SK278857B6 - Method for testing cytotoxicity and genotoxicity of chemical substances - Google Patents

Abstract

This patent describes cytotoxic and genotoxic properties of chemical substances which are being tested with the use of microorganism called Euglena gracilis so that, euglena cells are influenced by a chemical substance, at the temperature of 25 up to 39 Celsius degrees, in a stabile testing solution which is prepared as a mixture of solutions closed to Na2HPO4.12H2O and KH2PO4 in the such ration, in order the appropriate pH value of the environment to be reached, it means in a range of pH values related to 4,6 up to 8,5. The testing solution with the pH values in the interval of 3 up 4,5 is being prepared so that, it is acidified with the use of 0,5 M H2SO4.

Description

Technical field

The invention relates to a method for testing the cytotoxicity and genotoxicity of chemicals with Euglena gracilis in an environment having a pH of 3 - 8.5 and at a temperature of 25 to 39 ° C.

BACKGROUND OF THE INVENTION

The usefulness of the one-cell photosynthetic flagellate Euglena gracilis for the search for genotoxically active substances has been known for a long time (Science, 148, 1965, DR McCALLA, Chloroplast mutagenesis: Effect of N-methyl-N'-nitro-N-nitrosogunanidine and some other agents on Euglena, p. Acta FRN Univ Comen Microbiol., 1982, L. Ebringer, O. Chreo, Mutagenic action of nitrovines on Euglena gracilis and Salmonella typhimurium, pp. 1-8; Pathological Plant Physiology, IEBE, SAS , Bratislava, 1988, O. Chreňo, J. Jasik, Utilization of flagellate Euglena gracilis as a model organism for detecting mutagenicity and toxicity of various chemicals (pp. 531-540). Chemical compounds were tested at the physiological growth temperature of Euglena gracilis and in media having an accurate and unchanged pH. If these media were adjusted to a pH other than that determined, the environment became unsuitable for euglen cells to perform their vital functions and was toxic. A similar situation is found in other biological test models, where the properties of the test substances are monitored at only one unchanged pH value and at one temperature (e.g., Ames test, micronucleus test).

However, the nature of many biologically active chemical compounds varies depending on the pH of the environment and the half-life of the hydrolytic decay that is related to the temperature of the environment.

SUMMARY OF THE INVENTION

Summary of a method for testing the cytotoxicity and genotoxicity of chemical substances by the organism Euglena gracilis invention consists in that the cells of Euglena affect the chemical in a stable test solution prepared by mixing a solution of 0.03 M and 0.03 M NaiHPCý.lžHjO solution of KH ₂ PO ₄ in ratio to obtain the desired pH of the environment in the pH range of 4.6 to 8.5. It is a further object of the invention that a test solution having a pH of 3 to 4.5 is prepared by acidification of 0.5 MH ₂ SO ₄ . An advantage of the method for testing the cytotoxicity and genotoxicity of the chemicals of the invention is that it is possible to test the cytotoxic and genotoxic properties of the compounds with Euglena gracilis over a wide range of pH and ambient temperatures in short (up to 48 hours) or long term (over 48 hours to several days) ) intervals of action. The test solution is not toxic to the cells of this organism and does not die for at least two weeks at 25 to 30 ° C and pH values of 3 - 8.5. At temperatures of 30 to 39 ° C, substances can only be tested for short periods (up to 48 hours). The pH of the test solution does not change after autoclaving or at least 60 days of storage.

BRIEF DESCRIPTION OF THE DRAWINGS

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the effect of environmental pH and temperature on the cytotoxic and genotoxic effects of furantoin on flagella Euglena gracilis at pH 5; FIG. Fig. 2 shows the effect of environmental pH and temperature on the cytotoxic and genotoxic effects of furantoin on flagella Euglena gracilis at pH 7; Fig. 3 shows the effect of environmental pH and temperature on the cytotoxic and genotoxic effects of furantoin on flagella Euglena gracilis at pH 8.5; Fig. 4 shows the effect of environmental pH and temperature on the cytotoxic and genotoxic effects of acridine orange on flagella Euglena gracilis at pH 5; Fig. 5 shows the effect of environmental pH and temperature on the cytotoxic and genotoxic effects of acridine orange on flagella Euglena gracilis at pH 7; 6 shows the effect of environmental pH and temperature on the cytotoxic and genotoxic effects of acridine orange on flagella Euglena gracilis at pH 8.5.

The proportion of induced heterotrophic mutants is given in% following the effect of the mutagen on light of 111-1.1 µM, in the dark, at 37 ° C (H) or at 27 ° C (unlabeled). Thin continuous lines in the above samples in the graphs indicate the percentage of surviving cells in%.

DETAILED DESCRIPTION OF THE INVENTION

Detection of cytotoxic and genotoxic effects of furantoin and acridine orange by euglene test depending on the tested temperature and pH of the environment.

The cytotoxic and genotoxic effects of the compounds are evaluated in the eugenin assay by monitoring the number of surviving cells versus control untreated samples and evaluating the frequency of heterotrophic mutant formation after mutagenic treatment. In control samples, this frequency value equals zero (Pathological Plant Physiology, IEBE, SAS, Bratislava, 1988, O. Chreňo, J. Jásik, Utilization of flagellate Euglena gracilis as a model organism for detecting mutagenicity and toxicity of various chemicals, pages 531 to 53). 540).

To the test solutions prepared by mixing 0.03 M Na ₂ HPO ₄ .12H ₂ O solution and 0.03 M KH ₂ PO ₄ solution in such a ratio that solutions with pH values of 5, 7, 8.5 were sterilized after their sterilization. the washed inoculum washed cells were transferred by autoclaving to a final concentration in the test solutions of 5 x 10 ³ cells / ml. Cells of Euglena gracilis grew in Cramer-Myers medium (Arc. Microbiol. 17, 1952, M. Cramer, J. Myers, Growth and photosynthetic characteristics of Euglena gracilis, pp. 384-402), cells in inoculum derived from logarithmic growth phase at a concentration of 2.10 ⁵ cells / ml medium. The test substance - furantoin (acridine orange) was then added to the samples in all series except the control series and placed in thermostats at 27 ° C and 37 ° C. The test substance was dissolved in dimethylsulfoxide such that the solvent constituted 1% of the total volume of the test solution. After 24 hours treatment, euglene cells from each sample were washed again and transferred to fresh assay solution. The euglen cells were immediately seeded onto the surface of the agar broth in an amount such that, on a 10 cm petri dish, 500 euglen colonies were grown.

The numbers of mutated colonies were evaluated after 2-3 weeks of cultivation under light conditions at 27 ° C.

Furantoin was most effective in acidic environment (pH 5). In a neutral environment, there was a rapid increase in the proportion of surviving cells and a decrease in the frequency of heterotrophic euglen mutant formation at the same substance concentrations. In a basic environment (pH 8.5) the substance was only at the highest concentrations tested and only at 37 ° C. At all concentrations and in all pH environments, furantoin was more effective at higher test temperatures (37 ° C).

In the case of acridine orange, the results showed the opposite dependence on the biological activity of this substance depending on the pH environment used. It had the most potent cytotoxic and genotoxic effects in an alkaline environment (pH 8.5) and at concentrations at least a thousand times lower than those used in an acidic environment. In an acidic environment, no heterotrophic mutants were induced at the highest concentration tested (500 pg / ml) at all. In all series, the substance was more effective at a higher treatment temperature (37 ° C).

Industrial usability

The method of testing cytotoxicity and genotoxicity of chemicals in various pH environments from 3 to 8.5 and at different temperatures from 25 to 39 ° C by the organism Euglena gracilis of the invention is useful for determining the basic biological characteristics of novel synthesized compounds and drugs of interest , food and chemical industries. The test method can be used wherever it is important to determine the temperature and pH values at which the substance is the most and least biologically active.

Claims (2)

A method for testing cytotoxicity and genotoxicity of chemicals in environments with a pH value of 3 - 8.5 and at temperatures of 25 to 39 ° C using Euglena gracilis, characterized in that euglen cells are affected by the chemical stable test solution prepared by mixing 0.03 M Na ₂ HPO ₄ .12H ₂ O solution and 0.03 M KH ₂ PO ₄ solution in a ratio to obtain a pH of 4.6 to 8.5. Method for testing cytotoxicity and genotoxicity of chemicals according to claim 1, characterized in that the test solution having a pH value of 3 - 4.5 is prepared by acidification of 0.5 MH ₂ SO ₄ .