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Protocol no. 9
THE USE OF MEMBRANE PERMEABILITY AS A MEASURE OF CYTOTOXICITY IN PERFUSED CELL CULTURES

Membrane permeability of perfused cell cultures, as determined by the efflux of [3H]-2-deoxy-D-glucose-6- phosphate, is used as an indicator of the cytotoxic effect of chemicals.

CONTACT

Dr. Erik Walum
Unit of Neurochemistry and Neurotoxicology
University of Stockholm S-106 91 Stockholm Sweden
Tel: Sweden - 816 4272

RATIONALE

During the experimental period, cells are maintained in culture in a perfusion chamber which provides a more stable environment than traditional culture methods. The cytotoxicity of test chemicals is assessed by their ability to cause damage to the plasma membrane. This effect is, in turn, quantified by measuring the efflux of 2-deoxy- D-glucose-6-phosphate from the cells. The use of membrane permeability as an indication of cytotoxic damage The plasma membrane is the first barrier met by a toxic agent on reaching a cell. This factor, plus the many vital regulatory mechanisms inherent in the plasma membrane and its chemical composition, make it very susceptible to attack by many toxic compounds. Damage to the cell membrane has, therefore, been suggested as a good indicator of cytotoxicity. The use of [3H]-2-deoxy-D-glucose as a probe in the study of membrane permeability This probe has proved suitable as a tool to determine general cell membrane permeability changes in monolayer cultures. As an analogue of D-glucose, 2-deoxy-D-glucose is readily taken up into the cell via the glucose transport system and, as would be expected, it is then phosphorylated in a reaction involving hexokinase. The product - 2-deoxy-D-glucose-6-phosphate - accumulates in the cell for several reasons: hexokinase is not feedback inhibited the product is metabolically inert cell membrane permeability for this compound is very low. Thus, when the cell membrane is intact, the concentration of 2-deoxy-D-glucose-6-phosphate continues to rise in the cell. The original compound can be tritiated in such a way that the metabolite retains the [3H]-group. Incubation of cells with the tritiated substrate would, therefore, result in an accumulation of tritiated product within the cells. Subsequent removal of external contaminating tritium (and exposure to test chemicals) would enable detection of the product [3H]-2-deoxy-D-glucose-6-phosphate as it diffused out of the cells. This rate of diffusion would be expected to be very low unless the membrane was damaged in some way. The leakage of the product can, therefore, be used as an indicator of the extent of cell membrane damage which can then be related to the cytotoxicity of compounds.

BASIC PROCEDURE

Cells are grown in tissue culture grade dishes for 4 days prior to an experiment. After this time they are incubated in the absence of glucose, but in the presence of [3H]-2-deoxy-D-glucose, for 2 hours. The incubation is terminated, and all traces of [3H]-2-deoxy-D-glucose removed, by the addition of ice-cold PBS. The cultures are subsequently transferred to perfusion chambers and perfused at 37 C for 1 hour with PBS (containing D- glucose) which is supplemented with various concentrations of test compounds. The efflux-kinetics of the metabolite [3H]-2-deoxy-D-glucose-6-phosphate is determined by collecting fractions of the perfusate at 5 minute intervals in scintillation vials. At the end of the experiment cells are solubilised in NaOH, transferred into scintillation vials and neutralised with HCl. The radioactivity of perfusate samples and that of cells is determined by liquid scintillation. The radioactivity remaining in the cells at the end of the experiment can be related to that which was present at the beginning of the experiment, thus giving an indication of the leakage which may have occurred. Leakage can also be plotted against concentration of test chemical and against time by using the results obtained on analysing the radioactivity of perfusate samples. Increased leakage (i.e. higher levels of radioactivity in the perfusate or a greater apparent loss from the cells) from cells exposed to test chemicals, compared to control situations, is deemed indicative of a cytotoxic effect.

CRITICAL ASSESSMENT

This method provides a relatively simple means of maintaining and exposing cells to test compounds and assessing their cytotoxicity (as indicated by cell membrane damage). The procedure is readily adapted to different cell types. The assay method is sensitive and reproducible. The use of a perfusion chamber for the culture of cells in vitro offers several advantages over traditional culture procedures which often lead to an unstable cell environment: nutrient and pH levels fall, metabolites continually increase in their concentration, oxygen supply may become problematic (i.e. cells may receive insufficient oxygen with the result that respiratory oscillations may occur), etc. Many of these problems may be alleviated to some extent by the use of perfusion techniques which allow the continual passage of the end products of metabolism away from the cells, producing more constant pH levels and oxygen concentrations. These advantages have encouraged the use of perfusion chambers over the experimental period where the cells are exposed to test chemicals and the membrane permeability assessed (i.e. during measurement of efflux of [3H]-2-deoxy-D- glucose-6-phosphate). The perfusion apparatus discussed in this procedure has been modified and simplified in many ways compared to similar systems currently in use. The culture dish is an integrated part of the perfusion chamber. The chamber is easily handled being constructed of only 2 parts i.e. the perfusion block and the culture dish. Cells are grown on their normal substratum, i.e. in tissue culture grade plastic dishes. This allows results between perfused and non-perfused cultures to be easily compared. The high optical quality of the culture dishes and transparency of the perfusion block allows ease of observation and inspection in a standard inverted microscope. The perfusion chamber can be easily assembled in a laminar flow cabinet thus reducing the microbial contamination. The simplicity of the system allows for ease of use and increases reliability. [3H]-2-deoxy-D-glucose is a biochemically well characterised probe. It is rapidly taken up into cells where the phosphorylated metabolically inert product readily accumulates. It is a sensitive method for measurement of cell permeability (due to the high levels of accumulated intracellular radioactivity and comparatively small size of the compound).

TEST STATUS

This system was developed in-house, and has come to the point where the author feels that it is behaving satisfactorily. Research in this area has currently ceased, since the author does not have the economical means to carry out further work to validate the test.

CHEMICALS TESTED

Triton-X-100
HgCl2
CH3HgCl2
(C2H5)3SnCl
K2Cr2O7
Benzene
Phenol
Acrylamide
ORGANISATION USING THE SYSTEM
None at present.

REFERENCES

  1. Arrhenius, S. (1915) Quantitative laws in biological chemistry. Bell, London. Dulbecco, R. & Vogt, M. (1954) Plaque formation and isolation of pure cell lines with poliomyelitis viruses. J. Exp. Med., 199, 167-182.
  2. Kotyk, S. & Jan cek, K. (1976) Cell Membrane: Principles and Techniques. 240-244.
  3. Plenum, New York. Nyberg, E. & Walum, E. (1984) On the application of cultured neuronal cell lines in neurotoxicological studies: implications of acrylamide- induced neurite disintegration. ATLA, 11, 194-203.
  4. Peterson, A. & Walum, E. (1983) Growth and morphology of neuronal cell lines cultured in perfusion. In Vitro, 19(12), 875-880.
  5. Walum, E. (1975) Glucose uptake into cultured tumour cells from the nervous system. Phd thesis, University of Gothenburg, Sweden. Walum, E. (1982a) Membrane lesions in cultured mouse neuroblastoma cells exposed to metal compounds. Toxicology, 25, 67-74.
  6. Walum, E. (1982b) Temperature dependence of membrane permeability in cultured cells exposed to benzene and phenol. Biochemical and Biophysical Research Communications, 108(3), 948-952.
  7. Walum, E. & Marchner, H. (1983) Effects of mercuric chloride on the membrane integrity of cultured cell lines. Toxicology Letters, 18, 89-95.
  8. Walum, E. & Peterson, A. (1982) Tritiated 2-Deoxy-D-Glucose as a probe for cell membrane permeability studies. Analytical Biochemistry, 120, 8-11.

IP9 September 1992