) was purchased from Sigma-Aldrich (St. Louis, MO), and ICI 182,780 (ICI) was obtained from Tocris Bioscience (Ellisville, MO). The 12 EDCs used in this study were provided by the Midwest Research Institute (Kansas City, MO) via a contract with the National Toxicology Program. The chemical names, Chemical Abstracts Services Registry Numbers, and the sources are summarized in Supplemental Material, Table S1 (http://dx.doi.org/10.1289/ehp.1205951
EDC groups. The 12 EDCs were categorized into three groups based on their chemical and product classes (, ). Group 1 consists of BPA, BPAF, HPTE, and 4n-NP because of their shared bisphenol or phenol group. Dai, Gen, Kaem, Api, and Coum, all from natural products, comprise group 2; they each contain flavonoid, isoflavone, or phenol. Group 3 includes Endo, Kep, and 1-BP because they each contain organochlorine or organobromine in their chemical structures. Group 3 EDCs have traditionally been used as pesticides or chemical intermediates.
The chemical structures of EDCs tested in this study.
pcDNA vector plasmid was purchased from Promega (Madison, WI), pRL-TK vector plasmid from Invitrogen (Carlsbad, CA), and 7×AP-1 Luc from Stratagene (La Jolla, CA). pcDNA/mouse wild-type (WT)-ERα (pcDNA/ERα) and pcDNA/ΔNmERβ310G (former pcDNA/mouse WT-ERβ) have been described previously (Mueller et al. 2003
). The full-length mouse ERβ expression plasmid, pcDNA/WT-ERβ, was generated as described in Supplemental Material, p. 3 (http://dx.doi.org/10.1289/ehp.1205951
). The luciferase reporters 3×ERE (modified reporter) and pS2ERE (endogenous pS2 gene reporter) have been described previously (Hall et al. 2002
). The following reporters were gifts: pRSV/c-Jun (M. Karin, University of California, San Diego, La Jolla, CA), -73Col AP-1 Luc (D.P. McDonnell, Duke University, Durham, NC) and p21Sp1 Luc (J.L. Jameson, University of Pennsylvania, Philadelphia, PA).
Cell lines and tissue culture.
The HepG2 human hepatocellular cancer cell line and the HeLa cervical epithelial cancer cell line (both ER negative) were purchased from ATCC (Manassas, VA). The human endometrial adenocarcinoma stable cell lines Ishikawa/vector (Ishikawa/vec) and Ishikawa/WT ERα (Ishikawa/ERα) have been described previously (Burns et al. 2011
; Li et al. 2012
). HepG2 cells were maintained in phenol red–free minimum essential medium (MEM; Invitrogen) supplemented with 10% fetal bovine serum (FBS; Gemini Bio Products, West Sacramento, CA) and 4 mM l
-glutamine (Invitrogen). HeLa cells were maintained in phenol red–free Dulbecco’s modified Eagle medium (DMEM; Invitrogen) supplemented with 10% FBS and 4 mM l
-glutamine. The stable cell lines Ishikawa/vec and Ishikawa/ERα were maintained in phenol red–free DMEM:F12 medium (Invitrogen) supplemented with 10% FBS and geneticin (G418; 1 mg/mL; Invitrogen). For serum-starved conditions, 10% HyClone charcoal/dextran-stripped FBS (Thermo Scientific, Waltham, MA) was substituted for FBS in the medium (starve medium).
Transient transfection and luciferase assay. HepG2 and HeLa cells were seeded in 24-well plates with starve medium overnight. A total of 0.5 μg of DNA, including 0.2 μg of expression plasmid, 0.2 μg of reporter plasmid, and 0.1 μg of pRL-TK plasmid, were transfected overnight using Effectene transfection reagent (QIAGEN, Valencia, CA) according to the manufacturer’s protocol. E2, ICI, and EDCs were dissolved in 100% ethanol (EtOH) before being diluted in media. The final EtOH concentration was 0.01%. The cells were changed to fresh starve medium; after 8 hr, cells were treated with EtOH vehicle (control), 10 nM E2, 100 nM ICI, or EDCs for 18 hr. For experiments with pRSV/c-Jun on 7×AP-1 Luc, cells were transfected with a total of 0.7 μg of DNA, including 0.2 μg ERα or ERβ, 0.2 μg pRSV/c-Jun, 0.2 μg 7×AP-1 Luc, and 0.1 μg pRL-TK plasmids. Luciferase assays were performed using the Dual Luciferase Reporter Activity System (Promega, Madison, WI). Transfection efficiency was normalized by renilla luciferase using pRL-TK plasmid. All experiments were repeated at least three times. Data represent mean fold change (± SE; n = 3) relative to the control.
RNA extraction and real-time polymerase chain reaction (PCR).
Ishikawa/vec and Ishikawa/ERα cells were cultured in starve medium for 2 days and then treated with 10 nM E2
, 100 nM EDCs, or EtOH vehicle (control) for 18 hr. Total RNA was extracted using the RNeasy Mini Kit (QIAGEN). First-strand cDNA synthesis was performed using Superscript reverse transcriptase (Invitrogen) according to the manufacturer’s protocol. The mRNA levels of ER target genes were measured using SYBR green assays (Applied Biosystems, Carlsbad, CA). The Genbank accession numbers (http://www.ncbi.nlm.nih.gov/genbank/
) and sequences of primers used for real-time PCR were as follows: human PR
(NM_000926.4): forward 5´-GACGTGGAGGGCGCATAT-3´, reverse 5´-GCAGTCCGCTGTCCTTTTCT-3´; human pS2/TFF1
(NM_003225.2): forward 5´-GCCCTCCCAGTCTGCAAATA-3´, reverse 5´-CTGGAGGGACGTCGATGGTA-3´; human GREB1
(NM_014668): forward 5´-CAAAGAATAACCTGTTGGCCC-3´, reverse 5´-GACATGCCTGCGCTCTCATAC-3´; human SPUVE
(NM_007173): forward 5´-ATGCCCGAGCAGATGAAATT-3´, reverse 5´-CCAACCCTTGGGCACATG-3´; human WISP2
(NM_003881): forward 5´-TGAGCGGCACACCGAAGAC-3´, reverse 5´ACAGCCATCCAGCACCAG-3´; human SDF-1
(NM_000609): forward 5´-GTGGTCGTGCTGGTCCTC-3´, reverse 5´-GATGCTTGACGTTGGCTCTG-3´. Cycle threshold (Ct) values were obtained using the ABI PRISM 7900 Sequence Detection System and analysis software (Applied Biosystems, Foster City, CA). Each sample was normalized to its β-actin transcript content: forward 5´-GACAGGATGCAGAAGGAGATCAC-3´, reverse 5´-GCTTCATACTCCAGCAGG-3´. The experiments were repeated three times, and results are presented as the mean fold change (± SE; n
= 3) relative to control (vehicle-treated) Ishikawa/vec cells.
Statistical analysis. One-way analysis of variance (ANOVA) with Dunnett’s multiple comparison test and two-way ANOVA with Bonferroni posttests were performed using GraphPad Prism, version 6.00 (GraphPad Software Inc., La Jolla, CA).