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Protocol no. 33
YEAST GROWTH RATE CYTOTOXICITY TEST

The cytotoxic effect of chemicals upon yeast (Saccharomyces cerevisiae) cells in culture is determined by inhibition of cell proliferation, as measured by cell density.

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

The proliferation rate of the yeast, Saccharomyces cerevisiae, may be regarded as an overall indicator of the physiological status of the cell. Therefore, the effect of various toxic substances on different cell functions will be reflected by changes in the rate of proliferation. It is possible to determine the toxicity of a test substance simply by measuring cell density, since, the log EC20 of the growth rate in the logarithmic phase has been shown to correlate with the log EC20 of the cell density after 24 hours (0.99).

BASIC PROCEDURE

Yeast cells are cultured in fully complete media in the presence of various concentrations of test substance. Cell density is measured densitometrically every 30 minutes for 8 hours and again after 24 hours, and the proliferation rate determined from the logarithmic growth phase. A dose-response curve is obtained by plotting the relative growth rate versus the chemical concentration-from which concentrations leading to 20% and 50% inhibition of growth (EC20 and EC50 values) are determined.

CRITICAL ASSESSMENT

The maintenance of yeast cells such as Saccharomyces cerevisiae in culture, is a relatively simple and inexpensive technique. The application of such a culture to determine cytotoxicity enables the rapid, highly reproducible testing of many chemicals on a routine basis. Although toxicity of a chemical is determined during logarithmic phase of growth, since, some substances may also alter the duration of the lag-phase of growth, the proliferation rate should also be determined during this phase. The relative toxicity of chemicals on the growth rate is comparable to effects on physiological properties, such as the activity of the plasma membrane bound H+-ATPase. Results from this system also corresponds to those obtained from other test systems which use bacteria, fish and mammalian cell cultures (see Gies et al., 1988; Ahlers et al., 1991).

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.

ORGANISATIONS USING THE TEST

None at present.

CHEMICALS TESTED

4-Acetamidophenol Acetylsalicylic acid Aminoacetophenone 3-Aminobenzoic acid hydrazide 2-Amino-4-methylphenol 3-Aminophenol 4-Aminophenol Amitryptyline Aniline Benzidine 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 Diazepam 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 Digoxin 3,5-Dimethoxyphenol 3-Dimethylaminophenol 2,4-Dimethylaniline 2,4-Dinitroaniline Ethanol 2-Ethylaniline 4-Ethylaniline Ethylene glycol Ferrous sulphate 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 Paracetamol Pentachlorophenol Pentanol Phenol 1,2-Propanediol 1-Propanol 2-Propanol Sodium chloride 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. Rösick, E., Stadtlander, K. & Ahlers, J. (1985) Environmental chemicals and biomembranes. 1. Nitrilotriacetic acid. Chemosphere, 14, 529-544.
  2. Ahlers, J. & Rösick, E. (1986) Influence of nitrilotriacetic acid on cadmium uptake by yeast. Bull. Environ. Contam. Toxicol., 37, 96-105.
  3. Ahlers, J. & Rösick, E. (1986) Cadmium-uptake and influence on plasma membrane functions of yeast. Toxicol. Environ. Chem., 11, 291-300.
  4. 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.
  5. 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.
  6. 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.
  7. c) Yeast as ecotoxicological model system: Development of a validated test prescription for the determination of the toxicity of chemicals. Umweltforschungsplan des BMU 10603103.
  8. Ahlers, J. & Rösick, E. (1986) Cadmium-uptake and influence on plasma membrane functions of yeast. Toxicol. Environ. Chem., 11, 291-300.
  9. 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.
  10. 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.
  11. 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.
  12. Stadtlander, K. & Lawohnus, H. (1990) Yeast cells as a test systems for evaluating the toxicity of chemicals. In: The seventh scandinavian society for cell toxicology congress on in vitro toxicology. ATLA, 17, 203-206.

    13. IP-33 © August 1991