KR20190043862A - Biomarker Composition for Discriminating Remaining Endosulfan or Determining Toxicity of Ensodulfan Comprising Wax Ester, and Use thereof - Google Patents

Abstract

The present invention relates to a biomarker composition for determining remaining of endosulfan or a biomarker composition for confirming toxicity of endosulfan, including, as an active ingredient, one or more which are selected from a group consisting of wax ester (53:1) [WE(53:1)], wax ester (53:2) [WE(53:2)], wax ester (55:2) [WE(55:2)], wax ester (55:3) [WE(55:3)], wax ester (58:0) [WE(58:0)], and wax ester (58:1) [WE(58:1)]; and to a use thereof. Since increase of amounts of the wax ester (53:1) [WE(53:1)], the wax ester (53:2) [WE(53:2)], the wax ester (55:2) [WE(55:2)], the wax ester (55:3) [WE(55:3)], the wax ester (58:0) [WE(58:0)], and the wax ester (58:1) [WE(58:1)] of zebrafish is confirmed in an environment having endosulfan of 10 ppb or more, the present invention determines remaining of the endosulfan in the water and is useful as a biomarker for confirming toxicity of the endosulfan in an individual.

Description

[0001] The present invention relates to a biomarker composition for the determination of endosulfan residue or an endosulfan toxicity, which comprises a wax ester as an active ingredient, and a biomarker composition for use in the determination of endosulfan toxicity,

The present invention relates to a biomarker composition for judging the endosulfan residue or an endosulfan toxicity, comprising a wax ester as an active ingredient, a composition for determining the endosulfan residue or an endosulfan toxicity confirmation composition containing the measurable agent as an active ingredient, , A method for determining whether an endosulfan residue is present, and a method for confirming endosulfan toxicity.

Reducing the pollution caused by harmful chemical substances among agricultural products induces the people to secure safe food for the harmful substances and expects the effect of promoting the public health through reduction of exposure to the substance. Accordingly, it is necessary to carry out a survey on the intentional and non-intentional organic harmful chemical substances including agricultural chemicals in crop cultivation farmland and agricultural water continuously, and to ascertain the residual pattern and residue level to establish a safety management system There is a need.

However, when conducting surveys of residues and unintended organic toxic chemicals in agricultural and agricultural waters under general and classical methods, the analytical period is long and the analysis cost is high. In addition, There is a problem that it is necessary.

Therefore, there is a need to develop a monitoring system by developing a method for analyzing the actual condition of intentionally and unintentionally toxic chemical substances in aquatic ecosystem environment more quickly and inexpensively. As a result of this rapid analysis method, There is a method of analyzing the organic and / or organic contaminants present in the environment by causing an antibody reaction using a marker. Biomarkers of biomarkers for the toxicity of organic and inorganic contaminants in the environment are present in proteins and secondary metabolites. Recently, development of biomarkers of lipid biomarkers using metabolic techniques has been studied.

Endosulfan is particularly toxic to aquatic organisms and is an organochlorine insecticide that has been designated as a persistent organic pollutant (POPs) since 2012 and prohibited in the country. Although the number of detection frequencies is decreasing due to the prohibition of their use, the residuals of various environmental media are continuously reported in Korea, and the dynamics of these environments need to be continuously investigated.

Korean Patent No. 10-0752536 (registered on August 21, 2007).

It is an object of the present invention to provide a biomarker composition capable of effectively confirming the residual pattern and degree of the endosperm in an aqueous environment.

Another object of the present invention is to provide a composition capable of effectively confirming the residual pattern and degree of the endosulfan in the saucer environment.

It is still another object of the present invention to provide a kit which can effectively confirm the residual pattern and degree of the endoscope in the hand environment.

It is still another object of the present invention to provide a biomarker composition which can effectively confirm the toxicity of endosulfan.

It is another object of the present invention to provide a composition which can effectively confirm the toxicity of endosulfan.

It is still another object of the present invention to provide a kit which can effectively confirm toxicity of endosulfan.

It is yet another object of the present invention to provide a method for judging whether or not an endothelial plate remains in a water environment more effectively and accurately.

Another object of the present invention is to provide a method for more effectively and accurately confirming toxicity of endorphins.

In order to achieve the above object, the present invention provides a wax ester (53: 1), a wax ester (53: 2), a wax ester (55: 2) WE (55: 2)], wax ester (55: 3) [WE (55: 3)], wax ester (58: 58: 1)] as an active ingredient. The present invention also provides a biomarker composition for judging whether an endosulfan residue is present or not.

In order to achieve the above object, the present invention provides a wax ester (53: 1), a wax ester (53: 2), a wax ester (55: 2) WE (58: 0) and wax ester (58: 1) [WE (55: 2)], wax ester (55: 3) (58: 1)] as an active ingredient. The present invention also provides a composition for determining the endorsulfan residue.

According to another aspect of the present invention, there is provided a kit for determining whether or not an endothelium residue is contained in the composition.

In order to achieve the above another object, the present invention provides a wax ester (53: 1), a wax ester (53: 2) WE (55: 2)], wax ester (55: 3) [WE (55: 3)], wax ester (58: WE (58: 1)] as an active ingredient. The present invention also provides a biomarker composition for endotoxin toxicity confirmation.

In order to achieve the above another object, the present invention provides a wax ester (53: 1), a wax ester (53: 2) WE (55: 2)], wax ester (55: 3) [WE (55: 3)], wax ester (58: WE (58: 1)] as an active ingredient. The present invention also provides a composition for confirming endotoxin toxicity.

According to another aspect of the present invention, there is provided an endothelium toxicity-determining kit comprising the composition.

According to another aspect of the present invention, there is provided a method for measuring the residual amount of endosulfan, comprising the steps of: (1) administering zebrafish to an aqueous environment for measuring a residual amount of endosulfan; WE (53: 1), wax ester (53: 2) [WE (53: 2)] and wax ester (55: 2) were obtained from the samples separated from the zebrafish. WE (55: 2)], wax ester (55: 3) [WE (55: 3)], wax ester (58: WE (58: 1)]; (3) The wax ester (53: 1) [WE (53: 1)], the wax ester (53: 2) [WE (53: 2)], the wax ester (55: WE (58: 0)] and wax ester (55: 3) [WE (55: 3)], wax ester (58: Comparing the amount of one or more selected from the group consisting of the normal control sample with the normal control sample; WE (53: 2), wax ester (55: 2) [WE (55: 2)], and wax ester (53: WE (58: 0)] and wax ester (58: 1) [WE (58: 1)], wax ester (55: , And judging that the endo-sheet remains in the range of 10 ppb to 100 ppb when the amount of any one or more selected from the group consisting of sodium and potassium is higher than that of the control sample.

In order to achieve the above another object, the present invention provides a process for producing a wax ester (53: 1) [WE (53: 1)], a wax ester (53: WE (55: 3), wax ester (55: 2), wax ester (55: 2) ) And wax ester (58: 1) [WE (58: 1)]. WE (53: 2), wax ester (55: 2) [WE (55: 2)], and wax ester (53: WE (58: 0)] and wax ester (55: 3) [WE (55: 3)], wax ester (58: Comparing the amount of one or more selected from the group consisting of the normal control sample with the normal control sample; WE (53: 2)], wax ester (55: 2) [WE (55: 2)] and the wax ester (53: WE (58: 0)] and wax ester (58: 1) [WE (58: 1)], wax ester (55: And determining that the toxicity of the endosulfan is exhibited when the amount of the endosulfan is higher than that of the control sample.

The present invention relates to a process for the production of a wax composition comprising a total of six wax esters (WE (53: 1), WE (53: 2), WE (55: 2), WE (55: The present invention relates to a biomarker composition for use in determining an endothelium residue or an endotoxin toxicity-determining biomarker composition comprising the same as an active ingredient, and a composition, a kit, a method for determining an endosulfan residue or an endosulfan toxicity identification method using the same, The amount of the wax esters in the zebrafish of the present invention can be effectively used as a single biomarker or a combined biomarker to effectively confirm the residual state of the endosulfan and the toxicity of the endosulfan.

FIG. 1 is a chromatogram-lipid spectrum obtained from a Maltitol mass spectrometer after treatment of various concentrations of endosulfan in a zebrafish embryo according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail.

The inventors of the present invention have found that the endohedral residue of the environment is very important in aquatic ecosystem since it exhibits strong toxicity with the insecticidal toxicity of this substance. Therefore, in order to measure the residual toxicity of endosulfan rapidly and at low cost, The inventors of the present invention intend to develop biomarkers of lipid biomarkers which have been significantly or negatively produced after exposure and that the six kinds of wax ester compounds derived from zebrafish are significantly increased during the endosulfan treatment, Completed.

Therefore, the present invention relates to a process for the production of a wax ester (53: 1), a wax ester (53: 2), a wax ester (55: 2) WE (58: 0)] and wax ester (55: 3) [WE (55: 3)], wax ester (58: Wherein the biomarker composition comprises at least one selected from the group consisting of:

WE (53: 2)], wax ester (55: 2) [WE (55: 2)], and wax ester (53: WE (58: 0)], wax ester (55: 3) [WE (55: 3)], wax ester One or more selected from the group can be used as a biomarker for determining whether endosulfan is remaining or not, since the amount of the endosulfan increases from 10 ppb to 100 ppb when exposed to endosulfan.

In addition, the present invention relates to a process for the preparation of a wax ester (53: 1) [WE (53: 1)], a wax ester (53: 2) WE (58: 0)] and wax ester (58: 1) [WE (58: 1)], wax ester (55: As an active ingredient. The present invention also provides a composition for determining the endorsulfan residue.

In addition, the present invention provides a kit for determining whether or not an endosulfan residue is contained in the composition.

Further, the present invention relates to a wax ester (53: 1), a wax ester (53: 2), a wax ester (55: 2) WE (58: 0)] and wax ester (58: 1) [WE (58: 3)], wax ester (58: ] As an active ingredient. The present invention also provides a biomarker composition for endothelium toxicity determination, comprising at least one selected from the group consisting of:

That is, by utilizing the biomarker according to the present invention, it is possible not only to confirm the endosan remaining in the water, but also to confirm whether or not the toxicity of the endosan bean has appeared in the individual.

WE (53: 2)], wax ester (55: 2) [WE (55: 2)], and wax ester (53: WE (58: 0)], wax ester (55: 3) [WE (55: 3)], wax ester , The amount of the endosulfan increases from 10 ppb to 100 ppb, so that it can be used as a biomarker for confirming toxicity of endosulfan.

WE (53: 2), wax ester (55: 2) [WE (55: 2)], and wax ester (53: WE (58: 0)] and wax ester (55: 3) [WE (55: 3)], wax ester (58: The present invention provides a composition for confirming endotoxin toxicity, which comprises, as an active ingredient, a preparation capable of measuring at least one selected from the group consisting of

In addition, the present invention provides a kit for confirming endothelium toxicity comprising the above composition.

Further, the present invention provides a method for measuring the residual amount of endosulfan, comprising the steps of: (1) administering zebrafish to an aqueous environment for measuring the residual amount of endosulfan; WE (53: 1), wax ester (53: 2) [WE (53: 2)] and wax ester (55: 2) were obtained from the samples separated from the zebrafish. WE (55: 2)], wax ester (55: 3) [WE (55: 3)], wax ester (58: WE (58: 1)]; (3) The wax ester (53: 1) [WE (53: 1)], the wax ester (53: 2) [WE (53: 2)], the wax ester (55: WE (58: 0)] and wax ester (55: 3) [WE (55: 3)], wax ester (58: Comparing the amount of one or more selected from the group consisting of the normal control sample with the normal control sample; WE (53: 2), wax ester (55: 2) [WE (55: 2)], and wax ester (53: WE (58: 0)] and wax ester (58: 1) [WE (58: 1)], wax ester (55: , And judging that the endo-sheet remains in the range of 10 ppb to 100 ppb when the amount of any one or more selected from the group consisting of sodium and potassium is higher than that of the control sample.

The present invention also relates to a method for producing a wax ester (53: 1), a wax ester (53: 2), a wax ester (53: WE (58: 0)] and wax ester (58: 3) [55: 2] [WE (55: 2)], wax ester (55: : 1) [WE (58: 1)]; WE (53: 2), wax ester (55: 2) [WE (55: 2)], and wax ester (53: WE (58: 0)] and wax ester (55: 3) [WE (55: 3)], wax ester (58: Comparing the amount of one or more selected from the group consisting of the normal control sample with the normal control sample; WE (53: 2)], wax ester (55: 2) [WE (55: 2)] and the wax ester (53: WE (58: 0)] and wax ester (58: 1) [WE (58: 1)], wax ester (55: And determining that the toxicity of the endosulfan is exhibited when the amount of the endosulfan is higher than that of the control sample.

As used herein, the term " sample " refers to biomarkers such as wax ester (53: 1) [WE (53: 1)], wax ester (53: WE (58: 0) and wax ester (58: 1) [WE (55: 2)], wax ester (55: 3) But are not limited to, tissues, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid, or urine samples that differ from the control in the amount of the test compound (58: 1)

In one embodiment of the present invention, the wax ester (53: 1) [WE (53: 1)], the wax ester (53: 2) (WE (58: 0)] and wax ester (58: 1) [WE (58: 3)], wax ester (55: : 1)] can be, for example, a quantitative analysis using mass spectrometry, light absorption analysis, gas chromatography or emission spectroscopy, but the present invention is not limited thereto Do not.

The term " test substance " used in reference to the screening method of the present invention includes wax ester (53: 1) [WE (53: 1)], wax ester (53: WE (58: 0)] and wax ester (55: 2) [WE (55: 2)], wax ester (55: 58: 1) [WE (58: 1)] in the screening test. Such test materials include, but are not limited to, chemicals, peptides, nucleotides, DNA, antisense-RNA, small interference RNA (siRNA) and natural extracts.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. It is to be understood that both the foregoing description and the following detailed description are exemplary and explanatory and are intended to provide further explanation of the invention as claimed. no.

<Example 1> Evaluation of acute toxicity of endosulfan using adult of zebrafish

(1) Experimental method

The adult (3-5 cm) of the wild type zebrafish was purchased and the chlorine-free water was adjusted to a temperature of 26 ± 1 ° C., and the day and night lengths were adjusted to 16: 8. The toxicity test was conducted according to the OECD Guideline NO. 203, and 10 liters of purified zebrafish for each beaker were used for the experiment using 5 L of tap water with 5 L of beaker and chlorine removed. Endosulfan was treated with DMSO as the messenger and the concentration was adjusted so that the ratio to water did not exceed 1%. Each treatment group was repeated 3 times. The experiment was carried out for 96 hours at a temperature of 26 ± 1 ° C and day and night length of 16: 8. Lethal organisms were recorded for 96 hours in 24 hour units, and Lethal concentration 50 (LC ₅₀ ) was derived using SPSS.

(2) Experimental results

As a result of evaluating the acute toxicity of endosulfan to zebrafish, endoderm platelets had a half-life lethal dose of 4.587 ppb for 96 hours exposure to zebrafish, Which was 4.774 ppb. However, at 24 hours exposure, the value increased to 6.508 ppb. This is the result of proving that the exposure is exposed to a higher endosulfan concentration at short exposure times.

Time (h) Segment Lethal concentration (L L ^-1 , 95% Fiducial limits)





Endorphin

24 LC ₁₀ 0.468 (-5.214 to 2.668) LC ₂₀ 2.542 (-1.133-4.4688) LC ₅₀ 6.508 (4.584-10.002) LC ₉₅ 14.260 (10.526-26.049)

48 LC ₁₀ 0.903 (-1.859 to 2.354) LC ₂₀ 2.232 (0.197-3.534) LC ₅₀ 4.774 (3.468 ~ 6.451) LC ₉₅ 9.743 (7.692 to 14.323)

96 LC ₁₀ 1.021 (-2.106 ~ 2.505) LC ₂₀ 2.245 (0.022-3.609) LC ₅₀ 4.587 (3.199 to 6.441) LC ₉₅ 9.164 (7.09 to 14.296)

< Example  2> Endosulfan Zebra fish  Evaluation of effects on adult lipid production

(1) Experimental method

To investigate the lipid changes of the adult zebrafish exposed to endosulfan, two adult samples were homogenized in methanol - chloroform (1: 2, v / v) solution (15 mL) and shaken at room temperature for 20 minutes. After filtration using a cell strainer, 3 mL of 0.9% sodium chloride solution was added and mixed. The mixture was centrifuged at 2000 rpm for 5 minutes, and the lower layer (methanol-chloroform layer) was taken for analysis. At this time, the endosulfan was processed into zebrafish at the levels of 0, 1, 2.5, 10, and 25 ppb, respectively, from about 5 times to 4.5 times below the 4,587 ppb level of the endotoxin obtained in Example 1 Processed value.

1 μL lipid extract was placed on an MTP 384 ground steel target and dried at room temperature. After the sample was completely dried, 0.5 μL saturated ammoniacridine matrix solution (2-propanol / acetonitrile, 60/40, v / v) was placed on the dried sample and dried at room temperature and analyzed with a Malditi-Mass Spectrometer (MALDI- And analyzed the lipids. The results were repeated three times for each sample. The results were verified by using lipid database (www.lipidmaps.org).

Specifically, the MALDI-TOF MS analysis method was as follows: Zebrafish lipid extraction sample and sample binding buffer were diluted according to dilution drainage and stored at 4 ° C. To optimize the lipid analysis chip, 200 μl of the sample binding buffer was added to the wells, and the binding buffer was removed after stirring at 250 rpm for 5 minutes at room temperature. The above method was repeated one more time. 150 μl of the diluted lipid sample was added to the lipid-lipid analysis chip from which the binding buffer had been removed, and the mixture was stirred at 250 rpm for 30 minutes at room temperature. Cleavage of the lipid bound to the lipid analysis chip was performed by adding 200 μl of binding buffer to the wells and shaking at 250 rpm for 5 minutes at room temperature. This method was repeated twice. After washing twice with distilled water for HPLC, the remaining binding buffer was removed. After the lipid analysis chip was completely dried, 1 μl of EAM was added to form crystals, and the EAM addition was repeated one more time. As the EAM, supernatant obtained by dissolving CHCA easily in 1 to 15 kD analysis with 50% CHCA EAM solution (50% ACN, 0.25% TFA) and centrifuging at 10,000 rpm for 10 minutes at room temperature was used.

Repetition of MALDI-TOF MS analysis was performed with three replicates of lipid extraction. The setting of the laser energy was optimized by EAM-CHCA, and the peak detection and clustering analysis was performed using the Chip Data manager. The spectra were normalized from 500 Da to 1,000 Da. For peak detection, first-pass peak detection was obtained by setting the S / N ratio to 5 and the valley depth to 3, The detection of the second-pass peak was obtained by setting the S / N ratio to 2 and the valley depth to 2 signals. The measured peak (Estimated peak) was set as the At cluster center, and the lipid peak analysis was analyzed using the lipid Chip Data Manager Software provided by Bio Rad. However, the Kruskal-Wallis test was used to determine the significance of p value less than 0.01.

(2) Experimental results

Based on the results of Example 1 above, the results of analyzing zebrafish lipid metabolites at these concentrations by exposing zebrafish adults to five concentration gradients are shown in Table 2 below.

As shown in Table 2, lipid content of 769.8 m / z was decreased from 2.5 ppb to half of that of the control group, and then increased about 8 and 3 times at 10 ppb and 25 ppb, respectively, and 771.8 m / z Of the lipid was increased about 6.6 times in the zebrafish exposed to 10 ppb endosulfan compared to the control. In addition, the lipid of 795.84 m / z increased by a factor of 10 compared with that of the control, and the lipid of 841.86 m / z molecular weight increased by 42 times compared to the control. All of these were lipids belonging to the wax ester, which were newly generated lipids in zebrafish exposed to endosulfan, especially at a concentration level of 10 ppb, as compared to the control group, as shown in Fig.

Therefore, these wax ester lipids can be usefully used as lipid biomarkers that react when zebrafish is exposed to endopurdes of 10 ppb or more.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. Do. That is, the practical scope of the present invention is defined by the appended claims and their equivalents.

Claims (10)

WE (55: 2)], a wax ester (53: 1), a wax ester (53: 2) (58: 1) [WE (55: 3)], wax ester (58: 0) [WE (58: 0)] and wax ester Wherein the biomarker composition comprises at least one selected from the group consisting of: The method according to claim 1, wherein the wax ester (53: 1) [WE (53: 1)], the wax ester (53: 2) WE (58: 0)] and wax ester (58: 1) [WE (58: 3)], wax ester (55: )], The amount of which is increased when the endothelial plate is exposed to 10 ppb to 100 ppb of endothelium. WE (55: 2)], a wax ester (53: 1), a wax ester (53: 2) (58: 1) [WE (55: 3)], wax ester (58: 0) [WE (58: 0)] and wax ester Wherein the composition is capable of measuring any one or more of the endothelin residues. A kit for judging whether or not an endosulfan residue is contained in the composition according to claim 3. WE (55: 2)], a wax ester (53: 1), a wax ester (53: 2) (58: 1) [WE (55: 3)], wax ester (58: 0) [WE (58: 0)] and wax ester Wherein the biomarker composition is a biomarker composition for endothelium toxicity. The method of claim 5, wherein the wax ester (53: 1) [WE (53: 1)], the wax ester (53: 2) WE (58: 0)] and wax ester (58: 1) [WE (58: 3)], wax ester (55: )] Is increased when the endothelin of 10 ppb to 100 ppb is exposed to endosulfan. WE (55: 2)], a wax ester (53: 1), a wax ester (53: 2) (58: 1) [WE (55: 3)], wax ester (58: 0) [WE (58: 0)] and wax ester Or a pharmaceutically acceptable salt thereof, as an active ingredient. 7. An endothelium toxicity-confirming kit comprising the composition according to claim 7. (1) administering a zebrafish to a watery environment in which the residual amount of endosulfan is to be measured;
WE (53: 1), wax ester (53: 2) [WE (53: 2)] and wax ester (55: 2) were obtained from the samples separated from the zebrafish. WE (55: 2)], wax ester (55: 3) [WE (55: 3)], wax ester (58: WE (58: 1)];
(3) The wax ester (53: 1) [WE (53: 1)], the wax ester (53: 2) [WE (53: 2)], the wax ester (55: WE (58: 0)] and wax ester (55: 3) [WE (55: 3)], wax ester (58: Comparing the amount of one or more selected from the group consisting of the normal control sample with the normal control sample; And
WE (53: 2), wax ester (55: 2) [WE (55: 2)], and wax ester (53: WE (58: 0)] and wax ester (55: 3) [WE (55: 3)], wax ester (58: And judging that an endothelium remains at 10 ppb to 100 ppb in the saucer environment when at least one selected from the groups is higher than the control sample. WE (53: 1), wax ester (53: 2) [WE (53: 2)] and wax ester (55: 2) were obtained from the samples separated from the zebrafish. WE (58: 0) and wax ester (58: 1) [WE (55: 2)], wax ester (55: 3) (58: 1)]; measuring the amount of at least one selected from the group consisting of:
WE (53: 2), wax ester (55: 2) [WE (55: 2)], and wax ester (53: WE (58: 0)] and wax ester (55: 3) [WE (55: 3)], wax ester (58: Comparing the amount of one or more selected from the group consisting of the normal control sample with the normal control sample; And
(3) The wax ester (53: 1) [WE (53: 1)], the wax ester (53: 2) [WE (53: 2)], the wax ester (55: WE (58: 0)] and wax ester (55: 3) [WE (55: 3)], wax ester (58: And determining that the toxicity of the endothelin when the amount of at least one selected from the groups is higher than that of the control sample.

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