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. 2016 Jan;35(1):91-100.
doi: 10.1002/etc.3146. Epub 2015 Dec 9.

Development of a recombinant human ovarian (BG1) cell line containing estrogen receptor α and β for improved detection of estrogenic/antiestrogenic chemicals

Jennifer C Brennan  1 , Arzoo Bassal  1 , Guochun He  1 , Michael S Denison  1
Affiliations

Development of a recombinant human ovarian (BG1) cell line containing estrogen receptor α and β for improved detection of estrogenic/antiestrogenic chemicals

Jennifer C Brennan et al. Environ Toxicol Chem. 2016 Jan.

Erratum in

  • Corrigendum.
    [No authors listed] [No authors listed] Environ Toxicol Chem. 2017 May;36(5):1405. doi: 10.1002/etc.3528. Epub 2016 Jul 7. Environ Toxicol Chem. 2017. PMID: 28423203 No abstract available.

Abstract

Estrogenic endocrine-disrupting chemicals are found in environmental and biological samples, commercial and consumer products, food, and numerous other sources. Given their ubiquitous nature and potential for adverse effects, a critical need exists for rapidly detecting these chemicals. The authors developed an estrogen-responsive recombinant human ovarian (BG1Luc4E2) cell line recently accepted by the US Environmental Protection Agency (USEPA) and Organisation for Economic Co-operation and Development (OECD) as a bioanalytical method to detect estrogen receptor (ER) agonists/antagonists. Unfortunately, these cells appear to contain only 1 of the 2 known ER isoforms, ERα but not ERβ, and the differential ligand selectivity of these ERs indicates that the currently accepted screening method only detects a subset of total estrogenic chemicals. To improve the estrogen screening bioassay, BG1Luc4E2 cells were stably transfected with an ERβ expression plasmid and positive clones identified using ERβ-selective ligands (genistein and Br-ERβ-041). A highly responsive clone (BG1LucERβc9) was identified that exhibited greater sensitivity and responsiveness to ERβ-selective ligands than BG1Luc4E2 cells, and quantitative reverse-transcription polymerase chain reaction confirmed the presence of ERβ expression in these cells. Screening of pesticides and industrial chemicals identified chemicals that preferentially stimulated ERβ-dependent reporter gene expression. Together, these results not only demonstrate the utility of this dual-ER recombinant cell line for detecting a broader range of estrogenic chemicals than the current BG1Luc4E2 cell line, but screening with both cell lines allows identification of ERα- and ERβ-selective chemicals.

Keywords: BG1Luc ER TA; Environmental screening; Estrogen receptor bioassay; Estrogenic chemicals.

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Conflict of interest statement

The authors declare that they have no conflict or competing interests with regards to this manuscript.

Figures

Figure 1
Figure 1
ER subtype-specific induction of ER-dependent luciferase reporter gene expression in transiently transfected SKBR3 cells. Cells were cotransfected with the ER-responsive luciferase reporter plasmid pGudLuc7.ERE and the ERα or ERβ expression vector, incubated with the indicated concentration of (A) E2, (B) genistein, (C) Br-ERβ-041, or (D) PPT for 24 h, lysed and luciferase and total protein levels analyzed as described in Materials and Methods. Luciferase activity is expressed as a percent of maximum luciferase induction by 1 nM E2 and results represent the mean ± SD of triplicate determinations from each of three separate experiments after subtraction of the luciferase activity present in cells exposed to DMSO.
Figure 2
Figure 2
Concentration-dependent induction of luciferase in BG1LucERβ clone 9 and the parent cell line BG1Luc4E2 by estrogen. Cells were incubated with E2 (24 h), lysed, and luciferase levels measured and analyzed as described in Materials and Methods. Luciferase activity is expressed as relative light units (RLUs) and values represent the mean ± SD of triplicate determinations after subtraction of the luciferase activity obtained in cells exposed to DMSO and are representative of more than 15 different replicate experiments.
Figure 3
Figure 3
Concentration-dependent induction of luciferase in BG1LucERβc9 and BG1Luc4E2 cells by ERα and ERβ selective ligands. BG1LucERβc9 cells (closed circles) or BG1Luc4E2 cells (open circles) were incubated with the ERβ-selective ligands genistein (A) or Br-ERβ-041 (B) or with the ERα-selective ligand PPT (C) for 24 h, cells lysed, and luciferase levels measured and analyzed as described in Materials and Methods. Luciferase activity is expressed as a percent of maximum induction by E2 (0.1 nM) in each cell line and results represent the mean ± SD of triplicate determinations after subtraction of the background luciferase activity obtained in cells exposed to DMSO and values are representative of more than 8 replicate experiments.
Figure 4
Figure 4
Comparison of ERβ and ERα mRNA levels in BG1Luc4E2 and BG1LucERβc9 cells. (A) Threshold cycle values for ERα, ERβ, and β-actin mRNA levels in BG1Luc4E2 and BG1LucERβc9 cells. (B) Expression of ERβ and ERα mRNA in BG1LucERβc9 cells. Results represent the mean ± SD of four determinations, and ER mRNA levels were normalized to that of β-actin and expressed relative to levels in BG1Luc4E2 cells as described in Materials and Methods. Significant difference (p < 0.05, determined by Student’s t-test) in relative ERβ or ERα mRNA levels in BG1LucERβc9 cells from that of levels in BG1Luc4E2 cells is indicated by an asterisk *.
Figure 5
Figure 5
Screening results of a chemical library of common pesticides and industrial chemicals in BG1Luc4E2 and BG1LucERβc9 cells. Chemicals in the library were screened once in triplicate for ER agonist activity in BG1Luc4E2 or BG1LucERβc9 cells at a final concentration of 10 µM. Cells were incubated for 24 h, lysed, and luciferase levels measured and analyzed as described in Materials and Methods. Luciferase activity is expressed as a percent of maximal luciferase induction obtained with 1 nM E2 in each cell line. Each bar represents the mean of triplicate determinations.
Figure 6
Figure 6
Induction of luciferase activity by selected library chemicals in BG1Luc4E2 and BG1LucERβc9 cells. Cells were incubated with the indicated chemicals (10 µM) for 24 h, lysed, and luciferase levels measured and analyzed as described in Materials and Methods. Luciferase activity is expressed as a percent of maximum induction by E2 (0.1 nM) in each cell line. Results represent the mean ± SD of three individual experiments (with triplicate wells analyzed per chemical per experiment) after subtraction of the luciferase activity obtained in cells exposed to DMSO. Significant differences (p < 0.05, determined by Student’s t-test) in luciferase induction in BG1LucERβc9 cells above that of the BG1Luc4E2 cell line from chemical treatment is indicated by an asterisk (*).
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