Application of caenorhabditis elegans in detecting toxicity of novel brominated flame retardant

Abstract

The invention relates to the technical field of biology, in particular to application of caenorhabditis elegans to detection of toxicity of a novel brominated flame retardant. The invention provides a method for detecting the toxicity of a novel brominated flame retardant 1, 2-bis (2, 4, 6-tribromophenoxy) ethane based on caenorhabditis elegans. The toxicity of the novel brominated flame retardant is evaluated according to data differences of head swing, body bending, learning and memory chemotaxis behaviors, dopaminergic neuron damage and / or dopamine content of L1-stage caenorhabditis elegans exposed to a control group and an experimental group respectively; when the method is used for evaluating the toxicity of the novel brominated flame retardant, the method has the characteristics of simplicity in operation, simple process, low cost input, remarkably shortened experimental period and good repeatability, and has important significance for evaluating the toxicity of the novel brominated flame retardant; and a basic toxicity evaluation method is provided for researchers to find flame retardants with lower toxicity and lower harm.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to the application of Caenorhabditis elegans in detecting the toxicity of novel brominated flame retardants. Background Art

[0002] Due to the excellent flame retardant properties of brominated flame retardants (BFRs), they are used in various consumer products such as furniture, textiles, carpets, electronic castings, automotive parts, building materials, insulators, etc. to achieve the effect of fire prevention. However, with the wide application of brominated flame retardants, many research results show that the distribution of brominated flame retardants in the environment is characterized by universality and pervasiveness. Brominated flame retardants have low solubility and are hydrophobic and lipophilic, so they are not easily degraded in the environment and tend to exist in the environment for a long time. They have the potential for long-distance migration and are prone to bioaccumulation and biomagnification in the food chain and food web, and are very likely to accumulate in organisms, thereby having an adverse impact on organisms and even causing physical toxicity.

[0003] Therefore, as alternatives to the banned and restricted brominated flame retardants, some novel brominated flame retardants (NBFRs) that meet market demands have been introduced into the market. Some of the most common novel brominated flame retardants are decabromodiphenylethane (DBDPE), bis(2,4,6-tribromophenoxy)ethane (BTBPE), and tetrabromobisphenol A bis(2,3-dibromopropyl ether) (TBBPA-DBPE), which are used to replace decabromodiphenyl ether, octabromodiphenyl ether, and tetrabromobisphenol A (TBBPA), respectively.

[0004] 1,2-Bis(2,4,6-tribromophenoxy)ethane (BTBPE) is one of the common novel brominated flame retardants and has a wide range of applications in the fields of textiles, plastics, electronic devices, etc.; however, BTBPE does not chemically bond with the polymer matrix and can leach into various environmental matrices during its production, utilization, and disposal, and ultimately accumulate in environmental matrices and organisms, which may pose a potential threat to humans and the ecological environment. Therefore, developing a method for evaluating the toxicity of the novel brominated flame retardant 1,2-bis(2,4,6-tribromophenoxy)ethane (BTBPE) is crucial for studying its potential threat to humans and the ecological environment. Summary of the Invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide the application of Caenorhabditis elegans in detecting the toxicity of novel brominated flame retardants.

[0006] The present invention provides the application of Caenorhabditis elegans in detecting the toxicity of brominated flame retardants.

[0007] Furthermore, the brominated flame retardant includes 1,2-bis(2,4,6-tribromophenoxy)ethane.

[0008] The present invention provides a method for detecting the toxicity of brominated flame retardants, which determines whether there is a toxic effect of brominated flame retardants according to the data differences of the body indexes of Caenorhabditis elegans after exposure to the control group and the test group;

[0009] The body indexes include: head swing, body bend, learning and memory chemotaxis behavior, dopamine neuron damage and / or dopamine content;

[0010] The control group includes K solution and Escherichia coli;

[0011] The test group includes brominated flame retardant, K solution and Escherichia coli;

[0012] The K solution includes KCl and NaCl.

[0013] Furthermore,

[0014] The Caenorhabditis elegans is L1-stage Caenorhabditis elegans.

[0015] The exposure time is 72 h.

[0016] The concentration of the brominated flame retardant is 0.1 - 100 μg / L.

[0017] The Escherichia coli is uracil-deficient Escherichia coli E. coli OP50.

[0018] The K solution includes: 1 g / L - 4 g / L KCl and 1 g / L - 4 g / L NaCl.

[0019] The L1-stage Caenorhabditis elegans is obtained by incubating nematode eggs in M9 medium at 20 °C in the dark without food for 18 - 24 h.

[0020] Furthermore, the L1-stage Caenorhabditis elegans is obtained by using a lysis solution to lyse gravid nematodes to obtain a certain number of eggs, and then incubating them in M9 medium at 20 °C in the dark without food for 18 - 24 h.

[0021] The lysis solution includes a sodium hydroxide solution and a sodium hypochlorite stock solution with a volume ratio of (0.1 - 10):1, and the concentration of the sodium hydroxide solution is 0.5 - 5 mol / L. In some specific implementation manners, the lysis solution includes 0.5 mol / L NaOH and NaClO stock solution with a volume ratio of 4:1.

[0022] In the present invention, after the nematodes are subjected to the exposure treatment, the body indexes are measured, and the specific methods for measuring the body indexes are as follows:

[0023] The method for measuring the head swing is as follows: After the nematodes are placed in a 24-well plate containing 1 mL of buffer and allowed to freely recover for 1 min, the head swing of the nematodes is obtained by using a stereomicroscope connected to a CCD camera; the definition of the head swing is: When the head swings from the original direction to the other side at an angle greater than 90 degrees and then swings back to the original direction, it is counted as one time, and the number of head swings within 60 s is counted;

[0024] The method for measuring the body bend is as follows: After the nematodes are placed on culture plate 1 without food and allowed to recover for 1 min, the body bend of the nematodes is obtained by using a stereomicroscope connected to a CCD camera; the definition of the body bend is: In a complete sinusoidal motion, with the movement direction of the nematode as the x-axis and the swing direction of the body as the y-axis, the number of body bends of the nematode within 20 s is recorded.

[0025] The culture plate 1 can be NGM medium, and the present invention does not limit this.

[0026] The method for measuring the learning and memory chemotactic behavior is as follows: After washing the nematodes, place them on a NaCl agar plate without food and culture them at 20 °C for 4 hours to establish the learning and memory of hunger and NaCl. Subsequently, place the nematodes at the starting point of culture plate 2 and culture them in the dark at 20 °C for 30 minutes, then count the number of nematodes within a radius of 2 cm at the experimental point and the reference point, and calculate the chemotaxis index (Cl) = (number of nematodes at the experimental point - number of nematodes at the reference point) / total number of nematodes. The chemotaxis index is used as an evaluation parameter for the learning and memory chemotactic behavior.

[0027] The components of the medium of the culture plate 2 include: potassium phosphate, CaCl2, MgSO4, agar, pH 6.0; the starting point, the experimental point, and the reference point are arranged in an isosceles triangle on the culture plate 2, and the side length of the isosceles triangle is 4 cm; among them, the experimental point is obtained by placing a NaCl agar block, and the NaCl agar block is removed before the experiment; and 1 μL of 1 M NaN3 is added dropwise at the experimental point and the reference point for anesthesia treatment;

[0028] The method for measuring the damage of dopamine neurons is as follows: After washing the dopamine neuron-labeled nematodes, add them to a 60 μg / mL levamisole solution for anesthesia. After the nematodes are stationary, use a fluorescence microscope to take pictures of the neurons with green fluorescence labels on the heads of the nematodes under the conditions of Ex / Em = 488 nm / 525 nm; use Image-Pro Plus 6.0 software to process the pictures and calculate the fluorescence intensity (fluorescence intensity = IOD / area).

[0029] The method for determining the dopamine content is as follows: In this experiment, the dopamine content in nematodes was measured by enzyme-linked immunosorbent assay (ELISA). The specific steps are as follows: After washing the exposed nematodes, they were broken at low temperature, and the supernatant was taken by centrifugation as the nematode tissue fluid. Blank wells, standard wells, and sample wells to be tested were set on the enzyme-linked immunosorbent assay plate. After adding the corresponding reagents, they were incubated at 37°C for 30 minutes. After washing, enzyme-labeled reagents were added and incubated again for 30 minutes. After adding the chromogenic agent for 10 minutes of color development, the reaction was terminated by adding the termination solution. The absorbance at 450 nm was measured within 15 minutes, and the dopamine content was calculated according to the standard curve, and the data was normalized.

[0030] After treating the nematodes in the control group and the test group, the above physical indicators were measured according to the above method, and whether there was a toxic effect of the brominated flame retardant was determined based on the data differences of the physical indicators.

[0031] The standard for whether there is a toxic effect of the brominated flame retardant is as follows:

[0032] If the data difference between the experimental group and the control group is not significant (P>0.05), it is determined that the brominated flame retardant has no toxic effect;

[0033] If the data difference between the experimental group and the control group is significant (P<0.05), it is determined that the brominated flame retardant has a toxic effect.

[0034] The present invention provides a method for evaluating the toxicity effect of a novel brominated flame retardant, which exposes synchronized L1-stage Caenorhabditis elegans to a liquid medium containing the novel brominated flame retardant, and evaluates whether the sample has a toxic effect according to at least one of the indicators such as the body length, head swing, body bend, feeding rate, number of offspring, and / or egg hatching rate of Caenorhabditis elegans.

[0035] The present invention provides a method for detecting the toxicity of the novel brominated flame retardant 1,2-bis(2,4,6-tribromophenoxy)ethane based on Caenorhabditis elegans; it evaluates the toxicity of the novel brominated flame retardant according to the data differences of the head swing, body bend, learning and memory chemotactic behavior, dopamine neuron damage, and / or dopamine content of L1-stage Caenorhabditis elegans exposed to the control group and the experimental group respectively; using the method described in the present invention to evaluate the toxicity of the novel brominated flame retardant, it has the characteristics of simple operation, streamlined process, low cost investment, significantly shortened experimental period, and good repeatability, which is of great significance for the toxicity assessment of novel brominated flame retardants, and provides a basic toxicity assessment method for researchers to find flame retardants with lower toxicity and less harm. Description of the Drawings

[0036] Figure 1 Schematic diagram of the preparation of the culture plate for demonstrating the learning and memory chemotactic ability of nematodes;

[0037] Figure 2Characterization diagram of the effect of 1,2-bis(2,4,6-tribromophenoxy)ethane on the head swing of nematodes in the present invention;

[0038] Figure 3 Characterization diagram of the effect of 1,2-bis(2,4,6-tribromophenoxy)ethane on the body bending of nematodes in the present invention;

[0039] Figure 4 Characterization diagram of the effect of 1,2-bis(2,4,6-tribromophenoxy)ethane on the chemotactic ability of nematode learning and memory in the present invention;

[0040] Figure 5 Characterization diagram of the effect of 1,2-bis(2,4,6-tribromophenoxy)ethane on dopamine neurons of nematodes in the present invention;

[0041] Figure 6 Characterization diagram of the effect of 1,2-bis(2,4,6-tribromophenoxy)ethane on dopamine content of nematodes in the present invention. Detailed implementation manners

[0042] The present invention provides an application of Caenorhabditis elegans in detecting the toxicity of novel brominated flame retardants. Those skilled in the art can draw on the content of this article and appropriately modify the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and those skilled in the art can obviously make changes or appropriate modifications and combinations to the methods and applications in this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0043] Chemotaxis index (Cl, ranging from -1 to 1), which gradually increases within the range of -1 to 0, indicating that the chemotactic response of nematodes to specific chemical substances changes from negative chemotaxis to non-chemotaxis or positive chemotaxis.

[0044] BZ555 nematodes are a strain of Caenorhabditis elegans used to label dopaminergic neurons.

[0045] In the following examples, the wild-type Caenorhabditis elegans strain was the wild-type (Bristol N2) Caenorhabditis elegans generously provided by the Caenorhabditis Genetics Center (CGC), University of Minnesota, USA.

[0046] The 1,2-bis(2,4,6-tribromophenoxy)ethane (BTBPE) (CAS number: 37853-59-1, ≥98%) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0047] The dimethyl sulfoxide (DMSO) was purchased from Shanghai Sinopharm Chemical Reagent Co., Ltd.

[0048] Preparation of nematode growth medium (NGM): 400 mL of deionized water, 1 g of peptone, 1.2 g of NaCl, 6.8 g of agar. After mixing, it was sterilized at 121 ºC for 20 min. When the temperature dropped to about 60 ºC, 10 mL of K3PO4 buffer solution (prepared by dissolving 10.83 g of KH2PO4 and 4.66 g of K2HPO4∙3H2O in 100 mL of deionized water and sterilizing at 121 ºC for 20 min before use), 0.4 mL of 1 M CaCl2, 0.4 mL of 1 M MgSO4, and 0.4 mL of ethanol solution of 5 mg / mL cholesterol were added. After mixing, it was poured into petri dishes. After natural drying in a laminar flow hood, it was reserved for use.

[0049] The lysis solution includes: 800 mL of 0.5 M NaOH and 200 mL of NaClO.

[0050] The buffer solution is K solution, including 1.521 g of NaCl and 1.192 g of KCl.

[0051] The test materials used in the present invention are all ordinary commercially available products and can be purchased in the market. The present invention will be further described below in conjunction with the examples:

[0052] Example 1 Cultivation and exposure of Caenorhabditis elegans

[0053] The cultivation of nematodes needs to be carried out under constant temperature and light avoidance conditions. The temperature is set at a constant 20 ºC. The nematodes are cultivated on NGM plates with food (NGM plates containing Escherichia coli OP50 lawns). The plate transfer operation needs to be carried out in a timely manner according to the remaining amount of food to avoid starvation of the nematodes, which is not conducive to the survival and development of the nematodes. In order to obtain nematodes at the same growth stage and ensure the uniformity of the experiment, synchronization operation is usually adopted. During the nematode egg-laying stage, the outer wall can be broken by the lysis solution to obtain only eggs. The nematodes at the egg-laying stage are washed off the plate, allowed to settle naturally, and the supernatant is removed. After repeating the above operation 2-3 times, the lysis solution is added. The synchronized eggs are placed in M9 for cultivation and incubated in the dark without food at 20 ºC. After about 18-24 h, L1-stage larvae can be obtained for subsequent experiments.

[0054] For exposure, L1-stage nematodes obtained after synchronization are selected, and liquid exposure is used. The exposure time is 72 h, and some indicators are exposed for 48 h. The exposure medium is K solution. A 24-well plate is used as the exposure vessel, and each well is set as a 1 mL exposure system. The exposure system includes: bacterial solution, poison (BTBPE), K solution (ensuring the OD of the bacteria after addition 600In the range of 0.6 - 0.8, the concentration of the toxicant was set in a gradient within the range of 0.1 - 100 μg / L). BTBPE (solid powder) was dissolved in DMSO to prepare a BTBPE stock solution, and then diluted with K solution later. The concentration of DMSO was 0.01% (v / v), which had no effect on nematodes.

[0055] Example 2 Determination of the head swing of Caenorhabditis elegans

[0056] In this example, the head swing of nematodes was obtained according to the following steps: After exposing L1-stage Caenorhabditis elegans for 72 h, the nematodes were collected and washed, and then transferred to a 24-well plate containing 1 mL of buffer to facilitate their free swing in the liquid. After freely recovering for 1 min in the buffer, a stereomicroscope was connected to a CCD camera, and the head swing of the nematodes could be clearly observed. The head swing was defined as the head swinging from the original direction to the other side at an angle greater than 90 degrees and then swinging back to the original direction, which was recorded as one time, and the number of head swings within 60 s was counted.

[0057] The results are as Figure 2 shown. It can be seen from Figure 2 that compared with the control group, the head swing frequency of nematodes in the 0.1 - 100 μg / L experimental groups all showed a significant decrease (* indicates a significant difference, P < 0.05), and there was a dose effect. It was determined that BTBPE had an obvious toxic effect on the head swing of nematodes.

[0058] Example 3 Determination of the body bend of Caenorhabditis elegans

[0059] In this example, the body bend of nematodes was obtained according to the following steps: After exposing L1-stage Caenorhabditis elegans for 72 h, the exposed nematodes were collected and washed, and then transferred to an NGM plate without food to recover for 1 min on the plate. A stereomicroscope was connected to a CCD camera to observe the movement of the nematodes. A single body bend was defined as a complete sine movement. The movement direction of the nematode was regarded as the x-axis, and the swinging direction of the body was regarded as the y-axis, and the number of body bends of the nematodes within 20 s was recorded.

[0060] The results are as Figure 3 shown. It can be seen from Figure 3 that compared with the control group, the body bend frequency of nematodes in the 0.1 - 100 μg / L experimental groups all showed a significant decrease (* indicates a significant difference, P < 0.05), and there was a dose effect. It was determined that BTBPE had an obvious toxic effect on the body bend of nematodes.

[0061] Example 4 Determination of the chemotactic behavior of learning and memory of Caenorhabditis elegans

[0062] In this example, the chemotactic behavior of nematode learning and memory was obtained according to the following steps: Prepare the required petri dishes (φ: 9 cm) in advance. As Figure 1 shown, prepare the petri dishes according to the following ratio (5 mmol / L potassium phosphate, 1 mmol / L CaCl2, 1 mmol / L MgSO4, 20 g / L agar, pH 6.0). Place the NaCl agar block 3 cm away from the center of the petri dish and let it stand at 4 °C for 14 h to obtain a NaCl concentration gradient centered on the experimental point on the test petri dish. Remove the NaCl agar block 15 min before the experiment, set a reference point on the opposite side, about 4 cm away from the experimental point. Add 1 μL of 1 M NaN3 as anesthetic to the reference point and the experimental point respectively. After washing the exposed nematodes, place them on a NaCl agar plate without food and culture at 20 °C for 4 h to establish the learning and memory of hunger and NaCl for the nematodes. At this time, the chemotaxis experiment can be carried out. Place about 100 nematodes at the starting point and culture them in the dark at 20 °C for 30 min, then count the number of nematodes within a radius of 2 cm from the experimental point and the reference point. The chemotaxis index (Cl) is calculated by subtracting the number of nematodes near the control point from the number of nematodes near the NaCl experimental point and then dividing by the total number of nematodes on the culture plate.

[0063] The results are as Figure 4 shown. As can be seen from Figure 4 Figure, compared with the control group, there were significant differences in the chemotactic ability of learning and memory in the 10 μg / L and 100 μg / L experimental groups of nematodes (* indicates significant differences, P < 0.05). It was determined that BTBPE had an obvious toxic effect on the chemotactic ability of nematode learning and memory.

[0064] Example 5 Determination of damage to dopamine neurons in Caenorhabditis elegans

[0065] In this example, the damage to neurons in Caenorhabditis elegans was specifically measured according to the following method: Label the dopaminergic neurons of BZ555 nematodes, which are mainly distributed in the head and tail, and photograph the head of the nematodes with a 20-fold eyepiece. Collect the green fluorescent-labeled neuron BZ555 nematodes after exposure, wash them with PBS, take a glass slide, drop 20 μL of levamisole solution with a concentration of 60 μg / mL (diluted with M9) in the center, aspirate about 60 nematodes into the droplet, wait for 3 - 5 min until the nematodes stop moving, then cover the glass slide, observe the nematodes with a fluorescence microscope, observe the damage of the green fluorescent-stained neurons, photograph the nematodes under the condition of Ex / Em = 488 nm / 525 nm, photograph at least 30 nematodes for each concentration, and process the fluorescence images with Image-Pro Plus 6.0. Fluorescence intensity = IOD / Area. Set three parallels for each concentration group.

[0066] The results are as Figure 5As shown, from Figure 5 it can be seen that the dopamine neurons of nematodes in the experimental groups with concentrations of 1 - 100 μg / L showed fractures and deficiencies, and there were significant differences in relative fluorescence intensity (* indicates significant differences, P < 0.05), and there was a dose effect. It was determined that BTBPE had an obvious toxic effect on the dopamine neurons of nematodes.

[0067] Example 6 Determination of Dopamine Content in Caenorhabditis elegans

[0068] In this example, the content of dopamine (one of the important neurotransmitters) in nematodes was measured specifically according to the following method: Collect the exposed nematodes, wash them with PBS, transfer them to a 1.5 mL centrifuge tube, retain 300 μL of the worm solution, add the prepared grinding beads, place them in a high - speed tissue grinder, and break them at a frequency of 65 Hz and low temperature for 2 - 4 min. After breaking, centrifuge at 4°C and 6000 g for 2 min in a centrifuge, and transfer the supernatant to a pre - cooled centrifuge tube, which is the nematode tissue fluid. The standard product was serially diluted according to the concentration gradient with the diluent. Blank holes (no samples and enzyme - labeled reagents were added to the blank control holes, and the other operations were the same), standard holes, and sample holes to be measured were set respectively. Accurately add 50 μL of the standard product to the standard holes on the enzyme - labeled coated plate. First add 40 μL of the sample diluent to the sample holes to be measured, and then add 10 μL of the sample to be measured. Add the sample to the bottom of the enzyme - labeled plate wells, try not to touch the well walls, and gently shake to mix evenly. Seal the plate with a sealing film and place it in an incubator at 37°C for 30 min. Carefully remove the sealing film, discard the liquid, shake dry, fill each well with the washing solution, let it stand for 30 seconds and then discard, repeat this 5 times, and pat dry. Add 50 μL of the enzyme - labeled reagent to each well, except for the blank hole, and place it at 37°C for another 30 min of incubation, then shake dry and wash 5 times. First add 50 μL of chromogenic reagent A to each well, then add 50 μL of chromogenic reagent B, gently shake to mix evenly, develop color at 37°C in the dark for 10 minutes, add 50 μL of the stop solution to each well to terminate the reaction (at this time, the blue immediately turns yellow). Zero the absorbance of each well with the value of the blank hole, measure the absorbance (450 nm) of each well within 15 min, and calculate the dopamine content according to the standard curve drawn from the standard samples. Measure the protein concentration of the tissue fluid in each concentration group and normalize the experimental data.

[0069] The results are as Figure 6 shown, from Figure 6 it can be seen that compared with the control group, the dopamine content of nematodes in the experimental groups with concentrations of 0.1 μg / L - 100 μg / L was significantly reduced, showing significant differences (* indicates significant differences, P < 0.05). It was determined that BTBPE had an obvious toxic effect on the dopamine content of nematodes.

[0070] The above content is only the preferred implementation method of the present invention. It should be noted that those of ordinary skill in the art can make a series of improvements and optimizations without departing from the basic principles of the present invention. These improvements and refinements based on the present invention should also be included within the scope of protection of the present invention.

Claims

1. Application of Caenorhabditis elegans in detecting the toxicity of brominated flame retardants.

2. The use according to claim 1, characterized in that: The brominated flame retardant includes 1,2-bis(2,4,6-tribromophenoxy)ethane.

3. A method for detecting the toxicity of brominated flame retardants, characterized in that: Based on the data differences of physical indicators of Caenorhabditis elegans after exposure to the control group and the test group, it is determined whether there is a toxic effect of brominated flame retardants; The physical indicators include: head shaking, body bending, learning and memory chemotaxis behavior, dopamine neuron damage and / or dopamine content; The control group includes K solution, Escherichia coli and Caenorhabditis elegans; The test group includes brominated flame retardants, K solution, Escherichia coli and Caenorhabditis elegans; The K solution includes KCl and NaCl.

4. The detection method according to claim 3, characterized in that: The Caenorhabditis elegans is the Caenorhabditis elegans at the L1 stage.

5. The detection method according to claim 3, characterized in that: The exposure time was 72 h.

6. The detection method according to claim 3, characterized in that: The concentration of the brominated flame retardant is 0.1-100 μg / L.

7. The detection method according to claim 3, characterized in that: The Escherichia coli is uracil-deficient Escherichia coli OP50.

8. The detection method according to claim 3, characterized in that: The K solution includes: 1g / L~4g / L KCl and 1g / L~4g / L NaCl.

9. The detection method according to claim 3, characterized in that: The criteria for determination are: There was no significant difference in the data between the experimental group and the control group (P>0.05), which indicated that the brominated flame retardant had no toxic effect; The data of the experimental group and the control group were significantly different (P<0.05), indicating that brominated flame retardants have toxic effects.

10. The detection method according to claim 4, characterized in that: The L1 stage Caenorhabditis elegans is obtained by incubating nematode eggs in M9 medium at 20° C. in the dark and without food for 18 to 24 hours.

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