A method for evaluating health risks of human pesticide mixture exposure using a zebrafish embryo model

Abstract

The application discloses a method for evaluating health risks of human pesticide mixed exposure by using a zebrafish embryo model, and comprises the following steps: 1) configuring pesticide exposure solutions with different concentrations; 2) culturing zebrafish embryos in the pesticide exposure solutions for 96 hours; 3) recording development indexes of the zebrafish embryos during the culturing process; 4) calculating toxicity endpoints according to the development indexes in the step 3); and 5) extrapolating the toxicity endpoints to human exposure level reference values. The application uses zebrafish as a model, measures different physiological indexes after pesticide mixed exposure, screens the most sensitive indexes by using a mathematical model, and finally provides intake reference values for human exposure to pesticide mixed pollution by using dose extrapolation calculation.

Description

Technical Field

[0001] This invention relates to the field of pesticide toxicity, and more specifically to a method for evaluating the health risks of mixed exposure to pesticides in humans using a zebrafish embryo model. Background Technology

[0002] Currently, the relevant standards for pesticide residues in fresh food only consider the health risks of a single pesticide. Therefore, it is crucial to select a model that can evaluate the risk of mixed pesticide exposure and use extrapolation methods to provide a reference for human health risks.

[0003] Current methods for pesticide risk assessment often involve conducting experiments on mammals such as rats, mice, rabbits, or dogs to identify the most sensitive toxicity endpoints and then selecting an appropriate uncertainty coefficient (often 100) for conversion. This method is neither convenient for assessing the risk of mixed exposures nor aligns with the current trend of reducing the use of laboratory animals or replacing testing. Zebrafish models, on the other hand, offer advantages such as convenient testing, high-throughput screening, and the ability to withstand exposure to binary or ternary mixed toxicants. They are currently widely used for risk assessment of mixed chemical exposures. Furthermore, zebrafish toxicity endpoints are easily observed, facilitating the use of mathematical models to select the most sensitive indicators. Extrapolation and conversion can then be performed, ultimately providing a reference for human health risks. Summary of the Invention

[0004] The purpose of this invention is to provide a method for evaluating the health risks of mixed exposure to pesticides in humans using a zebrafish embryo model.

[0005] The method of the present invention for evaluating the health risks of human pesticide co-exposure using a zebrafish embryo model includes the following steps:

[0006] 1) Prepare pesticide exposure solutions of different concentrations;

[0007] 2) Zebrafish embryos were cultured in pesticide exposure solution for 96 hours;

[0008] 3) Record the developmental indicators of zebrafish embryos during the culture process;

[0009] 4) Calculate the toxicity endpoint based on the developmental indicators in step 3);

[0010] 5) Extrapolate toxicity endpoints to reference levels for human exposure.

[0011] The method for preparing the pesticide exposure solution in step 1) involves dissolving different weights of pesticides in dimethyl sulfoxide to prepare a stock solution, and then diluting it with Holt Buffer to prepare a treatment solution of the corresponding concentration, so that the final DMSO concentration is 0.1%. Holt Buffer is an aqueous solution containing 3.5 g / L NaCl, 0.05 g / L KCl, 0.1 g / L CaCl2, and 0.05 g / L NaHCO3.

[0012] The cultivation method in step 2) is as follows: 20 zebrafish embryos are cultured in 10 mL of pesticide exposure solution; 3 replicates are made for each concentration, the exposure solution is changed every 24 hours, and dead eggs are removed at the same time, and the exposure lasts for 96 hours.

[0013] The method for selecting zebrafish embryos in step 2) is as follows: 4 hours after fertilization, the eggs are placed under a stereomicroscope for observation, and unfertilized, coagulated, or damaged eggs are removed to obtain zebrafish embryos.

[0014] In step 3), the developmental indicators of zebrafish embryos during the culture process are recorded, specifically including: the mortality rate of zebrafish embryos at 24, 48, 72, and 96 hours after fertilization is statistically analyzed; the malformation rate of each group is statistically analyzed every 24 hours starting from 48 hours after fertilization; in addition, the hatching rate of embryos at 48 hours after fertilization is statistically analyzed.

[0015] 24 hours after fertilization, five embryos were selected in each parallel pair and observed under a stereomicroscope. Their spontaneous tail wagging was recorded for one minute, and the results were analyzed and statistically analyzed.

[0016] 48 hours after fertilization, five embryos were selected in each parallel group and observed under a stereomicroscope. Their heart rate was recorded for 30 seconds, and the heart rate data was analyzed and statistically analyzed.

[0017] In step 3), the criteria for death are egg coagulation and cardiac arrest; the criteria for deformity specifically include pericardial edema, yolk sac edema and tail deformity; and the criteria for hatching is complete separation of the larvae from the egg membrane.

[0018] The method for calculating the toxic endpoint in step 4) is as follows: statistically analyze the mortality rate and malformation rate, and calculate LC50 (lethal concentration 50%) and EC50 (half maximum effective concentration) of the malformation rate; substitute the dose-response relationship data of mortality rate and malformation rate into Expotential, Hill, and Linear models respectively for fitting, and select the model with the best fit to calculate the toxic endpoint that causes a 10% effect.

[0019] The method for extrapolating the toxicity endpoint to the reference value for human exposure level in step 4) is as follows: collect zebrafish toxicity endpoints and rat oral acute LD50 values ​​from publicly available databases. 50 The toxicity endpoints of zebrafish were standardized to the order of mg / L, and the LD50 was also determined. 50 The values ​​were uniformly converted to the mg / kg order of magnitude. After processing the values ​​by Log10, they were used as X and Y values ​​respectively. They were then substituted into SPSS for linear regression and plotted using GraphPad to obtain the linear regression formulas for X and Y. The toxicity endpoints obtained in the experiment were substituted into the formulas to calculate the mammalian toxicity endpoints. The results were then divided by the safety factor of 100 to obtain the final reference value for human health risks.

[0020] The method of this invention uses zebrafish as a model to measure different physiological indicators after exposure to mixed pesticides, and uses a mathematical model to screen the most sensitive indicators. Then, dose extrapolation is used to calculate the intake reference values ​​for human exposure to mixed pesticide pollution. Attached Figure Description

[0021] Figure 1 The quantitative relationship between zebrafish toxicity endpoints and acute oral LD50 in rats obtained by database comparison method. Detailed Implementation

[0022] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0023] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0024] Example 1

[0025] This invention provides a method for evaluating the health risks of mixed exposure to pesticides in humans using a zebrafish embryo model, comprising the following steps:

[0026] 1. Preparation of horsefish embryos

[0027] Wild-type Tu strain zebrafish (purchased from the National Zebrafish Resource Center) were raised in a zebrafish rearing system in a closed zebrafish room with a controlled light / dark cycle of 14h / 10h and a room temperature of 26±2℃. They were fed twice daily with freshly hatched brine shrimp larvae (China Crown brand).

[0028] On the afternoon before the experiment, select a number of mature male and female fish and place them in a breeding box at a ratio of two females to two males. Separate them with a partition. At the beginning of the photoperiod the next morning, remove the partition to allow the male and female fish to chase and mate freely. Collect the fertilized fish eggs within half an hour of removing the partition.

[0029] 2. Pesticide exposure in zebrafish embryos

[0030] Accurately weigh the pesticide and dissolve it in dimethyl sulfoxide (ThermoFischer, USA) to prepare a stock solution. Dilute it with Holt Buffer (containing 3.5 g / L NaCl, 0.05 g / L KCl, 0.1 g / L CaCl2, and 0.05 g / L NaHCO3, all reagents purchased from Shanghai Husheng Company) to prepare treatment solutions of the corresponding concentrations (different test concentrations can be selected according to specific experimental needs for different test chemicals) so that the final DMSO concentration is 0.1% (this concentration has been proven not to cause significant developmental toxicity). Select appropriate pesticide concentrations for single treatment and mixed exposure based on the preliminary test results, and select 0.1% DMSO as the solvent control group. Four hours after fertilization, collected oocytes were observed under a stereomicroscope (SZ-10, Olympus, Japan). Unfertilized, coagulated, and ruptured oocytes were removed. The oocytes were then placed in six-well plates with 10 mL of exposure solution pre-added to each well. Each well contained 20 oocytes, with three replicates per concentration. The exposure solution was changed every 24 hours, and dead oocytes were removed. Exposure continued for 96 hours. Embryo mortality and malformation rates were recorded at 96 hours post-fertilization. Malformations included spinal curvature, pericardial edema, and yolk sac edema.

[0031] 3. Developmental marker detection

[0032] The mortality rate of zebrafish embryos at 24, 48, 72, and 96 hours post-fertilization was statistically analyzed, with egg coagulation and cardiac arrest as the markers of death. Simultaneously, the malformation rate of each group was calculated every 24 hours starting from 48 hours post-fertilization. Specific malformations included pericardial edema, yolk sac edema, and tail malformations. Furthermore, the hatching rate of embryos at 48 hours post-fertilization was calculated, with hatching defined as complete separation of the larvae from the egg membrane.

[0033] 24 hours after fertilization, five embryos were selected in each parallel pair and observed under a stereomicroscope. Their spontaneous tail waggle was recorded for one minute, and the results were analyzed and statistically analyzed.

[0034] 48 hours after fertilization, five embryos were selected in each parallel group and observed under a stereomicroscope. Their heart rate was recorded for 30 seconds, and the heart rate data was analyzed and statistically analyzed.

[0035] 4. Statistical analysis and calculation of toxicity indicators

[0036] The mortality and malformation rates were statistically analyzed using SPSS 22.0 (IBM, USA) software, and the LC50 (Lethal Concentration 50%) and EC50 (Half Maximal Effective Concentration) of the malformation rate were calculated. The normality and homogeneity of variance of the embryonic development indicators were verified using the Shapiro-Wilk test and Levene's test, respectively. If the criteria were met (the standard for meeting the criteria was normality and homogeneity of variance distribution), one-way ANOVA was applied with a post-hoc LSD test. If the criteria were not met, the Kruskal-Wallis nonparametric test was used with a post-hoc Bonferroni test. A p-value < 0.05 was considered statistically significant. The original data were fitted to Expotential, Hill, and Linear models using statistical analysis software such as BMDS 3.2, and the model with the best fit was selected to calculate the toxic endpoint causing a 10% effect.

[0037] 5. Extrapolation of toxicity endpoints to reference levels for human exposure

[0038] Collect zebrafish toxicity endpoints and acute oral LD50 in rats for corresponding pesticides from publicly available databases. 50 This standardizes the toxicity endpoints of zebrafish to the same order of magnitude (ppm, equivalent to mg / L), and also reduces LD50. 50 The values ​​were uniformly converted to the mg / kg order of magnitude. After processing with Log10, they were used as X and Y values ​​respectively. These were then substituted into SPSS for linear regression, and graphed using GraphPad. The resulting linear regression formula for X and Y is Y = 0.3400X + 2.718 (e.g., ...). Figure 1 As shown, the solid line represents the fitted regression formula, and the two dashed lines represent the upper and lower limits of the 95% confidence interval of the regression formula. Substituting the toxicity endpoints obtained in the experiment into the formula, the mammalian toxicity endpoints are calculated and divided by the safety factor of 100 to obtain the final reference value for human health risks.

[0039] Example 2: Calculation of reference values ​​for human health risks of common mixed-polluting pesticide combinations, difenoconazole and dimethomorph.

[0040] Difenoconazole is a triazole fungicide with a broad spectrum of activity. It can be sprayed on crops or used to treat seeds and is widely used worldwide. Dimethomorph is a morpholine broad-spectrum fungicide with significant inhibitory effects on fungal spores. It is also widely used worldwide in the cultivation of various fruits and vegetables. Both fungicides have high detection rates in berries, grains, and other crops, which may pose a risk of co-exposure.

[0041] Accurately weigh the pesticide, dissolve it in DMSO to prepare a stock solution, and dilute it with HoltBuffer to the corresponding concentration of treatment solution, so that the final DMSO concentration is 0.1% (this concentration has been proven not to cause significant developmental toxicity). Referring to the experimental results of others and preliminary experiments, the final concentrations of difenoconazole were 0.50, 0.73, 1.08, 1.58, 2.32, 3.41, and 5.00 mg / L, and the final concentrations of dimethomorph were 2.00, 2.94, 4.31, 6.32, 9.28, 13.63, and 20.00 mg / L. Prepare mixed exposure solutions MIX (difenoconazole: dimethomorph = 1:5.92, v / v) according to the principle of equal toxicity ratio, with mixed concentrations of 1.38, 2.03, 2.98, 4.38, 6.42, 9.43, and 13.84 mg / L.

[0042] The above-prepared mixed exposure solution MIX was used to replace the exposure solution in step 2 of Example 1, while keeping other steps unchanged. The human health risk reference values ​​for the mixed contamination pesticide combination of difenoconazole and dimethomorph were calculated, and the results are shown in Table 1.

[0043] The most sensitive indicator for difenoconazole was calculated to be the deformity rate at 96 hpf, with a zebrafish toxicity endpoint of 0.3 mg / L. For dimethomorph, the most sensitive indicator was the deformity rate at 96 hpf, with a zebrafish toxicity endpoint of teratogenicity at 96 hpf and a toxicity endpoint of 1.1 mg / L. The most sensitive indicator for combined exposure to both was the juvenile heart rate at 48 hpf, with a zebrafish toxicity endpoint of 0.18 mg / L. The corresponding human health risk reference value is 0.29 mg / kg / day, with difenoconazole at 0.044 mg / kg / day and dimethomorph at 0.246 mg / kg / day.

[0044] Table 1. Toxicity endpoints and estimated risk reference values ​​for mixed exposure to difenoconazole and dimethomorph in zebrafish.

[0045]

[0046] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A method for evaluating health risks of human pesticide mixture exposure using a zebrafish embryo model, characterized in that, The method comprises the following steps: 1) preparing pesticide exposure solutions with different concentrations; 2) culturing zebrafish embryos in the pesticide exposure solutions for 96 hours; 3) recording the development indicators of the zebrafish embryos during the culturing process; 4) calculating the toxicity endpoints according to the development indicators in step 3); 5) extrapolating the toxicity endpoints to human exposure level reference values. The calculation method of the toxicity endpoint in step 4) is: statistics of mortality and malformation rate, and calculation of LC50 of mortality and EC50 of malformation rate; LC50 of mortality and EC50 of malformation rate are respectively substituted into Expotential, Hill, Linear model for fitting, and the toxicity endpoint causing 10% effect is calculated by selecting the model with the best fitting degree; the method of extrapolating the toxicity endpoint in step 5) to the human exposure level reference value is: collecting the toxicity endpoints of zebrafish and the acute oral LD 50 of rats in public databases, uniformly converting the toxicity endpoints of zebrafish into the order of mg / L and the acute oral LD 50 of rats into the order of mg / kg, and after Log10 processing, X value and Y value are substituted into SPSS for linear regression and GraphPad is used for drawing to obtain the linear regression formula of X and Y; the toxicity endpoint obtained in the experiment is substituted into the formula, the toxicity endpoint of mammals is calculated, and divided by the safety factor 100 to obtain the final human health risk reference value.

2. The method of claim 1, wherein, In step 1), the pesticide exposure solutions are prepared by dissolving different weights of pesticides in dimethyl sulfoxide to prepare stock solutions, and then diluting the stock solutions with Holt Buffer to prepare treatment solutions with corresponding concentrations, so that the final concentration of DMSO is 0.1%, wherein the Holt Buffer is an aqueous solution containing NaCl 3.5 g / L, KCl 0.05 g / L, CaCl2 0.1 g / L, and NaHCO3 0.05 g / L.

3. The method of claim 1, wherein, In step 2), the culturing method is as follows: 20 zebrafish embryos are cultured in each 10 mL of pesticide exposure solution; each concentration is repeated for three times; the exposure solution is replaced every 24 hours, and dead eggs are removed at the same time; and the exposure lasts for 96 hours.

4. The method of claim 3, wherein, In step 2), the selection method of zebrafish embryos is as follows: the eggs 4 hours after fertilization are observed under a stereomicroscope; the unfertilized, coagulated and damaged eggs are removed; and the zebrafish embryos are obtained.

5. The method of claim 1, wherein, In step 3), the development indicators of the zebrafish embryos during the culturing process are recorded, which specifically include: the mortality of the zebrafish embryos 24 hours, 48 hours, 72 hours and 96 hours after fertilization is counted; the malformation rate of each group is counted every 24 hours since 48 hours after fertilization; and the hatching rate of the embryos 48 hours after fertilization is counted. At 24 hours after fertilization, 5 embryos are selected from each parallel group, and their one-minute autonomous swinging is observed under a stereomicroscope and analyzed and counted. At 48 hours after fertilization, 5 embryos are selected from each parallel group, and their heart rate within 30 seconds is observed under a stereomicroscope and analyzed and counted.

6. The method of claim 5, wherein, In step 3), the death criteria are egg coagulation and heart stop; the malformation criteria specifically include pericardial edema, yolk sac edema and tail malformation; and the hatching criteria are that the larvae are completely separated from the egg membranes.

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