Factors influencing nominal effective concentrations of chemical compounds in vitro:: medium protein concentration

Quantitative data used to characterise biological activities of chemicals in vitro (e.g. EC50 values) are generally based on nominal concentrations and thus depend on factors influencing the availability of a compound. In this study, the impact of protein binding on the availability of chemicals in vitro is theoretically investigated and experimentally examined using a bovine sperm cell assay to measure the cytotoxic potency of selected compounds at different medium protein concentrations. In agreement with theoretical considerations, linear correlations between EC50 values and medium albumin concentrations were determined with 2,4-dichlorophenol, pentachloro phenol, p,p'-DDT and mercuric chloride. Ratios of EC50 values measured in the presence and absence of 4% (w/v) albumin varied between 500 (hexachlorophene), 258 (pentachlorophenol) and almost I (potassium cyanide, dextropropoxyphene). Calculated molar ratios of substance bound to albumin ranged from 0.05 (arsenic trioxide) and 0.1 (potassium cyanide) to 2.5 and 4.7 moles/mole for malathion and xylene, respectively. The fractions bound at 4% albumin varied between 11 and 15% for dextropropoxyphene and potassium cyanide, respectively, and more than 99% for hexachlorophene, pentachlorophenol and mercuric chloride, The results clearly demonstrate that the differing impact of protein binding on the bioavailability of chemicals considerably influences their nominal and relative potencies in the presence of albumin. (C) 2002 Elsevier Science Ltd. All rights reserved.