin immunoprecipitation assay.
Chromatin immunoprecipitation (ChIP) assays were performed using a ChIP assay kit (Upstate Biotechnology). HO23 Anti-AhR (Abcam) or anti-IgG (Santa Cruz) were used to immunoprecipitate DNA-containing complexes. PCR was done with primers complementary to the CYP1A1 (forward, 5'-TCC ATT CCG ATC CTT CAA TC-3'; and reverse, 5'-GGT TCC AAG GAA CTG TCA CC-3') and CYP1B1 (forward, 5'-CTC CAC TGA GGT GGC AAT TT-3', and reverse, 5'-CCT CAC AGC GTT GAG ATT GA-3') promoter region. Amplified products were loaded on to 2% agarose gels and stained by ethidium bromide.
Live Cell imaging.
Total internal reflection fluorescence microscopy was used to determine relative changes in cell size by means of a real-time cell imaging system. HO23 cells were transfected with pGFP-C1-AhR by LT1 (Mirus) transfection reagent. Cells were analysed contractile with real-time cell imaging microscopy was performed with or without BBP treatment. Cells were grown on coverslips and incubated overnight, followed by serum-free medium for 24 h. Data was recorded and analyzed using the Olympus Cell-R imaging software.
Immunoprecipitation, western blotting and flow cytometry assays.
For phthalates stimulation assays, cells were serum-free for 24 h, followed by treatment with BBP (10-6 M) for 24 h. To detect the physical interaction of AhR with ARNT in HO23 cells, nuclear and cytoplasm extracts were first immunoprecipitated with a goat polyclonal anti-AhR (Abcam) or anti-ARNT (Upstate). The precipitates were sequentially subjected to Western blotting using anti-AhR or anti-ARNT. For flow cytometry staining studies to detect intercellular protein expression in HO23 cells, cells were washed twice in ice-cold PBS and harvested with Trypsin-EDTA (Invitrogen). Cell pellets were eluted by PBS and added ice-cold 75% ethanol after centrifugation, followed by incubation for 24 h at -20°C to fix the cells. The following antibodies were used for intracellular staining: anti-AhR or anti-ARNT, anti-CYP1A1 (Santa cruz) and anti-CYP1B1 (Abcam), in accord with the manufacture's recommendations. Cells were stained for 1 h at room temperature with FITC or rhodamine-conjugated antibodies (Santa Cruz) before running the samples on an EPICS XL-MCL flow cytometer (Beckman). To determine apoptotic vs cell necrosis, the cells were identified using flow cytometric analysis of cells stained with PE-labeled annexin V to identify apoptotic cells and 7-aminoactinomycin D (7-AAD) to label permeable (dead) cells (BD Pharmingen), and staining was done according to the manufacturer's instructions.
The results are presented as means ± S.D. Statistical comparisons were performed using unpaired two-tailed t-tests where appropriate, with a probability value < 0.05 taken to indicate significance.
Effect of BBP on the expression of the AhR, ARNT, CYP1A1 and CYP1B1 genes in the HO23 cell lines and primary hGC
We first studied AhR, ARNT, CYP1A1 and CYP1B1 expression by RT-PCR in various subsets of the human ovarian granulosa cell line HO23 and primary granulosa cells treated with 10-6 M BBP for 24 hrs. This concentration of BBP was only to effects observed but not being toxic.10, 21 Treatment of HO23 and primary granulosa cells with BBP significantly increased mRNA levels both for AhR (2 fold), ARNT (1.4 fold) and CYP1B1 (1.4 fold)(Fig. 1a). AhR antagonist 3',4'-DMF and AhR siRNA knockdown significantly reduced AhR mRNA levels in BBP exposure group, compared to control siRNA. In addition, AhR siRNA not only suppressed the basal level of CYP1B1 mRNA but also prevented the BBP induction of CYP1B1. While 3',4'-DMF and AhR siRNA did not significantly effect the basal level of CYP1A1 mRNA or BBP-induced increase in CYP1A1 mRNA (Fig. 1b-1e). These data suggested that CYP1B1, but not CYP1A1, gene expression increased in response to BBP in a AhR-dependent fashion.
The increase also observed in protein levels by flow cytometry assays. The AhR (+37%), CYP1A1 (+28%) and CYP1B1 (+65%) protein all increased after BBP treatment in HO23 (Fig. 1f). Cotreated 3',4'-DMF or AhR siRNA with BBP, AhR and CYP1B1 protein would be suppressed (Fig. 1F). These data showed that the BBP-induced CYP1B1 increasing was AhR-depentent, but CYP1A1 was not AhR-depentent.
Exposure to BBP results in nuclear translocation of AhR protein
To determine if the degradation is preceded by translocation of AhR to the nucleus upon BBP treatment, we visualized the cellular localization of AhR in HO23 cells using a plasmid expressing a GFP-tagged AhR protein. In non-stimulated cells, GFP-tagged AhR was diffusely distributed in the cytoplasm in great majority of transfected cells, while BBP treatment resulted in the nuclear translocation of AhR-GFP as shown by green colored GFP. This demonstrates the fusion protein responses to the exogenous ligand treatment. BBP treatment caused the AhR-GFP fusion protein to translocate to nucleus (Fig. 2a).
Next we evaluated if the BBP nuclear translocated AhR heterodimerizes with ARNT. As expected, BBP treatment resulted in AhR heterodimerization with ARNT, as observed by immunoprecipitating AhR and immunoblotting with ARNT antibody (Fig. 2b). To asses nuclear translocated AhR after exposure to BBP if bind to the CYP1A1 or CYP1B1 transcription regulatory sites, cells were treated with BBP were subjected to ChIP assay. BBP treatment led to binding of AhR at the CYP1B1 transcription regulatory site, but not at CYP1A1 (Fig. 2c).
To understand how AhR regulates BBP-induced CYP1A1 and CYP1B1 regulation, we performed transient transfection of HO23 cells with plasmid constructed with the promoter region of CYP1A1 or CYP1B1 upstream to the luciferase reporter gene. Treatment with BBP resulted a time-dependent increase in CYP1B1 promoter-mediated GFP expression that was signiï¬cantly different to CYP1A1 promoter or control (Fig. 3). Cotreatment of 3',4'-DMF with BBP reduced the GFP expression compared to BBP treatment alone, suggesting that 3',4'-DMF exhibits inhibitory action to CYP1B1 mRNA expression via down-regulation the AhR binding to the CYP1B1 promoter. These results also suggested that BBP induced only CYP1B1 but not CYP1A1 via AhR.
Examination of BBP induced necrosis and apoptosis of the the HO23 cell lines
We ï¬rst determined if apoptotic events were induced in HO23 cells after exposure to BBP. The result of ï¬‚ow cytometry analysis revealed that the proportion of necrosis cells was signiï¬cantly increased at BBP-treated groups, but the proportion of apoptosis cells was not changed obviously (Fig. 4). In order to conï¬rm the role of AhR and CYP1B1 in effects of BBP on HO23 cells necrosis, we also used 3',4'-DMF or AhR siRNA to block AhR. The proportion of necrosis cells was signiï¬cantly reduced, this result suggested that BBP-induced HO23 cells necrosis due to AhR. To further understand if the CYP1B1 involved in BBP-induced HO23 cells necrosis, CYP1B1 siRNA knockdown significantly reduced the proportion of necrosis cells to BBP exposure group. These data suggested that BBP-induced increase necrosis in HO23 is AhR-dependent and via CYP1B1 induction.
Environmental pollutants that can conflict with endocrine functions become growing concerns. These endocrine-disrupting compounds may imitate, obstruct, or interfere with the synthesis and/or degradation of natural hormones. Some previous studies showed the endocrine disruption effect of phthalate on the ovarian granulosa cells.4, 22, 23, 24 DEHP was reported to inhibit oocyte maturation in the female Japanese medaka fish, and the anti-estrogenic activity was proposed as the cause.25 The same effect of impairment in oocyte maturation was investigated in the female rats by MEHP.26 Suppresed estradiol levels, ovulation and decreased granulosa cells numbers were also found in DEHP treated female rats.2,27 Two week repeated administration of DEHP was studied to be able to damage the female rat ovary.28
Due to the availabity of the specimen, most reports are conducted in the rodent experiments, only one report is originated from the human being.5 Though the animal and human material confirm the impairment of steroidogenesis pathway by the phthalate. Howevere, the mechanism remain unclear. Our present research indicates the novel finding that AhR activation mediates BBP effect on human granulose cells. It is interested that recent report incubation with organic extracts (main compounds are determined to be PAH and PAE) from Yangtze River induce expression of AhR-ARNT and CYP1A1.29,30 The above data indicated a possible mechanism underlying toxicity of the AhR signaling pathway. Many investigation demonstrated the expression of CYP1 genes can be induced by AhR in response to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure.31 Most genes responsed to AhR-mediated TCDD elicited expression in human liver cell were reported, include CYP1A1, CYP1B1, ALDH1A3 and SLC7A5 genes.32 MEHP was reported it activated AhR and CYP1B1 in rat ovarian granulosa cells.17 Our present study in the human granulosa cells show the same phenomenon. We investigate further more with siRNA, antagonist, and cell® image, reporter assay to confirm the pathway medicated by BBP activated AhR. In the ovarian granulosa cells, PAH can activate AhR and enhance the crosstalk between AhR and glucocorticoid receptor. The toxic effects by PAH in the ovaries are mediated dominantly by the AhR.33
CYP1B1 hydroxylates the estrogens to 4-hydroxy estrogens. The metabolic conversion has been postulated to act a part in carcinogenesis.34, 35, 36, 37, 38 CYP1B1 gene is inducible by PAHs and is regulated by several key transcriptional factors, including AhR and estrogen receptor.39 This effect also found in the ovary consequently to affect the oocyte quality, corpus luteum function, and ovarian carcinogenesis.40 Although CYP1B1 is also expressed in normal tissues, it is transcriptionally activated in potential carcinogens and found in many tumors at higher levels, especially in hormone-mediated cancers11, 41. Our data elicit the involvement of CYP1B1 in the BBP-induced cell necrosis of human granulosa cells. It suggested that CYP1B1 upregulation in human granulosa cells may cause the cell necrosis. We should take this result seriously for the sake of the potential health hazard effect of CYP1B1.
In conclusion, this study demonstrates that BBP exposure results in an increase in necrosis in human granulosa cells that is AhR-dependent and downstream of CYP1B1 induction. To our knowledge, no others have shown that CYP1B1 contributes to cell necrosis. Our results suggested that this might occur in human granulosa cells. Finally, this study provides insight into the BBP on the human ovaries. Future studies is needed to clarify the underlying mechanism and the role of CYP1B1-dependent granulosa cells necrosis in order to fully assess the risk of phthalate exposure to human reproductive health.