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Protocol no. 35
CHINESE HAMSTER OVARY CELL NA+/K+ -ATPASE TEST

The effect of chemicals on the activity of the plasma membrane-bound Na+/K+ -ATPase isolated from Chinese Hamster Ovary (CHO) cells is used as a measure of their toxicity.

CONTACT

Dr. Ingolf Cascorbi Free University of Berlin Institute of Biochemistry and Molecular Biology Ehrenbergstr. 26-28 D-W-1000 Berlin 33 Germany

RATIONALE

Many proteins which are important for the maintenance of the electrochemical gradient and metabolic status of the cell are located in the plasma membrane. Accumulation of lipophilic substances in the plasma membrane may affect the membrane lipid order and consequently affect the function of these proteins. Changes in the activity of the Na+/K+ -ATPase, which is the major active transport system responsible for the electrochemical potential in mammalian cells, can therefore be an indication of the effect that a chemical may have on the viability of the cell membrane and possibly the whole cell.

BASIC PROCEDURE

Plasma membranes are isolated from CHO cells which have been grown to confluency. They are incubated in the presence of test chemicals and ATP, and the activity of the ouabain-sensitive Na+/K+ ATPase is determined by measuring the amount of inorganic phosphate released over a 20 minute reaction period.

CRITICAL ASSESSMENT

The use of isolated plasma membranes eliminates the possibility of hydrophobic substances in the external medium or cell cytoplasm interfering with the assay results by altering membrane fluidity or directly inhibiting membrane-bound proteins. The test system is especially appropriate for the testing of lipophilic substances. Results so far have shown that EC20 and EC50 values obtained using this assay system correlate well with the lipophilicity of the test compounds (Cascorbi and Ahlers, 1989). Measurement of the activity of isolated plasma membrane Na+/K+- ATPase has the advantage of allowing an indirect insight into the unspecific impairment of plasma membranes.

TEST STATUS

Developed in-house. Validated by research program supported by the German Federal Environmental Agency.

CHEMICALS TESTED

4-Chlorophenol 2,4-Dichlorophenol 2,6-Dichlorophenol 3,5-Dimethoxyphenol 4-Methyl-2-nitrophenol 2,3,4,5-Tetrachlorophenol 2,4,6-Trichlorophenol 2,4,6-Triiodophenol Pentachlorophenol ORGANISATIONS USING THE SYSTEM None at present.

REFERENCES

  1. Ahlers, J., Cascorbi, I., Forêt, M., Gies, A., Köhler, M., Pauli, W., & Rösick, E. (1991) Interaction with functional membrane proteins - a common mechanism of toxicity for lipophilic environmental compounds? Comp. Physiol. Biochem. 100C, 111-113.
  2. Bradford, M. (1976) Rapid and sensitive method for quantisation of microgram quantities of protein utilizing principle of dye-binding. Anal. Biochem. 72, 248-254
  3. Cascorbi, I. (1989) Na+/K+ -ATPase of mammalian cells as a test system for predicting chemical hazard of environmental chemicals. A QSAR study. Biol. Chem. Hoppe Seyler 370, 617.
  4. Cascorbi, I & Ahlers, J. (1989) Correlation between the lipophilicity of substituted phenols and their inhibition of the Na+/K+ -ATPase of Chinese Hamster Ovary cells. Toxicology 58, 197-210
  5. Cascorbi, I & Forêt, M. (1991) Interaction of xenobiotics on the glucose-transport system and the Na+/K+ -ATPase of human skin fibroblasts. Ecotoxicol. Environ. Safety 21, 38-46
  6. Forêt, M. & Ahlers, J. (1988) Effect of phenols on growth rate and adenosine uptake of CHO cells. Ecotoxicol. Environ. Safety 16, 303-309.
  7. Ohnishi, T., Gall, R. & Mayer, M. (1975) An improved assay of inorganic phosphate in the presence of extralabile phosphate compounds.:Application of the ATPase assay in the presence of creatine. Anal. Biochem. 69, 261-267

IP-35 © November 1991