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Protocol no. 38
LS-L929 CYTOTOXICITY TEST

This simple cell culture-based cytotoxicity test (in which cell viability is determined by uptake of the dyes ethidium bromide and fluorescein acetate) has been developed as a general test for acute toxicity.

CONTACT

Dr. R.B. Kemp
Cell Biology Laboratory
Department of Biological Sciences
University College of Wales
Aberystwyth
Dyfed SY23 3DA UK
Tel: England - 0970 623111 Fax: England - 0970 622350

RATIONALE

This test is based on the premise that cell death is an unequivocal indication of acute toxicity. Mouse fibroblasts grown in suspension are exposed to water-soluble toxic chemicals over a range of concentrations for set periods of time. A resultant cytotoxic effect is quantified using two complementary fluorimetric assay procedures. After exposure to a test compound, viable cells may be identified by their ability to accumulate fluorescein on incubation with fluorescein diacetate (FDA). The nucleic acid of both viable and non-viable cells can be stained with ethidium bromide (EB). Non-viable cells (stained only with EB) may be distinguished from viable cells (stained with EB and with FDA) by the selective use of filters. Although originally developed as an alternative system to the Draize test, the assay has been found to have a more general application to cytotoxicity and to other forms of irritancy.

BASIC PROCEDURE

Mouse fibroblasts are maintained in continuous suspension culture, diluted when required and known volumes incubated in the presence of test material in a range of concentrations for 4 hours. Aliquots are then removed and added to an equal volume of physiological solution containing FDA and EB. The cells are then examined under epi-fluorescent illumination. An image analyser (together with the correct filter systems) enables differentiation between viable and non-viable cells. The endpoint is taken as 50% cell death (CD50) and this is used to rank the potential acute toxicity of test substances.

CRITICAL ASSESSMENT

Choice of endpoint The simplest and most elementary endpoint is cell death, which has been defined as the irreversible cessation of cellular activity and function coupled with irreparable disorganisation of structure (Dixon, 1967). The initial phases of injury are represented by fragmentation of the plasma membrane and changes in mitochondria and endoplasmic reticulum (Malinin & Perry, 1967; Trump et al., 1973). Damage to the cell membrane leads to release of intracellular enzymes (Trump et al., 1973), for instance lactate dehydrogenase (Cornelis et al., 1991), the occurrence of which often has been used as an indicator of cellular damage. It has been speculated, however, that over 100 enzymes may be essential for cell life. Thus, monitoring the absence or presence in culture medium of one of these is unlikely to provide an unequivocal indication of the cell's vital function. Methods of determining cell death include dye exclusion (Kemp et al., 1967), changes in cell morphology (Reinhardt et al., 1985) radioactive chromium release (Parish, 1985) and tritiated thymidine incorporation (Baserga, 1989), each of which present problems for the researcher. The first of these techniques introduces intra-observer variability in visually assessing whether or not the dye has been taken up by a cell. The second suffers from difficulties of inter-observer subjectivity while the other techniques often involve complex, expensive or time consuming assays, without great advantage. In advocating the use of a combination of fluorescent dyes in determining cell death (Kemp et al., 1983; Kemp et al., 1988) the needs for relatively low cost, high degree of accuracy, strict reliability, simplicity and potential for automation were borne in mind. The two complementary dyes are fluorescein diacetate (FDA) and ethidium bromide (EB). Both have been used as viability stains for many years. FDA is a non-polar compound which readily diffuses into the cell when incorporated into the culture medium. Intracellular esterases hydrolyse the dye to produce fluorescein (Rotman & Papermaster, 1966), a negatively charged and, therefore, highly polar molecule which only very slowly diffuses out from intact cells. It fluoresces green under ultra-violet excitation. Rotman & Papermaster (1966) postulated that the influx of FDA was much faster than the extrusion of fluorescein, resulting in its accumulation in cells with intact membranes - healthy cells. It was shown that Saponin-treated cells as well as those unable to form clones, that is to divide, were non-fluorochromatic because they had plasma membranes which were physically punctured and thus were unable to retain the dye. Such cells were deemed to be dead. It is interesting to note that erythrocytes and cells in primary culture originating from certain tissues always failed to exhibit fluorochromasia. A useful property of the reaction producing fluorescein from FDA is a broad pH maximum, pH 6.7-8.0 (Rotman & Papermaster, 1966). Fluorescein liberation only allows the number of viable cells to be determined but of course does not provide an indication of non-viable cells because they cannot be seen under ultra-violet light. For this purpose, the cells are also exposed to a red fluorescent compound, Ethidium Bromide. This binds covalently to nucleic acids and, in particular, intercalates with DNA (Gabby & Wilson, 1978), making it a powerful frame-shift mutagen. Thus, it stains nuclear material, especially chromatin and nucleoli, and is not present on nuclear and plasma membranes (Burns, 1972). It is taken up by both living and dead cells but its rate of penetration into the latter is of course faster, a fact which has been exploited in a spectrofluorimetric assay for cell viability using EB alone (Edidin, 1970). In combination with FDA (Takasugi, 1971), however, viable cells also contain fluorescein, the fluorescence of which partially masks the red colour. Quantitation of non-viable cells is achieved by the selective use of filters. Choice of test system The choice of cell type is, to a certain extent, arbitrary, once it is conceded that this test is a first-order one (Balls & Horner, 1985); that is, designed to rank the acute lethality of pure chemicals and chemical formulations to living matter. Cells are carefully chosen for specific physiological and biochemical properties to form the basis for second-order tests. It has been obviously suggested that primary cultures of human cells would provide ideal systems for assessing cytotoxicity, if results are to be extrapolated to the situation in man. For instance, primary cultures of human eye tissue should be used when potential ocular irritancy is the important parameter under consideration. Quite clearly, the widespread use of such a tissue source is not practicable. Primary culture of cells from selected tissues of other embryonic and adult animals might seem the best substitute because it could be reasoned that such cells would retain their characteristic physiological properties. This proposal has been criticised, however, because the necessary enzyme dissociation and other procedures can result in variability and friability without any scientific certainty of the validity of the model that the properties of animal tissues closely represent those of human tissues. Since cells in primary culture are likely to differ from those in vivo and obtaining them requires considerable use of animals, consideration should be given to utilizing established cells lines (Freshney, 1987), especially for first-order tests. While such cells do randomly lose certain physiological properties during the course of establishment, they subsequently remain constant having been adapted to specific culture conditions. The choice then lies between anchorage-dependent (monolayer) and anchorage-independent (suspension) types of cell. The former generally requires enzyme treatment for release from the substratum and a growth period of time before harvesting at confluency. The latter can be grown as a continuous culture with daily "milking" (generation time 22 hours) for experimentation. It is recommended that an anchorage-independent, suspension-adapted cell line be employed in first-order studies of acute lethality. The particular cell type, that is LS-L929 mouse fibroblasts originally cloned from areolar and adipose tissue, was selected for the ease with which it is maintained in continuous suspension culture. Many cytotoxicity tests require 48-72 hours of culture before determination of endpoint. This test was consciously designed for completion within the working day, so to give a rapid turnover of data. Modification of the endpoint A biochemical indication of toxicity has been incorporated within the procedure which increases the sensitivity, provides more accurate figures and allows automation. ATP measurement was chosen because ATP is the prime energy donor in the cell and is thus an ideal indicator of cellular health (Kemp et al., 1986; 1988). This method detects effects at lower irritant concentrations compared to the viability dye technique and, coupled with the greater intrinsic sensitivity of the luciferase bioluminescence system, provides a more efficient test procedure. Statistically reproducible effects can be obtained from as little as 104 cells (10 ml aliquots) leading to savings in culture costs and an increase in data output. Spectroscopic machines are now fully automated and can be interfaced with microcomputers reducing labour costs thus compensating for the greater cost of reagents. Testing of immiscible and poorly soluble substances The active ingredient of many commercial products is contained in a complex carrier system, e.g. lotion, cream, tablet, etc., at least some of which is insoluble and/or immiscible in aqueous culture medium. These excipients cause technical difficulties to the assay, visually interferring with endpoint estimations. This has necessitated modifications to the procedure involving extraction of water-soluble components of the formulation into culture medium before the 4 hour incubation. Extraction can be carried out in two ways (Kemp et al., 1985), by a variable volume technique or by a bulk method. Mostly, the former is preferred, but both rely upon mobilization of polar compounds into the culture medium.

REFERENCES

  1. Balls, M. & Horner, S.A. (1985) The FRAME interlaboratory programme on in vitro cytotoxicity. Fd. Chem. Toxic., 23, 209-213.
  2. Baserga, R. (1989) Cell Growth and Division Oxford; IRL Press. Bliss, C.I. (1937) The calculation of the time-mortality curve. Annals of Appl. Biol., 24 (4), 815-852.
  3. Burns, V.W. (1972) Localization and molecular characteristics of fluorescent complexes of ethidium bromide in the cell. Exp. Cell Res., 75, 200-206.
  4. Cornelis, M., Dupont, C. & Wepierre, J. (1991) In vitro cytotoxicity tests on cultured human skin fibroblasts to predict the irritation potential of surfactants. ATLA, 19, 324-336.
  5. Dixon, K.C. (1967) Events in dying cells. Proc. Royal Soc. Med., 60, 271-275. Edidin, M. (1970) A rapid quantitative fluorescence assay for cell damage by cytotoxic antibodies. J. Immunology, 104, 1303-1306.
  6. Freshney, R.I. (1987) Culture of Animal Cells and Manual of Basic Techniques. 2nd edition New York; A.R. Liss. Gabby, E.J. & Wilson, W.D. (1978) Intercalating agents as probes of chromatin structures. Methods Cell. Biol., 18, 351-384.
  7. Kemp, R.B., Jones, B.M., Cunningham, I.& James, M.C.M. (1967) Quantitative investigation on the effect of puromycin on the aggregation of trypsin- and versene-dissociated chick fibroblast cells. J. Cell Sci. 2, 323-340.
  8. Kemp, R.B., Meredith, R.W.J., Gamble, S. & Frost, M. (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. Cytobios., 36, 153-159.
  9. Kemp, R.B., Meredith, R.W.J. & Gamble, S.H. (1985) Toxicity of commercial products on cells in suspension culture : A possible screen for the Draize Eye Irritation Test. Fd. Chem. Toxic., 23, 267-270.
  10. Kemp, R.B., Cross, D.M. & Meredith, R.W.J. (1986) Adenosine triphosphate as an indicator of cellular toxicity in vitro. Fd. Chem. Toxic. 24, 465-466.
  11. Kemp, R.B., Cross, D.M. & Meredith, R.W.J. (1988) Comparison of cell death and adenosine triphosphate as indicators of acute toxicity in vitro. Xenobiotica 18, 633-639.
  12. Litchfield, J.T. Jr. (1949) A method of time - per cent effect curves. J. Pharm. Exp. Ther., 97, 399-406. Litchfield, J.T. Jr. & Wilcoxon, F. (1949) A simplified method of evaluating dose-effect curves. J. Pharm. Exp. Ther., 96, 99-113.
  13. Malinin, T.I. & Perry, P. (1967) A review of tissue and organ viability assay. Cryobiol., 4 (3), 104-115.
  14. Parish, W.E. (1985) Relevance of in vitro tests to in vivo acute skin inflammation: potential in vitro applications of skin keratome slices, neutrophils, fibroblasts, mast cells and macrophages. Fd. Chem. Toxic. 23, 275-288.
  15. Reinhardt, Ch.A., Pelli, D.A. & Zbinden, G. (1985) Interpretation of cell toxicity data for the estimation of potential irritation. Fd. Chem. Toxic. 23, 247-252. Rotman, B. & Papermaster, B.W. (1966) Membrane properties of living mammalian cells as studied by enzymatic hydrolysis of fluorogenic esters. Proc. Nat. Acad. Sci. (USA), 55, 134-141.
  16. Takasugi, M. (1971) An improved fluorochromatic cytotoxic test. Transplantation, 12, 148-151. Trump, B.F., Valigorsky, J. & Dees, J. (1973) The modernisation of the autopsy: applications of ultrastructural and biochemical methods to human disease. Med. Coll. Va. Q., 9, 323-333.
  17. Ulrich, K. & Moore, G.E. (1965) A vibrating mixer for agitation of suspension cultures of mammalian cells. Biotechnol. Bioeng., 7, 417.

IP-38 © July 1992