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 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)
Grady
L H
LH
Department of Veterinary Biomedical Sciences, University of Missouri, Columbia 65211.
Nonneman
D J
DJ
Rottinghaus
G E
GE
Welshons
W V
WV
D001943:Breast Neoplasms / Q000473:pathology*; D016923:Cell Death / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid; D004305:Dose-Response Relationship, Drug; D004340:Drug Contamination; D004958:Estradiol / Q000494:pharmacology; D004967:Estrogens / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010637:Phenolsulfonphthalein / Q000008:administration & dosage / Q000302:isolation & purification / Q000494:pharmacology*; D013629:Tamoxifen / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D004967:Estrogens; D013629:Tamoxifen; D004958:Estradiol; D010637:Phenolsulfonphthalein