CN110478365B

CN110478365B - Application of hydrogen-rich water in preparation of tritium-poisoning-promoting drinking water or medical liquid

Info

Publication number: CN110478365B
Application number: CN201910695966.4A
Authority: CN
Inventors: 李蓉; 王卫东; 李宏; 殷娅茹; 杨露勋; 卢丙慧; 刘晶; 曾一平; 高继宁; 贺尹娟; 粟永萍
Original assignee: Sichuan Cancer Hospital; Army Medical University
Current assignee: Third Military Medical University TMMU
Priority date: 2019-07-30
Filing date: 2019-07-30
Publication date: 2021-09-03
Anticipated expiration: 2039-07-30
Also published as: CN110478365A

Abstract

The invention relates to application of hydrogen-rich water in preparation of tritium-poisoning-promoting drinking water or medical liquid. The invention proves that the hydrogen-rich water can lead GSH (glutathione), SOD (superoxide dismutase) and 8-OHDG (OHDG) which are increased by the stimulation of cells to gradually fall back to normal level through experiments, and the tritium content in the cells is also obviously reduced due to the advanced protection of the hydrogen-rich water. The results also show that the effect of 0.6mmol/L hydrogen-rich water on cell protection and tritium excretion is better than that of 0.2mmol/L, which indicates that tritium in body tissues can be replaced by high-concentration hydrogen-1, and molecular hydrogen can also remove a large amount of free radicals generated in cells due to tritium damage, thereby improving the activity of the cells.

Description

Application of hydrogen-rich water in preparation of tritium-poisoning-promoting drinking water or medical liquid

Technical Field

The invention belongs to the field of medical application, and particularly relates to application of hydrogen-rich water in preparation of tritium-poisoning-promoting drinking water or medical liquid.

Background

Tritium (A)³H, trim) is widely used in the nuclear industry and defense industry as a main raw material for nuclear power plant operation and nuclear weapon manufacture, and the number of people affected is large. For example, the tritium occupational irradiation work range of a nuclear industry system is wide, the number of occupational irradiation workers is large, the most typical nuclear power system is that a certain amount of tritium is generated by both a light water pressurized water reactor and a heavy water pressurized water reactor, and particularly around a spent fuel water pool and a reactor water pool for refuelling and overhaul. In the running process of nuclear facilities, tritium is generated and discharged and then absorbed by human bodies, so that internal irradiation pollution of the tritium is caused, and the health of radiation personnel is directly influenced. Tritium is the most common artificial internal contamination nuclide detectable in the body of the radiator in a nuclear power plant. With the rapid development of national economy, China also needs to develop nuclear power vigorously to meet the demand for energy. The tritium occupational irradiation crowd is continuously expanded, and the concentration of tritium in the body of a radiation worker is obviously increased. In addition, tritium has important research value and wide application in various departments of national economic development, such as the fields of industry, agriculture, biology, medicine, environmental science, archaeology and the like, and the professional irradiation of tritium also exists in experimental piles for various researches, radioisotope production piles, associated mines, nuclear submarine power piles and some nuclear decommissioning facilities.

Tritium is mainly composed of tritium gas and tritium water (tritium oxide, HTO, T) in the environment₂O) form, and tritium gas in air generates tritium water through oxidation reaction and isotope exchange reaction, so that the content of the tritium gas and the content of the tritium water in the environment are 0.1% and 99% respectively. Tritium is easy to enter human body through respiration, digestive tract, skin and other ways, wherein tritium water has the greatest influence, the absorption capacity of human body to tritium water is 4 orders of magnitude higher than that of tritium gas, and the toxicity of tritium water is 520 times that of nuclide tritium. Only a small part of tritium gas is absorbed and combined by human body through inspiration, and the rest is discharged rapidly along with expiration. The biological absorption efficiency of gaseous tritium water is very high, and about 99% of inhaled tritium water is absorbed by a human body through blood circulation; the ingested liquid tritium water is also almost completely absorbed by the digestive tract and rapidly enters the blood circulation. Tissues with high water content and relatively high tritium content, such as liver and muscle tissues. By isotopic exchange or enzymatic reaction, in vivoTritium is incorporated into Organic molecules as tissue-bound tritium, wherein tritium, which is bound to oxygen, nitrogen, sulfur, and phosphorus in the Organic molecules, is easily exchanged again, and is called "exchangeable Organic bound tritium" (OBT); tritium bound to carbon is called "strongly organically bound tritium" because it is not readily exchanged out by enzymatic reactions. The metabolic kinetics of tritium water distribution in vivo can be described using a three-compartment model, see FIG. 14. The biological half-discharge period of tritium for human being is about 8-l 0 days, which can cause a certain internal irradiation hazard.

Tritium is a hazard classified as acute and chronic. Tritium emits beta particles with an average energy of 5.72kev, a maximum energy of 18.6kev, and an average range of 0.56 μm in air. During normal operation, the radiation hazard to personnel is mainly internal irradiation generated after tritium water in the air is absorbed into the human body. Tritium beta radiation causes ionization and excitation in body fluids, generating positive and negative ions and free radicals, which can cause radiation damage when in contact with tissue. The larger the surface area of organ tissue in unit mass in contact with body fluid, the faster the body fluid flows, the more chance the free radicals and positive and negative ions contact with the tissue, and the more serious radiation damage. Biological effect research shows that tritium water has acute radiation injury effect (similar to external radiation acute radiation sickness), chronic radiation sickness, reproductive effect, chromosome aberration and other deterministic effects on radiation damage of organisms; random effects such as carcinogenic effects and genetic effects were also confirmed. If researches indicate that low-level chronic tritium uptake can cause leukemia and other malignant tumors, mainly because the tritium is firmly combined with body tissues after being ingested and is organically combined with proteins, glycogen, DNA and other macromolecular sites for a long time. The contribution of organically bound tritium accounts for about 5% of total tritium and 10% of total tritium, but remains in the body for a long time, and produces a cumulative radiation effect with a large dose, and chronic radiation sickness can be caused by long-term contact with tritium.

Tritium is a radioactive isotope of hydrogen-1, both of which are similar in physicochemical properties. After the radioactive nuclide tritium enters the body, the radioactive nuclide tritium mainly participates in the metabolic process of the in vivo stable nuclide hydrogen-1, just as radioactive iodine participates in the metabolism of the in vivo stable nuclide iodine-127, and is finally accumulated in the thyroid. Similarly, tritium water has similar physicochemical properties to water, and regardless of the form in which it is absorbed by the body, it is generally distributed in 1-2h throughout all tissues and organs containing water in the body, exhibiting retention characteristics similar to water.

At present, tritium promotes excretion, and no better method is available for drinking water and diuretics at home and abroad. The tritium excretion promoting effect of traditional Chinese medicines such as tritium excretion tablets and the like on tritium is explored in China, and the mechanism of the tritium excretion promoting effect is to accelerate the excretion of tritium from the body through the diuresis effect of the traditional Chinese medicines.

Disclosure of Invention

The invention aims to provide application of Hydrogen Rich Water (HRW) in preparation of tritium-toxicity-discharge-promoting drinking water or medical liquid.

The concentration of hydrogen in the hydrogen-rich water is 1.2-3.0ppM, and the hydrogen-rich water is dissolved in purified water and prepared into solutions with different hydrogen concentrations for use according to requirements.

The tritium poisoning is the damage to human lymphocytes caused by exposure to tritiated water.

The human lymphocyte is an AHH-1 cell.

The tritium intoxication is a genetically altered insult.

The genetic material altered damage is increased myelophagocytic polycythemic micronucleus rate, damage to cell chromosomes and/or DNA caused by tritium water exposure.

The tritium intoxication is a hematological change.

The hematological change is a change in peripheral hemogram.

The peripheral hemogram changes to a decrease in leukocytes and/or platelets.

The medical liquid can be added with medically acceptable auxiliary materials in hydrogen-rich water; or mixing hydrogen-rich water with other medicines for preventing or treating tritium-expelling poisoning, and optionally adding pharmaceutically acceptable adjuvants.

The application of the invention adopts hydrogen-rich water to promote tritium discharge, and provides a good way for promoting tritium discharge. If administered with a high concentration of stable hydrogen-1 (molecular hydrogen)¹H₂) With its radioactive isotope tritium (³H) It should be a good way to compete for binding sites in the body or to exchange with exchangeable organic binding tritium to release tritium and promote the elimination of tritium. Hydrogen-rich water is a substance that generates a large amount of molecular hydrogen by high-pressure aeration, electrolysis of water, or the like (¹H₂) This water with a high molecular hydrogen content is called hydrogen-rich water. A large number of studies prove that molecular hydrogen can effectively prevent and treat various diseases and injuries, for example, a plurality of studies prove that hydrogen can resist radiation injury, improve spinal cord ischemia reperfusion injury, relieve renal injury induced by unilateral urinary tract obstruction, and improve rat striated muscle lysis and renal injury induced by glycerol; has protective effect on necrotizing enterocolitis and ulcerative colitis of newborn mice; relieving liver cirrhosis, improving life quality of cancer patients, treating periodontitis, sepsis and dermatosis, and preventing and treating acute oxidative stress, carbon monoxide poisoning, etc. Although the specific mechanism of action and the mechanism of targeting molecules are not clear, research has shown that the molecular hydrogen can react with the hydroxyl radical (. OH) and peroxynitrite anion (ONOO) with strong oxidizability^-) The like, and the nucleic acid cleavage, lipid peroxidation and protein activity loss caused by the reaction are reduced or prevented; in addition, it enhances the activity of antioxidase such as catalase, superoxide dismutase and oxygenase-1; hydrogen can have anti-apoptotic effect by inhibiting caspase-3 activity; in an inflammatory response induced by oxidative stress, hydrogen down-regulates the levels of the inflammatory response factors IL-1 β, IL-6, chemokines and TNF- α to have an anti-inflammatory response. Very small amount of H₂(0.04mM) can really regulate the expression of various genes and the phosphorylation process of proteins, and has more obvious protective and therapeutic effects. The most important hydrogen is a good antioxidant per se, and has the following advantages: the hydrogen molecule is very small, so that the hydrogen can quickly permeate to the whole body when entering the body, easily penetrates through cell membranes and enters organelles such as mitochondria, cell nucleus and the like; ② with OH in vivo^-、NO^-When the reaction condition of free radicals is mild enough, neutralized water is discharged out of the body without influencing other benign active oxygen (such as SOD and the like) and organ functions; ③ divideThe sub-hydrogen is almost non-toxic, on one hand, the human intestinal tract generates a small amount of hydrogen; on the other hand, hydrogen gas inhaled at a certain concentration is also applied to diving medicine. At present, hydrogen is considered to be relatively safe for human bodies, and even if the hydrogen with dozens of atmospheric pressures is breathed, the hydrogen does not cause toxic effect on the bodies and only has certain anesthetic effect. Thus, molecular hydrogen and water in hydrogen-rich water are preferred methods for tritium stimulation.

The invention proves that the hydrogen-rich water can lead GSH (glutathione), SOD (superoxide dismutase) and 8-OHDG (OHDG) which are increased by the stimulation of cells to gradually fall back to normal level through experiments, and the tritium content in the cells is also obviously reduced due to the advanced protection of the hydrogen-rich water. The results also show that the effect of 0.6mmol/L hydrogen-rich water on cell protection and tritium excretion is better than that of 0.2mmol/L, which indicates that tritium in body tissues can be replaced by high-concentration hydrogen-1, and molecular hydrogen can also remove a large amount of free radicals generated in cells due to tritium damage, thereby improving the activity of the cells.

Drawings

FIG. 1 is urine tritium, serum tritium, and tissue OBT levels at various time points in mice after HRW administration, where A is mouse urine output; b is the total amount of radioactivity excreted by the mice in urine at different times after exposure; c is the radioactivity of the mouse serum at different time points; d is the activity of the OBT in each mouse organ at day 7 after exposure. In the figure, p <0.05, p < 0.01;

FIG. 2 is a comparison of myeloeosinophilic polycythemic micronucleus rates of various groups of mice at different time points after HTO treatment; wherein, A is mouse marrow pleochromocyte micronucleus, B is the comparison of the mouse marrow pleochromocyte micronucleus rate at different time points after HTO treatment, p is less than 0.05, p is less than 0.01;

FIG. 3 is a trace whole blood comet assay for each group of mice, wherein A is a fluorescence picture of the trace whole blood comet assay for each group of mice, and DNA is stained with ethidium bromide; b, comparing the percentage content of DNA at the tail of the micro whole blood comet assay cells of each group of mice; p <0.05, p < 0.01; c is the comet cell rate of each group of mice in the trace whole blood comet experiment, and each group counts 1000 cells; p <0.05, p < 0.01;

FIG. 4 is a graph of the routine peripheral blood changes of groups of mice from 0 to 14 days after HTO treatment; wherein, A: peripheral blood leukocyte counts changed from 0 to 14 days after HTO treatment for each group of mice, # p <0.05, # p <0.01, compared to normal groups; b: peripheral platelet count changes from 0-14 days after HTO treatment for each group of mice, # p <0.05, # p <0.01, compared to normal groups;

FIG. 5 is a graph of the effect of HRW on the proliferative activity of tritium water exposed cells of different radioactivity; wherein, A: effect of HRW on cell proliferation activity of different concentrations of HTO exposure, p <0.05, p <0.01, compared to normal group; b: HRW detects the apoptosis of the cells exposed by different HTO concentrations, the early apoptotic cells are positioned in the lower right quadrant of the graph, and the late apoptotic or necrotic cells are positioned in the upper right quadrant of the graph; c: statistics of apoptosis of HRW on different concentrations of HTO exposure, including early apoptosis and late apoptosis, { fraction (p) < 0.05;

FIG. 6 is a graph of the effect of different HTO exposure doses on the micronucleus rate and DNA damage of AHH-1 cells in binuclear cells. Wherein, A: treating the micronucleus Gimsa staining pattern of AHH-1 cells with HRW for different HTO concentrations; b: HRW processes the micronucleus rate of AHH-1 cells for different HTO concentrations, each group counts 1000 binuclear cells, and calculates micronucleus rate, p is less than 0.01; c: comet electropherograms of AHH-1 cells treated with HRW for different HTO concentrations; d: statistics of tail DNA% in comet experiments with HRW treating AHH-1 cells for different HTO concentrations, # p < 0.01;

FIG. 7 is a graph of the effect of HRW on ROS in HTO-exposed cells. Wherein, A: total intracellular ROS levels of HRW 24h after exposure of different concentrations of HTO to AHH-1 cells; b: intracellular OH levels at different time points after HRW exposure to AHH-1 cells for different concentrations of HTO; c: intracellular O at different time points after exposure of HRW to AHH-1 cells for different concentrations of HTO₂ ^-Level,. about.p<0.05, compared to the same concentration HTO group at the same time point;

FIG. 8 is the effect of hydrogen rich water on the GSH content of tritium exposed cells, wherein<0.01，*p<0.05, compared to the normal group (normal group);^##p<0.01，^#p<0.05, compared to group 0;

FIG. 9 is the effect of hydrogen rich water on the SOD activity of tritium-exposed cells, wherein<0.01，*p<0.05, compared to the normal group;^##p<0.01，^#p<0.05, compared to group 0;

figure 10 is the effect of hydrogen rich water on the MDA content of tritium exposed cells, wherein p <0.01, p <0.05, compared to normal group; # p <0.01, # p <0.05, compared to group 0;

FIG. 11 is a graph showing the effect of hydrogen-rich water on the 8-OHDG content of tritium-exposed cells, wherein<0.01，*p<0.05, compared to the normal group;^##p<0.01，^#p<0.05, compared to group 0;

FIG. 12 is the effect of hydrogen rich water on mitochondrial membrane potential of tritium exposed cells, wherein<0.01，*p<0.05, compared to the normal group;^##p<0.01，^#p<0.05, compared to group 0;

FIG. 13 is a graph of the effect of hydrogen rich water on tritium exposure intracellular and medium tritium concentration, where (A) is intracellular tritium concentration; (B) is the medium tritium concentration. P<0.01，*p<0.05, compared to the normal group;^##p<0.01，^#p<0.05, compared to group 0;

FIG. 14 is a schematic of a three-compartment model of tritium metabolism in vivo.

Detailed Description

Reagent and instrument

Hydrogen-rich water (1mM) was purchased from Beijing active hydrogen source Co., Ltd and stored in an aluminum bag at 4 ℃;

HTO original concentration is 1mCi/mL (37MBq/mL), liquid scintillator is purchased from PE company in America, and the liquid scintillator is stored in a sealing way at room temperature;

AHH-1 cell lines were purchased from Milebo Biotech, Inc., Beijing.

Cell proliferation activity detection Kit (Cell Counting Kit-8, CCK-8) and apoptosis detection Kit (FITC-Annexin V/propidium iodide, PI) purchased from Dojindo, Japan;

fetal Bovine Serum (FBS), RPMI-1640 dry powder culture medium, superoxide anion (O)₂ ^.-) Fluorescent probe Dihydroethidium (DHE), low and normal melting point agarose, available from Invitrogen, USA;

Penicillin-Streptomycin solution (Penicillin-Streptomycin solution) was purchased from Hyclone in usa;

giemsa stain, Ethidium Bromide (EB), OH detection kit (2- [6- (4' -hydroxy) phenoxy-3H-xanthen-3-on-9-yl ] benzoic acid, HPF) and cytochalasin B were purchased from Sigma, USA;

reactive Oxygen Species (ROS) detection kit (2',7' -dichlorodihydrofluorescein diacetate, DCFH-DA), total SOD activity detection kit (WST-8 method), BCA protein concentration determination kit (enhanced type), lipid oxidation (MDA) detection kit and mitochondrial membrane potential detection kit (JC-1) are purchased from Shanghai Biyunnan biotechnology limited company.

A reduced Glutathione (GSH) assay kit (microplate method) and a human 8-hydroxydeoxyguanosine (8-OHDG) enzyme-linked immunoassay kit are purchased from Nanjing institute of bioengineering.

Second, the detailed description

Example 1 enhancement of urine tritium removal by Hydrogen-enriched Water (HRW)

HTO treatment of animals

Male BALB/c mice (4 weeks old, 14-16g) were purchased from the laboratory animal center of the Chinese people's liberation army military medical university and raised in the clean-grade animal laboratory. Mice were randomly divided into 4 groups: (1) normal control group: mice were fed with pure water without any treatment (n-10); (2) HTO group: mice were given a single i.p. injection of HTO (about 1mL) at 1.85MBq/kg and fed with purified water for 14 days (n-20); (3) h₂O control group: after the mice are infected with the virus, the mice are drenched with pure water according to the proportion of 1 mL/day/mouse, and the mice are fed with the pure water for 14 days (n is 20); (4) HRW group: after the mice were infected, they were gavaged with HRW at 1 mL/day/mouse and fed HRW for 14 days (n ═ 20). Mice were transferred to mouse metabolism cages (tai ni bais, italy) on the day after challenge and daily water intake and urine were recorded and collected for each group of mice. The cervical dislocation method is adopted to kill the mice (5 mice/group/time) at the

days

2, 4, 7 and 14 after HTO injection, and whole blood is taken from femoral artery for blood routine and comet trail experiments (alkaline single cell gel electrophoresis, the document: Hekinson, Jiyongxin, Sun Yulong, and the like, "the effect of active oxygen in the microwave radiation induction mouse bone marrow stromal cell adaptive reaction at 900 MHz" [ environmental and occupational medicine ], 2016, (2): 100-charge 104);taking femurs on two sides to perform a micronucleus experiment; liver, spleen, kidney, small intestine and testis were collected for OBT measurements.

The research and development and the preliminary application of a metabolism cage (experimental animals and comparative medicine, 2009, 29 (5): 319-324) of multifunctional large and small mouse physiological experiment such as Dingjiulong and Qianjin and the like) are adopted to record the daily water intake (or HRW drinking) and the urine discharge volume of the small mouse, the water intake of each group of small mice has no significant difference, about 2-2.5 mL/day, which indicates that the tritium water (HTO) is injected in the abdominal cavity or HRW (or H)₂O) gavage did not significantly affect the eating and drinking behavior of the mice.

As the additional drinking water is added in the stomach irrigation, H₂Mice in O and HRW groups (no significant difference between groups) had significantly higher urine output than Normal control (Normal group) and HTO groups (no significant difference between groups), averaging about 0.5-1mL higher.

The total beta activity of each group of mice excreted urine was further measured using a liquid scintillation counter, as shown in fig. 1A, 1B, with a background radioactivity of approximately 1.2 ± 0.3 Bq/d. The urine radioactivity increased significantly after HTO exposure, highest within 24h after HTO exposure and decreased rapidly with increasing time, until 4d, where it decreased substantially to normal. The gastric lavage increases the extra water intake of the mouse, thereby increasing the urine output, H₂The total beta radioactivity in urine voided in the first 4 days of mice in groups O and HRW was significantly higher than in the HTO group (total voided urine in the first 4 days was about 5X 10)⁴Bq). However, total activity of the front 4d discharge in HRW group mice (about 6.8X 10)⁴Bq) is significantly higher than H₂Group O (about 5.9X 10)⁴Bq)。

As a result: after the mice are exposed to HTO, the urine excretion is increased through multiple drinking water, and the excretion of urine tritium can be promoted; the HRW discharge promoting effect is obviously better than that of H₂O, the reason for which is in high concentration¹H competition³The binding site of H promotes tritium excretion by isotope exchange.

Example 2HRW promotes a decrease in levels of retained blood tritium.

After entering the body, HTO participates in water metabolism and is distributed to various tissues and organs mainly through blood circulation. By liquid scintillation countingThe beta-ion radioactivity in

serum

2, 4, 7 and 14d after the HTO exposure of mice was determined (comparison of the liquid scintillation counting tritium determination methods in Lixiaofeng, Caoan, Wuxianhai: "nuclear electronics and detection technology", 2015, 35 (7): 707 and 711). As shown in FIG. 1C, the background radioactivity concentration in the serum of normal mice was about 2.3. + -. 0.4 Bq/mL. Following HTO exposure, the mouse serum radioactivity increased significantly and decreased gradually over time. Consistent with the urine tritium assay, H₂The radioactive concentration in the serum of 7d in both O group and HW group is lower than that in HTO group, and HRW group and H group₂Compared with the group O, the tritium concentration in serum can be reduced.

As a result: multiple drinking water or HRW can reduce the retention of tritium in blood circulation and reduce the radioactive content in blood, and the HRW effect is superior to that of H₂O。

Example 3HRW promotes reduction of tissue-bound tritium (OBT) tritium content in tissue

Tissue bound tritium (OBT) of each tissue organ was determined by liquid scintillation counting after HTO exposure to mice for 7 d. Ashing mouse tissues by a muffle furnace, weighing the wet weight of the tissues before ashing and the ratio of the dry weight to the wet weight of the ashed tissues, and determining the water content of each organ of the mouse. Wherein the water content of testis, small intestine and kidney tissue is 80%, 78% and 75%, respectively, and the water content of liver and spleen is 70% and 65%, respectively. The measurement of the tissue homogenate radioactive concentration showed that the background radioactive levels of mouse testis, small intestine, spleen, kidney, and liver were about 10.5. + -. 0.8, 12.3. + -. 0.7, 13.3. + -. 0.6, 9.8. + -. 0.5, and 11.2. + -. 0.3Bq/g, respectively, as shown in FIG. 1D. After 7 days of HTO exposure, the radioactivity of the organs was significantly increased, with testis>Kidney (A)>Small intestine>Liver disease>Spleen, indicating that HTO is involved in water metabolism, primarily incorporated into tissues with higher water content. H₂After the mice are subjected to O or HRW intragastric administration, the OBT content of other organs of the mice except the spleen is obviously lower than that of the HTO group; and HRW group and H₂Compared with the O group, the OBT content of the tissue is further reduced remarkably.

As a result: multiple drinking water or hydrogen-rich water can reduce the tissue OBT content after HTO exposes mice, thereby reducing the radioactive damage of the OBT, and the HRW effect is better than that of H₂O。

Example 4HRW reduction of mouse genetic Material Damage following HTO Exposure

HRW significantly reduces mouse bone marrow polystaining erythrocyte micronucleus rate

To investigate the effect of HRW on HTO-induced chromosomal damage in mice, various groups of mice bone marrow MnPCEs were studied, as shown by MnPCE in fig. 2A arrows. After the mice were injected with HTO, according to micronucleus analysis (Amur, Wemeyan, perillium, etc. 'influence of 6 gelatin capsule shells on micronucleus rate of mouse bone marrow cells'; Guangdong medicine, 2015, 36(13):1982-1986), MnPCEs ‰ shows a tendency of rising first and then falling with time to the 4 th day, the highest. H₂The MnPCEs per mill of the bone marrow of the HTO infected mouse can be obviously reduced by the intragastric administration of O or HRW. And H₂In contrast to O, HRW had a stronger protective effect on HTO-induced chromosomal damage, as shown in fig. 2B.

HRW significantly reduces damage to mouse cell chromosomes and DNA

The damage condition of HTO treatment on the DNA of blood cells of each group of mice at different time points is researched by adopting a trace whole blood comet assay (alkaline single cell gel electrophoresis, the literature: Hekinina, Jiyongxing, Sun Yulong and the like, namely the effect of active oxygen in 900MHz microwave radiation induced bone marrow stromal cell adaptive reaction of mice, 2016 (2): 100-. As shown in fig. 12, DNA damage followed by HTO injection showed a first-to-last-drop trend, with damage at 4d being the most severe and with 14d having substantially recovered. 4d, H₂Comet tail fluorescence was significantly lower in cells of group O and HRW than in HTO, see fig. 3A; analysis using CASP software shows 4d, H after HTO injection₂The percentage content of DNA (Tail DNA%) in cells of the O group and the HRW group is obviously lower than that of the HTO group, and the damage of the DNA of the cells of the HRW group is obviously lower than that of the cells of the H group₂Group O is light; the number of comet cells of the HRW group is also significantly lower than that of the HTO group and H₂And group O, see FIGS. 3B and 3C.

Experiments prove that multiple drinking water or HRW can obviously reduce the damage of mouse cell chromosome and DNA caused by HTO exposure, and the HRW protective effect is stronger than that of H₂And O. This is probably due to the interaction with H₂Compared with the prior art, the HRW has the functions of selective oxidation resistance and apoptosis resistance, and can effectively protect cells from radioactive oxidative damage。

HRW effectively ameliorates routine changes in blood in mice due to HTO

After the mice were injected with HTO (as in example 1), their peripheral blood White Blood Cell (WBC) counts decreased and then increased, see fig. 4A; the total number of Platelets (PLTs) also showed a decrease followed by an increase, see fig. 4B. H₂The gastric lavage intervention of O and HRW can obviously improve the reduction of the total WBC and PLT in peripheral blood of mice caused by HTO, and the improvement of HRW on the conventional change of the blood of mice exposed to HTO is stronger than that of H₂Blood from O, HRW-treated mice was routinely substantially normal over the observation time.

Example 4 reduction of HTO-induced apoptosis of AHH-1 cells by HRW

Cells and treatments

Effect of HRW on cellular activity after HTO exposure of cells:

AHH-1 cells in RPMI-1640 medium (90% RPMI-1640 medium, 10% fetal bovine serum FBS, 1% penicillin-streptomycin solution), and 3000/hole in 96 hole cell culture plate. Normal group (Normal): without any treatment, 3 wells per group. Tritiated water group (HTO): each group was treated with 0.5, 2.5, 5, 25, 50, 100. mu. Ci/mL HTO, 3 wells. Hydrogen rich water group (HRW): treatment with HRW (1mM) for 5 minutes followed by the addition of 0.5, 2.5, 5, 25, 50, 100. mu. Ci/mL HTO, 3 wells per group. All cells were incubated for 48 hours. To each well, 10. mu.L of a CCK-8 solution (Cell Counting Kit-8, CCK-8)) was added, and the cells were cultured in the dark at 37 ℃ for 2 hours. The absorbance at 450nm of each well was measured by a multifunctional microplate reader (Spectra Max M4, Molecular Devices, USA) to calculate the cell proliferation activity.

Effect of HRW on apoptosis following HTO-exposed cells:

AHH-1 cells were cultured at 3X 10⁵Perwell in 6-well plates. Normal group (Normal): without any treatment, 3 wells per group. Tritiated water group (HTO): each group was treated with 10, 100. mu. Ci/mL HTO, 3 wells. Hydrogen rich water group (HRW): treatment with HRW (1mM) for 5 minutes followed by the addition of 10, 100. mu. Ci/mL HTO, 3 wells per group. All cells were cultured for 48 hours. Is carried out by an apoptosis detection kit (FITC-Annexin V/propidium iodide, PI)Line detection and final analysis by flow cytometry (C6, BD, USA). The mechanism of HRW for protecting radioactive damage caused by HTO exposure is discussed through detecting apoptosis by flow cytometry, and the influence of HRW on the proliferation activity of AHH-1 cells under HTO exposure is researched. As shown in fig. 5A, AHH-1 cells were exposed to HTO, and low concentrations of HTO exposure had little effect on cell proliferative activity; increasing HTO concentration, cell proliferation activity showed a concentration-dependent decrease of 100. mu. Ci/mL (3.7X 10)⁶Bq/mL) HTO treated cells, the proliferative activity was reduced to around 80%. Cells of hydrogen-rich water group (HRW) showed no significant change in proliferative activity. As shown in fig. 5B, flow cytometry detection of apoptosis revealed that the total apoptosis rate of AHH-1 cells increased with increasing HTO exposure dose to 100 μ Ci/mL, with a total apoptosis rate of about 20%; as shown in fig. 5C, the apoptosis rate of the hydrogen-rich water group (HRW) was significantly reduced. The experiment shows that HRW can effectively reduce AHH-1 cell apoptosis caused by HTO.

Example 5HRW reduction of HTO genotoxicity to AHH-1 cells

Cells and treatments

AHH-1 cells were cultured at 3X 10⁵Perwell in 6-well plates in RPMI-1640 medium (90% RPMI-1640 medium, 10% fetal bovine serum FBS, 1% penicillin-streptomycin solution). Normal group (Normal): without any treatment, 3 wells per group. Tritiated water group (HTO): each group was treated with 10, 100. mu. Ci/mL HTO, 3 wells. Hydrogen rich water group (HRW): treatment with HRW (1mM) for 5 minutes followed by the addition of 10, 100. mu. Ci/mL HTO, 3 wells per group.

Measurement of AHH-1 micronucleus rate (hummer, Wemeyan, perillium et al, "Effect of 6 gelatin capsule shells on micronucleus rate of mouse bone marrow cells"; Guangdong medicine, 2015, 36(13): 1982-.

The comet assay of AHH-1 cells adopts an alkaline single cell gel electrophoresis method (the document: Hekinina, Ji Neng, Sun Yulong, etc. 'the effect of active oxygen in the adaptive reaction of inducing the mouse bone marrow stromal cells by 900MHz microwave radiation'. 2016 (2): 100-104). The results show that as shown in fig. 6A, as HTO exposure dose increased, binuclear lymphocytes MN increased accordingly; as shown in fig. 6B, HRW significantly reduced the increase in MN due to HTO exposure. As shown in fig. 6C, comet experiments showed that HTO exposure for 48h, the fluorescence intensity of the cell tail increased dose-dependently, suggesting that the cell tail dna% gradually increased; as shown in fig. 6D, HRW significantly reduced the percentage of tail DNA exposed to AHH-1 cells at higher doses of HTO (100 μ Ci/mL), suggesting that HRW reduces radiation damage to cellular DNA by HTO.

Example six HRW reduction of HTO Exposure to AHH-1 induced increases in ROS production

Cells and treatments

AHH-1 cells in RPMI-1640 medium (90% RPMI-1640 medium, 10% fetal bovine serum FBS, 1% penicillin-streptomycin solution), and 3000/hole in 96 hole cell culture plate. Normal group (Normal): without any treatment, 3 wells per group. Tritiated water group (HTO): each group was treated with 0, 10, 100. mu. Ci/mL HTO, 3 wells. Hydrogen rich water group (HRW): treatment with HRW (1mM) for 5 minutes followed by addition of 0, 10, 100. mu. Ci/mL HTO, 3 wells per group of 3 wells.

HRW significantly reduced intracellular ROS production

The DCFH-DA method (literature: Tan Ling Li, Song Xue, Zhang Shirong, etc. 'influence of polystyrene nanoparticles on active oxygen of cells' Waxi pharmaceutical journal, 2017, 5: 493-. The total intracellular ROS production was determined 24h after exposure to different concentrations of HTO. Results as shown in fig. 7A, AHH-1 cells showed insignificant increase in total ROS production with increasing HTO exposure dose; but cells of HRW group were able to significantly reduce intracellular ROS production.

HRW significantly reduced intracellular OH production

Measuring the yield of intracellular OH (according to the method of the kit instruction) at different time points after different HTO exposure concentrations are applied to AHH-1 cells by using an OH detection kit (2- [6- (4' -hydroxy) phenoxy-3H-xanthen-3-on-9-yl ] benzoic acid, HPF), wherein the intracellular OH increases along with the increase of the HTO exposure dose and the exposure time as shown in FIG. 7B; the cells of the HRW group had a significant decrease in intracellular OH at different time points after HTO exposure at different doses.

HRW treatment on intracellular O2^-

Using superoxide anion (O)₂ ^.-) Fluorescent probe Dihydroethidium (DHE) for determination of intracellular O at various time points₂ ^.-Content of (a), intracellular O2-study, as shown in FIG. 7C, intracellular O2^-Dose and time of exposure did not increase significantly with HTO exposure, and HRW treatment was on intracellular O2^-The results show that the content influence is not great.

The results demonstrate that HRW reduces AHH-1 cell damage due to HTO exposure, primarily due to its selective antioxidant function, selectively eliminates the intracellular OH increase due to HTO exposure, and has no significant effect on other free radicals such as O2-.

And (4) conclusion: HRW intragastric administration can obviously increase the urine tritium content of the mice exposed by HTO, reduce the serum tritium content and effectively reduce the tissue OBT; increasing white blood cell and platelet depletion from HTO exposure, decreasing the incidence of MnPCE in mouse bone marrow and alleviating damage to blood cell DNA. In vitro experiments prove that compared with a pure HTO group, the AHH-1 cells cultured under the hydrogen-rich culture condition have the advantages that the apoptosis rate is obviously reduced, the micronucleus rate of binuclear lymphocytes is obviously reduced, comet experiments show that the DNA damage degree is obviously reduced, the total ROS and hydroxyl free radicals (. OH) in the cells are obviously reduced, and the superoxide anions (O2.-) in the cells are not obviously changed.

Example mechanism of Hydrogen-Rich Water vs. tritium Water stimulated emission

Cells and treatments

The influence of hydrogen-rich water on the content of GSH, MDA and 8-OHDG in AHH-1 cells, SOD activity and mitochondrial membrane potential after tritium excretion promotion: different substances of AHH-1 are given, and the substances are divided into a normal control group, a tritium water group and a hydrogen-rich water group. . Normal control group: normal control group (Normal) without addition of hydrogen rich water and tritiated water, 3 wells per group. Tritiated water group: tritiated water was added to give final concentrations of 1. mu. Ci/mL, 10. mu. Ci/mL, and 100. mu. Ci/mL, 3 wells per group, respectively. Hydrogen-rich water group: hydrogen-rich water with hydrogen concentration of 0, 0.2mmol/L and 0.6mmol/L is added into the cells respectively, and after 5min, tritium water is added to make the final concentration of the tritium water be 1 mu Ci/mL, 10 mu Ci/mL and 100 mu Ci/mL, and each group has 3 holes. After 24h of culture, cells are collected by 3500rpm/min centrifugation for 10min, 100 mu L PBS is added into the cells to crack the cells by a repeated freeze thawing method, and supernatant is taken by 3500rpm/min centrifugation for 10min and is respectively detected by GSH, MDA, 8-OHDG, a total SOD activity detection kit and a mitochondrial membrane potential detection kit (operation according to kit instructions).

1. Changes in intracellular GSH

The content of reduced Glutathione (GSH) in the cell homogenate was measured using a microplate method and a BCA protein concentration measurement kit (enhanced type) (the operation was performed according to the kit description), and the results are shown in fig. 8, in comparison with the normal control group, after tritiated water of various concentrations was administered, the cells were stimulated to generate various radicals, and in order to scavenge these radicals, the cells produced a large amount of GSH, so that the GSH content in the cells was significantly increased, and the GSH content of each group was increased with the increase in the concentration of tritiated water. The GSH content of each group, to which 0.6mmol/L hydrogen-rich water was previously administered, decreased very significantly, while the GSH level of each group, to which 0.2mmol/L hydrogen-rich water was previously administered, did not decrease. When tritium water is added, cells are stimulated to generate a large amount of free radicals, and cells without hydrogen-rich water can generate a large amount of GSH to eliminate the free radicals; after the hydrogen-rich water is given to the cells in advance, the cells can absorb a large amount of molecular hydrogen, and the molecular hydrogen has a scavenging effect on free radicals, so that the concentration of the generated GSH is not higher than that of the tritium water group.

2. Changes in intracellular SOD enzyme Activity

SOD enzyme activity in the cell homogenate was measured (by the kit instructions) using a total SOD activity detection kit (WST-8 method) and a BCA protein concentration measurement kit (enhanced type), and the results are shown in FIG. 9. Compared with a normal control group (normal), after tritium water with various concentrations is given, cells are stimulated to generate superoxide anion free radicals, and the cells combine into a large amount of SOD enzyme for scavenging the superoxide anion free radicals, so that the SOD activity is increased, and the increase is more obvious in two concentration groups of 1 mu Ci/mL and 10 mu Ci/mL. When hydrogen-rich water is given in advance, the activity of each group of SOD is reduced to a certain degree, but still is obviously higher than that of a normal control group, wherein the higher the concentration of molecular hydrogen is, the stronger the capability of eliminating superoxide anions is, and the activity of the generated SOD is not obviously improved as compared with that of a tritium water group.

3. Changes in intracellular MDA levels

The lipid oxidation (MDA) detection kit and the BCA protein concentration determination kit (enhanced type) are adopted to determine (operate according to the kit description) the MDA content in the cell homogenate, and the results are shown in figure 10, compared with a normal control group, after tritium water with various concentrations is given, the MDA content of two tritium water groups of 10 mu Ci/mL and 100 mu Ci/mL slightly rises, and the MDA content of a hydrogen-rich water group slightly falls, but no statistical difference exists, and the figure 10 shows that. The reason is as follows: possibly at a lower concentration than the tritium water set, not to such an extent as to cause cell damage to a significant rise in MDA.

4. Changes in intracellular 8-OHDG levels

8-hydroxydeoxyguanosine (8-hydroxy-2 deoxyguaosine, 8-OHDG) is the most common biomarker for DNA oxidative damage at present and can reflect the degree of DNA damage after tritiated water irradiates cells. The result shows that compared with a normal control group, the tritium water group causes the content of the intracellular 8-OHDG to be remarkably increased due to the radiation damage of cells; and the content of the intracellular 8-OHDG is obviously reduced compared with that of the tritium water group along with the increase of the concentration of molecular hydrogen in the hydrogen-rich water group, and the figure 11 shows. Indicating that hydrogen molecules in the hydrogen-rich water can protect tritium water from oxidative damage to cell DNA.

5. Changes in the level of mitochondrial membrane potential in cells

The decrease in the transmembrane potential within the mitochondria, the earliest event in the apoptotic cascade, causes a cascade of biochemical changes in the mitochondrial membrane leading to apoptosis.

Compared with a normal control group, the mitochondrial membrane potential of the tritium water group and the 0.2mmol/L hydrogen-rich water group is obviously increased; while the mitochondrial membrane potential of the 0.6mmol/L hydrogen-rich water group cells dropped significantly back to normal levels, see fig. 12.

6. Intracellular tritium concentration and Medium tritium concentration

The concentration of tritium in the cells and the concentration of tritium in a culture medium are measured by adopting a liquid scintillation counting method, and the result shows that the concentration of the tritium in the cells is obviously increased after tritium water with various concentrations is given compared with a normal control group; the cell tritium concentration of each concentration group of hydrogen-rich water was reduced compared to the tritium water group, with each group given 0.6mmol/L hydrogen-rich water showing a more significant reduction trend, see fig. 13A.

The concentration of tritium in the medium was significantly increased after administration of tritium water of various concentrations, compared with the normal group. The concentration of medium tritium was significantly higher for each concentration group than for the tritium water group after administration of hydrogen-rich water, which is consistent with the trend of decreasing cell tritium concentration, see fig. 13B.

And (4) conclusion: after human lymphocytes and experimental animals are given hydrogen-rich water in advance, molecular hydrogen can be competitively combined with organic matters in the cells with tritium, so that tritium is not easy to form organic combined tritium, and the discharge of the tritium is promoted.

Claims

1. Application of hydrogen-rich water in preparing drinking water or medical liquid for promoting discharge of tritium poisoning in vivo.

2. Use according to claim 1, characterized in that: the hydrogen concentration of the hydrogen-rich water is 1.2-3.0ppM, and the hydrogen-rich water is dissolved in purified water or distilled water.

3. Use according to claim 1, wherein the tritium intoxication is damage to human lymphocytes caused by tritium water exposure.

4. The use of claim 3, wherein said human lymphocytes are AHH-1 cells.

5. Use according to claim 1, characterized in that the tritium intoxication is a damage of genetic material alteration.

6. Use according to claim 5, wherein the altered damage of genetic material is an increase in the micronucleus rate of myelodophil red blood cells, damage to cell chromosomes and/or DNA caused by exposure to tritium water.

7. Use according to claim 1, characterized in that the tritium intoxication is a hematological change.

8. Use according to claim 7, wherein the haematological change is a peripheral haematological change.

9. Use according to claim 8, wherein the peripheral hemogram change is a decrease in leukocytes and/or platelets.

10. Use according to claim 1, wherein the medical liquid is prepared by adding a medically acceptable excipient to the hydrogen-rich water; or mixing hydrogen-rich water with other medicines for preventing or treating tritium-expelling poisoning, and optionally adding pharmaceutically acceptable adjuvants.