JPH03228697A - Method for testing drug-sensitivity of cell membrane - Google Patents

Abstract

PURPOSE:To easily and quickly determine the influence of drug upon a cell membrane by carrying out a coupled reaction of a specific enzyme with an intracellular enzyme group capable of supplying reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H]. CONSTITUTION:An enzymatic reaction system (A) is produced by adding a quinone such as menadione (vitamin K3: 2-methyl-1,4-naphthoquinone) and a cytotoxic substance such as dimethyl sulfoxide to a living cell system such as a yeast suspension containing an NAD(P)H:quinone oxidoreductase of cell membrane and NAD(P)H-supplying enzyme group in the cell. The reaction system A is made to react at about 30 deg.C for about 2 min to generate H2O2 (B). The component B is added with perylene, etc., to effect the emission of fluorescence and the amount of H2O2 is determined at an accuracy of 10<-7> to 10<-3>mol/l by fluorometry. The drug-sensitivity of a cell membrane can be examined by this procedure.

Description

[Detailed description of the invention] [Purpose of the invention]

[Industrial Application Field] The present invention relates to a method for rapidly measuring the effect of a drug on the cell membrane of a living cell or a living tissue or organ containing a living cell. (Left below) [Prior art] (1) Background Conventionally, toxicity tests for drugs or drugs have mainly been conducted using animals (
In particular, in vivo experiments using mice, rats, guinea pigs, etc. have been used. However, animal experiments are expensive, especially pure-bred animals with good reproducibility, so in recent years, toxicity testing using cultured animal cells has been promoted as an alternative toxicity testing method, including from the perspective of animal recipients. Effectiveness is attracting attention. Examples of such test methods include;
Measurement of colony formation rate by biological cells, ■ Discrimination between live and dead cells by staining method and measurement of survival rate, ■ Measurement of dye uptake due to membrane damage, ■ Measurement of free enzyme amount due to membrane damage, ■ Viability Measurement of the rate of dye reduction by cell mitochondria, etc. +21 Problems with the Prior Art However, the above-mentioned known methods cannot be said to be excellent in terms of quantitative performance (quantitative accuracy of results), speed and simplicity of operation, etc. That is, all of these known methods are based on the principle of determining the survival rate or mortality rate of cells after incubation for several days after adding a drug, etc., so the fact remains that incubation takes time. , therefore, it is not suitable for rapid measurement. [Problem to be solved by the invention] The present invention significantly shortens cell incubation time in drug toxicity tests at the cellular level, and causes mild damage to cell membranes that does not cause death of living cells. By making it possible to quantitatively understand the effects of drugs, we can investigate drug toxicity at the cellular level.
The purpose is to provide a means for rapid and precise implementation.

[Structure of the invention]

[Means for Solving the Problems] In order to solve the above problems, the drug sensitivity testing method according to the present invention uses NAD(P)H:quinone oxidoreductase in the cell membrane and NAD(P)H supplying enzyme in the cell. It is characterized by the use of conjugate reactions with groups. Below, various matters related to the configuration of the invention will be explained. (1) Quinone The quinone in the present invention includes quinones such as benzoquinone, naphthoquinone, diphenoquinone, anthraquinone, and derivatives thereof, but it is preferable that the quinone does not have an easily oxidizable moiety other than a quinone group. All quinones can be used for inventive purposes, but quinones, known as vitamins, are particularly suitable as physiological substances.
-Methyl 1,4-naphthoquinone (menadione). (2) Quantification of hydrogen peroxide In the present invention, the production of reduced quinone can be measured by various known analytical techniques, but what seems to be pointed out at this point is that it is produced by the reaction between reduced quinone and dissolved oxygen. It is to quantify hydrogen peroxide. The hydrogen peroxide generated in the reaction system reacts with an excitation source material such as an aryl ester of oxalic acid and changes into an excited material such as 1,2-dioxetanedione, and the latter is converted into an excited material such as 1,2-dioxetanedione, which is converted into an excited material such as perylene with π electrons. By raising the energy level of a fluorescent substance rich in phosphors, it emits light. The reaction when oxalic acid diphenyl ester is used as an excitation source material and perylene is used as a fluorescent material is summarized by the following formula. (1,2 dioxetanedione) -0 (In the formula, Φ represents a phenyl group.) Quinone added to the reaction system changes cyclically from oxidized form to reduced form and from reduced form to oxidized form. , acts as a hydrogen peroxide production catalyst. The quantitative accuracy of hydrogen peroxide by the above luminescent reaction is 10-
7 to 10-3 mol/liter and the required time is about 1
It is 0 seconds. On the other hand, the time required for hydrogen peroxide production by cells depends on the physiological state of each individual cell, but for example yeast (
At 10 μg/m) g, detectable hydrogen peroxide is produced in 30 seconds. Therefore, it is possible to measure the number of live cells and vitality of yeast within about 40 seconds in total. (3) Target of measurement In the cell sensitivity measurement method according to the present invention, as described in the [Effect] section below, oxidized quinone present in the matrix outside living cells is used to detect NAD(P)H in the cell membrane. It is based on the phenomenon that quinone oxidoreductase and intracellular NAD(P)H-supplying enzymes convert into reduced quinone through a coupled reaction, and that this change changes depending on the permeability or vitality of the cell membrane. It is. Therefore, changes in cell membrane permeability or changes in the cytoplasm itself due to drugs are the subject of measurement, and means that can objectively measure the production of such reduced quinones can be used for measurement purposes. However, as mentioned above, at present, from the viewpoint of accuracy and simplicity, it is preferable to quantify hydrogen peroxide produced by the production of reduced quinone using a luminescent reaction. [Action] NAD(P)H (reduced nicotinamide adenine dinucleotide (phosphate)): Quinone oxidoreductase is present in the cytoplasm and reduces oxidized quinone to non-toxic diol quinone. It is known that this serves to prevent the production of toxic active oxygen (02 and H20□) from dissolved oxygen. However, as a result of research, the present inventor found that externally added quinone reacts with an as-yet-unidentified NAD(P)H:quinone oxidoreductase in the cell membrane, resulting in the production of H2O2 outside the cell. to promote the reduction of the redox indicator, and when this quinone additive H2O
The amount of 2 produced and the reduction rate of the redox indicator determine the vitality of the cell,
That is, we have discovered that there is a correlation with N A D (P) H supply ability. Therefore, this phenomenon is
499 (Japanese Patent Application No. 62319677) and the method for measuring stress sensitivity of cells according to Japanese Patent Application No. 1-96176. Furthermore, when a drug that damages cell membranes is added, H
Since the amount of 2O2 produced and the reduction rate of the redox indicator fluctuate sharply, by measuring these fluctuations, it is possible to test the toxicity of drugs that cause damage to cell membranes and denaturation of membrane proteins. [Example] Hereinafter, embodiments of the invention will be described using examples, but the examples are merely for explanation and are not intended to limit the idea of the invention. Example 1 (amount of peroxygen 60 mg of TCP○ (bis(
1,4,6-)lichlorophenyl)oxalate) and 1
A luminescent reaction solution was prepared by dissolving 0 mg of olefin. Sample solution 1- was mixed in 1r11 with this TCPO solution, and the luminescence count was measured for 5 seconds. 2 (Toxicity of dimethyl sulfoxide: )2.5 XIO
Dimethyl sulfoxide was added to a yeast suspension (solvent: 20mM imidazole/nitric acid pH 7.0) at various concentrations and incubated at 30°C for 15 minutes. Thereafter, menadione was added at a concentration of 0.5 mM, and after incubation at 30°C for 2 minutes, the generated hydrogen peroxide was quantified. As seen in the accompanying Figure 1, as the concentration of dimethyl sulfoxide increased, the production of hydrogen peroxide was inhibited exponentially. Concanavalin A (a T-cell mitogen derived from sea cucumber) was added to a suspension of 2.5 Xl06 yeast protoplasts/d (solvent: 1.2 M sorbitol + 25 mM imidazole + nitric acid pH 7.0). 30
After incubation for 10 minutes at 0C, the hydrogen peroxide generated was quantified. As seen in FIG. 2, concanavalin A had the effect of promoting the production of hydrogen peroxide. In this example, concanavalin A changes the membrane structure by binding to the sugar chain region of the glycoprotein exposed on the cell membrane surface, and induces the catalytic action of menadione or NAD (
P)H: It is thought to activate quinone oxidoreductase. Sodium dodecyl sulfate was added to a yeast suspension of 106 cells/d (solvent 30mM imidazole/nitric acid pH 7.0), and after incubation at 33°C for 2 minutes, the generated hydrogen peroxide was quantified. . As seen in Figure 3, sodium dodecyl sulfate is
It promoted the production of hydrogen peroxide up to 5m14, and on the contrary showed a suppressive effect above that. Sodium dodecyl sulfate is a well-known anionic surfactant that is used to denature proteins, but Sakoku 3 shows that this substance has the effect of causing changes in membrane structure and membrane protein structure. There is. Example 5 (Nystatin Hibiki) Nystatin (Str, no.
ursei (an antibiotic produced by C. ursei) was added and incubated at 33℃ for 1 hour.
Incubated for hours. Then, menadione was added to a concentration of 0.5M,
After incubation for 2 minutes at 33°C, the hydrogen peroxide generated was quantified. As seen in FIG. 4, Nyscutin inhibited the production of hydrogen peroxide. Nyscutin is known to have the property of reacting with sterols in cell membranes of Candida and the like to cause perforation in the abdominal cavity, causing cell contents to flow out. Therefore, substances with strong biochemical destructive power against cell membranes,
It is found that it damages cell membranes and acutely inhibits the production of hydrogen peroxide.

【Effect of the invention】

As explained above, the present invention provides a means for easily and quickly measuring the effect of drugs on the membranes of living cells, thereby improving the toxicity and irritation of drugs in cosmetics, shampoos, pharmaceuticals (e.g. eye drops), etc. Contribute to new product development through scales and speed up research on sex.

[Brief explanation of drawings]

FIG. 1 is a graph showing the relationship between the concentration of dimethyl sulfoxide and the amount of hydrogen peroxide generated, and FIG. 2 is a graph showing the relationship between the concentration of concanavalin A and the amount of hydrogen peroxide generated.
FIG. 3 is a graph showing the relationship between the concentration of sodium dodecyl sulfate and the amount of hydrogen peroxide generated, and FIG. 4 is a graph showing the relationship between the concentration of Nyscutin and the amount of hydrogen peroxide generated. Figure 2 Funcanapal 1 A (PH7) Sodium dodecyl sulfate (eM) Nystatin (Ato 10゛)

Claims (1)

[Scope of Claims] 1. A drug sensitivity testing method characterized by utilizing a coupled reaction between a cell membrane NAD(P)H:quinone oxidoreductase and an intracellular NAD(P)H supplying enzyme group. 2. The test method according to claim 1, wherein menadione is used as the quinone. 3. Claim 1 or 2, wherein hydrogen peroxide is determined by a luminescence method.
Method described.