Method for evaluating thyroid toxicity of pregnadiene ketone through zebra fish

Abstract

The invention relates to the technical field of toxicity analysis, in particular to a method for evaluating the thyroid toxicity of gestodienone through zebra fish, which analyzes the influence of gestodienone on zebra fish through a molecular level, a cellular level and an individual form. Specifically, the movement behavior, reproduction, hormone content and tissue slices of zebrafish treated by gestodienone are analyzed. The result shows that the gestodienone exposure medium not only has potential negative effects on the growth and development of the zebrafish, but also possibly causes serious threats to the long-term survival and reproductive capacity of the zebrafish. The invention provides an important scientific basis for further understanding the potential risk of gestodienone and other environmental hormones on aquatic organisms, also discloses the application value of zebrafish as a model organism in environmental toxicology research, and has important guiding significance for making environmental protection policies and reducing environmental pollution. Compared with the prior art, the method has a wide application prospect.

Description

Technical Field

[0001] The invention relates to the technical field of toxicity analysis, and in particular to a method for evaluating the thyroid toxicity of gestodene through zebrafish. Background Art

[0002] Synthetic progestins are widely used in various fields, including hormone therapy in medicine and increasing meat production in animal husbandry. However, with the increase in progestin production and consumption in recent years, unused progestins may be discharged into domestic wastewater with feces, and some progestins cannot be treated and effectively removed by sewage treatment plants.

[0003] Gestodene, levonorgestrel and nortestosterone are considered the three progestogens of greatest concern in the aquatic environment, of which gestodene is the most active in humans, is often used as a hormonal therapy or contraceptive, and is widely consumed.

[0004] Zebrafish are a common model organism that is cheap to feed and maintain and easy to breed indoors on a large scale. As a small fish, zebrafish are small, easy to obtain, easy to raise, and have a relatively short life cycle, making them a more ideal test organism than frogs, mice, etc. At present, zebrafish have become a popular research tool at home and abroad, especially in the field of toxicology, such as using zebrafish to explore the toxic effects of progesterone.

[0005] In the prior art, for example, patent document CN109900884A discloses a method for studying the toxic effects of short-chain chlorinated paraffin on zebrafish embryos based on metabolomics. In this invention, zebrafish embryos are first exposed to short-chain chlorinated paraffins, and the metabolites in the embryos are analyzed using liquid chromatography-high-resolution mass spectrometry, and then statistical processing and metabolic pathway enrichment analysis are combined to reflect the effects of short-chain chlorinated paraffins on the metabolic pathways of zebrafish embryos. This invention is based on the study of zebrafish using metabolomics, which is low-cost and easy to operate, but it is rather one-sided to analyze the effects of environmental endocrine disruptors on zebrafish only at the molecular level.

[0006] Therefore, based on the above-mentioned related technologies, it is urgent to develop a method for evaluating the thyroid toxicity of gestodene using zebrafish. Summary of the invention

[0007] In view of this, the purpose of the present invention is to propose a method for evaluating the thyroid toxicity of gestodene by zebrafish, so as to solve the problem that the prior art is relatively one-sided in analyzing the effects of environmental endocrine disruptors on zebrafish.

[0008] Based on the above objectives, the present invention provides a method for evaluating the thyroid toxicity of gestodene using zebrafish.

[0009] A method for evaluating gestodene thyroid toxicity in zebrafish, comprising the following steps:

[0010] Step S1. 40 adult female zebrafish of similar size were selected and placed in a water tank for 14 days, and then divided into four parts, and then transferred to fish tank A, fish tank B, fish tank C and fish tank D respectively, and raised for 60 days, respectively, to obtain zebrafish group A, zebrafish group B, zebrafish group C and zebrafish group D;

[0011] Step S2. Randomly select a female zebrafish from each of the zebrafish group A, zebrafish group B, zebrafish group C and zebrafish group D and place them in a water tank individually, observe for 10-20 minutes and use a zebrafish tracking system to record its movement behavior, average speed, total movement distance and total bottom residence time of the zebrafish;

[0012] Step S3. randomly selecting a female zebrafish from each of the zebrafish group A, the zebrafish group B, the zebrafish group C, and the zebrafish group D, taking blood from the fish tail after cryo-anesthesia, obtaining a plasma sample after the first centrifugation, and obtaining a supernatant of the zebrafish group A, the zebrafish group B, the zebrafish group C, and the zebrafish group D by a second centrifugation, and then measuring the contents of acetylcholinesterase, thyroid hormone T3, thyroid hormone T4, and thyroid-stimulating hormone in the supernatant of each group of zebrafish;

[0013] Step S4. randomly selecting one zebrafish from each of the zebrafish group A, the zebrafish group B, the zebrafish group C and the zebrafish group D, and placing the intestine thereof in Bonn fixative, dehydrating, making transparent and dipping in wax, and then staining, sealing and photographing under a microscope, and then counting the length of the intestinal villi of the zebrafish in each group;

[0014] Step S5. Randomly select one adult female zebrafish and two unexposed adult male zebrafish from zebrafish group A, zebrafish group B, zebrafish group C and zebrafish group D, respectively, and place them on both sides of the partition of the breeding pool. First place them in dark conditions for 10-12 hours, then remove the partition, and use light to stimulate the female zebrafish to lay eggs. After 1.5-2 hours, collect the fertilized eggs and place them in E3 culture medium for incubation for 144-148 hours, and record the hatching rate, survival rate and deformity rate of each group of zebrafish.

[0015] Preferably, the adult female zebrafish in step S1 is three months old, and the breeding conditions in the water tank are feeding with brine shrimp larvae twice a day, and the residual food is removed after 5 minutes each feeding;

[0016] The concentration of gestodene in the fish tank A is 0 ng / L;

[0017] The concentration of gestodene in the fish tank B is 5 ng / L;

[0018] The concentration of gestodene in the fish tank C is 50 ng / L;

[0019] The concentration of gestodene in the fish tank D is 500 ng / L.

[0020] Preferably, the water temperature in the fish tank in step S1 is set to 25-27°C, the pH is 6.9-7.5, the dissolved oxygen is 6-15 mg / L, the light-dark cycle ratio is 12.5-14:10-11.5, and the light intensity is 54-324 lux.

[0021] Preferably, the fish tank in step S1 is reared under the conditions of feeding shelled brine shrimp eggs twice a day, changing half of the water every 24 hours and adding gestodene to keep the concentration of gestodene in the water consistent.

[0022] Preferably, the first centrifugation speed in step S3 is set to 2000-2500 rpm, the centrifugation time is 15-20 min, and the centrifugation temperature is 2-5°C.

[0023] Preferably, the second centrifugation speed in step S3 is set to 1500-2000 rpm, the centrifugation time is 10-15 min, and the centrifugation temperature is 2-5°C.

[0024] Preferably, the Bonn fixative in step S4 is obtained by mixing 72-75 mL of a saturated solution of picric acid with a mass concentration of 1.22%, 22-25 mL of formaldehyde with a mass concentration of 40%, and 3-5 mL of glacial acetic acid.

[0025] Preferably, the light intensity in step S5 is 100-240 lux, and the E3 culture medium is obtained by mixing sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, methylene blue and deionized water.

[0026] Preferably, during the incubation in step S5, the temperature is set to 27.5-28.5° C., the pH is 6.9-7.5, the dissolved oxygen is 6-15 mg / L, and the light-dark cycle ratio is 12.5-14:10-11.5.

[0027] Beneficial effects of the present invention:

[0028] The present invention provides a method for evaluating the thyroid toxicity of gestodene by zebrafish. The present invention analyzes the effect of gestodene on zebrafish at the molecular level, cellular level and individual morphology, and specifically analyzes the movement behavior, reproduction, hormone content and tissue sections of zebrafish after gestodene treatment. The results show that gestodene damages the reproductive system and nervous system of zebrafish and affects thyroid function, further indicating that gestodene exposure media not only has a potential negative impact on the growth and development of zebrafish, but also may pose a serious threat to their long-term survival and reproductive capacity.

[0029] Therefore, the present invention provides an important scientific basis for further understanding the potential risks of environmental hormones such as gestodene to aquatic organisms. In addition, it also reveals the application value of zebrafish as a model organism in environmental toxicology research. Through careful observation of its behavior, physiology and tissue structure, we can have a deeper understanding of the mechanism of influence of environmental pollutants on organisms. It has important guiding significance for formulating environmental protection policies and reducing environmental pollution. Compared with the prior art, it has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the technical solutions in the present invention or 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 in the following description are only for the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0031] Figure 1 This is a comparison chart of the thyroid hormone T3 content of each group of zebrafish in Example 1 of the present invention;

[0032] Figure 2 This is a comparison chart of the thyroid hormone T4 content of each group of zebrafish in Example 1 of the present invention;

[0033] Figure 3 This is a comparison chart of the thyroid stimulating hormone content of each group of zebrafish in Example 1 of the present invention;

[0034] Figure 4 This is a comparison chart of the acetylcholinesterase content of each group of zebrafish in Example 1 of the present invention;

[0035] Figure 5 The heat map of the behavior of each group of zebrafish in Example 1 of the present invention;

[0036] Figure 6 This is a comparison chart of the movement behavior performance of each group of zebrafish in Example 1 of the present invention;

[0037] Figure 7 This is a comparison diagram of zebrafish intestinal villus sections in each group in Example 1 of the present invention;

[0038] Figure 8 This is a comparison diagram of the length of zebrafish intestinal villi in each group in Example 1 of the present invention;

[0039] Fig. 9 Schematic diagram of the zebrafish behavior measurement experiment in the present invention. DETAILED DESCRIPTION

[0040] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments.

[0041] The sources and properties of some of the raw materials used in the present invention are as follows:

[0042] Bonn fixative was purchased from Wuhan Kanos Technology Co., Ltd.; hematoxylin-eosin was purchased from Yunnan Haoxin Biomedical Technology Co., Ltd.; E3 culture medium was purchased from Shanghai Huicheng Biotechnology Co., Ltd.; and gestodene was purchased from Hubei Weideli Chemical Technology Co., Ltd.

[0043] Example 1: A method for evaluating gestodene thyroid toxicity by zebrafish, comprising the following steps:

[0044] S1. 40 adult female zebrafish of similar size at three months of age were selected and placed in a water tank. They were fed with brine shrimp larvae twice a day. The remaining food was removed after 5 minutes of each feeding. They were raised for 14 days to adapt to the experimental environment. They were divided into four parts and then transferred to 15L fish tank A containing 0 ng / L gestodene concentration, fish tank B containing 5 ng / L gestodene concentration, fish tank C containing 50 ng / L gestodene concentration and fish tank C containing 15L gestodene concentration. In the fish tank D with a gestodene concentration of 500 ng / L, the water temperature in the fish tank was 25°C, the pH was 6.9, the dissolved oxygen was 6 mg / L, the light-dark cycle ratio was 12.5:11.5, the light intensity was 54 lux, and the fish were fed with shelled brine shrimp eggs twice a day. Half of the water was changed every 24 hours and gestodene was added to keep the concentration of gestodene in the water consistent. After 60 days, zebrafish group A, zebrafish group B, zebrafish group C and zebrafish group D were obtained;

[0045] S2. Randomly select a female zebrafish from each of the zebrafish groups A, B, C, and D and place them in a water tank. Observe for 10 min and use a zebrafish tracking system to record their movement behavior. The water tank size is 30 cm × 23 cm × 25 cm, and is divided into the upper part (up zone) and the bottom (down zone) with the half-height 15 cm as the dividing line. Record the average speed, total moving distance, and total time spent at the bottom of the zebrafish.

[0046] S3. A female zebrafish was randomly selected from each of the zebrafish group A, zebrafish group B, zebrafish group C and zebrafish group D, and blood was collected from the fish tail after cryo-anesthesia. The blood was centrifuged at a speed of 2000 rpm and a temperature of 2°C for 15 min to obtain a plasma sample. The blood was then centrifuged at a speed of 1500 rpm and a temperature of 2°C for 10 min to obtain the supernatant of the zebrafish group A, the supernatant of the zebrafish group B, the supernatant of the zebrafish group C and the supernatant of the zebrafish group D. The contents of acetylcholinesterase, thyroid hormone T3, thyroid hormone T4 and thyroid-stimulating hormone in the supernatant of each group of zebrafish were measured respectively.

[0047] S4. Randomly select one zebrafish from each of the zebrafish group A, zebrafish group B, zebrafish group C, and zebrafish group D, and place its intestine in Bonn fixative. After dehydration, transparency, and wax treatment, use a rotary slicer to make continuous sections, then stain with hematoxylin-eosin, dry and seal with gum, and examine and photograph under an optical microscope. Use CaseViewer software to analyze the micrographs, and count the length of the intestinal villi of each group of zebrafish.

[0048] S5. One adult female zebrafish and two unexposed adult male zebrafish were randomly selected from zebrafish group A, zebrafish group B, zebrafish group C and zebrafish group D and placed on both sides of the partition of the breeding pool. They were first placed in dark conditions for 10 hours, and then the partition was removed and the female zebrafish were stimulated to lay eggs with light. After 1.5 hours, the fertilized eggs were collected and placed in E3 medium for incubation for 144 hours. The hatching rate, survival rate and deformity rate of zebrafish in each group were recorded.

[0049] Example 2: A method for evaluating gestodene thyroid toxicity by zebrafish, comprising the following steps:

[0050] S1. 40 adult female zebrafish of similar size at three months of age were selected and placed in a water tank. They were fed with brine shrimp larvae twice a day. The residual food was removed after 5 minutes each feeding. They were raised for 14 days to adapt to the experimental environment, and then divided into four parts. They were then transferred to 15L fish tank A with a gestodene concentration of 0 ng / L, fish tank B with a gestodene concentration of 5 ng / L, fish tank C with a gestodene concentration of 50 ng / L, and fish tank D with a gestodene concentration of 500 ng / L. The water temperature in the fish tank was 26°C, pH was 7.0, dissolved oxygen was 8 mg / L, the light-dark cycle ratio was 13:11, and the light intensity was 125 lux. They were fed with molted brine shrimp eggs twice a day. Half of the water was changed every 24 hours and gestodene was added to keep the concentration of gestodene in the water consistent. After 60 days, zebrafish group A, zebrafish group B, zebrafish group C, and zebrafish group D were obtained.

[0051] S2. Randomly select a female zebrafish from each of the zebrafish groups A, B, C, and D and place them in a water tank. Observe for 15 min and use a zebrafish tracking system to record their movement behavior. The water tank is 30 cm × 23 cm × 25 cm in size and is divided into the upper part (up zone) and the bottom (down zone) with the half-height of 15 cm as the dividing line. Record the average speed, total moving distance, and total time spent at the bottom of the zebrafish.

[0052] S3. A female zebrafish was randomly selected from each of the zebrafish group A, zebrafish group B, zebrafish group C and zebrafish group D, and blood was collected from the fish tail after cryo-anesthesia. The blood was centrifuged at a speed of 2200 rpm and a temperature of 3°C for 17 min to obtain a plasma sample, and then centrifuged at a speed of 1700 rpm and a temperature of 3°C for 12 min to obtain the supernatant of the zebrafish group A, the supernatant of the zebrafish group B, the supernatant of the zebrafish group C and the supernatant of the zebrafish group D, and then the contents of acetylcholinesterase, thyroid hormone T3, thyroid hormone T4 and thyroid stimulating hormone in the supernatant of each group of zebrafish were measured respectively;

[0053] S4. Randomly select one zebrafish from each of the zebrafish group A, zebrafish group B, zebrafish group C, and zebrafish group D, and place its intestine in Bonn fixative. After dehydration, transparency, and wax treatment, use a rotary slicer to make continuous sections, then stain with hematoxylin-eosin, dry and seal with gum, and examine and photograph under an optical microscope. Use CaseViewer software to analyze the micrographs, and count the length of the intestinal villi of each group of zebrafish.

[0054] S5. One adult female zebrafish and two unexposed adult male zebrafish were randomly selected from zebrafish group A, zebrafish group B, zebrafish group C and zebrafish group D, and placed on both sides of the partition of the breeding pool. They were first placed in dark conditions for 11 hours, and then the partition was removed. The female zebrafish were stimulated to lay eggs with light. After 1.5 hours, the fertilized eggs were collected and placed in E3 medium for incubation for 145 hours. The hatching rate, survival rate and deformity rate of zebrafish in each group were recorded.

[0055] Example 3: A method for evaluating gestodene thyroid toxicity by zebrafish, comprising the following steps:

[0056] S1. 40 adult female zebrafish of similar size at three months of age were selected and placed in a water tank. They were fed with brine shrimp larvae twice a day. The remaining food was removed after 5 minutes of each feeding. They were raised for 14 days to adapt to the experimental environment. They were divided into four parts and then transferred to 15L fish tank A containing 0 ng / L gestodene concentration, fish tank B containing 5 ng / L gestodene concentration, fish tank C containing 50 ng / L gestodene concentration and fish tank C containing 15L gestodene concentration. In fish tank D with gestodene concentration of 500 ng / L, the water temperature in the fish tank was 26°C, the pH was 7.2, the dissolved oxygen was 12 mg / L, the light-dark cycle ratio was 13.5:10.5, the light intensity was 245 lux, and the shelled brine shrimp eggs were fed twice a day. Half of the water was changed every 24 hours and gestodene was added to keep the concentration of gestodene in the water consistent. After 60 days, zebrafish group A, zebrafish group B, zebrafish group C and zebrafish group D were obtained;

[0057] S2. Randomly select a female zebrafish from each of the zebrafish groups A, B, C, and D and place them in a water tank. Observe for 15 min and use a zebrafish tracking system to record their movement behavior. The water tank is 30 cm × 23 cm × 25 cm in size and is divided into the upper part (up zone) and the bottom (down zone) with the half-height of 15 cm as the dividing line. Record the average speed, total moving distance, and total time spent at the bottom of the zebrafish.

[0058] S3. A female zebrafish was randomly selected from each of the zebrafish group A, zebrafish group B, zebrafish group C and zebrafish group D, and blood was collected from the fish tail after cryo-anesthesia. The blood was centrifuged at a speed of 2400 rpm and a temperature of 4°C for 18 min to obtain a plasma sample, and then centrifuged at a speed of 1800 rpm and a temperature of 4°C for 14 min to obtain the supernatant of the zebrafish group A, the supernatant of the zebrafish group B, the supernatant of the zebrafish group C and the supernatant of the zebrafish group D, and then the contents of acetylcholinesterase, thyroid hormone T3, thyroid hormone T4 and thyroid stimulating hormone in the supernatant of each group of zebrafish were measured respectively;

[0059] S4. Randomly select one zebrafish from each of the zebrafish group A, zebrafish group B, zebrafish group C, and zebrafish group D, and place its intestine in Bonn fixative. After dehydration, transparency, and wax treatment, use a rotary slicer to make continuous sections, then stain with hematoxylin-eosin, dry and seal with gum, and examine and photograph under an optical microscope. Use CaseViewer software to analyze the micrographs, and count the length of the intestinal villi of each group of zebrafish.

[0060] S5. One adult female zebrafish and two unexposed adult male zebrafish were randomly selected from zebrafish group A, zebrafish group B, zebrafish group C and zebrafish group D and placed on both sides of the partition of the breeding pool. They were first placed in dark conditions for 11 hours, and then the partition was removed and the female zebrafish were stimulated to lay eggs with light. After 2 hours, the fertilized eggs were collected and placed in E3 medium for incubation for 146 hours. The hatching rate, survival rate and deformity rate of zebrafish in each group were recorded.

[0061] Example 4: A method for evaluating gestodene thyroid toxicity by zebrafish, comprising the following steps:

[0062] S1. 40 adult female zebrafish of similar size and three months old were selected and placed in a water tank. They were fed with brine shrimp larvae twice a day. The residual food was removed after 5 minutes each feeding. They were raised for 14 days to adapt to the experimental environment, and then divided into four parts. They were then transferred to 15L fish tank A with a gestodene concentration of 0 ng / L, fish tank B with a gestodene concentration of 5 ng / L, fish tank C with a gestodene concentration of 50 ng / L, and fish tank D with a gestodene concentration of 500 ng / L, respectively. The water temperature was 27°C, pH was 7.5, dissolved oxygen was 15 mg / L, the light-dark cycle ratio was 14:10, the light intensity was 324 lux, and they were fed with molted brine shrimp eggs twice a day. Half of the water was changed every 24 hours and gestodene was added to keep the concentration of gestodene in the water consistent. After 60 days, zebrafish group A, zebrafish group B, zebrafish group C, and zebrafish group D were obtained.

[0063] S2. Randomly select a female zebrafish from each of the zebrafish groups A, B, C, and D and place them in a water tank. Observe for 20 min and use a zebrafish tracking system to record their movement behavior. The water tank size is 30 cm × 23 cm × 25 cm, and is divided into the upper part (up zone) and the bottom (down zone) with a half-height line of 15 cm as the dividing line. Record the average speed, total moving distance, and total time spent at the bottom of the zebrafish.

[0064] S3. A female zebrafish was randomly selected from each of the zebrafish group A, zebrafish group B, zebrafish group C and zebrafish group D, and blood was collected from the fish tail after cryo-anesthesia. The blood was centrifuged at a speed of 2500 rpm and a temperature of 5°C for 20 min to obtain a plasma sample, and then centrifuged at a speed of 2000 rpm and a temperature of 5°C for 15 min to obtain the supernatant of the zebrafish group A, the supernatant of the zebrafish group B, the supernatant of the zebrafish group C and the supernatant of the zebrafish group D, and then the contents of acetylcholinesterase, thyroid hormone T3, thyroid hormone T4 and thyroid stimulating hormone in the supernatant of each group of zebrafish were measured respectively;

[0065] S4. Randomly select one zebrafish from each of the zebrafish group A, zebrafish group B, zebrafish group C, and zebrafish group D, and place its intestine in Bonn fixative. After dehydration, transparency, and wax treatment, use a rotary slicer to make continuous sections, then stain with hematoxylin-eosin, dry and seal with gum, and examine and photograph under an optical microscope. Use CaseViewer software to analyze the micrographs, and count the length of the intestinal villi of each group of zebrafish.

[0066] S5. One adult female zebrafish and two unexposed adult male zebrafish were randomly selected from zebrafish group A, zebrafish group B, zebrafish group C and zebrafish group D, and placed on both sides of the partition of the breeding pool. They were first placed in dark conditions for 12 hours, and then the partition was removed. The female zebrafish were stimulated to lay eggs with light. After 2 hours, the fertilized eggs were collected and placed in E3 medium for incubation for 148 hours. The hatching rate, survival rate and deformity rate of zebrafish in each group were recorded.

[0067] Data analysis

[0068] Table 1 Summary of the breeding data of zebrafish in each group in Example 1

[0069] project Hatching rate / % Survival rate / % Deformity rate / % Zebrafish Group A 83.4 94.2 0.8 Zebrafish Group B 79.9 88.5 1.4 Zebrafish Group C 63.2 75.2 5.6 Zebrafish Group D 46.7 62.8 8.9

[0070] As can be seen from Table 1, the hatching rate, survival rate and deformity rate of zebrafish B treated with 5ng / L gestodene were not significantly changed compared with zebrafish A not treated with gestodene, while the hatching rate, survival rate and deformity rate of zebrafish C and zebrafish D treated with 50ng / L and 500ng / L gestodene were significantly reduced compared with zebrafish A not treated with gestodene. The results show that high concentrations of gestodene exposure medium have an adverse effect on the reproduction of zebrafish;

[0071] Depend on Figure 1 and Figure 2 It can be seen that the zebrafish B and zebrafish C treated with 5ng / L and 50ng / L gestodene have a certain increase in the content of thyroid hormone T3 and thyroid hormone T4 compared with the zebrafish A not treated with gestodene, and the zebrafish D treated with 500ng / L gestodene has a significant increase in the content of thyroid hormone T3 and thyroid hormone T4 compared with the zebrafish A not treated with gestodene, indicating that gestodene exposure medium can stimulate the synthesis and release of thyroid hormones in zebrafish, and may cause symptoms of hyperthyroidism in zebrafish, such as weight loss, anxiety behavior, etc.

[0072] Depend on Figure 3 It can be seen that the zebrafish B and zebrafish C treated with 5ng / L and 50ng / L gestodene have a certain increase in the content of thyroid stimulating hormone compared with the zebrafish A not treated with gestodene, and the zebrafish D treated with 500ng / L gestodene has a significantly increased content of thyroid stimulating hormone compared with the zebrafish A not treated with gestodene, indicating that gestodene exposure medium may cause hyperpituitarism in zebrafish, promote the secretion of thyroid stimulating hormone, and then promote the secretion of thyroid hormone;

[0073] Depend on Figure 4It can be seen that the acetylcholinesterase content of zebrafish B, zebrafish C and zebrafish D treated with 5ng / L, 50ng / L and 500ng / L gestodene increased compared with zebrafish A that was not treated with gestodene. Excessive acetylcholinesterase content will cause the neurotransmitter acetylcholine to metabolize too quickly, leading to hyperfunction of the nervous system. Therefore, exposure to gestodene may cause zebrafish to suffer from hyperthyroidism or mania and other hyperactive diseases;

[0074] Depend on Figure 5 and Figure 6 It can be seen that the behavior speed and movement distance of zebrafish C and D treated with 50ng / L and 500ng / L gestodene were significantly changed compared with zebrafish A not treated with gestodene, while the behavior speed and movement distance of zebrafish B treated with 5ng / L gestodene were not significantly changed compared with zebrafish A not treated with gestodene. This experiment quantified the degree of mania of zebrafish by detecting the speed index in the movement behavior of zebrafish. The results show that high concentrations of gestodene exposure medium can cause damage to the nervous system of zebrafish and produce violent phenomena;

[0075] The length of intestinal villi is related to the body's absorption and digestion functions. The main causes of intestinal villus atrophy are immune diseases and malnutrition. Figure 7 and Figure 8 It can be seen that the zebrafish D treated with 500ng / L gestodene has obvious atrophy of intestinal villi compared with the zebrafish A not treated with gestodene. The results show that high concentration of gestodene exposure medium has certain damage to the immune system of zebrafish, which may be manifested as abnormal hormone secretion or tumors, and the presence of gestodene exposure medium affects the absorption of food by zebrafish, thereby affecting the weight and length of zebrafish.

[0076] Those skilled in the art should understand that the discussion of any of the above embodiments is merely illustrative and is not intended to imply that the scope of the present invention is limited to these examples. Under the concept of the present invention, the technical features in the above embodiments or different embodiments may be combined, the steps may be implemented in any order, and there are many other variations of the different aspects of the present invention as described above, which are not provided in detail for the sake of simplicity.

[0077] The present invention is intended to cover all such substitutions, modifications and variations that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for evaluating gestodene thyroid toxicity by zebrafish, characterized in that: The following steps are involved: Step S1. 40 adult female zebrafish of similar size were selected and placed in a water tank for 14 days, and then divided into four parts, and then transferred to fish tank A, fish tank B, fish tank C and fish tank D respectively, and raised for 60 days, respectively, to obtain zebrafish group A, zebrafish group B, zebrafish group C and zebrafish group D; Step S2. Randomly select a female zebrafish from each of the zebrafish group A, zebrafish group B, zebrafish group C and zebrafish group D and place them in a water tank individually, observe for 10-20 minutes and use a zebrafish tracking system to record its movement behavior, average speed, total movement distance and total bottom residence time of the zebrafish; Step S3. randomly selecting a female zebrafish from each of the zebrafish group A, the zebrafish group B, the zebrafish group C, and the zebrafish group D, taking blood from the fish tail after cryo-anesthesia, obtaining a plasma sample after the first centrifugation, and obtaining a supernatant of the zebrafish group A, the zebrafish group B, the zebrafish group C, and the zebrafish group D by a second centrifugation, and then measuring the contents of acetylcholinesterase, thyroid hormone T3, thyroid hormone T4, and thyroid-stimulating hormone in the supernatant of each group of zebrafish; Step S4. randomly selecting one zebrafish from each of the zebrafish group A, the zebrafish group B, the zebrafish group C and the zebrafish group D, and placing the intestine thereof in Bonn fixative, dehydrating, making transparent and dipping in wax, and then staining, sealing and photographing under a microscope, and then counting the length of the intestinal villi of the zebrafish in each group; Step S5. Randomly select one adult female zebrafish and two unexposed adult male zebrafish from zebrafish group A, zebrafish group B, zebrafish group C and zebrafish group D, respectively, and place them on both sides of the partition of the breeding pool. First place them in dark conditions for 10-12 hours, then remove the partition, and use light to stimulate the female zebrafish to lay eggs. After 1.5-2 hours, collect the fertilized eggs and place them in E3 culture medium for incubation for 144-148 hours, and record the hatching rate, survival rate and deformity rate of various zebrafish.

2. The method for evaluating gestodene thyroid toxicity by zebrafish according to claim 1, characterized in that The adult female zebrafish described in step S1 is three months old and is kept in a water tank under the condition of feeding with brine shrimp larvae twice a day, and removing residual food after 5 minutes each feeding; The concentration of gestodene in the fish tank A is 0 ng / L; The concentration of gestodene in the fish tank B is 5 ng / L; The concentration of gestodene in the fish tank C is 50 ng / L; The concentration of gestodene in the fish tank D is 500 ng / L.

3. The method for evaluating gestodene thyroid toxicity by zebrafish according to claim 1, characterized in that The water temperature in the fish tank in step S1 is set to 25-27°C, pH to 6.9-7.5, dissolved oxygen to 6-15 mg / L, light-dark cycle ratio to 12.5-14:10-11.5, and light intensity to 54-324 lux.

4. The method for evaluating gestodene thyroid toxicity by zebrafish according to claim 1, characterized in that The fish tank in step S1 is reared under the conditions of feeding molted brine shrimp eggs twice a day, changing half of the water every 24 hours and adding gestodene to keep the concentration of gestodene in the water consistent.

5. The method for evaluating gestodene thyroid toxicity by zebrafish according to claim 1, characterized in that The first centrifugation speed in step S3 is set to 2000-2500 rpm, the centrifugation time is 15-20 min, and the centrifugation temperature is 2-5°C.

6. The method for evaluating gestodene thyroid toxicity by zebrafish according to claim 1, characterized in that The second centrifugation speed in step S3 is set to 1500-2000 rpm, the centrifugation time is 10-15 min, and the centrifugation temperature is 2-5°C.

7. The method for evaluating gestodene thyroid toxicity by zebrafish according to claim 1, characterized in that The Bonn fixative in step S4 is obtained by mixing 72-75 mL of saturated picric acid solution with a mass concentration of 1.22%, 22-25 mL of formaldehyde with a mass concentration of 40%, and 3-5 mL of glacial acetic acid.

8. The method for evaluating gestodene thyroid toxicity by zebrafish according to claim 1, characterized in that The illumination intensity in step S5 is 100-240 lux, and the E3 culture medium is obtained by mixing sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, methylene blue and deionized water.

9. The method for evaluating gestodene thyroid toxicity by zebrafish according to claim 1, characterized in that During the incubation in step S5, the temperature is set to 27.5-28.5° C., the pH is set to 6.9-7.5, the dissolved oxygen is set to 6-15 mg / L, and the light-dark cycle ratio is set to 12.5-14:10-11.5.

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