Lipophilic impurities, not phenolsulfonphthalein, account for the estrogenic activity in commercial preparations of phenol red

Previously, we found that Phenol Red, a pH indicator dye commonly used in tissue culture media, had weak estrogenic activity, demonstrable by competitive binding to the estrogen receptor, stimulation of the growth rate of human breast cancer (MCF-7) cells, and elevation of progesterone receptor levels in these cells. We have now examined in more detail the source of this estrogenic activity, present in commercially available preparations of Phenol Red. By high performance liquid chromatography and solvent partitioning, we find that the receptor binding and growth promoting activity does not correspond to the indicator dye itself (phenolsulfonphthalein), but rather to more lipophilic impurities present in these preparations. There are numerous such impurities, many of which show some competitive binding activity, but the major receptor binding activity is accounted for by a single impurity component. Commercial preparations of Phenol Red can be purified by ether extraction of the sodium salt, whereby 95-99% of the lipophilic estrogenic impurities are removed, and the growth stimulating activity towards MCF-7 cells is reduced.