Zebra fish-based gatifloxacin water ecotoxicity and risk assessment method and application

Abstract

The invention discloses a gatifloxacin water ecotoxicity and risk assessment method based on zebra fish and application, the method comprises the following steps: S1, establishing a zebra fish experimental model, selecting zebra fish fry 3 days after fertilization, and culturing according to a standardized culture program; s2, toxicity evaluation: calculating the maximum non-lethal concentration (MNLC) and 10% lethal concentration (LC10) of the GTFX; s3, evaluating hepatotoxicity indexes; s4, carrying out histopathologic analysis; s5, oxidative stress reaction state detection; s6, detecting the relative expression quantity of the PPAR-gamma, CYP1A1 and CYP1B1 genes through qPCR (quantitative polymerase chain reaction), and exploring a molecular mechanism; and S7, data analysis. According to the method, morphological, histological, oxidative stress reaction and molecular mechanism research are integrated, a multi-scale evaluation system is provided for toxicity monitoring of trace drugs in the water environment, and the water ecological environment and public health safety can be better protected.

Description

Technical Field

[0001] The invention belongs to the technical field of ecological environment assessment research, and in particular relates to a zebrafish-based gatifloxacin water ecological toxicity and risk assessment method and application. 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[0002] Gatifloxacin (GTFX) is a fourth-generation fluoroquinolone antibiotic that was withdrawn from the Canadian and US markets in 2006 due to its association with serious adverse reactions (such as hypoglycemia and hyperglycemia).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 fluoroquinolones are not completely removed during wastewater treatment, resulting in the accumulation of these substances in surface waters. In addition, even after photolysis treatment, GTFX still has a significant inhibitory effect on Escherichia coli, indicating that its degradation products may still have an impact on microbial communities in the aquatic environment. This continued environmental presence has increased concerns about its ecotoxicology, especially its potential harm to aquatic organisms.

[0003] Zebrafish are an ideal model for environmental toxicology studies because of their genetic homology with humans, rapid development, and transparent embryos that are easy to observe. The zebrafish liver begins to develop at 24 hpf (hours post fertilization) and becomes functional after 72 hours, making it an ideal subject for studying hepatotoxicity. Therefore, zebrafish have 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 information on its effects on aquatic vertebrates is still very limited. Summary of the invention

[0004] In response to the above problems, the present invention provides a zebrafish-based gatifloxacin aquatic ecotoxicity and risk assessment method and application, aiming to reveal the correlation between GTFX-induced hepatotoxicity in aquatic animals through studies on zebrafish liver morphology, histopathological changes, oxidative stress response and molecular mechanisms, provide important information for the formulation of water environment quality standards and risk assessment of GTFX, provide a multi-scale evaluation method for toxicity monitoring of trace drugs in aquatic environments, and help to better protect aquatic ecology and public health safety.

[0005] The present invention is achieved in that:

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

[0007] S1, zebrafish experimental model was established, 30 larvae 3 days post fertilization (3 dpf) were used in each experiment, and the breeding method was natural pair mating;

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

[0009] S3, evaluation of hepatotoxicity indicators, analysis and statistics of zebrafish liver area, liver average opacity and yolk sac absorption delay area were performed to evaluate the effects of GTFX exposure on zebrafish liver;

[0010] S4, histopathological analysis of stained zebrafish liver sections;

[0011] S5, oxidative stress detection, fluorescent probe was used to detect the level of reactive oxygen species (ROS) to evaluate whether GTFX induced oxidative stress in zebrafish;

[0012] S6, relative expression levels of PPAR-γ, CYP1A1, and CYP1B1 genes were detected by qPCR;

[0013] S7, Data analysis.

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

[0015] Furthermore, the S2 method for evaluating the effect of GTFX on zebrafish liver is as follows: 210 3dpf zebrafish were randomly selected and distributed into a six-well plate, 30 zebrafish were placed in each well, and 0.5, 1.0, 2.0, 3.0, 4.0 and 5.0 mg / mL of GTFX solution were given respectively, with a capacity of 3 mL of GTFX solution in each well. A normal control group was set up at the same time, and the death of zebrafish in each experimental group was observed and recorded 48 hours after the drug treatment, and the concentration-mortality effect curve was drawn using Origin 8.0.

[0016] Furthermore, step S3 randomly selected 150 3dpf zebrafish, distributed them into a six-well plate, placed 30 zebrafish in each well, and administered 1 / 9MNLC, 1 / 3MNLC, MNLC, and LC, respectively. 10 Concentration of GTFX aqueous solution, and a normal control group was set up at the same time. The volume of each well was 3 mL. After 48 hours of GTFX treatment, 10 zebrafish were randomly selected from each experimental group, photographed under a dissecting microscope, and data were collected.

[0017] Furthermore, the S4 method for histopathological analysis of zebrafish liver sections after staining is as follows: 60 3dpf zebrafish are randomly selected and distributed into a six-well plate, 30 zebrafish are placed in each well, and LC 10 The zebrafish were treated with a GTFX aqueous solution of 1% concentration, and a normal control group was set up with a capacity of 3 mL in each well. After 48 hours of GTFX treatment, the zebrafish were fixed with 4% paraformaldehyde and then transferred to 70% ethanol. The zebrafish were dehydrated, embedded, sliced, stained and sealed in sequence, and the stained zebrafish sections were subjected to histopathological analysis.

[0018] Furthermore, the S5 is specifically as follows: 150 3dpf zebrafish were randomly selected and distributed into six-well plates, 30 zebrafish were treated in each well, and 1 / 9MNLC, 1 / 3MNLC, MNLC and LC were given respectively. 10 The concentration of GTFX aqueous solution was 100 μg / mL, and a normal control group was set up at the same time. The volume of each well was 3 mL. After 48 hours of treatment, the reactive oxygen species ROS detection reagent CM-H2DCFDA was added, and the zebrafish were placed in an incubator at 28°C for another 1 hour. After that, fluorescence (F) analysis was performed on each experimental group. The fluorescence values ​​of 10 zebrafish were randomly selected from each experimental group for statistical processing, and the relative change rate of ROS level was calculated at the same time:

[0019]

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

[0021] Furthermore, the zebrafish used in the S5 test were 450 randomly selected 3dpf zebrafish, distributed into a six-well plate, 30 per well, and given 1 / 9MNLC, 1 / 3MNLC, MNLC, and LC, respectively. 10 A GTFX aqueous solution of 1.50 μg was added to the wells, and 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 zebrafish total RNA was extracted, 20 μL of cDNA was synthesized, and the cDNA was stored at -20°C for later use.

[0022] Another aspect of the present invention is to use the above-mentioned zebrafish-based gatifloxacin aquatic ecotoxicity and risk assessment method to determine and evaluate the impact of drug residues on aquatic ecology.

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

[0024] The present invention is further explained below in conjunction with the accompanying drawings and specific implementation methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 GTFX's "concentration-mortality" effect curve;

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

[0027] Figure 3 Effects of GTFX on zebrafish yolk sac absorption delay area, liver area, and liver opacity;

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

[0029] Figure 5 Relative fluorescence values ​​and relative change rates of ROS levels in the GTFX-exposed and control groups;

[0030] Figure 6 Relative expression levels of PPAR-γ, CYP1A1, and CYP1B1 mRNA. DETAILED DESCRIPTION

[0031] The meanings of some abbreviations involved in the present invention are as follows:

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

[0033] 3dpf: means 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), anhydrous 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] UV-visible spectrophotometer (Nanodrop2000, Thermo, USA);

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

[0042] Low-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 (Cat. No. 1725124, Bio-rad, America);

[0045] FastQuant RT Kit (With gDNase) (Cat. No. KR106, TIANGEN, China);

[0046] RNA-Quick Purification Kit (RNA Quick Extraction Kit) (Cat. No. RN001, YiShan Biotech, China).

[0047] 2. The steps of the water ecotoxicity and risk assessment method of gatifloxacin based on zebrafish of the present invention are as follows:

[0048] S1. Establishment of zebrafish experimental model

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

[0050] Farming conditions: According to standardized farming procedures, water quality parameters are maintained within a specific range, including conductivity 480-510μS / cm, pH 6.9-7.2, hardness 53.7-71.6mg / L CaCO3, and a constant temperature of 28℃.

[0051] Wild-type AB zebrafish were raised in aquaculture water at 28.0±0.5℃. The water quality parameters were as follows: 200mg instant sea salt was added to each 1L of reverse osmosis water, the conductivity was 480-510μS / cm; pH was 6.9-7.2; hardness was 53.7-71.6mg / LCaCO3. The experimental animal use license number is: SYXK (Zhejiang) 2012-0171. The feeding and management were in accordance with the requirements of the international AAALAC certification.

[0052] Zebrafish were bred in natural pairs, and 30 3dpf (3 days after fertilization) fry were used in each experimental group for hepatotoxicity studies. After the experiment, zebrafish of all developmental stages were treated with 0.3mg / mL tricaine methanesulfonate for hyperanesthesia. The anesthesia procedure was strictly carried out in accordance with the American Veterinary Medical Association (AVMA) animal anesthesia guidelines.

[0053] S2, Toxicity Concentration Assessment

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

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

[0056] GTFX was stored at 4°C and prepared into a stock solution with a concentration of 5 mg / mL using fish water before use. 210 3dpf zebrafish were randomly selected and distributed into six-well plates, with 30 zebrafish placed in each well (experimental group). GTFX was administered in water at 0.5, 1.0, 2.0, 3.0, 4.0 and 5.0 mg / mL, respectively. A normal control group (zebrafish treated with fish water) was also set up. The capacity of each well was 3 mL. After 48 hours of drug treatment, the mortality of zebrafish in each experimental group was observed and recorded. Origin 8.0 was used to draw the concentration-mortality effect curve, and the maximum non-lethal concentration MNLC and 10% lethal concentration LC of GTFX for zebrafish were calculated. 10 .

[0057] S3, Assessment of hepatotoxicity indicators: liver transparency, area and yolk sac absorption: quantitative assessment of the hepatotoxic effects of gatifloxacin at different concentrations.

[0058] 150 3dpf zebrafish were randomly selected and distributed into six-well plates, with 30 zebrafish placed in each well. They were given 1 / 9MNLC, 1 / 3MNLC, MNLC, and LC, respectively. 10 The concentration of GTFX aqueous solution was 3 mL, and a normal control group was set up at the same time. The capacity of each well was 3 mL. After 48 h of GTFX treatment, 10 zebrafish were randomly selected from each experimental group, photographed and collected under a dissecting microscope (Nikon, SMZ18, Tokyo, Japan), and the liver area, average liver opacity and yolk sac absorption delay area of ​​zebrafish were analyzed and statistically analyzed to evaluate the effect of gatifloxacin on zebrafish liver.

[0059] S4, histopathological analysis of the stained zebrafish sections: HE staining technique: Microscopic observation of zebrafish liver sections after GTFX exposure was performed to record structural damage such as hepatocyte swelling and vacuolization.

[0060] 60 3dpf zebrafish were randomly selected and distributed in a six-well plate, with 30 zebrafish placed in each well, and LC 10 The zebrafish were treated with GTFX aqueous solution of 100 μg / mL and a normal control group was set up at the same time. The volume of each well was 3 mL. After 48 h of GTFX treatment, the zebrafish were fixed with 4% paraformaldehyde and then transferred to 70% ethanol. The zebrafish were dehydrated, embedded, sliced, stained and sealed in sequence. The stained zebrafish liver sections were subjected to histopathological analysis.

[0061] S5, oxidative stress status detection

[0062] Measurement of ROS relative fluorescence value: The relative fluorescence value of reactive oxygen species (ROS) in zebrafish cells was measured to determine whether GTFX caused toxicity by inducing oxidative stress.

[0063] 150 3dpf zebrafish were randomly selected and distributed into six-well plates, with 30 zebrafish treated in each well. 1 / 9MNLC, 1 / 3MNLC, MNLC, and LC were given to each well. 10 A GTFX aqueous solution of 100 μg / ml was added to each well, and a normal control group was set up at the same time. The volume of each well was 3 mL. After 48 h of treatment, the ROS detection reagent CM-H2DCFDA was added. The zebrafish were placed in an incubator at 28 °C for another 1 h, and then fluorescence (F) analysis was performed on each experimental group using a multifunctional microplate reader (Mithras LB940, Berthold Technologies, Stuggart, Germany). The fluorescence values ​​of 10 zebrafish were randomly selected from each experimental group for statistical processing to evaluate whether GTFX induced oxidative stress in zebrafish. At the same time, the relative change rate of ROS was calculated.

[0064]

[0065] In the formula, F Sample Represents the relative fluorescence value (AU) of the experimental group, F Control The fluorescence intensity (AU) of the control group was expressed as ROS (%), and the relative change rate of reactive oxygen species was expressed as ROS (%).

[0066] S6, Molecular Mechanism Exploration

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

[0068] 450 3dpf zebrafish were randomly selected and distributed into six-well plates, with 30 zebrafish treated in each well. 1 / 9MNLC, 1 / 3MNLC, MNLC, and LC were given 10 Concentration of GTFX aqueous solution, and set up a normal control group at the same time, each well volume of 3mL, incubated at 28 ℃ for 48h, using RNA rapid extraction kit to extract total RNA of zebrafish in each experimental group, and using UV-visible spectrophotometer Nanodrop 2000, Thermo Fisher Scientific, USA) to determine the concentration and purity of total RNA. Take 2μg of zebrafish total RNA, follow the instructions of cDNA first-strand synthesis kit, synthesize 20μL of cDNA, and store at -20℃.

[0069] The relative expression of PPAR-γ, CYP1A1 and CYP1B1 genes was detected by qPCR. β-actin was used as the internal reference gene, and the relative expression of PPAR-γ, CYP1A1 and CYP1B genes was calculated using the 2^-ΔΔCt method. The primer sequences are shown in Table 1.

[0070] Table 1 Primer sequence list

[0071]

[0072] S7, Data analysis:

[0073] All data are average ± standard error (SE), one-way analysis of variance (ANOVA) and Dunnett's test were used to compare the differences among the groups, and p < 0.05 was considered a significant difference. The data were 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 The "concentration-mortality" effect curve of GTFX reflects the mortality of zebrafish larvae (3dpf) treated with different concentrations of GTFX for 2-5 days. As the concentration of GTFX increased, the mortality rate of zebrafish gradually increased. 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) of GTFX 10 ) were 1.7 mg / mL and 2.3 mg / mL, respectively. The subsequent experimental concentrations were set at 0.2 mg / mL (1 / 9MNLC), 0.6 mg / mL (1 / 3MNLC), 1.7 mg / mL (MNLC) and 2.3 mg / mL (LC 10 ).

[0077] (2) Phenotypic observation

[0078] Figure 2 The figure shows the morphological changes of the liver of zebrafish larvae (3 dpf) after being treated with different concentrations of GTFX for 48 hours. 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 complete morphology and no deformity. Figure 2 As shown in (a), the liver area of ​​the GTFX-treated group gradually decreased with the increase of concentration. At 0.2 mg / mL, the liver area was slightly reduced. Figure 2 As shown in (b), as the concentration continues to increase, the liver area continues to decrease, the liver opacity gradually increases, and the edge becomes blurred, as shown in Figure 2 As shown in (c), (d) and (e).

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

[0080] Zebrafish treated with 0.6 mg / mL GTFX showed delayed yolk sac absorption and a significant increase in yolk sac area (p < 0.05). In the MNLC (1.7 mg / mL) exposure group, the delayed yolk sac absorption was particularly significant (p < 0.001). Figure 3 (a), and the liver area was significantly smaller than that of the control group (p < 0.001). Figure 3 As shown in b, liver damage is aggravated. 10 (2.3 mg / mL) the liver area is the smallest and the liver edge is blurred, such as Figure 2 In addition, compared with the control group, LC 10 The liver area, liver transmittance and yolk sac area of ​​the two groups were significantly different (p < 0.001).

[0081] Overall, zebrafish exposed to GTFX showed a dose-dependent reduction in liver area, increased liver opacity, and increased yolk sac area. These results suggest that GTFX-induced liver morphological changes may lead to liver structural abnormalities, seriously threatening the survival of zebrafish and having a serious impact on aquatic ecology. At the end of the experiment, LC 10 Exposure to GTFX at a concentration of 2.3 mg / mL directly resulted in 3.3% mortality in zebrafish.

[0082] (3) Histopathological analysis

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

[0084] (4) Oxidative stress assessment

[0085] Figure 5 a is the relative fluorescence value of the GTFX-treated group and the control group, and the data are expressed as mean ± standard error (SE). Figure 5 b is the relative change of ROS between the GTFX-treated group and the control group. CM-H2DCFDA was used to evaluate the intracellular ROS level. The results showed that the relative fluorescence values ​​of zebrafish under 0.2, 0.6, 1.7 and 2.3 mg / mL GTFX treatment were 28449, 16085, 20962 and 17617 pixels, respectively. Figure 5 Compared with the relative fluorescence value of the control group (23826 pixels), the relative change rates of ROS in the GTFX exposure group were 19.4%, -32.5%, -12.0% and -26.1%, respectively. Figure 5 b. Therefore, there was no significant difference between the GTFX-exposed group and the control group (p>0.05), indicating that GTFX did not induce oxidative stress in zebrafish under the experimental concentration conditions.

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

[0087] like Figure 6 As shown in the figure, the relative expression levels of PPAR-γ, CYP1A1 and CYP1B1 mRNA are shown. In the figure, * compared with the control group, p < 0.05. According to the calculation of the relative expression of genes, the relative expression of PPAR-γ gene in the normal control group is 1.00. When the concentration of GTFX is 0.2, 0.6, 1.7 and 2.3 mg / mL, the relative expression of PPAR-γ gene is 1.39, 1.41, 1.50 and 1.97, respectively. Compared with the normal control group (1.00), the p values ​​are p>0.05, p>0.05, p>0.05 and p<0.05, respectively. Among them, there is 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 PPAR-γ gene, as shown in Figure 6 (a) shown.

[0088] Similarly, the relative expression of CYP1A1 gene in the normal control group was 1.01. When the concentration of GTFX was 0.2, 0.6, 1.7 and 2.3 mg / mL, the relative expression of CYP1A1 gene was 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 upregulated the expression of CYP1A1 gene, such as Figure 6 (b) as shown.

[0089] For the CYP1B1 gene, the relative expression of the normal control group was 1.10. When the GTFX concentration was 0.2, 0.6, 1.7 and 2.3 mg / mL, the relative expression of the CYP1B1 gene was 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 a concentration of 1.7 mg / mL, the expression of the CYP1B1 gene was significantly different from that of the control group (p<0.05), indicating that GTFX significantly upregulated the expression of the CYP1B1 gene, such as Figure 6 (c) as shown.

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

[0091] 4. Analysis and discussion

[0092] (1) Effects on zebrafish liver morphology

[0093] Liver size, opacity, and yolk sac absorption are common indicators for evaluating zebrafish hepatotoxicity. GTFX can induce delayed yolk sac absorption, reduced liver area, and liver degeneration in zebrafish, leading to significant changes in liver morphology, such as 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 shown.

[0094] In zebrafish hepatotoxicity assessment, a decrease in liver area usually indicates hepatocyte atrophy or apoptosis, which is the liver's response to exogenous stress. Changes in liver opacity reflect potential changes in liver composition, such as increased fat deposition or development of fibrosis, suggesting disruption of normal liver function and potential cellular damage. The occurrence of yolk sac edema indicates metabolic disturbances, impaired liver ability to regulate metabolic processes, and impaired function.

[0095] For LC 10 Histopathological analysis of the livers of the two groups revealed swelling and vacuolization of hepatocytes, such as Figure 4 This provided deeper insights into the cellular changes that occur following exposure, highlighting the structural changes within the liver tissue. Notably, the presence of hepatocyte swelling and vacuolization is often associated with intracellular stress.

[0096] (2) Effects on oxidative stress

[0097] Given the significant changes in liver morphology, in order to understand the underlying cellular mechanisms, the oxidative stress levels of zebrafish exposed to GTFX were analyzed. Potential oxidative damage was assessed by measuring ROS levels. Contrary to expectations, ROS levels in zebrafish treated with GTFX did not change significantly compared with those in the control group. Figure 5 As shown, this suggests that GTFX induces hepatotoxicity through a pathway independent of oxidative stress. The observation that ROS levels remained stable while liver morphology was significantly altered suggests that the expression of PPAR-γ, CYP1A1, and CYP1B1 may disrupt cellular homeostasis through pathways different from traditional oxidative stress, which provides new insights into the environmental toxicity of fluoroquinolones.

[0098] (3) Molecular mechanism

[0099] In zebrafish exposed to GTFX, ROS levels did not increase, suggesting that non-oxidative pathways may be responsible for the hepatotoxicity. The above studies showed that the expression of genes related to lipid metabolism and stress response, PPAR-γ, CYP1A1, and CYP1B1, was 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 response. 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 elevated PPAR-γ expression is an important indicator of fatty liver disease. CYP1A1 and CYP1B1 are important members of the cytochrome P450 enzyme family, responsible for the metabolism of a variety of endogenous and exogenous compounds, including environmental pollutants, drugs, and carcinogens.

[0100] Normally, activation of PPAR-γ helps maintain energy balance and regulates intracellular lipid levels. When PPAR-γ expression is enhanced, it may indicate that lipid handling and storage processes are disturbed. However, the increase in PPAR-γ under GTFX exposure reflects a compensatory mechanism that attempts to correct the imbalance of lipid metabolism caused by the drug. Similarly, the increased expression of CYP1A1 and CYP1B1 highlights the enhanced metabolic activity in GTFX exposure. The enzymes encoded by these genes are essential for metabolizing xenobiotics and can convert lipophilic compounds into hydrophilic products that are more easily excreted. Although these enzymes are traditionally associated with the generation of ROS, in the above studies, the increased expression of CYP1A1 and CYP1B1 was not accompanied by an increase in ROS levels, suggesting that their main role may be more focused on metabolic adaptation rather than causing oxidative stress.

[0101] In summary, the method of the present invention combines morphological observation, histopathological analysis and molecular biology to find that GTFX significantly reduces the liver area of ​​zebrafish and delays yolk sac absorption. At the same time, GTFX interferes with lipid metabolism by upregulating PPAR-γ, CYP1A1 and CYP1B1 gene expression, but does not significantly induce oxidative stress. This indicates that its toxic mechanism mainly depends on non-oxidative stress pathways.

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

[0103] In addition, the zebrafish-based aquatic ecotoxicity and risk assessment method of gatifloxacin described in the present invention can be used to determine and evaluate the impact of gatifloxacin and other drug residues on aquatic ecology. This work lays the foundation for future exploration of the long-term effects of GTFX at lower concentrations, and can provide a reference for a wider range of aquatic ecological risks related to common drug exposure in aquatic environments.

[0104] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention (such as adjustment of the sequence of experimental steps, etc.) can be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. A method for aquatic ecotoxicity and risk assessment of gatifloxacin based on zebrafish, characterized in that: The method steps are as follows: S1, zebrafish experimental model was established, 30 larvae 3 days post fertilization (3 dpf) were used in each experiment, and the breeding method was natural pair mating; S2, toxicity concentration assessment, zebrafish were exposed to a gradient concentration of gatifloxacin (GTFX) of 0.5-5.0 mg / mL, and the maximum non-lethal concentration (MNLC) and 10% lethal concentration (LC) were calculated using the concentration-mortality curve. 10 ; S3, evaluation of hepatotoxicity indicators, analysis and statistics of zebrafish liver area, liver average opacity and yolk sac absorption delay area were performed to evaluate the effects of GTFX exposure on zebrafish liver; S4, histopathological analysis of stained zebrafish liver sections; S5, oxidative stress detection, fluorescent probe was used to detect the level of reactive oxygen species (ROS) to evaluate whether GTFX induced oxidative stress in zebrafish; S6, relative expression levels of PPAR-γ, CYP1A1, and CYP1B1 genes were detected by qPCR; S7, Data analysis.

2. The method for aquatic ecotoxicity and risk assessment of gatifloxacin based on zebrafish according to claim 1, characterized in that: The water quality parameters for zebrafish breeding in S1 are: 200 mg of instant sea salt is added to every 1 L of reverse osmosis water, the conductivity is 480-510 μS / cm; the pH is 6.9-7.2; the hardness is 53.7-1.6 mg / LCaCO3, and the temperature is constant at 28°C.

3. The method for aquatic ecotoxicity and risk assessment of gatifloxacin based on zebrafish according to claim 1, characterized in that: The S2 method for evaluating the effect of GTFX on zebrafish liver is as follows: 210 3dpf zebrafish were randomly selected and distributed into a six-well plate, 30 zebrafish were placed in each well, and 0.5, 1.0, 2.0, 3.0, 4.0 and 5.0 mg / mL of GTFX solution were respectively administered, with a capacity of 3 mL of GTFX solution in each well. A normal control group was set up at the same time, and the death of zebrafish in each experimental group was observed and recorded 48 hours after the drug treatment, and the concentration-mortality effect curve was drawn using Origin 8.

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4. The method for aquatic ecotoxicity and risk assessment of gatifloxacin based on zebrafish according to claim 1, characterized in that: In step S3, 150 3dpf zebrafish were randomly selected and distributed into a six-well plate, with 30 zebrafish placed in each well, and 1 / 9MNLC, 1 / 3MNLC, MNLC and LC were given to each well. 10 Concentration of GTFX aqueous solution, and a normal control group was set up at the same time. The volume of each well was 3 mL. After 48 hours of GTFX treatment, 10 zebrafish were randomly selected from each experimental group, photographed under a dissecting microscope, and data were collected.

5. The method for aquatic ecotoxicity and risk assessment of gatifloxacin based on zebrafish according to claim 1, characterized in that: The method for performing histopathological analysis on zebrafish liver sections after staining in S4 is as follows: 60 3dpf zebrafish were randomly selected and distributed into a six-well plate, 30 zebrafish were placed in each well, and LC 10 The zebrafish were treated with a GTFX aqueous solution of 1% concentration, and a normal control group was set up with a capacity of 3 mL in each well. After 48 hours of GTFX treatment, the zebrafish were fixed with 4% paraformaldehyde and then transferred to 70% ethanol. The zebrafish were dehydrated, embedded, sliced, stained and sealed in sequence, and the stained zebrafish sections were subjected to histopathological analysis.

6. The method for aquatic ecotoxicity and risk assessment of gatifloxacin based on zebrafish according to claim 1, characterized in that: Specifically, S5 is as follows: 150 3dpf zebrafish were randomly selected and distributed into six-well plates, 30 zebrafish were treated in each well, and 1 / 9MNLC, 1 / 3MNLC, MNLC and LC were given to each well. 10 The concentration of GTFX aqueous solution was 100 μg / mL, and a normal control group was set up at the same time. The volume of each well was 3 mL. After 48 hours of treatment, the reactive oxygen species ROS detection reagent CM-H2DCFDA was added, and the zebrafish were placed in an incubator at 28°C for another 1 hour. After that, fluorescence (F) analysis was performed on each experimental group. The fluorescence values ​​of 10 zebrafish were randomly selected from each experimental group for statistical processing, and the relative change rate of ROS level was calculated at the same time: In the formula, F Sample Represents the relative fluorescence value (AU) of the experimental group, F Control represents the relative fluorescence value (AU) of the control group, and ROS (%) represents the relative change rate of reactive oxygen species.

7. The method for aquatic ecotoxicity and risk assessment of gatifloxacin based on zebrafish according to claim 1, characterized in that: The zebrafish used in the test of S5 were 450 zebrafish at 3 dpf randomly selected and distributed into a six-well plate, with 30 fish per well, and given 1 / 9MNLC, 1 / 3MNLC, MNLC and LC, respectively. 10 A concentration of GTFX aqueous solution was added, and a normal control group was set up at the same time. The volume of each well was 3 mL. After incubation at 28°C for 48 h, 2 μg of zebrafish total RNA was extracted, 20 μL of cDNA was synthesized, and it was stored at -20°C for later use.

8. The zebrafish-based gatifloxacin aquatic ecotoxicity and risk assessment method according to any one of claims 1 to 7 is used to determine and evaluate the impact of drug residues on aquatic ecology.

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