Endocrinology, Vol 129, 3321-3330, Copyright © 1991 by Endocrine Society

ARTICLES

pH-dependent cytotoxicity of contaminants of phenol red for MCF-7 breast cancer cells

LH Grady, DJ Nonneman, GE Rottinghaus and WV Welshons
Department of Veterinary Biomedical Sciences, University of Missouri, Columbia 65211.

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 micrograms/ml) was present in the medium. Because damaged/killed cells detached from the substratum (greater than 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 micrograms 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 micrograms/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 micrograms/ml, while the estrogenic activities, as half-maximal stimulation of estrogen-dependent proliferation, were identical at 2 micrograms/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.(ABSTRACT TRUNCATED AT 250 WORDS)