Detection of oestrogenic activity of steroids present during mammalian gestation using oestrogen receptor alpha- and oestrogen receptor beta-specific in vitro assays

Abstract

Numerous steroid hormones are present in the foetus but their potential to activate oestrogen receptor (ER) and/or is largely unknown. In this study, in vitro assays were developed to rapidly and specifically detect ER or ER activation by these steroid hormones. Our results showed that several oestrogen precursors and androgens are able to activate both ER and ER . Of special interest is that some of these precursors are able to activate ER and ER at concentrations that are present during human gestation. Moreover, some precursors (dehydroepiandrosterone (DHEA) and 17-hydroxylated pregnenolone sulphate) and androgens (5-androsten-3 ,16 ,17 -triol and testosterone) showed a more than 100-fold relative preference for ER transactivation over ER transactivation when compared with 17 -oestradiol. Due to their relatively high levels, the precursor steroids DHEA and pregnenolone may be of particular importance in the regulation of ER activity in vivo. To obtain information about the oestrogenic activity of the total pool of steroid hormones present during mammalian gestation, steroids were extracted from mouse embryos at different prenatal stages and assayed for oestrogenic activity in the established in vitro assays. Oestrogenic activity was detected in steroid extracts from all stages tested. This study has demonstrated that oestrogen receptor agonists are present in the murine embryo and that oestrogen precursors may contribute to the total pool of agonists during foetal life.

Is There a Receptor for Dehydroepiandrosterone or Dehydroepiandrosterone Sulfate?

“…These studies are summarized in Table 3. [56][57][58][59][60][61][62][63] Binding assays with purified recombinant ER or AR have shown that the affinity of DHEA for ER or AR is approximately 1000-fold weaker than for the cognate ligand. The transcriptional activation of ER or AR by DHEA also requires approximately 1000-fold higher concentrations than for the cognate ligand.…”

In Vivo Imaging of Activated Estrogen Receptors in Utero by Estrogens and Bisphenol A

“…Very low levels of estrogens have been described to be present in steroid extracts of mouse embryo homogenates as determined in estrogenic activity measurements (Lemmen et al 2002). These levels may, however, be too low to activate endogenous ERs or are not able to activate ERs in vivo because of such different factors as tissue distribution and inactivation through binding proteins.…”

“…Stable 239HEK cell lines containing human ER-α(hER-α) or hER-βand an estrogen-responsive reporter construct—similar to the one introduced in the transgenic animals, but without the flanking insulator sequences—were used and cultured as previously described (Lemmen et al 2002). Briefly, cells were plated in 96-well tissue culture plates (NUNC, Life Technologies, Breda, The Netherlands) in medium consisting of phenol red–free Dulbecco’s modified Eagle’s medium/F12 (1:1) medium containing 3 × 10 −8 M selenite, 10 μg/mL transferrin, 0.2% (wt/vol) bovine serum albumin, and 5% (wt/vol) dextran-coated charcoal-stripped fetal calf serum.…”

Risk Assessment for Dioxins and Related Compounds

“…Other effect-based bioassays are also available, e.g. the YES yeast-based oestrogen assay and the CALUX ER assay [42]. Whilst all such assays have the advantage of covering a range of chemicals with similar modes of action and are usually rapid and relatively easy to implement, they all have similar drawbacks to those mentioned above for VTG monitoring.…”