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Method for evaluating cytotoxicity of environmental pollutants by adopting HPLC-MS/MS (high performance liquid chromatography and mass spectrometry or mass spectrometry) and metabolic flux analysis system

A technology for metabolic flow analysis and environmental pollutants, applied in the field of environmental pollutant cytotoxicity assessment, can solve the problems of cumbersome test methods, limited information, long analysis cycle, etc., achieve short time-consuming, simple pre-treatment, shortened analysis the effect of time

Active Publication Date: 2013-10-23
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Application Information

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Problems solved by technology

2008, 565-569) However, the traditional cell metabolic toxicity evaluation method has the disadvantages of long analysis period, cumbersome test method, the combination of multiple instruments, and the limited amount of information obtained, so it is urgent to develop an efficient, fast and informative method. To evaluate the metabolic toxicity of cells, HPLC-MS / MS has the advantages of accurate quantification and wide testing range (Monique P, Christine V.S, Konstantinos P, et al. Rapid Commun Mass Spectrom, 2003, 17: 1297-1311) , the metabolic flow analysis system can use the changes in extracellular metabolites to calculate the corresponding changes in intracellular metabolites (Jens N, Fozia N, Elmar H. Toxicol Appl Pharm, 2009, 240: 327-336), the two organic It is of great significance for the rapid and efficient study of the cellular metabolic toxicity of environmental pollutants

Method used

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  • Method for evaluating cytotoxicity of environmental pollutants by adopting HPLC-MS/MS (high performance liquid chromatography and mass spectrometry or mass spectrometry) and metabolic flux analysis system
  • Method for evaluating cytotoxicity of environmental pollutants by adopting HPLC-MS/MS (high performance liquid chromatography and mass spectrometry or mass spectrometry) and metabolic flux analysis system
  • Method for evaluating cytotoxicity of environmental pollutants by adopting HPLC-MS/MS (high performance liquid chromatography and mass spectrometry or mass spectrometry) and metabolic flux analysis system

Examples

Experimental program
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Effect test

Embodiment 1

[0032] Liver cancer cell HepG2 cells in the logarithmic phase of growth were cultured in an incubator with cell culture medium DMEM, and cultured at 37°C for 48 hours. 2 air; pipette 10 μL of cell culture solution cultured for 48 hours into 1 ml of methanol and water mixture (volume ratio 90:10), vortex for 2 minutes, and then 15000 r min -1 After centrifugation for 5 min, the supernatant was passed through a 0.22 μm needle filter to obtain a metabolite extract. In this method, protein removal and sample dilution are completed in one step, and the sampling volume is only 10 μL. The results showed that the repeatability RSD of this method was less than 2.88%, and the recovery rate was 91.27%-115.07%.

Embodiment 2

[0034] The metabolite extract obtained in Example 1 was loaded and analyzed, and the HPLC-MS / MS analysis method was established. The liquid chromatographic column was selected as an Atlantis C18 reverse-phase chromatographic column (waters, 150mm × 2.1mm, 3.0 μm); the mobile phase A was Pure water, mobile phase B is methanol; the total flow rate is 200 μL min -1 , the temperature of the column oven was 20°C, and the injection volume was 10 μL. The gradient elution program was as follows: the volume of methanol used was 5% during 0-2 minutes; the amount of methanol was increased to 40% after 3 minutes and kept for 5 minutes; then the amount of methanol was reduced to 5% within 1 minute and then kept for 9 minutes.

[0035] The ionization mode of mass spectrometer is electrospray ion, and the sheath gas and auxiliary gas are both nitrogen. The mass spectrometry scan mode adopts selective reaction monitoring (SRM) detection mode, and data acquisition is carried out in positive i...

Embodiment 3

[0045] According to the bioinformatics analysis (KEEG) database of metabolic pathway, according to the metabolic flux balance simplified metabolic network of 23 kinds of metabolites obtained in embodiment 2, draw the metabolic network (metabolic contour map) of HepG2 cell, as figure 2 shown. The drawn metabolic network includes 71 cellular biochemical reaction pathways, including 5 biomacromolecular cellular synthesis reactions (nucleic acid-DNA and RNA, proteins, lipids, and organic carbon), 41 intracellular reactions, and 24 extracellular reactions. Protein synthesis comes from 20 kinds of amino acids, which are not marked in the figure. The unframed part in the figure is the material exchange between extracellular metabolites and the external environment. The metabolic pathways included in the entire metabolic network are: glycolysis, gluconeogenesis, pentose phosphate pathway, glycerol metabolism, tricarboxylic acid cycle, urea cycle, amino acid metabolism and lipid meta...

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Abstract

The invention relates to a method for evaluating the cytotoxicity of environmental pollutants. The method adopts a HepG2 cell as a test cell and comprises the following steps: (a) establishing a pretreatment method for extracting metabolites from a cell culture fluid rapidly; (b) analyzing the 23 metabolites in the cell culture fluid quantitatively by adopting HPLC-MS / MS (high performance liquid chromatography and mass spectrometry or mass spectrometry); (c) establishing the metabolic profile chart of the HepG2 cell; and (d) analyzing the influence of the environmental pollutants on all the metabolic pathways of the HepG2 cell successfully by adopting a metabolic flux analysis system according to data obtained by adopting the HPLC-MS / MS. The pretreatment method is simple, various metabolites in the cell culture fluid can be quantitated accurately by adopting the HPLC-MS / MS, and the influence of the environmental pollutants on the metabolic pathways of the HepG2 cell can be reflected intuitively by combining the metabolic flux analysis system. The method provided by the invention has the advantages of simple operation, short analysis time, high sensitivity, good reproducibility and the like, and comprehensive information can be obtained.

Description

technical field [0001] The present invention is a method for evaluating the cytotoxicity of environmental pollutants, specifically a method for evaluating the influence of environmental pollutants on cellular metabolic toxicity by using HPLC-MS / MS analysis technology and metabolic flow system. The method has the advantages of simple pretreatment , short analysis time, high sensitivity, good reproducibility, and comprehensive information. Background technique [0002] The important purpose of toxicology research is to protect human health. However, there are still few basic data available for human health risk assessment for many new organic pollutants and persistent organic pollutants in the environment. Relevant research is very weak, making environmental decision-making and environmental management lack of scientific basis. Especially for persistent organic pollutants in the environment, such as dioxins, polychlorinated biphenyls, etc., it is not enough to support the conc...

Claims

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Application Information

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IPC IPC(8): G01N30/36
Inventor 张保琴张海军金静陈吉平
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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