TOXICOLOGY AND APPLIED PHARMACOLOGY 115, 147-155 ( 1992) A Novel Cytotoxicity Screening Assay Using a Multiwell Fluorescence Scanner’ ANNA-LIISA NIEMINEN, GREGORY J. GORES,’ JOHN M. BOND, ROBERTOIMBERTI,~ BRIAN HERMAN, AND JOHN J. LEMASTERS~ Laboratories .for Cell Biology: Department yf Cell Biology & Anatomy. School qf‘kfedicine. Universitv of North Carolina ut Chapel Hill, Chapel Hill. North Carolina 27599-7090 Received December 5. 199 I; accepted March 23. I992 A Novel Cytotoxicity Screening AssayUsing a Multiwell Flu- orescenceScanner. NIEMINEN, A.-L., GORES,G. J., BOND, J.M., IMBERTI,R., HERMAN,B., ANDLEMASTERS, J.J,(1992). Toxicol. Appl. Pharmacol. 115, 147-155. A new assayusing a multiwell fluorescence scannerwas de- veloped for screeningcytotoxicity to cells cultured in 96well microtiter plates. The assay is basedon binding of propidium iodide to nuclei of cells whoseplasma membranes have become permeable due to cell death. Fluorescence of propidium iodide measuredwith a multiwell fluorescencescanner increased in proportion to the number of permeabilizedcells. After ATP de- pletion of hepatocytes and neonatal cardiac myocyteswith met- abolic inhibitors (“chemical hypoxia”), and exposureof Madine Darby canine kidney cells to the toxic chemical, HgClz, propid- ium iodide fluorescenceprogressively increased. Increases of fluorescence were linearly proportional with releaseof lactate dehydrogenase into the culture medium. Employing this cyto- toxicity screeningassay, protection by various agents against lethal injury was evaluatedin cultured hepatocytes during chem- ical hypoxia. Inhibitors of cysteine proteases (i.e., antipain, leu- peptin, E-64), serine proteases (i.e., PMSF), and aspartic acid proteases (i.e., pepstatin A) did not protect against chemical hypoxia. In contrast, l,lO-phenanthroline, an inhibitor of me- talloprotease,markedly protected againstthe onsetof cell death during chemicalhypoxia. Half-maximal protection after 60 min occurred at 0.5 PM. Phospholipase inhibitors, chlorpromazine (50 pM) and mepacrine(50 PM), also substantially retarded cell killing. U74006F, an inhibitor of lipid peroxidation, slowed cell ’ This work was supported. in part, by Grants AGO72 I8 and DK-30874 from the National Institutes of Health and Grant J- 1433 from the Office of Naval Research. Preliminary reports of portions of this work were presented at theSeventy-third Annual Meeting of theFederation of American Societies for Experimental Biology, New Orleans, LA, March 19-23. 1989 (Nieminen et a/.. 1989). and atthe Thirtieth Annual Meeting of the Society ofToxicology. Dallas. TX. February 25-March 1, 1991 (Lemasters et al., 1991). ’ Current address: Center for Basic Research in Digestive Diseases, Mayo Clinic, Rochester. MN 55905. ’ Current address: II Rianimazione IRCCS Policlinico S. Matteo, 27 100 Pavia. Italy. 4 To whom correspondence should be addressed at Laboratories for Cell Biology. Department of Cell Biology & Anatomy, School of Medicine. Uni- versity of North Carolina. Campus Box 7090,236 Taylor Hall. Chapel Hill. NC 27599-7090. killing to a lesser extent during chemical hypoxia and after ox- idative stress with t-butyl hydroperoxide. Calciphor, a dimer of prostaglandin B,, did not protect against ceil killing during chemical hypoxia or t-butyl hydroperoxide toxicity. In conclu- sion, this high capacity cytotoxicity assay for cells cultured in 96-well microtiter plates is suitable for rapid screeningof po- tential cytoprotective agents in a variety of cell types. ,C 1992 Academic Press. Inc. Each year, thousands of new chemical compounds are synthesized by the chemical and pharmaceutical industries. Compounds showing economic promise must be screened for toxicity. Initial screening determines the dose that pro- duces 50% killing of test animals. Such testing is expensive, consumeslarge numbers of animals, and has been criticized as inhumane. In response to these issues, manufacturers of pharmaceuticals, pesticides,cosmetics, and household prod- ucts are evaluating methods to reduce the number of animals used in toxicity screening (Holden, 1989). Increasingly, manufacturers view methods that employ cell and tissuecul- ture techniques as economically and scientifically preferable. In particular, the Ames test is widely employed as a prelim- inary assay for mutagenicity and carcinogenicity (Ames et al., 1972). A variety of biochemical and morphological tests have been employed to assess cytotoxicity of chemical substances in vitro. Because the plasma membrane is essential to cell function and viability, most common indicators of cell vi- ability monitor plasmamembrane integrity. Nuclear staining by membrane-impermeant dyes (e.g.. trypan blue, ethidium bromide. propidium iodide), releaseof trapped cytoplasmic probes (e.g., fluorescein, “Cr-labeled protein) and leakage of intracellular enzymes (e.g., lactate dehydrogenase, trans- aminases, creatine phosphokinase) all have been accepted as appropriate indicators of irreversible cell damage (Spangberg, 1973: Bhuyan et al.. 1976; Story et al., 1983; Green et al.. 1984: Cheung et al., 1985: Gores et al., 1988). Indeed, the plasma membrane literally ruptures at the onset of cell death. This irreversible event is followed quickly by loss of cytosolic probes and enzymes and by nuclear labeling with extracel- 147 0041-008X/92 $5.00 Copyright Gl 1992 by Academic Press. Inc. All rights of reproduction in any form reserved.