CN103364510B - Method for evaluating cytotoxicity of environmental pollutants by adopting HPLC-MS/MS (high performance liquid chromatography and mass spectrometry or mass spectrometry) - Google Patents
Method for evaluating cytotoxicity of environmental pollutants by adopting HPLC-MS/MS (high performance liquid chromatography and mass spectrometry or mass spectrometry) 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, obtains the advantages such as quantity of information is comprehensive.
Background technology
The free-revving engine of toxicologic study is protection health; but the persistence organic pollutant existed in current many novel organic contaminants and environment to can be used for the basic data that human health risk evaluates also little; very weak to the correlative study of Health Impact in toxicologic study; Environmental Decision-making and environmental management is made to lack scientific basis. especially for the persistence organic pollutant in environment; as bioxin; polychlorinated biphenyl etc.; be not enough to support to draw the conclusion (Yang Yongbin, the ecotoxicological journal that are of practical significance.2006,105-115), utilize metabolism group to be new tool (Liu Peng, Dalian Medical Univ's journal of pollutant toxicity research to Cytotoxicity evaluation.2008, but it is long to there is analytical cycle in 565-569) traditional cellular metabolism methods of Toxicity Assessment, method of testing is loaded down with trivial details, multiple instrument is needed to combine, and the shortcomings such as quantity of information is limited obtained, therefore be badly in need of development a kind of efficient fast and the analysis means of informative assesses the metabolism toxicity of cell, HPLC-MS/MS exists quantitatively accurately, advantage (the Monique P that test specification is wide, Christine V.S, Konstantinos P, et al.Rapid Commun Mass Spectrom, 2003, 17:1297-1311), Metabolic flux analysis system can utilize the variable quantity of extracellular metabolic product to calculate situation of change (the Jens N of corresponding born of the same parents' intracellular metabolite thing, Fozia N, Elmar H.Toxicol Appl Pharm, 2009, 240:327-336), the two combines, cellular metabolism toxicity for research environment pollutant rapidly and efficiently has great importance.
Summary of the invention
The object of the invention is to develop a kind of simple to operate, the cycle is short, obtain the Cytotoxicity evaluation method of informative.
For solving the problems of the technologies described above, the technical solution adopted in the present invention is:
Utilize HPLC-MS/MS to detect the 23 kinds of metabolins analyzed in cell culture fluid, build HepG2 cellular metabolism profile diagram, carry out the toxicity evaluation in cellular metabolism path
A) adopt liquid-liquid extraction method to extract the metabolin in cell culture fluid, Sample Dilution and removing protein one step are completed;
B) the HPLC-MS/MS quantivative approach simultaneously analyzing 23 kinds of metabolic products in cell culture fluid is established;
C) according to analyzing the Metabolic profiling requiring to construct HepG2 cell;
D) Metabolic flux analysis system is in conjunction with the cellular metabolism toxicity of HPLC-MS/MS successful analysis environmental contaminants.
In step a, cell culture fluid and solvent load are than being 5-20 μ l:1ml;
The Simultaneous Quantitative Analysis of 23 kinds of metabolic products is achieved by optimization chromatographiccondition and mass spectrometry parameters, the especially separation of isomers in step b;
In step c, mainly glycolysis is included to the structure of the Metabolic profiling of HepG2 cell, tricarboxylic acid cycle, amino acid metabolism, the steps such as urea cycle.
Extraction solvent in step a is methanol/water, and this Extraction solvent can also match with the mobile phase in chromatogram except can removing a large amount of albumen in cell culture fluid, is more conducive to chromatographic resolution.
In step b, the application of the methyl alcohol of HPLC-MS/MS is advantageously in the separation of isomers.
In step b, liquid chromatogram mobile phase is methanol/water, elution requirement is gradient elution, the selection of methyl alcohol and the change of elution requirement are not adding the separation that can reach isomers under ion-pairing agent and organic acid condition on the one hand, can shorten analysis time on the other hand.
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.), one or two or more kinds in a series of materials that cellular metabolism can be caused to change such as novel organic contaminant (chloro palycyclic aromatic), its concentration 0.001nM-1 μM in cell culture fluid;
In step a, extract is methanol/water, 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;
Being separated of 23 kinds of metabolic products is achieved with mass spectrometry parameters, the especially separation of isomers by optimizing chromatographiccondition in 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;
In step c, mainly glycolysis is included to the structure of the Metabolic profiling of HepG2 cell, tricarboxylic acid cycle, amino acid metabolism, the steps such as urea cycle.
In step a, after liquid-liquid extraction, solution needs 15000 revs/min of centrifugal 3-5min, to reach the object of protein precipitation.
Mobile phase in step b is: first alcohol and water, and relative to acetonitrile, methyl alcohol is more conducive to the separation of isomers compound, is also to match with the medium of cell extract on the other hand.
In step b, the mass spectrum ionization mode of HPLC-MS/MS is electron spray ion, and sheath gas and assisted gas are nitrogen.Scanning of the mass spectrum pattern adopts selective reaction to detect (SRM) detecting pattern, and data acquisition is carried out in the positive-ion mode.Mass Spectrometry Conditions: ion source temperature is 300 DEG C; 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 ion scanning time is spaced apart 0.05s.
In step b, the elution requirement of liquid chromatography is gradient elution, and this elution requirement is not adding the separation that can reach isomers under ion-pairing agent and organic acid condition on the one hand, can accelerate analysis time on the other hand.
In step c, the structure of metabolic profile can require the structure metabolism network of selection according to the analysis of oneself, but can not omit in profile diagram for metabolic intermediate.
Tool of the present invention has the following advantages:
(1) pre-treatment is simple, and after sample mix with extract, a needs is simple shakes centrifugal and filters and namely can be used for HPLC-MS/MS and analyze; (2) quantitatively accurate, consuming time short: HPLC-MS/MS can carry out Simultaneous Quantitative Analysis to kind of the metabolic product of 23 in cell culture fluid, substantially reduce analysis time by optimizing chromatographic condition; (3) HPLC-MS/MS and Metabolic flux analysis system combination get up by the method; (4) analysis result computational analysis can also go out the change of intracellular products except accurately can obtaining the situation of change of metabolic product in cell culture fluid, and then obtains the situation of change in cellular metabolism path.
First the present invention utilizes HPLC-MS/MS to analyze 23 kinds of metabolins in cell culture fluid, and builds the profile diagram of HepG2 cell, utilizes Metabolic flux analysis with having developed a kind of method for environmental contaminants Cytotoxicity evaluation.
Accompanying drawing explanation
Fig. 1, for adopting the inventive method, have studied 2,3,7 of variable concentrations, 8-TCDD impacts on cellular metabolism path (HepG2 cellular metabolism stream).
Fig. 2 is used for the HepG2 cellular metabolism network chart that metabolic flux calculates.(non-Framed mark represents Extracellular metabolism)
Embodiment
The present invention utilizes HepG2 cell for supplying examination cell, by the cytotoxicity of HPLC-MS/MS in conjunction with Metabolic flux analysis systematic quantification analysis environments pollutant.
Embodiment 1:
Adopt cell culture fluid DMEM in incubator, carry out growing the hepatocellular carcinoma H22 cell chulture of logarithmic phase, cultivate 48 hours in 37 DEG C, in incubator, in incubation, pass into the air of volume content 5%CO2; Draw in cell culture fluid 10 μ L to the 1ml methyl alcohol and water mixed liquid (volume ratio 90:10) cultivating 48h, whirlpool concussion 2min, then 15000rmin
-1centrifugal 5min, 0.22 μm of syringe filters crossed by supernatant, obtains metabolic product extract.The method removing protein and Sample Dilution step through, sampling amount only needs 10 μ L.Result shows, the repeated RSD<2.88% of the method, and the recovery is 91.27%-115.07%.
Embodiment 2:
Metabolic product extract loading analysis embodiment 1 obtained, sets up HPLC-MS/MS analytical approach, and liquid-phase chromatographic column selects Atlantis C18 reverse-phase chromatographic column (waters, 150mm × 2.1mm, 3.0 μm); Mobile phase A is pure water, and Mobile phase B is methyl alcohol; Overall flow rate is 200 μ Lmin
-1, column oven temperature is 20 DEG C, and sample size is 10 μ L.Gradient elution program is: during 0-2min, methyl alcohol volumetric usage is 5%; After 3min, methanol usage brings up to 40%, keeps 5min; Then in 1min, methanol usage drops to 5%, and then keeps 9min.
Mass spectrum ionization mode is electron spray ion, and sheath gas and assisted gas are nitrogen.Scanning of the mass spectrum pattern adopts selective reaction to detect (SRM) detecting pattern, and data acquisition is carried out in the positive-ion mode.Mass Spectrometry Conditions: ion source temperature is 300 DEG C; 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 ion scanning time is spaced apart 0.05s.The mass spectrometry parameters of 20 seed amino acids, glycerine, urea and lactic acid is as table 1.The detection limit of method, by the hybrid standard sample of practical measurement series low concentration, is 3 to determine with signal to noise ratio (S/N ratio) (S/N).Test the recovery by standard addition method defining method, add 50 μ g/L respectively in processed good cell culture fluid sample, the potpourri standard solution of 100 μ g/L and 500 μ g/L.Continuous sample introduction 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 set up, the recovery of standard addition of each compound reaches between 91%-105%, and the recovery is good.The detection limit of 23 kinds of metabolins, the range of linearity, related coefficient, precision are in table 2.Table 3 lists the metabolic fluxes of 23 kinds of metabolic products.
The mass spectrometry parameters of table 1 compound
The detection limit (LOD) of table 2 23 kinds of metabolins, the range of linearity, related coefficient (r2), precision (RSD in peak area) and the recovery.
The metabolic flux of each metabolic pathway that the metabolic fluxes of table 3 23 kinds of metabolic products and computational analysis obtain
Embodiment 3:
According to bioinformatic analysis (KEEG) database of metabolic pathway, metabolic flux balance according to the 23 kinds of metabolins obtained in embodiment 2 simplifies metabolism network, draw the metabolism network (Metabolic profiling) of HepG2 cell, as shown in Figure 2.The metabolism network drawn 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 react outward.The synthesis of protein derives from 20 seed amino acids, does not mark in figure.In figure, non-Framed mark part is 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 basis with quasi-stable state, supposes that intracellular intermediate metabolites is all in quasi-stable state, and namely its concentration change speed is 0 (Jens N, Fozia N, Elmar H.Toxicol Appl Pharm, 2009,240:327-336).
Embodiment 4:
Grow the hepatocellular carcinoma H22 of logarithmic phase at 37 DEG C, 5%CO
2, cultivate containing in the DMEM nutrient culture media of 10% hyclone, difference from Example 1 is, adds 2,3,7,8-TCDD of variable concentrations respectively in DMEM nutrient solution, uses 2,3,7,8-TCDD (0.001n molL of variable concentrations respectively
-1, 0.01n molL
-1, 0.1n molL
-1with 1.0n molL
-1) process 48h.Draw in cell culture fluid 10 μ L to the 1ml methyl alcohol and water mixed liquid (volume ratio 90:10) cultivating 48h, whirlpool concussion 2min, then 15000rmin
-1centrifugal 5min, 0.22 μm of syringe filters crossed by supernatant.
Adopt HPLC-MS/MS to analyze the situation of change of Extracellular metabolism concentration by the method for embodiment 2, calculate the change degree in born of the same parents' intracellular metabolite path according to the Metabolic profiling of embodiment 3, result as shown in Figure 1.
This invention pre-treating method is simple, utilizes the multiple metabolin in HPLC-MS/MS energy accurate quantification cell culture fluid, can reflect the impact of environmental contaminants on cellular metabolism path intuitively in conjunction with Metabolic flux analysis system; Have simple to operate, analysis time is short, highly sensitive, favorable reproducibility, obtains the advantages such as quantity of information is comprehensive.
Claims (7)
1. the 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) not add the cell culture fluid of environmental contaminants for standard items, environmental contaminants are put into cell culture fluid DMEM as testing sample, HepG2 cell chulture is carried out respectively in incubator, cultivate 6-72 hour in 37 DEG C, in incubator, in incubation, pass into the air of volume content 5%CO2; Get cell culture fluid, adopt liquid-liquid extraction method to extract the metabolin in cell culture fluid, obtain metabolic product extract;
B) the metabolic product extract loading will obtained, carries out HPLC-MS/MS quantitative test, obtains the metabolic flux of 23 kinds of metabolic products in cell culture fluid;
Gradient elution program is: during 0-2min, methanol usage is 5%; After 3min, methanol usage brings up to 40%, keeps 5min; In 1min, methanol usage drops to 5%, and then keeps 9min;
C) according to the bioinformatic analysis KEEG database of metabolic pathway, according to quasi-stable state hypothesis, the metabolic flux balance of the 23 kinds of metabolins obtained is simplified metabolism network, constructs the Metabolic profiling of HepG2 cell;
D) Metabolic profiling that testing sample and standard items obtain is contrasted, calculated the situation of change of metabolic pathway by metabolic flux, know the metabolism toxicity profile of environmental contaminants to cell.
2., according to the Cytotoxic appraisal procedure of environmental contaminants according to claim 1, it is characterized in that:
The environmental contaminants that the method relates to mainly comprise persistence organic pollutant, excellent control pollutant, one or two or more kinds in a series of material that cellular metabolism can be caused to change of novel organic contaminant, its concentration 0.001nM-1 μM in cell culture fluid;
In step a, extract is methanol/water, and the two volume ratio is 70:20-95:5, and the amount ratio of cell culture fluid and extract is 5-20 μ l:1ml;
Being separated of 23 kinds of metabolic products and being separated of isomers is achieved by optimizing chromatographiccondition with mass spectrometry parameters in 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;
In step c, mainly glycolysis is included to the structure of the Metabolic profiling of HepG2 cell, tricarboxylic acid cycle, amino acid metabolism, the steps such as urea cycle.
3. according to the Cytotoxic appraisal procedure of environmental contaminants according to claim 1, it is characterized in that: in step a, after liquid-liquid extraction, solution needs 15000 revs/min of centrifugal 3-5min, to reach the object of protein precipitation.
4. according to the Cytotoxic appraisal procedure of environmental contaminants according to claim 1, it is characterized in that: the mobile phase in step b is: first alcohol and water.
5. according to the Cytotoxic appraisal procedure of environmental contaminants according to claim 1, it is characterized in that: in step b, the mass spectrum ionization mode of HPLC-MS/MS is electron spray ion, and sheath gas and assisted gas are nitrogen; Scanning of the mass spectrum pattern adopts selective reaction to detect SRM detecting pattern, and data acquisition is carried out in the positive-ion mode; Mass Spectrometry Conditions: ion source temperature is 300 DEG C; 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 ion scanning time is spaced apart 0.05s.
6. according to the Cytotoxic appraisal procedure of environmental contaminants according to claim 1, it is characterized in that: in step b, the elution requirement of liquid chromatography is gradient elution.
7. according to the Cytotoxic appraisal procedure of environmental contaminants according to claim 1, it is characterized in that: in step c, the structure of metabolic profile does not omit metabolic intermediate.
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