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Protocol no. 6
HUMAN LYMPHOCYTE CYTOTOXICITY ASSAY

This method measures the leakage of DNA and lactate dehydrogenase (LDH, EC. 1.1.1 27) from lymphocytes into the surrounding medium as an indicator of cytotoxicity. This method also includes an assay of intracellular (mitochondrial) diaphorase as a measure of cellular activity (MTT assay).

CONTACT

Professor Jorgen Clausen
Institute for Life Sciences and Chemistry
Roskilde University Postbox 260 DK-4000 Roskilde Denmark
Tel: Denmark - 46 75 77 11
Direct: Denmark - 46 75 77 81 Fax: Denmark - 46 75 74 01

RATIONALE

Acute toxicity can be detected in several cellular systems including the MIT-24 system using HeLa cells and other cell lines. Human lymphocytes offer a more sensitive approach of assessing cytotoxicity especially with a sensitive endpoint such as lactate dehydrogenase loss from intact cells. Lymphocytes are a mixture of B- and T- lymphocytes. They both react to foreign antigens or to antigen presenting cells. The B-cells synthesizes immunoglobulin whereas T-cells (suppressor, helper and killer cells) have regulating and other functions. The lymphocyte Interleukin-2 receptor is stimulated by phytohaemagglutinin. As a result T-cells, in particular suppressor cells, leave the G phase and enter the S phase of the cell cycle and start dividing. Consequently after several days in culture suppressor T-cells are prevalent. Lymphocytes can be held in culture for several weeks under these conditions. Lymphocytes are not that easy to grow and the use of fluorometric methods, which are more sensitive than photometric methods, minimises the number of cells required (essential considering the difficulties encountered during their growth and culture). The basis of this test is that a toxic chemical will disrupt the cellular membrane resulting in an increase in LDH and DNA found in the external medium. The resulting toxicity can then be expressed in terms of the chemical concentration which causes 50% of the DNA or LDH to leak from the damaged cells.

BASIC PROCEDURE

Lymphocytes are isolated from anticoagulated, normal, human blood samples. The cells are grown for 5 days, centrifuged and resuspended in complete medium. 1ml samples of cell suspension are then aliquoted into 24-well plates and the test chemical at an appropriate dilution is added. The cells are incubated for 24 hours before aliquots of cell suspension are removed. The suspension is then centrifuged and LDH and DNA leakage assays as well as the MTT assay, performed. LC50 values are then calculated for each assay.

CRITICAL ASSESSMENT

Both DNA leakage and lactate dehydrogenase activity can be measured fluorimetrically, which is 1000x more sensitive than conventional photometric procedures. LDH activity is proportional to the cell number over 24 hours, with a lower limit of detection of approximately 1000 cells. Assessment of DNA leakage shows a similar proportionality but with a lower limit of detection, 10 times higher that required for the LDH assay. The LD50 values obtained using the LDH assay are generally lower than those found in the DNA assay. This is not totally surprising because cells would need to be damaged more extensively for DNA leakage to occur. Lymphocytes cultures are a little more sensitive than cultures of cells from cell lines such as BCL-D1 to most of the chemicals tested so far, but there appear to be no significant differences with HeLa cells (morphological endpoint) because in general LC50 values are lower in the lymphocyte culture, one exception being ouabain. The correlations between crude data on human LD values and the LC50 values from the MTT, LDH and DNA assays was fairly good (coefficients of correlation, r = 0.68, r = 0.67 and r = 0.68, respectively), with the MTT values giving the closest correspondence.

TEST STATUS

Interlaboratory validation

CHEMICALS TESTED

Amitriptyline
Benzylpenicillin
Caffeine
Chloroquine
Cycloheximide
Dinitrophenol
Imipramine
Lidocaine
Lithium chloride
Methyldopa
Nicotine
Noscapine
Ouabain
Phenol
Phenylbutazone
Prilocaine
Procaine
Promethazine
Quinine
Sodium fluoride
Theophylline
Verapamil
The chemicals were obtained form either Sigma Chemical Co., St. Louis, MO, USA or Merck, Darmstadt, FRG, and were of the highest obtainable purity. Theophylline, noscapine, amitriptyline, lidocaine, phenylbutazone and promethazine were gifts from DAK, Copenhagen, Denmark. Verapamil and prilocain were gifts from Erchopharm, Copenhagen and Astra, Sweden, respectively.

REFERENCES

  1. Balls, M & Horner, S.A. (1985) The FRAME interlaboratory programme on in vitro cytotoxicology. Food and Chemical Toxicology, 23, 209-13.
  2. Bergmeyer, H.U. (1970) In: Methoden der Enzymatischen Analyse, 2nd edn. Ed. Trautschold, I. & Löffler, G, pp. 182-183,
  3. Verlag Chemie Weinheim. Ekwall, B. (1980) Preliminary Studies on the Validity of In Vitro Measurement of Drug Toxicity Using HeLa Cells. II. Drug Toxicity in the MIT-24 System Compared with Mouse and Human Lethal Dosage of 53 Drugs. Toxicology Letters, 5, 309-17.
  4. Ekwall, B. & Johanson, A. (1980) Preliminary Studies on the Validity of In Vitro Measurement of Drug Toxicity Using HeLa Cells. I. Comparative In Vitro Cytotoxicity of 27 Drugs. Toxicology Letters, 5, 299-307.
  5. Fotina, M., Merson, E.J. & Allan, F.H. (1971) Micromethod for rapid separation of lymphocytes from peripheral blood. Annals of Clinical and Laboratory Science, 1, 131-33.
  6. Holt, P.S., Buckley, S. & DeLoach, J.R. (1987) Detection of the lethal effects of T-2 mycotoxin on cultured cells using a rapid colorimetric viability assay. Toxicology Letters, 39, 301-
  7. Holt, P.S., Buckley, S., Norman, J.O. & DeLoach, J.R. (1987) Cytotoxic effect of T-2 mycotoxin on cells in cultures as determined by a rapid colorimetric bioassay. Toxicon, 26(6), 549-58. Kissane, J.M. & Robins, E. (1958) The fluorometric measurement of deoxyribonucleic acid in animal tissues with special reference to the central nervous system. J. Biol. Chemistry, 33, 184-88.
  8. Skaanild, M.T. & Clausen, J. (1989) Estimation of LC50 values by Assay of Lactate Dehydrogenase and DNA redistribution in Human Lymphocyte Cultures. ATLA, 16, 293-296.

IP-6 © May 1991