Cytotoxicity Studies of Anchorage-independent LS-L-929 Mouse Fibroblasts Using Membrane Integrity,ATP Content and ATP/ADP Ratio as Determinants: Including the Xenobiotic Effects of Components in an Experimental Shampoo Formulation
Restricted accessResearch articleFirst published online October, 1993
Cytotoxicity Studies of Anchorage-independent LS-L-929 Mouse Fibroblasts Using Membrane Integrity,ATP Content and ATP/ADP Ratio as Determinants: Including the Xenobiotic Effects of Components in an Experimental Shampoo Formulation
Cell death (CD50), as measured by loss of membrane integrity using fluorescein diacetate and ethidium bromide, and reduction in ATP content (ATP50), were used as endpoints in a comparative study of the cytotoxicity of 40 chemicals (supplied by FRAME) to anchorage-independent LS-L-929 mouse fibroblasts. The rank order correlation of the two tests was strong (r = 0.98), but the ATP50 values only showed a weak correlation with rat acute lethality (LD50) data (r = 0.43). More-mechanistic endpoints were sought in order to characterise cellular energy status, and the effects of acetaminophen, acetylsalicylic acid, diazepam, digoxin and ethanol on ATP/ADP ratio, phosphorylation potential and adenylate energy charge were examined in detail. The first two ratios were very sensitive to toxic insult to the cells and gave results which more closely mimicked differences in human acute oral lethality than did the ATP50 and CD50. In addition, there was evidence that digoxin and diazepam affected oxidative phosphorylation, which suggested an alteration to mitochondrial function. Energy charge appeared less sensitive to the xenobiotics, probably owing to the dampening effect of the denominator in the ratio.
The effects of individual components of an experimental detergent-based formulation on cell death were examined. It was found that one detergent gave different results, depending on the presence or absence of a second detergent. A commercial cream containing varying amounts of glyceryl monostearate contaminated with varying levels of sodium dodecyl sulphate gave results which reinforced the point that cytotoxic assays are valuable in quality assurance.
BallsM., and BridgesJ. (1984). The FRAME Research Programme on in vitro cytotoxicology. In Alternative Methods in Toxicology, Vol II, Acute Toxicity Testing: Alternative Approaches (ed. GoldbergA.M.), pp. 61–75. New York: Mary Ann Liebert.
2.
BallsM., and HornerS.A. (1985). The FRAME interlaboratory programme on in vitro cytotoxicology.Food and Chemical Toxicology23, 209–213.
3.
KnoxP., UphillP.F., FryJ.R., BenfordD., and BallsM. (1986). The FRAME multicentre project on in vitro cytotoxicology.Food and Chemical Toxicology24, 457–463.
4.
ClothierR.H., HulmeL., AhmedA.B., ReevesH.L., SmithM., and BallsM. (1988). In vitro cytotoxicity of 150 chemicals to 3T3-L1 cells, assessed by the FRAME kenacid blue method.ATLA16, 84–95.
5.
ShopsisC., and SatheS. (1984). Uridine uptake as a cytotoxicity test.Toxicology29, 195–204.
6.
ShawA.J., ClothierR.H., and BallsM. (1990). Loss of trans-epithelial impermeability of a confluent monolayer of Madin-Darby canine kidney (MDCK) cells as a determinant of ocular irritancy potential.ATLA18, 145–151.
7.
BorenfreundE., and PuernerJ.A. (1985). Toxicity determined in vitro by morphological alterations and neutral red absorption.Toxicology Letters24, 119–124.
8.
BabichH., and BorenfreundE. (1990). Applications of the neutral red cytotoxicity assay to in vitro toxicology.ATLA18, 129–144.
9.
ReinhardtC.A., PelliD.A., and ZbindenG. (1985). Interpretation of the cell toxicity data for the estimation of potential irritation.Food and Chemical Toxicology23, 247–252.
10.
KempR.B., MeredithR.W.J., GambleS., and FrostM. (1983). A rapid cell culture technique for assessing the toxicity of detergent-based products in vitro as a possible screen for eye irritancy in vivo.Cytobios36, 153–159.
11.
DraizeJ.H., WoodwardG., and CalveryH.O. (1944). Methods for the study of irritation and toxicity of substances applied topically to the skin and mucous membrane.Journal of Pharmacology and Experimental Therapeutics82, 377–390.
12.
TakasugiM. (1971). An improved fluorochromatic cytotoxic test.Transplantation12, 148–151.
13.
KempR.B. (1993). Cytotoxicity of an anchorage-independent fibroblast cell line as measured by a combination of fluorescent dyes. In In Vitro Toxicity Testing Protocols (ed. AtterwillC.K., & O'HareS.O.). New York: Humana Press, in press.
14.
KempR.B., MeredithR.W.J., and GambleS.H. (1985). Toxicity of commercial products on cells in suspension culture: a possible screen for the Draize eye irritation test.Food and Chemical Toxicology23, 267–270.
15.
KempR.B., CrossD.M., and MeredithR.W.J. (1986). Adenosine triphosphate as an indicator of cellular toxicity in vitro.Food and Chemical Toxicology24, 465–466.
16.
KempR.B., CrossD.M., and MeredithR.W.J. (1988). Comparison of cell death and adenosine triphosphate content as indicators of acute toxicity in vitro.Xenobiotica18, 633–639.
17.
PurchaseI.F.H., FarrarD.G., and WhitakerLA. (1987). Toxicology profiles on substances used in the FRAME cytotoxicology research project.ATLA14, 184–242.
18.
AtkinsonD.E. (1977). Cellular Energy Metabolism and its Regulation, 121 pp. New York: Academic Press.
19.
ErecińskaM., and WilsonD.F. (1978). Homeostatic regulation of cellular energy metabolism.Trends in the Biochemical Sciences3, 219–223.
20.
BondessonI., EkwallB., HellbergS., RomertsL., StenbergK., and WalumE. (1989). MEIC: a new international multicenter project to evaluate the relevance to human toxicity of in vitro cytotoxicity tests.Cell Biology and Toxicology5, 331–347.
21.
StarkD.M., ShopsisC., BorenfreundE., and BabichH. (1986). Progress and problems in evaluating and validating alternative assays in toxicology.Food and Chemical Toxicology24, 449–455.
22.
DaveyC.L., KellD.B., KempR.B., and MeredithR.W.J. (1988). On the audio- and radio-frequency dielectric behaviour of anchorage-independent mouse L929-derived LS fibroblasts.Bioelectrochemistry and Bioenergetics20, 83–90.
23.
RotmanB., and PapermasterB.W. (1966). Membrane properties of living mammalian cells as studied by enzymatic hydrolysis of fluorogenic esters.Proceedings of the National Academy of Sciences of the United States of America55, 134–141.
24.
EdidinM. (1970). A rapid quantitative fluorescence assay for cell damage by cytotoxic antibodies.Journal of Immunology104, 1,303–1,306.
25.
LitchfieldJ.T.Jr., WilcoxonF. (1949). A simplified method of evaluating dose-effect experiments.Journal of Pharmacology and Experimental Therapeutics96, 99–107.
26.
DeLucaM.A., and McElroyW.D. (1981). Bioluminescence and Chemiluminescence, 122 pp. New York and London: Academic Press.
27.
ThoreA., ÅnsehnA., LundinA., and BergmanS. (1975). Detection of bacteriuria by luciferase assay of adenosine triphosphate.Journal of Clinical Microbiology1, 1–8.
DunnJ.P.D., and KempR.B. (1975). Isotachophoretic studies of adenosine phosphates and divalent cations of perfused mouse liver cells.Protides of the Biological Fluids22, 727–732.
30.
EkwallB., BondessonI.K., CastellJ.V., Gómez-LechónM.J., HellbergS., HögbergL, JoverR., PonsodaX., RomertL., StenbergK., and WalumE. (1989). Cytotoxicity evaluation of the first ten MEIC chemicals: acute lethal toxicity in man predicted by cytotoxicity in five cellular assays and by oral LD50 tests in rodents.ATLA17, 83–100.
31.
HarewoodK., and WolffJ.S. (1973). Detection of sodium dodecyl sulphates in PAGE eluates.Analytical Biochemistry55, 573–579.
32.
KempR.B. (1982). Toxicity of commercial products in vitro. In The Responsibilities and Ethics of Product Testing (ed. HarrisonR.), pp. 87–104. London: SCS.
33.
KempR.B., MeredithR.W.J., GambleS., and FrostM. (1983). Toxicity of detergent-based commercial products on cells of a mouse line in suspension culture as a possible screen for the Draize rabbit eye irritation test.ATLA11, 15–21.
34.
ScaifeM.C. (1982). An investigation of detergent action on cells in vitro and possible correlations with in vivo data.International Journal of Cosmetic Science4, 179–193.
35.
ScaifeM.C. (1985). An in vitro cytotoxicity test to predict the ocular irritation potential of detergents and detergent products.Food and Chemical Toxicology23, 253–258.
36.
AeschbackerM., ReinhardtC.A., and ZbindenG. (1986). A rapid cell membrane permeability test using fluorescent dyes and flow cytometry.Cell Biology and Toxicology2, 247–258.
37.
GettingsS.D., and McEwenG.N. (1990). Development of potential alternatives to the Draize eye test. The CTFA Evaluation of Alternatives Program.ATLA17, 317–324.
38.
PlattK.L., UteschD., Gemperlein-MertesI., GlattH.R., and OeschF. (1986). Metabolizing systems in short-term in vitro tests for carcinogenicity.Food and Chemical Toxicology24, 721–729.
39.
BabichH., Martin-AlguacilN., and BorenfreundE. (1987). Mediating role of metabolic activation in in vitro cytotoxicity assays.Molecular Toxicology1, 363–372.
40.
MarinovichM., VivianiB., and GalliC.L. (1990). Reversibility of TBT-induced protein synthesis inhibition after ATP recovery in HEL/30 cells.Toxicology Letters52, 311–317.
41.
WilcoxD.K., and BrunerL.H. (1990). In vitro alternatives to ocular safety testing: an outline of assays and possible future developments.ATLA18, 117–128.
42.
HellbergS., ErikssonL., JonssonJ., LindgrenD., SjöstromM., and WoldS. (1990). Analogy models for prediction of human toxicity.ATLA18, 103–116.
43.
SeibertH., GüldenM., KolossaM., and SchepersG. (1992). Evaluation of the relevance of selected in vitro toxicity test systems for acute systemic toxicity.ATLA20, 240–245.
44.
GuldenM., and FingerJ. (1992). Effects of the first twenty MEIC reference chemicals on viability, glucose consumption and spontaneous contractility of primary cultured rat skeletal muscle cells.ATLA20, 222–225.
45.
ErecińskaM., StubbsM., MiyataY., DitreC.M., and WilsonD.F. (1977). Regulation of cellular metabolism by intracellular phosphate.Biochimica et Biophysica Acta462, 20–35.
46.
LehningerA.L. (1982). Principles of Biochemistry, 174 pp. New York: Worth.
47.
CassidyS.L., HenryJ.A., and HafiziS. (1989). Antagonist studies using the erythrocyte osmotic fragility membrane stabilization assay.Toxicology in Vitro3, 1–5.
48.
Anon. (1990). Directive 6397/04/P2T/92. European Community DGXIV, July 1990, 92 pp. Brussels, Belgium.
49.
IndoratoA.M., SnyderK.B., and UsinowiczP.J. (1984). Toxicity screening using Microtox™ analyses.Drug Chemistry and Toxicology1, 37–53.
50.
FrazierJ.M., GadS.C., GoldbergA.M., and McCulleyJ.P. (1987). In Alternative Methods in Toxicology, Vol. IV, A Critical Evaluation of Alternatives to Ocular Limitation Testing (ed. GoldbergA.M.). New York: Mary Ann Liebert.
51.
KempR.B., BeckN.F.G., MeredithR.W.J., and GambleS.A. (1990). Bull sperm as a potential model system for cytotoxicity in vitro.Toxicology in Vitro4, 47–50.
