CN101580869A - Cigarette mainstream smoke genotoxicity measurement method - Google Patents
Cigarette mainstream smoke genotoxicity measurement method Download PDFInfo
- Publication number
- CN101580869A CN101580869A CNA200910065337XA CN200910065337A CN101580869A CN 101580869 A CN101580869 A CN 101580869A CN A200910065337X A CNA200910065337X A CN A200910065337XA CN 200910065337 A CN200910065337 A CN 200910065337A CN 101580869 A CN101580869 A CN 101580869A
- Authority
- CN
- China
- Prior art keywords
- cell
- cigarette mainstream
- genotoxicity
- mainstream smoke
- cigarette
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Abstract
A cigarette mainstream smoke genotoxicity measurement method is characterized in that: the cultured cells are directly placed in cigarette mainstream smoke diluted by pure air to expose, the exposed cells are cultured, centrifuged and fixed by cold methanol, acridine orange solution is used for the fluorescent staining for the cells, the stained cells are observed in low power lens to select the region which is distributed evenly and stained well and calculate the number of cells with microkernel(including mononuclear, binuclear and multinuclear) of 500 cells in each sample under fluorescent microscope, the samples is compared with the control sample exposed in pure air to judge whether the cigarette mainstream smoke can increase the cell micronucleus and judge the genotoxicity of the cigarette mainstream smoke. The genotoxicity of the cigarette mainstream smoke can be quantitative evaluated by the invention and the invention has simple operation, high sensitivity and reliable result, thus establishing a new method for measuring cigarette mainstream smoke genotoxicity.
Description
Technical field
The present invention relates to cigarette mainstream smoke genotoxicity and estimate the field, be a kind of cigarette mainstream smoke genotoxicity measurement method specifically, be by with cellular exposure under cigarette mainstream flue gas, the ratio that measuring cell micronucleus that main flume causes increases is carried out genotoxicity measurement.
Background technology
The aerosol that cigarette smoke is made up of several thousand kinds of chemical substances, wherein many components all have certain toxic action for cell or human body.Scientists has been carried out a lot of genotoxicity researchs about the chemical ingredients in cigarette smoke or the flue gas, and the genotoxicity of suitable mammalian cell with micronucleus test method evaluating cigarette flue gas recommended to select for use by the external toxicology of CORESTA ad hoc working group.The purpose of genotoxicity test mainly is structure or the function potential negative effect of test exogenous material for DNA.At present, genotoxicity has been generally acknowledged that the cancer with animal and human's class is closely related.Micronucleus test is be most widely used during the genotoxicity of chemical substance or mixture is tested a kind of.Bibliographical information is exposed to increase [the reference 1:Channarayappa that all can obviously observe micronucleus in the V79 cell of smoke condensate, J Nath, T Ong.Clastogenic and aneuploidogeniceffects of cigarette smoke condensate, Mitomycin C and vincristine sulfate[J] .Mutagenesis, 1992,7 (6): 457-260. reference 2:Veltel D, A Hoheneder.Characterizat ion of cigarettesmoke-induced micronuclei in vitro[J] .Exp.Toxic Pathol, 1996,48:548-550.].This is external to be exposed to increase [the reference 3:MooreM M that also can find micronucleus in the BALB/c-3T3 cell of smoke condensate significantly, Honma M, Clements J.Mouse lymphoma thymidine kinase locus gene mutation assay:InternationalWorkshop on Genotoxicity Test Procedures Workgroup Report[J] .Environmental and Molecular Mutagenesis, 2000,35:185-190.]
But the object of above research test is the main flume granule phase substance, and main flume is captured with cambridge filter, and the main flume granule phase substance that will trap cambridge filter then extracts, and carries out micronucleus test again.Such testing method will spend a large amount of time, complex operation, and, can not reflect mainstream smoke genotoxicity truly because the just cigarette mainstream flue gas granule phase substance of test is not tested the gaseous component in the main flume.
Summary of the invention
Purpose of the present invention is only tested the deficiency of the genotoxicity of the main flume granule phase substance pair cell that traps on the cambridge filter just at above-mentioned prior art, and a kind of cigarette mainstream smoke genotoxicity measurement method that is used for of special exploitation, present method also can be omitted and carry out loaded down with trivial details solvent extraction process after flue gas is collected, the genotoxicity that can directly also comprehensively reflect cigarette mainstream flue gas, this method is simple to operate, highly sensitive, reliable results.
The objective of the invention is to be achieved through the following technical solutions: elder generation directly inserts cultured cell in the cigarette mainstream flue gas that dilutes through uncontaminated air and exposes, the cell that exposed is through cultivating, centrifugal and cold methanol is fixed, use the fluorescent dye of acridine orange solution then, under low power lens (10 times), observe earlier, selection is evenly distributed, dyeing is the zone preferably, under fluorescent microscope, count again, contain micronucleus in 500 cells of each sample counting and (comprise monokaryon, double-core and multinuclear) cell quantity, the control sample that exposes with uncontaminated air compares, judge that cigarette mainstream flue gas causes the quantity that cell micronucleus increases, and then judge the size of the genotoxicity of cigarette mainstream flue gas.
Concrete measuring method of the present invention may further comprise the steps:
A. cell cultures: test cell is inoculated in the cell culture fluid, at CO
2Cultivate down for 37 ℃ in the cell culture incubator, when cell proliferation is counted during to finite concentration, and with cell transfer to the T-75mL culturing bottle, concentration is 5.0 * 10
5-10.0 * 10
5Cell/bottle adds nutrient solution to cumulative volume 20mL, in CO
2Cultivate 24h down for 37 ℃ in the cell culture incubator;
B.+S9 organizes cell cultures: each culturing bottle sucking-off 10ml nutrient solution, and with these nutrient solution preparation S9 mixtures (53ml nutrient solution+45ml NADPH (coenzyme II)+2ml S9 liver tissue homogenate liquid), each culturing bottle adds 10ml S9 mixture;
C. the cell main flume exposes: smoking cigarette under the iso standard condition, the main flume that produces dilutes with uncontaminated air, fume exposure concentration is that index is controlled with total particulate matter (TPM) concentration in the flue gas after diluting, flue gas after the dilution directly import the long culturing bottle that cell arranged (comprise-the S9 group and+S9 organizes two groups of samples, + S9 represents to be added with liver tissue homogenate's mixture, and-S9 represents not add liver tissue homogenate's mixture) in carry out main flume and expose.Exposure duration is 60min, and whole process-exposed is controlled according to the TPM/L value of monitoring, the normal control group (comprise-S9 group and+S9 organizes two groups of samples) feed the uncontaminated air exposure.
D. micronucleus test: behind the cell cultures 2h that exposed, the sucking-off nutrient solution is also used D-PBS (containing Ca2+, the Mg2+ phosphate buffered saline buffer) rinsing 2 times of 20ml preheating respectively, adds 3ml trysinization liquid, treats to add the 2ml nutrient solution behind the cell dissociation.Cell suspension is changed in the centrifuge tube, the centrifugal 5min of 2000rpm, abandoning supernatant adds the cell culture fluid that 1ml contains 6 μ g/ml cytochalasin Bs, mixes, and carries out cell counting and calculates total cell quantity.Add the cell culture fluid that contains 6 μ g/ml cytochalasin Bs and regulate cell concn about 0.1 * 10
6Cell/ml pipettes 3.5ml to the cavate slide glass after mixing, cultivate 20h.Remove nutrient solution and cavity,, remove gasket, dry naturally or air-dry with the fixing slide 10min of 90% cold methanol.Air-dry slide is soaked 3min in the acridine orange solution (with the preparation of D-PBS solution) of 10mg/ml, use distilled water flushing slide 30s then, dark place nature dries or is air-dry.Observe under low power lens (10 times) earlier, select to be evenly distributed, dyeing is the zone preferably, counts under fluorescent microscope again, contains the micronucleus cell quantity of (comprising monokaryon, double-core and multinuclear) in 500 cells of each sample counting.
E. data processing and analysis, the evaluating cigarette mainstream smoke genotoxicity.
In the present invention, the S9 mixture is by formulated (53% nutrient solution+45%NADPH+2%S9) of cell culture fluid and NADPH (coenzyme II) and S9 liver tissue homogenate liquid.The cell culture fluid of 6 μ g/ml cytochalasin Bs is to be mixed in proportion formulated by cytochalasin B and cell culture fluid.90% cold methanol is to be prepared by volume and preserved at refrigerator (20 ℃) by distilled water and methyl alcohol.The acridine orange solution of 10mg/ml is formulated in proportion by acridine orange and D-PBS.
The test philosophy of foundation of the present invention is: cultured cell is directly inserted in the cigarette mainstream flue gas expose, the cell that exposed through cultivate, centrifugal and cold methanol fixes, use the fluorescent dye of acridine orange solution then, the cell quantity that under fluorescent microscope, contains micronucleus again in the counting cells, the control sample that exposes with uncontaminated air compares, judge that cigarette mainstream flue gas causes the quantity that cell micronucleus increases, and then judge the size of the genotoxicity of cigarette mainstream flue gas.
This experimental design has reflected the toxic action of cigarette mainstream flue gas more objectively, and the genotoxicity of energy quantitative evaluation main flume, compared with prior art have simple to operate, highly sensitive, the characteristics of reliable results have been started a kind of new method that is used for cigarette mainstream smoke genotoxicity measurement.
Description of drawings
Fig. 1 is the synoptic diagram of the used instrument of process of the test.
Among the figure: 1 is smoking machine, and 2 is under meter, and 3 is pump, and 4 is Tissue Culture Flask.
Fig. 2 causes the histogram of the micronucleus increment rate of 3T3 cell for embodiment 1 cigarette smoke.
Fig. 3 causes the histogram of the micronucleus increment rate of 3T3 cell for embodiment 2 cigarette smokes.
Fig. 4 causes the histogram of the micronucleus increment rate of 3T3 cell for embodiment 3 cigarette smokes.
Embodiment
The present invention is described further with the following Examples, but does not limit the present invention.
(box mark tar 15mg) estimates to homemade certain cigarette.The sample cigarette aspirates under the iso standard condition, and the suction capacity is every mouthful of 35ml, time length 2s, and the per minute suction is flatly.The main flume that cigarette burning produces dilutes with clean air, and adjusting flue gas total particulate matter concentration to 41.5 μ g/L, 53.8 μ g/L and 67.3 μ g/L, with the flue gas after the dilution directly import the long culturing bottle that cell arranged (comprise-the S9 group and+S9 organizes two groups of samples) expose, each concentration exposure duration is 60min.In the test used smoking machine be connected with Tissue Culture Flask and the gas fume control mode referring to Fig. 1.
In addition, with the long culturing bottle that cell arranged (comprise-the S9 group and+S9 organizes two groups of samples) feed uncontaminated air sample in contrast.After exposing end, the nutrient solution in the culturing bottle is changed into fresh nutrient solution and places CO
2Cultivate 2h in the incubator.The sucking-off nutrient solution is also used the D-PBS rinsing 2 times of 20ml preheating respectively, adds 3ml trysinization liquid, treats to add the 2ml nutrient solution behind the cell dissociation.Cell suspension is changed in the centrifuge tube, the centrifugal 5min of 2000rpm, abandoning supernatant adds the cell culture fluid that 1ml contains 6 μ g/ml cytochalasin Bs, mixes, and carries out cell counting and calculates total cell quantity.Add the cell culture fluid that contains 6 μ g/ml cytochalasin Bs and regulate cell concn about 0.1 * 10
6Cell/ml pipettes 3.5ml to the cavate slide glass after mixing, cultivate 20h.Remove nutrient solution and cavity,, remove gasket, dry naturally or air-dry with the fixing slide 10min of 90% cold methanol.Air-dry slide is soaked 3min in the acridine orange solution (with the preparation of D-PBS solution) of 10mg/ml, use distilled water flushing slide 30s then, dark place nature dries or is air-dry.Observe under low power lens (10 times) earlier, select to be evenly distributed, dyeing is the zone preferably, counts under fluorescent microscope again, contains the micronucleus cell quantity of (comprising monokaryon, double-core and multinuclear) in 500 cells of each sample counting.The results are shown in Figure 2.Along with the increase of fume exposure concentration, cigarette smoke causes that the probability of cell micronucleus enlarges markedly, and is tangible dosage-response relation as can be seen from Figure 2.
Present embodiment is same as embodiment 1 substantially, only is that cigarette sample is different with fume exposure concentration.Cigarette sample is homemade certain cigarette of box mark tar 8mg, and fume exposure concentration is 24.2 μ g/L, 50.6 μ g/L and 98.7 μ g/L, the results are shown in Figure 3.
Embodiment 3
Present embodiment is same as embodiment 1 substantially, only is that cigarette sample is different with fume exposure concentration.Cigarette sample is a box mark tar 9mg import cigarette, and fume exposure concentration is 57.5 μ g/L and 90.5 μ g/L, the results are shown in Figure 4.
Claims (7)
1, a kind of cigarette mainstream smoke genotoxicity measurement method, it is characterized in that: this measuring method may further comprise the steps: cultured cell is directly inserted in the cigarette mainstream flue gas through uncontaminated air dilution expose, the cell that exposed is through cultivating, centrifugal and cold methanol is fixed, use the fluorescent dye of acridine orange solution then, under low power lens, observe earlier, selection is evenly distributed, dyeing is the zone preferably, under fluorescent microscope, count again, the cell quantity that contains micronucleus in 500 cells of each sample counting, the control sample that exposes with uncontaminated air compares, judge that cigarette mainstream flue gas causes the quantity that cell micronucleus increases, and then judge the size of the genotoxicity of cigarette mainstream flue gas.
2, cigarette mainstream smoke genotoxicity measurement method according to claim 1 is characterized in that: the concrete steps of this measuring method are as follows:
A. cell cultures: test cell is inoculated in the cell culture fluid, at CO
2Cultivate down for 37 ℃ in the cell culture incubator, when cell proliferation is counted during to finite concentration, and with cell transfer to the T-75mL culturing bottle, concentration is 5.0 * 10
5-10.0 * 10
5Cell/bottle adds nutrient solution to cumulative volume 20mL, in CO
2Cultivate 24h down for 37 ℃ in the cell culture incubator;
B.+S9 organizes cell cultures: each culturing bottle sucking-off 10ml nutrient solution, and with these nutrient solution preparations S9 mixture, each culturing bottle adds 10ml S9 mixture;
C. the cell main flume exposes: smoking cigarette under the iso standard condition, the main flume that produces dilutes with uncontaminated air, fume exposure concentration is that index is controlled with total particulate matter (TPM) concentration in the flue gas after diluting, flue gas after the dilution directly import the long culturing bottle that cell arranged (comprise-the S9 group and+S9 organizes two groups of samples) in carry out main flume and expose, exposure duration is 60min, whole process-exposed is controlled according to the TPM/L value of monitoring, the normal control group (comprise-S9 group and+S9 organizes two groups of samples) feed the uncontaminated air exposure;
D. micronucleus test: behind the cell cultures 2h that exposed, the sucking-off nutrient solution is also used the D-PBS rinsing 2 times of 20ml preheating respectively, add 3ml trysinization liquid, treat to add the 2ml nutrient solution behind the cell dissociation, cell suspension is changed in the centrifuge tube, the centrifugal 5min of 2000rpm, abandoning supernatant, adding 1ml contains the cell culture fluid of 6 μ g/ml cytochalasin Bs, mix, carry out cell counting and calculate total cell quantity, add the cell culture fluid that contains 6 μ g/ml cytochalasin Bs and regulate cell concn about 0.1 * 10
6Cell/ml, pipette 3.5ml after mixing to the cavate slide glass, cultivate 20h, remove nutrient solution and cavity, with the fixing slide 10min of 90% cold methanol, remove gasket, naturally dry or air-dry,, use distilled water flushing slide 30s then air-dry slide immersion 3min in the acridine orange solution (with the preparation of D-PBS solution) of 10mg/ml, dark place nature dries or is air-dry, observe under low power lens (10 times) earlier, select to be evenly distributed, dyeing is the zone preferably, under fluorescent microscope, count again, contain the cell quantity of micronucleus in 500 cells of each sample counting;
E. data processing and analysis, the evaluating cigarette mainstream smoke genotoxicity.
3, cigarette mainstream smoke genotoxicity measurement method according to claim 2 is characterized in that: the S9 mixture is made of jointly cell culture fluid+NADPH (coenzyme II)+S9 liver tissue homogenate liquid.
4, cigarette mainstream smoke genotoxicity measurement method according to claim 3 is characterized in that: the S9 mixture is to be mixed by 53% nutrient solution+45%NADPH (coenzyme II)+2%S9 liver tissue homogenate liquid.
5, cigarette mainstream smoke genotoxicity measurement method according to claim 2 is characterized in that: the cell culture fluid of 6 μ g/ml cytochalasin Bs is to be mixed in proportion formulated by cytochalasin B and cell culture fluid.
6, cigarette mainstream smoke genotoxicity measurement method according to claim 2 is characterized in that: 90% cold methanol is to be prepared by volume and preserved at refrigerator (20 ℃) by distilled water and methyl alcohol.
7, cigarette mainstream smoke genotoxicity measurement method according to claim 2 is characterized in that: the acridine orange solution of 10mg/ml is formulated in proportion by acridine orange and D-PBS.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200910065337XA CN101580869B (en) | 2009-07-01 | 2009-07-01 | Cigarette mainstream smoke genotoxicity measurement method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200910065337XA CN101580869B (en) | 2009-07-01 | 2009-07-01 | Cigarette mainstream smoke genotoxicity measurement method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101580869A true CN101580869A (en) | 2009-11-18 |
| CN101580869B CN101580869B (en) | 2011-09-21 |
Family
ID=41363183
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN200910065337XA Active CN101580869B (en) | 2009-07-01 | 2009-07-01 | Cigarette mainstream smoke genotoxicity measurement method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101580869B (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102206572A (en) * | 2011-03-11 | 2011-10-05 | 云南烟草科学研究院 | Device for exposing cigarette mainstream smoke |
| CN102676382A (en) * | 2012-05-29 | 2012-09-19 | 云南烟草科学研究院 | Cell gas-liquid interface contact type cigarette whole smoke exposure device |
| CN103792218A (en) * | 2014-02-21 | 2014-05-14 | 中国烟草总公司郑州烟草研究院 | Method for detecting genetic toxicity of total particulate matters in main stream smoke of cigarette by adopting binuclear method |
| CN103983478A (en) * | 2014-05-23 | 2014-08-13 | 中国烟草总公司郑州烟草研究院 | Trapping device and trapping method suitable for smoke released by tobacco material at heating non-burning state |
| WO2020004470A1 (en) * | 2018-06-28 | 2020-01-02 | ダイキン工業株式会社 | Method for evaluating cytotoxicity of gas |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7662565B2 (en) * | 2004-05-12 | 2010-02-16 | Vector Tobacco, Inc. | Approaches to identify less harmful tobacco and tobacco products |
| US8394592B2 (en) * | 2006-06-12 | 2013-03-12 | The Board Of Regents Of The University Of Texas System | Methods for assessing cancer susceptibility to carcinogens in tobacco products |
| CN101251536A (en) * | 2008-04-01 | 2008-08-27 | 中国烟草总公司郑州烟草研究院 | Small rat in-vivo microkernel analysis testing method for evaluating cigarette flue gas toxity |
-
2009
- 2009-07-01 CN CN200910065337XA patent/CN101580869B/en active Active
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102206572A (en) * | 2011-03-11 | 2011-10-05 | 云南烟草科学研究院 | Device for exposing cigarette mainstream smoke |
| CN102676382A (en) * | 2012-05-29 | 2012-09-19 | 云南烟草科学研究院 | Cell gas-liquid interface contact type cigarette whole smoke exposure device |
| CN103792218A (en) * | 2014-02-21 | 2014-05-14 | 中国烟草总公司郑州烟草研究院 | Method for detecting genetic toxicity of total particulate matters in main stream smoke of cigarette by adopting binuclear method |
| CN103792218B (en) * | 2014-02-21 | 2016-05-04 | 中国烟草总公司郑州烟草研究院 | Adopt double-core method to detect the genotoxic method of cigarette mainstream smoke total particulate matter |
| CN103983478A (en) * | 2014-05-23 | 2014-08-13 | 中国烟草总公司郑州烟草研究院 | Trapping device and trapping method suitable for smoke released by tobacco material at heating non-burning state |
| WO2020004470A1 (en) * | 2018-06-28 | 2020-01-02 | ダイキン工業株式会社 | Method for evaluating cytotoxicity of gas |
| JP2020000101A (en) * | 2018-06-28 | 2020-01-09 | ダイキン工業株式会社 | Method for evaluating cytotoxicity of gas |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101580869B (en) | 2011-09-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101580869B (en) | Cigarette mainstream smoke genotoxicity measurement method | |
| CN101393190A (en) | Cell toxicity determination method in cigarette mainstream flue gas | |
| CN103792218B (en) | Adopt double-core method to detect the genotoxic method of cigarette mainstream smoke total particulate matter | |
| AU2015266027B2 (en) | Fixative composition for cell-comprising liquid samples | |
| CN103710434B (en) | A kind of making method of marrow chromosome G band | |
| CN102980925A (en) | Sandwich type electrochemical immunosensor, preparation method and application thereof | |
| CN102495183B (en) | Detection method for cytotoxicity of gas-liquid contact type cigarettes under full-smoke exposure | |
| CN102140489A (en) | Method for testing cytotoxicity in full smoke contamination of cigarette | |
| CN104569397A (en) | Quality control sample for detecting breast cancer and preparation method of quality control sample | |
| CN104865250A (en) | Dry chemical analysis reagent membrane for atypia histiocytes and preparing method thereof | |
| CN102520154B (en) | Method for detecting dioxins in environment | |
| CN101671733A (en) | Improved cigarette smoke in-vitro contamination method | |
| CN104726533B (en) | Culture medium solidifying agent that a kind of cold water can coagulate and preparation method and application | |
| CN105938065A (en) | Application of Pap pen with fast operation speed in cell growing slide immunohistochemical detection | |
| CN105510543A (en) | Method for detecting genotoxic agent in water by utilization of Pseudorasbora parva | |
| WO2023221509A1 (en) | Method for separating, extracting and quickly classifying and counting living algae cells of soil/sediment | |
| CN103898188B (en) | LC-MS studies sample preparation and the detection method of Pyricularia oryzae metabolism group | |
| CN103558063A (en) | Full-automatic atmosphere nitrogen dry and wet deposition collecting device as well as sample collecting and detection method | |
| CN108048401A (en) | People's biliary tract cancerous cell line and application | |
| CN102174465A (en) | Method for separating enriched target cells from tissues | |
| CN103267752A (en) | Method for determining proportion of number of A cells to number of B cells in pancreatic islets | |
| CN107576713B (en) | A kind of method and its utilization detecting earthworm coelomocyte DNA damage | |
| Zhen et al. | Determination of the in situ growth rate of Microcystis based on carbon and nitrogen stable isotope fractionation | |
| CN105241878B (en) | A kind of aldehydes rapidly measuring device part based on capillarity and Nano Silver recognition principle and its preparation method and application | |
| CN114577774B (en) | Mitochondrial probe fluorescence intensity correction method and cell concentration detection method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |