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Metabonomics technology-based persistent organic pollutant cytotoxicity action mechanism research method

A technology for organic pollutants and cytotoxicity, applied in the field of molecular biology-based evaluation, it can solve the problems of low throughput, resource consumption, and low sensitivity of toxicity testing, and achieve rapid detection, easy operation, and good repeatability.

Inactive Publication Date: 2018-01-30
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The human health risk assessment of POPs is generally assessed by means of traditional toxicology methods, but traditional toxicity testing has the characteristics of low throughput, long cycle, low sensitivity and resource consumption, which obviously cannot meet the requirements of toxicity testing for these environmental pollutants. need

Method used

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  • Metabonomics technology-based persistent organic pollutant cytotoxicity action mechanism research method
  • Metabonomics technology-based persistent organic pollutant cytotoxicity action mechanism research method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] HepG2 cells growing in the logarithmic phase were seeded into six-well plates at a cell density of 3×10 5 cells / well, the volume of culture medium in each well of the six-well plate is 2.5mL; culture in the six-well plate for 24-48 hours, when the cells cover about 80% of the area covered by the culture medium on the well plate, expose the cells with pollutants After 24 hours, samples of cells and extracellular culture fluid were collected. The culture medium is high-sugar DMEM of GIBCO, containing 4500mg / l glucose, containing 10% (v / v) of fetal bovine serum and 1% (v / v) of penicillin-streptomycin; the conditions for culturing in the incubator are 37°C, 5% (v / v) CO in air 2 Expose with commercial hexabromocyclododecane, that is, when the cells are covered with about 80% of the area covered by the culture solution on the orifice plate, the original culture solution is discarded, and each hole is added with DMSO containing 0.05% (v / v), 2.5 mL of culture solution contain...

Embodiment 2

[0032] The liquid-liquid extraction method extracts small molecular metabolites in the intracellular and extracellular culture medium. The extraction is divided into two steps: the first step is to collect the extracellular culture medium in each well of the six-well plate into a separate centrifuge tube, and then quench it with liquid nitrogen. The adherent cells were destroyed, and the extracellular culture medium and the six-well plate were stored at -80°C; then, 1 mL of deionized water was added to each well of the cells, and the cells were ultrasonically disrupted in an ice-water bath for 5 minutes to obtain a broken suspension. The broken suspensions in the wells were placed in separate centrifuge tubes; take 200 μL extracellular culture medium into a new centrifuge tube, and freeze-dry together with the centrifuge tube containing the broken cell suspension; the second step, after freeze-drying Add 500 μL extract solution (80% methanol / water, containing six internal stand...

Embodiment 3

[0034] The UHPLC / Q-Trap MS analysis method was established for quantitative analysis of intracellular and extracellular metabolites. Due to the different molecular properties of metabolites, it was divided into two scanning modes, positive and negative ions. The chromatographic conditions are as follows. Positive ion mode: ACQUITY UPLC BEH C8 (1.7μm, 2.1×100mm) is used for chromatographic column, mobile phase A is superstored water containing 0.1% (v / v) formic acid, phase B is containing 0.1% formic acid (v / v) of acetonitrile. The mobile phase gradient elution program is: start at 90% A, drop to 60% at 3 minutes, drop to 0% at 15 minutes and maintain for 5 minutes, return to 90% A at 20.1 minutes, and balance for 3 minutes; the flow rate is 0.35mL / min , the column temperature was 50°C, and the injection volume was 10 μL. Negative ion mode: ACQUITY UPLC HSS T3 (1.8μm, 2.1×100mm) for chromatographic column, mobile phase A contains NH 4 HCO 3 5mmol / L ultrapure water, phase B ...

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Abstract

The present invention discloses a metabonomics technology-based persistent organic pollutant cytotoxicity action mechanism research method. According to the method, HepG2 cells are exposed to organicpollutants, the intracellular and extracellular culture liquid metabolites can be effectively analyzed by using UHPLC / Q-Trap MS, the influence of the organic pollutants on the cell metabolism pathwaycan be intuitively reflected by combining statistical analysis and metabolic pathway analysis, and by using a biological detection method, the activity of rate-limiting enzyme related to the metabolicpathway is further rapidly analyzed. According to the present invention, the organic pollutant cytotoxicity action mechanism is clarified according to the intracellular metabolite content change andthe activity change of the rate-limiting enzyme in the metabolic pathway, and the pollutant toxicity action mechanism evaluation method is constructed based on molecular biology, and the metabonomicstechnology-based persistent organic pollutant cytotoxicity action mechanism research method has advantages of easy operation, rapid detection, high sensitivity, strong selectivity, good repeatability,comprehensive information acquisition and the like.

Description

technical field [0001] The invention belongs to the field of pollutant toxicity evaluation, and relates to an evaluation method based on molecular biology, which evaluates the cytotoxic action mechanism of small-dose environmental pollutants through the influence of pollutants on cell metabolism pathways. Background technique [0002] Persistent organic pollutants (POPs) have the characteristics of environmental persistence, bioaccumulation, and semi-volatility, and are extremely difficult to degrade and highly toxic. They can migrate globally through environmental media such as the atmosphere, rivers, and oceans. Therefore, POPs pollution has become a global environmental hotspot that has attracted much attention. So far, the compounds listed in POPs by Stockholm include aldrin, chlordane, DDT, dieldrin, endrin, heptachlor, mirex, toxaphene, hexachlorobenzene, dioxin, furan, PCBs, alpha-HCH, beta-HCH, gamma-HCH, pentachlorobenzene, chlordecone, hexabromobiphenyl, tetrabrom...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/50G01N30/02
Inventor 王菲迪陈吉平张海军耿柠波张保琴任晓倩
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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