A new assay using a multiwell fluorescence scanner was developed for screening cytotoxicity to cells cultured in 96-well microtiter plates. The assay is based on binding of propidium iodide to nuclei of cells whose plasma membranes have become permeable due to cell death. Fluorescence of propidium iodide measured with a multiwell fluorescence scanner increased in proportion to the number of permeabilized cells. After ATP depletion of hepatocytes and neonatal cardiac myocytes with metabolic inhibitors ("chemical hypoxia"), and exposure of Madine Darby canine kidney cells to the toxic chemical, HgCl2, propidium iodide fluorescence progressively increased. Increases of fluorescence were linearly proportional with release of lactate dehydrogenase into the culture medium. Employing this cytotoxicity screening assay, protection by various agents against lethal injury was evaluated in cultured hepatocytes during chemical hypoxia. Inhibitors of cysteine proteases (i.e., antipain, leupeptin, E-64), serine proteases (i.e., PMSF), and aspartic acid proteases (i.e., pepstatin A) did not protect against chemical hypoxia. In contrast, 1,10-phenanthroline, an inhibitor of metalloprotease, markedly protected against the onset of cell death during chemical hypoxia. Half-maximal protection after 60 min occurred at 0.5 μm. Phospholipase inhibitors, chlorpromazine (50 μm) and mepacrine (50 μm), also substantially retarded cell killing. U74006F, an inhibitor of lipid peroxidation, slowed cell killing to a lesser extent during chemical hypoxia and after oxidative stress with t-butyl hydroperoxide. Calciphor, a dimer of prostaglandin B1, did not protect against cell killing during chemical hypoxia or t-butyl hydroperoxide toxicity. In conclusion, this high capacity cytotoxicity assay for cells cultured in 96-well microtiter plates is suitable for rapid screening of potential cytoprotective agents in a variety of cell types.
Bibliographical noteFunding Information:
A variety of biochemical and morphological tests have been employed to assescsy totoxicity of chemical substances in vitro. Because the plasma membrane is essential to cell ’ This work was supported. in part, by Grants AGO72 I8 and DK-30874 function and viability, most common indicators of cell vi-from the National Institutes of Health and Grant J-1433 from the Office of ability monitor plasma membrane integrity. Nuclear staining NavalR esearcPh.r eliminaryre portso f portionso f this work were presented by membrane-impermeant dyes (e.g.. trypan blue, ethidium at theSeventy-thirAdn nualM eetingo f theFederatioonf AmericanS ocieties bromide. propidium iodide), release of trapped cytoplasmic et a/.. 1989)a. nda t theT hirtiethA nnuaMl eetingo f theS ocietyo f Toxicology. for Experimental Biology, New Orleans, LA, March 19-23. 1989 (Nieminen probes (e.g., fluorescein, “Cr-labeled protein) and leakage Dallas. TX. February 25-March 1, 1991 (Lemasters et al., 1991). of intracellular enzymes (e.g., lactate dehydrogenase, trans-’ Current address: Center for Basic Research in Digestive Diseases, Mayo aminases,c reatine phosphokinase) all have been accepted as Clinic, Rochester. MN 55905. appropriate indicators of irreversible cell damage (Spangberg, ’ Current address: II Rianimazione 1973: Bhuyan et al.. 1976; Story et al., 1983; Green et al.. 4 To whom correspondence should be addressed at Laboratories for Cell Pavia. Italy. 1984: Cheung et al., 1985: Gores et al., 1988). Indeed, the Biology. Department of Cell Biology & Anatomy, School of Medicine. Uni- plasma membrane literally ruptures at the onset of cell death.