title: In vitro toxicity testing with microplate cell cultures: Impact of cell binding.
abstract: In vitro generated data on toxic potencies are generally based on nominal concentrations. However, cellular and extracellular binding and elimination processes may reduce the available free fraction of a compound. Then, nominal effective concentrations do not represent appropriate measures of toxic exposure in vitro and underestimate toxic potencies. In this study it was investigated whether cell binding can affect the availability of chemicals in microplate based toxicity assays. To this end the cytotoxicity of compounds like mercury chloride, digitonin and alcohol ethoxylates, accumulated by cells via different modes, was investigated in 96-well microplate cultures with varying concentrations of Balb/c 3T3 cells. The median effective nominal concentrations of all but one of the tested compounds depended linearly from the cell concentration. Applying a previously developed equilibrium distribution model cell concentration-independent median effective extracellular concentrations and cell burdens, respectively, could be calculated. The compounds were accumulated by the cells with bioconcentration factors, BCF, between 480 and ≥ 25,000. Cell binding of the alcohol ethoxylates was correlated with their lipophilicity. The results show that significant cell binding can occur even at the small cell volume fractions (∼ 1 × 10(-5) to 3 × 10(-3) L/L) encountered in microplate assays. To what extent cell binding affects the bioavailability depends on the BCF and the cell volume fraction. EC50 measurements in the presence of at least two different cell concentrations allow for excluding or detecting significant cell binding and for determining more appropriate measures of toxic exposure in vitro like median effective extracellular (free) concentrations or cell burdens.
authors.affiliation: Institute for Toxicology and Pharmacology for Natural Scientists, University Hospital Schleswig-Holstein, Campus Kiel, Brunswiker Str. 10, 24105 Kiel, Germany. Electronic address: guelden@toxi.uni-kiel.de.
authors.forename: Michael
authors.initials: M
authors.lastname: Gülden
authors.affiliation: Institute for Toxicology and Pharmacology for Natural Scientists, University Hospital Schleswig-Holstein, Campus Kiel, Brunswiker Str. 10, 24105 Kiel, Germany.
authors.forename: Jeannine
authors.initials: J
authors.lastname: Schreiner
authors.affiliation: Institute for Toxicology and Pharmacology for Natural Scientists, University Hospital Schleswig-Holstein, Campus Kiel, Brunswiker Str. 10, 24105 Kiel, Germany.
authors.forename: Hasso
authors.initials: H
authors.lastname: Seibert
mesh_terms: D016475:3T3 Cells; D000438:Alcohols / Q000378:metabolism / Q000633:toxicity; D000826:Animal Testing Alternatives; D000818:Animals; D001682:Biological Availability; D001692:Biological Transport; D002470:Cell Survival / Q000187:drug effects; D004072:Digitonin / Q000378:metabolism / Q000633:toxicity; D004305:Dose-Response Relationship, Drug; D004867:Equipment Design; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D020128:Inhibitory Concentration 50; D016014:Linear Models; D008627:Mercuric Chloride / Q000378:metabolism / Q000633:toxicity; D051379:Mice; D008904:Miniaturization; D008954:Models, Biological; D018675:Toxicity Tests / Q000295:instrumentation / Q000379:methods*
chemical_list: D000438:Alcohols; D008627:Mercuric Chloride; D004072:Digitonin
keywords: Bioavailability in vitro; Cell binding; Cell burden; Free concentration in vitro; In vitro biokinetics; In vitro toxicity testing