Method for evaluating toxicity effects of low-dose combined exposure of organic pollutants based on metabonomics technology

A technology for organic pollutants and toxic effects, applied in the field of molecular biology-based evaluation, it can solve the problems of limited information, data stability, poor repeatability, and incomparable analytical throughput, and achieves reduced variance and high sensitivity. , selective effect

Inactive Publication Date: 2019-06-14
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although non-targeted methods have the advantages of high analytical throughput and rich data information, they have shortcomings such as narrow quantitative linear range, poor data stability and repeatability, which often lead to unsatisfactory or even questionable results.
Although targeted analysis methods represented by triple quadrupole mass spectrometry m

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  • Method for evaluating toxicity effects of low-dose combined exposure of organic pollutants based on metabonomics technology
  • Method for evaluating toxicity effects of low-dose combined exposure of organic pollutants based on metabonomics technology
  • Method for evaluating toxicity effects of low-dose combined exposure of organic pollutants based on metabonomics technology

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Experimental program
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Embodiment 1

[0037] HepG2 cells were cultured at 37°C in a cell incubator containing 5% CO2, and the medium was DMEM cell culture medium containing 10% FBS and 1% penicillin-streptomycin. Inoculate the cells grown in the logarithmic phase into a 6-well plate at a seeding density of 3×105 cells / well. After about 80% of the area of ​​the orifice plate was covered by the culture solution, the individual and combined exposure tests of PAHs and SCCPs were carried out, and the cell samples were collected after 24 hours of exposure. PAHs and SCCPs were introduced into the cell culture medium by dissolving in DMSO, and the exposure concentration was set as follows: SCCP mixed standard sample (the total concentration ΣSCCPs was 100 μg / L), PAH mixed standard sample (each PAH concentration was 10 μg / L, and the total concentration ΣPAHs was 160μg / L), a mixture of PAHs and SCCPs (the concentrations of PAHs and SCCPs in the mixed standard sample are: ΣPAHs: 160μg / L and ΣSCCPs: 100μg / L). The exposure do...

Embodiment 2

[0039] Intracellular small molecule metabolites were analyzed using a pseudo-targeted metabolomics approach. First, Agilent UHPLC / Q-TOF MS was used for non-targeted analysis of cell samples, which were divided into positive and negative ion scanning modes, and the chromatographic conditions were 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, phas...

Embodiment 3

[0042] Statistical analysis was performed on the mass spectrometry data of intracellular metabolites obtained from UHPLC / Q-Trap MS analysis. The analysis was processed based on the online http: / / www.metaboanalyst.ca. / , and PLS-DA and ANOVA analysis were performed on intracellular metabolites , a total of 246 differential metabolites were found (P1). Among them, the molecular structures of 115 metabolites were characterized by MS / MS analysis and further confirmed with standards. In order to further explore the potential links between differential metabolites, the metabolic pathways involved in 115 differential metabolites were first identified through the metabolic pathway online database (KEGG, http: / / www.genome.jp / kegg / ). Then, through the calculation of the Pearson correlation coefficient (Pearson correlation coefficient) between the differential metabolites, the metabolites that participate in the same metabolic pathway and are significantly correlated (correlation coeffici...

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Abstract

The invention discloses a method for evaluating toxicity effects of low-dose combined exposure of organic pollutants based on a metabonomics technology. Firstly, single exposure and combined exposureare performed to HepG2 cells by two organic pollutants, unbiased and accurate metabolite information can be obtained based on quasi-targeted metabolomics analysis, and multiple comparative analysis and enrichment analysis of metabolic pathways can directly reveal the impact of the organic pollutants on cellular metabolites and the metabolic pathways. By calculation of the metabolic impact level index, the combined exposure effect type of the combined exposure on overall metabolism and specific metabolic pathways can be quantitatively described. A biological detection method can further determine the activity of metabolic pathway rate-limiting enzymes, and confirm the change direction of the metabolic pathways and the main risk-driven compounds in a mixture. The method has the advantages ofeasy operation, fast detection, high sensitivity, strong selectivity, good repeatability and obtaining of systematic and comprehensive information.

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 joint exposure toxicity effect of small doses of environmental pollutants through the influence of pollutants on cell metabolism pathways. Background technique [0002] In recent years, environmental pollution has received more and more attention, and there are many kinds of environmental chemical pollutants, often in the form of low-concentration mixtures, which inevitably cause complex and diverse health hazards to humans, namely potential joint toxic effects. Therefore, the low dose and combined toxic effects of environmental pollutants have become a hot issue in toxicology research. Chemical pollutants with similar or different mechanisms of action can affect each other's toxic effects, which may cause a series of synergistic, additive or antagonistic effects on different biological response indica...

Claims

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Application Information

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IPC IPC(8): C12Q1/02G01N27/62
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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