Optimization of a yeast estrogen screen and its applicability to study the release of estrogenic isoflavones from a soygerm powder

Here we describe a redesigned protocol of the yeast estrogen screen developed by Routledge and Sumpter. The redesigned test comprises two steps. First, a large a-mount of yeast with estrogenic compounds is incubated for 24 hr. Subsequently, a mixture of cycloheximide and the chromogenic substrate chlorophenol red-beta -D-galactopyranoside (CPRG) is added. The cycloheximide stops protein synthesis and allows for an end-point measurement of P-galactosidase activity generated during the first 24 hr. CPRG is converted to chlorophenol red and reflects beta -galactosidase activity, which is indicative of the estrogenic activity. The modifications shorten the duration of the assay at least I day and avoid interference of the estrogenic CPRG or chlorophenol red. The redesigned and the original protocol were used to study the estrogenic activity of bisphenol A, methoxychlor, p,p ' -DDT, and isoflavones (genistein, daidzein, and glycitein). Bisphenol A, methoxychlor, and genistein triggered higher levels of beta -galactosidase activity in the redesigned protocol. Estrogenic activity of p,p ' -DDT could only be demonstrated with the redesigned protocol. Glycitein and daidzein failed to give a response with both protocols. We also studied deconjugation of beta -glycosidic isoflavones present in soygerm powder. Treatment of the soygerm powder with beta -glycosidase released isoflavones. The estrogenic response of the samples was confirmed with the redesigned protocol and correlated with the amount of genistein present. The release of isoflavones under conditions prevailing in the intestines was studied. Bacterial beta -glycosidase present in the large intestine released isoflavones, and moderate estrogenic activity could lie demonstrated