PH-DEPENDENT CYTOTOXICITY OF CONTAMINANTS OF PHENOL RED FOR MCF-7 BREAST-CANCER CELLS

The pH indicator phenol red (phenolsulfonphthalein) is present in most tissue culture media. Contaminants of this indicator have shown substantial estrogenic activity for estrogen-dependent cells in culture, including the human breast cancer-derived MCF-7 cell line. In the course of other studies, we observed that brief (1- to 4-h) incubations of these cells at 37 C in serum-free medium (Hanks' or Earle's Balanced Salts Solution) could be toxic to MCF-7 cells when the pH was increased above 7.4, but only if phenol red (10-mu-g/ml) was present in the medium. Because damaged/killed cells detached from the substratum (> 98% of detached cells stained with trypan blue), we used DNA assay of the cells remaining after treatment and wash (98% of the remaining cells were dye excluding) to further assess cytotoxicity. The MCF-7 cells were more susceptible to the cytotoxicity at lower cell densities, so further characterization of phenol red cytotoxicity was performed at cell densities of 1-10-mu-g DNA/2-cm2 well, or approximately 40,000-400,000 cells/ml medium. In the pH range of 7.0-8.2, 50% cell death was observed in the presence of phenol red at pH as low as 7.6-7.7, with nearly 100% of the cells killed by pH 8.0. Little effect was seen in phenol red-free medium at any part of the tested pH range or in medium that contained phenol red at pH less-than-or-equal-to 7.4. In time-course studies of cytotoxicity at pH 8.0 (phenol red, 10-mu-g/ml), greater than 50% cell damage could be observed after less than 1 h, and little cell recovery was observed if the pH was restored to 7.4. For phenol red samples from two major commercial sources, the concentration for half-maximal cytotoxicity (TD50) in dose-responses after 4 h at pH 8.0 showed TD50 values of 2 and 6-mu-g/ml, while the estrogenic activities, as half-maximal stimulation of estrogen-dependent proliferation, were identical at 2-mu-g/ml. Both the cytotoxic and estrogenic activities could be removed from the phenol red by extraction with diethyl ether. A number of contaminants of the commercial phenol red were detected by reverse phase C18 HPLC. Cytotoxicity and estrogen bioassays of each of the HPLC fractions indicated that the pH-dependent cytotoxicity was separate from the estrogenic activity and confirmed that neither activity was associated with the phenol red itself. Although to date only MCF-7 cells have been affected, the pH-dependent cytotoxicity of phenol red may need to be considered as a factor in many studies of physiological response and pH that have been performed in tissue culture with phenol red present, especially those that have involved alkaline pH (> 7.4) and low cell density. Even very brief elevations of pH in phenol red-containing media and salts solutions may produce detrimental effects on MCF-7 cells. This cytotoxicity for a hormone-dependent breast cancer cell line may have application in anticancer therapy. In view of the hormonal and now complicated cytotoxic activities present in contaminants of this pH indicator, the use of phenol red that has not been highly purified should be carefully considered, especially in endocrine studies in tissue culture.