SU1659851A1 - Method for determination of membrane-toxic action of chemicals - Google Patents

Abstract

The invention relates to medicine, medical and microbiological industries, ecology, and specifically to research methods and quantitative assessment of the potential hazard of chemicals, and can be used in toxicology to determine the maximum concentration of the membrane-toxic effect of chemical compounds in industrial hygiene to determine the maximum permissible level of toxic substances. in the environment. The purpose of the invention is to accelerate the method for determining the membrane-toxic effect of chemicals. The essence of the invention is that cells of luminescent bacteria are incubated with the test substance, centrifuged, and the membrane-toxicity of the compound is determined by the appearance of the marker protein, luciferase, in the supernatant, and its activity is determined. The rate of determination of the membrantotoxic effect of substances increases 9-fold compared with the prototype. 2 tab. cl

Description

The invention relates to medicine, medical and microbiological industries, ecology, and specifically to research methods and quantitative assessment of the potential hazard of chemicals, can be used in toxicology to determine the maximum concentration of the membrane-toxic effect of chemical compounds, in industrial hygiene to determine the maximum permissible level of toxic substances in environment.

The purpose of the invention is the acceleration of the method.

The method is carried out as follows.

To a suspension of ph.Leiognathf cells, a chemical test substance is added, incubated and centered. The supernatant is analyzed for luciferase content. For this purpose, 0.1 ml of the supernatant is added to the cuvette of the luminometer and added. 0.02 ml of a 0.01% alcohol solution of myristic aldehyde. The cuvette is placed in a luminometer. The reaction was started by injecting 0.5 ml of 0.05 mM reduced flavin mononucleotide. The maximum light flash recorded by a recorder or digital device and expressed in microamperes. The degree of the membrane-toxic effect of chemicals is determined by the magnitude of the luciferase activity in the supernatant.

EXAMPLE 1: A culture suspension of ph. Lelognathl is poured into a series of test tubes according to

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1 ml and poured 10 µl butyl methane acetate in final concentrations of 0.5; 1.0; 2; four; eight; sixteen; 32 mM As a control, use 10 μl of the solvent, on which a solution of the studied chemical was prepared. The mixture thus prepared is incubated for 5 min at 2b ° C. After incubation, intact cells and cell windings are precipitated by centrifugation. 0.1 ml of the supernatant is analyzed for the content of luciferase enzyme released from the cells of luminescent bacteria by the luminescent reaction in vitro.

The amount of released luciferase under the action of butyl methacrylate at a dose of 1 mM was minimal (0.23 used units ± 0.03) and was not significantly different from the control. At dose

2 mM amount of luciferase was 1.05 ± 0.08 used, which was significantly different from the control (P 0.01). The data obtained indicate that the threshold of the membrane-toxic effect of butyl methacrylate is 2 mM.

. Example 2. Determination of 50% of the membrane-lymphatic dose (EDEo). The culture suspension of ph.Lelognathi is poured into duplicates in a series of 1 ml tubes. Double tubes are poured into both tubes at a rate of 10 μl of a specific concentration of xenobiotic. In this case, it is an alcohol solution of butyl acrylate in final concentrations of 4; eight; sixteen; 32; 64 mM As a first control of complete cell lysis, a membranotropic antibiotic that does not act on the luminescent system, polimixin, is poured at a final concentration of 100 units / ml to 1 ml of bacterial culture. The second control for the study of luminescence quenching directly tested concentrations of xenobiotics are duplicate tubes with xenobiotics and the culture of phototobacteria, to which polymyxin is added at a final concentration of 100 units / ml. Control and experimental samples are incubated for 5 min at 28 ° C, and cells and cell debris and 0.1 ml are precipitated by centrifugation. supernatants are examined for luciferase content by an in vitro luminescent reaction.

The results of a study of 50% of the membranolytic dose for butyl acrylate are presented in Table 1.

According to the results of the study, the calculation of the true values of the amount of luciferase released from the cells, i.e. considering inhibition of luminescent

systems directly xenobiotic, conducted as follows. The luminescence of the test samples is multiplied by an inhibition ratio equal to the ratio of control 1 / control 2. Certain values are calculated for butyl acrylate. From the calculated values, 50% of the membranolytic dose (ED) is calculated. The calculated dose for butyl acrylate is 7.4 mm (Table 2).

The specific EDU value for the prototype butyl acrylate was 12 mM.

Example When comparing the exposure time of photobacteria with butyl acrylate at a concentration of 7.4 mM with time

exposure of erythrocytes with the same substance at a concentration of 12 mM found that in the first case, the maximum effect is achieved in 5 minutes, while in the second, the maximum output of hemoglobin from red blood cells is reached in 45 minutes.

Thus, the time to determine the membrane-toxic effect of chemicals is significantly reduced.

Claims (1)

Invention Formula The method of determining the membrane-toxic effect of chemicals, including the incubation of indicator cells with the chemical substances under investigation, centrifuging and quantifying the released intracellular contents of the appearance of a marker protein in the supernatant, characterized in that, in order to speed up the method, luminescent bacteria serve as indicator cells, and luciferase is used as a marker protein and its activity is determined in the supernatant. Table 1 table 2