Method for evaluating influence of OBS environment exposure on zebra fish fry

Abstract

The invention belongs to the technical field of aquatic organism and environment relevance research, and particularly relates to a method for evaluating the influence of OBS environment exposure on zebrafish fry. The method comprises the following steps: preparing zebra fish larvae subjected to an OBS exposure test; carrying out a high-concentration acute exposure test to obtain the safe concentration of OBS to the zebra fish larvae; setting a sub-lethal concentration based on the safe concentration; obtaining a zebra fish larvae detection sample under the sub-lethal concentration exposure test; the method comprises the following steps: determining oxidative stress and immune related parameters of zebra fish larvae and expression levels of an antioxidant system, an immune system, NF-kB and TLR signal channels and endocrine related genes in the zebra fish larvae, and sorting the determined parameters, so as to analyze and research a toxic effect and a molecular mechanism caused by OBS. The influence of the actual OBS exposure concentration in the environment on organisms and the water ecological environment is evaluated.

Description

Technical Field

[0001] The invention belongs to the technical field of aquatic organism and environment correlation research, and particularly relates to a method for evaluating the impact of OBS environmental exposure on zebrafish larvae. Background Art

[0002] Perfluorooctane sulfonic acid (PFOS) is one of the most common and widely used PFASs. It has good chemical stability, surface activity and thermal stability, and is therefore widely used in industrial production and human consumer products. However, large-scale production and use have led to the widespread detection of PFOS in various environmental media including air, water, sediments and soil, and even in wild animals and human samples in high concentrations, indicating that PFOS has environmental persistence and strong bioaccumulation, becoming a typical persistent organic pollutant (POPs). Sodium perfluorononenyloxybenzene sulfonate (OBS) is a PFOS substitute, mainly used in the production of fire extinguishing agents, metal etchants, detergents and nickel plating brighteners for use in fire protection, oil extraction and electroplating industries.

[0003] In recent years, a large number of studies have shown that OBS is continuously detected in different environmental media. It is difficult to completely remove OBS by conventional means, and advanced oxidation technology is required to partially convert it into other substances, which causes a large amount of OBS in the sewage system to enter various environmental media. OBS also shows a certain degree of bioaccumulation. Laboratory studies have found that short-term exposure of zebrafish embryos can significantly enrich OBS, and field surveys have also found that OBS can be present in high concentrations in the blood, liver, gonads and muscles of wild crucian carp. OBS enriched in the body may cause potential harm to organisms, and is potentially toxic to organisms under conditions of high-concentration OBS pollution. Therefore, it is necessary to conduct detailed research on the toxic effects, molecular mechanisms and characteristics of organisms exposed to OBS environmental pollution.

[0004] Currently, the OBS exposure concentrations in most toxicity studies are relatively high, which is inconsistent with the actual low concentrations of OBS in the environment. In addition, the model organisms selected are usually adult zebrafish and mice, which cannot more accurately evaluate the potential risks of OBS environmental exposure to sensitive stages of organisms (such as the initial hatching period). Summary of the invention

[0005] 1. Technical issues to be resolved

[0006] In view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides a method for evaluating the impact of OBS environmental exposure on zebrafish larvae, which uses hatched zebrafish larvae as test subjects to study the impact of short-term exposure to lower concentrations of OBS on important functional systems of zebrafish in the early stages, including the antioxidant, immune and endocrine systems, so as to better evaluate the potential risks of OBS to aquatic organisms and aquatic ecosystems.

[0007] (II) Technical solution

[0008] A method for evaluating the effects of OBS environmental exposure on zebrafish larvae, comprising:

[0009] (1) Place the normally developed cleavage-stage zebrafish embryos in a culture dish containing zebrafish embryo culture medium, and then culture them in a constant temperature and light incubator with a light-dark cycle of 14h:10h and a temperature of 27±1°C; replace the culture medium every day and remove the egg membrane after hatching. After 3 days of culture, select the newly hatched zebrafish larvae that have hatched and developed normally for exposure testing;

[0010] (2) The safe concentration of OBS for zebrafish larvae was calculated using a high-concentration acute exposure test. The safe concentration for zebrafish larvae is SC = 0.1 × 96h-LC 50 ;

[0011] (3) Based on the safe concentration, multiple sublethal concentrations were set as the OBS concentrations in the exposure experiment, and the sublethal concentration did not exceed the safe concentration; newly hatched zebrafish larvae were randomly grouped into multiple parallel culture dishes, with the group exposed to the sublethal concentration of OBS working solution as the treatment group, and the group exposed to 0.01wt% DMSO as the control group; during the exposure period, the exposure solution was changed every day, and the exposure time was 4 days; after the exposure, the zebrafish larvae of each group were washed with zebrafish embryo culture medium, and the zebrafish larvae were collected into centrifuge tubes, quickly frozen in liquid nitrogen, and stored at -80°C for later use;

[0012] (4) taking samples from one of the centrifuge tubes for oxidative stress and immune-related parameter detection: adding 8-12 times the volume of ice-cold physiological saline to homogenize, then centrifuging at 4°C, 10,000×g for 5-15 min, and aspirating the supernatant into a new centrifuge tube; measuring oxidative stress parameters and immune-related parameters in the supernatant sample; the oxidative stress parameters include ROS, T-AOC, CAT, T-SOD, GSH and MDA, and the immune-related parameters include TNOS, iNOS and NO, and the protein concentration in each sample is determined according to the BCA method; taking samples from another part of the centrifuge tubes for RNA extraction, and detecting the expression levels of antioxidant system, immune system, NF-κB and TLR signaling pathways and endocrine-related genes in zebrafish larvae;

[0013] (5) Based on the detection results of step (4), perform data statistical analysis to evaluate the effect of OBS exposure on zebrafish larvae.

[0014] According to a preferred embodiment of the present invention, in step (1), adult male and female zebrafish are cultured in a zebrafish circulation culture system with a light-dark cycle of 14h:10h, a water temperature of 27±1°C, a pH value of 7.5±0.5, and an electrical conductivity of 500±50μS, and are fed with Artemia twice a day; the male and female zebrafish are mated to obtain zebrafish embryos, and the normally developed cleavage-stage embryos are evenly distributed into a culture dish containing zebrafish embryo culture medium, and then placed in a constant temperature and light incubator for culture with a light-dark cycle of 14h:10h and a temperature of 27±1°C; the culture medium is replaced every day and the egg membranes after hatching are picked out, and after culturing for 3 days, the hatched and normally developed zebrafish newly hatched larvae are selected for subsequent exposure tests.

[0015] According to a preferred embodiment of the present invention, in step (2),

[0016] In the high-concentration acute exposure test, 80 mg / L, 60 mg / L, 40 mg / L, 20 mg / L, 10 mg / L, 8 mg / L, 6 mg / L, 4 mg / L and 2 mg / L OBS working solutions were prepared in sequence to conduct acute exposure tests of high-concentration OBS;

[0017] The experimental steps are as follows: newly hatched zebrafish larvae are randomly distributed into multi-well culture plates, with 10 larvae in each well and 2.5 mL of the above-mentioned OBS working solution of different concentrations is added, and a solvent control group of 0.8 wt% DMSO is set up, with 3 parallel wells in each group; then the culture dish is placed in a constant temperature light incubator for culture, with a light-dark cycle of 14h:10h and a temperature of 27±1°C. During the exposure period, the exposure solution is replaced every day, the number of deaths of zebrafish larvae is recorded and the dead larvae are removed in time. The exposure time is 4 days. The number of deaths of zebrafish larvae in each treatment group after 4 days of exposure is counted, and then the 96h-LC is calculated using the Probit analysis method, the Trimmed Spearman-Karber (TSK) method and the nonlinear fitting method. 50 The safe concentration of OBS for zebrafish larvae was calculated based on the value of SC = 0.1 × 96h-LC 50 .

[0018] Through experimental tests, it was determined that the concentration of OBS that causes the death of half of the zebrafish fry is about 10 mg / L, and its safe concentration is 1 mg / L; therefore, when preparing the OBS working solution subsequently, the OBS concentration in each working solution should not exceed this safe concentration.

[0019] According to a preferred embodiment of the present invention, step (3) is: using zebrafish embryo culture medium to prepare OBS working solutions with concentrations of 0.1 μg / L, 10 μg / L and 1000 μg / L respectively; randomly distributing newly hatched zebrafish larvae into culture dishes, with 100 larvae distributed to each culture dish, and 5 parallel culture dishes in each group; changing the exposure solution every day during the exposure period, and the exposure time is 4 days; after the exposure, washing the zebrafish larvae in each treatment group with zebrafish embryo culture medium, collecting all the zebrafish larvae in each treatment group into centrifuge tubes, with 30 larvae in each centrifuge tube, and quickly freezing the zebrafish larvae in liquid nitrogen and storing them at -80°C as test samples for later use.

[0020] According to a preferred embodiment of the present invention, step (5) includes: combining all detection parameters to perform a comprehensive biomarker response analysis and draw a star map to comprehensively reflect the effects of OBS on the antioxidant and immune systems of zebrafish larvae.

[0021] According to a preferred embodiment of the present invention, step (5) also includes: all data are expressed as mean ± standard error in the bar graph, and expressed as the change fold of gene expression in the heat map; the Kolmogorov-Smirnov and Levene methods are used to test the normal distribution of data and the homogeneity of variance; the statistical analysis is performed using the one-way analysis of variance (ANOVA) or two-tailed Student's-t test method in GraphPad Prism 8.3.0 software, *P<0.05, **P<0.01 and ***P<0.001 indicate that there are significant differences between the OBS exposure group and the control group.

[0022] (III) Beneficial effects

[0023] The zebrafish genome is highly similar to that of humans and shares multiple signaling pathways, making it a model organism for assessing the toxicity of PFASs. The sensitivity of zebrafish to pollutant exposure varies at different life stages (embryos, larvae, young fish, and adults), and newly hatched zebrafish larvae are more sensitive to OBS than zebrafish embryos and adults. Therefore, they can be better used to study the toxic effects and molecular mechanisms of OBS on organisms, which is conducive to more accurately assessing the potential risks of OBS environmental exposure to organisms at sensitive stages (such as the initial hatching period).

[0024] After zebrafish larvae were exposed to OBS, the ROS content in their bodies increased. Excessive production of ROS generally occurs simultaneously with inflammatory diseases. Therefore, the present invention further detected changes in immune-related enzyme activity indicators and inflammation-related gene levels. Based on the changes in immune-related enzyme activity indicators and inflammation-related gene levels, the toxic effects and molecular mechanisms caused by OBS were analyzed and studied, indicating that OBS had a significant effect on the immune system of zebrafish larvae.

[0025] The conclusion of the study showed that short-term exposure to OBS significantly induced the expression of the estrogen receptor gene er in zebrafish larvae, indicating the estrogen-like effect of OBS; the CYP19 enzyme can catalyze the conversion of androgens to estrogens, and the induced expression of the cyp19a1 gene in the OBS exposure group may lead to the sexual differentiation of zebrafish after long-term exposure to OBS and affect the population structure. In addition, after OBS exposure, the gene expression level of vitellogenin vtg in zebrafish larvae also increased significantly, as a key biomarker of estrogen endocrine disruption, indicating that short-term exposure to OBS can significantly interfere with the estrogen effect of zebrafish larvae and affect the normal function of the endocrine system. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The mortality of newly hatched zebrafish larvae after exposure to different concentrations of OBS solution for 4 days and the LC calculated by different methods 50 Values; in the figure, OBS exposure concentration 0 represents the solvent control group (0.8wt% DMSO).

[0027] Figure 2 Figure 3 Changes in the levels of oxidative stress-related parameters (ROS, T-AOC, CAT, T-SOD, GSH, MDA) and immune-related parameters (TNOS, iNOS, NO) in zebrafish larvae after 4 days of exposure to sublethal concentrations of OBS (a), as well as the star chart of integrated biomarker response (IBR) analysis (b) and the cumulative IBR values ​​of each exposure group (c); In the figure, Control represents the solvent control group (0.01wt% DMSO), * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001.

[0028] Figure 3 Figure 4 shows the changes in the expression levels of genes related to immunity (a), nuclear transcription factor pathway (b), Toll-like receptor pathway (c), antioxidant system (d) and endocrine system (e) in zebrafish larvae after 4 days of exposure to sublethal concentrations of OBS. In the figure, OBS exposure concentration 0 is the solvent control group (0.01wt% DMSO), * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001. DETAILED DESCRIPTION

[0029] In order to better explain the present invention and facilitate understanding, the present invention is described in detail below through specific implementation modes in conjunction with the accompanying drawings.

[0030] The invention comprises preparing zebrafish larvae for OBS exposure test, performing high concentration acute exposure test to obtain safe concentration of OBS for zebrafish larvae, setting multiple groups of sublethal concentrations based on the safe concentration and obtaining zebrafish larvae test samples under sublethal concentration exposure test, measuring oxidative stress and immune-related parameters in zebrafish larvae test samples and expression levels of antioxidant system, immune system, NF-κB and TLR signaling pathways and endocrine-related genes in zebrafish larvae, and finally arranging the measured parameters, thereby analyzing and studying the toxic effects and molecular mechanisms caused by OBS, and evaluating the effects of actual OBS exposure concentration in the environment on organisms and aquatic ecological environment. The contents of each part are described below, and the following instruments, reagents and materials will be used in the experiment.

[0031] Experimental instruments: zebrafish recirculation culture system (ZebTEC Active Blue, Tenebris, Italy), constant temperature and light incubator (LRH-150F, Shanghai Yiheng), high-throughput tissue grinder (Scientz-48, Ningbo Xinzhi), refrigerated centrifuge (CF1524R, Cellococcus, USA), microplate reader (SpectraMax iD3, Meigu Molecular Instruments, USA) and fluorescence quantitative PCR instrument (CFX96 Touch, Bio-Rad, USA), etc.

[0032] Reagents and materials: OBS powder (purity 97%, CAS 87-56-8, Guangdong Wengjiang Chemical Reagent), dimethyl sulfoxide (DMSO, purity 99.8%, chromatographic grade, Shanghai Aladdin), accurately weigh OBS powder and dissolve it in DMSO to prepare 10g / L OBS stock solution, which was stored at 4°C. Zebrafish embryo culture medium was purchased from Nanjing Yishulihua, reactive oxygen species (ROS), total antioxidant capacity (T-AOC), catalase (CAT), total superoxide dismutase (T-SOD), glutathione (GSH), malondialdehyde (MDA), nitric oxide (NO) and nitric oxide synthase (NOS) typing test kits were purchased from Nanjing Jiancheng Bioengineering Institute, BCA protein concentration determination kit was purchased from GlpBio, USA, RNA extraction kit was purchased from Beijing Kangwei Century, PrimeScript TM RT reagent and real-time fluorescence quantitative PCR detection kit were purchased from Dalian Takara.

[0033] 1. Preparation of Zebrafish Larvae for OBS Exposure Assay

[0034] Adult male and female zebrafish (wild-type AB strain) were purchased from Nanjing Yishulihua and cultured in a zebrafish recirculation culture system. The breeding and exposure test procedures were in accordance with the ethical standards for zebrafish. The light-dark cycle was 14h:10h, the water temperature was 27±1℃, the pH value was 7.5±0.5, the conductivity was 500±50μS, and Artemia was fed twice a day. Male and female zebrafish were mated to obtain zebrafish embryos, and the normally developed cleavage-stage embryos (2hpf) were evenly distributed into a culture dish containing zebrafish embryo culture medium, and then placed in a constant temperature and light incubator for culture, with a light-dark cycle of 14h:10h and a temperature of 27±1℃. The culture medium was replaced every day and the egg membranes after hatching were picked out. After 3 days of culture, the newly hatched zebrafish larvae with normal hatching and development were selected for subsequent exposure tests.

[0035] 2. High-concentration acute exposure test to obtain the safe concentration of OBS for zebrafish larvae

[0036] The 10g / L OBS stock solution was gradiently diluted with zebrafish embryo culture fluid to prepare 80, 60, 40, 20, 10, 8, 6, 4 and 2mg / L OBS working solutions in sequence, and the acute exposure test of high concentration OBS was carried out according to the national fish acute toxicity test standard. The test steps are as follows: the newly hatched zebrafish larvae were randomly assigned to six-well culture plates, 10 larvae per well and 2.5mL of the above-mentioned OBS working solution of different concentrations were added, and a solvent control group (0.8wt% DMSO) was set up, with 3 parallel wells in each group (n=3). The culture dish was then placed in a constant temperature light incubator for culture (light and dark cycle 14h:10h, temperature 27±1℃), the exposure solution was changed every day during the exposure period, the number of deaths of zebrafish larvae was recorded and the dead larvae were removed in time, and the exposure time was 4 days. The number of zebrafish larvae that died in each treatment group after 4 days of exposure was used to calculate the 96h-LC 50 The safe concentration of OBS for zebrafish larvae was calculated as SC = 0.1 × 96h-LC 50 .

[0037] like Figure 1 The figure shows the mortality rate of zebrafish larvae at different exposure concentrations after 4 days of exposure in a high-concentration acute exposure test of OBS on newly hatched zebrafish larvae. The results show that with the increase of OBS exposure concentration, the mortality rate of zebrafish larvae gradually increased and reached 100% in the 40 mg / L group. Different methods were used to estimate the 96h-LC of OBS on zebrafish larvae. 50The values ​​were as follows: among them, the estimated value by Probit method was 9.66 mg / L, the estimated value by TSK method was 10.10 mg / L, and the estimated value by linear fitting was 9.21 mg / L. It was preliminarily determined that the concentration of OBS that causes the death of half of the zebrafish larvae is about 10 mg / L, and its safe concentration is 1 mg / L.

[0038] 3. Set the sublethal concentration of OBS exposure based on the above safety concentration, and obtain zebrafish larvae test samples under the sublethal concentration exposure test

[0039] Based on the safety concentration obtained in the above-mentioned high-concentration acute exposure test, the OBS working solution with OBS concentrations of 0.1, 10 and 1000 μg / L was set in the sublethal concentration exposure experiment (the 10 g / L OBS stock solution was gradiently diluted with zebrafish embryo culture medium to prepare the corresponding concentration of OBS working solution). The newly hatched zebrafish larvae were randomly distributed into culture dishes, each culture dish included 100 larvae, and a solvent control group (0.01wt% DMSO, Control group) was set up, with 5 parallel culture dishes in each group. The exposure solution was changed every day during the exposure period, and the exposure time was 4 days.

[0040] After the exposure, the zebrafish larvae in each treatment group were first washed with zebrafish embryo culture medium, and all the zebrafish larvae in each treatment group were collected into 1.5 mL centrifuge tubes, with 30 larvae in each centrifuge tube. The water was dried, and all the zebrafish larvae were quickly frozen in liquid nitrogen and stored at -80°C as test samples for subsequent experiments.

[0041] 4. Determination of oxidative stress and immune-related parameters of zebrafish larvae test samples and the expression levels of antioxidant system, immune system, NF-κB and TLR signaling pathways and endocrine-related genes in zebrafish larvae

[0042] (1) Detection of oxidative stress and immune-related parameters in zebrafish larvae test samples

[0043] Four centrifuge tubes (n=4) containing zebrafish larvae were randomly selected from each treatment group, homogenized after adding 10 volumes of ice-cold saline, and then centrifuged at 4°C, 10,000×g for 10 min, and the supernatant was aspirated into a new centrifuge tube. The levels of oxidative stress parameters (ROS, T-AOC, CAT, T-SOD, GSH, and MDA) and immune-related parameters (TNOS, iNOS, and NO) in the supernatant samples were determined according to the instructions of the kit. The protein concentration in each sample was determined according to the BCA method and used to standardize each parameter.

[0044] (2) Detection of the expression levels of antioxidant system, immune system, NF-κB and TLR signaling pathways, and endocrine-related genes in zebrafish larvae

[0045] Six centrifuge tubes containing zebrafish larvae were randomly selected (n=6), and the RNA extraction, concentration detection, cDNA synthesis and real-time fluorescence quantitative PCR (qRT-PCR) were performed according to the corresponding kits to detect the expression levels of antioxidant system, immune system, NF-κB and TLR signaling pathways and endocrine-related genes in zebrafish larvae. The primers for the reference gene β-actin and all target genes were synthesized by Sangon Biotech (Shanghai) Co., Ltd., and the primer sequences are shown in Table 1.

[0046] Table 1: Primer sequence information of reference genes and target genes

[0047]

[0048]

[0049] 5. Data Statistical Analysis

[0050] All data are expressed as mean ± standard error (Mean ± SEM) in the bar graph and as the fold change of gene expression in the heat map. The Kolmogorov-Smirnov and Levene methods were used for data normal distribution and variance homogeneity test. Statistical analysis was performed using one-way analysis of variance (ANOVA) or two-tailed Student's-t test in GraphPad Prism 8.3.0 software. *P<0.05, **P<0.01 and ***P<0.001 indicated significant differences between the OBS-exposed group and the control group. GraphPad Prism 8.3.0 was used to draw bar graphs and heat maps, and Microsoft Office Excel 2019 was used to draw star graphs. The experimental results are shown in the figure. Figure 2 and Figure 3 shown.

[0051] like Figure 2 As shown in the figure, after newly hatched zebrafish larvae were exposed to 0.1, 10, and 1000 μg / L OBS solutions for 4 days, the levels of oxidative stress-related parameters and immune-related parameters in zebrafish larvae were measured. Figure 2As shown in (a), the levels of oxidative stress-related parameters in zebrafish larvae in the OBS exposure group increased. Among them, the ROS content increased significantly in the zebrafish larvae in the three OBS exposure groups, the T-AOC content increased significantly in the 10μg / L exposure group, the CAT enzyme activity did not change significantly, the T-SOD enzyme activity was significantly enhanced in the OBS exposure group, and the GSH and MDA contents were significantly increased in the 1000μg / L and 0.1μg / L exposure groups, respectively. Among the immune-related parameters, the TNOS enzyme activity did not change significantly, while the iNOS enzyme activity was significantly enhanced in the 0.1 and 1000μg / L exposure groups, and the NO content was significantly increased in the 1000μg / L exposure group. Further IBR analysis results showed that the cumulative IBR value increased with the increase of OBS exposure concentration (such as Figure 2 b and c), indicating that the comprehensive effect of OBS on the above parameters has a significant concentration-effect relationship.

[0052] like Figure 3 As shown in Figure 2, after newly hatched zebrafish larvae were exposed to 0.1, 10, and 1000 μg / L OBS solutions for 4 days, the expression levels of genes related to multiple functional systems in zebrafish larvae were detected. Figure 3 As shown in a, the expression levels of inflammatory factors il1β and il8 increased significantly in the 0.1 and 1000 μg / L exposure groups, and the expression levels of other immune-related genes il12, tnfα, lyz, cc-chem and cxcl-clc increased significantly only in the 1000 μg / L exposure group, while the expression level of inos decreased significantly in all three exposure groups; in the genes related to the NF-κB pathway (such as Figure 3 b), the expression of p65 was significantly decreased in the 0.1 and 10 μg / L exposure groups, the expression of traf6 was significantly decreased in the 0.1 μg / L exposure group, and the expression of nfkbiaa was significantly increased in all three exposure groups. In addition, the expression of ptgs2 was also significantly induced; the expression levels of genes related to the Toll-like receptor pathway were induced after OBS exposure (such as Figure 3 c), among which, tlr2 and irak4 were significant in the 10 and 1000 μg / L exposure groups, tlr4a, p38a, p38b, erk2, erk3 and jnk were significant only in the 1000 μg / L exposure group, and c3b was significant only in the 0.1 μg / L exposure group; among the antioxidant system related genes (such as Figure 3 d), the expression of cat increased significantly in the 10 and 1000 μg / L exposure groups, the expression of sod and gst increased significantly in the 1000 μg / L exposure group, while the expression of nrf was inhibited and showed significance in the 0.1 μg / L exposure group; OBS exposure induced the expression levels of endocrine disruption-related genes (such as Figure 3e), among which erα, erβ1, cyp19a1, vtg1 and vtg2 were significant in the 1000μg / L exposure group.

[0053] Previous studies have shown that many types of organic pollutants have an LC- 50 The values ​​were lower than the LC for embryos or adult fish. 50 The value indicates that zebrafish larvae are more sensitive to pollutants than embryos and adults, and have a higher mortality rate. For example, previous studies have found that OBS has a LC value of 1. 50 The value is 25.5mg / L; the present invention found that OBS has an effect on the 96h-LC 50 The value is about 10 mg / L, which is lower than the LC for adult fish. 50 The values ​​indicate that newly hatched zebrafish larvae are more sensitive to OBS than adult fish, that is, OBS is more toxic to newly hatched zebrafish larvae. Based on the fact that the safe concentration of OBS for zebrafish larvae is 1 mg / L, the OBS concentrations in the sublethal concentration exposure test in the present invention were set at 0.1, 10, and 1000 μg / L to explore the toxic effects of OBS safety concentration exposure on zebrafish larvae in the early developmental stage.

[0054] The antioxidant system and immune system are the first line of defense and important protective systems to deal with external environmental stimuli. Excessive production of reactive oxygen species (ROS) in organisms may induce oxidative stress and participate in many pathological processes. After zebrafish larvae were exposed to sublethal concentrations of OBS for 4 days, the increase in ROS content in the body required more ROS scavenging enzymes, which led to a significant increase in SOD activity and a significant increase in sod gene levels; although CAT activity did not increase significantly, the expression level of its encoding gene cat was significantly induced; and the GSH content increased significantly and the expression level of the gst gene was significantly induced, which collectively indicated that the oxidative stress response caused by OBS activated the antioxidant system of zebrafish larvae to ensure the normal development of the body.

[0055] The overproduction of ROS generally occurs simultaneously with inflammatory diseases, therefore, the present invention further detects changes in immune-related enzyme activity indicators and inflammation-related gene levels. After OBS exposure, although there was no significant change in the total synthase activity of NO in zebrafish larvae, the activity of the inducible synthase iNOS increased significantly, thereby increasing the NO level in the body, indicating the activation of the body's immune system. However, the expression of the iNOS coding gene inos was significantly reduced, which was inconsistent with the increase in iNOS activity, which may be due to the negative feedback regulation mechanism formed by the overproduction of NO. OBS exposure also significantly induced the expression of lyz in zebrafish larvae, indicating that OBS caused a pro-inflammatory response in zebrafish larvae.

[0056] In addition, in the fish immune system, a variety of cytokines including interleukin IL, chemokine CXC and tumor necrosis factor TNF play an important role. After OBS exposure, the expression levels of proinflammatory factors il1β, il8, il12, tnfα, cc-chem and cxcl-clc in zebrafish larvae increased significantly. At the same time, short-term exposure to F-53B can also significantly induce the expression of il1β, il8, tnfα and cxcl-clc in zebrafish larvae, indicating the generation of proinflammatory response, which is consistent with the effect caused by increased lyz gene expression.

[0057] The study of the present invention shows that short-term exposure to OBS significantly induced the expression of the estrogen receptor gene er in zebrafish larvae, indicating the estrogen-like effect of OBS; in addition, the induced expression of the cyp19a1 gene in the OBS exposure group may lead to the sexual differentiation of zebrafish after long-term exposure to OBS, thereby affecting the population structure. In addition, after OBS exposure, the gene expression level of vitellogenin vtg in zebrafish larvae also increased significantly. This study comprehensively shows that short-term exposure to OBS can significantly interfere with the estrogen effect of zebrafish larvae and affect the normal function of the endocrine system.

[0058] Finally, in the present study, it was found that short-term exposure to OBS at a concentration of 0.1 μg / L can have a significant impact on the relevant indicators of multiple important functional systems of newly hatched zebrafish larvae. According to surveys, the actual highest concentration of OBS in the water environment is 3.2 μg / L, and the concentration of sewage treatment plant effluent or surface water in some areas can reach 57.26 ng / L and 193 ng / L respectively; this shows that OBS has significant toxicity to aquatic organisms in these water bodies.

[0059] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for evaluating the effects of OBS environmental exposure on zebrafish larvae, characterized in that: include: (1) Place the normally developed cleavage-stage zebrafish embryos in a culture dish containing zebrafish embryo culture medium, and then culture them in a constant temperature and light incubator with a light-dark cycle of 14h:10h and a temperature of 27±1°C; replace the culture medium every day and remove the egg membrane after hatching. After 3 days of culture, select the newly hatched zebrafish larvae that have hatched and developed normally for exposure testing; (2) The safe concentration of OBS for zebrafish larvae was calculated using a high-concentration acute exposure test. The safe concentration for zebrafish larvae is SC = 0.1 × 96h-LC 50 ; (3) Based on the safety concentration, multiple sublethal concentrations were set as the OBS concentrations in the exposure experiment, and the sublethal concentrations did not exceed the safety concentration; The newly hatched zebrafish larvae were randomly divided into multiple parallel culture dishes, with the group exposed to sublethal concentration of OBS working solution as the treatment group, and the group exposed to 0.01wt% DMSO as the control group; during the exposure period, the exposure solution was changed every day, and the exposure time was 4 days; after the exposure, the zebrafish larvae of each group were washed with zebrafish embryo culture solution, and the zebrafish larvae were collected into centrifuge tubes, quickly frozen in liquid nitrogen, and stored at -80°C for later use; (4) taking samples from one of the centrifuge tubes for oxidative stress and immune-related parameter detection: adding 8-12 times the volume of ice-cold physiological saline to homogenize, then centrifuging at 4°C, 10,000×g for 5-15 min, and aspirating the supernatant into a new centrifuge tube; measuring oxidative stress parameters and immune-related parameters in the supernatant sample; the oxidative stress parameters include ROS, T-AOC, CAT, T-SOD, GSH and MDA, and the immune-related parameters include TNOS, iNOS and NO, and the protein concentration in each sample is determined according to the BCA method; taking samples from another part of the centrifuge tubes for RNA extraction, and detecting the expression levels of antioxidant system, immune system, NF-κB and TLR signaling pathways and endocrine-related genes in zebrafish larvae; (5) Based on the detection results of step (4), perform data statistical analysis to evaluate the effect of OBS exposure on zebrafish larvae.

2. The method according to claim 1, characterized in that In step (1), adult male and female zebrafish are cultured in a zebrafish recirculating culture system with a light-dark cycle of 14h:10h, a water temperature of 27±1°C, a pH value of 7.5±0.5, and an electrical conductivity of 500±50μS, and are fed with Artemia twice a day; the male and female zebrafish are mated to obtain zebrafish embryos, and the normally developed cleavage-stage embryos are evenly distributed into a culture dish containing zebrafish embryo culture medium, and then placed in a constant temperature and light incubator for culture with a light-dark cycle of 14h:10h and a temperature of 27±1°C; the culture medium is replaced every day and the egg membrane after hatching is picked out, and after culturing for 3 days, the hatched and normally developed zebrafish newly hatched larvae are selected for subsequent exposure tests.

3. The method according to claim 1, characterized in that: In step (2), in the high concentration acute exposure test, 80 mg / L, 60 mg / L, 40 mg / L, 20 mg / L, 10 mg / L, 8 mg / L, 6 mg / L, 4 mg / L and 2 mg / L of OBS working solutions were prepared in sequence, and an acute exposure test of high concentration OBS was carried out; The experimental steps are as follows: newly hatched zebrafish larvae are randomly distributed into multi-well culture plates, with 10 larvae in each well and 2.5 mL of the above-mentioned OBS working solution of different concentrations is added, and a solvent control group of 0.8 wt% DMSO is set up, with 3 parallel wells in each group; then the culture dish is placed in a constant temperature light incubator for culture, with a light-dark cycle of 14h:10h and a temperature of 27±1°C. During the exposure period, the exposure solution is replaced every day, the number of deaths of zebrafish larvae is recorded and the dead larvae are removed in time. The exposure time is 4 days. The number of deaths of zebrafish larvae in each treatment group after 4 days of exposure is counted, and then the 96h-LC is calculated using the Probit analysis method, the Trimmed Spearman-Karber (TSK) method and the nonlinear fitting method. 50 The safe concentration of OBS for zebrafish larvae was calculated based on the value of SC = 0.1 × 96h-LC 50 .

4. The method according to claim 1, characterized in that: Step (3) is: using zebrafish embryo culture medium to prepare OBS working solutions with concentrations of 0.1 μg / L, 10 μg / L and 1000 μg / L respectively; randomly distributing newly hatched zebrafish larvae into culture dishes, with 100 larvae distributed to each culture dish, and 5 parallel culture dishes in each group; changing the exposure solution every day during the exposure period, and the exposure time is 4 days; after the exposure, washing the zebrafish larvae in each treatment group with zebrafish embryo culture medium, collecting all the zebrafish larvae in each treatment group into centrifuge tubes, with 30 larvae in each centrifuge tube, and quickly freezing the zebrafish larvae in liquid nitrogen and storing them at -80°C as test samples for later use.

5. The method according to claim 1, characterized in that Step (5) includes: combining all detection parameters to conduct a comprehensive biomarker response analysis and draw a star map to comprehensively reflect the effects of OBS on the antioxidant and immune systems of zebrafish larvae.

6. The method according to claim 5, characterized in that Step (5) also includes: all data are expressed as mean ± standard error in the bar graph and as the change fold of gene expression in the heat map; the Kolmogorov-Smirnov and Levene methods are used to test the normal distribution of data and the homogeneity of variance; the statistical analysis is performed using the one-way analysis of variance (ANOVA) or two-tailed Student's-t test method in GraphPad Prism 8.3.0 software, *P<0.05, **P<0.01 and ***P<0.001 indicate that there are significant differences between the OBS exposure group and the control group.

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