CN109342709A - A method of based on caenorhabditis elegan detection plasticiser more than generation cumulative toxicity - Google Patents
A method of based on caenorhabditis elegan detection plasticiser more than generation cumulative toxicity Download PDFInfo
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Abstract
The invention belongs to technical field of analysis and detection, in particular to a kind of based on caenorhabditis elegan detection plasticiser more than generation cumulative toxicity method, including preparing the caenorhabditis elegan worm's ovum solution in contemporaneity and then obtaining larva solution, prepare Escherichia coli OP50 bacterium solution, concentration gradient carries out contamination culture and calculates the LC50 of plasticiser sample, it obtains P0 generation, F1 generation, F2 generation, the service life in F3 generation, body length, body bending and head oscillation Frequency Index respectively under the plasticiser sample concentration of 1/1000 concentration of LC50, evaluates the generation cumulative toxicity classification of plasticiser sample.The present invention provides a kind of method of intergenerational cumulative toxicity convenient and efficient, low in cost, easy to operate with caenorhabditis elegan measurement phthalate plasticiser, and the toxicity research method under simulation actual environment exposure situation is supplemented and extended.
Description
Technical field
It is the invention belongs to technical field of analysis and detection, in particular to a kind of mostly to be stored from generation to generation based on caenorhabditis elegan detection plasticiser
The method of product toxicity.
Background technique
Caenorhabditis elegan (Caenorhabditis elegans) is that a kind of pair of environmental factor changes very sensitive mode life
Object has the advantages that the breeding cycle is short, is easy to cultivate, is widely used in the Environmental Toxicological of heavy metal, nano material in recent years
It learns in evaluation.Caenorhabditis elegan genetic background understands there is similar cellular elements structure and signal path with higher mammal, can
To parse the toxic effect and mechanism of action of environmental poisonous substance from macroscopic view and molecular level；Movement rate, growth and development and life cycle
Etc. indexs easily surveyed due to simple, frequently as the index in toxic effect evaluation.
Phthalate plasticiser is a kind of high molecular material auxiliary agent, extensive because the flexibility of plastics can be improved
Applied in the products such as plastic toy, various packaging materials, sebific duct, vinyl flooring.With caenorhabditis elegan measurement plasticiser toxicity
Method has possessed some special knowledge, and method is related to plasticiser to behaviors such as caenorhabditis elegan motor behavior, metabolism, Fat Accumulation and service life
With many aspects such as the influence of metabolic index.Plasticiser is the reality of a kind of pollutant existed in the environment, the mankind and organism
The border way of contact is more generations, repetition or continues, the contact of low dosage, Study on Acute Toxic Effects will not occur, but long-term low concentration connects
Touching may generate unexpected effect, research and the long-term toxicity for repeating contact poison and acting on compound for determining compound
It is most important in Effect Evaluation.Existing research usually only receives exposure in the certain time period of caenorhabditis elegan life cycle, not
It is the exposure of entire life span, does not account for the sensitization time section from egg development to adult, it is more not account for caenorhabditis elegan yet
The real environmental conditions that a generation persistently exposes, thus can not also judge toxic effect caused by the plasticiser in true environment and
Whether ecological risk, being unable to characterize plasticiser has cumulative toxicity effect to the long term of environmental organism and its filial generation.And such as
Fruit is then that can not embody more generation sustained low doses using lethal dose as assessment endpoint using traditional cumulative toxicity method
The actual environment exposure level of contact.
Summary of the invention
The present invention provides a kind of caenorhabditis elegan measurement phthalic acid ester convenient and efficient, low in cost, easy to operate
The method of the intergenerational cumulative toxicity of class plasticiser mends the toxicity research method under simulation actual environment exposure situation
It fills and extends.
The present invention provides a kind of method based on generation cumulative toxicity more than caenorhabditis elegan detection plasticiser, including following steps
It is rapid:
Egg-laying season caenorhabditis elegan is cleaned with kaliumphosphate buffer and collected to S1, culture caenorhabditis elegan to the egg-laying season, then with splitting
Solution liquid cracking caenorhabditis elegan parent simultaneously collects the worm's ovum for having resistivity to lysate, is cleaned with kaliumphosphate buffer, phosphorus is added
Sour potassium buffer adjustment caenorhabditis elegan worm's ovum concentration is 10 pieces/μ L, obtains the worm's ovum solution in contemporaneity；
Worm's ovum hydroponics 12-18h in S1 is made egg hatch but is maintained at L1 larva period, L1 larva is trained by S2
It supports to L4 larva period, being cleaned with kaliumphosphate buffer and adjusting L4 larva concentration is 19-21 item/100 μ L, obtains larva solution；
Escherichia coli OP50 solution absorbance 1.2-1.4 in wavelength 570nm is cleaned and adjusted with kaliumphosphate buffer, obtains bacterium solution；
S3 is arranged different plasticiser sample concentration gradients to be checked, sequentially adds in S2 in each concentration plasticiser sample
Larva solution and bacterium solution, contamination culture 48h at 20 DEG C, 4h/ record polypide death toll, the LC50 of calculating plasticiser sample；
Wherein, plasticiser sample: larva solution: the volumetric usage ratio of bacterium solution is 2:1:1；
S4, prepares the plasticiser sample of 1/1000 concentration of LC50, is added the worm's ovum solution in S1, the bacterium solution in S2, and 20
Contamination culture 48h, is denoted as P0 generation at DEG C, and then measurement P0 refers to for service life, body length, body bending and head oscillation frequency respectively
Mark；
Wherein, plasticiser sample: worm's ovum solution: the volumetric usage ratio of bacterium solution is 2:1:1；
In P0 generation in S4, is cleaned and is collected with kaliumphosphate buffer, repeated S1, S4 and obtain F1 generation, F2 generation, F3 respectively by S5
The service life in generation, body is long, body is bent and head oscillation Frequency Index；
S6, if F1 generation and P0 are statistically significant for any one of each indicator difference, plasticiser sample has explicitly
Generation cumulative toxicity；If F1 generation and P0 are not statistically significant for each indicator difference, and F2 generation with P0 in each indicator difference
Any one is statistically significant, then plasticiser sample has medium generation cumulative toxicity；
If F1 generation and P0 generation, F2 generation with P0 it is not statistically significant for each indicator difference, and F3 generation with P0 for each index
Any one of difference is statistically significant, then plasticiser sample has slight generation cumulative toxicity；If F1 generation and P0 generation, F2 generation
Not statistically significant for each indicator difference with P0 with P0 generation, F3 generation, then plasticiser sample is without clear generation cumulative toxicity.
Preferably, above-mentioned plasticiser sample is phthalate plasticiser.
Preferably, the condition of culture of caenorhabditis elegan is equal in above-mentioned S1, S2 are as follows: cultivates in NGM culture medium at 20 DEG C.
Preferably, the KCl of the NaCl and 32mmol/L in above-mentioned kaliumphosphate buffer containing 53mmol/L.
Preferably, in above-mentioned S1 lysate composition: the NaOH and mass concentration of 0.45mol/L be 2%NaClO.
Preferably, the dimethyl sulfoxide solution for being 1% using volumetric concentration when above-mentioned preparation plasticiser sample solution is solvent.
Preferably, concentration≤20mg/mL of above-mentioned plasticiser sample solution.
Compared with prior art, beneficial effects of the present invention:
The present invention provides a kind of caenorhabditis elegan measurement phthalic acid ester convenient and efficient, low in cost, easy to operate
The method of the intergenerational cumulative toxicity of class plasticiser, more close to plasticiser actual environment exposure situation, to simulation actual environment
Toxicity research method under exposure situation is supplemented and has been extended；Plasticiser is carried out using caenorhabditis elegan to cover P0 generation, F1
Generation, F2 generation, F3 to detect the long term toxicity effect of the plasticiser, and judge to be somebody's turn to do for the persistence exposure in multiple successive generations
Whether poisonous effect has cumulative toxicity effect under long-term intergenerational exposure condition.
Detailed description of the invention
Fig. 1 is influence diagram of the DEHP to the caenorhabditis elegan service life of 1/1000 concentration of LC50 in the embodiment of the present invention 1；
Fig. 2 is the DEHP of 1/1000 concentration of LC50 in the embodiment of the present invention 1 influence diagram long to caenorhabditis elegan body；
Fig. 3 is shadow of the DEHP to caenorhabditis elegan body crooked behavior of 1/1000 concentration of LC50 in the embodiment of the present invention 1
Ring figure；
Fig. 4 is shadow of the DEHP to caenorhabditis elegan head oscillation behavior of 1/1000 concentration of LC50 in the embodiment of the present invention 1
Ring figure；
Fig. 5 is influence of the DBP of 1/1000 concentration of LC50 in the embodiment of the present invention 2 to the caenorhabditis elegan different generations service life
Figure；
Fig. 6 is the DBP of 1/1000 concentration of LC50 in the embodiment of the present invention 2 influence long to caenorhabditis elegan different generations body
Figure；
Fig. 7 is that the DBP of 1/1000 concentration of LC50 in the embodiment of the present invention 2 is bent row to caenorhabditis elegan different generations body
For influence diagram；
Fig. 8 is the DBP of 1/1000 concentration of LC50 in the embodiment of the present invention 2 to caenorhabditis elegan different generations head oscillation row
For influence diagram；
Wherein, " * " in Fig. 2-Fig. 4, Fig. 6-Fig. 8 indicates that P < 0.05, " * * " indicate P < 0.01.
Specific embodiment
The specific embodiment of the present invention is described in detail in 1-8 with reference to the accompanying drawing, it is to be understood that this hair
Bright protection scope is not limited by the specific implementation.
Embodiment 1
A method of based on caenorhabditis elegan detection plasticiser more than generation cumulative toxicity, which is characterized in that including following step
It is rapid:
S1 cultivates caenorhabditis elegan at 20 DEG C to the egg-laying season in conventional NGM culture medium, is cleaned and received with kaliumphosphate buffer
Collect egg-laying season caenorhabditis elegan, then crack caenorhabditis elegan parent with lysate and collect the worm's ovum for having resistivity to lysate, uses
Kaliumphosphate buffer cleaning, it is 10 pieces/μ L that kaliumphosphate buffer adjustment caenorhabditis elegan worm's ovum concentration, which is added, is obtained in contemporaneity
Worm's ovum solution；
The composition of lysate: the NaOH and mass concentration of 0.45mol/L is 2%NaClO.
S2 will cultivate 12-18h in conventional NGM culture medium at 20 DEG C of worm's ovum solution in S1, make egg hatch but holding
In L1 larva period, L1 larva was cultivated to L4 larva period, being cleaned with kaliumphosphate buffer and adjusting L4 larva concentration is 19-
21/100 μ L, obtain larva solution；
Escherichia coli OP50 is cultivated into 24-48h, 4000rpm under the conditions of 37 DEG C, 200rpm with existing LB liquid medium
It is centrifuged 5-10min, supernatant is abandoned and retains precipitating, cleaned with kaliumphosphate buffer and adjust Escherichia coli OP50 solution in wavelength
Absorbance 1.2-1.4 when 570nm, obtains bacterium solution；
S3 waits logarithms spacing that 10 plasticiser sample concentration gradients to be checked are arranged, in each concentration plasticiser sample successively
The larva solution in S2 and bacterium solution is added, contamination culture 48h at 20 DEG C, 4h/ times record polypide death toll (is seen under stereomicroscope
Survey, polypide is stiff, regards as death), using log concentration as abscissa, the death rate is ordinate, is calculated using method of linear interpolation
The half lethal concentration of plasticiser sample, i.e. LC50；
If the death rate caused by set concentration gradient is respectively less than 50%, keep sample to be tested minimum concentration constant,
It is appropriate to increase used equal logarithms spacing, set higher concentration gradient；If death caused by set concentration gradient
Rate is all larger than 50%, then keeps the maximum concentration of sample to be tested constant, appropriate to increase used equal logarithms spacing, sets lower
Concentration gradient；Then contamination culture is carried out, until obtaining LC50.If maximum concentration has reached 20mg/mL and the death rate still
Lower than 50%, then with 20mg/mL calculating standard subsequent；
Wherein, plasticiser sample in S3: larva solution: the volumetric usage ratio of bacterium solution is 2:1:1；
S4, prepares the plasticiser sample of 1/1000 concentration of LC50, is added the worm's ovum solution in S1, the bacterium solution in S2, and 20
Contamination culture 48h (worm's ovum has grown up to adult), is denoted as P0 generation at DEG C, then respectively measurement P0 for the service life, body is long, body bending and
Head oscillation Frequency Index；
Wherein, plasticiser sample in S4: worm's ovum solution: the volumetric usage ratio of bacterium solution is 2:1:1；
S5 is cleaned with kaliumphosphate buffer and collects the P0 generation in S4 in the egg-laying season and cleaned and collect, repeats S1, S4 simultaneously
F1 generation, F2 generation, the service life in F3 generation, body length, body bending and head oscillation Frequency Index are obtained respectively；
S6, if F1 generation and P0 are statistically significant for any one of each indicator difference, plasticiser sample has explicitly
Generation cumulative toxicity；If F1 generation and P0 are not statistically significant for each indicator difference, and F2 generation with P0 in each indicator difference
Any one is statistically significant, then plasticiser sample has medium generation cumulative toxicity；
If F1 generation and P0 generation, F2 generation with P0 it is not statistically significant for each indicator difference, and F3 generation with P0 for each index
Any one of difference is statistically significant, then plasticiser sample has slight generation cumulative toxicity；If F1 generation and P0 generation, F2 generation
Not statistically significant for each indicator difference with P0 with P0 generation, F3 generation, then plasticiser sample is without clear generation cumulative toxicity.
It should be noted that being put with Data Analysis Software SPSS to the service life of two eposides, body length, body bending and head
When any of dynamic frequency index does variance analysis, if P < 0.05, illustrate that the index is statistically significant.
The KCl of NaCl and 32mmol/L in above-mentioned kaliumphosphate buffer containing 53mmol/L；Prepare plasticiser sample solution
When be 1% using volumetric concentration dimethyl sulfoxide solution as solvent.
To verify accuracy of the invention, the plasticiser sample of embodiment 1 uses DEHP (phthalic acid two (2- ethyl)
Own ester), the plasticiser sample concentration gradient to be checked of setting are as follows: 0 μ g/mL, 0.02 μ g/mL, 0.08 μ g/mL, 0.32 μ g/mL,
1.28 μ g/mL, 5.12 μ g/mL, 20.48 μ g/mL, 81.92 μ g/mL, 327.68 μ g/mL, 1310.72 μ g/mL, DEHP plasticisers
Sample is added in 96 orifice plates with 100 holes μ L/, and larva solution, worm's ovum solution and bacterium solution are added in 96 orifice plates with 50 holes μ L/,
The LC50 of DEHP is 204.8 μ g/mL.DEHP influences such as Fig. 1-Fig. 4 to caenorhabditis elegan different generations service life, body length and motor behavior
It is shown, as can be seen from the figure 0.2 μ g/mL DEHP exposure after the caenorhabditis elegan service life with exposure the generation extension gradually contract
Short, growth and development is affected, and body length becomes smaller, and motor behavior ability is impaired, and head oscillation and the curved frequency of body are with exposure
The extension in epoch and decline and become apparent, F1 generation has significant difference, P < 0.05, therefore 0.2 μ g/mL compared with P0
DEHP has specific generation accumulating effect to caenorhabditis elegan.
Embodiment 2
The step of embodiment 2 and embodiment 1 and reagent usage and dosage are all identical, and difference is that embodiment 2 is (adjacent using DBP
Dibatyl phithalate) it is plasticiser sample to be checked, the LC50 of DBP is 211.7 μ g/mL.DBP is to the caenorhabditis elegan different generations longevity
Life, body are long and motor behavior influences as shown in Figure 5-Figure 8, as can be seen from the figure caenorhabditis elegan after the DBP exposure of 0.2 μ g/mL
Service life is gradually shortened with the extension of exposure generation, and growth and development is affected, and body length becomes smaller, and motor behavior ability is impaired, head
Portion is swung and the curved frequency of body declines and becomes apparent with the extension in exposure epoch, and F1 generation has conspicuousness compared with P0 generation
Difference, P < 0.05, therefore the DEHP of 0.2 μ g/mL have specific generation accumulating effect to caenorhabditis elegan.
It should be noted that the step method used in claims of the present invention is same as the previously described embodiments, in order to anti-
Only repeating, the present invention describes preferred embodiment, once a person skilled in the art knows basic creative concept,
Then additional changes and modifications can be made to these embodiments.So it includes being preferably implemented that the following claims are intended to be interpreted as
Example and all change and modification for falling into the scope of the invention.
Obviously, various changes and modifications can be made to the invention without departing from essence of the invention by those skilled in the art
Mind and range.In this way, if these modifications and changes of the present invention belongs to the range of the claims in the present invention and its equivalent technologies
Within, then the present invention is also intended to include these modifications and variations.
Claims (7)
1. it is a kind of based on caenorhabditis elegan detection plasticiser more than generation cumulative toxicity method, which comprises the following steps:
S1, culture caenorhabditis elegan is cleaned with kaliumphosphate buffer to the egg-laying season and collects egg-laying season caenorhabditis elegan, then uses lysate
Cracking caenorhabditis elegan parent simultaneously collects the worm's ovum for having resistivity to lysate, is cleaned with kaliumphosphate buffer, potassium phosphate is added
It is 10 pieces/μ L that buffer, which adjusts caenorhabditis elegan worm's ovum concentration, obtains the worm's ovum solution in contemporaneity；
Worm's ovum hydroponics 12-18h in S1 is made egg hatch but is maintained at L1 larva period by S2, by L1 larva cultivate to
In L4 larva period, being cleaned with kaliumphosphate buffer and adjusting L4 larva concentration is 19-21 item/100 μ L, obtains larva solution；Use phosphorus
Sour potassium buffer solution for cleaning simultaneously adjusts Escherichia coli OP50 solution absorbance 1.2-1.4 in wavelength 570nm, obtains bacterium solution；
S3 is arranged different plasticiser sample concentration gradients to be checked, the children in S2 is sequentially added in each concentration plasticiser sample
Worm solution and bacterium solution, contamination culture 48h at 20 DEG C, 4h/ record polypide death toll, the LC50 of calculating plasticiser sample；
Wherein, plasticiser sample: larva solution: the volumetric usage ratio of bacterium solution is 2:1:1；
S4, prepares the plasticiser sample of 1/1000 concentration of LC50, is added the worm's ovum solution in S1, the bacterium solution in S2, at 20 DEG C
Contamination culture 48h, is denoted as P0 generation, then measures P0 respectively for service life, body length, body bending and head oscillation Frequency Index；
Wherein, plasticiser sample: worm's ovum solution: the volumetric usage ratio of bacterium solution is 2:1:1；
In P0 generation in S4, is cleaned and is collected with kaliumphosphate buffer, repeated S1, S4 and obtain F1 generation, F2 generation, F3 generation respectively by S5
Service life, body is long, body is bent and head oscillation Frequency Index；
S6, if F1 generation and P0 are statistically significant for any one of each indicator difference, plasticiser sample has the specific generation
Cumulative toxicity；If F1 generation and P0 are not statistically significant for each indicator difference, and F2 generation with P0 for any in each indicator difference
Item is statistically significant, then plasticiser sample has medium generation cumulative toxicity；
If F1 generation and P0 generation, F2 generation with P0 it is not statistically significant for each indicator difference, and F3 generation with P0 for each indicator difference
Any one of it is statistically significant, then plasticiser sample has slight generation cumulative toxicity；If F1 generation and P0 generation, F2 generation and P0
In generation, F3 generation, are not statistically significant for each indicator difference with P0, then plasticiser sample is without clear generation cumulative toxicity.
2. as described in claim 1 based on caenorhabditis elegan detection plasticiser more than generation cumulative toxicity method, which is characterized in that
Plasticiser sample is phthalate plasticiser.
3. as described in claim 1 based on caenorhabditis elegan detection plasticiser more than generation cumulative toxicity method, which is characterized in that
The condition of culture of caenorhabditis elegan is equal in S1, S2 are as follows: cultivates in NGM culture medium at 20 DEG C.
4. as described in claim 1 based on caenorhabditis elegan detection plasticiser more than generation cumulative toxicity method, which is characterized in that
The KCl of NaCl and 32mmol/L in kaliumphosphate buffer containing 53mmol/L.
5. as described in claim 1 based on caenorhabditis elegan detection plasticiser more than generation cumulative toxicity method, which is characterized in that
The composition of lysate in S1: the NaOH and mass concentration of 0.45mol/L is 2%NaClO.
6. as described in claim 1 based on caenorhabditis elegan detection plasticiser more than generation cumulative toxicity method, which is characterized in that
The dimethyl sulfoxide solution for being 1% using volumetric concentration when preparing plasticiser sample solution is solvent.
7. as described in claim 1 based on caenorhabditis elegan detection plasticiser more than generation cumulative toxicity method, which is characterized in that
Concentration≤20mg/mL of plasticiser sample solution.
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| CN114085884A (en) * | 2021-11-17 | 2022-02-25 | 中国科学技术大学 | Application of caenorhabditis elegans in detection of nano plastic and nano silver generation combined toxicity |
| CN115097088A (en) * | 2022-06-13 | 2022-09-23 | 山东农业大学 | Toxicity evaluation method of fluoride ether bacteria amide |
| CN115097088B (en) * | 2022-06-13 | 2024-02-13 | 山东农业大学 | Toxicity evaluation method of fluoroether bacteria amide |
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