A method for assessing the aquatic ecological toxicity and risk of gatifloxacin based on zebrafish and its application

Abstract

The present invention discloses a method and application for the aquatic ecological toxicity and risk assessment of gatifloxacin based on zebrafish. The steps of the method include: S1 establishing a zebrafish experimental model, selecting zebrafish larvae 3 days after fertilization, according to a standardized breeding procedure; S2, toxicity assessment, calculating the maximum non-lethal concentration (MNLC) and 10% lethal concentration (LC 10 ); S3, assessment of hepatotoxicity indicators; S4, histopathological analysis; S5, detection of the oxidative stress response status; S6, exploring the molecular mechanism by detecting the relative expression levels of PPAR-γ , CYP1A1 and CYP1B1 genes by qPCR; S7, data analysis. The method of the present invention combines morphological, histological, oxidative stress response and molecular mechanism studies, provides a multi-scale evaluation system for the toxicity monitoring of trace drugs in the water environment, and helps to better protect the water ecological environment and public health safety.

Description

Technical Field

[0001] The present invention belongs to the technical field of ecological environment assessment research, and particularly relates to a method and application for assessing the aquatic ecological toxicity and risk of gatifloxacin based on zebrafish. Technical Background

[0002] Gatifloxacin (GTFX) is a fourth-generation fluoroquinolone antibiotic that was withdrawn from the Canadian and US markets in 2006 due to its association with serious adverse reactions (such as hypoglycemia and hyperglycemia). However, GTFX is still used in some developing countries due to its excellent antibacterial properties. Studies have shown that fluoroquinolone drugs are not completely removed during wastewater treatment, leading to the accumulation of these substances in surface waters. In addition, even after photolysis treatment, the inhibitory effect of GTFX on Escherichia coli remains significant, indicating that its degradation products may still affect the microbial community in the water environment. This continuous environmental presence has increased concerns about its ecotoxicology, especially the potential harm to aquatic organisms.

[0003] Due to its genetic homology with humans, rapid development, and easy observation of transparent embryos, zebrafish has become an ideal model for environmental toxicology research. The liver of zebrafish begins to develop at 24 hpf (hours post fertilization) and becomes functional after 72 h, making it an ideal object for studying hepatotoxicity. Therefore, zebrafish has been widely used to evaluate the toxicity of substances such as pesticides, nanoparticles, and pharmaceuticals. The neurotoxicity and reproductive system damage caused by fluoroquinolone antibiotics in zebrafish have also attracted the attention of scientists. However, previous studies have mainly focused on the toxicity of GTFX to mammals and algae, and the information on its impact on aquatic vertebrates is still very limited. Summary of the Invention

[0004] In view of the above problems, the present invention provides a method and application for assessing the aquatic ecological toxicity and risk of gatifloxacin based on zebrafish, aiming to reveal the correlation between the hepatotoxicity induced by GTFX in aquatic animals through the study of zebrafish liver morphology, histopathology changes, oxidative stress response, and molecular mechanisms, provide important information for formulating water environmental quality standards and risk assessments of GTFX, and provide a multi-scale evaluation method for the toxicity monitoring of trace drugs in the water environment, which helps to better protect water ecology and public health safety.

[0005] The present invention is implemented as follows:

[0006] A method for assessing the aquatic ecological toxicity and risk of gatifloxacin based on zebrafish, the steps of the method are as follows:

[0007] S1. Establish a zebrafish experimental model. Use 30 larvae at 3 days post-fertilization (3dpf) in each group of experiments, with the natural paired mating reproduction method.

[0008] S2. Toxicity concentration assessment. Expose zebrafish to gradient concentrations of gatifloxacin (GTFX) from 0.5 to 5.0 mg / mL, and calculate the maximum non-lethal concentration (MNLC) and 10% lethal concentration (LC) through the concentration-mortality curve. 10 ;

[0009] S3. Assessment of hepatotoxicity indicators. Analyze and statistically process the liver area, average liver opacity, and delayed yolk sac absorption area of zebrafish to evaluate the effect of GTFX exposure on the zebrafish liver.

[0010] S4. Conduct histopathological analysis on the stained zebrafish liver sections.

[0011] S5. Oxidative stress detection. Detect the level of reactive oxygen species (ROS) with a fluorescent probe to evaluate whether GTFX induces an oxidative stress response in zebrafish.

[0012] S6. Detect the relative expression levels of PPAR-γ, CYP1A1, and CYP1B1 genes by qPCR.

[0013] S7. Data analysis.

[0014] Furthermore, the water quality parameters for zebrafish breeding in S1 are as follows: Add 200 mg of instant sea salt to every 1 L of reverse osmosis water, with the conductivity being 480 - 510 μS / cm; pH being 6.9 - 7.2; hardness being 53.7 - 1.6 mg / L CaCO3, and maintaining a constant temperature of 28°C.

[0015] Furthermore, the method for S2 to evaluate the effect of GTFX on the zebrafish liver is as follows: Randomly select 210 zebrafish at 3dpf, distribute them into six-well plates, with 30 zebrafish placed in each well. Respectively administer GTFX solutions at concentrations of 0.5, 1.0, 2.0, 3.0, 4.0, and 5.0 mg / mL, with the volume of each well being 3 mL of GTFX solution. At the same time, set up a normal control group. After 48 hours of drug administration treatment, observe and record the death situation of zebrafish in each experimental group, and use Origin 8.0 to plot the concentration-mortality effect curve.

[0016] Furthermore, in step S3, randomly select 150 zebrafish at 3dpf, distribute them into six-well plates, with 30 zebrafish placed in each well. Respectively administer aqueous GTFX solutions at concentrations of 1 / 9 MNLC, 1 / 3 MNLC, MNLC, and LC 10 concentration. At the same time, set up a normal control group, with the volume of each well being 3 mL. After 48 hours of GTFX treatment, randomly select 10 zebrafish from each experimental group, take pictures and collect data under a dissection microscope.

[0017] Further, the method for histopathological analysis of the stained zebrafish liver sections in S4 is as follows: Randomly select 60 3-dpf zebrafish and distribute them into a six-well plate, with 30 zebrafish placed in each well. Administer the GTFX aqueous solution at the LC 10 concentration. Meanwhile, set a normal control group. The volume of each well is 3 mL. After 48 h of GTFX treatment, fix the zebrafish with 4% paraformaldehyde, then transfer the zebrafish to 70% ethanol, and successively perform dehydration, embedding, sectioning, staining, and coverslipping treatments, and conduct histopathological analysis on the stained zebrafish sections.

[0018] Further, S5 is specifically as follows: Randomly select 150 3-dpf zebrafish, distribute them into a six-well plate, with 30 zebrafish treated in each well. Respectively administer the GTFX aqueous solution at the 1 / 9 MNLC, 1 / 3 MNLC, MNLC, and LC 10 concentrations. Meanwhile, set a normal control group. The volume of each well is 3 mL. After 48 h of treatment, add the reactive oxygen species (ROS) detection reagent CM-H2DCFDA. Place the zebrafish in an incubator at 28 °C and continue to incubate for 1 h, then perform fluorescence (F) analysis on each experimental group. Randomly select the fluorescence values of 10 zebrafish in each experimental group for statistical processing, and simultaneously calculate the relative change rate of the ROS level:

[0019]

[0020] In the formula, F Sample represents the relative fluorescence value (A.U.) of the experimental group, F Control represents the relative fluorescence value (A.U.) of the control group, and ROS (%) represents the relative change rate of the reactive oxygen species.

[0021] Further, the zebrafish used in S5 for testing are randomly selected 450 3-dpf zebrafish, distributed into a six-well plate, with 30 zebrafish in each well. Respectively administer the GTFX aqueous solution at the 1 / 9 MNLC, 1 / 3 MNLC, MNLC, and LC 10 concentrations. Meanwhile, set a normal control group. The volume of each well is 3 mL. After incubating at 28 °C for 48 h, extract 2 μg of total RNA from the zebrafish, synthesize 20 μL of cDNA, and store it at -20 °C for standby.

[0022] Another aspect of the present invention is to use the gatifloxacin aquatic ecotoxicity and risk assessment method based on zebrafish described in one of the above to judge and evaluate the impact of drug residues on the aquatic ecosystem.

[0023] The beneficial effects of the present invention are as follows: The method of the present invention uses zebrafish as the experimental object, comprehensively combines the studies of morphology, histology, oxidative stress response and molecular mechanisms, evaluates the ecological toxicity effects of gatifloxacin in the aquatic environment, provides a multi-scale evaluation method system for the toxicity monitoring of trace drugs in the water environment, provides a practical reference for the future water pollution treatment and the formulation of ecological protection strategies, and helps to better protect the water ecological environment and public health safety. At the same time, it lays a foundation for the future exploration of the long-term effects of GTFX at lower, environmentally relevant concentrations, and can provide a reference for the more extensive ecological risks related to the exposure of common drugs in the water environment.

[0024] The following further elaborates on the present invention in conjunction with the accompanying drawing descriptions and specific embodiments. Description of the Drawings

[0025] Figure 1 The "concentration-mortality" effect curve of GTFX;

[0026] Figure 2 Phenotype map of GTFX-induced liver toxicity in zebrafish (L: Liver, Y: Yolk sac);

[0027] Figure 3 The effects of GTFX on the delayed absorption area of the yolk sac, liver area and liver opacity in zebrafish;

[0028] Figure 4 Histopathological examination of the liver of zebrafish after GTFX exposure;

[0029] Figure 5 The relative fluorescence values and relative change rates of ROS levels in the GTFX exposure group and the control group;

[0030] Figure 6 The relative expression levels of PPAR-γ, CYP1A1 and CYP1B1 mRNAs. Specific Embodiments

[0031] The meanings represented by some abbreviations involved in the present invention:

[0032] GTFX: Gatifloxacin, which is a fourth-generation fluoroquinolone antibiotic;

[0033] 3dpf: indicating 3 days after fertilization;

[0034] ROS: Reactive Oxygen Species.

[0035] 1. Instruments and materials used in the present invention:

[0036] Gatifloxacin (GTFX, CAS No: 121577-32-0), absolute ethanol, 4% paraformaldehyde, hematoxylin, eosin, and xylene were purchased from Aladdin Holding Group (Shanghai, China).

[0037] PCR instrument (T100, BIO-RAD, USA), fluorescence quantitative PCR instrument (CFX Connect, BIO-RAD, USA);

[0038] High-speed refrigerated centrifuge (PIC017 / 21, Thermo, USA);

[0039] 6-well plate (Nest Biotech, China);

[0040] Ultraviolet-visible spectrophotometer (Nanodrop2000, Thermo, USA);

[0041] Microplate mini centrifuge (BE-6100, Qilinbeier Instrument Manufacturing Co., Ltd., Haimen, China);

[0042] Low-profile skirt 96-well plate (transparent) (HSP9601, Bio-rad, America);

[0043] Optical adhesive sealing film B (MSB1001, Bio-rad, America);

[0044] iTaq Universal SYBR Green Supermix (product number 1725124, Bio-rad, America);

[0045] FastQuant RT Kit (With gDNase) kit (product number KR106, TIANGEN, China);

[0046] RNA-Quick Purification Kit (RNA rapid extraction kit) (product number RN001, Yishan Biotech, China).

[0047] 2. The steps of a method for evaluating the aquatic ecological toxicity and risk of gatifloxacin based on zebrafish in the present invention are as follows:

[0048] S1. Establishment of zebrafish experimental model

[0049] Experimental materials: Zebrafish larvae at 3 days after fertilization were selected.

[0050] Cultivation conditions: According to the standardized cultivation procedure, keep the water quality parameters within a specific range, including conductivity of 480 - 510 μS / cm, pH value of 6.9 - 7.2, hardness of 53.7 - 71.6 mg / L CaCO3, and maintain a constant temperature of 28°C.

[0051] Wild-type AB strain zebrafish were raised in fish culture water at 28.0 ± 0.5°C, and the water quality parameters were as follows: 200 mg of instant sea salt was added to every 1 L of reverse osmosis water, with conductivity of 480 - 510 μS / cm; pH of 6.9 - 7.2; hardness of 53.7 - 71.6 mg / L CaCO3. The experimental animal use license number was: SYXK(Zhe)2012 - 0171. The feeding management complied with the requirements of international AAALAC certification.

[0052] Zebrafish were reproduced by natural paired mating. 30 larvae at 3 dpf (3 days after fertilization) were used in each experimental group to conduct the hepatotoxicity study. After the experiment, all stages of zebrafish were treated with 0.3 mg / mL tricaine methanesulfonate for over-anesthesia. The anesthesia procedure was strictly carried out in accordance with the animal anesthesia specifications of the American Veterinary Medical Association (AVMA).

[0053] S2, Toxicity concentration assessment

[0054] Concentration gradient exposure: Zebrafish were exposed to GTFX at a gradient concentration (0.5 to 5.0 mg / mL).

[0055] Toxicity threshold calculation: Calculate the MNLC (maximum non-lethal concentration) and LC 10 (10% lethal concentration) through the "concentration - mortality" curve to provide a reference value for the risk assessment of water environment pollutants.

[0056] GTFX was stored at 4°C. Before use, it was prepared into a stock solution with a concentration of 5 mg / mL using fish culture water and reserved. 210 zebrafish at 3 dpf were randomly selected and allocated into six-well plates, with 30 zebrafish placed in each well (experimental group). GTFX at concentrations of 0.5, 1.0, 2.0, 3.0, 4.0, and 5.0 mg / mL was given by water solution respectively. At the same time, a normal control group (zebrafish treated with fish culture water) was set up. The volume of each well was 3 mL. After 48 h of drug administration, the death situation of zebrafish in each experimental group was observed and recorded, and the concentration - mortality effect curve was drawn using Origin 8.0 to calculate the maximum non-lethal concentration MNLC and 10% lethal concentration LC of GTFX to zebrafish 10 .

[0057] S3, Assessment of hepatotoxicity indicators: Liver transparency, area, and yolk sac absorption: Quantitatively evaluate the hepatotoxicity effect of gatifloxacin at different concentrations.

[0058] Randomly select 150 3-dpf zebrafish and distribute them into six-well plates, with 30 zebrafish placed in each well. Treat them with GTFX aqueous solutions at concentrations of 1 / 9MNLC, 1 / 3MNLC, MNLC, and LC respectively, and set up a normal control group at the same time. The volume of each well is 3 mL. After 48 h of GTFX treatment, randomly select 10 zebrafish from each experimental group, take pictures and collect data under a dissection microscope (Nikon, SMZ18, Tokyo, Japan), and analyze and statistically process the liver area, average opacity of the liver, and delayed yolk sac absorption area of the zebrafish to evaluate the effect of gatifloxacin on the zebrafish liver. 10 After 48 h of GTFX treatment, randomly select 10 zebrafish from each experimental group, take pictures and collect data under a dissection microscope (Nikon, SMZ18, Tokyo, Japan), and analyze and statistically process the liver area, average opacity of the liver, and delayed yolk sac absorption area of the zebrafish to evaluate the effect of gatifloxacin on the zebrafish liver.

[0059] S4. Histopathological analysis of stained zebrafish sections: HE staining technique: Microscopically observe the liver sections of zebrafish after GTFX exposure and record structural damages such as hepatocyte swelling and vacuolization.

[0060] Randomly select 60 3-dpf zebrafish and distribute them into six-well plates, with 30 zebrafish placed in each well. Treat them with GTFX aqueous solution at LC concentration, and set up a normal control group at the same time. The volume of each well is 3 mL. After 48 h of GTFX treatment, fix the zebrafish with 4% paraformaldehyde, then transfer the zebrafish to 70% ethanol, and successively perform dehydration, embedding, sectioning, staining, and mounting. Perform histopathological analysis on the stained zebrafish liver sections. 10 After 48 h of GTFX treatment, fix the zebrafish with 4% paraformaldehyde, then transfer the zebrafish to 70% ethanol, and successively perform dehydration, embedding, sectioning, staining, and mounting. Perform histopathological analysis on the stained zebrafish liver sections.

[0061] S5. Detection of oxidative stress status

[0062] Measurement of relative fluorescence value of ROS: Measure the relative fluorescence value of reactive oxygen species (ROS) in zebrafish cells to determine whether GTFX induces toxicity by triggering oxidative stress.

[0063] Randomly select 150 3-dpf zebrafish and distribute them into six-well plates, with 30 zebrafish treated in each well. Treat them with GTFX aqueous solutions at concentrations of 1 / 9MNLC, 1 / 3MNLC, MNLC, and LC respectively, and set up a normal control group at the same time. The volume of each well is 3 mL. After 48 h of treatment, add the ROS detection reagent CM-H2DCFDA. Place the zebrafish in an incubator at 28 °C and continue to incubate for 1 h. Then, use a multifunctional microplate reader (Mithras LB940, Berthold Technologies, Stuggart, Germany) to perform fluorescence (F) analysis on each experimental group. Statistically process the fluorescence values of 10 randomly selected zebrafish from each experimental group to evaluate whether GTFX induces oxidative stress response in zebrafish. At the same time, calculate the relative change rate of ROS. 10 After 48 h of treatment, add the ROS detection reagent CM-H2DCFDA. Place the zebrafish in an incubator at 28 °C and continue to incubate for 1 h. Then, use a multifunctional microplate reader (Mithras LB940, Berthold Technologies, Stuggart, Germany) to perform fluorescence (F) analysis on each experimental group. Statistically process the fluorescence values of 10 randomly selected zebrafish from each experimental group to evaluate whether GTFX induces oxidative stress response in zebrafish. At the same time, calculate the relative change rate of ROS.

[0064]

[0065] In the formula, F Sample represents the relative fluorescence value (A.U) of the experimental group, and F Control represents the relative fluorescence value (A.U) of the control group, and ROS(%) represents the relative change rate of reactive oxygen species.

[0066] S6, Exploration of molecular mechanism

[0067] qPCR technology: Use qPCR to detect the expression levels of key metabolic genes (PPAR-γ, CYP1A1, CYP1B1), and analyze the effects of GTFX on lipid metabolism and hepatocyte metabolic load.

[0068] Randomly select 450 zebrafish at 3 dpf and distribute them into six-well plates, with 30 zebrafish in each well. Treat them with GTFX aqueous solutions at concentrations of 1 / 9 MNLC, 1 / 3 MNLC, MNLC, and LC 10 respectively. At the same time, set a normal control group. The volume of each well is 3 mL. After incubating at 28 °C for 48 h, use a rapid RNA extraction kit to extract the total RNA of zebrafish in each experimental group, and use a UV-visible spectrophotometer (Nanodrop 2000, Thermo Fisher Scientific, USA) to measure the concentration and purity of the total RNA. Take 2 μg of zebrafish total RNA, operate according to the instructions of the cDNA first-strand synthesis kit to synthesize 20 μL of cDNA, and store it at -20 °C.

[0069] Detect the relative expression levels of PPAR-γ, CYP1A1, and CYP1B1 genes by qPCR. Using β-actin as an internal reference gene, use the 2^-ΔΔCt method to calculate the relative expression levels of PPAR-γ, CYP1A1, and CYP1B genes. The primer sequences are shown in Table 1.

[0070] Table 1 Primer sequence table

[0071]

[0072] S7, Data analysis:

[0073] All data are expressed as mean ± standard error (SE), and one-way analysis of variance (ANOVA) and Dunnett test are used to compare the differences between groups. A p < 0.05 is considered a significant difference. The data are completed using GraphPad Prism 6.00 (GraphPad Software, San Diego, CA, USA).

[0074] 3. The analysis conclusions are as follows:

[0075] (1) MNLC and LC 10

[0076] Figure 1 This is the "concentration - mortality" effect curve of GTFX, which reflects the lethality of zebrafish larvae (3 dpf) treated with different concentrations of GTFX for 2 - 5 days. As the concentration of GTFX increases, the mortality rate of zebrafish gradually increases. At a concentration of 2.0 mg / mL, GTFX did not show any lethal effect; however, when the concentration increased to 3.0 mg / mL, the mortality rate increased significantly to 46.7%, and at 5.0 mg / mL, the mortality rate reached 100%. According to the curve fitting results, the maximum non - lethal concentration (MNLC) and 10% lethal concentration (LC 10 ) of GTFX were 1.7 mg / mL and 2.3 mg / mL, respectively. The subsequent experimental concentrations were set at 0.2 mg / mL (1 / 9 MNLC), 0.6 mg / mL (1 / 3 MNLC), 1.7 mg / mL (MNLC), and 2.3 mg / mL (LC 10 ).

[0077] (2) Phenotypic observation

[0078] Figure 2 This shows the morphological changes of the liver in zebrafish larvae (3 dpf) after being treated with different concentrations of GTFX for 48 h. The L part in the figure is the liver area, and the Y part is the yolk sac area. After 2 days of GTFX exposure, the liver of zebrafish in the control group was transparent, with a complete and normal morphology without deformities, as shown in Figure 2 (a); in the GTFX - treated group, as the concentration increased, the liver area gradually decreased. At 0.2 mg / mL, the liver area decreased slightly, as shown in Figure 2 (b); as the concentration continued to increase, the liver area continued to decrease, the opacity of the liver also gradually increased, and the edges became blurred, as shown in Figure 2 (c), (d), and (e).

[0079] Figure 3 This shows the quantitative evaluation of the delayed yolk sac absorption (a), liver area (b), and liver turbidity (c) in zebrafish larvae (3 dpf) after being treated with different concentrations of GTFX for 48 h. Ten zebrafish were randomly selected from each experimental concentration group, photographed under a dissection microscope, and data were collected. Statistical analysis was performed using analysis of variance and Dunnett's T - test. Compared with the normal control group, ***p < 0.001 indicates a significant difference.

[0080] Zebrafish treated with 0.6 mg / mL GTFX showed delayed yolk sac absorption, with a significantly increased yolk sac area (p < 0.05). In the MNLC (1.7 mg / mL) exposure group, the delay in yolk sac absorption was particularly significant (p < 0.001), as shown in Figure 3 (a). At the same time, the liver area was significantly smaller than that of the control group (p < 0.001), as shown in Figure 3 b, indicating aggravated liver injury. The liver area was the smallest at LC 10 (2.3 mg / mL), and the liver margin was blurred, as shown in Figure 2 . In addition, compared with the control group, the liver area, liver light transmittance, and yolk sac area in the LC 10 group were all significantly different (p < 0.001).

[0081] Overall, zebrafish exposed to GTFX showed dose-dependent reduction in liver area, increased liver opacity, and enlarged yolk sac area. These results suggest that the morphological changes in the liver caused by GTFX may lead to abnormal liver structure, seriously threatening the survival of zebrafish and having a severe impact on the aquatic ecosystem. At the end of the experiment, GTFX exposure at the LC 10 concentration (2.3 mg / mL) directly caused the death of 3.3% of the zebrafish.

[0082] (3) Histopathological analysis

[0083] Figure 4 shows the histopathological examination of the liver of zebrafish larvae (3 dpf) after 48 h of treatment with 2.3 mg / mL GTFX. Magnification: 400×, GTFX concentration: 2.3 mg / mL. The liver tissue of the control group of zebrafish showed intact cell morphology and uniform cytoplasmic characteristics. In contrast, obvious swelling of some hepatocytes (indicated by the blue arrow in the figure) and liver vacuolization (indicated by the yellow arrow in the figure) were found in the LC 10 exposure group, indicating that GTFX exposure led to damage to the liver structure. It shows that morphological changes may lead to abnormal liver structure and threaten the survival of zebrafish.

[0084] (4) Oxidative stress assessment

[0085] Figure 5 a shows the relative fluorescence values of the GTFX treatment group and the control group. The data are expressed as mean ± standard error (SE). Figure 5 b shows the relative changes in ROS between the GTFX treatment group and the control group. The intracellular ROS level was evaluated using CM-H2DCFDA. The results showed that the relative fluorescence values of zebrafish treated with 0.2, 0.6, 1.7, and 2.3 mg / mL GTFX were 28449, 16085, 20962, and 17617 pixels, respectively, as shown inFigure 5 As shown in a. Compared with the relative fluorescence value (23,826 pixels) of the control group, the relative change rates of ROS in the GTFX exposure groups were 19.4%, -32.5%, -12.0%, and -26.1% respectively, as shown in Figure 5 b. Therefore, there was no significant difference between the GTFX exposure group and the control group (p > 0.05), indicating that under the experimental concentration conditions, GTFX did not induce oxidative stress response in zebrafish.

[0086] (5) Effects of GTFX on the expression levels of PPAR-γ and P450-related genes

[0087] As Figure 6 shown, the relative expression level graphs of PPAR-γ, CYP1A1, and CYP1B1 mRNAs. In the figure, * compared with the control group, p < 0.05. According to the calculation of the relative gene expression, the relative expression of the PPAR-γ gene in the normal control group was 1.00. When the GTFX concentrations were 0.2, 0.6, 1.7, and 2.3 mg / mL, the relative expressions of the PPAR-γ gene were 1.39, 1.41, 1.50, and 1.97 respectively. Compared with the normal control group (1.00), the p values were p > 0.05, p > 0.05, p > 0.05, and p < 0.05 respectively. Among them, there was a significant difference between the 2.3 mg / mL experimental group and the control group (p < 0.05), indicating that GTFX up-regulated the expression of the PPAR-γ gene, as shown in Figure 6 (a).

[0088] Similarly, the relative expression of the CYP1A1 gene in the normal control group was 1.01. When the GTFX concentrations were 0.2, 0.6, 1.7, and 2.3 mg / mL, the relative expressions of the CYP1A1 gene were 1.34, 1.82, 2.41, and 2.49 respectively. Compared with the normal control group (1.01), the p values were p > 0.05, p > 0.05, p > 0.05, and p < 0.05 respectively. Among them, there was a significant difference between the 2.3 mg / mL experimental group and the control group (p < 0.05), indicating that GTFX up-regulated the expression of the CYP1A1 gene, as shown in Figure 6 (b).

[0089] For the CYP1B1 gene, the relative expression level in the normal control group was 1.10. When the GTFX concentrations were 0.2, 0.6, 1.7, and 2.3 mg / mL, the relative expression levels of the CYP1B1 gene were 1.82, 1.96, 2.81, and 2.19, respectively. Compared with the normal control group (1.10), the p-values were p > 0.05, p > 0.05, p < 0.05, and p > 0.05, respectively. At the concentration of 1.7 mg / mL, there was a significant difference in the expression of the CYP1B1 gene compared with the control group (p < 0.05), indicating that GTFX significantly up-regulated the expression of the CYP1B1 gene, as shown in Figure 6 (c).

[0090] The above results indicate that within the experimental concentration range, GTFX induced zebrafish hepatotoxicity by promoting the expression of liver toxicity-related genes PPAR-γ, CYP1A1, and CYP1B1.

[0091] 4. Analysis and Discussion

[0092] (1) Effects on the morphology of zebrafish liver

[0093] Liver size, opacity, and the absorption of the yolk sac are common indicators for evaluating zebrafish hepatotoxicity. GTFX can induce delayed absorption of the yolk sac, reduced liver area, and liver degeneration in zebrafish, resulting in significant changes in liver morphology, as shown in Figure 2 . These changes were more obvious when treated with high concentrations of GTFX (0.6, 1.7, and 2.3 mg / mL), as shown in Figure 3 .

[0094] In the evaluation of zebrafish hepatotoxicity, a reduction in liver area usually indicates hepatocyte atrophy or apoptosis, which is a response of the liver to exogenous stress. Changes in liver opacity reflect potential alterations in liver components, such as increased fat deposition or the development of fibrosis, indicating the interruption of normal liver function and potential cell damage. The occurrence of yolk sac edema indicates metabolic disorders, impaired ability of the liver to regulate metabolic processes, and damaged function.

[0095] For the LC 10 group, histopathological analysis of the liver found hepatocyte swelling and vacuolization, as shown in Figure 4 . This provides deeper insights into the cellular changes that occur after exposure, highlighting the structural changes within the liver tissue. It is worth noting that the presence of hepatocyte swelling and vacuolization is usually associated with intracellular stress.

[0096] (2) Effects on the oxidative stress status

[0097] Given the significant changes in liver morphology, to understand the underlying cellular mechanisms, the oxidative stress levels in zebrafish exposed to GTFX were analyzed, and potential oxidative damage was evaluated by measuring ROS levels. Contrary to expectations, the ROS levels in GTFX-treated zebrafish did not change significantly compared to the control group, as Figure 5 shown, indicating that GTFX induces hepatotoxicity through a pathway independent of oxidative stress. Since stable ROS levels were observed while significant changes in liver morphology occurred, it is suggested that the expression of PPAR-γ, CYP1A1, and CYP1B1 may disrupt cellular homeostasis through a pathway different from traditional oxidative stress, providing new insights into the environmental toxicity of fluoroquinolones.

[0098] (3) Molecular mechanism

[0099] In zebrafish exposed to GTFX, the ROS levels did not increase, indicating that non-oxidative pathways may be the cause of hepatotoxicity. The above studies showed that the expressions of genes related to lipid metabolism and stress response, PPAR-γ, CYP1A1, and CYP1B1, were significantly upregulated. Peroxisome proliferator-activated receptor γ (PPAR-γ) is a member of the nuclear receptor superfamily and mainly regulates lipid metabolism, glucose homeostasis, and inflammatory responses. Although its expression in liver tissue is lower than that in adipose tissue, PPAR-γ plays an important regulatory role in hepatic lipogenesis. It has been reported that an increase in PPAR-γ expression is an important indicator of fatty liver disease. CYP1A1 and CYP1B1 are important members of the cytochrome P450 enzyme family and are responsible for metabolizing a variety of endogenous and exogenous compounds, including environmental pollutants, drugs, and carcinogens.

[0100] Normally, activating PPAR-γ helps maintain energy balance and regulate intracellular lipid levels. When the expression of PPAR-γ is enhanced, it may indicate that the processes of lipid processing and storage are disrupted. The increase in PPAR-γ under GTFX exposure reflects a compensatory mechanism that attempts to correct the drug-induced lipid metabolism imbalance. Similarly, the increased expressions of CYP1A1 and CYP1B1 highlight the enhanced metabolic activity during GTFX exposure. The enzymes encoded by these genes are crucial for metabolizing exogenous substances and can convert lipophilic compounds into more hydrophilic products that are more easily excreted. Although these enzymes are traditionally associated with the production of ROS, in the above studies, the increased expressions of CYP1A1 and CYP1B1 were not accompanied by an increase in ROS levels, indicating that their main role may be more focused on metabolic adaptation rather than causing oxidative stress.

[0101] In summary, through the combined methods of morphological observation, histopathological analysis, and molecular biology, the method of the present invention found that GTFX significantly reduced the liver area of zebrafish and delayed the absorption of the yolk sac. Meanwhile, GTFX interfered with lipid metabolism by upregulating the gene expressions of PPAR-γ, CYP1A1, and CYP1B1, but did not significantly induce oxidative stress. This indicates that its toxicity mechanism mainly depends on the non-oxidative stress pathway.

[0102] The present invention provides a method for aquatic ecotoxicity and risk assessment based on a zebrafish model, aiming to evaluate the ecotoxicity impact of Gatifloxacin (GTFX) in the aquatic environment. The method of the present invention integrates morphological, histological, oxidative stress response, and molecular mechanism studies, providing a multi-scale evaluation system for the toxicity monitoring of trace drugs in the water environment and a practical reference for the formulation of future water pollution control and ecological protection strategies.

[0103] In addition, the method for aquatic ecotoxicity and risk assessment of gatifloxacin based on zebrafish described in the present invention can be used to judge and evaluate the impact of drug residues such as gatifloxacin on the aquatic ecosystem. This work lays a foundation for exploring the long-term effects of GTFX at lower concentrations in the future and can provide reference for more extensive aquatic ecological risks related to the exposure of common drugs in the water environment.

[0104] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention (such as the adjustment of the sequence of experimental steps, etc.) can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for assessing the aquatic ecological toxicity and risk of gatifloxacin based on zebrafish, characterized in that, The method steps are as follows: S1. Establish a zebrafish experimental model. Use 30 larvae 3 days after fertilization in each group of experiments, with the natural paired mating and reproduction method. S2, Toxicity concentration assessment: zebrafish were exposed to gradient concentrations of gatifloxacin (GTFX) ranging from 0.5 to 5.0 mg / mL, and the maximum non-lethal concentration (MNLC) and 10% lethal concentration (LC) were calculated through the concentration-mortality curve. 10 ; S3. Evaluate liver toxicity indicators. Analyze and statistically process the liver area, average liver opacity, and delayed yolk sac absorption area of zebrafish to evaluate the impact of GTFX exposure on the zebrafish liver. S4. Conduct histopathological analysis on the stained zebrafish liver sections. S5. Detect oxidative stress. Use a fluorescent probe to detect the level of reactive oxygen species (ROS) to evaluate whether GTFX induces an oxidative stress response in zebrafish. S6. Detect the relative expression levels of PPAR-γ, CYP1A1, and CYP1B1 genes by qPCR. S7. Conduct data analysis.

2. The method for evaluating the aquatic ecological toxicity and risk of gatifloxacin based on zebrafish according to claim 1, characterized in that, The water quality parameters for zebrafish breeding in S1 are as follows: Add 200 mg of instant sea salt to every 1 L of reverse osmosis water, with the conductivity being 480 - 510 μS / cm; the pH is 6.9 - 7.2; the hardness is 53.7 - 1.6 mg / L CaCO3, and keep the temperature at 28°C constantly.

3. The method for assessing the aquatic ecological toxicity and risk of gatifloxacin based on zebrafish according to claim 1, characterized in that, The method for evaluating the impact of GTFX on the zebrafish liver in S2 is as follows: Randomly select 210 zebrafish at 3 dpf and distribute them into six-well plates, with 30 zebrafish placed in each well. Administer GTFX solutions at concentrations of 0.5, 1.0, 2.0, 3.0, 4.0, and 5.0 mg / mL respectively, with the volume of each well being 3 mL of GTFX solution. At the same time, set up a normal control group. After 48 hours of drug treatment, observe and record the death situation of zebrafish in each experimental group, and use Origin 8.0 to draw a concentration-mortality effect curve.

4. The method for assessing the aquatic ecological toxicity and risk of gatifloxacin based on zebrafish according to claim 1, wherein Step S3 is to randomly select 150 zebrafish at 3 dpf, distribute them into a six-well plate, with 30 zebrafish placed in each well, and respectively administer GTFX aqueous solutions with concentrations of 1 / 9 MNLC, 1 / 3 MNLC, MNLC, and LC. 10 Meanwhile, a normal control group is set up, with a volume of 3 mL in each well. After 48 h of GTFX treatment, 10 zebrafish are randomly selected from each experimental group, photographed under a dissecting microscope, and data is collected.

5. The method for assessing the aquatic ecological toxicity and risk of gatifloxacin based on zebrafish according to claim 1, wherein The histopathological analysis method of the stained zebrafish liver sections in S4 is as follows: randomly select 60 zebrafish at 3 dpf and distribute them into six-well plates, with 30 zebrafish placed in each well. Treat them with an aqueous solution of GTFX at the LC 10 concentration, and set up a normal control group at the same time. The volume of each well is 3 mL. After 48 h of GTFX treatment, fix the zebrafish with 4% paraformaldehyde, then transfer the zebrafish to 70% ethanol, and successively perform dehydration, embedding, sectioning, staining and coverslipping treatments, and conduct histopathological analysis on the stained zebrafish sections.

6. The method for evaluating the aquatic ecological toxicity and risk of gatifloxacin based on zebrafish according to claim 1, wherein, Specifically, S5 is as follows: Randomly select 150 3-dpf zebrafish and distribute them into six-well plates, with 30 zebrafish treated in each well. Then, administer GTFX aqueous solutions with concentrations of 1 / 9 MNLC, 1 / 3 MNLC, MNLC, and LC respectively. At the same time, set up a normal control group. The volume of each well is 3 mL. After 48 hours of treatment, add the reactive oxygen species (ROS) detection reagent CM-H2DCFDA. Place the zebrafish in an incubator at 28 °C and continue to incubate for 1 hour. Then, perform fluorescence F analysis on each experimental group. Randomly select the fluorescence values of 10 zebrafish in each experimental group for statistical processing, and calculate the relative change rate of the ROS level simultaneously: 10 ROS level relative change rate = (experimental group fluorescence value - normal control group fluorescence value) / normal control group fluorescence value × 100%. where F Sample represents the relative fluorescence value of the experimental group, F Control represents the relative fluorescence value of the control group, and ROS(%) represents the relative change rate of reactive oxygen species.

7. The method for assessing the aquatic ecological toxicity and risk of gatifloxacin based on zebrafish according to claim 1, wherein The zebrafish used for the S6 test were randomly selected, with 450 3-dpf zebrafish allocated to six-well plates, 30 in each well, and given GTFX aqueous solutions at concentrations of 1 / 9 MNLC, 1 / 3 MNLC, MNLC, and LC respectively. At the same time, a normal control group was set up. The volume of each well was 3 mL. After incubation at 28 °C for 48 h, 2 μg of total zebrafish RNA was extracted, 20 μL of cDNA was synthesized, and it was stored at -20 °C for standby. 10 Concentrations of the GTFX aqueous solution, while setting up a normal control group, with a volume of 3 mL per well. After incubation at 28 °C for 48 h, 2 μg of total zebrafish RNA was extracted, 20 μL of cDNA was synthesized, and it was stored at -20 °C for standby.

8. Use the gatifloxacin aquatic ecotoxicity and risk assessment method based on zebrafish described in any one of claims 1 - 7 to judge and evaluate the impact of drug residues on the aquatic ecosystem.

Images