CN113241131B - Nitrosamine TD based on fluorescent yeast sensor 50 Prediction method - Google Patents

Nitrosamine TD based on fluorescent yeast sensor 50 Prediction method Download PDF

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CN113241131B
CN113241131B CN202110664434.1A CN202110664434A CN113241131B CN 113241131 B CN113241131 B CN 113241131B CN 202110664434 A CN202110664434 A CN 202110664434A CN 113241131 B CN113241131 B CN 113241131B
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郑枫
何颖
柳文媛
凌育栋
丁昊天
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Abstract

The invention discloses a nitrosamine TD based on a fluorescent yeast sensor 50 A prediction method, comprising: by monitoring the expression level of GFP fusion protein in real time after exposure to nitrosamine compounds using a DNA damage responsive fluorescent yeast sensor capable of expressing GFP fusion protein, the genetic toxicity was quantified using the total protein expression level TEL, based on the TEL of nitrosamine compounds 1.5 For TD-free 50 TD of nitrosamine Compounds reported in data 50 And (5) predicting. The method has the advantages of low cost, time saving, reliable information and small sample consumption, can be widely applied, and is beneficial to improving the research and development speed and quality of the medicine.

Description

Nitrosamine TD based on fluorescent yeast sensor 50 Prediction method
Technical Field
The invention belongs to the field of medicine analysis and detection, and relates to nitrosamine TD based on a fluorescent yeast sensor 50 A prediction method.
Background
During the production and storage of pharmaceutical products, potentially mutagenic and carcinogenic impurities, i.e., genotoxic impurities (Genotoxic Impurities, GTIs), may be introduced. Genotoxic impurities typically cause induction of DNA adducts, strand breaks, point mutations, and structural and numerical changes in chromosomes by interacting with DNA or other targets that control apoptosis [1-3] . Genotoxic impurities can induce genetic mutation and genetic material loss at low concentrationInjury and further cause the occurrence of cancer [4] 。
Drug recall events associated with nitrosamine impurities have raised concerns in the global pharmaceutical industry and in global drug regulatory authorities, as are the associated regulations of genotoxic impurities, represented by nitrosamine impurities [5-7] . Nitrosamine compounds are compounds containing nitroso functional groups directly attached to the nitrogen atom in the structure [8] . Nitrosamine compounds are metabolized in vivo by cytochrome P450 enzymes and activate to form alpha-hydroxynitrosamines, which decompose to form highly reactive carbonium or diazonium species, which can lead to alkylation of DNA [9-11] . Nitrosamine compounds are generally highly carcinogenic, N-Nitrosodimethylamine (NDMA) and N-Nitrosodiethylamine (NDEA) are classified by the international agency for research on cancer (International Agency for Research on Cancer, IARC) as group 2A, i.e. these agents may be carcinogenic to humans. Research on genotoxicity of nitrosamine compounds can be traced back to 1956, where NDMA was first tested for carcinogenicity in rats [12] . Currently, there is a large gap in research on genotoxicity and carcinogenicity of nitrosamine compounds.
The genotoxicity evaluation of drug impurities faces the problems of low impurity purity and high impurity variety. At present, carcinogenicity data TD is measured by using a mammalian experiment 50 Is the best mode for evaluating the carcinogenicity of genotoxic impurities, but animal experiments have the defects of long experimental period, large test amount and high cost, and most of impurities can not detect TD at present 50 . Genotoxic compounds can cause genetic material damage upon interaction with DNA, and thus damage at the gene level is the molecular basis for cancer production. Bacterial back mutation test (Bacterial Reverse Mutation Test, ames) based on detection of base mutation is the first choice method for evaluating compound genotoxicity, but has the defects of large test object dosage and long test period [13] . Comet assay (Comet assay) was established by Ostleg and Johansson in 1984 and was able to detect the extent of damage to DNA strands at the single cell level [14] . The method is carried out by observing damaged DNA after the action of the compoundMigration levels in electrophoresis to evaluate genotoxicity suffer from the disadvantage of limited types of detection lesions. The computer simulation evaluation based on the compound warning structure is a commonly used drug impurity genetic toxicity evaluation method in actual production at present, but the method is easy to generate false positive or false negative results, so that the research and development cost is increased sharply, and the drug quality is hidden danger is caused.
Therefore, in order to improve the development speed and quality of drugs, development of a genotoxicity assessment method with lower cost, faster speed and more reliable information is needed.
Reference to the literature
[1]He Y，Xia XY，Wei JL,et al.A highly sensitive yeast cell sensor constructed by overlap PCR was used to evaluate genotoxic compounds[J].J China Pharm Univ,2021,52(2):236-471.
[2]Friedberg EC.DNA damage and repair[J].Nature,2003,421(6921):436-440.
[3]Jarque S,Bittner M,Blaha L,et al.Yeast biosensors for detection of environmental pollutants:current state and limitations[J].Trends in Biotechnology,2016,34(5):408-419.
[4]Bercu JP,Dobo KL,Gocke E,et al.Overview of genotoxic impurities in pharmaceutical development[J].International Journal of Toxicology,2009,28(6):468-478.
[5]European Medicines Agency.CHMP Assessment report for viracept[S].2007.
[6]European Medicines Agency.Sartan Medicines:Companies to Review Manufacturing Processes to Avoid Presence of Nitrosamine Impurities[S].2019.
[7]U.S.Food and Drug Administration.Recalls of Angiotensin II Receptor Blockers(ARBs)Including Valsartan,Losartan and Irbesartan[S].2020.
[8]López-Rodríguez R,Mcmanus J A,Murphy N S,et al.Pathways for N-nitroso compound formation:secondary amines and beyond[J].Organic Process Research&Development,2020,24(9):1558-1585.
[9]Guttenplan J B.N-nitrosamines:bacterial mutagenesis and in vitro metabolism[J].Mutation Research.1987,186(2):81-134.
[10]Lijinsky,W.Carcinogenicity and Mutagenicity of N-Nitroso Compounds[J].Molecular Toxicology.1987,1(1):107-119.
[11]Suzuki H,Takasawa H,Kobayashi K,et al.Evaluation of a liver micronucleus assay with 12chemicals using young rats(II):a study by the Collaborative Study Group for the Micronucleus Test/Japanese Environmental Mutagen Society–Mammalian Mutagenicity Study Group[J].Mutagenesis,2008,24(1):9-16.
[12]Magee P N,Barnes J M.The production of malignant primary hepatic tumours in the rat by feeding dimethylnitrosamine[J].British Journal of Cancer,1956,10(1):114.
[13]Hu M,Ren F,Luo H,et al.Progress of genetic toxicity test[J].Journal of Food Safety&Quality,2011,2(2):76-83.
[14]Collins AR.Measuring oxidative damage to DNA and its repair with the comet assay[J].Biochimica Et Biophysica Acta-General Subjects,2014,1840(2):794-800.
Disclosure of Invention
Aiming at the defects and drawbacks of the existing genotoxicity assessment method, the invention applies the fluorescent yeast sensor for expressing the fusion protein of the green fluorescent protein (Green fluorescent protein, GFP) to the genotoxicity assessment of nitrosamine impurities, and provides a rapid, convenient and low-cost nitrosamine compound TD for new drug research and development units and institutions 50 A prediction method.
The technical scheme of the invention is as follows:
nitrosamine TD based on fluorescent yeast sensor 50 A prediction method, comprising: by monitoring the expression level of GFP fusion protein in real time after exposure to nitrosamine compounds using a DNA damage responsive fluorescent yeast sensor capable of expressing GFP fusion protein, the total protein expression level (TEL) was used to quantify the genotoxicity, based on the TEL of nitrosamine compounds 1.5 For TD-free 50 TD of nitrosamine Compounds reported in data 50 Making predictions。
Specifically, a nitrosamine TD based on a fluorescent yeast sensor 50 A prediction method, comprising:
step (1), obtaining GFP fluorescence response and OD 600 Value: inoculating DNA damage response fluorescent yeast sensor into culture medium, shake culturing at speed of 160-240 rpm and temperature of 25-32 deg.C until initial OD 600 The value reaches 0.14 to 0.2; adding yeast suspension/hole into black wall transparent bottom micro-porous plate, adding PBS buffer solution or nitrosamine compound solution to be tested with PBS equal volume into each hole, respectively as control group and test group, and setting blank group at the same time: referring to the control group, the yeast suspension in each well was replaced with an equal volume of medium; placing 96-hole black transparent bottom micro-pore plate in enzyme labeling instrument, oscillating for 1min, measuring GFP fluorescence response and OD every 3-6 min under dark condition 600 The value is measured continuously within 1.5-3 h;
step (2), calculation of total protein expression level:
(a)、OD 600 and GFP raw data correction:
GFP i, t-correction ＝GFP i, t-test -GFP i, t-blank ；
OD i, t-correction ＝OD i, t-test -OD i, t-blank ；
Wherein GFP is i, t-test Fluorescence data for each GFP fusion protein i of the test panel at time t are shown; GFP (Green fluorescent protein) i, t-blank Fluorescence data for each GFP fusion protein i in the blank at time t are shown; OD (optical density) i, t-test Represents the OD of each GFP fusion protein i of the test group at time t 600 Data; OD (optical density) i, t-blank Represents the OD of each GFP fusion protein i in the blank at time t 600 Data;
(b) Calculation of fusion protein expression level:
P i,t ＝GFP i, t-correction /OD i, t-correction ；
Wherein P is i,t Expression level of GFP fusion protein i at time point t;
(c) Calculation of fold induction of fusion protein expression:
FI i,t ＝P i, t-test /P i, t-control ；
Wherein Pi, t- Testing Expression level of GFP fusion protein i, pi, t- Control The expression level of GFP fusion protein i at time t for the control group; FI (FI) i,t Indicating the change in expression of GFP fusion protein i at time point t due to chemical exposure compared to the control group;
(d) Calculation of total levels of fusion protein expression:
wherein, AP i Indicating the total level of expression of the accumulated GFP fusion protein i over the exposure time (i.e. the time of measurement), which reflects the level of activation of a particular DNA damage repair pathway;
(e) Calculation of total expression level of DNA damage repair related protein:
wherein n represents the amount of GFP fusion protein in the fluorescent yeast sensor set; w (w) i Indicating a damage repair path weight factor corresponding to the GFP fusion protein i, wherein all weights are assigned to be 1; TEL represents the total expression level of the DNA damage repair related protein, TEL reflects the genotoxicity intensity of the nitrosamine compound;
step (3), fitting a concentration-TEL response curve of the nitrosamine compound in GraphPad Prism software by using a four-parameter logarithmic nonlinear regression model; setting the genetic threshold to be 1.5, and calculating the corresponding concentration TEL of the nitrosamine compound when the TEL reaches 1.5 according to the fitted curve 1.5 ；TEL 1.5 Smaller indicates greater genotoxicity of the nitrosamine compound, and conversely, indicates less genotoxicity of the nitrosamine compound;
step (4), soft in GraphPad PrismTEL of para-nitrosamine Compounds in Member 1.5 With TD 50 Performing correlation analysis;
step (5) according to step (1) -step (4), determining TD-free using a DNA damage responsive fluorescent Yeast sensor 50 GFP fluorescence response and OD of nitrosamine Compounds reported by the data 600 Value, based on calculated TEL 1.5 TD for the nitrosamine Compound 50 And (5) predicting.
In the step (1), the DNA damage response fluorescent yeast sensor is a Saccharomyces cerevisiae strain capable of expressing GFP-fusion protein, which is constructed by systematically labeling by inserting the Victoria jellyfish GFP gene after the chromosome gene open reading frame (Open Reading Frame, ORF). The marked gene is regulated by an endogenous promoter, and can express GFP fusion protein with fluorescent markers, so that GFP signals can reflect protein expression. For the seven currently known damage repair pathways, a specific gene marked by GFP is respectively selected for indication, and the change of the expression level of GFP fusion protein can indicate the response level of different DNA damage related repair pathways. When nitrosamine compounds cause cellular DNA damage, the corresponding damage repair pathway is activated and the specific GFP fusion protein is expressed via an endogenous promoter. The level of expression of GFP fusion protein can be quantified by detecting the fluorescent signal of a fluorescent yeast sensor and indicating the level of response of the DNA damage repair pathway. The fluorescent yeast sensor is distinguished from other compounds by the in vitro genotoxicity assessment method: the yeast and the human cells responding to DNA damage react similarly, share a plurality of conservative biochemical pathways, take the live yeast cells as experimental models, can capture all types of damage signals and directly reflect the DNA damage condition in the cell life activities, have obvious biological advantages, and simultaneously, the optical sensing signals are suitable for high-flux detection, and the amount of required test objects is very small.
The different DNA damage repair pathways and GFP fusion proteins selected are shown below:
the black wall transparent bottom micro-pore plate is a 96-hole black wall transparent bottom micro-pore plate.
The culture medium is SD/-His liquid culture medium.
The nitrosamine compound to be detected is N-nitrosodimethylamine, N-nitrosodiethylamine, N-nitrosodi-N-propylamine, N-nitrosodi-N-butylamine, N-nitroso-N-methylaniline, N-nitrosomorpholine, 1-nitrosopyrrolidine and N-nitrosodiethanolamine.
The concentration of the nitrosamine compound in the nitrosamine compound solution to be detected is the concentration of the nitrosamine compound when the cell survival rate of the DNA damage response fluorescent yeast sensor is more than or equal to 95%, so that the high concentration with cytotoxicity is eliminated, and the subsequent genotoxicity test is carried out under the concentration which does not show cytotoxicity. The nitrosamine compound solution to be tested can be a series of sample solutions of concentration, or can be a single sample solution of concentration.
The PBS buffer solution is PBS buffer solution with pH of 7.2-7.4.
Each group (namely, test group: a certain nitrosamine compound corresponding to a certain DNA damage response fluorescent yeast sensor, a certain nitrosamine compound concentration; blank group; control group) is set in 3 parallels.
The conditions for measuring GFP fluorescence response were: excitation wavelength 485nm, emission wavelength 535nm.
Specifically, GFP fluorescence response and OD were obtained 600 The method of the value is as follows: inoculating DNA damage response fluorescent yeast sensor into SD/-His liquid culture medium, shake culturing at 220rpm and 30deg.C until the initial OD 600 Values up to 0.15; 190. Mu.L of yeast suspension/well was added to a 96-well black-wall transparent bottom microplate, and 10. Mu.L of PBS buffer or nitrosamine compound solution to be tested was added to each well as a control group and a test group, respectively, while a blank group was set: 190. Mu.L of medium and 10. Mu.L of PBS buffer were added to each well; placing 96-well black transparent bottom microplate in an enzyme-labeling instrument, oscillating for 1min, and measuring GFP fluorescence response and OD under dark condition every 5min 600 The value was measured continuously over 2h.
In the step (3), in the four-parameter logarithmic nonlinear regression model, four parameters are Bottom, top, EC and HillSlope, and values of four parameters are given when a curve is fitted.
The fitting equation is Y=bottom+ (Top-Bottom)/(1+10+ (LogEC 50-LogX) HillSlope)
In step (4), TEL 1.5 With TD 50 The linear fitting equation is: y=0.3009 x-0.04101.
In step (5), according to step (1) -step (4), no TD is determined using a DNA damage responsive fluorescent Yeast sensor 50 GFP fluorescence response and OD of nitrosamine Compounds reported by the data 600 Value, based on calculated TEL 1.5 To TEL (TEL) 1.5 With TD 50 Linear fitting equation for TD of the nitrosamine compound 50 And (5) predicting.
Compared with the prior art, the invention has the following beneficial effects when evaluating the nitrosamine compounds:
the invention relates to nitrosamine TD based on fluorescent yeast sensor 50 The prediction method has the advantages of low cost, time saving, reliable information and small sample consumption, can be widely applied, and is beneficial to improving the research and development speed and quality of the medicine. The concrete steps are as follows:
(1) The saccharomyces cerevisiae is easy to culture and grow rapidly, has tough cell walls and good tolerance, and is an excellent biosensing material; as eukaryotes, the basic response of mammalian cells to DNA damage is similar; the genome is small, the genetic background is relatively simple, and the genetic operation is easy to carry out.
(2) The saccharomyces cerevisiae strain used for expressing GFP-fusion protein is commercialized and is easy to obtain; the strain can be repeatedly used after passage, the prepared glycerinum strain can be stored for a long time, and the measurement process can be realized by only an enzyme-labeled instrument, so that the method has the advantages of low cost, convenience in operation and time saving.
(3) The serial compound solutions are realized by stepwise dilution, and the sample dosage for preparing the standard solution is only tens of milligrams at the minimum, so that the method has the advantage of low sample dosage.
(4) The fluorescent yeast sensor can directly reflect the vital activity of cells, capture various types of DNA damage signals and is similar to the physiological activity of mammalian cells.
(5) The signal identification and transduction unit of the fluorescent yeast sensor is integrated, can be compatible with a high-flux microplate mode, and is suitable for high-flux screening of various impurity compounds.
Drawings
FIG. 1 Compounds were tested for 24 hours incubationn=3) (a: N-Nitrosodimethylamine (NDMA), B: N-Nitrosodiethylamine (NDEA), C: N-nitrosodi-N-propylamine (NDPA), D: N-nitrosodi-N-butylamine (NDBA), E: N-nitroso-N-methylaniline (NMPhA), F: N-Nitrosomorpholine (NMOR), G: 1-Nitrosopyrrolidine (NPYR), H: N-Nitrosodiethanolamine (NDELA), I: salicylic acid, J: bisphenol a).
Fig. 2. Real-time DNA damage repair protein expression profile of n-Nitrosodimethylamine (NDMA), natural logarithm of fold induction (ln FI, n=3) indicates the magnitude of change in protein expression, black indicates upregulation of GFP fusion protein expression level, and white indicates downregulation of GFP fusion protein expression level. Top of X axis: the concentration of each compound; x-axis bottom: testing time; y axis: GFP fusion protein indicative of DNA damage repair pathways.
Fig. 3. Real-time DNA damage repair protein expression profile of n-Nitrosodiethylamine (NDEA), natural logarithm of fold induction (ln FI, n=3) indicates the magnitude of change in protein expression, black indicates upregulation of GFP fusion protein expression level, and white indicates downregulation of GFP fusion protein expression level.
Fig. 4 shows the real-time DNA damage repair protein expression profile of n-Nitrosodipropylamine (NDPA), the natural logarithm of the fold induction (ln FI, n=3) indicates the magnitude of the change in protein expression, black indicates the up-regulation of GFP fusion protein expression level, and white indicates the down-regulation of GFP fusion protein expression level.
Fig. 5. Real-time DNA damage repair protein expression profile of n-Nitrosodibutylamine (NDBA), natural logarithm of fold induction (ln FI, n=3) indicates the magnitude of change in protein expression, black indicates upregulation of GFP fusion protein expression level, and white indicates downregulation of GFP fusion protein expression level.
Fig. 6, real-time DNA damage repair protein expression profile of N-nitroso-N-methylaniline (NMPhA), natural logarithm of fold induction (ln FI, n=3) indicates the magnitude of change in protein expression, black indicates upregulation of GFP fusion protein expression level, and white indicates downregulation of GFP fusion protein expression level.
Fig. 7. Real-time DNA damage repair protein expression profile of n-Nitrosomorpholine (NMOR), natural logarithm of fold induction (ln FI, n=3) indicates the magnitude of change in protein expression, black indicates upregulation of GFP fusion protein expression level, and white indicates downregulation of GFP fusion protein expression level.
Fig. 8.1-Nitrosopyrrolidine (NPYR) real-time DNA damage repair protein expression profile, natural logarithm of fold induction (ln FI, n=3) indicates magnitude of change in protein expression, black indicates upregulation of GFP fusion protein expression level, and white indicates downregulation of GFP fusion protein expression level.
Fig. 9 shows the real-time DNA damage repair protein expression profile of n-Nitrosodiethanolamine (NDELA), the natural logarithm of the fold induction (ln FI, n=3) indicates the magnitude of the change in protein expression, black indicates the up-regulation of GFP fusion protein expression level, and white indicates the down-regulation of GFP fusion protein expression level.
Fig. 10. Real-time DNA damage repair protein expression profile of salicylic acid, natural logarithm of fold induction (ln FI, n=3) indicates magnitude of change in protein expression, black indicates upregulation of GFP fusion protein expression level, and white indicates downregulation of GFP fusion protein expression level.
Fig. 11, real-time DNA damage repair protein expression profile of bisphenol a, natural logarithm of fold induction (ln FI, n=3) indicates the magnitude of change in protein expression, black indicates upregulation of GFP fusion protein expression level, and white indicates downregulation of GFP fusion protein expression level.
FIG. 12 concentration-response curve of compoundn＝3)。
FIG. 13 TEL 1.5 And TD (time division) 50 And (5) correlation analysis.
Detailed Description
1.1. Instrument for measuring and controlling the intensity of light
THZ-100 thermostatic incubator (Shanghai-Heng science instruments Co., ltd.); spectraMax M2e multifunctional microplate reader (America Molecular Devices instruments Co., ltd.); GI54DS autoclave (Zealway instruments ltd, usa); AB135-S type electronic analytical balance (d=0.01 mg) (METTLER tolio group, switzerland).
1.2. Reagent(s)
N-nitrosodimethylamine (NDMA, 99%), N-nitrosodiethylamine (NDEA, 99%), N-nitrosodipropylamine (NDPA, 98%), N-nitrosodibutylamine (NDBA, 99%), N-nitroso-N-methylaniline (NMPhA, > 98%) and 1-nitrosopyrrolidine (NPYR, 99%) were purchased from Shanghai Michelin Biochemical technologies Co., ltd; n-nitrosomorpholine (NMOR, > 99%) and N-nitrosodiethanolamine (NDELA, > 97%) were purchased from Shanghai Ala Biochemical technologies Co., ltd; dimethyl sulfoxide (DMSO, 99%), salicylic acid (. Gtoreq.99.5%) and bisphenol A (BPA,. Gtoreq.99.5%) were purchased from Nanjing reagent Co., ltd; PBS buffer (pH 7.4) was purchased from Shanghai Biotechnology Co., ltd; SD/-His medium was purchased from Shanghai Ai Li Biotechnology Co. All chemicals were used without further purification.
1.3. Strain
DNA damage responsive fluorescent yeast strain (No. 95702, ATCC 201388) was purchased from Invitrogen, inc., USA. The selected yeast cell collection consists of 8 DNA damage response fluorescent yeast strains, and 7 DNA damage repair paths are covered.
TABLE 1 Yeast strains used
The different DNA damage repair pathways and GFP fusion proteins selected are shown in table 2:
TABLE 2 different DNA damage repair pathways and selected GFP fusion proteins
1.4. Yeast agar puncture recovery
Preparing SD/-His liquid culture medium: 5.58g of SD/-His culture medium solid powder is weighed, dissolved in 200mL of ultrapure water, sterilized at 121 ℃ for 15min to prepare SD/-His liquid culture medium for later use.
SD/-His liquid medium was added to the sterilized tubes. Dipping each microzyme clone with a sterilized toothpick, and immersing the microzyme into the liquid below the liquid surface of the test tube; the test tube was subjected to shaking culture at 30℃and 220 rpm. Culturing the bacterial liquid to OD 600 When the value is 0.4, the standby is realized.
1.5. Preparation of saccharomycetes
Preparing a plurality of sterile centrifuge tubes, and adding a certain volume of 50% (v/v) glycerol solution for later use; adding resuscitated saccharomycete liquid into each of the separation pipes, uniformly mixing, sealing, and ensuring that the final concentration of glycerol is 12.5%; taking glycerol bacterial tube, standing at 4 ℃ for several hours, standing at-20 ℃ for several hours, and finally placing in a refrigerator at-80 ℃ for freezing preservation.
1.6. Preparation of test compound solutions
Accurately weighing 8 nitrosamine compounds and 2 negative controls (salicylic acid, bisphenol A) with proper amount, dissolving in 100% dimethyl sulfoxide (DMSO), and preparing into stock solution; the stock solution was serially diluted with PBS solution until serial diluted sample solutions were obtained.
Serial diluted sample solutions:
NDMA：1×10 -6 、1×10 -5 、1×10 -4 、1×10 -3 、1×10 -2 、1×10 -1 、1、10、1×10 2 、1×10 3 μg/mL；
NDEA：1×10 -6 、1×10 -5 、1×10 -4 、1×10 -3 、1×10 -2 、1×10 -1 、1、10、1×10 2 、1×10 3 μg/mL；
NDPA：5×10 -6 、5×10 -5 、5×10 -4 、5×10 -3 、5×10 -2 、5×10 -1 、5、50、5×10 2 、5×10 3 μg/mL；
NDBA：1×10 -5 、1×10 -4 、1×10 -3 、1×10 -2 、1×10 -1 、1、10、1×10 2 、1×10 3 、1×10 4 μg/mL；
NMPhA：5×10 -6 、5×10 -5 、5×10 -4 、5×10 -3 、5×10 -2 、5×10 -1 、5、50、5×10 2 、5×10 3 μg/mL；
NMOR：5×10 -6 、5×10 -5 、5×10 -4 、5×10 -3 、5×10 -2 、5×10 -1 、5、50、5×10 2 、5×10 3 μg/mL；
NPYR：1×10 -5 、1×10 -4 、1×10 -3 、1×10 -2 、1×10 -1 、1、10、1×10 2 、1×10 3 、1×10 4 μg/mL；
NDELA：5×10 -5 、5×10 -4 、5×10 -3 、5×10 -2 、5×10 -1 、5、50、5×10 2 、5×10 3 、5×10 4 μg/mL；
salicylic acid: 1X 10 -5 、1×10 -4 、1×10 -3 、1×10 -2 、1×10 -1 、1、10、1×10 2 、1×10 3 、1×10 4 μg/mL；
BPA：1×10 -5 、1×10 -4 、1×10 -3 、1×10 -2 、1×10 -1 、1、10、1×10 2 、1×10 3 、1×10 4 μg/mL。
1.7. Compound cytotoxicity test
Cytotoxicity assays were performed with 8 nitrosamine compounds (NDMA, NDEA, NDPA, NDBA, NMPhA, NMOR, NPYR and NDELA) and two negative controls (salicylic acid and BPA) as test compounds.
Preparing SD/-His solid medium: weighing 5.58g of SD/-His culture medium solid powder, dissolving in 200mL of ultrapure water, adding 20g of purified agar, and sterilizing at 121 ℃ for 15min; pouring the mixture into a plastic culture dish when the temperature is cooled to 55 ℃ and cooling the mixture until the liquid is solidified to prepare an SD/-His solid culture medium for later use; taking saccharomycetes for plate streaking, picking single colony, inoculating the single colony into a fresh SD/-His liquid culture medium, and placing the liquid culture medium at 30 ℃ and 220rpm for shake culture overnight for later use.
Taking overnight cultured yeast liquid, replacing fresh SD/-His liquid culture medium, and culturing to OD 600 A value of about 0.15; a volume of 190. Mu.L yeast solution was added to each well of a 96-well fully transparent microplate, and 10. Mu.L PBS or sample solution was added as a control group and a test group, respectively, to set a blank group: add 190. Mu.L of medium and 10. Mu.L of PBS per well; each set was set up with 3 parallels.
Placing the microplate in a dark condition at 30 ℃ and 100rpm for shake culture; taking out the micro-porous plate after 24 hours, and measuring OD 600 The values, cell viability, were calculated as follows:
cell viability= (OD Testing -OD Blank space )/(OD Solvent(s) -OD Blank space )×100％
Figure 1 shows that the test compounds all showed some cell growth inhibition at high incubation concentrations and caused cytotoxicity in a dose-dependent manner in fluorescent yeast sensors. Cytotoxicity levels were quantified at a concentration at which the viability curve reached 95% and the cytotoxicity of the test compounds was ranked as follows: NDELA < NPYR < BPA < NDBA < NMOR < NDPA < NDMA < NMPhA < NDEA < salicylic acid.
The maximum non-cytotoxic concentration is defined as the concentration of test compound at which the cell viability is greater than or equal to 95%. High concentrations of cytotoxicity were knocked out and subsequent genotoxicity testing was performed at concentrations that did not exhibit cytotoxicity.
GFP fusion protein expression assay
Taking overnight cultured saccharomycete liquid (same as '1.7. Compound cytotoxicity test'), replacing fresh SD/-His liquid culture medium, and culturing until OD600 value is about 0.15; to a 96 Kong Heibi clear bottom microplate (black wall is used to avoid fluorescence interference of wells, clear bottom is used to measure absorbance) 190 μl yeast liquid/well, 10 μl PBS or sample solution is added per well as control and test groups, respectively, and a blank group is set: add 190. Mu.L of medium and 10. Mu.L of PBS per well; each set was set up with 3 parallels.
According to the compound cytotoxicity test determination result, the concentration of the sample solution is as follows:
NDMA：1×10 -4 、1×10 -3 、1×10 -2 、1×10 -1 、1、10μg/mL；
NDEA：1×10 -4 、1×10 -3 、1×10 -2 、1×10 -1 、1、10μg/mL；
NDPA：5×10 -4 、5×10 -3 、5×10 -2 、5×10 -1 、5、50μg/mL；
NDBA：1×10 -3 、1×10 -2 、1×10 -1 、1、10、1×10 2 μg/mL；
NMPhA：5×10 -6 、5×10 -5 、5×10 -4 、5×10 -3 、5×10 -2 、5×10 -1 μg/mL；
NMOR：5×10 -3 、5×10 -2 、5×10 -1 、5、50、5×10 2 μg/mL；
NPYR：1×10 -2 、1×10 -1 、1、10、1×10 2 、1×10 3 μg/mL；
NDELA：5×10 -1 、5、50、5×10 2 、5×10 3 、5×10 4 μg/mL；
salicylic acid: 1X 10 -4 、1×10 -3 、1×10 -2 、1×10 -1 、1、101μg/mL；
BPA：1×10 -3 、1×10 -2 、1×10 -1 、1、10、1×10 2 μg/mL。
Placing 96 Kong Heibi transparent bottom microplate in enzyme labeling instrument, oscillating for 1min at intervals of 5min measurement of primary GFP fluorescence response (excitation wavelength 485nm, emission wavelength 535 nm) and OD under dark conditions 600 The value was continued for 2h.
As shown in fig. 2-11, the real-time DNA damage repair protein expression profile shows the damage mechanism and damage level of different compounds. The expression of a particular protein is concentration dependent, and the intensity of the damage repair response is related to the test concentration of the compound, generally with increasing concentration, the magnitude of the protein expression increases. For certain compounds, specific protein expression, such as MLH2-GFP in NDEA and RAD30-GFP in NDPA, at the highest exposure concentrations, the up-regulation of protein expression was reduced in magnitude, and even became down-regulated. This may be due to the conversion of the intracellular response from a specific chemical response to a cytotoxic response at high exposure concentrations.
Nitrosamine compounds have a degree of activation for all seven DNA damage repair pathways. High expression levels of PHR1-GFP and MAG1-GFP fusion proteins indicate that the Direct Reverse Repair (DRR) and Base Excision Repair (BER) pathways are activated. This is consistent with the metabolism of N-nitrosamine compounds to form alpha-hydroxynitrosamines, which upon decomposition alkylate genetic material. RAD2-GFP and RAD52-GFP fusion proteins are highly expressed, representing the Nucleotide Excision Repair (NER) and Double Strand Break Repair (DSBR) pathways, respectively, consistent with the ability of nitrosamine compounds to cause DNA breaks. The activation information of the DNA damage repair protein expression profile obtained by the invention is consistent with the genetic toxicity mechanism reported in the literature, so that the DNA damage mechanism of the nitrosamine compound which is not fully researched can be analyzed. After exposure to all of the nitrosamine compounds tested, the expression of CHK1-GFP fusion proteins indicating DNA Damage Signaling (DDS) was up-regulated to a lesser extent, indicating that the level of activation of DDS by nitrosamine compounds in DNA damage repair may not be high, whereas the general up-regulation of RAD30-GFP fusion proteins reflects general activation of cross-damaged DNA synthesis (TLS). The lower magnitude of upregulation of MLH2-GFP fusion proteins in exposure experiments with NDEA, NDPA, NMPhA, NMOR and NDELA, indicative of Mismatch Repair (MR), suggests that mismatch may not be the primary DNA damage mechanism for most nitrosamine compounds. For most of the nitrosamine compounds tested (e.g., NDMA, NDEA, NDBA, NMPhA and NMOR), the levels of upregulation of YKU-GFP fusion proteins, indicative of non-homologous end joining (NHEJ), were generally weaker than those of other proteins such as RAD52-GFP, indicating that the nitrosamine compound induced DSBR had a stronger Homologous Recombination (HR) response than NHEJ.
The nitrosamine compounds tested are all highly active in DSBR, which is not only associated with direct disruption of DNA structure, but may be indirectly affected by other DNA damage activation. Nitrosamine compounds induce the activation of a more extensive DNA damage repair pathway, suggesting that their genotoxicity is related to the structure of nitrosamines, which typically lead to disruption of cellular DNA structure. The manner and extent of DNA damage by different derivatives varies, which may be related to substituents in their structure.
The expression levels of most GFP fusion proteins in both the salicylic acid and BPA negative controls were down-regulated, and the down-regulation amplitude increased with increasing exposure concentration, indicating that the DNA repair pathway of the negative control was not activated.
1.9. Data processing
1. Calculation of total protein expression level:
(a)、OD 600 and GFP raw data correction:
GFP i, t-correction ＝GFP i, t-test -GFP i, t-blank ；
OD i, t-correction ＝OD i, t-test -OD i, t-blank ；
Wherein GFP is i, t-test Fluorescence data for each GFP fusion protein i of the test panel at time t are shown; GFP (Green fluorescent protein) i, t-blank Fluorescence data for each GFP fusion protein i in the blank at time t are shown; OD (optical density) i, t-test Represents the OD of each GFP fusion protein i of the test group at time t 600 Data; OD (optical density) i, t-blank Represents the OD of each GFP fusion protein i in the blank at time t 600 Data;
(b) Calculation of fusion protein expression level:
P i,t ＝GFP i, t-correction /OD i, t-correction ；
Wherein P is i,t Is shown at the time pointExpression level of GFP fusion protein i at t;
(c) Calculation of fold induction of fusion protein expression:
FI i,t ＝P i, t-test /P i, t-control ；
Wherein Pi, t- Testing Expression level of GFP fusion protein i, pi, t- Control The expression level of GFP fusion protein i at time t for the control group; FI (FI) i,t Indicating the change in expression of GFP fusion protein i at time point t due to chemical exposure compared to the control group;
(d) Calculation of total levels of fusion protein expression:
wherein, AP i Indicating the total level of expression of the accumulated GFP fusion protein i over a 2 hour exposure period, reflecting the level of activation of a particular DNA damage repair pathway;
(e) Calculation of total expression level of DNA damage repair related protein:
wherein n represents the amount of GFP fusion protein in the fluorescent yeast sensor collection, n=8; w (w) i Indicating a damage repair path weight factor corresponding to the GFP fusion protein i, wherein all weights are assigned to be 1; TEL represents the total expression level of the DNA damage repair related protein, and TEL reflects the genotoxicity intensity of the nitrosamine compound.
2. Fitting a concentration of nitrosamine compound-TEL response curve in GraphPad Prism software using a four-parameter logarithmic nonlinear regression model; the fitting equation is:
Y＝Bottom+(Top-Bottom)/(1+10^((LogEC50-LogX)*HillSlope))
setting the genetic threshold to be 1.5, and calculating the corresponding concentration of the nitrosamine compound when the TEL reaches 1.5 according to the fitted curveDegree TEL 1.5 ；TEL 1.5 Smaller indicates greater genotoxicity of the nitrosamine compound, and conversely, indicates less genotoxicity of the nitrosamine compound;
the TEL-value dose response curves for each nitrosamine compound exhibited a symmetrical S-shape, and the non-linear regression model fitting results are shown in fig. 12. TEL quantifies the overall expression effect of a test compound in a range of test concentrations on DNA damage repair pathway related proteins induced during exposure time. TELmax represents the highest total expression level of the protein pool over the range of concentrations tested. For eight nitrosamine compounds, the TELmax obtained from the dose response curve exceeded the threshold of 1.5, indicating that all nitrosamine compounds tested were genotoxic. Accordingly, the TELmax of the dose response curves for both negative controls was less than 1.5. The TEL values of the nitrosamine compounds at low concentrations were all below 1.5, with increasing concentrations, the TEL values increasing progressively, consistent with the trend of real-time protein expression profiles.
3.TEL of nitrosamine Compound 1.5 With TD 50 Performing correlation analysis
TD 50 Is the dosage that causes 50% of mammal carcinogens in long-term carcinogenicity test, and is a reliable standard for evaluating the carcinogenic potential of carcinogens.
TABLE 3 TEL of test compounds 1.5 And TD (time division) 50 Data
Note that: a represents data from the Lhasa carcinogenicity database (https:// carbdb. Lhasalimited org /); ND represents undetected; -the representation is not applicable.
TEL of para-nitrosamine Compounds in GraphPad Prism software 1.5 With TD 50 And performing correlation analysis.
Fig. 13 shows that: TEL (TEL) 1.5 And TD (time division) 50 The fitted linear regression equation is: y=0.3009X-0.04101, wherein X is lg (TEL 1.5 ) Y is lg (TD 50 ). Both exhibit a strong correlation of statistical significance(rP＝0.8，P<0.05). The evaluation result of the genetic toxicity of the nitrosamine compound is consistent with the cancerogenic result of mammals, and the test based on the fluorescent yeast sensor can be used for predicting the TD of nitrosamine 50 Is reliable: firstly, the fluorescent yeast sensor is adopted to test TD-free 50 Data reported nitrosamine compounds, recalculated TEL of nitrosamine compounds 1.5 Values, combined with linear regression equations, to achieve TD for them 50 And (5) predicting data.
TEL based on the invention 1.5 The DNA damage efficacy of the nitrosamine compounds tested was ranked as: NDELA (non-uniform abrasive article)<NPYR<NDPA<NMOR<NDBA<NMPhA<NDEA <NDMA. Since nitrosamine compounds have the same molecular skeleton N-nitroso group, the difference in genotoxicity is mainly affected by the substituents thereof. Overall, nitrosamines with alkane substituents exhibit higher genotoxicity, while nitrosamines with heterocyclic or heteroatom substituents exhibit lower genotoxicity. This may be related to the bulky substituents formed after nitrosamine metabolism affecting the formation of carbocations or diazo compounds and further hindering the latter from alkylating genetic material. In addition, nitrosamine compounds having hydroxyl substituents may be detrimental to carbocation or diazonium compound formation due to hydroxyl oxidation.

Claims (9)

1. Nitrosamine TD based on fluorescent yeast sensor 50 The prediction method is characterized in that: comprising the following steps: by monitoring the expression level of GFP fusion protein in real time after exposure to nitrosamine compounds using a DNA damage responsive fluorescent yeast sensor capable of expressing GFP fusion protein, the genetic toxicity was quantified using the total protein expression level TEL, based on the TEL of nitrosamine compounds 1.5 For TD-free 50 TD of nitrosamine Compounds reported in data 50 Predicting; comprising the following steps:
step (1), obtaining GFP fluorescence response and OD 600 Value: inoculating DNA damage response fluorescent yeast sensor into culture medium, shake culturing at speed of 160-240 rpm and temperature of 25-32 deg.C until initial OD 600 The value reaches 0.14 to 0.2The method comprises the steps of carrying out a first treatment on the surface of the Adding yeast suspension/hole into black wall transparent bottom micro-porous plate, adding PBS buffer solution or nitrosamine compound solution to be tested with PBS equal volume into each hole, respectively as control group and test group, and setting blank group at the same time: referring to the control group, the yeast suspension in each well was replaced with an equal volume of medium; placing 96-hole black transparent bottom micro-pore plate in enzyme labeling instrument, oscillating for 1min, measuring GFP fluorescence response and OD every 3-6 min under dark condition 600 The value is measured continuously within 1.5-3 h;
step (2), calculation of total protein expression level:
(a)、OD 600 and GFP raw data correction:
GFP i, t-correction ＝GFP i, t-test -GFP i, t-blank ；
OD i, t-correction ＝OD i, t-test -OD i, t-blank ；
Wherein GFP is i, t-test Fluorescence data for each GFP fusion protein i of the test panel at time t are shown; GFP (Green fluorescent protein) i, t-blank Fluorescence data for each GFP fusion protein i in the blank at time t are shown; OD (optical density) i, t-test Represents the OD of each GFP fusion protein i of the test group at time t 600 Data; OD (optical density) i, t-blank Represents the OD of each GFP fusion protein i in the blank at time t 600 Data;
(b) Calculation of fusion protein expression level:
P i,t ＝GFP i, t-correction /OD i, t-correction ；
Wherein P is i,t Expression level of GFP fusion protein i at time point t;
(c) Calculation of fold induction of fusion protein expression:
FI i,t ＝P i, t-test /P i, t-control ；
Wherein Pi, t- Testing Expression level of GFP fusion protein i, pi, t- Control The expression level of GFP fusion protein i at time t for the control group; FI (FI) i,t Represents GFP fusion protein i at time t compared to the control groupExpression changes;
(d) Calculation of total levels of fusion protein expression:
wherein, AP i Indicating the total level of expression of the accumulated GFP fusion protein i over the exposure time;
(e) Calculation of total expression level of DNA damage repair related protein:
wherein n represents the amount of GFP fusion protein in the fluorescent yeast sensor set; w (w) i The damage repair path weight factors corresponding to GFP fusion protein i are represented, and all weight factors are assigned to be 1; TEL represents the total expression level of the DNA damage repair related protein, TEL reflects the genotoxicity intensity of the nitrosamine compound;
step (3), fitting a concentration-TEL response curve of the nitrosamine compound in GraphPad Prism software by using a four-parameter logarithmic nonlinear regression model; calculation of the corresponding concentration of nitrosamine Compound TEL at which TEL reached 1.5 from the fitted Curve 1.5 ；
Step (4), TEL of the para-nitrosamine Compound in GraphPad Prism software 1.5 With TD 50 Performing correlation analysis;
step (5) according to step (1) -step (4), determining TD-free using a DNA damage responsive fluorescent Yeast sensor 50 GFP fluorescence response and OD of nitrosamine Compounds reported by the data 600 Value, based on calculated TEL 1.5 TD of para-nitrosamine Compound 50 And (5) predicting.
2. Nitrosamine TD based on fluorescent Yeast sensor according to claim 1 50 The prediction method is characterized in that: the DNA damage response fluorescent yeast sensor canA strain of Saccharomyces cerevisiae expressing GFP fusion protein is constructed by systematic tagging of the inserted jellyfish GFP gene after the chromosomal gene open reading frame.
3. Nitrosamine TD based on fluorescent Yeast sensor according to claim 1 50 The prediction method is characterized in that: the GFP fusion proteins in the DNA damage responsive fluorescent yeast sensor were as follows:
4. nitrosamine TD based on fluorescent Yeast sensor according to claim 1 50 The prediction method is characterized in that: in the step (1), the black wall transparent bottom micro-pore plate is a 96-hole black wall transparent bottom micro-pore plate.
5. Nitrosamine TD based on fluorescent Yeast sensor according to claim 1 50 The prediction method is characterized in that: in the step (1), the culture medium is SD/-His liquid culture medium.
6. Nitrosamine TD based on fluorescent Yeast sensor according to claim 1 50 The prediction method is characterized in that: in the step (1), the nitrosamine compound to be detected is N-nitrosodimethylamine, N-nitrosodiethylamine, N-nitrosodi-N-propylamine, N-nitrosodi-N-butylamine, N-nitroso-N-methylaniline, N-nitrosomorpholine, 1-nitrosopyrrolidine, N-nitrosodiethanolamine; the concentration of the nitrosamine compound in the nitrosamine compound solution to be detected is the concentration of the nitrosamine compound when the cell survival rate of the DNA damage response fluorescent yeast sensor is more than or equal to 95%.
7. Nitrosamine TD based on fluorescent Yeast sensor according to claim 1 50 The prediction method is characterized in that: in step (1), the PBSThe buffer is PBS buffer with pH of 7.2-7.4.
8. Nitrosamine TD based on fluorescent Yeast sensor according to claim 1 50 The prediction method is characterized in that: in step (1), the conditions for measuring GFP fluorescence response are: excitation wavelength 485nm, emission wavelength 535nm.
9. Nitrosamine TD based on fluorescent Yeast sensor according to claim 1 50 The prediction method is characterized in that: in step (4), TEL 1.5 With TD 50 The linear fitting equation is: y=0.3009 x-0.04101.
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