Uridine uptake inhibition assay: an automated micromethod for the screening of cytotoxicity.
The uridine uptake inhibition assay is a sensitive microassay for measuring cytotoxicity. This assay is normally performed with Hela S3 cells, which lack metabolic activity. In an earlier study, we adapted the test to HepG2 cells, a human hepatoma cell line that retains many hepatocyte characteristics, such as functional metabolic enzymes. This study describes a new automated protocol for the assay that makes it much more rapid. In the previous protocol, after the cells were treated with the test compounds and allowed to take up uridine for 30 min, samples were taken manually one by one and spotted onto 3MM Whatman paper. After drying, the paper sheet was then chromatographed in 5% (P/V) TCA for 2 h in order to precipitate and measure the total amount of RNA. In the new method, instead of paper chromatography, samples are transferred onto a 96-well microplate equipped with GF/C glass filters. Then, RNA precipitation by TCA is carried out with a manifold system, and the amount of radiolabeled uridine taken up by the cells is counted directly with a radioactivity microplate reader. This method makes it possible to screen many compounds simultaneously for cytotoxicity. To evaluate its sensitivity, we compared the IC(50) values obtained with new and original protocol for each eight toxic compounds. We found an excellent correlation between the two methods (r(2)=0.99). With the automated protocol, the uridine uptake inhibition assay is both sensitive and rapid enough for high-throughput daily screening.
Valentin-Severin
Isabelle
I
ENSBANA, UMR (0938) Toxicologie Alimentaire, 1 Esplanade Erasme, 21000, Dijon, France.
Laignelet
Laurence
L
Lhuguenot
Jean Claude
JC
Chagnon
Marie Christine
MC
D001331:Automation; D002460:Cell Line / Q000187:drug effects; D006801:Humans; D020128:Inhibitory Concentration 50; D012313:RNA / Q000096:biosynthesis; D012044:Regression Analysis; D012680:Sensitivity and Specificity; D013997:Time Factors; D018675:Toxicity Tests / Q000379:methods*; D014316:Tritium; D014529:Uridine / Q000037:antagonists & inhibitors / Q000378:metabolism*
D014316:Tritium; D012313:RNA; D014529:Uridine