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Protocol no. 15
THE FRAME CYTOTOXICITY TEST (KENACID BLUE)

The cytotoxic effect of chemicals upon cells in culture is measured by the change in total cell protein arising from the inhibition of cell proliferation (Kenacid Blue R dye binding method).

CONTACT

Dr. Richard H.
Clothier Department of Human Morphology
Medical School
Queen's Medical Centre
Clifton Boulevard Nottingham, NG7 2UH UK
Tel: England - 0602 709431 Fax: England - 0602 709732

RATIONALE

Healthy 3T3-L1 cells (an established cell-line, ATCC CCL92.1), when maintained in culture continuously divide and multiply over time. The basis of this test is that a cytotoxic chemical will interfere with this process and, thus, result in a reduction of the growth rate and therefore in reduced cell numbers compared with control cultures. This difference is reflected in the total protein content of the culture. Cells in continuous culture proliferate at a known optimal rate, and this would be reduced by chemicals which affect one or more essential functions, such as mitochondrial activity, DNA synthesis, maintenance of membrane integrity, or protein synthesis. The degree of inhibition of growth, related to the concentration of the test compound, provides an indication of toxicity.

BASIC PROCEDURE

3T3-L1 cells are maintained in culture and exposed to test compounds over a range of concentrations. The cultures are visually examined after 24, 48 and 72 hours, and the number of viable cells and/or the total cell protein content determined, after either 24 or 72 hour exposure, by the Kenacid Blue method. This assay may be performed on cells previously used for the Neutral Red uptake assay as described in INVITTOX Protocol 3a. The number of cells in the presence of test chemicals is compared to that observed in control cultures and the percentage inhibition of growth calculated. The ID20, ID50 and ID80 concentrations (i.e. the concentrations producing 20, 50 and 80% inhibition of growth) are determined and expressed as mg/ml or mM. These values enable a comparison to be made of the relative cytotoxicity of the test compounds.

CRITICAL ASSESSMENT

Cell culture procedure The maintenance and culture of a cell line such as 3T3-L1 cells is a relatively simple and inexpensive technique. The use of such cultures permits rapid, highly reproducible cytotoxicity testing of many chemicals on a routine basis. There are certain limitations of the technique, some of which concern the type of compounds being be tested: Volatile chemicals tend to evaporate under the conditions of the test, thus the ID50 value may be variable, especially when the toxicity of the compound is fairly low. This has been overcome to some extent by adapting the procedure for use in 96-well plates rather than in 24-well plates (Knox et al., 1986; Riddell et al., 1986) as the smaller surface area of the well in these dishes reduces the extent of evaporation. In addition, a method for sealing the plates has been published (Smith et al., 1992). This involves adding 100 ml of mineral oil (Sigma M3516) on top of the culture medium once the test substance has been added to the cultures. This does not affect the normal growth of the cells but does reduce the evaporation rate. It is necessary to take into account the likely partition of the test compound between the aqueous medium and the oil. The respective octanol/water partition coefficient should indicate whether partition is likely to cause problems. Other chemicals which are difficult to test include those which are unstable or explosive in water. Insoluble substances are also unsuitable for testing, although some such compounds have been tested using mineral oil as the solvent. The mineral oil containing the dissolved test compound is layered on top of the cultures. Other difficulties are related to the nature of the cell line, which consists of rapidly growing, non-differentiating cells of very low metabolic activity. This gives rise to problems of direct extrapolation of results to the in vivo situation. The system is likely to underestimate the toxicity of chemicals which require metabolic activation to a toxic intermediary or product. Substances which specifically attack dividing cells may appear to be of a much higher order of toxicity than they would be in vivo. The toxicity of substances which bind to serum proteins (i.e. such as those found in newborn calf serum) may also be underestimated. 24 versus 72 hr exposure period: The procedure may be adapted to enable determination of cytotoxicity of chemicals after an exposure period of 24 or 72 hr. It must be stressed, however, that the longer exposure period should be used routinely. Kenacid Blue R Dye Binding Assay Since Kenacid Blue R is no longer available, Coomassie Brilliant Blue R-250 has now been substituted. One of the drawbacks of this assay is that the dye may, on occasion, precipitate out. The likelihood of this occurring increases as the length of handling time increases, therefore 96-well plates should be agitated regularly and inspected visually for uneven blue colour. The process is, however, readily reversed by agitation, so any odd reading should be retested after trituration to obviate the possibility of precipitation. Another problem which may occur, is the deposition of a ring of dried protein around the walls of the well, at the air/medium interface. This arises if the culture medium is not properly removed through excessive evaporation. Such precipitated protein will give an inaccurate assessment of total cellular protein. Total protein measurement does not make allowances for necrotic cells which may still be attached to the culture dish and, therefore, may underestimate the toxicity of a compound. It should be noted, however, that the occurrence of adhering dead or dying cells is very rare. Advantages of this system include the fact that it can be repeated more than once on the same cells. Furthermore, the cells can be fixed and the staining performed later. The cell distribution can easily be seen with the naked eye after staining and before desorbing, thus giving a rapid indication of the success of the assay. Despite its limitations, the system provides a simple screen for the rapid assessment of the toxicity of compounds. Validation studies have produced results for a wide range of compounds. Data for 150 compounds have been published by Clothier et al. (1988). It would appear that the correlation to the in vivo situation (especially when problems concerning metabolic activation, etc., are taken into account) is fair. When the in vitro cytotoxicities i.e. ID50 values, of 59 chemicals were compared with rat oral and mouse intraperitoneal LD50 values, correlation coefficients of 0.76 and 0.80, respectively, were obtained (Clothier et al., 1989). Under certain conditions, a direct comparison of the Kenacid Blue and the Neutral Red Uptake methods may be of value, for example, certain chemicals such as chloroquine sulphate and other anti-malarial agents which target lysosomes may give different results in the two test systems. In such cases performing both methods will yield extra information.

TEST STATUS

The cytotoxicity test system has undergone a period of in-house development and, more recently, an investigation into its interlaboratory validation has been performed in a collaborative study involving four different centres. The results of this study are presented by Knox et al. (1986). The authors of this paper note that the initial cytotoxicity test employed a human embryonic cell line, BCL-D1, but now uses a mouse embryonic cell line, 3T3-L1. At present, a number of research groups are evaluating the current procedure and comparing its performance to a variety of other test systems in co-operative initiatives set up by the European Commission, and by the Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) scheme organised by the Scandinavian Society of Cell Toxicology.

CHEMICALS TESTED

300 pure chemicals and 200 formulations have now been tested in the system at the FRAME Alternatives Laboratory in Nottingham. The experimental data for 150, together with in vivo comparisons for 59 of these chemicals have been published (Clothier et al., 1988; Clothier et al., 1987; Hulme et al., 1987). The ID20, ID50 and ID80 values (as determined by the Kenacid Blue method) for all 150 chemicals are presented in this Protocol.

REFERENCES

  1. Balls, M., Riddell, R.J., Horner, S.A. & Clothier, R.H. (1987) The FRAME approach to the Development, Validation, and Evaluation of In Vitro Alternative Methods. In: In vitro Methods in Toxicology - Approaches to Validation (ed. Goldberg, A.M.), Alternative Methods in Toxicology 5, (Published Mary Ann Liebert, Inc., New York, USA), pp.45-58.
  2. Clothier, R.H., Hulme, L., Ahmed, A.B.; Reeves, H.L., Smith, M. & Balls, M. (1988) In vitro cytotoxicity of 150 chemicals to 3T3-L1 cells, assessed by the FRAME Kenacid Blue Method. ATLA, 16, 84-95.
  3. Clothier, R.H., Hulme, L., Smith, M. & Balls, M. (1987) A comparison of the in vitro cytotoxicities and acute in vivo toxicities of 59 chemicals. Molecular Toxicology, 1, 571-577.
  4. Hulme, L., Reeves, H.L., Clothier, R.H., Smith, M. & Balls, M. (1987) An assessment of two alternative methods for predicting the in vivo toxicities of metallic compounds. Molecular Toxicology, 1, 589-596.
  5. (N.B. "Molecular Toxicology" although cited as being published in 1987, was published in 1989) Knox, P., Uphill, P.F., Fry, J.R., Benford, D.J. & Balls, M. (1986) The FRAME Multicentre Project on In Vitro Cytotoxicology. Fd. Chem. Tox., 24, 457-463.
  6. Riddell, R.J.; Clothier, R.H. & Balls, M. (1986) An evaluation of three in vitro cytotoxicity assays. Fd. Chem. Toxicol., 24, 469-471. Riddell, R.J., Panacer, D.S., Wilde, S.M., Clothier, R.H. & Balls, M. (1986) The importance of exposure period and cell type in in vitro cytotoxicity tests. ATLA, 14, 86-92.
  7. Smith, L.M., Clothier, R.H., Hillidge, S. & Balls, M. (1992) Modification of the FRAME Kenacid Blue method for cytotoxicity tests on volatile materials. ATLA, 20, 230-234.

IP-3b © July 1992