A screening method for estrogens using an array-type DNA glass slide.
A new screening assay was described for the determination of endocrine disrupting chemicals (EDCs), such as synthetic estrogens, with an array-type DNA glass slide having characteristics of 1) a high sample throughput, 2) a compact size allowing a small sample volume, and 3) a sensitive determination based on the estrogen-dependent binding of the human estrogen receptor a (hERalpha) with its estrogen responsive element (ERE; Vit. A2 gene promoter). We devised a glass slide on which a thin agarose gel was mounted. Avidin was then covalently immobilized on each well of the glass slide after the gel was activated by a NaIO4 solution. Also, the biotinylated ERE as a DNA probe was immobilized on the gel layer through avidin-biotin binding. After the estrogen-dependent binding of a yellow fluorescent protein-fused hERalpha (YFP-hERalpha) to ERE on the gel layer, the fluorescence intensity of YFP-hERalpha quantitatively extracted into the gel was directly determined with a fluorescence microplate reader. Pre-incubation of YFP-hERalpha with estrogen at 37 degrees C for 30 min enhanced the estrogen-dependent hERalpha-ERE binding. The determined hormonal activities of estrogens on the interaction of YFP-hERalpha with ERE were as follows in their decreasing order: diethylstilbestrol (DES) > 17beta-estradiol (E2) <==> ethynylestradiol (EE2) > 4-hydroxy tamoxifen (OHT) > clomiphene (Clo). The present method provides a sensitive estrogen-dependent dose-response curve down to approximately 10(-13) M in the case of DES. This method will become a competitive alternative to the conventional in vitro assays, such as a DNA-binding assay using radioisotopes.
Kim
Sung Bae
SB
Department of Chemistry, School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Ozawa
Takeaki
T
Umezawa
Yoshio
Y
D001360:Avidin / Q000737:chemistry; D001426:Bacterial Proteins / Q000032:analysis; D001483:Base Sequence; D004247:DNA / Q000737:chemistry*; D004864:Equipment and Supplies; D047628:Estrogen Receptor alpha; D004967:Estrogens / Q000032:analysis*; D008164:Luminescent Proteins / Q000032:analysis; D011960:Receptors, Estrogen / Q000032:analysis; D011993:Recombinant Fusion Proteins / Q000032:analysis; D012685:Sepharose / Q000737:chemistry
D001426:Bacterial Proteins; D047628:Estrogen Receptor alpha; D004967:Estrogens; D008164:Luminescent Proteins; D011960:Receptors, Estrogen; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria; D001360:Avidin; D004247:DNA; D012685:Sepharose