Protein precipitation In Vitro as a measure of chemical-induced cytotoxicity: an EDIT sub-programme.
As a priority area of the Evaluation-Guided Development of In Vitro Toxicity and Toxicokinetic Tests (EDIT) programme, an in vitro protein precipitation (PP) assay was used on the 50 reference chemicals of the Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) project, to confirm and extend the MEIC results. Dose-response curves were generated for only 30 of the chemicals, and the concentrations causing 10% (EC10) and 50% (EC50) protein precipitation versus the positive control were chosen as endpoints. The number of chemicals with a positive response increased to 46 when a new endpoint, the minimum effect concentration (MEC) that induces protein precipitation with respect to the negative control, was used. When the results were correlated with in vitro cytotoxicity in human cell lines, a similarly good correlation was found between the various endpoints of the PP assay at 5 hours and the 24-hour IC50 average cytotoxicity in human cell lines, even though the number of chemicals included in the correlation was larger for the MEC. Using the prediction error, the endpoint that gave the best correlation between the PP assay and human cell cytotoxicity was once more found to be the 5-hour MEC, and this was chosen for the PP assay. The sensitivity of the PP assay is lower than that of the in vitro cell-line cytotoxicity assay, possibly due to its shorter exposure period and because precipitation is the ultimate event in the sequence of a protein disturbance. It is expected that earlier denaturation steps would give better sensitivity. However, this simple, inexpensive and rapid assay could be useful in the early stages of testing chemicals.
Novillo
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A
Environmental Toxicology, CISA-INIA, Valdeolmos 28130, Madrid, Spain.
Ekwall
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B
Castaño
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A
D002460:Cell Line; D011232:Chemical Precipitation; D004305:Dose-Response Relationship, Drug; D006801:Humans; D066298:In Vitro Techniques; D020128:Inhibitory Concentration 50; D010047:Ovalbumin / Q000737:chemistry*; D018675:Toxicity Tests / Q000379:methods*
D010047:Ovalbumin