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 Protocol no. 3.

THE FRAME MODIFIED NEUTRAL RED UPTAKE CYTOTOXICITY TEST

The cytotoxic effect of chemicals upon cells in culture is measured by cell viability (neutral red uptake) 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 

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 (regardless of site or mechanism of action) will interfere with this process and, thus, result in a reduction of the growth rate as reflected by cell number. 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 hours exposure, by the Neutral Red Uptake method. The nature of the assay is such that both this and the kenacid blue assay can be performed on the same cultures provided that the Neutral Red Uptake (an indication of the number of viable cells) determination is performed first. 
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 & 80% inhibition of growth) are determined and expressed as mg/ml or mM. These values enable a comparison 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 application of such cultures to determine cytotoxicity enables the rapid, highly reproducible, testing of many chemicals on a routine basis. There are certain limitations of the technique, some of which concern the character of the compounds to 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 rather than 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. Other chemicals which are difficult to test include those which are unstable or explosive in water. Insoluble substances are also unsuitable for testing, although the author has adapted the method for use with some compounds, using vegetable oil as the solvent. Other difficulties are related to the nature of the cell line, i.e. rapidly growing, non-differentiating cells of very low metabolic activity, hence raising 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 be also underestimated.24 versus 72 hour exposure period. The procedure may be adapted to enable determination of cytotoxicity of chemicals after an exposure period of either 24 or 72 hours. The authors would stress, however, that they consider the longer exposure period should be used routinely.Neutral Red Uptake Assay Neutral Red is preferentially taken up into the lysosomes/endosomes of the cell. Any chemical having a localised effect upon the lysosomes/endosomes will, therefore, result in an artificially low (or possibly high) reflection of cell viability and cell number. This factor does, however, make the system useful to detect those chemicals which selectively affect the lysosomes, especially when it is used in conjunction with other tests capable of determining cell number.

One major drawback of the assay is the precipitation of the Neutral Red dye into readily visible, fine, needle-like crystals. When this occurs it is almost impossible to reverse, thus producing inaccurate readings. Some chemicals induce this precipitation, therefore, a visual inspection stage in the procedure is very important, (especially when carrying out the assay in 96-well plates due to increased handling time). The advantages and disadvantages of the Neutral Red Uptake assay are presented above. A direct comparison of the Kenacid Blue and the Neutral Red methods may also be of value to someone considering choice of endpoint: Once initiated the Neutral Red Uptake assay must be completed, i.e. once the cells have been incubated with the Neutral Red and the dye is taken up into the lysosomes, the process of fixing and destaining must follow on immediately.
One disadvantage of the Neutral Red assay is the possibility that deceptively low cell viability or cell number readings will result in those cases where a chemical has a relatively selective effect upon the lysosomes/endosomes of the cell. An example of this would be chloroquine sulphate which alters the pH of lysosomes/endosomes, an effect which inhibits Neutral Red uptake. One advantage of Neutral Red assay is that it detects only viable cells.It is possible to perform both the Kenacid Blue and the Neutral Red assays on the same culture, i.e. Neutral Red estimates can be obtained and, as the cells are by then fixed, protein determination can be made using the Kenacid Blue method. Performing both assays would provide a means of checking the sensitivity of the Neutral Red assay, when a chemical is suspected of affecting the lysosomes.

TEST STATUS

The Neutral Red Uptake cytotoxicity test system is at present used by a number of research groups. The test is being used in co-operative schemes to compare different results, including those run by the European Commission, and the Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) scheme organised by the Scandinavian Society of Cell Toxicology.

CHEMICALS TESTED

100 pure chemicals and 20 formulations have now been tested in the system by these authors. Results have been published using this method by a number of authors; our results are included under Reddell et al., 1986.


Dibutyltin dichloride 
Tri-n-butyltin chloride 
Benzalkonium chloride 
Silver nitrate 
Mercury (II) chloride 
Brij 35 
SDS 
n-Hexane 
Fluorescein 
Toluene 
Chloroform 
Acetaldehyde 
Triethanolamine 
Triacetin 
Acetic acid 
Sodium hydroxide 
n-Butanol 
2-Methox8yethanol 
Methyl sulphoxide 
Glycerol 
2-Butoxethyl acetate 

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. Borenfreund, E. & Borrero, O. (1984) In vitro cytotoxicity assays: potential alternatives to the Draize ocular irritancy test. Cell Biol. Toxicol., 1, 55-65.
  3. Borenfreund, E. & Puerner, J.A. (1985) Toxicity determined in vitro by morphological alterations and Neutral Red absorption. Toxicology Lett., 24, 119-124.
  4. Riddell, R.J., Clothier, R.H. & Balls, M. (1986) An evaluation of three in vitro cytotoxicity assays. Fd. Chem. Toxicol., 24, 469-471.
  5. 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. 
IP-3A© 1991