A novel cytotoxicity screening assay using a multiwell fluorescence scanner

https://doi.org/10.1016/0041-008X(92)90317-LGet rights and content

Abstract

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.

References (50)

  • T. Mosmann

    Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays

    J. Immunol. Methods

    (1983)
  • A.-L. Nieminen et al.

    Calcium dependence of bled formation and cell death in hepatocytes

    Cell Calcium

    (1988)
  • A.-L. Nieminen et al.

    Toxic injury from mercuric chloride in rat hepatocytes

    J. Biol. Chem.

    (1990)
  • A.-L. Nieminen et al.

    Toxic injury from mercuric chloride in rat hepatocytes

    J. Biol. Chem.

    (1990)
  • A.-L. Nieminen et al.

    Protection by acidotic pH and fructose against lethal injury to rat hepatocytes from mitochondrial inhibitors, ionophores and oxidant chemicals

    Biochem. Biophys. Res. Commun.

    (1990)
  • C.A. Rahn et al.

    Assessment of mitochondrial membrane potential as an indicator of cytotoxicity

    Fundam. Appl. Pharmacol.

    (1991)
  • P.O. Seglen

    Preparation of isolated rat liver cells

    Methods Cell Biol.

    (1976)
  • L. Spangberg

    Kinetic and quantitative evaluation of material cytotoxicity in vitro

    Oral Surg.

    (1973)
  • J.M. Weinberg et al.

    Mitochondrial bioenergetics during the initiation of mercuric chloride-induced renal injury

    J. Biol. Chem.

    (1982)
  • J.R. Whitaker et al.

    Chemical modification of papain. 1. Reaction with the chloromethyl ketones of phenylalanine and lysine and with phenylmethylsulfonyl fluoride

    Arch. Biochem. Biophys.

    (1968)
  • D. Adkison et al.

    Role of free radicals in ischemia-reperfusion injury to the liver

    Acta Physiol. Scand. Suppl.

    (1986)
  • B.N. Ames et al.

    Carcinogens as frameshift mutagens: Metabolites and derivatives of 2-Acetylaminofluorene and other aromatic amine carcinogens

  • J.M. Bond et al.

    Recovery of cultured rat neonatal myocytes from hypercontracture after chemical hypoxia

    Res. Commun. Chem. Pathol. Pharmacol.

    (1991)
  • E. Borenfreund et al.

    A simple quantitative procedure using monolayer cultures for cytotoxicity assays

    J. Tissue Cult. Methods

    (1984)
  • J.M. Braughler et al.

    Novel 21-amino steroids as potent inhibitors of iron-dependent lipid peroxidation

    J. Biol. Chem.

    (1987)
  • Cited by (0)

    This work was supported, in part, by Grants AG-07218 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 preseted at the Seventy-third Annual Meeting of the Federation of American Societies for Experimental Biology, New Orleans, LA, March 19–23, 1989 (Nieminen et al., 1989), and at the Thirtieth Annual Meeting of the Society of Toxicology, Dallas, TX, February 25–March 1, 1991 (Lemasters et al., 1991).

    2

    Current address: Center for Basic Research in Digestive Diseases, Mayo Clinic, Rochester, MN 55905.

    3

    Current address: II Rianimazione IRCCS Policlinico S. Matteo, 27100 Pavia, Italy.

    View full text