Ultrasensitive Cell-Based Bioassay for the Measurement of Global Estrogenic Activity of Flavonoid Mixtures Revealing Additive, Restrictive, and Enhanced Actions in Binary and Higher Order Combinations

Abstract

Flavonoids present in food, botanicals, and body fluids occur as complex mixtures, and data on their combinatorial estrogenic effects are sparse. Human cell lines that permanently express estrogen receptor (ER) alpha and ERbeta proteins were developed for the measurement of the global estrogenicity of flavonoids in such complex mixtures. The presence of estrogenic ligands, known and unknown, in these mixtures can be detected by activation of an ER-driven luciferase reporter gene. We also examined the effect of hydroxylation on the estrogenic activities of four common flavonoids-apigenin, kaempferol, luteolin, and quercetin, alone and in combination. An inverse relationship was observed between the number of hydroxyl groups in flavonoids and ERalpha bioactivity. When submaximal doses of apigenin, luteolin, kaempferol, genistein, and estradiol were combined in binary and higher order mixtures, the experimental estrogenic effects matched those obtained by summing effects extrapolated from dose-response curves of individual compounds. The estrogenic activities of mixtures containing quercetin were observed to deviate from additivity, suggesting that it was a partial agonist/antagonist. Our assay reveals superagonistic, additive, and antagonistic ERalpha or ERbeta actions of flavonoids and adds to our understanding of the estrogenic effects of phytoestrogens in complex mixtures.

Molecular and pharmacodynamic properties of estrogenic extracts from the traditional Chinese medicinal herb, Epimedium

“…Furthermore, estrogenic effects of Epimedium brevicornum were small, due perhaps to the limited numbers of animals used in this exploratory study. Nevertheless our data indicate that ER␣ and MCF-7 cell bioassays, and related ultrasensitive methods recently developed (Wong et al, 2007), have utility to improve our understanding of the pharmacokinetics/pharmacodynamics of Epimedium extracts. Such knowledge is a critical prerequisite for the development of safe and efficacious traditional medicines.…”

AroER tri-screen™ is a novel functional assay to estimate both estrogenic and estrogen precursor activity of chemicals or biological specimens

“…Rather, the mixture activated the ER pathway in a more complicated manner. Congruently, there are a few reports that demonstrate the complexity of multi-component exposure [27, 28]. This emphasizes the importance of determining the total biological activity of a sample properly (i.e., the combined effects of all constituents).…”

Serum estrogen receptor bioactivity and breast cancer risk among postmenopausal women

“…Biological activity of estrogens in serum was assessed using a validated estrogen-driven recombinant cell bioassay (Li, et al 2009; Wong, et al 2007). Human uterine cervical HeLa cells [ATCC (Manassas, VA, USA)] were transformed to stably express either ERα (ESR1) or ERβ (ESR2).…”