KR20030033518A - Method for determining radiation sensitivity of animal by activities of glutathione related enzymes - Google Patents

Abstract

PURPOSE: A method of determining the radiation sensitivity or radiation resistance of an animal by measuring the activity level of an enzyme associated with glutathione from a biological sample is provided, thereby predicting the radiation sensitivity of a patient prior to radiotherapy and effectively preventing of adverse effects caused by radiation. CONSTITUTION: A method of determining the radiation sensitivity of an animal comprises: collecting a biological sample from an animal; measuring the activity level of one or two enzymes selected from the group consisting of glutathione peroxidase, glutathione reductase and glucose 6-phosphate dehydrogenase; and determining the radiation sensitivity thereof.

Description

METHOD FOR DETERMINING RADIATION SENSITIVITY OF ANIMAL BY ACTIVITIES OF GLUTATHIONE RELATED ENZYMES}

The present invention relates to a method for determining the animal's sensitivity to radiation. Specifically, the present invention measures the activity of glutathione-related enzymes glutathione peroxidase, glutathione reductase and glucose 6-phosphate dehydrogenase from animal biological samples. The present invention relates to a method for determining the sensitivity to radiation.

Ionizing radiation (IR), such as X-rays and gamma rays, ionizes water and produces excitation products. Animals contain water at 55-80% of their body weight. Therefore, when exposed to radiation, such as hydroxy radical (OH ^· ), hydrogen peroxide (H ₂ O ₂ ), alkoxy (RO ^· ), peroxy (ROO ^· ), superoxide (O 2 ^·- ) Reactive oxygen species (ROS) are produced in the body. The production of free radicals, the product of radiolysis of water, increases even more when oxygen is dissolved in water. Because reactive oxygen species are very reactive, they react with almost all kinds of biomolecules in the cell, resulting in protein oxidation, cell membrane lipid peroxidation, DNA chain cleavage and oxidation, and so on.

Treatment of cancer using the cellular damaging effects of radiation is commonplace. Radiation therapy is a cancer treatment that uses high energy radiation to kill cancer cells. Radiation can affect both cancerous cells and healthy cells, but many methods and techniques are used to treat cancer by destroying many cancer cells while less affecting normal tissues. When radiation is irradiated to cancer, it does not kill cancer cells immediately, but destroys their ability to divide and proliferate, preventing new cancer cells from dividing and producing, and cancer cells that do not divide anymore die at the end of their lifespan. With every treatment, more cells die and the dead cells are broken down and transported to the blood, where they are released out of the body, reducing the size of the tumor. Most healthy cells recover, but some do not, and can cause side effects of radiation therapy. In other words, radiotherapy may inevitably result in weakening or loss of normal tissue function. Secondary cancers may occur in areas that have undergone radiation treatment years or decades after treatment. In particular, in children with the development of each organ, serious side effects such as delayed intelligence development and disordered bone development may occur due to radiotherapy.

Until now, much research has been conducted to develop a means for effectively treating patients with radiation, and there are no studies on minimizing side effects that can be caused by radiation before radiation therapy. US Patent No. 6,261,795 discloses a method for measuring the radiation sensitivity of cancer cells by irradiating the cancer cells and then culturing and proliferating the cancer cells, but the object of the present invention is that cancer cells can be killed by radiation to some extent. Was to figure out.

In this regard, there is an urgent need for a method that can minimize the side effects of radiation to the patient by accurately predicting how much radiation can affect the patient depending on the patient's individual constitution before the patient is given radiation therapy. .

The inventors of the present invention have studied the sensitivity or resistance of radiation to the activity of animals while studying the sensitivity of the radiation to the activity of glutathione peroxidase, glutathione peroxidase, glutathione reductase and glucose-6 phosphate dehydrogenase. It has been found that by determining can be determined quickly and accurately.

The present invention collects a biological sample from an animal, and optionally irradiates the sample with radiation, and then measures the activity level of one or two enzymes selected from the group consisting of glutathione peroxidase and glutathione reductase from the sample. The present invention relates to a method for determining the sensitivity of an animal to radiation, characterized in that it is determined as radiation sensitivity when the activity level is low, whereas radiation resistance is determined when the measured activity level is high.

In addition, the present invention collects a biological sample from the animal, and optionally irradiated with the collected sample, and then measure the activity level of glucose phosphate dehydrogenase from the sample, if the measured activity level is low is determined as radiation resistance A method for determining the sensitivity of an animal to radiation is characterized by determining radiation sensitivity when the measured activity level is high.

1 is a schematic diagram showing the oxidation / reduction reaction of glutathione.

Figure 2 is a schematic diagram showing the flow of glutathione in vivo.

Figure 3 is a graph comparing the activity of glutathione peroxidase by radiation of radiation resistant C3H mice and radiation sensitive C57BL / 6 mice.

4 is a graph comparing the activity of glutathione reductase by irradiation of radiation resistant C3H mice and radiation sensitive C57BL / 6 mice.

5 is a graph comparing the activity of glucose 6-phosphate dehydrogenase by irradiation of radiation-resistant C3H mice and radiation-sensitive C57BL / 6 mice.

As used herein, the term "irradiation" means exposure to the action of electron radiation or alpha or beta particles, including, for example, X-rays, ultraviolet light, alpha particles and gamma rays.

The term "Gy" is the dose of radiation capable of releasing one joule from 1 kg of biological tissue.

The term “sensitivity to radiation” generally refers to the propensity of a cell to respond to radiation by being sensitive or resistant to radiation.

The term "radiation sensitive" generally refers to the propensity of cells that are easily affected by irradiation, for example, a reduced proportion of cells in the mitotic stage of cell restoration or cell growth that is inhibited after irradiation. It is used to indicate the propensity of a sample, and more specifically, due to the low activity of enzymes involved in the antioxidant system in vivo, which functions to remove reactive oxygen species generated in vivo, It indicates the tendency of the cell to be easily affected by the generated active oxygen body.

The term "radiation resistant" generally refers to the propensity of a cell not to be easily affected by irradiation and that no change in cell restoration or proliferation occurs when exposed to radiation. Specifically, the activity of the enzymes involved in the antioxidant system in vivo, which acts to remove the active oxygen bodies generated in vivo, and thus the tendency of the cells not affected by the active oxygen bodies generated in large quantities by radiation are shown. Point.

The term "animal" refers to a mammal and includes, for example, livestock, primates, especially humans.

The term "patient" applies to all mammals and in particular refers to a mammal suffering from a disease in which radiation therapy may be effective.

Glutathione (γ-glutamylcysteinylglycine) is a tripeptide sulfhydryl compound present in cells, at concentrations of several to tens of mM, in animals, plants as well as some bacteria. Glutathione is an antioxidant molecule that protects living organisms from radiation, ultraviolet and photoactive effects (Arrick BA et al., J Biol Chem, 57, 1231-1247 (1982); Connor MJ anc Weeler LA, Photochem Photobiol 46, 239-245 ( 1987)). Glutathione exists in reduced form (reduce glutathione, GSH) and oxidized form (oxidized glutathione or glutathione disulfide, GSSG) depending on the oxidation / reduction state of the cell. In a normal cellular environment, glutathione is mostly present in reduced form (GSH) and in very small amounts of oxidized form (GSSG) (Sies, H., Oxidative stress, 73-90, New York, Academic; Thom, SR et al. , J. Appl. Physio. 82, 1424-1432 (1997)). However, when the cells become oxidized, enzymatic action occurs to prevent the oxidation of major biomolecules in the tissue, and glutathione is converted to the oxidized form. Oxidation and reduction of glutathione are regulated through a series of reactions as shown in FIG. 1 and include glutathione peroxidase, glutathione reductase, and glucose 6-posphate dehydrogenase. do. Glutathione is produced in the liver and secreted in the form of reduced glutathione (GSH) in the blood. The kidney degrades glutathione by the γ-glutamyl transpeptidase reaction to either absorb GSH from the blood or directly into the kidney cells (FIG. 2).

Glutathione's antioxidant system occurs through the following processes. When reactive oxygen species (ROS) are produced and cells become oxidized by various factors such as irradiation, pathogenic tolerance, and drug effects, glutathione acts on the action of glutathione peroxidase (Gpx). By reacting with hydrogen peroxide (H ₂ O ₂ ) is oxidized to oxidized glutathione (GSSG) to reduce the ratio of reduced glutathione / oxidized glutathione (GSH / GSSG). The generated GSSG is reduced by NADPH under the action of glutathione reductase (Grd) and then reduced to GSH or discharged out of the cell. That is, the oxidation / reduction cycling of glutathione by the action of glutathione peroxidase and glutathione reductase is an important determinant for removing the active oxygen generated by irradiation. At this time, since NADPH is required as a reducing agent to reduce intracellular glutathione, the degree of activity of glucose 6-phosphate dehydrogenase (G6PDH), which produces NADPH, is another factor that determines the reduction of glutathione. . The reaction scheme of the glutathione metabolism-related enzyme is as follows.

Biological samples for use in the methods of the present invention can be obtained from kidney tissue, lung tissue, liver tissue, which can be selected for the determination of radiation sensitivity. Especially preferably, lung tissue is selected as a determination object. This is because in the lungs, dissolved oxygen is present at a high level and thus the cell damage caused by free radicals is most severe.

In addition, biological samples may include biopsy specimens of mammals, solid tissue samples such as tissue culture or cells derived therefrom and their progeny. The samples also include reagent treatment, solubilized samples or cultured cells, cell supernatants and cell lysates. More specifically, biological samples for the purposes of the present invention are obtained from tissues in which glutathione metabolism occurs, such as by excisional surgery, biopsy, aspiration or other methods known in the art.

According to the present invention, the activity of glutathione peroxidase, glutathione reductase and glucose hexaphosphate dehydrogenase from biological samples of radiation-sensitive animals before and after radiation of radiation-sensitive and radiation-resistant animals, Comparing the activity of glutathione peroxidase, glutathione reductase and glucose hexaphosphate dehydrogenase from biological samples, there is a significant difference between the biological samples of the two animals. In other words, biological samples obtained from radiation-resistant animals before and after irradiation showed significantly increased activity of glutathione peroxidase and glutathione reductase than biological samples obtained from radiation-sensitive animals before and after radiation, whereas glucose 6-phosphate dehydrogenase Much higher activity is seen in biological samples obtained from radiation sensitive animals before and after irradiation than in biological samples obtained from radiation resistant animals before and after irradiation.

Similarly, according to the present invention, biological samples from radiation-sensitive and radiation-resistant animals are irradiated and irradiated, and the activity and radiation resistance of glutathione peroxidase, glutathione reductase and glucose hexaphosphate dehydrogenase from biological samples of radiation-sensitive animals. Comparing the activity of glutathione peroxidase, glutathione reductase and glucose hexaphosphate dehydrogenase from biological samples from animals, there is a significant difference between the irradiated biological samples of the two animals. In other words, after taking biological samples from radiation-sensitive and radiation-resistant animals, the irradiated samples showed significantly increased activity of glutathione peroxidase and glutathione reductase than biological samples obtained from radiation-sensitive animals before and after irradiation, whereas glucose Hexaphosphate dehydrogenase shows much higher activity in biological samples obtained from radiation-sensitive animals before and after irradiation than in biological samples obtained from radiation-resistant animals before and after irradiation.

As an exemplary experiment, radiation-resistant C3H mice (Department of Experimental Radiation Oncology, University of Texas MD Anderson Cancer Center) and radiation-sensitive C57BL / 6 mice (Jackson Laboratory) before and after irradiation were exposed to lungs over time. Tissues were extracted to measure glutathione peroxidase and glutathione reductase activity. As a result, prior to irradiation, the activity of glutathione peroxidase and glutathione reductase was much higher in lung tissue extracts from radiation-resistant C3H mice than in lung tissue extracts from radiation-sensitive C57BL / 6 mice. Immediately after irradiation, both mice temporarily increased both the activity of glutathione peroxidase and glutathione reductase, but still radiation resistant mice showed much higher enzyme activity than radiation sensitive mice.

In contrast to the activity of glutathione peroxidase and glutathione reductase, glucose hexaphosphate dehydrogenase (G6PDH) activity was more in lung tissue extracts of radiation-sensitive C57BL / 6 mice than in lung tissue extracts from radiation-resistant C3H mice prior to irradiation. High activity was measured, and after irradiation, glucose hexaphosphate dehydrogenase activity from lung tissue extracts of radiation-resistant C3H mice was increased and peaked at 2 hours after irradiation, whereas decreased in lungs of radiation-sensitive C57BL / 6 mice. In the case of the tissue extract, the glucose hexaphosphate dehydrogenase activity was decreased temporarily after irradiation, showed the lowest activity after 6 hours, and then increased again, except for the intermediate transient period. High in Lung Extracts of Sensitive C57BL / 6 Mice Clique 6 phosphate dehydrogenase activity exhibited.

Significant differences in activity of glutathione metabolism-related enzymes appearing from biological samples of radiation resistant animals and biological samples of radiation sensitive animals as described above can be applied to determine the sensitivity of animals to radiation in accordance with the present invention so that patients may receive radiation It may be useful to minimize the patient's side effects to radiation by determining if the patient is resistant or sensitive to radiation before receiving therapy.

In general, the most commonly used method for assaying the activity of glutathione peroxidase from biological samples of animals is the method of analyzing glutathione peroxidase (GSSG), a product of the glutathione peroxidase reaction, using glutathione reductase (coupled assay). As a result, it can be analyzed by measuring the rate of production of NADP ⁺ , the end product of metabolism. The reaction scheme is summarized as follows.

In general, the most commonly used method for assaying the activity of glutathione reductase from biological samples in animals can be analyzed by measuring the rate of reduction of oxidized glutathione (GSSG) by the action of glutathione reductase from the rate of decrease of NADPH. . The reaction scheme is summarized as follows.

In general, the most commonly used method for assaying glucose-6 dehydrogenase activity from biological samples from animals is to obtain the oxidation of glucose 6-phosphate by the action of glucose 6-phosphate dehydratase from the rate of NADPH production. Is as follows.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are illustrative of the present invention, and the content of the present invention is not limited by the examples.

<Example>

Example 1

Preparation of Samples for Measuring Activity of Glutathione Metabolism-Related Enzymes During Irradiation

Radiation-resistant C3H mice and radiation-sensitive C57BL / 6 mice (male, 4 weeks, 20-30 g) were irradiated with 10 Gy of whole-body with a 4 MV X-ray machine (Siemens, MeVatron). After 2 hours, 6 hours, 1 day, and 4 days after irradiation, animals were sacrificed, and lungs and kidneys were immediately removed, compressed with forceps cooled to -70 ° C, rapidly frozen, and stored at -70 ° C until analysis. . The animals were fed standard feed and water and maintained a 12 hour light / dark cycle for one week. 20 mg of the lung tissue was ground by adding 0.5 ml of 10 mM phosphate buffer solution (pH 7.4). The ground lung tissue extract was centrifuged at 12000 g at 4 ° C. for 30 minutes to obtain a supernatant, which was used for enzyme activity analysis.

Example 2

Determination of Glutathione Peroxidase Activity

Glutathione peroxidase activity following irradiation of radiation-sensitive and radiation-resistant mice was investigated in the following manner (Lawrence RA and Burk RF, Biochem Biophys Res Commun 71, 952-958 (1976)).

The sample prepared in Example 1 contains 1 mM EDTA, 1 mM NaN ₃ , 0.2 mM NADPH, 1 unit / ml glutathione reductase (Sigma, USA), 1 mM reduced glutathione (GSH, Sigma, USA) 50 mM potassium phosphate buffer (pH 7.4) was added in the same amount and incubated for 5 minutes at room temperature. Then, the reaction was started by adding 0.25 mM H ₂ O _2. Upon completion of the reaction, the absorbance at 340 nm was measured. Ε ₃₄₀ = 6.22 / mM · cm of NADPH, from which the change in absorbance was converted into enzymatic activity (nmol / min / mg protein).

As a result, the activity of glutathione peroxidase by irradiation was temporarily increased in both species, but higher in radiation-resistant C3H mice (FIG. 3). At this time, the change in enzyme activity was temporally different. C3H mice showed the highest activity after 2 hours of irradiation, and the radiation-sensitive C57BL / 6 showed the highest activity after 1 day and then began to decrease. This resulted in higher glutathione peroxidase activity and higher radiation activity in radiation resistance than radiation-sensitive species.

Example 3

Determination of Glutathione Reductase Activity

Glutathione reductase activity following irradiation of radiation-sensitive and radiation-resistant mice was investigated in the following manner (Tabatabaie T and Floyd RA, Arch Biochem Biophys, 314, 112-119 (1994)).

To the sample of Example 1, 100 mM potassium phosphate buffer (pH 7.4) containing 0.6 mM EDTA and 2 mM oxidized glutathione (GSSG) was added in the same amount, followed by incubation for 5 minutes at room temperature, and 0.1 mM of NADPH. The reaction was initiated. After the reaction was completed, absorbance at 340 nm was measured. The NADPH was ε ₃₄₀ = 6.22 / mM · cm, from which the change in absorbance was converted into enzymatic activity (nmol / min / mg protein).

As a result, the activity of glutathione reductase by irradiation was temporarily increased in both species, but higher in radiation-resistant C3H mice (FIG. 4). At this time, the change in enzyme activity was temporally different. C3H mice showed the highest activity after 2 hours of irradiation, and the radiation-sensitive C57BL / 6 showed the highest activity after 1 day and then began to decrease. The radiation-resistant species showed higher glutathione reductase activity than the radiation-sensitive species, and the increase in activity by irradiation showed a faster response.

Example 4

Measurement of Glucose-6 Phosphate Dehydrogenase Activity

The activity of glucose-6 phosphate dehydrogenase following irradiation in radiation-sensitive and radiation-resistant mice was investigated in the following manner.

The absorbance at 340 nm was measured by reacting the sample of Example 1 with 50 mM Tris-HCl buffer solution (pH 8.0) containing 50 mM MgCl ₂ , 2 mM NADP ⁺ , 4 mM glucose 6-phosphate. . The NADPH was ε ₃₄₀ = 6.22 / mM · cm, from which the change in absorbance was converted into enzymatic activity (nmol / min / mg protein).

The results showed that glucose 6-phosphate dehydrogenase activity was higher in radiation-sensitive C57BL / 6 mice before irradiation, but increased in radiation-resistant C3H mice after irradiation. In radiation-resistant C3H mice, the highest enzyme activity was decreased after 2 hours of irradiation and then decreased. On the other hand, in the case of radiation-sensitive C57BL / 6 mice, the enzyme activity was decreased temporarily after irradiation, showing the lowest activity after 6 hours, and increasing again (FIG. 5).

The method of determining the sensitivity to radiation in an animal according to the present invention is characterized by measuring the activity of glutathione peroxidase, glutathione reductase and / or glucose 6 phosphate dehydrogenase, an enzyme related to glutathione from a biological sample of the animal, thereby preventing the animal from radiation. There is an advantage that it is possible to quickly and accurately determine whether sensitive or sensitive to. Accordingly, the method of the present invention is expected to be very useful for preventing side effects caused by radiation of a patient by providing a means of predicting the patient's sensitivity to radiation prior to administering radiation therapy to the patient.

Claims (8)

Taking a biological sample from the animal, measuring the activity level of one or two enzymes selected from the group consisting of glutathione peroxidase and glutathione reductase from the sample, and determining the radiation sensitivity if the activity level is low. On the other hand, if the measured activity level is high, characterized in that it is determined as radiation resistance, the method of determining the sensitivity of the animal to radiation. The method of claim 1, wherein the biological sample is selected from animal lung tissue, liver tissue and kidney tissue. The method of claim 1 wherein the animal is a human. The method of claim 1, wherein the biological sample taken from the animal is irradiated with radiation. Taking biological samples from animals, measuring the activity level of glucose 6-phosphate dehydrogenase from the collected samples, and determining the radiation resistance if the measured activity level is low, and determining the radiation sensitivity if the activity level is high. Characterized in that it determines the sensitivity of the animal to radiation. 6. The method of claim 5, wherein the biological sample is selected from animal lung tissue, liver tissue and kidney tissue. The method of claim 5 wherein the animal is a human. The method of claim 5, wherein the biological sample taken from the animal is irradiated with radiation.