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Protocol no. 34

YEAST PLASMA MEMBRANE H+ -ATPASE TOXICITY TEST

The effect of chemicals on the activity of the plasma membrane-bound H ATPase, isolated from yeast (Saccharomyces cerevisiae) 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 Tel: Germany - 838 3171

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, many of which may be environmental chemicals, affects the membrane lipid order and consequently affects the functions of these proteins. Since, the function of important cellular proteins, such as the H+-ATPase strongly depends upon the integrity of the lipid bilayer, the activity of the H+-ATPase may be used as a sensitive indicator of the effect that a chemical may have on the viability of the cell.

BASIC PROCEDURE

Plasma membranes are isolated from the yeast Saccharomyces cerevisiae. The cell wall is initially digested by helicase, followed by hypoosmotic lysis and homogenization. Membranes are prepared by subsequent differential centrifugation. The activity of the H+-ATPase is then determined by measuring the amount of inorganic phosphate released from ATP.

CRITICAL ASSESSMENT

The maintenance of yeast cells such as Saccharomyces cerevisiae in culture, is a relatively simple and inexpensive technique. It has been shown that the activity of the H+-ATPase is a very good indicator for the unspecific toxic effects of environmental chemicals, and that EC20 and EC50 values obtained using this test system correlate well with the octanol/water partition coefficient for the majority of chemicals tested. However, it should be noted that unpredicted effects may occur with compounds which specifically interact with the enzyme. Interference has also been found to occur, when the phosphate reagent reacts with the compound under test. Comparison with Saccharomyces cerevisiae proliferation assay It has been shown that the results obtained using this procedure correspond well with those obtained with the yeast growth rate cytotoxicity assay, for instance, for 56 chemicals including phenols, anilines and aliphatic alcohols, a correlation of r = 0.91 was obtained when log EC20 (H+-ATPase) was compared with log EC20 (cell growth). The H+-ATPase assay has over the proliferation assay since it is of relatively short duration, and requires very small amounts of test substance. It may also be used as a mechanistic tool, since, the inhibition of the H+-ATPase gives an insight into unspecific plasma membrane perturbations as indicated by a high dependency of the octanol/water partition coefficient of the test compound.

TEST STATUS

Developed in-house. The test system has been validated in research studies supported by the German Federal Environmental Agency. The yeast cytotoxicity test has been validated in the following research projects:

a) Environmental Chemicals and Biomembranes.

    • Umweltforschungsplan des BMI 10603035/02. Rösick, E., Stadtlander, K. & Ahlers, J. (1985) Environmental chemicals and biomembranes. 1. Nitrilotriacetic acid. Chemosphere, 14, 529-544.
    • Ahlers, J. & Rösick, E. (1986) Influence of nitrilotriacetic acid on cadmium uptake by yeast. Bull. Environ. Contam. Toxicol., 37, 96-105.
    • Ahlers, J. & Rösick, E. (1986) Cadmium-uptake and influence on plasma membrane functions of yeast. Toxicol. Environ. Chem., 11, 291-300.
    • Ahlers, J.; Rösick, E., & Stadtlander, K. (1986) Environmental chemicals and biomembranes. Kinetics of uptake and influence on membrane functions. In: Functional testing of aquatic biota for estimating hazards of chemicals. (eds. Cairns, Jr. J.& Pratt, J.R.) American society for testing and materials, ASTM, 988, Philadelphia.

b) Determination of multi-variate structure-toxicity relationships between biotest-systems and chemical classes as defined by federal chemical law - yeast assays. Umweltforschungsplan des BMU 10603045/04.

    • Ahlers, J., Benzig, M., Gies, A., Pauli, W. & Rösick, E. (1988) Yeast as a unicellular model system in ecotoxicology and xenobiochemistry. Chemosphere, 17, 1603-1615.
    • Ahlers, J., Cascorbi, I., Foret, M., Gies, A., Köhler, M., Pauli, W. & Rösick, E. (1991) Interaction with functional membrane proteins - a common mechanism of toxicity for lipophilic environmental chemicals? Comp. Physiol. Biochem., 100C, 111-113.

c) Yeast as ecotoxicological model system: Development of a validated test prescription for the determination of the toxicity of chemicals. Umweltforschungsplan des BMU 10603103.

ORGANISATIONS USING THE TEST

None at present.

CHEMICALS TESTED

4-Acetamidophenol 3-Aminobenzoic acid hydrazide 2-Amino-4-methylphenol 3-Aminophenol 4-Aminophenol Amitryptyline Aniline 2-Bromoaniline 2-Bromophenol 1-Butanol 2-Butanol iso-Butanol tert-Butanol Cadmium chloride 2-Chloroaniline 3-Chloroaniline 4-Chloroaniline 5-Chloro-2,4-dimethoxyaniline 4-Chloro-3,5-dimethylphenol 2-Chloroethanol 3-Chloro-4-methylaniline 4-Chloro-2-nitroaniline 4-Chlorophenol 2,3-Dibromopropanol 2,4-Dichloroaniline 2,6-Dichloroaniline 2,3-Dichlorophenol 2,4-Dichlorophenol 2,6-Dichlorophenol 3,5-Di-(1,1-dimethylethyl)phenol 3,5-Dimethoxyphenol 3-Dimethylaminophenol 2,4-Dimethylaniline 2,4-Dinitroaniline Ethanol 2-Ethylaniline 4-Ethylaniline Hydroxybenzoic acid 2-Hydroxy-5-methylaniline 4-Hydroxyphenol 3-Hydroxyphenylurea Methanol 4-Methoxyaniline 4-Methylaniline 4-Methyl-2-nitrophenol 2-Methyl-4,6-dinitrophenol 2-Nitrophenol 4-Nitrophenol Pentachlorophenol Pentanol Phenol 1,2-Propanediol 1-Propanol 2-Propanol 2,3,4,5-Tetrachlorophenol 1,1,1-Trichloroethane 2,2,2-Trichloroethanol 2,4,5-Trichlorophenol 2,4,6-Trichlorophenol 2,4,6-Trifluoromethylaniline 3-(Trifluoromethyl)phenol 2,4,6-Triiodophenol 2,4,6-Trinitrophenol

REFERENCES

  1. Ahlers, J. & Rösick, E. (1986) Cadmium-uptake and influence on plasma membrane functions of yeast. Toxicol. Environ. Chem., 11, 291-300. Ahlers, J., Benzig, M., Gies, A., Pauli, W. & Rösick, E. (1988) Yeast as a unicellular model system in ecotoxicology and xenobiochemistry. Chemosphere, 17, 1603-1615.
  2. Ahlers, J., Cascorbi, I., Foret, M., Gies, A., Köhler, M., Pauli, W. & Rösick, E. (1991) Interaction with functional membrane proteins - a common mechanism of toxicity for lipophilic environmental chemicals? Comp. Physiol. Biochem., 100C, 111-113.
  3. Bradford, M. (1976) Rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle of dye-binding. Anal. Biochem., 72, 248-254.
  4. Gies, A., Rösick, E., Pauli, W., Köhler, M., Cascorbi, I., Berger, S. & Wiegener, B. (1988) Determination of multi-variate structure-toxicity relationships between biotest-systems and chemical classes as defined by federal chemical law - Yeast assays. Umweltforschungsplan des BMU 10603045/04.
  5. Ohnishi, T., Gall, R. & Mayer, M. (1975) An improved assay of inorganic phosphate compounds: Applications to the ATPase assay in the presence of phosphocreatine. Anal. Biochem., 69, 261-267.
  6. Wach, A., Ahlers, J. & Gräber, P. (1990) The H+-ATPase of the plasma membrane from yeast. Kinetics of ATP hydrolysis in native membranes, isolated and reconstituted enzymes. Eur. J. Biochem., 189, 675-682.

    IP-34 © August 1991