A method for rapid evaluation of intestinal toxicity of pollutants using the Drosophila melanogaster model

Abstract

The present invention provides a method for rapidly evaluating the intestinal toxicity of pollutants using a Drosophila melanogaster model, which belongs to the technical field of pollutant intestinal toxicity evaluation. Specifically, the method comprises placing Drosophila melanogaster in an environment containing environmental pollutants and a non-pollutant environment, and measuring the average survival rate reduction ratio, intestinal length shortening rate, intestinal wall thickness reduction rate, Smurf phenotype rate increase ratio, fecal point number reduction rate, and intestinal metabolic rate reduction ratio of the fruit flies after culturing for 7 to 15 days; then calculating a toxicity evaluation value Y by a formula. If Y ≥ 10, it indicates that the pollutant has intestinal toxicity, and the larger the Y value, the greater the intestinal toxicity; if Y < 10, it indicates that the pollutant has no intestinal toxicity; if a certain indicator has no significant difference compared with the control, the result is directly judged as 0; if the three indicators B, C, and D have no significant difference, it is directly judged that the pollutant has no intestinal toxicity. The method for evaluating the intestinal toxicity of pollutants has the characteristics of being simple and rapid, highly accurate, and highly practical.

Description

Technical Field

[0001] The present invention belongs to the technical field of enterotoxicity evaluation of pollutants, and in particular relates to a method for rapidly evaluating the enterotoxicity of pollutants using Drosophila melanogaster. Background Art

[0002] Drosophila, a species of the genus Drosophila in the family Drosophilidae, is one of the most important model organisms in biological research. Characterized by its short lifespan, ease of care, rapid reproduction, few chromosomes, numerous mutations, and small size, Drosophila is one of the most important model organisms in biological research. As a model organism for studying human disease, Drosophila not only shares similarities with mammals in basic biology, physiology, and nervous system function, but also possesses unique advantages as a model organism. Its clear genetic background and ease of experimental manipulation have made it an irreplaceable tool in diverse fields, including genetics, developmental biology, biochemistry, and molecular biology.

[0003] Currently, a large number of chemical pollutants are constantly being detected in the environment, many of which have no relevant toxicity data. However, long-term contact with these substances in people's lives can cause harm to human health. The intestine is an important barrier that protects the body from external harmful substances. In recent years, research on the effects of pollutants on intestinal homeostasis and intestinal barrier integrity has become the focus of attention of many scholars at home and abroad. Compared with traditional mammalian intestinal toxicity evaluation methods for pollutants, the model organism Drosophila also has the characteristics of highly conserved intestinal structure and function of tissues and organs such as the intestine and the human intestine, which makes it suitable for rapid evaluation of the intestinal toxicity of pollutants. The current method for detecting the intestinal toxicity of fruit flies accurately determines the toxicity of various substances. The inventor has conducted experimental tests and set up a method for quickly judging the intestinal toxicity of pollutants. According to current tests, this method accurately determines the intestinal toxicity of an unknown substance. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a method for rapidly evaluating the enteric toxicity of pollutants using Drosophila melanogaster. The method provided by this method can rapidly and accurately evaluate the enteric toxicity of pollutants, providing a basis for rapid and effective enteric toxicity dose and model determination for industries such as industry, agriculture, and food.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following experimental scheme:

[0006] The present invention provides a method for rapidly evaluating the intestinal toxicity of pollutants using a Drosophila melanogaster model. The method comprises: placing Drosophila melanogaster on filter paper sheets containing different concentrations of 3-MCPD and culturing them for 7 days, observing the intestines of the fruit flies, and when the length of the fruit fly intestines is shortened, the intestinal wall thickness is reduced, and the Smurf phenotype rate increase ratio is increased, and the above indicators or phenotypes are changed (not all indicators), it indicates that the pollutant has damaged the intestinal structure; when the number of fecal spots and the intestinal metabolic rate of the fruit flies exposed to 3-MCPD are reduced, and the above indicators or phenotypes are changed (not all indicators), it indicates that the pollutant has damaged the intestinal function, and in summary, the pollutant has intestinal toxicity.

[0007] A method for rapidly evaluating the intestinal toxicity of pollutants using a Drosophila melanogaster model comprises the following steps:

[0008] Drosophila melanogaster were placed in an environment containing environmental pollutants and in an environment without pollutants. The experimental group was cultured in the environment containing environmental pollutants, and the control group was cultured in the environment without pollutants. The following indicators were measured after 7 to 15 days of culture:

[0009] A-survival rate, the average survival rate reduction ratio of the experimental group compared with the control group was measured respectively; it is represented by A in the formula;

[0010] B-intestinal morphological changes, respectively measuring the shortening rate of the fruit fly intestinal length and the decreasing rate of the fruit fly intestinal wall thickness in the experimental group compared with the control group, which are represented by B1 and B2 in the formula;

[0011] C-intestinal structural changes, respectively measured the experimental group compared with the control group to obtain the Drosophila Smurf phenotype rate increase ratio, represented by C in the formula;

[0012] D-intestinal function changes, the reduction rate of fruit fly fecal points and the reduction rate of fruit fly intestinal metabolic rate obtained by the experimental group compared with the control group were measured respectively, and were expressed as D1 and D2 in the formula respectively;

[0013] The calculation formula for toxicity evaluation Y is:

[0014] Y=0.1(200A-10)+0.3×200(B1+B2)+0.3(200C-10)+0.3×200(D1+D2);

[0015] If Y ≥ 10, the pollutant is enterotoxic, and the larger the Y value, the greater the enterotoxicity. If Y < 10, the pollutant is non-enterotoxic. If a certain indicator is not significantly different from the control, the result is directly determined to be 0. In addition, if there is no significant difference in the three indicators B, C, and D, the pollutant is directly determined to be non-enterotoxic.

[0016] The average survival rate reduction ratio=(average survival rate of fruit flies in the control group-average survival rate of fruit flies in the experimental group) / average survival rate of fruit flies in the control group.

[0017] The shortening rate of the fruit fly intestinal length=(intestinal length of the fruit flies in the control group-intestinal length of the fruit flies in the experimental group) / intestinal length of the fruit flies in the control group.

[0018] The reduction rate of the fruit fly intestinal wall thickness=(intestinal wall thickness of the fruit flies in the control group-intestinal wall thickness of the fruit flies in the experimental group) / intestinal wall thickness of the fruit flies in the control group.

[0019] The increase ratio of the fruit fly Smurf phenotype rate=(the Smurf phenotype rate of the fruit flies in the experimental group-the Smurf phenotype rate of the fruit flies in the control group) / the Smurf phenotype rate of the fruit flies in the control group.

[0020] The reduction rate of the number of fruit fly feces spots=(the number of fruit fly feces spots in the control group-the number of fruit fly feces spots in the experimental group) / the number of fruit fly feces spots in the control group.

[0021] The reduction ratio of the fruit fly intestinal metabolic rate=(the intestinal metabolic rate of the fruit flies in the experimental group-the intestinal metabolic rate of the fruit flies in the control group) / the intestinal metabolic rate of the fruit flies in the control group.

[0022] The culture conditions of the fruit fly are: 24-26° C., a light-dark ratio of 12h:12h, and a relative humidity of 55-65%.

[0023] The fruit fly is specifically the W1118 strain of Drosophila melanogaster.

[0024] The environment containing environmental pollutants is specifically to expose fruit flies by evenly soaking a sucrose solution containing acrylamide (Acr), perfluorooctane sulfonate (PFOS) and cadmium chloride (CdCl2) on a filter paper in a tube.

[0025] Beneficial effects

[0026] By studying the relationship between enterotoxic substances and various indicators of Drosophila melanogaster and building on a large number of mathematical models established previously, our research group has developed a method for evaluating the enterotoxicity of pollutants using Drosophila melanogaster. This method involves culturing Drosophila melanogaster on filter paper containing varying concentrations of pollutants for 7 to 15 days. The method then measures the reduction in average survival rate, intestinal length reduction, intestinal wall thickness reduction, Smurf phenotype increase, fecal dot count reduction, and intestinal metabolic rate reduction. The toxicity evaluation formula, Y, is then calculated based on the proportion of each indicator: Y = 0.1(200A-10) + 0.3×200(B1+B2) + 0.3(200C-10) + 0.3×200(D1+D2). If Y ≥ 10, the pollutant is enterotoxic, and a larger Y value indicates greater enterotoxicity. If Y < 10, the pollutant is non-enterotoxic. If an indicator shows no significant difference compared to the control, the result is directly determined as 0. In addition, if there is no significant difference among the three indicators B, C, and D, it can be directly determined that the pollutant has no intestinal toxicity.

[0027] Finally, the research team substituted the concentration of the known pollutant 3-MCPD of 0.001-0.016% into the formula provided by the present invention, and the result obtained was that the greater the toxicity, the larger the Y value, thus verifying the accuracy of the disclosure provided by the present invention.

[0028] The method provided by this invention can quickly and accurately evaluate the enterotoxicity of the pollutant 3-MCPD, providing theoretical and experimental data support for safe dosages and concentrations for industry, agriculture, and the food industry. Furthermore, the method was validated using three pollutants known to be enterotoxic: acrylamide (Acr), perfluorooctane sulfonate (PFOS), and cadmium chloride (CdCl2). BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The effects of exposure to different concentrations of 3-MCPD pollutants on the survival of Drosophila;

[0030] Figure 2 The effect of exposure to different concentrations of 3-MCPD pollutants on the intestinal length of Drosophila;

[0031] Figure 3 The effect of exposure to different concentrations of 3-MCPD pollutants on the intestinal wall thickness of Drosophila;

[0032] Figure 4 Effects of exposure to different concentrations of 3-MCPD pollutants on the intestinal permeability of Drosophila;

[0033] Figure 5 The effect of exposure to different concentrations of 3-MCPD pollutants on the number of fruit fly fecal spots;

[0034] Figure 6 Effects of exposure to different concentrations of 3-MCPD pollutants on food metabolism of Drosophila;

[0035] Figure 7 The effects of exposure to pollutants known to be enterotoxic on the intestinal length of Drosophila melanogaster;

[0036] Figure 8 The effects of exposure to known enterotoxic pollutants on intestinal permeability in Drosophila;

[0037] Figure 9 The effect of exposure to pollutants known to be enterotoxic on the number of fecal spots in Drosophila;

[0038] Figure 10 The effects of exposure to pollutants known to be enterotoxic on food metabolism in Drosophila; DETAILED DESCRIPTION

[0039] The present invention provides a method for rapidly evaluating the intestinal toxicity of pollutants using a Drosophila melanogaster model, comprising: placing Drosophila melanogaster on filter paper containing different concentrations of 3-MCPD and culturing them for 7 days. The intestinal tract of the fruit flies is then observed. When the fruit fly intestinal tract shows a shortening of length, a decrease in intestinal wall thickness, or changes in the aforementioned indicators or phenotypes (not all indicators), it indicates that the pollutant has damaged intestinal morphology. An increase in the Smurf phenotype ratio indicates that the pollutant has damaged intestinal structure. When the fruit flies exposed to 3-MCPD show a decrease in fecal spots and an increase in the ratio of fruit flies with altered intestinal contents, and changes in the aforementioned indicators or phenotypes (not all indicators), it indicates that the pollutant has damaged intestinal function. The final concentration of the filter paper containing the pollutant 3-MCPD is 0.001 to 0.016%. A significant difference P value of less than 0.05 is *, a P value of less than 0.01 is **, a P value of less than 0.001 is ***, and no significant difference is ns.

[0040] In the present invention, the Drosophila melanogaster is preferably selected from the W1118 wild-type Drosophila melanogaster. The present invention has no special limitation on the source of the W1118 wild-type Drosophila melanogaster, and conventional commercially available ones are used.

[0041] In the present invention, preferably, newly emerged virgin Drosophila melanogaster flies and male flies are separately raised for 3 days; preferably, 15 to 20 male and female Drosophila flies are placed on filter paper containing 3-MCPD for culture.

[0042] In the present invention, the concentration of the filter paper containing 3-MCPD is 0.004-0.016%, specifically 0.004%, 0.008% and 0.016%.

[0043] In the present invention, the source of the pollutant 3-MCPD is not particularly limited, and a conventional commercial product can be used. Specifically, it was purchased from Shanghai McLean Biochemical Technology Co., Ltd. with a purity of 98%.

[0044] The present invention does not particularly limit the method for observing the intestinal length and intestinal wall thickness of fruit flies, and conventional methods can be used. Specifically, preferably, the fruit flies are anesthetized with carbon dioxide, dissected in 1×PBS, fixed with 4% paraformaldehyde solution for 10 minutes, and then mounted with 50% glycerol. The intestinal morphology is observed under an inverted fluorescence microscope, and the length and intestinal wall thickness are calculated.

[0045] In the present invention, the filter paper of 3-MCPD uses 5% sucrose as solvent.

[0046] In the present invention, the fruit fly culture is carried out in a constant temperature incubator, and the conditions preferably include: the culture temperature is preferably 25° C.; the light-dark ratio of the culture is 12h:12h; and the culture environment humidity is 55-65%.

[0047] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0048] Reagent configuration:

[0049] 1) 5% sucrose: 5 g sucrose (food grade), purchased from a large supermarket, was dissolved in 95 ml triple-distilled water.

[0050] 2) 3-MCPD: (analytical grade), purity: 98%, purchased from Shanghai McLean Biochemical Technology Co., Ltd.

[0051] 3) Brilliant Blue: (experimental reagent), purity: 85%, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0052] Example 1

[0053] Experiment 1

[0054] Method for determining the survival rate of Drosophila

[0055] Culture conditions: 25°C, light-dark ratio of 12h:12h, relative humidity of 60%.

[0056] Experimental animals: Drosophila melanogaster strain W1118.

[0057] Exposure conditions: Set up four experimental groups, each with five replicates, with 15 fruit flies per tube. Use a pipette to evenly distribute 100 μl of the prepared 5% sucrose solution containing varying concentrations of 3-MCPD onto the filter paper within the tube. Expose the fruit flies. The tubes were rotated every 12 hours, and the number of fruit flies killed at each concentration was counted daily until all fruit flies in the control group had died.

[0058] Index detection: Record the number of fruit flies killed at each concentration every day and then perform survival analysis to calculate the survival rate of fruit flies at each concentration. Figure 1 shown.

[0059] Experiment 2

[0060] Methods for measuring intestinal morphological impairment

[0061] Culture conditions: 25°C, light-dark ratio of 12h:12h, relative humidity of 60%.

[0062] Experimental animals: Drosophila melanogaster strain W1118.

[0063] Exposure conditions: Four experimental groups, each with five replicates, were set up, with 15 flies per tube. 100 μl of the prepared 5% sucrose solution containing varying concentrations of 3-MCPD was pipetted evenly onto the filter paper in the tube. The flies were exposed for 7 days. After feeding, the flies were anesthetized with CO2 and dissected in 1x PBS.

[0064] Index detection: Observe the intestinal length and intestinal wall thickness under a microscope. Figure 2 、 Figure 3 shown.

[0065] Experiment 3

[0066] Method for measuring intestinal structural damage (Smurf method)

[0067] Culture conditions: 25°C, light-dark ratio of 12h:12h, relative humidity of 60%.

[0068] Experimental animals: Drosophila melanogaster strain W1118.

[0069] Exposure conditions: Set up four experimental groups, each with five replicates, with 15 flies per tube. Use a pipette to evenly distribute 100 μl of the prepared 5% sucrose solution containing varying concentrations of 3-MCPD onto a filter paper slip within the tube. Expose the flies for 7 days. After CO2 anesthesia, place the flies in a tube containing 1% (wt / vol) agar (with water), starve them for 3 hours, and then place them on a paper slip containing brilliant blue dye solution and feed them for 12 hours. After CO2 anesthesia, the flies were re-anesthetized.

[0070] Index detection: Observe under a microscope to see if there is brilliant blue dye penetration in addition to intestinal staining. Figure 4 shown.

[0071] Experiment 4

[0072] Pipe wall feces point counting method

[0073] Culture conditions: 25°C, light-dark ratio of 12h:12h, relative humidity of 60%.

[0074] Experimental animals: Drosophila melanogaster strain W1118.

[0075] Exposure conditions: Four experimental groups, each with five replicates, were set up, with 15 flies per tube. 100 μl of the prepared 5% sucrose solution containing varying concentrations of 3-MCPD was pipetted onto a filter paper slip in the tube. The flies were exposed for 7 days. After 7 days of feeding, the flies were anesthetized with CO2 and placed in a tube containing 1% (wt / vol) agar (with water). After starvation for 3 hours, the flies were transferred to a paper slip containing brilliant blue dye solution and fed for 12 hours. The flies were then anesthetized with CO2, and fecal spots on the tube walls were counted.

[0076] Index detection: fecal point count on pipe wall. Figure 5 shown.

[0077] Experiment 5

[0078] Brilliant Blue Dye Complete Metabolism Drosophila Counting Method

[0079] Culture conditions: 25°C, light-dark ratio of 12h:12h, relative humidity of 60%.

[0080] Experimental animals: Drosophila melanogaster strain W1118.

[0081] Exposure conditions: Four experimental groups were set up, with five replicates per group, and 15 fruit flies were placed in each tube. A 5% sucrose solution containing varying concentrations of 3-MCPD was pipetted and evenly applied to the filter paper in the tube. The flies were exposed for 7 days. After feeding, the flies were anesthetized with CO2 and placed in tubes containing 1% (wt / vol) agar (with water). They were starved for 3 hours and then transferred to a paper strip containing Brilliant Blue dye solution. The flies were then fed for 12 hours. The flies were then transferred to standard corn culture medium and fed for 3 hours. The flies were then anesthetized with CO2 and observed for residual Brilliant Blue in their abdomens under a stereomicroscope. The percentage of flies that had fully metabolized the Brilliant Blue dye in each concentration group was calculated.

[0082] Indicator monitoring: Brilliant blue dye complete metabolism fruit fly count. Specific results are shown in Figure 6 shown.

[0083] The following is the technical plan for the verification experiment.

[0084] In this verification experiment, newly emerged virgin Drosophila melanogaster flies and male flies were selected and raised separately for 3 days; 15 to 20 male Drosophila flies were cultured on filter paper sheets containing Acr, PFOS and CdCl2.

[0085] In this verification experiment, the concentration of the filter paper containing Acr was 10 mg / kg, the concentration of the filter paper containing PFOS was 12.5 mg / kg, and the concentration of the filter paper containing CdCl2 was 12.5 mg / kg.

[0086] Verification Example 1

[0087] Culture conditions: 25°C, light-dark ratio of 12h:12h, relative humidity of 60%.

[0088] Experimental animals: Drosophila melanogaster strain W1118.

[0089] Exposure conditions: Four experimental groups, each with four replicates, were set up, with 15 flies per tube. A 5% sucrose solution containing Acr, PFOS, and CdCl₂ was pipetted onto a filter paper sheet in the tube, evenly dissecting the fruit flies. The flies were reared for 7–15 days. After completion of the rearing period, the flies were anesthetized with CO₂ and dissected in 1× PBS.

[0090] Index detection: Observe the intestinal length under a microscope. Figure 7 shown.

[0091] Verification Example 2

[0092] Culture conditions: 25°C, light-dark ratio of 12h:12h, relative humidity of 60%.

[0093] Experimental animals: Drosophila melanogaster strain W1118.

[0094] Exposure conditions: Set up four experimental groups, each with four replicates, with 15 fruit flies per tube. Use a pipette to evenly apply 100 μl of the prepared 5% sucrose solution containing Acr, PFOS, and CdCl2 onto a filter paper slip in the tube. Expose the fruit flies for 7-15 days. After feeding, anesthetize the flies with CO2 and place them in a tube containing 1% (wt / vol) agar (with water). Starve them for 3 hours, then transfer them to a paper slip containing brilliant blue dye solution and feed them for 12 hours. After feeding, anesthetize them with CO2.

[0095] Index detection: Observe under a microscope to see if there is brilliant blue dye penetration in addition to intestinal staining. Figure 8 shown.

[0096] Verification Example 3

[0097] Culture conditions: 25°C, light-dark ratio of 12h:12h, relative humidity of 60%.

[0098] Experimental animals: Drosophila melanogaster strain W1118.

[0099] Exposure conditions: Set up four experimental groups, each with four replicates, with 15 fruit flies per tube. Use a pipette to evenly apply 100 μl of the prepared 5% sucrose solution containing Acr, PFOS, and CdCl2 onto a filter paper slip in the tube. Expose the fruit flies for 7-15 days. After feeding, anesthetize the flies with CO2 and place them in tubes containing 1% (wt / vol) agar (with water). Starve them for 3 hours, then transfer them to a paper slip containing brilliant blue dye solution and feed them for 12 hours. Then, anesthetize the flies with CO2 and count the fecal spots on the tube walls.

[0100] Index detection: fecal point count on pipe wall. Figure 9 shown.

[0101] Verification Example 4

[0102] Culture conditions: 25°C, light-dark ratio of 12h:12h, relative humidity of 60%.

[0103] Experimental animals: Drosophila melanogaster strain W1118.

[0104] Exposure conditions: Four experimental groups were set up, with five replicates per group, and 15 fruit flies were placed in each tube. A 5% sucrose solution containing Acr, PFOS, and CdCl2 was pipetted and evenly soaked onto a filter paper slip in the tube. The fruit flies were exposed for 7 to 15 days. After feeding, the flies were anesthetized with CO2 and placed in a tube containing 1% (wt / vol) agar (with water). They were starved for 3 hours and then transferred to a paper slip containing Brilliant Blue dye solution. The flies were then transferred to a standard corn culture medium and fed for 3 hours. The flies were then anesthetized with CO2 and observed for residual Brilliant Blue in their abdomens under a stereomicroscope. The proportion of flies that had fully metabolized the Brilliant Blue dye in each treatment group was calculated.

[0105] Indicator monitoring: Brilliant blue dye complete metabolism fruit fly count. Specific results are shown in Figure 10 shown.

[0106] Example 2

[0107] Based on the magnitude of the influencing factors of some experiments in Example 1 and calculations, this example provides a method for rapidly evaluating the intestinal toxicity of pollutants using the Drosophila melanogaster model, comprising the following steps:

[0108] Place Drosophila melanogaster on filter paper containing different concentrations of environmental pollutants and culture for 7 to 15 days, then observe whether the following results appear:

[0109] A-survival rate, the average survival rate reduction ratio of the experimental group compared with the control group was measured respectively; it is represented by A in the formula;

[0110] B-intestinal morphological changes, measured by the shortening rate of the fruit fly intestinal length and the decreasing rate of the fruit fly intestinal wall thickness in the experimental group compared with the control group; represented by B1 and B2 in the formula respectively;

[0111] C-intestinal structural changes, measured by the increase rate of the Smurf phenotype in the experimental group compared with the control group; represented by C in the formula;

[0112] D-intestinal function changes, measured by the rate of decrease in the number of fecal spots and the rate of increase in the ratio of fruit flies to intestinal contents changes in the experimental group compared with the control group; represented by D1 and D2 in the formula respectively;

[0113] The toxicity evaluation calculation formula is:

[0114] Y=0.1(200A-10)+0.3×200(B1+B2)+0.3(200C-10)+0.3×200(D1+D2)

[0115] If Y ≥ 10, the pollutant is enterotoxic, and the larger the Y value, the greater the enterotoxicity. If Y < 10, the pollutant is non-enterotoxic. If there is no significant difference between an indicator and the control, the result is directly determined to be 0. In addition, if there is no significant difference between indicators B, C, and D, the pollutant is directly determined to be non-enterotoxic.

[0116] Average survival rate reduction ratio = (average survival rate of fruit flies in the control group - average survival rate of fruit flies in the experimental group) / average survival rate of fruit flies in the control group.

[0117] Drosophila intestinal length shortening rate = (intestinal length of control group Drosophila - intestinal length of experimental group Drosophila) / intestinal length of control group Drosophila.

[0118] The reduction rate of the intestinal wall thickness of fruit flies = (intestinal wall thickness of fruit flies in the control group - intestinal wall thickness of fruit flies in the experimental group) / intestinal wall thickness of fruit flies in the control group.

[0119] The increase ratio of the Smurf phenotype rate of fruit flies=(the Smurf phenotype rate of fruit flies in the experimental group-the Smurf phenotype rate of fruit flies in the control group) / the Smurf phenotype rate of fruit flies in the control group.

[0120] The reduction rate of the number of fruit fly feces spots = (the number of fruit fly feces spots in the control group - the number of fruit fly feces spots in the experimental group) / the number of fruit fly feces spots in the control group.

[0121] The reduction ratio of the intestinal metabolic rate of fruit flies = (intestinal metabolic rate of fruit flies in the experimental group - intestinal metabolic rate of fruit flies in the control group) / intestinal metabolic rate of fruit flies in the control group.

[0122] Verification experiment 1:

[0123] Whether 0.004% 3-MCPD exposure is intestinal toxic according to the following experiments and evaluation criteria:

[0124] 1. Analysis of experimental results: A-survival rate, the experimental group decreased by approximately 25% compared with the control group; B1-intestinal length, the experimental group decreased by approximately 15% compared with the control group; B2-intestinal wall thickness, the experimental group decreased by approximately 24% compared with the control group; C-Smurf phenotype rate increase ratio, the experimental group had no significant difference compared with the control group; D1-fecal point count, the experimental group had a decrease of approximately 10% compared with the control group; D2-metabolic rate, the experimental group had no significant difference compared with the control group;

[0125] 2. Calculate according to the toxicity evaluation formula:

[0126] Y=0.1(200×0.25-10)+0.3×200(0.15+0.24)+(200×0-10)+0.3×200(0.01+0)=25>10

[0127] The above results indicate that 0.004% 3-MCPD has intestinal toxicity.

[0128] Verification experiment 2:

[0129] Whether 0.008% 3-MCPD exposure is intestinal toxic according to the following experiments and evaluation criteria:

[0130] Analysis of experimental results: A-survival rate, the experimental group decreased by about 47% compared with the control group; B1-intestinal length, the experimental group decreased by about 30% compared with the control group; B2-intestinal wall thickness, the experimental group decreased by about 43% compared with the control group; C-Smurf phenotype rate increase ratio, the experimental group increased by about 30% compared with the control group; D1-fecal point count, the experimental group decreased by about 20% compared with the control group; D2-metabolic rate, the experimental group decreased by about 22% compared with the control group;

[0131] Calculated according to the toxicity evaluation formula:

[0132] Y=0.1×(200×0.47-10)+0.3×200(0.3+0.43)+0.3×(200×0.3-10)+0.3×200(0.2+0.22)=92.4>10

[0133] The above results indicate that 0.008% 3-MCPD has intestinal toxicity.

[0134] Verification experiment three:

[0135] Whether 0.016% 3-MCPD exposure is intestinal toxic according to the following experiments and evaluation criteria:

[0136] Analysis of experimental results: A-survival rate, the experimental group decreased by about 70% compared with the control group; B1-intestinal length, the experimental group decreased by about 40% compared with the control group; B2-intestinal wall thickness, the experimental group decreased by about 50% compared with the control group; C-Smurf phenotype rate increase ratio, the experimental group increased by about 40% compared with the control group; D1-fecal point number, the experimental group decreased by about 50% compared with the control group; D2-metabolic rate, the experimental group decreased by about 44% compared with the control group;

[0137] Calculated according to the toxicity evaluation formula:

[0138] Y=0.1×(200×0.7-10)+0.3×200(0.4+0.5)+0.3×(200×0.4-10)+0.3×200(0.5+0.44)=144.4>10

[0139] The above results indicate that 0.016% 3-MCPD has intestinal toxicity.

[0140] Verification experiment 4:

[0141] Determine whether Acr exposure is intestinal toxic based on the following experiments and evaluation criteria:

[0142] Analysis of experimental results: A-survival rate, not tested; B1-intestinal length, the experimental group decreased by about 25% compared with the control group; B2-intestinal wall thickness, not tested; C-Smurf phenotype rate increase ratio, the experimental group increased by about 90% compared with the control group; D1-fecal point number, there was no significant difference between the experimental group and the control group; D2-metabolic rate, the experimental group decreased by about 70% compared with the control group;

[0143] Calculated according to the toxicity evaluation formula:

[0144] Y=0.1×(200×0-10)+0.3×200(0.25+0)+0.3×(200×0.9-10)+0.3×200(0+0.7)=107>10

[0145] The above results indicate that Acr has intestinal toxicity.

[0146] Verification experiment five:

[0147] Determine whether PFOS exposure is intestinal toxic based on the following experiments and evaluation criteria:

[0148] Analysis of experimental results: A-survival rate, not tested; B1-intestinal length, the experimental group decreased by about 25% compared with the control group; B2-intestinal wall thickness, not tested; C-Smurf phenotype rate increase ratio, the experimental group increased by about 140% compared with the control group; D1-fecal point number, there was no significant difference between the experimental group and the control group; D2-metabolic rate, the experimental group decreased by about 73% compared with the control group;

[0149] Calculated according to the toxicity evaluation formula:

[0150] Y=0.1×(200×0-10)+0.3×200(0.25+0)+0.3×(200×1.4-10)+0.3×200(0+0.73)=138.8>10

[0151] The above results indicate that PFOS has intestinal toxicity.

[0152] Verification experiment six:

[0153] Determine whether CdCl2 exposure is intestinal toxic based on the following experiments and evaluation criteria:

[0154] Analysis of experimental results: A-survival rate, not tested; B1-intestinal length, the experimental group decreased by about 25% compared with the control group; B2-intestinal wall thickness, not tested; C-Smurf phenotype rate increase ratio, the experimental group increased by about 160% compared with the control group; D1-fecal point count, the experimental group decreased by about 60% compared with the control group; D2-metabolic rate, the experimental group decreased by about 85% compared with the control group;

[0155] Calculated according to the toxicity evaluation formula:

[0156] Y=0.1×(200×0-10)+0.3×200(0.25+0)+0.3×(200×1.6-10)+0.3×200(0.6+0.85)=194>10

[0157] The above results indicate that CdCl2 has intestinal toxicity.

[0158] Verification Experiment 7:

[0159] Determine whether 5% sucrose exposure is intestinal toxic based on the following experiments and evaluation criteria:

[0160] Analysis of experimental results: A-survival rate, not tested; B1-intestinal length, no significant difference between the experimental group and the control group; B2-intestinal wall thickness, not tested; C-Smurf phenotype rate increase ratio, no significant difference between the experimental group and the control group; D1-fecal point number, no significant difference between the experimental group and the control group; D2-metabolic rate, no significant difference between the experimental group and the control group;

[0161] Calculated according to the toxicity evaluation formula:

[0162] Y=0.1×(200×0-10)+0.3×200(0+0)+0.3×(200×0-10)+0.3×200(0+0)=-4<10

[0163] The above results indicate that 5% sucrose has no intestinal toxicity.

[0164] This experiment used Acr, PFOS, and CdCl2, three pollutants known to be intestinal toxic, to validate this method. Using the experimental method and exposure duration of the present invention, exposure concentrations known to be intestinal toxic were selected to test phenotypes such as Drosophila intestinal length, Smurf phenotype rate increase ratio, fecal point number, and metabolic rate. The experimental results showed that compared with the control, the intestines of Drosophila in the exposed group were shortened, the Smurf phenotype rate increase ratio increased significantly, the fecal point number showed an upward trend, and the metabolic rate also decreased significantly, suggesting the toxic effects of pollutant exposure on the Drosophila intestine. The inventors also calculated the influence weight of each factor based on the changes in each characteristic at each concentration. The formula also avoided the deviation caused by the influence of some natural non-toxic pollutants on intestinal data. The entire verification experiment also demonstrated the accuracy and reliability of the present invention.

[0165] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for rapidly assessing the intestinal toxicity of pollutants using Drosophila melanogaster, characterized by: The steps include: Drosophila melanogaster were placed in an environment containing environmental pollutants and in an environment without pollutants. The experimental group was cultured in the environment containing environmental pollutants, and the control group was cultured in the environment without pollutants. The following indicators were measured after 7 to 15 days of culture: A-survival rate, the average survival rate reduction ratio of the experimental group compared with the control group was measured respectively; it is represented by A in the formula; B-intestinal morphological changes, measured by the shortening rate of the fruit fly intestinal length and the decreasing rate of the fruit fly intestinal wall thickness in the experimental group compared with the control group; represented by B1 and B2 in the formula respectively; C-intestinal structural changes, respectively measured the rate of increase in the Smurf phenotype of Drosophila in the experimental group compared with the control group; represented by C in the formula; D-intestinal function changes, measured by the rate of decrease in the number of fecal spots and the rate of increase in the ratio of fruit flies to intestinal contents changes in the experimental group compared with the control group; represented by D1 and D2 in the formula respectively; The calculation formula for toxicity evaluation Y is: Y=0.1(200A-10)+0.3×200(B1+B2)+0.3(200C-10)+0.3×200(D1+D2); If Y ≥ 10, it means that the pollutant has enterotoxicity, and the larger the Y value, the greater the enterotoxicity; if Y < 10, it means that the pollutant has no enterotoxicity. In addition, if there is no significant difference in the three indicators B, C, and D, it is directly determined that the pollutant has no enterotoxicity. The average survival rate reduction ratio = (average survival rate of fruit flies in the control group - average survival rate of fruit flies in the experimental group) / average survival rate of fruit flies in the control group; Among them, the shortening rate of fruit fly intestinal length = (the fruit fly intestinal length of the fruit fly in the control group - the fruit fly intestinal length of the fruit fly in the experimental group) / the fruit fly intestinal length of the fruit fly in the control group; Among them, the reduction rate of the fruit fly intestinal wall thickness = (the fruit fly intestinal wall thickness of the fruit flies in the control group - the fruit fly intestinal wall thickness of the fruit flies in the experimental group) / the fruit fly intestinal wall thickness of the fruit flies in the control group; The increase rate of the fruit fly Smurf phenotype rate = (the fruit fly Smurf phenotype rate of the fruit flies in the experimental group - the fruit fly Smurf phenotype rate of the fruit flies in the control group) / the fruit fly Smurf phenotype rate of the fruit flies in the control group; Among them, the reduction rate of the number of fruit fly feces spots = (the number of fruit fly feces spots in the control group - the number of fruit fly feces spots in the experimental group) / the number of fruit fly feces spots in the control group; The increasing rate of the ratio of fruit flies with changed intestinal contents of fruit flies = (the ratio of fruit flies with changed intestinal contents of fruit flies in the experimental group - the ratio of fruit flies with changed intestinal contents of fruit flies in the control group) / the ratio of fruit flies with changed intestinal contents of fruit flies in the control group.

2. The method for rapidly evaluating the intestinal toxicity of pollutants using Drosophila melanogaster according to claim 1, characterized in that: The culture conditions of the fruit fly are: 24-26° C., a light-dark ratio of 12h:12h, and a relative humidity of 55-65%.

3. The method for rapidly evaluating the intestinal toxicity of pollutants using Drosophila melanogaster according to claim 1, characterized in that: The fruit fly is specifically the W1118 strain of Drosophila melanogaster.

4. The method for rapidly evaluating the intestinal toxicity of pollutants using Drosophila melanogaster according to claim 1, characterized in that: The environment containing environmental pollutants specifically comprises: uniformly soaking a sucrose solution containing Acr, PFOS and Cdcl2 on a 3-MCPD filter paper in a tube and exposing fruit flies.

Images