An animal model of ionizing radiation-induced reproductive developmental harm and an ionizing radiation hazard evaluation system

Abstract

The present invention provides an animal model of ionizing radiation-induced reproductive developmental harm and an ionizing radiation hazard evaluation system. The present invention constructs a zebrafish model of ionizing radiation-induced reproductive developmental harm, comprehensively examines the risk of IR (ionizing radiation) harm to the reproductive health of zebrafish, screens the main biomarkers and molecular targets, and compares the sensitivity differences of IR to female and male reproductive health harm, which can be applied to the study of radiation-induced reproductive harm mechanism and the development of related protective drugs; at the same time, a complete radiation hazard evaluation system is constructed by comprehensively considering various radiation impact indicators and gender differences, and the radiation hazard evaluation system can be used for ecological monitoring or biological early warning to evaluate the radiation impact of the environment in which the sample is located.

Description

Technical Field

[0001] The invention relates to the field of animal model technology and radiation hazard assessment, in particular to an animal model of ionizing radiation-induced reproductive developmental harm and an ionizing radiation hazard assessment system. Background Art

[0002] In recent years, basic research related to radiation damage and protection has received increasing attention. Although the harmfulness and potential health risks of ionizing radiation in the marine environment to aquatic ecosystems have always been highly valued, the toxicological effects of ionizing radiation on aquatic organisms, such as developmental toxicity, reproductive toxicity, neurotoxicity, and carcinogenicity, still lack favorable research data support. At present, there is a lack of zebrafish animal models for evaluating IR (ionizing radiation) reproductive health hazards, which has brought difficulties to the research on the mechanism of radiation-induced reproductive hazards and the development of related protective drugs. Therefore, the inventors constructed a zebrafish animal model to provide model support for the study of IR reproductive development hazards. Summary of the invention

[0003] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and to provide an animal model of ionizing radiation-induced reproductive developmental harm and an ionizing radiation hazard evaluation system. The technical solutions adopted by the present invention are as follows:

[0004] In a first aspect, an animal model of ionizing radiation-induced reproductive developmental damage is provided, wherein the method for constructing the animal model comprises the following steps:

[0005] S1. Irradiate adult sexually mature female and male zebrafish using a biological irradiator, and set up paired groups of male and female zebrafish with different irradiation doses;

[0006] S2. Male and female zebrafish pairs were mated within 16-48 hours after irradiation, with one female and one male placed in each mating box;

[0007] S3. Collect embryos produced by mating of male and female zebrafish in each group and evaluate their egg production and fertilization rate;

[0008] S4. The embryos produced were observed 8 to 120 hours after fertilization, and the embryo hatching rate, heart rate, malformation rate and major malformation phenotypes were statistically analyzed.

[0009] Preferably, the major deformity phenotypes include abnormal development of the embryonic head, spinal curvature, abnormal yolk sac, unopened sac, and tail deformity.

[0010] Preferably, the abnormal phenotype also includes systemic apoptosis of the embryo, and the apoptosis phenomenon is determined by acridine orange staining analysis of embryonic tissue.

[0011] Preferably, the deformed phenotype further includes oxidative damage in the visceral region, and the oxidative damage in the visceral region is determined by reactive oxygen species (ROS) fluorescent dye staining analysis.

[0012] Preferably, the malformation phenotype also includes cardiovascular developmental malformation, embryonic inflammatory response and neurodevelopmental malformation.

[0013] Preferably, the ovarian immune stress genes myhz1, MHCII, mhc2d8, mhc2dab, b2m, and membrane and ovarian development regulatory genes vtg, apoa2b, and fabp of female zebrafish are used as ion radiation sensitive genes; the cell conduction genes p4ha3, ahcyl2a, nt5c2l1, metabolism-related genes cyp, ugt family genes, ferroptosis genes gpx4a, gsto2 and gsta2, and heme binding genes hbba2, hbaa2, and hbba1 of male zebrafish are used as ion radiation sensitive genes; and the degree to which the animal model is affected by ion radiation is evaluated based on the expression of the ion radiation sensitive genes.

[0014] Specifically, in the above step S1, a non-irradiated control group in which the irradiation dose of zebra and parents is zero, an IR group of parents in which the irradiation dose is not zero, a paternal IR group in which only male fish are irradiated, and a maternal IR group in which only female fish are irradiated are set.

[0015] In a second aspect, an ion radiation hazard assessment system is provided, which is constructed based on the above-mentioned zebrafish animal model, and the ion radiation hazard assessment system includes a female fish assessment system and a male fish assessment system;

[0016] According to the animal models of the paternal IR group and the maternal IR group, a comparison table of radiation impact indicators of female and male zebrafish under different irradiation doses is constructed respectively, wherein the radiation impact indicators include at least one of the following: egg laying amount, fertilization rate, embryo hatching rate, deformity rate, main deformity phenotype, and expression of ion radiation sensitivity gene; one or more statistical results are tested for significance, and whether the sample has been affected by radiation is judged according to whether they are significant;

[0017] The female fish evaluation system uses the zebrafish female sample to be evaluated to mate with uncontaminated male fish, and the male fish evaluation system uses the zebrafish male sample to be evaluated to mate with uncontaminated female fish. The above-mentioned radiation impact index evaluation table is used to evaluate whether the sample to be evaluated is affected by ion radiation and the degree of radiation impact on the sample to be evaluated and its offspring embryos.

[0018] The above ion radiation hazard assessment system can also be applied to biological early warning to construct a three-level assessment system to assess the ion radiation hazard level of the environment;

[0019] First-level evaluation: After the zebrafish sample to be evaluated mates with the uncontaminated zebrafish, one or more of the following items are counted: egg laying volume, fertilization rate, embryo hatching rate, external deformity rate, and juvenile mortality rate. Based on the statistical results, it is determined whether the results are significant compared with the control group whose parents are not contaminated. If multiple evaluations are conducted, if two of the multiple items are significant, then the overall results are determined to be significant.

[0020] Secondary evaluation: After the zebrafish samples to be evaluated mate with uncontaminated zebrafish, the main deformity phenotypes and / or oxidative damage in the visceral regions of the fry are counted. The main deformity phenotypes include abnormal embryonic head development, spinal curvature, abnormal yolk sac, unopened swim sac, tail deformity, cardiovascular developmental deformity, embryonic inflammatory response, and neurodevelopmental deformity. The incidence of the main deformity phenotypes and / or oxidative damage in the visceral regions of the fry is used to determine whether they are significant relative to the control group whose parents are not contaminated. If multiple evaluations are performed, if two of the multiple evaluations are significant, then the overall evaluation is considered significant.

[0021] Level 3 evaluation: The ovarian immune stress genes myhz1, MHCII, mhc2d8, mhc2dab, b2m, and membrane and ovarian development regulatory genes vtg, apoa2b, and fabp of female zebrafish were set as ion radiation sensitive genes; the cell conduction genes p4ha3, ahcyl2a, nt5c2l1, metabolism-related genes cyp, ugt family genes, ferroptosis genes gpx4a, gsto2, and gsta2, and heme binding genes hbba2, hbaa2, and hbba1 of male zebrafish were set as ion radiation sensitive genes; the differential expression rates of the above ion radiation sensitive genes were used to determine whether they were significant relative to the uncontaminated control group of the parents;

[0022] When the sample is a male fish, if both the primary and secondary evaluations are not significant, it is judged that the environment where the sample is located has no radiation hazard or no biological hazard;

[0023] When the sample is a male or female fish, if either the primary evaluation or the secondary evaluation is significant, the environment where the sample is located is judged to be biologically hazardous and may be exposed to radiation hazards;

[0024] When the sample is a male or female fish, both the first-level and second-level evaluations are significant, and the third-level evaluation can be selected to determine whether the sample is indeed a radiation hazard;

[0025] When the sample is a female fish, if both the first-level evaluation and the second-level evaluation are not significant, the third-level evaluation is performed. If the third-level evaluation is significant, it is judged that the environment where the sample is located may be subject to radiation hazard. If the third-level evaluation is not significant, it is judged that the environment where the sample is located has no radiation hazard or no biological hazard.

[0026] Under the same conditions, male zebrafish samples were preferentially selected to mate with uncontaminated females for evaluation.

[0027] The beneficial effects of the present invention are as follows: the present invention constructs a zebrafish model of ionizing radiation-induced reproductive developmental harm, comprehensively examines the risk of IR (ionizing radiation) harm to the reproductive health of zebrafish, screens the main biomarkers and molecular targets, and compares the sensitivity differences of IR to female and male reproductive health hazards, which can be applied to the study of radiation-induced reproductive harm mechanisms and the development of related protective drugs; at the same time, a complete radiation hazard evaluation system is constructed by comprehensively considering various radiation impact indicators and gender differences, and the radiation hazard evaluation system can be used for ecological monitoring or biological early warning to evaluate the radiation impact of the environment in which the sample is located. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, without paying creative labor, other drawings obtained based on these drawings still belong to the scope of the present invention.

[0029] Figure 1 Identification of the dose effect of IR reproductive damage; A is the egg-laying index; B is the fertilization rate index; N = 3-5, *P<0.05, **P<0.001 compared with the control group;

[0030] Figure 2 Among them, A is the egg-laying index; B is the fertilization rate index; C is the hatching rate of embryos at 48 hpf; D is the hatching rate of embryos at 72 hpf; E is the heart rate of embryos at 48 hpf; F is the cumulative deformity rate of embryos at 96 hpf, N=3-5 for A and B; N=6*20 for C, D, and F; N=20 for E. *P<0.05, **P<0.001 compared with the control group;

[0031] Figure 3 AC are representative images of embryonic development at 24 hpf, 72 hpf and 120 hpf; D is a representative image of embryos stained with AO apoptosis; E is a representative image of juvenile fish stained with ROS; F is a quantitative analysis of apoptotic cells stained with AO, N=3~5; G is a quantitative analysis of ROS staining intensity, N=3~5. Asterisks point to the location of head deformities, s: spine; ye: yolk cyst; mt: tail deformity; usb: swim sac not opened; b: brain; e: eye; t: body; l: liver; g: intestine; N=20. *P<0.05, **P<0.001 compared with the control group;

[0032] Figure 4 In the figure, A is a 48hpf vascular development diagram; B is a 72hpf immunoreaction diagram; C is a 120hpf neural development diagram. D is a quantitative analysis of the vascular development malformation rate, N=3 replicates (each replicate contains 10 zebrafish samples); E is a quantitative analysis of the number of cells with MPO immunoreaction, N=5~8 replicates; F is a ratio analysis of neural development malformations, N=3 replicates (each replicate contains 10 samples). Asterisks point to the location of vascular development inhibition; y: yolk sac; tr: body; t: tail; g: intestine; sm: spinal nerve malformations. N=20.*P<0.05, **P<0.001 compared with the control group;

[0033] Figure 5 In the figure, A is the principal component analysis (PCA) of transcriptome sequencing, B is the heat map of differentially expressed genes in transcriptome sequencing, and C is the enrichment pathway analysis diagram of transcriptome sequencing;

[0034] Figure 6 Transcriptome sequencing enrichment pathway analysis; A is the GO analysis of female ovarian tissue; B is the GO analysis of male testicular tissue; C is the female TOP20-KEGG analysis; D is the male TOP20-KEGG analysis;

[0035] Figure 7 Signaling pathways affected by transcriptome sequencing; A. Female ovarian tissue mainly affects genes of membrane, MHC protein complex, and lipid transport activity; B. Male testicular tissue mainly affects genes of intracellular signal transduction, heme binding, oxygen transport, and oxygen binding;

[0036] Figure 8 A brief flowchart and embryonic phenotype for the construction of each group of animal models in the present invention. DETAILED DESCRIPTION

[0037] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below in conjunction with embodiments and drawings.

[0038] Example

[0039] 1.1 Experimental Materials and Instruments

[0040] Instruments: zebrafish incubator, incubator, RXZ intelligent artificial climate chamber (Ningbo Jiangnan Instrument Factory), stereo microscope (Nikon SMZ1500, Shanghai Qianxin Instrument Co., Ltd.), 12-well plate, culture dish, 6-well plate, X-RAD 320 biological irradiation instrument.

[0041] 1.2 Experimental animals

[0042] Wild-type AB strain and transgenic strains of fli:EGFP (marking blood vessels), MPO:EGFP (marking the immune system) and Oligo2:dsRED (marking the nervous system) (Delov, V., Muth-Köhne, E., Schäfers, C. andFenske, M. 2014. Transgenic fluorescent zebrafish Tg(fli1:EGFP)y¹ for theidentification of vasotoxicity within the zFET. Aquat Toxicol 150, 189-200.) zebrafish were used as experimental subjects. Zebrafish were cultured using the feeding method described in the Zebrafish Book (Westerfield. The zebrafish book : a guidefor the laboratory use of zebrafish (Danio rerio). 1995.). The water temperature was 26±1℃, the pH value was 6.5~7.5, sea salt was added to the water, the conductivity was kept in the range of 450-1000uS / cm, and the photoperiod was 14L: 10D. The night before the experiment, the normally developed female and male fish were paired in a 1:1 ratio and placed in the incubator before turning off the lights. After turning on the lights in the morning of the next day, the male and female fish spontaneously mated to obtain the embryos required for the experiment. The embryo development phenotype observation and analysis reference (Kimmel CB, Ballard WW, Kimmel SR, et al. Stages of Embryonic Development of the Zebrafish. Developmental dynamics 1995, 203:253-310.).

[0043] 1.3 Zebrafish IR irradiation and mating experiments

[0044] The present invention uses an X-RAD 320 biological irradiation instrument to perform zebrafish irradiation (IR) experiments. Adult zebrafish use a 2L fish tank, put in 400mL of newly configured rearing water, add 3 females and 3 males for irradiation, and set three replicates. The irradiation dose is set to four groups of 0, 1, 5, and 10 Gy, and a dose rate of 1Gy / min is used. It is carried out according to the experimental design and instrument operation rules. A mating experiment is carried out on the evening of the IR treatment, and each mating box is placed with 1 female and 1 male. A non-irradiated control group and three biological replicates are set. The embryos produced are collected on the morning of the next day and their egg production (total number of embryos) and fertilization rate (proportion of successfully fertilized embryos) are evaluated. On this basis, a suitable single IR dose is determined, and a subsequent sensitivity analysis is carried out, specifically including a non-irradiated control group, a parental IR group, a paternal IR group, and a maternal IR group. Three replicates are set, and the egg production and fertilization rate are also evaluated.

[0045] 1.4 Evaluation of IR toxicity on offspring embryonic development and neurobehavioral toxicity

[0046] The embryos produced were placed in six-well plates at 8 hours after fertilization (8 hpf) for observation experiments. During the embryonic development, the hatching rate, heart rate, malformation rate and the determination of the main malformation phenotypes were evaluated. The hatching rate mainly observed the difference in the hatching rate of embryos at 48 and 72 hpf, the heart rate was analyzed at 48hpf, and the malformation rate was analyzed at 120hpf. The different malformation phenotypes produced by embryos in different treatment groups were photographed and analyzed to determine the typical malformation phenotype. Acridine orange (AO) staining analysis was performed at 24hpf to determine the effect of cell apoptosis, and ROS fluorescent dye staining analysis was performed at 120hpf to determine oxidative damage. The transgenic lines were used to evaluate the effect of IR on the development of specific systems, mainly by evaluating the effect of fluorescence expression in offspring embryos at specific developmental periods.

[0047] 1.5 RNA-Seq transcriptome sequencing analysis of IR on gonadal differentially expressed genes and information pathways

[0048] Transcriptome sequencing analysis was performed using testes or female ovaries of zebrafish in the control and IR treatment groups, and samples were collected 0.5 h after IR treatment. Total RNA was extracted from 3 gonads using Trizol reagent (Thermofisher, catalog # 15596018) according to the manufacturer's procedures, and three biological replicates were set up for each group. Total RNA quantity and purity were analyzed using Bioanalyzer 2100 and RNA6000 Nano LabChip Kit (Agilent, CA, USA, catalog # 5067-1511). High-quality RNA samples with a RIN number > 7.0 were sequenced for mRNA expression using the Illumina Novaseq™ 6000 platform according to the manufacturer's instructions (LC-Bio Technology Co., Ltd., Hangzhou, China). The obtained mRNA-Seq was aligned to the zebrafish genome (DanRer10) using tophat2 after removing the sequence adapter sequencer. Zebrafish gene annotations were obtained from Ensembl. After counting the reads of each gene, principal component analysis (PCA) was used to analyze sample variation. DESeq2 with q < 0.05 and absolute fold change ≥ 2 was used as a significant cutoff and was called a differentially expressed gene. Gene ontology (GO) functional enrichment analysis and KEGG pathway enrichment analysis were performed on the differentially expressed genes.

[0049] 1.6 Data Analysis

[0050] Data analysis and graphics were performed using Prism 6.0. One-way analysis of variance (ANOVA) and Tukey's multiple comparison test were used to compare the differences between different treatment groups, and the data met the assumptions of parametric tests. In mRNA-Seq analysis and Atr treatment analysis, t-test was used to analyze differentially expressed genes. All data are reported as mean ± standard error (SEM).

[0051] 2.1 Identification of dose effects of IR reproductive damage

[0052] In the dose range of 1-10 Gy, IR significantly inhibited the egg production (1A) and fertilization rate (1B) with increasing doses. The lowest dose of 1 Gy was used to ensure that enough viable embryos were obtained for further evaluation and analysis.

[0053] 2.2 Gender sensitivity analysis of IR reproductive damage

[0054] Under different IR parental treatment conditions, we found that both parental IR and paternal IR groups significantly affected zebrafish reproduction (2A, 2B) and offspring development, such as increased deformity rates ( Figure 2 C,D) Heart rate increases ( Figure 2E) and increased deformity rate (2F). However, the effect of maternal IR group was not significant.

[0055] It was observed that IR irradiation mainly caused abnormal head development (3A), spinal curvature and yolk sac abnormalities (3B), unopened swim sac and tail deformity (3C) in offspring embryos. At the same time, IR treatment induced systemic apoptosis in embryos, which was concentrated in the tail, head eyes and brain areas (3C, 3F). IR treatment caused oxidative damage in the visceral areas of offspring larvae (3E, 3G).

[0056] Transgenic fluorescence observations revealed that IR irradiation inhibited cardiovascular development in offspring embryos (4A, 4D), induced embryonic inflammatory responses (4B, 4E) and neurodevelopmental malformations (4C, 4F). The effects were more significant in the parental IR and paternal IR groups.

[0057] 2.3 Molecular mechanisms of reproductive damage caused by IR

[0058] RNA-Seq analysis was performed on the gonads of adult fish treated with IR to identify the reproductive toxic genes that mainly respond to IR and their differences between sexes. mRNA-Seq sequencing analysis found that the PCA of the main components of the samples in the IR-treated group and the control group in the female ovarian tissue was not much different, while the difference between the two groups in the male testicular tissue was very obvious (5A). Correspondingly, the heat map analysis of differentially expressed genes also found that IR caused 52 differentially expressed genes (22 up-regulated and 30 down-regulated) in the ovarian tissue, while IR caused 898 differentially expressed genes (437 up-regulated and 461 down-regulated) in the testis (5B). GO analysis found that the ovary mainly affected the membrane sites of cell components, while the testis had a more extensive impact, including the membrane, nucleus and cytoplasm of cell components, as well as protein binding and iron ion binding of molecular functions (5C).

[0059] Specifically, the GO enrichment bubble map showed that the ovary mainly affected membranes, antigen processing and presentation, MHC class II protein complexes, and lipid transporter activity (6A), while the testis mainly affected intracellular signal transduction, heme binding, oxygen transport, oxygen binding, and hemoglobin complexes (6B). TOP 20 KEGG signaling pathway analysis found that the ovary mainly affected the intestinal immune network to produce IgA, progesterone-mediated oocyte maturation, gap junctions, cell adhesion molecules, oocyte meiosis, and vascular smooth muscle contraction (6C), while the testis mainly affected cytokine-cytokine receptor interactions, folate biosynthesis, metabolic pathways, ECM-receptor interactions, amino sugar and nucleotide sugar metabolism, and ferroptosis (6D).

[0060] The differentially expressed genes were extracted to construct a PPI relationship network for the main affected signal pathways and biological processes. It was found that the main affected genes in the ovary were immune stress genes such as myhz1, MHCII, mhc2d8, mhc2dab and b2m, membrane and ovarian development regulatory genes vtg, apoa2b, fabp, etc. (7A), while the main affected genes in the testis were cell conduction genes such as p4ha3, ahcyl2a, nt5c2l1, metabolism-related genes cyp, ugt family genes, ferroptosis genes gpx4a, gsto2 and gsta2, and blood red binding genes hbba2, hbaa2, hbba1, etc. (7B).

[0061] In summary, the present invention has discovered that cell apoptosis can be used as a specific biological sensitive marker for evaluation of IR, and indicators for the morphological development of offspring embryos can also illustrate the effects of IR on different systems and their extent. For example, the effect on heart rate can reflect the impact on heart development, the effect on the spine can reflect the impact on the nervous system, and the visualization of oxidative stress ROS indicators also shows that IR has obvious developmental damaging effects on the liver and intestinal system.

[0062] The present invention also studies the intrinsic reasons for the differences in sensitivity of IR to reproductive harm of different sexes through transcriptome sequencing, and finds that differential expression of genes in gonadal tissue can be caused 0.5 hours after IR, among which the sensitivity to the testis is much greater than that to the ovary, because the differentially expressed genes caused are 17 times as many as those in the ovary. From the changed genes, it can be seen that the regulatory genes that mainly affect the development of the membrane and yolk protein in the ovary may also be the protective effect of the ovarian outer membrane that slows down its radiation damage, while many biological signals in the testis are affected, which also indirectly shows that the outer mold protection of the testicular tissue is relatively weak, and the testicular genome is more susceptible to damage.

[0063] The present invention determined the effects of IR irradiation during zebrafish pregnancy on the reproductive system and offspring development and the differences in radiation sensitivity of different gamete irradiations. At the same time, the dose effect and different response analyses at the biological and molecular levels can provide the most favorable reference value for the evaluation of the reproductive health effects of IR.

[0064] In summary, the present invention comprehensively examined the risk of IR (ionizing radiation) to the reproductive health of zebrafish, constructed an animal model and screened the main biomarkers and molecular targets, and also compared the differences in sensitivity of IR to the reproductive health of females and males, which can be applied to the study of radiation-induced reproductive harm mechanisms and the development of related protective drugs; at the same time, a complete radiation hazard evaluation system was constructed by comprehensively considering various radiation impact indicators and gender differences. The radiation hazard evaluation system can be used for ecological monitoring or biological early warning to evaluate the radiation impact of the environment in which the sample is located.

[0065] The above disclosure is only the preferred embodiment of the present invention, which certainly cannot be used to limit the scope of the present invention. Therefore, equivalent changes made according to the claims of the present invention are still within the scope of the present invention.

Claims

1. An application method of an ion radiation hazard assessment system in biological early warning, characterized in that: First, we constructed a zebrafish animal model of ionizing radiation-induced reproductive developmental damage: S1. Irradiate adult sexually mature female and male zebrafish using a biological irradiator, and set up paired groups of male and female zebrafish with different irradiation doses; S2. Male and female zebrafish pairs were mated within 10 to 48 hours after irradiation, with one female and one male placed in each mating box; S3. Collect embryos produced by mating of male and female zebrafish in each group and evaluate their egg production and fertilization rate; S4. Observe the embryos produced 8 to 120 hours after fertilization and calculate the embryo hatching rate, heart rate, malformation rate and major malformation phenotypes; Furthermore, in step S1, a non-irradiated control group in which the irradiation dose of zebrafish and parents is zero, an IR group of parents in which the irradiation dose is not zero, a paternal IR group in which only male fish are irradiated, and a maternal IR group in which only female fish are irradiated are set; According to the animal models of the paternal IR group and the maternal IR group, an evaluation table of irradiation impact indexes of female and male zebrafish is constructed respectively, wherein the radiation impact indexes include one or more of the following: egg laying amount, fertilization rate, embryo hatching rate, deformity rate, major deformity phenotype, juvenile mortality rate, and expression of ion radiation sensitivity genes; The ion radiation hazard assessment system includes a female fish assessment system and a male fish assessment system; The female fish evaluation system uses the zebrafish female fish sample to be evaluated to mate with the uncontaminated male fish, and the male fish evaluation system uses the zebrafish male fish sample to be evaluated to mate with the uncontaminated female fish, and the above-mentioned radiation impact index evaluation table is used to evaluate whether the sample to be evaluated is affected by ion radiation and the degree of radiation impact of the sample to be evaluated and its offspring embryos; The application method of the ion radiation hazard assessment system in biological early warning is: Construct a three-level evaluation system to assess the ionizing radiation hazard level of the environment; Level 1 evaluation: After the zebrafish sample to be evaluated mates with uncontaminated zebrafish, one or more of the following items are counted: egg laying volume, fertilization rate, embryo hatching rate, external deformity rate, and juvenile mortality rate. Based on the statistical results, it is determined whether the results are significant compared with the control group whose parents are not contaminated. Based on whether the results are significant, it is determined whether the sample has been affected by radiation. If multiple evaluations are conducted, if two of the multiple evaluations are significant, then the overall evaluation is determined to be significant. Secondary evaluation: After the zebrafish samples to be evaluated mate with uncontaminated zebrafish, the main deformity phenotypes and / or oxidative damage in the visceral regions of the fry are counted. The main deformity phenotypes include abnormal embryonic head development, spinal curvature, abnormal yolk sac, unopened swim sac, tail deformity, cardiovascular developmental malformation, embryonic inflammatory response, and neurodevelopmental malformation. The incidence of the main deformity phenotypes and / or oxidative damage in the visceral regions of the fry is used to determine whether they are significant relative to the control group whose parents are not contaminated. Whether the sample has been affected by radiation is determined based on whether it is significant. If multiple evaluations are performed, if two of the multiple evaluations are significant, then the overall evaluation is determined to be significant. Level 3 evaluation: The ovarian immune stress genes myhz1, MHCII, mhc2d8, mhc2dab, b2m, and membrane and ovarian development regulatory genes vtg, apoa2b, and fabp of female zebrafish are set as ion radiation sensitive genes; the cell conduction genes p4ha3, ahcyl2a, nt5c2l1, metabolism-related genes cyp, ugt family genes, ferroptosis genes gpx4a, gsto2 and gsta2, and heme binding genes hbba2, hbaa2, and hbba1 of male zebrafish are set as ion radiation sensitive genes; the difference in expression rate of the above ion radiation sensitive genes is judged whether it is significant compared with the uncontaminated control group of the parents, and whether the sample has been affected by radiation is judged based on whether it is significant; When the sample is a male fish, if both the primary and secondary evaluations are not significant, it is judged that the environment where the sample is located has no radiation hazard or no biological hazard; When the sample is a male or female fish, if either the primary evaluation or the secondary evaluation is significant, the environment where the sample is located is judged to be biologically hazardous and may be exposed to radiation hazards; When the sample is a male or female fish, both the first-level and second-level evaluations are significant, and the third-level evaluation can be selected to determine whether the sample is indeed a radiation hazard; When the sample is female fish, if both the first-level evaluation and the second-level evaluation are not significant, a third-level evaluation will be performed. If the third-level evaluation is significant, it is judged that the environment in which the sample is located may be subject to radiation hazard. If the third-level evaluation is not significant, it is judged that the environment in which the sample is located has no radiation hazard or no biological hazard.

2. The method for applying the ionizing radiation hazard assessment system in biological early warning according to claim 1 is characterized in that: The abnormal phenotype also includes systemic apoptosis of embryos, and the apoptosis phenomenon is determined by acridine orange staining analysis of embryonic tissues.

3. The method for applying the ionizing radiation hazard assessment system in biological early warning according to claim 1 is characterized in that: The deformed phenotype also includes oxidative damage in the visceral region, which is determined by staining analysis using reactive oxygen species fluorescent dye.

4. The method for applying the ionizing radiation hazard assessment system in biological early warning according to claim 1 is characterized in that: Male zebrafish samples were selected and mated with uncontaminated females for evaluation.

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