Method for evaluating biotoxicity of endosulfan

A technology of biotoxicity and endosulfan, applied in the biological field, can solve the problems of false positive, cell membrane damage, cell membrane structure damage, etc., and achieve the effect of simple method and high correlation

Inactive Publication Date: 2014-09-10
DALIAN MARITIME UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these are all quantitative descriptions of the content of endosulfan, and the content alone cannot reflect the effect of endosulfan on organisms
[0015] For a long time, lactate dehydrogenase LDH has been used as a susceptibility effect marker to detect the cytotoxicity of endosulfan. The principle is that the release of LDH can be regarded as an important indicator of cell membrane integrity. When endosulfan exposure induces apoptosis Or necrosis causes damage to the cell membrane structure, which will cause the release of LDH, but there are many problems in this method
Firstly, the LDH enzyme itself is sensitive to temperature and is easily inactivated and unstable; secondly, the release of LDH is affected by the degree and density of cell growth, centrifugation speed, and the temperature difference between inside and outside the incubator, which may easily cause false positives; and when the concentration of endosulfan When it is too low to damage the cell membrane, the release of LDH will not be detected; in addition, there is a problem of high background in the detection of LDH by colorimetry. Toxic Gene Regulatory Mechanisms

Method used

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  • Method for evaluating biotoxicity of endosulfan
  • Method for evaluating biotoxicity of endosulfan
  • Method for evaluating biotoxicity of endosulfan

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] The effect of embodiment 1 endosulfan on the cell proliferation of HUVEC-C

[0035] HUVEC-C (human umbilical vein endothelial cells) cells were exposed to endosulfan, and the experiment was divided into three groups: normal cell group (C), DMSO control group (dimethyl sulfoxide, D), endosulfan experimental group ( endosulfan, ES). Control group DMSO1% concentration, endosulfan exposure concentration is (20,40,60 μ M), exposure time is 48 hours, collects cells and carries out cell counting, as figure 1 The results showed that endosulfan exposure caused inhibition of cell proliferation in a dose-dependent manner. Compared with the control group, endosulfan significantly inhibited cell growth at a low concentration of 20 μM (P*<0.05), and significantly induced cell proliferation at a higher concentration (40-60 μM) Inhibition (P**<0.01).

Embodiment 2

[0036] The effect of embodiment 2 endosulfan on HUVEC-C cell cycle

[0037] HUVEC-C cells were exposed to endosulfan for 48 hours, and the cells in the normal cell group, DMSO group, and endosulfan experimental group (20, 40, 60 μM) were collected, fixed with 70% cold ethanol overnight, and then stained with PI and RNase treatment, using flow cytometry (BD FACSCalibur) for cell cycle determination, such as figure 2 The results showed that when endosulfan was at a high concentration of 60 μM, compared with the control group, there was a G1 / S phase arrest, which was significant (P**<0.01).

Embodiment 3

[0038] Example 3 Exposure to endosulfan causes changes in the level of IL-6 secretion by cells

[0039] HUVEC-C cells have the characteristics of autocrine IL-6. First, in terms of dose effect, HUVEC-C cells were exposed to endosulfan for 12 hours, and the normal cell group, DMSO group, and endosulfan experimental group (20,40 , 60 μM) of the cell culture supernatants of the three groups were used to measure the level of IL-6 secreted by the cells using the IL-6human ELISA Kit (Abcam). image 3 A is the standard curve that utilizes ELISA method to measure IL-6, image 3 B is the effect of different concentrations of endosulfan on the secretion level of IL-6. image 3 The results of A and 3B show that exposure to endosulfan can increase the expression of IL-6, which is dose-dependent and statistically significant. Secondly, in terms of time effect, HUVEC-C cells were treated with different exposure times (4, 6, 8, 12, 24, 48 h), and the cell culture supernatants of the DMSO g...

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Abstract

The invention discloses a method for evaluating biotoxicity of endosulfan. The method includes the step of detecting biomarker expression of biological samples, wherein the biomarker is IL-6 and/or IL-8. The method is simple and convenient, high in correlation and capable of rapidly evaluating whether a living organism is exposed to endosulfan or not and whether cytotoxicity is caused or not. The method discusses that endosulfan toxicity is related to expression changes of inflammatory factors IL-6 and IL-8 from the aspect of inflammation, hints that the IL-6 and the IL-8 can serve as the biological indicators for evaluating endosulfan cytotoxicity, reflects the relationship between the concentration of endosulfan exposure and effect of endosulfan exposure, has significance in judging influences of endosulfan biotoxicity, especially inflammation damage on the living organism, and is beneficial for revealing the molecular mechanism of environmental persistence organic pollutant inflammatory damage.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for the biological toxicity of environmental pollutants, in particular to a method for evaluating endosulfan inflammatory damage. Background technique [0002] With the advancement of science and technology, the improvement of the level of industrialization, and the expansion of human activity areas, various chemical agents are constantly entering people's lives. Among them, the hazards of persistent organic environmental pollutants (POPs) to the environment and the impact on human health have become more and more serious. Can not be ignored, including dioxins (PCDD / Fs), polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), organochlorine pesticide endosulfan (endosulfan), etc. [0003] Although the existing chemical analysis methods can quantitatively describe the content of pollutants in the environment or in organisms, the content alone cannot reflect t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/50G01N33/68
CPCG01N33/50G01N33/68
Inventor 徐丹孙野青
Owner DALIAN MARITIME UNIVERSITY
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