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Protocol no. 39
V79 CYTOTOXICITY TEST FOR MEMBRANE DAMAGE

The cytotoxic effect of test chemicals in V79 cell culture can be determined by assessing damage to the plasma membrane as determined by a nucleic acid leakage assay.

CONTACT

Professor Vera Bianchi Dept. of Biology (Cellular Biology Section) University of Padova Via Trieste 75 35121 Padova Italy Tel: Italy - 49 8286272 Fax: Italy - 49 8286261

RATIONALE

The plasma membrane is the first point of contact between cells and xenobiotics. Impairment of its molecular organisation with consequent changes in permeability is a direct indicator of damage. The size of the intracellular components which leak out from the cells relates to the degree of damage that has occurred and can therefore form the basis of tests designed to assess membrane-directed toxic effects. Preincubation of cells with [3H] adenine results in the tracer being incorporated into the ATP pool via the salvage enzyme adenine phosphoribosyl-transferase, and ultimately into RNA and DNA following the reduction of ADP to dADP by ribonucleotide reductase. Radioactivity is thus distributed between the soluble nucleotide pool and the macromolecular nucleic acids. By varying the duration and timing of this preincubation with respect to exposure of cells to the test substance, it is possible to assess the degree of membrane damage caused. If exposure follows on immediately after a short preincubation, the label will be present mainly within the soluble nucleotides and will be released into the medium after only moderate membrane damage. However if preincubation is timed so as to allow most of the isotope to be incorporated into macromolecular nucleic acids, the presence of radioactivity in the medium will indicate more extensive lesions. Furthermore, a comparison of isotope distribution between the various subcellular fractions after exposure to the test substance may provide information on the mechanism of cytotoxicity.

BASIC PROCEDURE

Hamster cell monolayers are subjected to one of two protocols. In protocol A they are preincubated with [3H] adenine for 1 hour and then exposed immediately to the test substance. In protocol B they are preincubated with radioactive adenine for 24 hours and left in non-radioactive medium for a further 24 hours before being exposed to the test substance. After exposure, the distribution of radioactivity is determined in the following fractions: medium (both in total medium and in the acid-precipitate); intracellular soluble nucleotide pool; intracellular macromolecules. The distribution of radioactivity between these fractions in test and control cultures is compared in order to estimate the degree of membrane damage.

CRITICAL ASSESSMENT

The use of tritiated adenine in the procedure described here results in an assay sensitive enough to detect toxic effects even at very low levels of membrane damage which would not be detectable by other methods. The protocol is technically simple, highly reproducible and suited to any chemical that affects the integrity of the plasma membrane. However, the procedure does necessarily bear the disadvantages associated with the handling of radioactive materials. The use of a cultured cell line allows multiple assays to be performed rapidly and under consistent conditions. When using the protocol that requires only a short preincubation with the adenine, followed immediately by exposure to the test substance, the use of exponentially growing cells is recommended. This is because nucleotide metabolism is more active in such cells, and thus precursor uptake is more efficient. In addition, the authors have noticed that V79 cells treated with LAS are more resistant to the detergent at high cell density, probably due to less of the cell surface actually being exposed. Assessment of membrane damage Tests which identify membrane damage by assessing changes that have occurred in membrane permeability may be grouped into two main categories: dye exclusion/retention tests, and those measuring the release of cellular components into the incubation medium. The use of e.g. Trypan blue staining will provide gross estimates of alterations in membrane permeability in a population of cells, but changes may occur with respect to small molecules before the cells lose their ability to exclude the dye. Furthermore, cells that are taking in the dye may exhibit different degrees of membrane lesion. The second category is able to provide more information since the size of the leaked material indicates the size of the "holes" in the membrane, and identification of the types of molecules that are being lost may add to interpretation of the toxic effect that is taking place. While some currently available techniques, such as high pressure liquid chromatography and atomic absorption spectrometry, can detect leakage of small molecules and ions without the use of a tracer, such methods are time-consuming and thus not appropriate for routine use in large-scale screening of chemicals. More suitable for this purpose are assays which measure radioactivity released into the medium from cells which have been preincubated with labelled physiological precursors such as nucleosides, bases, amino acids, or non-metabolisable analogues. A nucleic acid precursor was chosen as a tracer for several reasons. There is no competition with medium ingredients, as occurs for example with the glucose analogues that have been used in leakage tests (Walum & Peterson, 1982; Malik et al., 1983). The test may therefore be carried out in culture medium rather than in saline, which ensures that the cell metabolism remains as normal as possible. The spontaneous release back into the medium that occurs with tracers that are not metabolised, e.g. the amino acid analogue amino-isobutyric acid (used by Thelestam & M”llby, 1975b; Malik et al., 1983) is not a great problem because this tracer is quickly phosphorylated and further metabolised. Although polymerisation of the labelled precursor into macromolecular nucleic acids removes radioactivity from the soluble nucleotide pool, this may be used to advantage since a further group of damage indicators, i.e. labelled nucleic acids of high molecular weight, is available to detect more extensive membrane damage. This may substitute for the use of enzymes, especially where low endogenous levels of the indicator enzyme or the exposure conditions for a given test chemical do not allow the use of an enzyme leakage assay, e.g. in the case of V79 cells exposed to LAS (Bianchi & Fortunati, 1990). Adenine was chosen in preference to other nucleic acid precursors because it is incorporated into the ATP pool which is the largest nucleotide pool and therefore makes a larger quantity of radioactivity available for release into the medium after membrane damage. In itself, the presence of radioactivity in the extracellular environment does not prove loss of adenine nucleotides. However, this may be inferred from the presence of acid-precipitable, RNase-sensitive labelled material in the medium which demonstrates RNA leakage accompanied by nucleotide release. Quantitative determination of nucleic acid release The two experimental protocols described allow the detection of different degrees of membrane damage. They are modified from those of Thelestam & M”llby (1975a,1976). The modified procedure is faster and, besides indicating the size of "functional holes" in the membrane, provides a detailed picture of cytotoxicity with respect to the stability and metabolism of nucleic acids. In protocol A exposure to test chemicals takes place immediately after the cells have been incubated for a short period with tritiated adenine. At this stage, and under the described conditions, 75% of total cell radioactivity is found within the soluble ATP pool. Radioactivity in the medium after exposure to the test substance indicates leakage of small molecules, i.e. minor membrane damage. It was found that the counts in the medium fraction exceeded those lost from the nucleotide pool because leakage of macromolecular nucleic acids had also occurred. However, since exposure had taken place while the cells were still metabolising the tracer, a direct estimate of the amount of RNA lost was not possible. For this reason, a second protocol was developed. In protocol B, the cells are preincubated for 24 hours with the label, and then kept for a further 24 hours in non-radioactive medium before being exposed to the test substance. This allows for about 85% of the cell radioactivity to be incorporated into the macromolecular nucleic acids (DNA and RNA) and for the metabolism of the radioactive precursor to reach a plateau. Since incorporation of the label is complete, its distribution between the various cell fractions will be the same in all cells. Changes in this distribution will provide information on the effects of the test substance on the various cell components (RNA, DNA, soluble nucleotides). The amount of radioactivity present in the DNA of the cell monolayers is a direct measure of the cells remaining attached to the substrate, i.e. it indicates whether any cells became detached after exposure to the test substance. The counts lost from the RNA fraction are directly related to the degree of cytotoxicity as exposure occurs when RNA labelling is homogenous in all the cultures. The acid-precipitated labelled material in the medium indicates more extensive membrane damage, however the counts of this fraction would in themselves tend to underestimate the damage. If the counts of this fraction cannot account for the counts lost from intracellular macromolecular RNA, it is possible to assume that exposure to the test substance has resulted in degradation of RNA, for example by the release of lysosomal nucleases.

TEST STATUS

In-house development.

CHEMICALS TESTED

Linear alkyl benzene sulphonate
Sodium dodecyl sulphonate
Triton-X-100
Potassium dichromate
Ethyl methane sulphonate

REFERENCES

  1. Thelestam, M. & M”llby, R. (1975a) Determination of toxin-induced leakage of different-sized nucleotides through plasma membrane of human diploid fibroblasts. Infect. Imm., 11, 640-648.
  2. Thelestam, M. & M”llby, R. (1975b) Sensitive assay for detection of toxin-induced damage to the cytoplasmic membrane of human diploid fibroblasts. Infect. Imm. 12, 225-232.
  3. Thelestam, M., and M”llby, R. (1976) Cytotoxic effects on the plasma membrane of human diploid fibroblasts : A comparative study of leakage tests. Med. Biol. 54, 39-49.
  4. Walum, E. & Peterson, A. (1982) Tritiated 2-deoxy-D-glucose as a probe for cell membrane permeability studies. Anal. Biochem., 120, 8-11. Malik, J.K., Schwartz, L.R. & Wiebel, F.J. (1983) Assessment of membrane damage in continuous culture of mammalian cells. Chem. Biol. Interact., 45, 29-42.
  5. Fortunati, E. & Bianchi, V. (1989) Plasma membrane damage detected by nucleic acid leakage. Mol. Toxicol., 1, 27-38.
  6. Bianchi, V. & Fortunati, E. (1990) Cellular effects of an anionic surfactant detected in V79 fibroblasts by different cytotoxicity tests. Toxicol. In Vitro, 4, 4-16.

IP-39 © June 1990