Factors influencing nominal effective concentrations of chemical compounds in vitro: cell concentration.

In vitro potency data (e.g. EC(50) values), used to characterise the biological activity of chemicals, are generally based on nominal effective concentrations and thus depend on any factor influencing the availability of a compound. In this study the significance of cell binding for the availability of chemicals in vitro is (i) theoretically investigated by means of a simple equilibrium distribution model and (ii) experimentally examined using a bull sperm assay to measure the cytotoxic potency of selected compounds at different 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 cell binding other than partitioning into cellular lipids. With the exception of xylene, the EC(50) values increased with increasing cell concentration. The ratios of EC(50) values determined at about 120 x 10(6) and 15 x 10(6) cells/ml were: pentachlorophenol. 1.2, 1-nitronaphthalene: 1.9, thioridazine: 2.7, dieldrin: 4.1, hexachlorophene: 4.1, digitonin: 5.1, methylmercury chloride: 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 well predicted by the equilibrium distribution model, except for p,p'-DDE. The results show that cell binding can significantly affect the availability of compounds in vitro and thus toxic potencies and toxic equivalency factors.

[1]  H Seibert,et al.  Influence of protein binding and lipophilicity on the distribution of chemical compounds in in vitro systems. , 1997, Toxicology in vitro : an international journal published in association with BIBRA.

[2]  P. Allen,et al.  Physiological model for the pharmacokinetics of methyl mercury in the growing rat. , 1993, Toxicology and applied pharmacology.

[3]  E. C. Slater,et al.  The allosteric binding of antimycin to cytochrome b in the mitochondrial membrane. , 1972, Biochimica et biophysica acta.

[4]  S. Rottem,et al.  Isolation of Mycoplasma Membranes by Digitonin , 1972, Journal of bacteriology.

[5]  K. Krishnan,et al.  A tissue composition-based algorithm for predicting tissue:air partition coefficients of organic chemicals. , 1996, Toxicology and applied pharmacology.

[6]  H. Seibert,et al.  A Short-term Bovine Sperm Cell Assay for the Evaluation of the In Vitro Cytotoxicity of Chemicals , 1990 .

[7]  P. Seeman.  III. A method for distinguishing specific from nonspecific hemolysins , 1966 .

[8]  Jerome O. Nriagu,et al.  The Biogeochemistry of mercury in the environment , 1979 .

[9]  Helmut Segner,et al.  MEIC Evaluation of Acute Systemic Toxicity , 1998 .

[10]  Joop L. M. Hermens,et al.  Solid phase microextraction as a tool to determine membrane/water partition coefficients and bioavailable concentrations in in vitro systems. , 1997, Chemical research in toxicology.

[11]  R. Obach,et al.  Prediction of human clearance of twenty-nine drugs from hepatic microsomal intrinsic clearance data: An examination of in vitro half-life approach and nonspecific binding to microsomes. , 1999, Drug metabolism and disposition: the biological fate of chemicals.

[12]  H. Iwamura,et al.  Structural factors of antimycin A molecule required for inhibitory action. , 1994, Biochimica et biophysica acta.

[13]  O. H. Lowry,et al.  Protein measurement with the Folin phenol reagent. , 1951, The Journal of biological chemistry.

[14]  A. Leo,et al.  Substituent constants for correlation analysis in chemistry and biology , 1979 .

[15]  N. Ballatori,et al.  Mechanisms of hepatic methylmercury uptake. , 1995, Journal of toxicology and environmental health.

[16]  R. Braddock,et al.  Prediction of the partition coefficient of lipophilic compounds in the air-mammal tissue system. , 1993, The Science of the total environment.

[17]  J. Hermens,et al.  Measurement of the Free Concentration Using Solid-Phase Microextraction: Binding to Protein , 1996 .

[18]  J. S. Rieske.  Inhibitors of respiration at energy-coupling site 2 of the respiratory chain. , 1980, Pharmacology & therapeutics.

[19]  Hasso Seibert,et al.  Inclusion of Physicochemical Data in Quantitative Comparisons of In Vitro and In Vivo Toxic Potencies , 1994 .