CN114755366A - Method for comprehensively analyzing motor neurotoxicity of chemicals by using caenorhabditis elegans - Google Patents
Method for comprehensively analyzing motor neurotoxicity of chemicals by using caenorhabditis elegans Download PDFInfo
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Abstract
The invention provides a method for comprehensively analyzing the motor neurotoxicity of chemicals by using caenorhabditis elegans, which is used for culturing and synchronizing the caenorhabditis elegans, exposing the caenorhabditis elegans based on the environment-related concentration of exogenous pollutants of the chemicals, detecting indexes related to the motor neurotoxicity, and evaluating the motor neurotoxicity of the chemicals by using a comprehensive biomarker response index system. On the basis of the traditional caenorhabditis elegans movement index, the caenorhabditis elegans full-automatic imaging and behavior tracker is adopted to further analyze a plurality of movement behavior indexes, and the indexes are subjected to comprehensive biomarker response analysis, so that the motor neurotoxicity of chemicals is systematically evaluated. The method integrates microscopic observation and automatic monitoring, and the detection result has higher accuracy and reliability and is suitable for popularization and use.
Description
Technical Field
The invention relates to the field of environmental health and chemical ecotoxicology analysis, in particular to a method for comprehensively analyzing the motor neurotoxicity of a chemical by utilizing caenorhabditis elegans.
Background
For a long time, the environmental pollution of China has the characteristics of structural property, compressibility and compositeness, and the environmental pollution is low in dosage of toxic and harmful pollutants and difficult to eliminate in a short time when exposed to ecological environment and public health risks for a long time. 4.5 thousands of production and use records in China, 3000 of which are listed in a dangerous chemical directory. Major chemical accidents occur all over the world due to the reasons of non-standard safety management of chemicals, technical defects of production, storage and transportation, insufficient emergency plans for sudden accidents and the like. Along with the migration and transformation of a large amount of chemicals in the environment, the chemicals exist in water, soil, atmosphere and other environment media through volatilization, abrasion, leaching and other ways, and further enter the human body along with breathing exposure, skin contact, food accumulation and other ways, finally threat the health of the human body and interfere the stability of an ecosystem. The lancet report in 2017 indicates that chemical pollution is an important influence factor of global disease burden, so that environmental health and chemical risk management has risen to national strategic height, and the toxicity detection of chemicals is a key means for accurately evaluating environmental hazards and health risks.
Caenorhabditis elegans (Caenorhabditis elegans) is an invertebrate which lives in soil and interstitial water and can reflect the stability of a soil ecosystem, and has the advantages of short life cycle, small volume, easiness in culture, semitransparent body, convenience in fluorescent marking and microscopic observation and the like. In addition, the caenorhabditis elegans has complete nervous system, simple neuron structure and conserved function, known whole gene sequence and 60-80% homology with human gene, and is an ideal model organism for nerve toxicology research. The caenorhabditis elegans is very sensitive to the stimulation of the external environment and exogenous chemical pollutants, and can indirectly reflect the ecological toxicology and environmental health risks of chemicals, wherein abnormal motor behaviors are direct expression symptoms of most nervous system diseases, and the change of the motor behaviors is taken as the most direct expression form of animals to the external stimulation, is mainly regulated by the nervous system, and can intuitively reflect whether the exogenous pollutants have the potential risks of motor neurotoxicity.
Although the motor behavior indexes of caenorhabditis elegans are mature at present, in the research of primarily detecting motor neurotoxicity induced by exogenous pollutants, researchers usually only select 1-2 indexes for evaluation, and the motor behavior indexes are mutually independent for data analysis, so that the detection accuracy is relatively low, the evaluation error of toxicity effect is large, and the result is not reliable enough. In addition, most of the existing detection of the movement behaviors of caenorhabditis elegans depends on artificial judgment under a common optical microscope, a great deal of time and energy of researchers are consumed, the movement behaviors of a plurality of caenorhabditis elegans cannot be tracked simultaneously, the detection speed is low, artificial counting is prone to be inaccurate, and the result is not accurate and reliable enough.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a method for comprehensively analyzing the motor neurotoxicity of chemicals by using caenorhabditis elegans. The method has short detection period, integrates microscopic observation and full-automatic monitoring, can intuitively and vividly display the change condition of each index under different exposure conditions, and effectively improves the accuracy and reliability of risk evaluation of the chemical induced motor neurotoxicity.
The technical scheme of the invention provides a method for comprehensively analyzing the motor neurotoxicity of chemicals by using caenorhabditis elegans, which comprises the following steps:
step 2, preparing a solid culture medium special for culturing nematodes, placing the solid culture medium in a high-pressure steam sterilization pot, sterilizing the solid culture medium for 30min at 120 ℃, preparing a culture plate, and cooling the culture plate for later use;
step 3, preparing a lysate, performing synchronization treatment on the caenorhabditis elegans in the pregnancy period by using the lysate, centrifuging to remove supernatant, transferring collected eggs to the center of a new solid culture medium, and incubating for 16-18h at 20 ℃;
Step 4, determining a real environment medium of the chemical, and preparing chemical exposure solutions with different concentration gradients under the environment concentration; wherein the environmental medium comprises the concentration ranges of soil, water, sediment and organisms;
step 5, collecting successfully-incubated larvae of caenorhabditis elegans in L1 stage, uniformly adding the larvae into prepared exposure solutions with different concentration gradients, carrying out exposure culture in a constant-temperature incubator at 20 ℃ for 72h under the dark condition, and regularly feeding a specific amount of food;
step 6, collecting the caenorhabditis elegans subjected to exposure of different concentration gradients, cleaning for 3 times by using a k liquid, placing the caenorhabditis elegans on a solid culture medium without food, placing the solid culture medium under a stereoscopic microscope, observing and recording the head swinging frequency, the body bending frequency and the number of times of pumping throat of the caenorhabditis elegans within 20s, and calculating the head swinging frequency, the body bending frequency and the frequency of pumping throat;
step 7, collecting the caenorhabditis elegans subjected to exposure of different concentration gradients, cleaning for 3 times by using a k liquid, and detecting the movement wavelength, the movement amplitude, the movement speed and the movement distance of the caenorhabditis elegans by using a caenorhabditis elegans full-automatic imaging and behavior tracker;
and 8, carrying out comprehensive biomarker response Index (IBR) analysis on the multiple detection index results at different concentrations, displaying the multiple biomarker results through a star chart, calculating the area of the star chart to obtain the comprehensive biomarker response index, and systematically evaluating the motor neurotoxicity of the chemicals at the environmental concentration.
Further, in the step 1, 10g of peptone, 5g of yeast powder and 5g of NaCl are added into 1L of distilled water for constant volume, and after autoclaving, an LB liquid medium is prepared.
Further, in the step 2, 3g of NaCl, 17g of agar powder, 2.5g of peptone and 0.24g of MgSO (MgSO) were added4And 0.111g of CaCL is added into 1L of distilled water for constant volume and is prepared into the NGM solid culture medium after autoclaving.
Further, in the step 3, the centrifugation speed is 2500-.
Further, in the step 3, 0.4g of NaOH and 10ml of NaCLO are added, and then distilled water is added to the mixture to reach a constant volume of 40ml, so as to prepare the nematode lysate.
Furthermore, in the step 6, 2.386g of KCl and 2.98g of NaCl are added into 1L of distilled water for constant volume, and a k liquid is prepared.
Compared with the prior art, the invention has the following advantages:
firstly, the invention utilizes the caenorhabditis elegans full-automatic imaging and behavior tracker to automatically monitor various motion behavior related indexes such as motion wavelength, motion amplitude, motion speed, motion distance and the like, and the visual observation is more visual and accurate than the conventional visual observation under a microscope.
Secondly, the observation of the caenorhabditis elegans by a microscope is combined with full-automatic monitoring, a star-shaped graph of monitoring results of various indexes is drawn, the results of various motor indexes are linked by adopting a comprehensive biomarker response index analysis method, the influence of different concentrations of chemical pollutants on various motor nerve indexes is reflected visually and vividly, and the accuracy and the reliability of risk evaluation of the chemical induced motor nerve toxicity are improved.
And thirdly, the caenorhabditis elegans is exposed under the condition of the actual environmental concentration of chemicals, so that a more reasonable data basis which is closer to the actual environmental exposure situation can be provided for the motor neurotoxicity evaluation and risk management and control of chemical pollutants, and more reliable data can be provided for the environmental health and the ecotoxicology analysis of the chemicals.
Drawings
FIG. 1 shows the effect of DBDPE exposure to decabromodiphenylethane at ambient concentrations on the motor behavior index of C.elegans;
FIG. 2 shows a schematic representation of the comprehensive biomarker response index IBR analysis of environmental concentrations of DBDPE exposure to C.elegans.
Detailed Description
The invention is further explained in detail with reference to the attached drawings and the detailed description. The described embodiments are only some embodiments of the present invention, and the technical solution is not limited to the specific implementation methods listed below.
Examples
The embodiment provides a method for comprehensively analyzing the motor neurotoxicity of a chemical by using caenorhabditis elegans, which specifically comprises the following steps:
In the embodiment, an escherichia coli liquid is prepared, 10g of peptone, 5g of yeast powder and 5g of nacl are added into an erlenmeyer flask to fix the volume in 1L of distilled water, the erlenmeyer flask is placed into a high-pressure steam sterilization pot to be sterilized for 30min at 120 ℃, after cooling, escherichia coli colonies are picked up to be placed in a culture medium, and the escherichia coli colonies are shaken for 16h in a shaking incubator at 37 ℃ and 220 rpm.
Step 2, preparing a solid culture medium special for culturing nematodes, placing the solid culture medium in a high-pressure steam sterilization pot, sterilizing the solid culture medium for 30min at 120 ℃, preparing a culture plate, and cooling the culture plate for later use;
in the embodiment, a special NGM solid culture medium for caenorhabditis elegans growth is prepared, 3g of NaCL, 17g of agar powder, 2.5g of peptone, 0.24g of MgSO4 and 0.111g of CaCl are added into 1L of distilled water for constant volume, the mixture is placed in a high-pressure steam sterilization pot for sterilization at 120 ℃ for 30min, and then a sterile culture plate is poured and cooled for later use;
step 3, preparing a lysis solution, performing synchronization treatment on the caenorhabditis elegans in the pregnancy period by using the lysis solution, centrifuging to remove a supernatant, transferring the collected worm eggs to the center of a new solid culture medium, and incubating for 16-18h at 20 ℃;
in step 4, the designed and prepared concentration range is within the actual environment concentration range, and the designed highest concentration is higher than the current actual environment concentration under the condition of increasing the annual output and the use amount of chemicals;
In the embodiment, the concentration ranges of chemicals in real environmental media such as soil, water, sediments, organisms and the like are determined through literature research, and chemical exposure solutions with different concentration gradients under the environmental concentration are prepared. Taking decabromodiphenylethane (DBDPE) as an example, the concentration range of DBDPE in the actual water body environment and organism is from several ng/L to hundreds of mu g/L, so that the chemical exposure concentration range is preliminarily determined to be 0, 1, 10, 100, 500, 1000 mu g/L, and the final concentration of DMSO is 0.01%, and according to the description of < 0.1% DMSO solution in the 2012 article published by Food Chem journal of Gonz alezmann, S, and the like, the DMSO solution does not have influence on nematodes;
weighing 0.01g of DBDPE solid powder by using a balance, adding 1mL of dimethyl sulfoxide for dissolving, performing ultrasonic-assisted dissolution to obtain a DBDPE mother solution, adding a k solution for dilution to prepare a DBDPE solution with the concentration of 0, 1, 10, 100, 500 or 1000 mu g/L, uniformly placing the exposure solution in a sterile 6-hole plate, adding 8mL of the exposure solution into each hole, and setting 3 repeated holes in each concentration;
step 5, collecting successfully-incubated larvae of the L1-stage caenorhabditis elegans, uniformly adding the larvae into prepared exposure liquid with different concentration gradients, performing exposure culture for 72 hours in a constant-temperature incubator at 20 ℃ under the dark condition, and regularly feeding a specific amount of food;
In the step, the caenorhabditis elegans with different concentration gradient exposure is collected, washed for 3 times by using a k liquid, 5 mu L of a caenorhabditis elegans solution is dripped on an NGM culture medium without being coated with bacteria, and the head swing times of the caenorhabditis elegans within 20s are observed and counted under a stereoscopic microscope. As shown in FIG. 1, exposure to higher concentrations of 500 and 1000. mu.g/L of DBDPE significantly inhibited C.elegans head wobble frequency compared to the control group, with a decrease in 20s head wobble frequency of about 19% (p <0.01) and 26% (p <0.01), respectively.
Step 6, setting the sinusoidal motion of the nematode along the x axis, wherein the head swing frequency is defined as the change frequency of the caenorhabditis elegans in one period along the y axis bending direction within 1min, the body bending frequency is defined as the movement frequency of one wavelength along the x axis within 1min, and the frequency of the pump pharynx is defined as the frequency of one-time pump pharynx jumping and returning to the original position within 20 s;
and collecting the caenorhabditis elegans with different concentration gradient exposure, washing for 3 times by using a k liquid, adding the caenorhabditis elegans to an uncoated NGM plate, and placing under a stereoscopic microscope to count the number of times of body bending of the caenorhabditis elegans within 20s after all the solution is volatilized. As shown in FIG. 1, exposure to 1000. mu.g/L of DBDPE significantly suppressed C.elegans body bending frequency compared to the control group, with a decrease in 20s body bending times of about 34% (p <0.01), respectively.
And collecting the caenorhabditis elegans with different concentration gradient exposure, cleaning for 3 times by using a k liquid, placing the caenorhabditis elegans on an NGM culture medium coated with escherichia coli, enabling the caenorhabditis elegans to reach a stable feeding state after about 1 hour, and observing and counting 20s endocervical dysphagia of the caenorhabditis elegans under a stereoscopic microscope. As shown in fig. 1, exposure to higher concentrations of 500 and 1000 μ g/L DBDPE significantly inhibited the number of puffs in the nematodes, which decreased by about 15% (p <0.01) and 18% (p <0.01) within 20s, respectively, compared to the control group.
Step 7, collecting the caenorhabditis elegans subjected to exposure of different concentration gradients, cleaning for 3 times by using a k liquid, and detecting the movement wavelength, the movement amplitude, the movement speed and the movement distance of the caenorhabditis elegans by using a caenorhabditis elegans full-automatic imaging and behavior tracker;
in the step, the caenorhabditis elegans subjected to exposure of different concentration gradients is collected, washed for 3 times by using a k liquid, added onto an NGM plate without being coated with bacteria, a caenorhabditis elegans full-automatic imaging and behavior tracker is used for recording a caenorhabditis elegans movement video within 20s, and based on the video, a caenorhabditis elegans behavior tracking analysis system is used for counting the nematode movement wavelength. As shown in fig. 1, the wavelength of motion dropped 17%, 23% and 15% in the 100, 500, 1000 μ g/LDBDPE exposure groups, respectively.
After the culture is finished, nematodes are collected and added onto an NGM plate without being coated with bacteria, the movement video of the nematodes in 20s is recorded by using a caenorhabditis elegans full-automatic imaging and behavior tracker, and the movement amplitude is counted by analysis software. As shown in FIG. 1, the amplitude of the movement was significantly inhibited at 100-1000 μ g/L exposure (p <0.05) compared to the control group, with a decrease of about 18% in the average amplitude.
After the culture is finished, the nematodes are collected and added onto an NGM plate without being coated with bacteria, the movement video of the nematodes in 20s is recorded by utilizing a caenorhabditis elegans full-automatic imaging and behavior tracker, and the movement speed is counted by analysis software. As shown in FIG. 1, when the concentration is 500. mu.g/L or more, the movement velocity is significantly suppressed, and decreased by 27% -34% (p <0.01) in the high concentration group studied
After the culture is finished, the nematodes are collected and added onto an NGM plate without being coated with bacteria, the movement video of the nematodes in 20s is recorded by utilizing a caenorhabditis elegans full-automatic imaging and behavior tracker, and the movement distance is counted by analysis software. As shown in FIG. 1, the nematode locomotor distance was significantly reduced by 39% and 36% compared to the control group when the DBDPE exposure concentration was 500. mu.g/L and 1000. mu.g/L, respectively. (p < 0.01).
And 8, carrying out comprehensive biomarker response Index (IBR) analysis on the multiple detection index results at different concentrations, displaying the multiple biomarker results through a star chart, calculating the area of the star chart to obtain the comprehensive biomarker response index, and systematically evaluating the motor neurotoxicity of the chemicals at the environmental concentration.
In this step, the comprehensive biomarker response index of DBDPE exposure to C.elegans at Environmental Toxicology & Chemistry journal was calculated by referring to a comprehensive biomarker response index calculation method established in Belieff and Burgeot et al 2002 (Integrated biomarker response: A useful tool for Environmental risk assessment).
Firstly, calculating the total average value (m) of the head swing, body bending, pumping throat, movement speed, amplitude, speed and distance of the movement behavior indexes at all concentrations to be 46.8, 9.5, 55.8, 308.8, 43.0, 144.1 and 3105.3 respectively and the standard deviation(s) to be 5.29, 1.82, 5.26, 30.31, 6.32, 22.15 and 1451.66 respectively according to the average value (Xi) of each movement behavior index in each concentration group of 0, 1, 10, 100, 500 and 1000 mug/L;
carrying out standardized calculation on the average value Xi value to obtain a processed value Yi, wherein the calculation formula is that Yi is (Xi-m)/s;
if the exercise behavior index is in positive correlation with the exposure concentration, Z is equal to Yi, otherwise Z is equal to-Yi. Calculating a score S ═ Z + min |, of the athletic performance index at each exposure concentration according to the analysis result of the athletic performance index in the embodiment, wherein | min |, the smallest absolute value of Z is 0.13, 0.05, 0.27, 0.5, 0.38, 0.03 and 0.16 respectively;
The S value is used as a star chart as shown in FIG. 2, the area of the star chart of each exposure concentration is an IBR value, and the calculation formula is as follows:
wherein: IBR (Integrated Biomarker Response index) refers to the integrated marker Response index;
Airepresenting the area of a triangle formed by the surrounding of the biomarker i on two adjacent radiation lines;
Siand Si+1Represents the S value of the biomarker i on two radial lines which are continuous in the clockwise direction;
α ═ 2 π/n, which refers to the angle between two adjacent rays, n being the number of biomarkers;
the calculation results show that the comprehensive biomarker response index IBR values are 0.61, 0.41, 0.45, 3.22 and 3.77 respectively at different DBDPE exposure concentrations of 0, 1, 10, 100, 500 and 1000 mug/L. Wherein, the response indexes of the comprehensive biomarkers of the DBDPE exposure groups of 500 and 1000 mug/L are far higher than those of the comprehensive biomarkers of other concentration groups, which indicates that the motor behavior indexes of the caenorhabditis elegans under the DBDPE exposure condition of the higher concentration group are obviously influenced, have stronger motor neurotoxicity to the caenorhabditis elegans and have potential nervous system hazard risks which are worthy of attention.
Claims (9)
1. A method for comprehensively analyzing the motor neurotoxicity of chemicals by using caenorhabditis elegans, which comprises the following steps:
Step 1, preparing a uracil-deficient escherichia coli liquid culture medium, placing the uracil-deficient escherichia coli liquid culture medium in a high-pressure steam sterilization pot, sterilizing the uracil-deficient escherichia coli liquid culture medium for 30min, selecting colonies, placing the colonies in the culture medium, and performing shake culture in a shake culture box at 37 ℃ and 220rpm for 16h to serve as nematode food for later use;
step 2, preparing a solid culture medium special for culturing nematodes, placing the solid culture medium in a high-pressure steam sterilization pot, sterilizing the solid culture medium for 30min at 120 ℃, preparing a culture plate, and cooling the culture plate for later use;
step 3, preparing a lysis solution, performing synchronization treatment on the caenorhabditis elegans in the pregnancy period by using the lysis solution, centrifuging to remove a supernatant, transferring the collected worm eggs to the center of a new solid culture medium, and incubating for 16-18h at 20 ℃;
step 4, determining a real environment medium of the chemicals, and preparing chemical exposure solutions with different concentration gradients under the environment concentration; wherein the environmental medium comprises the concentration ranges of soil, water, sediment and organisms;
step 5, collecting successfully-incubated larvae of caenorhabditis elegans in L1 stage, uniformly adding the larvae into prepared exposure solutions with different concentration gradients, carrying out exposure culture in a constant-temperature incubator at 20 ℃ for 72h under the dark condition, and regularly feeding a specific amount of food;
step 6, collecting the caenorhabditis elegans subjected to exposure of different concentration gradients, cleaning for 3 times by using a k liquid, placing the caenorhabditis elegans on a solid culture medium without food, placing the solid culture medium under a stereoscopic microscope, observing and recording the head swinging frequency, the body bending frequency and the number of times of pumping throat of the caenorhabditis elegans within 20s, and calculating the head swinging frequency, the body bending frequency and the frequency of pumping throat;
Step 7, collecting the caenorhabditis elegans subjected to exposure of different concentration gradients, cleaning for 3 times by using a k liquid, and detecting the movement wavelength, the movement amplitude, the movement speed and the movement distance of the caenorhabditis elegans by using a caenorhabditis elegans full-automatic imaging and behavior tracker;
and 8, carrying out comprehensive biomarker response Index (IBR) analysis on the results of the multiple detection indexes at different concentrations, displaying the results of the multiple biomarkers through a star chart, calculating the area of the star chart to obtain the comprehensive biomarker response index, and systematically evaluating the motor neurotoxicity of the chemicals at the environmental concentration.
2. The method of claim 1, wherein the concentration range of the reagent prepared in step 4 is designed to be within the range of practical environmental concentrations, and the highest concentration of the reagent is designed to be higher than the current practical environmental concentration with the increase of the annual chemical yield and chemical consumption.
3. The method of claim 1, wherein in step 6, the nematode is set to move sinusoidally along the x-axis, the number of head oscillations is defined as the number of changes of the C.elegans in one cycle along the y-axis bending direction within 1min, the number of body bends is defined as the number of movements of one wavelength along the x-axis within 1min, and the number of pumpingh is defined as the number of pumpingh beats once and returns to its original position within 20 s.
4. The method of claim 1, wherein in step 7, the nematode is set to move sinusoidally along the x-axis, the movement wavelength is defined as 2 times the distance between adjacent peaks and valleys, the movement amplitude is defined as the maximum displacement of the midpoint of the nematode along the y-axis, the movement distance is defined as the absolute length of the forward and backward movement of the midpoint of the caenorhabditis elegans within a certain time, and the movement velocity is defined as the absolute length of the forward and backward movement of the midpoint of the caenorhabditis elegans per unit time.
5. The method for the integrated analysis of the motor neurotoxicity of chemicals of caenorhabditis elegans as claimed in claim 1, wherein in step 1, 10g of peptone, 5g of yeast powder and 5g of NaCl are added to 1L of distilled water for volume determination, and after autoclaving, LB liquid medium is prepared.
6. The method for the integrated analysis of the motor neurotoxicity of chemicals by caenorhabditis elegans as claimed in claim 1, wherein 3g of NaCl and 17g of agar are added in step 2Powder, 2.5g peptone, 0.24g MgSO4And 0.111g of CaCL is added into 1L of distilled water for constant volume and is prepared into the NGM solid culture medium after autoclaving.
7. The method for comprehensively analyzing the motor neurotoxicity of chemicals by using caenorhabditis elegans as claimed in claim 1, wherein in step 3, the centrifugation speed is 2500-.
8. The method of claim 1, wherein the solution of step 3 is prepared by adding NaOH 0.4g and NaCLO 10ml, and adding distilled water to a volume of 40 ml.
9. The method for comprehensively analyzing the motor neurotoxicity of chemicals by caenorhabditis elegans as claimed in claim 1, wherein 2.386g of KCl and 2.98g of NaCl are added to 1L of distilled water to a constant volume in step 6, and the solution is prepared as a k solution.
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