CN103364510A - 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 - Google Patents
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 Download PDFInfo
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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
The present invention is the Cytotoxic appraisal procedures of a kind of environmental contaminants, specifically a kind of HPLC-MS/MS analytical technology and metabolic flux system evaluation environmental contaminants of utilizing are to the method for cellular metabolism toxic effect, it is simple that the method has pre-treatment, analysis time is short, highly sensitive, favorable reproducibility, the advantage such as the acquired information amount is comprehensive.
Background technology
The free-revving engine of toxicologic study is the protection health; yet the persistence organic pollutant that exists in many novel organic contaminants and the environment at present can be used for basic data that human health risk estimates also seldom; correlative study to Health Impact in toxicologic study is very weak; so that Environmental Decision-making and environmental management lack scientific basis. especially for the persistence organic pollutant in the environment; such as bioxin; polychlorinated biphenyl etc.; be not enough to support to draw conclusion (Yang Yongbin, the ecological toxicology newspaper that is of practical significance.2006,105-115), assessment is new tool (Liu Peng, Dalian Medical Univ's journal of pollutant toxicity research to cytotoxicity to utilize metabolism group.2008,565-569) still traditional cellular metabolism methods of Toxicity Assessment exists analytical cycle long, method of testing is loaded down with trivial details, need multiple instrument to combine, and the shortcomings such as the quantity of information that obtains is limited, therefore be badly in need of a kind of analysis means fast efficient and that quantity of information is abundant of development and assess the metabolism toxicity of cell, HPLC-MS/MS exists quantitatively accurately, the advantage that test specification is wide (Monique P, Christine V.S, Konstantinos P, et al.Rapid Commun Mass Spectrom, 2003,17:1297-1311), the Metabolic flux analysis system can utilize the variable quantity of extracellular metabolic product to calculate situation of change (Jens N, Fozia N, the Elmar H.Toxicol Appl Pharm of corresponding born of the same parents' intracellular metabolite thing, 2009,240:327-336), the two combines, and has great importance for the cellular metabolism toxicity of rapidly and efficiently research environment pollutant.
Summary of the invention
The object of the invention is to develop a kind of simple to operate, the cycle short, the acquired information amount is abundant cytotoxicity appraisal procedure.
For solving the problems of the technologies described above, the technical solution adopted in the present invention is:
Utilize HPLC-MS/MS to detect and analyzed 23 kinds of metabolins in the cell culture fluid, make up HepG2 cellular metabolism profile diagram, carry out the toxicity evaluation in cellular metabolism path
A) adopt liquid-liquid extraction method that the metabolin in the cell culture fluid is extracted so that Sample Dilution with finish except one step of albumen;
B) set up the HPLC-MS/MS quantivative approach of analyzing simultaneously 23 kinds of metabolic products in the cell culture fluid;
C) according to analyzing the Metabolic profiling that requires to have made up the HepG2 cell;
D) the Metabolic flux analysis system is in conjunction with the cellular metabolism toxicity of HPLC-MS/MS successful analysis environmental contaminants.
Cell culture fluid is 5-20 μ l: 1ml with the solvent load ratio among the step a;
Realized the separation of Simultaneous Quantitative Analysis, the especially isomers of 23 kinds of metabolic products among the step b by optimizing chromatographiccondition and mass spectrum parameter;
Structure to the Metabolic profiling of HepG2 cell among the step c has mainly comprised the steps such as glycolysis, tricarboxylic acid cycle, amino acid metabolism, urea cycle.
Extraction solvent among the step a is methanol/water, this extract solvent except can removing a large amount of albumen in the cell culture fluid can also with chromatogram in mobile phase match, be more conducive to chromatographic resolution.
The application of the methyl alcohol of HPLC-MS/MS is conducive to the separation of isomers more among the step b.
Liquid chromatogram mobile phase is methanol/water among the step b, elution requirement is gradient elution, the selection of methyl alcohol and the change of elution requirement can be shortened analysis time on the other hand on the one hand not adding the separation that can reach isomers under ion-pairing agent and the organic acid condition.
The environmental contaminants that the method relates to mainly comprise persistence organic pollutant (bioxin, polychlorinated biphenyl, hexachloro-benzene etc.), excellent control pollutant (palycyclic aromatic etc.), novel organic contaminants (chloro palycyclic aromatic) etc. are a series of to cause in the material that cellular metabolism changes one or two or more kinds, its concentration 0.001nM-1 μ M in cell culture fluid;
Extract is methanol/water among the step a, and the two ratio is 70: 20-95: 5, and the amount ratio of cell culture fluid and extract is 5-20 μ l: 1ml;
Separating of 23 kinds of metabolic products, the especially separation of isomers have been realized by optimizing chromatographiccondition with the mass spectrum parameter among the step b; 23 kinds of metabolic products are urea, glycerine, lactic acid glycocoll, alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophane, serine, tyrosine, halfcystine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine and histidine;
Structure to the Metabolic profiling of HepG2 cell among the step c has mainly comprised the steps such as glycolysis, tricarboxylic acid cycle, amino acid metabolism, urea cycle.
15000 rev/mins of centrifugal 3-5min of solution needs after the liquid-liquid extraction among the step a are to reach the purpose of protein precipitation.
Mobile phase among the step b is: the first alcohol and water, with respect to acetonitrile, methyl alcohol more is conducive to the separation of isomers compound, also is on the other hand to match for the medium with cell extract.
The mass spectrum ionization mode of HPLC-MS/MS is the electron spray ion among the step b, and sheath gas and assisted gas are nitrogen.The scanning of the mass spectrum pattern adopts selective reaction to detect (SRM) detecting pattern, and data acquisition is carried out under positive ion mode.The mass spectrum condition: ion source temperature is 300 ℃; Sheath gas and assisted gas pressure are respectively 20psi and 5psi, and electron spray voltage is 3000V, and sweep limit is m/z 50～300, and the ion scan time interval is 0.05s.
The elution requirement of liquid chromatography is gradient elution among the step b, and this elution requirement can accelerate analysis time on the other hand on the one hand not adding the separation that can reach isomers under ion-pairing agent and the organic acid condition.
The structure of metabolic profile can require the structure metabolism network of selection according to the analysis of oneself among the step c, but can not omit in profile diagram for metabolic intermediate.
The present invention has following advantage:
(1) pre-treatment is simple, and only the simple concussion of needs was centrifugal and filter and namely can be used for the HPLC-MS/MS analysis after sample mixed with extract; (2) quantitatively accurately, weak point consuming time: HPLC-MS/MS can carry out Simultaneous Quantitative Analysis to 23 kinds of metabolic products in the cell culture fluid, has greatly shortened analysis time by optimizing chromatographic condition; (3) the method is got up HPLC-MS/MS and Metabolic flux analysis system combination; (4) analysis result can also computational analysis go out the change of intracellular products except the situation of change that can accurately obtain metabolic product in the cell culture fluid, and then obtains the situation of change in cellular metabolism path.
The present invention at first utilizes HPLC-MS/MS to analyze 23 kinds of metabolins in the cell culture fluid, and makes up the profile diagram of HepG2 cell, utilizes Metabolic flux analysis with having developed a kind of method for the assessment of environmental contaminants cytotoxicity.
Description of drawings
Fig. 1 has studied 2,3,7 of variable concentrations for adopting the inventive method, and 8-TCDD is on the impact of cellular metabolism path (HepG2 cellular metabolism stream).
Fig. 2 is used for the HepG2 cellular metabolism network chart that metabolic flux calculates.(the Framed mark does not represent born of the same parents' extra-metabolite)
Embodiment
The present invention utilizes the HepG2 cell for supplying the examination cell, by the cytotoxicity of HPLC-MS/MS in conjunction with Metabolic flux analysis systematic quantification analysis environments pollutant.
Embodiment 1:
Adopt in incubator, the grow hepatoma carcinoma cell HepG2 cell of logarithmic phase of cell culture fluid DMEM to cultivate, cultivated 48 hours in 37 ℃, in incubator, pass into volume content 5%CO in the incubation
2Air; Draw in cell culture fluid 10 μ L to the 1ml methyl alcohol and water mixed liquid (volume ratio 90: 10) of cultivating 48h whirlpool concussion 2min, then 15000rmin
-1Centrifugal 5min, supernatant cross 0.22 μ m syringe filters, obtain the metabolic product extract.The method is except albumen and Sample Dilution step through, and sampling amount only needs 10 μ L.The result shows, the repeated RSD of the method＜2.88%, and the recovery is 91.27%-115.07%.
Embodiment 2:
The metabolic product extract loading that embodiment 1 obtains is analyzed, set up the HPLC-MS/MS analytical approach, liquid-phase chromatographic column is selected Atlantis C18 reverse-phase chromatographic column (waters, 150mm * 2.1mm, 3.0 μ m); Mobile phase A is pure water, and Mobile phase B is methyl alcohol; Overall flow rate is 200 μ Lmin
-1, the column oven temperature is 20 ℃, sample size is 10 μ L.The gradient elution program is: the methyl alcohol volumetric usage is 5% during 0-2min; Methanol usage brings up to 40% behind the 3min, keeps 5min; Then methanol usage drops to 5% in 1min, and then keeps 9min.
Mass spectrum ionization mode is the electron spray ion, and sheath gas and assisted gas are nitrogen.The scanning of the mass spectrum pattern adopts selective reaction to detect (SRM) detecting pattern, and data acquisition is carried out under positive ion mode.The mass spectrum condition: ion source temperature is 300 ℃; Sheath gas and assisted gas pressure are respectively 20psi and 5psi, and electron spray voltage is 3000V, and sweep limit is m/z 50～300, and the ion scan time interval is 0.05s.Mass spectrum parameter such as the table 1 of 20 seed amino acids, glycerine, urea and lactic acid.The detection limit of method is by the hybrid standard sample of practical measurement series low concentration, determines as 3 take signal to noise ratio (S/N ratio) (S/N).Experiment is determined the recovery of method by standard addition method, adds respectively 50 μ g/L, the potpourri standard solution of 100 μ g/L and 500 μ g/L in the cell culture fluid sample of having handled well.Continuous sample introduction is 6 times successively, calculates the RSD value of each chromatographic peak retention time and SRM chromatographic peak peak area.Adopt the LC-MS/MS analytical approach of setting up, the recovery of standard addition of each compound has reached between the 91%-105%, and the recovery is good.The detection limit of 23 kinds of metabolins, the range of linearity, related coefficient, precision see Table 2.Table 3 has been listed the metabolism stream of 23 kinds of metabolic products.
The mass spectrum parameter of table 1 compound
The detection limit (LOD) of 23 kinds of metabolins of table 2, the range of linearity, related coefficient (r
2), precision (in the RSD of peak area) and the recovery.
The metabolic flux of each metabolic pathway that the metabolism stream of 23 kinds of metabolic products of table 3 and computational analysis obtain
Embodiment 3:
According to bioinformatic analysis (KEEG) database of metabolic pathway, simplify metabolism network according to the metabolic flux balance of 23 kinds of metabolins that obtain among the embodiment 2, draw the metabolism network (Metabolic profiling) of HepG2 cell, as shown in Figure 2.The metabolism network of drawing comprises 71 cellular biochemical reaction paths, is respectively 5 biomacromolecule cell synthetic reactions (nucleic acid-DNA and RNA, protein, lipid and organic carbon), 41 intramicellar reactions, 24 born of the same parents and reacts outward.Synthetic 20 seed amino acids that derive from of protein do not mark among the figure.Among the figure not the Framed mark part be the mass exchange of the outer metabolin of born of the same parents and external environment.The metabolic pathway that whole metabolism network comprises has: glycolysis, gluconeogenesis, pentose phosphate pathway, glycerol metabolism, tricarboxylic acid cycle, urea cycle, amino acid metabolism and lipid metabolism.The calculating of metabolic flux is assumed to be the basis with quasi-stable state, supposes that intracellular intermediate metabolites all is in quasi-stable state, namely its concentration change speed be 0 (Jens N, FoziaN, Elmar H.Toxicol Appl Pharm, 2009,240:327-336).
Embodiment 4:
The growth logarithmic phase hepatoma carcinoma cell HepG2 at 37 ℃, 5%CO
2, containing in the DMEM nutrient culture media of 10% hyclone and cultivate, difference from Example 1 is that respectively at adding 2,3,7 of variable concentrations in the DMEM nutrient solution, 8-TCDD uses respectively 2,3,7 of variable concentrations, 8-TCDD (0.001nmo lL
-1, 0.01n molL
-1, 0.1n molL
-1With 1.0n molL
-1) processing 48h.Draw in cell culture fluid 10 μ L to the 1ml methyl alcohol and water mixed liquid (volume ratio 90: 10) of cultivating 48h whirlpool concussion 2min, then 15000rmin
-1Centrifugal 5min, supernatant cross 0.22 μ m syringe filters.
The method of pressing embodiment 2 adopts HPLC-MS/MS to analyze the situation of change of born of the same parents' extra-metabolite concentration, calculates the change degree in born of the same parents' intracellular metabolite path according to the Metabolic profiling of embodiment 3, and the result as shown in Figure 1.
This invention pre-treating method is simple, utilizes the multiple metabolin in the HPLC-MS/MS energy accurate quantification cell culture fluid, can reflect intuitively that in conjunction with the Metabolic flux analysis system environmental contaminants are on the impact in cellular metabolism path; Have simple to operately, analysis time is short, and is highly sensitive, favorable reproducibility, the advantage such as the acquired information amount is comprehensive.
Claims (7)
1. Cytotoxic appraisal procedure of environmental contaminants is characterized in that:
Utilize HPLC-MS/MS in conjunction with the cytotoxicity of Metabolic flux analysis systematic quantification analysis environments pollutant, its operation steps is as follows:
A) take the cell culture fluid that do not add environmental contaminants as standard items, environmental contaminants are put into cell culture fluid DMEM as testing sample, cultivate respectively at carrying out the HepG2 cell in the incubator, in 37 ℃ of cultivations 6-72 hour, in incubator, pass into volume content 5%CO in the incubation
2Air; Get cell culture fluid, adopt liquid-liquid extraction method that the metabolin in the cell culture fluid is extracted, obtain the metabolic product extract;
B) with the metabolic product extract loading that obtains, carry out the HPLC-MS/MS quantitative test, obtain the metabolic flux of 23 kinds of metabolic products in the cell culture fluid;
C) according to bioinformatic analysis (KEEG) database of metabolic pathway, according to the quasi-stable state hypothesis, the metabolic flux balance of 23 kinds of metabolins obtaining is simplified metabolism network, made up the Metabolic profiling of HepG2 cell;
D) Metabolic profiling that testing sample and standard items is obtained compares, and calculates the situation of change of metabolic pathway by metabolic flux, knows that environmental contaminants are to the metabolism toxicity situation of cell.
According to claimed in claim 1 to the property appraisal procedure, it is characterized in that:
The environmental contaminants that the method relates to mainly comprise persistence organic pollutant, excellent control pollutant, and novel organic contaminant is a series of to cause in the material that cellular metabolism changes one or two or more kinds, its concentration 0.001nM-1 μ M in cell culture fluid;
Extract is methanol/water among the step a, and the two ratio is 70: 20-95: 5, and the amount ratio of cell culture fluid and extract is 5-20 μ l: 1ml;
Separating of 23 kinds of metabolic products, the especially separation of isomers have been realized by optimizing chromatographiccondition with the mass spectrum parameter among the step b; 23 kinds of metabolic products are urea, glycerine, lactic acid glycocoll, alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophane, serine, tyrosine, halfcystine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine and histidine;
Structure to the Metabolic profiling of HepG2 cell among the step c has mainly comprised the steps such as glycolysis, tricarboxylic acid cycle, amino acid metabolism, urea cycle.
3. according to methods of Toxicity Assessment claimed in claim 1, it is characterized in that: 15000 rev/mins of centrifugal 3-5min of solution needs after the liquid-liquid extraction among the step a, to reach the purpose of protein precipitation.
4. according to methods of Toxicity Assessment claimed in claim 1, it is characterized in that: the mobile phase among the step b is: the first alcohol and water, with respect to acetonitrile, methyl alcohol more is conducive to the separation of isomers compound, also is on the other hand to match for the medium with cell extract.
5. according to methods of Toxicity Assessment claimed in claim 1, it is characterized in that: the mass spectrum ionization mode of HPLC-MS/MS is the electron spray ion among the step b, and sheath gas and assisted gas are nitrogen.The scanning of the mass spectrum pattern adopts selective reaction to detect (SRM) detecting pattern, and data acquisition is carried out under positive ion mode.The mass spectrum condition: ion source temperature is 300 ℃; Sheath gas and assisted gas pressure are respectively 20psi and 5psi, and electron spray voltage is 3000V, and sweep limit is m/z 50～300, and the ion scan time interval is 0.05s.
6. according to methods of Toxicity Assessment claimed in claim 1, it is characterized in that: the elution requirement of liquid chromatography is gradient elution among the step b, this elution requirement can accelerate analysis time on the other hand on the one hand not adding the separation that can reach isomers under ion-pairing agent and the organic acid condition.
7. according to methods of Toxicity Assessment claimed in claim 1, it is characterized in that: the structure of metabolic profile can require the structure metabolism network of selection according to the analysis of oneself among the step c, but can not omit in profile diagram for metabolic intermediate.
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| CN103675129A (en) * | 2013-12-05 | 2014-03-26 | 柳州联海科技有限公司 | Gas chromatography-mass spectrometry detection method for intracellular carbohydrates and organic acids |
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| CN106324159A (en) * | 2015-07-02 | 2017-01-11 | 中国科学院大连化学物理研究所 | Pretreatment method for detection and analysis of micromolecular metabolites in adherent cells |
| CN106324165A (en) * | 2015-07-02 | 2017-01-11 | 中国科学院大连化学物理研究所 | Method for detecting free fatty acids in trace amount of cell culture fluid |
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| CN110907575A (en) * | 2018-09-14 | 2020-03-24 | 中国科学院大连化学物理研究所 | Deep annotation method of hydroxycinnamic acid amide in plants |
| CN110907575B (en) * | 2018-09-14 | 2021-10-08 | 中国科学院大连化学物理研究所 | Deep annotation method of hydroxycinnamic acid amide in plants |
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