Factors influencing nominal effective concentrations of chemical compoundsin vitro: cell concentration

In vitro potency data (e.g. EC50 values), used to characterise the biologic al activity of chemicals, are generally based on nominal effective concentr ations and thus depend on any factor influencing the availability of a comp ound. In this study the significance of cell binding for the availability o f chemicals in vitro is (i) theoretically investigated by means of a simple equilibrium distribution model and (ii) experimentally examined using a bu ll sperm assay to measure the cytotoxic potency of selected compounds at di fferent cell concentrations. Compounds were selected either to cover a wide range of hydrophobicity (log K-ow = 2.52-5.69) or to represent modes of ce ll binding other than partitioning into cellular lipids. With the exception of xylene, the EC50 values increased with increasing cell concentration. T he ratios of EC50 values: determined at about 120x10(6) and 15x10(6) cells/ ml were: pentachlorophenol. 1.2, 1-nitronaphthalene: 1.9, thioridazine: 2.7 , dieldrin: 4.1, hexachlorophene: 4.1, digitonin: 5.1, methylmercury chlori de: 7.9, antimycin A: 10.1 and p,p'-dichlorodiphenyl dichloroethylene (p,p' -DDE): > 19.1. The influence of partitioning into cell lipids was rather we ll predicted by the equilibrium distribution model, except for p,p'-DDE. Th e results show that cell binding can significantly affect the availability of compounds in vitro, and thus toxic potencies and toxic equivalency facto rs. (C) 2001 Elsevier Science Ltd. All rights reserved.