Estrogenic activity of estradiol and its metabolites in the ER‐CALUX assay with human T47D breast cells^Note

Abstract

A number of metabolites of 17beta-estradiol were tested for their estrogenic activity using the ER-CA-LUX assay based on the increased expression of luciferase in exposed T47D breast cancer cells. E2beta and estrone showed similar potencies in the test, whereas E2alpha was 100 times less active. Incubation of cells with estrone (0.35 microM) resulted in the formation of E2beta, whereas the reverse reaction was observed for E2beta. The resulting equilibrium may explain the similar estrogenic potency of estrone in the test. The synthetic 17-hydroxy benzoate ester of E2beta was 3 times less active than the parent compound. The 17-hydroxy palmitate and oleate esters of E2beta, were respectively 25 and 200 times less active than the parent compound. The 2-hydroxy metabolites of E2beta and estrone showed a 5,000 to 10,000 fold lower activity. The 4-hydroxy metabolites were more potent than the 2-hydroxy metabolites, showing only a 20-200 times lower activity. The 2- and 4-methoxyesters of estrone were 700 times less active. It is concluded that the estrogenic potency of metabolites formed in cattle after treatment with E2beta, like estrone, E2alpha and especially the esters of E2beta, may be significant with respect to the potential risk of the use of estradiol for growth promotion in domestic animals in certain countries.

Analysis of steroidal estrogen residues in food and environmental samples

“…This may lead to cancer, reproductivesystem disorders, decreases in sperm counts, and the disturbances in the balance of the ecosystem. Based on daily excretion of estrogens by humans and animals, dilution factor, and previous observations by other authors, ng L À1 levels of estrogens are expected to be present in aqueous environmental samples [25,26,30,33,34]. The analytical difficulties associated with the determination of such low estrogen concentrations in complex aqueous matrices have limited extensive surveys on the occurrence of estrogens in the aquatic environment.…”

“…The log K ow of 3.1-4.7 indicates that estrogens are rather lipophilic and should appreciably adsorb into sediment and sludge [29,30]. This assumption is underscored by the detection of high concentrations of estrogens in water released by dewatering sewage sludge.…”

“…The CALUX assay, which was initially developed for the detection of dioxins and dioxin-like PCBs [203], is a suitable choice for screening new and existing chemicals, as well as complex environmental samples for unexpected estrogenic activity that may be further identified by chemical analysis [30,[204][205][206]. In the CALUX assay, chemicals are added to sterile medium containing serum with lipids and proteins, which mediate the cellular availability and membrane transport of test chemicals.…”

In vitro bioassays for the study of endocrine-disrupting food additives and contaminants

“…This ability to metabolize compounds has led to varying results in studies on the relative estrogenic potency (REP) of estrone and estriol in comparison to 17b-estradiol (17b-E2). However, conversion of estrone to 17b-E2 and vice versa was confirmed as the reason [47,48]. Differences have also been highlighted between the estrogenic and anti-estrogenic responses in the ER-CALUX assay and the YES recombinant yeast assay [49].…”

“…However, other cell lines (e.g., the U2-OS human osteosarcoma), which lack steroid receptors, have been transfected with the androgen receptor (AR) and ERa to produce newer forms of CALUX assays designed to overcome this cross-talk issue [46]. Mammalian RGAs can identify compounds that require metabolism for activation into their active state [40,43,47]. This ability to metabolize compounds has led to varying results in studies on the relative estrogenic potency (REP) of estrone and estriol in comparison to 17b-estradiol (17b-E2).…”