THE EFFECTIVE FREE FRACTION OF ESTRADIOL AND XENOESTROGENS IN HUMAN SERUM MEASURED BY WHOLE-CELL UPTAKE ASSAYS - PHYSIOLOGY OF DELIVERY MODIFIES ESTROGENIC ACTIVITY

The biological activity of natural estrogens is influenced by the degr ee to which they bind to serum proteins. To determine directly how ser um affected the uptake of estradiol, we compared the whole cell uptake of [H-3]estradiol in intact MCF-7 human breast cancer cells from seru m-free medium with the uptake from 100% serum from adult men. In estro gen receptor saturation assays, 28.9 times more estradiol was required in serum to occupy the same number of estrogen receptors as was requi red in serum-free medium (SFM), suggesting that the effective free fra ction of estradiol in adult male serum was 3.46% (1 divided by 28.9). Since most xenoestrogens are not available in tritium-labeled form, th e cell uptake of unlabeled xenoestrogens could not be measured directl y with saturation analysis. Therefore, we developed the relative bindi ng affinity-serum modified access (RBA-SMA) assay to determine the eff ect of serum on the access of nonradioactive xenoestrogens to estrogen receptors within intact MCF-7 cells. Serum modified access (SMA) was calculated by dividing the relative binding affinity (RBA, relative to estradiol) measured in 100% serum, by the RBA measured in serum-free medium. An SMA > 1 Indicated that the xenoestrogen had greater access to estrogen receptors than estradiol from serum. In contrast, an SMA < 1 indicated that the xenoestrogen had less access to estrogen recepto rs from serum than did estradiol. The synthetic estrogen diethylstilbe strol (DES) binds poorly to sex hormone binding globulin (SHBG), and D ES showed enhanced access in serum, SMA = 6.2. Additional calculations through the K-I (inhibition constant) indicated that this corresponde d to an effective free fraction of 26.9% for DES in serum. The phytoes trogens, coumestrol, genistein, and equol, showed substantial enhanced access in serum, over 10-fold relative to estradiol (SMA = 12.1, 10.3 , and 11.3, respectively), and effective free fractions in serum of 47 .8, 45.8, and 49.7%, respectively. Since most in vitro assays of xenoe strogens do not address how serum influences their bioactivity, the es trogenic activity of these phytoestrogens would be underestimated. Con versely, biochanin A showed decreased access from serum (SMA = 0.44) a nd had an effective free fraction of 2.4%; its estrogenic activity wou ld be overestimated in serum-free assays.