KR20030033519A - Method for determining radiation sensitivity of animal by activities of antioxidative enzyme - Google Patents

Abstract

PURPOSE: A method for determining radiation sensitivity of animals by measuring the activity of catalase or superoxide dismutase is provided, thereby predicting the radiation sensitivity of a patient required for radiation therapy prior to the radiation therapy, and preventing adverse effects of the radiation therapy. CONSTITUTION: A method for determining radiation sensitivity of animals by measuring the activity of catalase or superoxide dismutase comprises the steps of: collecting a sample from an animal; measuring the activity of catalase or superoxide dismutase in the sample; and determining the radiation sensitivity of a tested animal as "radiation sensitive" when the level of the catalase or superoxide dismutase activity is low or as "radiation resistant" when the level of the catalase or superoxide dismutase activity is high, wherein the sample includes a lung tissue, a liver tissue and a kidney tissue of the animal; and the animal includes a human being.

Description

METHOD FOR DETERMINING RADIATION SENSITIVITY OF ANIMAL BY ACTIVITIES OF ANTIOXIDATIVE ENZYME}

The present invention relates to a method for determining the animal's sensitivity to radiation. Specifically, the present invention relates to a method for determining the animal's sensitivity to radiation by measuring the activity of antioxidant catalase and superoxide dismutase from a biological sample of the animal.

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 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 of the healthy cells recover, but some do not recover, which can lead to 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 may occur due to radiation therapy, such as delayed intelligence development or disorders in bone development.

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. To figure out whether

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 can quickly and accurately determine the sensitivity or resistance of an animal to the radiation while measuring the activity level of antioxidant catalase and superoxide dismutase from an animal biological sample. It was found.

The present invention collects a biological sample from an animal, optionally irradiates the sample with radiation, and then measures the activity level of the catalase enzyme from the sample, and if the measured activity level is low, it is determined as radiation sensitivity while the measured activity level is And a method of determining the sensitivity of the animal to radiation, characterized in that it is determined to be radiation resistant if high.

In addition, the present invention is to collect a biological sample from an animal, and optionally irradiate the sample with radiation, to measure the activity level of the superoxide dismutase enzyme from the sample, and to determine the radiation resistance if the measured activity level is low On the other hand, when the measured activity level is high, the radiation sensitivity is characterized in that it relates to a method for determining the animal's sensitivity to radiation.

1 is a graph comparing the activity of catalase by radiation in radiation resistant C3H mice and radiation sensitive C57BL / 6 mice.

Figure 2 is a graph comparing the activity of superoxide dismutase by radiation in radiation resistant C3H mice and radiation sensitive C57BL / 6 mice.

As used herein, the term "irradiation" means exposure to the action of electromagnet 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 the cell to whether the cell is sensitive or resistant by irradiation.

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.

Catalase and superoxide dismutases are representative defense enzymes for lipid peroxidation. Catalase and superoxide dismutase are both mitochondrial medulla of erythrocyte membranes and hepatocytes (Nohl H and Hegner D, Eur. J. Biochem, 82, 563 (1978)) and cytoplasm (Tappel AL Adv. Exp. Med. Biol. 97, 111 (1978)) and superoxide anion O₂ ^-) To prevent the peroxidation of lipids. Ie O by SOD₂ ^-end Dismutation to H₂O₂Is generated catalase H₂O₂Remove Therefore, such an enzyme is expected to play a crucial role in preventing tissue damage when free radicals are produced in the body by stimulation such as irradiation.

Catalase is a heme protein that exists in all living organisms and is an enzyme that catalyzes the reaction of hydrogen peroxide disproportionately divided into water and molecular oxygen.

Superoxide dismutase catalyzes the disproportionation of peroxide anions generated in aerobic tissues.₂ ^-of It is an antioxidant enzyme that protects organisms from toxicity. The higher animal SOD molecular weight is 32,000 and consists of two subunits, each of which has Cu²⁺And Zn²⁺Each of them contains 1 atom. In general, human lung has been reported to contain 7500 units / g wet wt of CuZnSOD and 86 units / g wet wt of MnSOD (Halliwell, B. and Gutterridge, JMC in Free radicals in Biology and Medicine, 3rd). ed. Oxford, (1999).

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 solid tissue samples such as body fluids of mammals (eg, blood, plasma), biopsy specimens, tissue cultures, or cells derived therefrom and their progeny. The samples also include reagent treatment, solubilized samples or cultured cells, cell supernatants and cell lysates.

According to the present invention, the activity of catalase and superoxide dismutase from a biological sample of a radiation-sensitive animal and a catalase from a biological sample of a radiation-resistant animal before and after irradiation of a biological sample of a radiation-sensitive animal and a radiation-resistant animal Comparison of the activity of the superoxide dismutase shows 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 catalase activity than biological samples obtained from radiation-sensitive animals before and after irradiation, whereas superoxide dismutases showed radiation resistance before and after irradiation. Much higher activity is seen in biological samples obtained from radiation-sensitive animals before and after irradiation than in biological samples obtained from animals.

Similarly, according to the present invention, biological samples are taken from radiation-sensitive and radiation-resistant animals and irradiated, and the activity of catalase and superoxide dismutase from biological samples of radiation-sensitive animals and from biological samples of radiation-resistant animals. Comparing the activity of catalase and superoxide dismutase shows a significant difference between the irradiated biological samples of the two animals. In other words, after biological samples from radiation-sensitive and radiation-resistant animals, the irradiated samples show significantly increased activity of catalase than biological samples obtained from radiation-sensitive animals before and after irradiation, whereas in the case of superoxide dismutase 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. The tissue was extracted and the catalase activity was measured. As a result, prior to irradiation, the lung tissue extract from radiation-resistant C3H mice showed much higher activity of catalase than in the lung tissue extract from radiation-sensitive C57BL / 6 mice. Immediately after irradiation, both mice transiently increased the activity of catalase, but still radiation-resistant mice showed higher enzymatic activity than radiation-sensitive mice.

In contrast to the activity of catalase, the activity of superoxide dismutase was determined to be higher in the lung tissue extracts of radiation-sensitive C57BL / 6 mice than in the lung tissue extracts from radiation-resistant C3H mice before irradiation. Later, superoxide dismutase activity from lung tissue extracts in radiation-resistant C3H mice increased and decreased after peak activity at 2 hours post-irradiation, whereas lung tissue extracts in radiation-sensitive C57BL / 6 mice showed post-irradiation 2 At time the catalase activity is rather temporarily lower than in lung tissue extracts of resistant C3H mice and continues to increase, decreasing after 24 hours showing higher peak activity than lung tissue extracts in resistant C3H mice. Except for the intermediate transient periods, lung tissue extracts from radiation-sensitive C57BL / 6 mice continued to show higher superoxide dismutase activity than lung tissue extracts from radiation-resistant C3H mice.

The significant difference in activity of glutathione metabolism related enzymes 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 of assaying the activity of catalase enzymes from biological samples from animals is a method of measuring the rate of hydrogen peroxide reduction due to the activity of catalase.

In general, the most commonly used method of assaying the activity of superoxide dismutase enzymes from biological samples from animals produces superoxides in various assay systems and reduces the rate of superoxide reduction by superoxide dismutase. It is a method using the principle of measurement. For example, cytochrome C (McCord, JM and Fridovich. IJ Biol. Chem., 244, 6049-6055 (1969)), nitroblue tetrazoliu (NBT) (Beauchamps, C. and Fidovich, I, Anal.Biochem., 44, 276-287 (1971)), lactate dehydrogenase (LDH) and NADH (Chan, PC and Bielski, BHJ, J. Biol. Chem., 249, 1317) -1319 (1974)) and O ₂ ⁻ production in a reaction system using any one selected from epinephrine (Misra, H. P and Fridovich. IJ Biol. Chem., 247, 3170-3175 (1972)). There is a method of determining the amount inhibited by this superoxide dismutase. At this time, as a source for providing the superoxide (xanthine) / xanthine oxidase (xanthine oxidase) and illuminated flavin (illuminated flavin) can be used. In addition, SOD inhibits the oxidation of pyrogallol and adrenaline by superoxide produced by autooxidation such as pyrogallol and adrenalin. How to measure the degree to

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 Sample for Measurement of Antioxidant Enzyme Activity by 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 irradiator (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 day / night cycle for one week. For enzyme activity analysis, 0.5 ml of 10 mM phosphate buffer solution (pH 7.4) was added to about 20 mg of the lung tissue, followed by grinding . After centrifugation at 4 ° C. for 30 minutes, the supernatant was taken and used as an enzyme source.

Example 2

Catalase Enzyme Activity Measurement

Catalase enzyme activity following irradiation of radiation-sensitive and radiation-resistant mice was analyzed by measuring the degree of decrease in absorbance due to the decomposition of hydrogen peroxide by this enzyme (Aebi, H. Catalase. In Bergmyer, HU, ed.Methods in enzymatic analysis.vol. 2. New York, Academic Press, 671-678 (1974).

50 mM potassium phosphate buffer solution (pH 7.0) containing 10 mM H ₂ O ₂ was added to the sample of Example 1 and reacted at room temperature to measure absorbance at 240 nm. At this time, the enzyme activity of catalase was converted into H ₂ O ₂ removal rate / mg protein as ε ₂₄₀ = 43.6 / M · cm of hydrogen peroxide (H ₂ O ₂ ).

As a result, the catalase activity before irradiation was higher in radiation-resistant lungs of C3H mice than in radiation-sensitive C57BL / 6 mice. At this time, the enzyme activity was observed according to the time difference between the two species, the radiation-resistant mice showed the highest activity after 2 hours of irradiation and the radiation-sensitive mice had the highest activity after 1 day of irradiation (Fig. 1). .

Example 3

Determination of Superoxide Dismutase Enzyme Activity

Superoxide dismutase enzyme activity following irradiation in radiation-sensitive and radiation-resistant mice was measured by the following method (McCord, J. M. J. Biol. Chem. 244, 6049-6055 (1969)).

The rate of reduction of cytochrome C by peroxide was determined from the absorbance change of 550 nm while generating superoxide by xanthine / xanthine oxidase reaction, and then the plasma of Example 1 was added and exceeded. It was determined by measuring the extent of reduction of the cytochrome c reduction caused by removal of excess oxide by oxide dismutase. At this time, the amount of excess oxide dismutase that can reduce the reduction rate of cytochrome C by 50% was set to 1 unit. That is, the same amount of 50 mM potassium phosphate buffer (pH7.5) containing 0.1 mM xanthine, 0.5 mM xanthine oxidase, 0.1 mM EDTA, and 10 μM cytochrome C was added to the sample of Example 1 and reacted at room temperature to react at 550 nm. Absorbance was measured.

As a result, the superoxide dismutase activity before irradiation was higher in radiation-resistant lungs of C3H mice than in radiation-sensitive C57BL / 6 mice, and the enzyme activity increased after irradiation. At this time, the enzyme activity was observed according to the time difference between the two species, radiation-resistant mice showed the highest activity after 2 hours of irradiation and radiation-sensitive mice had the highest activity after 1 day of irradiation (FIG. 2). .

The method for determining the sensitivity of an animal to radiation according to the present invention is characterized by measuring the activity of the antioxidant enzyme catalase and the superoxide dismutase from a biological sample of the animal to determine whether the animal is resistant to radiation or sensitive. There is an advantage that can be quickly and accurately determine the sensitivity. 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 the catalase enzyme from the sample, determining the radiation sensitivity if the measured activity level is low, and determining the radiation resistance if the activity level is high. , How to determine the sensitivity of animals 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 a biological sample from the animal, measuring the activity level of the superoxide dismutase enzyme from the sample, 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 the method for determining 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.