CN109991354A - A kind of method of NNK and its metabolite content and prediction NNK exposure in measurement blood - Google Patents
A kind of method of NNK and its metabolite content and prediction NNK exposure in measurement blood Download PDFInfo
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Abstract
The invention belongs to test and analyze technical field, and in particular to a method of NNK and its metabolite content and prediction NNK exposure in measurement blood.The method of prediction NNK exposure of the invention is the following steps are included: measurement NNK first exposes the content of NNK and its metabolite in the blood of animal, then the 'alpha '-hydroxylation coefficient of NNK is determined using the content of NNK in blood and its metabolite, thus the 'alpha '-hydroxylation extent of reaction after assessing NNK exposure in Different Individual.Present invention utilizes the dual indexes of 'alpha '-hydroxylation coefficient and indirect product content to reflect NNK in the intracorporal 'alpha '-hydroxylation extent of reaction of machine, assessment result is accurate, reliable, it the advantages such as is comparable, establishes a kind of new method that can be used for assessing NNK early stage exposure.
Description
Technical field
The invention belongs to test and analyze technical field, and in particular to NNK and its metabolite content in a kind of measurement blood
With the method for prediction NNK exposure.
Background technique
TSNAs is distinctive one group of N- nitrous acid amine harmful components in tobacco, wherein 4- (N- methyl nitroso) -1-
(3- pyridyl group) -1- butanone (NNK) is classified as I anthropoid carcinogen by international cancer research institution, causes with strong organ
Cancer activity, it is related with the generation of cancers such as lung cancer, cancer of pancreas.It is now recognized that the carcinogenesis of TSNAs is by vivo
Metabolism activation and generate, therefore study TSNAs metabolic fate in vivo and seem particularly necessary.
Studies have shown that NNK is mainly metabolized by three approach in vivo, i.e., carbonyl reduction, pyridine-N- are aoxidized
And 'alpha '-hydroxylation.Into intracorporal NNK under the action of microsomal enzyme or carbonyl reductase, can quickly it be formed equally with carcinogenic
4- (N- methyl nitroso) -1- (3- pyridyl group)-n-butyl alcohol (NNAL) of effect, NNAL pass through pyridine--glycosides acid again
Change can form NNAL- glucosiduronate, and NNK and NNAL can also be respectively formed NNK-N-Oxide by pyridine-N- oxidation
And NNAL-N-Oxide；Under normal conditions, N- oxidation and glucoside acidification are considered as the detoxification processes of TSNAs in vivo.
On the other hand, 'alpha '-hydroxylation reaction (including Alpha-Methyl hydroxyl of the NNK and NNAL under Cytochrome P450 serial enzymes mediation
Change and alpha-methylene hydroxylating) it is considered as main metabolic activated channel.NNK is raw first by Alpha-Methyl hydroxylation metabolism approach
At alpha-hydroxymethyl NNK, alpha-hydroxymethyl NNK is spontaneous to slough a formaldehyde generation pyridine oxygen-butyl diazohydroxide, this compound
It can be reacted with water and generate 4- hydroxyl -1- (3- pyridyl group) -1- butanone (HPB)；NNK passes through alpha-methylene hydroxylation metabolism approach
Firstly generate α-hydroxy methylene NNK, α-hydroxy methylene NNK spontaneous generation alkyl diazohydroxide and keto-aldehyde, alkyl diazonium
Hydroxide attacks DNA and generates a series of methyl DNA adducts, and keto-aldehyde ultimately generates ketone acid by further conversion
(OPBA).The 'alpha '-hydroxylation metabolic pathway of NNAL is similar with NNK, and pyrrole can be generated by Alpha-Methyl hydroxylation metabolism approach in NNAL
Methyl DNA can be generated by alpha-methylene hydroxylation metabolism approach in pyridine hydroxybutyl DNA adduct and glycol (PBD), NNAL
Adduct and carboxylic acid (HPBA).The electrophilic intermediate that NNK and NNAL is formed by 'alpha '-hydroxylation metabolic pathway is with DNA adduction
Carcinogenic key.Wherein, the methyl DNA adduct of formation mainly has O6Methyl guanine (O6- mG), 7- methyl guanine and
O43 kinds of forms of methyl thymine, and with O6- mG is relatively conventional.
Although the metabolic pathway and carcinogenic effect of NNK are more clear, research in relation to NNK at present or concentrate on is adopted
The toxicology mechanism after NNK exposes directly is investigated with model in vitro or in vivo or stays in description NNK in different biological models
In metabolic characteristics, and the quantizating index that the potential Toxic activity of NNK is directly assessed from metabolic analysis angle is still not definitely established.
Trace in a kind of animal blood is disclosed in the Chinese invention patent file that Authorization Notice No. is CN102012409B
The analysis method of the distinctive N- nitrosamine of tobacco, the content of a variety of nitrosamine in blood is able to detect by this method.But its
The object of test is N- nitrosamine, rather than the metabolite of N nitrosamine, can not reflect N nitrosamine metabolic condition, it is even more impossible to predict
N nitrosamine exposure.
Summary of the invention
The purpose of the present invention is to provide a kind of methods for predicting NNK exposure, can be from metabolic analysis by this method
Angle directly assesses the exposure of NNK.
The object of the invention is also to provide a kind of method of NNK and its metabolite content in measurement blood, this method
Detection accuracy is high.
To achieve the above object, the technical scheme adopted by the invention is as follows:
A method of prediction NNK exposure, comprising the following steps:
(1) blood sample of NNK exposure animal is centrifuged, supernatant is taken to be uniformly mixed with internal standard compound, acetonitrile,
It is then centrifuged for separating, takes supernatant detection NNK and its metabolite content；
(2) according to the content of NNK and its metabolite in the animal blood sample of different time after exposure relative to the time
Concentration time curve is made, and calculates AUC value, the 'alpha '-hydroxylation coefficient of NNK is then calculated, according to 'alpha '-hydroxylation coefficient prediction
NNK exposure.
The method of prediction NNK exposure of the invention is to evaluate NNK exposure from metabolism activation path analysis.Using
NNK exposes animal model, then measures the content of the direct metabolite of NNK in blood, is obtaining entering for NNK and its metabolin
It is opposite with the NNK of body circulation is entered according to 'alpha '-hydroxylation metabolic pathway products total amount in blood on the basis of body circulation relative quantity
The ratio ('alpha '-hydroxylation coefficient) of amount assesses the 'alpha '-hydroxylation extent of reaction in Different Individual after NNK exposure, and then to comment
The potential exposure risk of valence NNK provides quantizating index.
This method is that (such as cell toxicity test, mutagenicity test, rodent toxicity are tried using end-point method toxicology test
Test) useful supplement is provided from the risk assessment that biological effect angle carries out NNK, it can be in NNK exposure in early days from metabolism
Analytic angle provides the quantizating index that can refer to for the potential Toxic activity of NNK.
The metabolite is HPB, PBD, HPBA and OPBA.HPB, PBD, HPBA and OPBA are the 'alpha '-hydroxylation of NNK
It is high to assess the accuracy of 'alpha '-hydroxylation degree by measuring the content of direct metabolite for direct metabolite.
For the detection order of accuarcy for improving NNK and its metabolite content, step (1) the detection NNK and its metabolism are produced
The detection method of object content is using NNK and its generation in the blood sample of different time after UHPLC-Q Exactive measurement exposure
Thank to the content of product.
The chromatographic condition of the UHPLC-Q Exactive are as follows: chromatographic column is HILIC column；Column temperature: 25 DEG C；5 μ L of sample volume,
Flow velocity 0.5mL/min；Mobile phase includes A phase and B phase, and A phase is 10mmol/mL ammonium formate solution, and B phase is acetonitrile；Gradient:
The volume fraction of B phase drops to 85% by 95% in 0~2min, is down to 75% by 85% in 2~6min, in rising in 0.1min
95%, maintain 2min；Mass Spectrometry Conditions are as follows: heating esi ion source, positive ion mode, spray voltage 3.5kV；Sheath gas is
9.9L/min, secondary air speed are 3.3L/min, and capillary heating temperature is 320 DEG C, and heter temperature is 300 DEG C；Scan mould
Formula uses Full MS/ddMS2, level-one full scan resolution ratio is 70000, and range is 100~300m/z, and automatic growth control is
1e6, ion maximum injection length are 50ms；Second level scanning is that data dependence scans, resolution ratio 17500, automatic growth control
For 1e5, maximum injection length is 50ms, and normalized collision energy is 35%.
The 'alpha '-hydroxylation coefficient is ratio of the sum of the AUC of NNK metabolite with the sum of the AUC of NNK and NNAL.According to
'alpha '-hydroxylation metabolic pathway products total amount and enter the ratio of NNK relative quantity of body circulation in blood and determine 'alpha '-hydroxylation system
Number, can reduce experimental error.
The method of the prediction NNK exposure is further comprising the steps of: O in detection NNK exposure animal's liver tissue6-
MG content, according to 'alpha '-hydroxylation coefficient and O6- mG content prediction NNK exposure.
One aspect of the present invention reflects NNK in the intracorporal average Alpha-hydroxy of machine using the ratio of metabolite direct in blood
Change extent of metabolism, is on the other hand also confirmed using the generation situation of indirect metabolic product caused by the metabolism of 'alpha '-hydroxylation approach
DNA damage degree in main metabolic organ-tissue establishes and directly assesses the potential Toxic activity of NNK from metabolic analysis angle
Quantizating index, analysis result is accurate and reliable, and assay NNK is had in the intracorporal 'alpha '-hydroxylation extent of metabolism of different machines
Comparativity achieves obvious progress.It can quantify the exposure of the animal model NNK under different condition using technical solution of the present invention
'alpha '-hydroxylation extent of metabolism afterwards establishes a kind of new method that can be used for assessing NNK early stage exposure.
O in the detection NNK exposure animal's liver tissue6- mG content the following steps are included:
1) DNA in liver organization is extracted, is then hydrolyzed, is uniformly mixed with internal standard compound and is then separated by solid-liquid separation；
2) liquid after being separated by solid-liquid separation step 1) crosses solid-phase extraction column, elutes, and eluent removes solvent and obtains solid-like
Product；
3) it dissolves the solid sample that step 2) obtains to obtain test sample with acetonitrile, test sample is used into UHPLC-Q
Exactive test analysis O6- mG content.
O in detection NNK exposure animal's liver tissue of the invention6The detection accuracy of the method for-mG content is high, quantitative standard
Really.
To further increase sensitivity for analysis, it is preferred that step 3) is described to use UHPLC-Q Exactive test analysis
When chromatography condition are as follows: spectrum column be C18 column；50 DEG C of column temperature；Mobile phase A is that volume fraction is 0.01% aqueous formic acid, flowing
Phase B is acetonitrile, and the two volume ratio is 30:70；Isocratic elution, flow velocity 0.20mL/min；10 μ L of sample volume.
The method of NNK and its metabolite content in measurement blood of the invention, the technical solution taken includes following step
It is rapid: blood sample being centrifuged, supernatant is taken to be uniformly mixed with internal standard compound, acetonitrile, is then centrifuged for separating, takes supernatant
Detect NNK and its metabolite content.
The detection accuracy of the method for NNK and its metabolite content is high in measurement blood of the invention, according to NNK in blood
And its content results of metabolite assess the 'alpha '-hydroxylation extent of reaction in Different Individual after NNK exposure, for from metabolism
Activated channel assay NNK exposure lays the foundation.
Detailed description of the invention
Fig. 1 is in rabbit blood in the embodiment 1 of the method for NNK and its metabolite content in measurement blood of the invention
The Concentration-time change curve of NNK；
Fig. 2 is in rabbit blood in the embodiment 1 of the method for NNK and its metabolite content in measurement blood of the invention
The Concentration-time change curve of NNAL；
Fig. 3 is in rabbit blood in the embodiment 1 of the method for NNK and its metabolite content in measurement blood of the invention
The Concentration-time change curve of HPB；
Fig. 4 is in rabbit blood in the embodiment 1 of the method for NNK and its metabolite content in measurement blood of the invention
The Concentration-time change curve of PBD；
Fig. 5 is in rabbit blood in the embodiment 1 of the method for NNK and its metabolite content in measurement blood of the invention
The Concentration-time change curve of OPBA；
Fig. 6 is in rabbit blood in the embodiment 1 of the method for NNK and its metabolite content in measurement blood of the invention
The Concentration-time change curve of HPBA；
Fig. 7 is that different groups are small in the embodiment 2 of the method for NNK and its metabolite content in measurement blood of the invention
The mean concentration-time change curve of NNK in mouse blood；
Fig. 8 is that different groups are small in the embodiment 2 of the method for NNK and its metabolite content in measurement blood of the invention
The mean concentration-time change curve of NNAL in mouse blood；
Fig. 9 is that different groups are small in the embodiment 2 of the method for NNK and its metabolite content in measurement blood of the invention
The mean concentration-time change curve of HPB in mouse blood；
Figure 10 is different groups in the embodiment 2 of the method for NNK and its metabolite content in measurement blood of the invention
The mean concentration-time change curve of PBD in mouse blood；
Figure 11 is different groups in the embodiment 2 of the method for NNK and its metabolite content in measurement blood of the invention
The mean concentration-time change curve of OPBA in mouse blood；
Figure 12 is different groups in the embodiment 2 of the method for NNK and its metabolite content in measurement blood of the invention
The mean concentration-time change curve of HPBA in mouse blood.
Specific embodiment
In measurement blood of the invention NNK and its when metabolite content every 90~110 μ L supernatant it is corresponding be added 200~
300 μ L acetonitriles.
In measurement blood of the invention NNK and its when metabolite content every 90~110 μ L supernatant it is corresponding be added 15~
25 μ L internal standard substance solutions.The internal standard substance solution is containing NNK-d4、NNAL-d3And HPB-d4Acetonitrile solution, NNK-d4、NNAL-
d3And HPB-d4Concentration be 100ng/mL.
Present invention NNK and its when metabolite content in measuring blood, determines NNK, NNAL using standard working curve
And its content of metabolite HPB, PBD, OPBA and HPBA.The determination of the standard working curve is the following steps are included: accurate
Each standard items of NNK, NNAL, HPB, PBD, OPBD and HPBA and internal standard compound are weighed, after the dissolution of a small amount of methanol, with acetonitrile constant volume,
Compound concentration is the standard reserving solution of 100 μ g/mL, -20 DEG C of preservations respectively.Each Standard Stock solutions are taken, are mixed according to a certain percentage
It closes, (is made of the ammonium formate solution and acetonitrile of 10mmol/mL, wherein the volume fraction of acetonitrile with the mobile phase under primary condition
For 95%) dilution prepare respectively a series of concentration gradient of containing the internal standards standard solution (NNK, NNAL in standard solution, HPB,
The concentration of each standard items of PBD, OPBD and HPBA is 0.1,0.5,1.0,5,10,50,100,500ng/mL, with NNK-d4For
NNK internal standard compound, NNAL-d3For NNAL internal standard compound, HPB-d4For the internal standard compound of remaining several target analytes, internal standard compound final concentration
It is 5ng/mL).Then each standard solution loading being carried out the analysis of liquid matter, (parameter used in instrument is this hair when the analysis test of liquid matter
Bright parameter used), the mass spectrometry parameter of NNK and each target analytes is as shown in table 1.With the chromatographic peak face of each target analytes
Long-pending each target analyte concentration that compares with internal standard peak area carries out linear regression analysis, and the standard for obtaining each target analytes is bent
Line regression equation and linearly dependent coefficient, method detection limit between 0.013~0.079ng/mL, the range of linearity 0.5~
Between 500ng/mL.
Present invention O in measurement NNK exposure animal's liver tissue6When-mG content, O is determined using standard working curve6-mG
Content.The determination of the standard working curve the following steps are included: accurately weigh O respectively6- mG and internal standard compound [CD3]-O6-
MG, acetonitrile constant volume after methanol dissolution are each formulated into the standard reserving solution that concentration is 100 μ g/mL.Liver after acidolysis of learning from else's experience neutralization
A certain amount of standard reserving solution is added in tissue DNA extracting solution, and the O of containing the internal standard object is prepared with dilution in acetonitrile6- mG concentration be 0.05,
0.25, the series of standards working solution of 0.5,1.0,5.0,10.0,25,100ng/mL, the concentration of internal standard compound are 2ng/mL.It will
Above-mentioned standard working solution loading carries out the analysis of liquid matter (parameter used in instrument is parameter used in the present invention when analysis test of liquid matter),
The mass spectrometry parameter of each object is as shown in table 1.Object standard working curve and related coefficient are obtained using internal standard method,
O6- mG is linear good within the scope of 0.05~100.00ng/mL, detection limit and quantitative limit be respectively 0.011ng/mL and
0.037ng/mL。
The mass spectrometry parameter of 1 NNK of table and each target analytes
Combined with specific embodiments below and attached drawing 2 the invention will be further described.
It is selected in NNK and its metabolite content and prediction NNK exposure experimentation in measurement blood of the invention
Animal is rabbit and mouse.Wherein rabbit is the adult Japan big-ear rabbit of health, 2.5~3.0kg of weight, using only
Vertical isolated rearing cage tool raising；Mouse is C57BL/6 healthy mature mouse.
Measure the embodiment 1 of the method for NNK and its metabolite content in blood
The present embodiment is exposure animal with rabbit, in blood the measuring method of NNK and its metabolite content include with
Lower step:
(1) two rabbit are chosen, number is divided into A, B.Overnight fast, then next day auricular vein injects NNK solution
(0.5mg/kg, normal saline).5,15,30,60,120,240 and 360min different time points, venous blood sampling after exposure
About 0.5mL is in the anticoagulant centrifuge tube of 1.5mL, with the centrifugal force 10min of 2000g under the conditions of 4 DEG C.Then 100 μ of supernatant is taken
In L to 1.5mL centrifuge tube, the mixing internal standard substance solution (NNK-d that 20 μ L concentration are 100ng/mL is added4、NNAL-d3And HPB-d4
Concentration is 100ng/mL) and 280 μ L acetonitriles, vortex 30s mix well after in 4 DEG C of centrifugal forces with 12000g
10min.Supernatant is taken, loading UHPLC-Q Exactive detection NNK and its metabolism produce after being filtered with 0.22 μm of syringe filter
Object content.
(2) chromatographic condition when UHPLC-Q Exactive is tested and analyzed: Waters Atlantis HILIC silica
Column (3.0mm i.d. × 100mm, 3.0 μm) chromatographic column；Column temperature: 25 DEG C；Sample volume 5 μ L, flow velocity 0.5mL/min；Flowing
Phase A is 10mmol/mL ammonium formate solution, and Mobile phase B is acetonitrile；Gradient: in 0~2min the volume fraction of Mobile phase B by
95% drops to 85%, and 2~6min is interior to be down to 75% by 85%, in rising to 95% in 0.1min, maintains 2min.Mass Spectrometry Conditions:
Electric heating esi ion source (HESI), positive ion mode, spray voltage 3.5kV.Sheath gas is 9.9L/min, secondary air speed
For 3.3L/min, capillary heating temperature is 320 DEG C, and heter temperature is 300 DEG C.Scan pattern uses Full MS/ddMS2,
Level-one full scan resolution ratio is 70000, and range is 100~300m/z, and automatic growth control (AGC) is 1e6, the injection of ion maximum
Time is 50ms.Second level scanning is that data dependence scans (Data dependent), resolution ratio 17500, and AGC 1e5 is maximum
Injection length is 50ms, and normalized collision energy (NCE) is 35%.
(3) standard working curve of NNK and its metabolite in result and blood is analyzed according to UHPLC-Q Exactive,
Determine the content of NNK and its metabolite in different time points blood.
The Concentration-time drawn according to the content and exposure duration of NNK in two groups of blood of rabbit A, B and its metabolite
Change curve is as shown in Fig. 1, and area under a curve (AUC) is as shown in table 2.
Area under the Concentration-time change curve of NNK and its metabolite in 2 blood of table
| Target analytes | AUC (the ngmL of rabbit A-1·min) | AUC (the ngmL of rabbit B-1·min) |
| NNK | 2023 | 1468 |
| NNAL | 2970 | 3026 |
| HPB | 160 | 165 |
| PBD | 83 | 89 |
| OPBA | 891 | 986 |
| HPBA | 1236 | 1347 |
Measure the embodiment 2 of the method for NNK and its metabolite content in blood
The present embodiment is exposure animal with mouse, and the method for NNK and its metabolite content in blood that measures includes following
Step:
(1) two groups of C57BL/6 healthy mature mouse are chosen, normal group and (the following letter of alcoholic fatty liver group are randomly divided into
Claim alcohol group), every group 5.Alcohol group mouse feeds with high-sugar-fat-diet (normal diet 83wt%+ cholesterol 2wt%+ lard
10wt%+ sucrose 5wt%), free No alcoholic beverages [15% (mass ratio) aqueous sucrose solution+alcohol], alcohol concentration is pressed
According to 5% (volume ratio, similarly hereinafter), 10%, 15%, 20%, 25%, 30%, 35% and 40% is successively improved, and is maintained 40%
2 weeks.Normal group mouse feeds with normal diet, free water.Two groups of mouse are to be freely eaten, and the duration 8 weeks.
(2) mouse is overnight fast before experiment, and NNK solution (2.0mg/kg, normal saline) is injected intraperitoneally in next day.In
5,15,30,60,120 and 240min different time points after exposure pluck eyeball and take blood about 0.5mL in the anticoagulant centrifuge tube of 1.5mL,
Subsequent processing and continuous mode are identical referring to the embodiment 1 of the method for NNK and its metabolite content in measurement blood.
It is drawn according to normal group and the content and exposure duration of NNK, NNAL and its metabolite in two groups of blood of alcohol group
For the mean concentration-time change curve of system as shown in Fig. 7~Figure 12, area under a curve (AUC) is as shown in table 3.
Area (AUC) under the mean concentration-time change curve of 3 NNK of table and each object in mouse blood
| Target analytes | Normal group AUC (ngmL-1·min) | AUC (the ngmL of alcohol group1·min) |
| NNK | 3314 | 1997 |
| NNAL | 4357 | 1563 |
| HPB | 136 | 156 |
| PBD | 55 | 75 |
| OPBA | 637 | 1013 |
| HPBA | 764 | 1126 |
Predict the embodiment 1 of the method for NNK exposure
According to the two groups of rabbit of A, B measured in the embodiment 1 of the method for NNK and its metabolite content in measurement blood
Area (AUC) calculates under the Concentration-time change curve of NNK and its metabolite in the blood of different time points after NNK exposure
'alpha '-hydroxylation coefficient (α-hydroxylation ratio, HR), its calculation formula is HRNNK=(AUCHPB+AUCPBD+AUCHPBA+
AUCOPBA)/(AUCNNK+AUCNNAL), calculated result is as shown in table 4.
The 'alpha '-hydroxylation coefficient results of 4 rabbit of table
| Index | Rabbit A | Rabbit B |
| HR | 0.47 | 0.58 |
As shown in Table 4, the HR value of rabbit B blood is greater than rabbit A, illustrates that NNK is more likely in rabbit B body to Alpha-hydroxy
Change metabolic pathway conversion.The intracorporal 'alpha '-hydroxylation extent of reaction of rabbit B is higher, the potential carcinogenic risk after implying NNK exposure
It is bigger.This may be derived from the active difference of key metabolic enzymes between rabbit individual, show that genetic background difference is to consider TSNAs exposure
An important factor for risk.This method provides the reference that can quantify to compare for the individuation difference that NNK early stage exposure is assessed
Index.
Predict the embodiment 2 of the method for NNK exposure
According to normal group and the alcohol measured in the embodiment 2 of the method for NNK and its metabolite content in measurement blood
The evaluation Concentration-time change curve of NNK and its metabolite in the blood of different time points after the NNK exposure of two groups of mouse of group
Lower area (AUC) calculates 'alpha '-hydroxylation coefficient (α-hydroxylationratio, HR), and its calculation formula is HRNNK=(AUCHPB
+AUCPBD+AUCHPBA+AUCOPBA)/(AUCNNK+AUCNNAL), calculated result is as shown in table 5.
The 'alpha '-hydroxylation coefficient results of 5 mouse of table
| Index | Normal group | Alcohol group |
| HR | 0.21 | 0.67 |
As shown in Table 5, the HR in alcohol group Mice Body after NNK exposure is 3.2 times of normal group mouse, is illustrated in alcohol group
The degree that 'alpha '-hydroxylation metabolism occurs for NNK in Mice Body further enhances, and Chronic Alcohol induction increases potential after NNK exposes
Carcinogenic risk.
Predict the embodiment 3 of the method for NNK exposure
DNA adduct yield in the 'alpha '-hydroxylation coefficient and Rabbit Liver tissue of NNK in the comprehensive rabbit blood of the present embodiment
(DNA-adduct productivity, DP) predicts NNK exposure.Wherein in rabbit blood NNK 'alpha '-hydroxylation coefficient
For the 'alpha '-hydroxylation coefficient in the embodiment 1 of the method for predicted exposure risk.DP is O in liver organization6The content value of-mG.Its
O in middle liver organization6The content of-mG is measured by following methods:
Rabbit in measurement blood in the embodiment 1 of the method for NNK and its metabolite content is taken a blood sample in last time
It puts to death afterwards and takes out hepatic tissue rapidly, will be cleaned through physiological saline after weighing, extract tissue DNA using DNA extraction kit, use
2000 ultramicron spectrophotometer of NanoDrop detects DNA content at 260nm, and (for double-stranded DNA, 1 absorbance units is 50
μ g/mL) it moves in centrifuge tube later, the HCl solution of 100 μ L 1mol/L is added, vortex 15s is simultaneously incubated in 80 DEG C of water-baths
30min；The NaOH solution that 100 μ L 1mol/L are then added adjusts pH to 7.0, and the internal standard compound of 20 μ L 100ng/mL is added
[CD3]-O6- mG solution (standard reserving solution when by drafting standard curve is formed with dilution in acetonitrile), vortex 15s are allowed to sufficiently mixed
It is even；Then first use 0.22 μm of organic phase pin type membrane filtration, then by by 2mL methanol and 2mL ultrapure water activation after
Oasis HLB solid-phase extraction column, loading flow velocity are 1~2mL/min；Then methanol successively is put into for 10% with 3mL volume fraction
Water elution, the elution of 2mL methanol；Eluent is redissolved after being dried with nitrogen with 1mL acetonitrile, loading UHPLC-Q after vortex 15s
Exactive analyzes O6- mG content.
Chromatographic condition is Agilent Zorbax Eclipse XDB-C18 (150mm × 2.1mm, 5 μm) chromatography when analysis
Column；50 DEG C of column temperature；Mobile phase A is that volume fraction is 0.01% aqueous formic acid, and Mobile phase B is acetonitrile (A:B=30:70 (volume
Than))；Isocratic elution, flow velocity 0.20mL/min；10 μ L of sample volume.NNK and its metabolite in mass spectrographic parameter and measurement blood
The parameter of the embodiment 1 of the method for content is identical.According to chromatographic peak area and O6The standard working curve of-mG determines liver organization
In O6The content of-mG, with O6For the content of-mG as DNA adduct yield, test result is as shown in table 6.
DP result in 6 Rabbit Liver tissue of table
| Index | Rabbit A | Rabbit B |
| DP | 41.3pg/mgDNA | 53.2pg/mgDNA |
According to table 4 and table 6 it is found that DP is equally higher than rabbit A while the 'alpha '-hydroxylation coefficient of rabbit B is higher than rabbit A,
Further demonstrate rabbit B suffers from cancer risk height.
Predict the embodiment 4 of the method for NNK exposure
DP predicts that NNK is sudden and violent in the 'alpha '-hydroxylation coefficient and mouse liver tissue of NNK in the comprehensive mouse blood of the present embodiment
It divulges a secret danger.The 'alpha '-hydroxylation coefficient of NNK is the 'alpha '-hydroxylation system in the embodiment 2 of the method for predicted exposure risk in mouse blood
Number；O in liver organization6O of the measurement of-mG content referring to the embodiment 3 of the method for prediction NNK exposure6- mG assay
Process, measurement result are as shown in table 7.
DP result in 7 mouse liver tissue of table
| Index | Normal group | Alcohol group |
| DP | 5036±363pg/mg DNA | 6390±814pg/mg DNA |
According to table 5 and table 7, DP average value is 1.3 times of normal group mouse in alcohol group Mice Body, is further proved
Chronic Alcohol induction increases the potential carcinogenic risk after NNK exposure.
Claims (9)
1. a kind of method for predicting NNK exposure, which comprises the following steps:
(1) blood sample of NNK exposure animal is centrifuged, takes supernatant to be uniformly mixed with internal standard compound, acetonitrile, then
Centrifuge separation takes supernatant detection NNK and its metabolite content；
(2) it is made according to the content of NNK and its metabolite in the animal blood sample of different time after exposure relative to the time
Concentration time curve, and AUC value is calculated, the 'alpha '-hydroxylation coefficient of NNK is then calculated, it is sudden and violent according to 'alpha '-hydroxylation coefficient prediction NNK
It divulges a secret danger.
2. it is according to claim 1 prediction NNK exposure method, which is characterized in that the metabolite be HPB,
PBD, HPBA and OPBA.
3. the method for prediction NNK exposure according to claim 1, which is characterized in that step (1) the detection NNK
And its detection method of metabolite content is the blood sample using different time after UHPLC-Q Exactive measurement exposure
The content of middle NNK and its metabolite.
4. the method for prediction NNK exposure according to claim 3, which is characterized in that the UHPLC-Q
The chromatographic condition of Exactive are as follows: chromatographic column is HILIC column；Column temperature: 25 DEG C；Sample volume 5 μ L, flow velocity 0.5mL/min；Mobile phase
Including A phase and B phase, A phase is 10mmol/mL ammonium formate solution, and B phase is acetonitrile；Gradient: the volume of B phase point in 0~2min
Number drops to 85% by 95%, and 2~6min is interior to be down to 75% by 85%, in rising to 95% in 0.1min, maintains 2min；Mass spectrum item
Part are as follows: heating esi ion source, positive ion mode, spray voltage 3.5kV；Sheath gas is 9.9L/min, and secondary air speed is
3.3L/min, capillary heating temperature are 320 DEG C, and heter temperature is 300 DEG C；Scan pattern uses Full MS/ddMS2, one
Grade full scan resolution ratio is 70000, and range is 100~300m/z, automatic growth control 1e6, and ion maximum injection length is
50ms；Second level scanning is that data dependence scans, resolution ratio 17500, automatic growth control 1e5, and maximum injection length is
50ms, normalized collision energy are 35%.
5. the method for prediction NNK exposure according to claim 1, which is characterized in that the 'alpha '-hydroxylation coefficient is
The ratio of the sum of the AUC of the sum of AUC of NNK metabolite and NNK and NNAL.
6. the method for prediction NNK exposure according to claim 1, which is characterized in that further comprising the steps of: detection
O in NNK exposure animal's liver tissue6- mG content, according to 'alpha '-hydroxylation coefficient and O6- mG content prediction NNK exposure.
7. the method for prediction NNK exposure according to claim 6, which is characterized in that the detection NNK exposure animal
O in liver organization6- mG content the following steps are included:
1) DNA in liver organization is extracted, is then hydrolyzed, is uniformly mixed with internal standard compound and is then separated by solid-liquid separation；
2) liquid after being separated by solid-liquid separation step 1) crosses solid-phase extraction column, elutes, and eluent removes solvent and obtains solid sample；
3) it dissolves the solid sample that step 2) obtains to obtain test sample with acetonitrile, test sample is used into UHPLC-
QExactive test analysis O6- mG content.
8. the method for prediction NNK exposure according to claim 7, which is characterized in that the step 3) use
The condition of chromatography when UHPLC-Q Exactive test analysis are as follows: spectrum column is C18 column；50 DEG C of column temperature；Mobile phase A is volume fraction
For 0.01% aqueous formic acid, Mobile phase B is acetonitrile, and the two volume ratio is 30:70；Isocratic elution, flow velocity 0.20mL/min；Into
10 μ L of sample amount.
9. a kind of method of NNK and its metabolite content in measurement blood, which comprises the following steps: by blood
Sample is centrifuged, and supernatant is taken to be uniformly mixed with internal standard compound, acetonitrile, is then centrifuged for separating, take supernatant detection NNK and
Its metabolite content.
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