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

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, there is a critical need for rapidly detecting these chemicals. We developed an estrogen-responsive recombinant human ovarian (BG1Luc4E2) cell line recently accepted by the USEPA and OECD as a bioanalytical method to detect estrogen receptor (ER) agonists/antagonists. Unfortunately, these cells appear to contain only one of the two 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 qRT-PCR 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.

The effect of individual and mixtures of mycotoxins and persistent organochloride pesticides on oestrogen receptor transcriptional activation using in vitro reporter gene assays

“…It is well established that ZEN, α-ZOL and β-ZOL activate ERα and ERβ both in vitro and in vivo, with α-ZOL showing the highest oestrogenic potency (EFSA, 2016(EFSA, , 2017Tatay et al, 2018). The estrogenic potential of dichlorodiphenyltrichloroethane (DDT) and its isomers have also been investigated with 1,1,1-trichloro-2,2-bis(p-chlorophenyl) ethane (p,p'-DDT) and 1,1-dichloro-2,2-bis(p-chlorophenyl) ethylene (p,p'-DDE) showing low agonistic activities on ERs whereas o, p'-DDT (l,l,l-trichloro-2(p-chlorophenyl)-2-(o-chlorophenyl) ethane) had strong oestrogenic activity (Chen et al, 1997;Brennan et al, 2016). However,…”

“…transcriptional activity. Since human reproductive organs such as testis, prostate, ovary, premenopausal and foetal uteri, and endometrium contain significant amounts of both ER-α and ER-β receptors (Mosselman et al 1996;Brandenberger et al, 1997Brandenberger et al, , 1998Brandenberger et al, , 1999, and given the critical role that ER-β plays in modulating the functional activity and levels of ER-α, the use of reporter cell lines with one ER subtype (ER-α or ER-β) may underestimate the overall estrogenic potency of a sample (Brennan et al, 2016). This is true as oestrogenic chemicals that were not detected using BG1Luc4E2 cells expressing only ER-α were identified by the BG1LucERβc9 cells which constitutively expresses both ER-α and ER-β (Brennan et al, 2016).…”

Assessment of pesticides in surface water samples from Swedish agricultural areas by integrated bioanalysis and chemical analysis

“…The cell line VM7Luc4E2, based on human breast cancer MCF-7 cells, contains a stably transfected firefly luc reporter construct, controlled by estrogen response elements, which will detect substances with in vitro ER agonist activity. This cell line was a generous gift from Michael Denison, University of California at Davis [40,41]. This cell line was previously named BG1Luc4E2, but due to a misidentification of the cell line, the name has been changed into VM7Luc4E2 (rational for this name change can be found in the corrigendum to Ref.…”

“…This cell line was previously named BG1Luc4E2, but due to a misidentification of the cell line, the name has been changed into VM7Luc4E2 (rational for this name change can be found in the corrigendum to Ref. [41]). The cells were cultured as a monolayer in a humidified environment at 37 °C and 5% CO 2 .…”

Relationship between the molecular composition, visible light absorption, and health-related properties of smoldering woodsmoke aerosols

“…Screening of WSA extracts for AhR-or ER-active compounds were carried out using recombinant mouse hepatoma (H1L6.1c2) cells containing a stably integrated AhR-responsive luciferase reporter gene plasmid (pGudLuc6.1) and human breast carcinoma (VM7Luc4E2) cells containing a stably integrated ER-responsive luciferase reporter gene plasmid (pGudLuc7ere), as previously described (He et al, 2014;Brennan et al, 2016). Briefly, for AhR analysis, H1L6.1c2 cells in growth medium (alpha minimal essential media (aMEM) containing 10% FBS) were plated (75,000 cells/well) into white, clear-bottomed 96-well tissue culture plates and incubated at 37°C for 24 h prior to the addition of WSA extracts.…”