CN101393190A - Cell toxicity determination method in cigarette mainstream flue gas - Google Patents
Cell toxicity determination method in cigarette mainstream flue gas Download PDFInfo
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- CN101393190A CN101393190A CNA2008102306590A CN200810230659A CN101393190A CN 101393190 A CN101393190 A CN 101393190A CN A2008102306590 A CNA2008102306590 A CN A2008102306590A CN 200810230659 A CN200810230659 A CN 200810230659A CN 101393190 A CN101393190 A CN 101393190A
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- dimethyl diaminophenazine
- diaminophenazine chloride
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Abstract
The invention relates to a method for mensurating cytotoxicity of mainstream cigarette smoke. The method is characterized in comprising the following steps: firstly, a cultured cell is directly put to the mainstream cigarette smoke diluted by clean air and is exposed; secondly, the cell is subjected to neutral red staining; according to the situation that the cell is ingested with neutral red, a spectrophotometer of a readable porous culturing plate is utilized to mensurate an absorbance value; and the cell is compared with a contrastive sample exposed in the clean air, thereby judging the toxicity of the mainstream cigarette smoke. The method is based on a testing principle that the cultured cell is directly put to the mainstream cigarette smoke and is exposed; the level for ingesting the neutral red by the cell is in positive proportion to the number of viable cells; when the toxic effect of the mainstream cigarette smoke is larger, the survival cells exposed in the mainstream cigarette smoke is fewer, correspondingly the dye absorption amount is less and the absorbance value is lower. The method can quantitatively evaluate the cytotoxicity of a tested object, has the characteristics of simple operation, high sensitivity and reliable result, and creates a new method for mensurating the cytotoxicity of the mainstream cigarette smoke.
Description
Technical field
The present invention relates to cigarette mainstream flue gas Cytotoxic evaluation field, be a kind of cell toxicity determination method in cigarette mainstream flue gas specifically, be by with cellular exposure under cigarette mainstream flue gas, measure the cell mortality that main flume causes and carry out cytotoxic assay.
Background technology
The gasoloid 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 Study of cytotoxicity about the chemical constitution in cigarette smoke or the flue gas, the cytotoxicity of suitable mammalian cell with dimethyl diaminophenazine chloride method evaluating cigarette flue gas recommended to select for use by the external toxicology of CORESTA task force, the dimethyl diaminophenazine chloride evaluation method has been widely used in evaluating cytotoxicity [the Putnam KP with the smoke condensate of more different tar contents and dissimilar cigarette, Bombick DW, Doolittle DJ.Evaluation of eight in vitro assaysfor assessing the cytotoxicity of cigarette smoke condensate.ToxicologyIn Vitro[J] 2002,16 (5): 599-607].In addition, the dimethyl diaminophenazine chloride method is applied to also that some have the cytotoxic assay of cytotoxic compound in the cigarette smoke, and be used for measuring relatively cytotoxicity [the Roemer E. that adds and do not add cigarette additive cigarette smoke granule phase substance and gas phase thing by tobacco productive corporation, F.J Tewes, T.J.Meisgen, et al.Evaluation of the potential effects ofingredients added to cigarettes.Part 3:in vitro genotoxicity andcytotoxicity.Food Chem Tox[J] .2002,40:105-111.].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 the cytotoxicity test again.Such method of testing will spend a large amount of time, complex operation, and, can not reflect main flume cytotoxicity 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 just at above-mentioned existing in prior technology problem, and a kind of cell toxicity determination method in cigarette mainstream flue gas that is used for of special exploitation, this method is at only testing the Cytotoxic weak point of main flume granule phase substance that traps on the cambridge filter in the above-mentioned cigarette smoke cytotoxicity test, can also reduce significantly simultaneously and carry out loaded down with trivial details solvent extraction process after flue gas is collected, can directly also comprehensively reflect the cigarette mainstream flue gas cytotoxicity, 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 pure air and exposes, dye by dimethyl diaminophenazine chloride then, take in the situation of dimethyl diaminophenazine chloride according to cell, utilizing readable porous culture plate spectrophotometer to carry out absorbance measures, expose control sample relatively with pure air, and then judge cigarette mainstream flue gas toxicity.
Concrete assay method of the present invention may further comprise the steps:
A. cellular incubation: 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 culture flask, 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. the cell main flume exposes: smoking cigarette under the iso standard condition, the main flume that produces dilutes with pure air, fume exposure concentration is that index is controlled with TPM (TPM) concentration in the flue gas after diluting, and the flue gas after the dilution directly imports to grow and carries out the main flume exposure in the culture flask that cell is arranged.Exposure duration is 60min, and whole process-exposed is controlled according to the TPM/L value of monitoring, and the normal control group feeds pure air and exposes.
C. cellular incubation after the fume exposure: expose finish after, change the nutrient solution in the culture flask into fresh nutrient solution, and place CO
2Cultivate 24h down for 37 ℃ in the incubator, contrast parallel sample into feeding pure air and cultivating through the same terms;
D. dimethyl diaminophenazine chloride dyeing: behind the cellular incubation 24h that fume exposure is crossed, the sucking-off nutrient solution is with the Ca that contains of 37 ℃ of following preheatings
2+, Mg
2+Phosphate buffer 1 0mL-30mL rinsing 2 times adds 20mL dimethyl diaminophenazine chloride nutrient solution, puts into CO
2Cultivate 3h down for 37 ℃ in the incubator, then, remove the dimethyl diaminophenazine chloride nutrient solution, add 5mL-15mL 1% formaldehyde fixed liquid, place 2min-10min under the room temperature, immobile liquid is removed in suction, add 20mL dimethyl diaminophenazine chloride lysate, place 2min-10min under the room temperature, put upside down culture flask several times, dimethyl diaminophenazine chloride is fully dissolved, draw the 0.1mL-0.3mL lysate, place 96 well culture plates,, air exposure sample blank with dimethyl diaminophenazine chloride lysate work compares, with the light absorption value of readable 96 well culture plate spectrophotometric determination lysates at the 540nm place;
E. data processing and analysis, evaluating cigarette main flume cytotoxicity.
In the present invention, the dimethyl diaminophenazine chloride nutrient solution be by dimethyl diaminophenazine chloride dye liquor and cell culture fluid by volume 1:99 ratio mixed preparing form, need preparation on the same day and filtration sterilization.Formaldehyde fixed liquid be by formaldehyde and distilled water by volume 1:99 mix.The dimethyl diaminophenazine chloride lysate is that 50:49:1 is formulated by volume by ethanol, water and glacial acetic acid.
The test philosophy of foundation of the present invention is: cultured cell is directly inserted in the cigarette mainstream flue gas expose, the level that cell is taken in dimethyl diaminophenazine chloride is directly proportional with the quantity of living cells, the toxic action of cigarette mainstream flue gas is big more, it is just few more to be exposed to the cell of surviving in the cigarette mainstream flue gas, the dyestuff uptake is also corresponding few more, and its absorbance is just low.This experimental design has reflected the toxic action of cigarette mainstream flue gas more objectively, and the energy quantitative evaluation is tried the cytotoxicity of thing, 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 the cigarette mainstream flue gas cytotoxic assay.
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 flowmeter, and 3 is pump, and 4 is Tissue Culture Flask.
Fig. 2 is embodiment 1 cigarette smoke cytotoxicity dose response curve figure.
Fig. 3 is embodiment 2 cigarette smoke cytotoxicity dose response curve figure.
Fig. 4 is embodiment 3 cigarette smoke cytotoxicity dose response curve figure.
Embodiment
The present invention is described further with the following Examples, but does not limit the present invention.
Embodiment 1
(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, duration 2s, and the per minute suction is flatly.The main flume that cigarette burning produces dilutes with clean air, and adjusting flue gas TPM concentration to 30 μ g/L, 60 μ g/L, 90 μ g/L and 120 μ g/L, flue gas after the dilution directly imported long has the culture flask of cell to expose, and each concentration exposure duration is 60min.In the test used smoking machine be connected with Tissue Culture Flask and the flue gas control mode referring to Fig. 1.
In addition, there is the culture flask of cell to feed pure air sample in contrast with long.After exposing end, the nutrient solution in the culture flask is changed into fresh nutrient solution and places CO
2Cultivate 24h in the incubator.Sucking-off nutrient solution and with the Ca that contains of preheating then
2+, Mg
2+ phosphate buffer 20mL rinsing 2 times adds 20mL dimethyl diaminophenazine chloride nutrient solution (add the dimethyl diaminophenazine chloride configuration by nutrient solution and form, dispose the same day and filtration sterilization), puts into CO
2Cultivate 3h in the incubator.Then the dimethyl diaminophenazine chloride nutrient solution is removed, add 5mL-15mL 1% formaldehyde fixed liquid and place 2min-5min, immobile liquid is removed in suction, add 20mL dimethyl diaminophenazine chloride lysate (ethanol: water: glacial acetic acid: 50:49:1), put upside down culture flask several times after placing 2min-5min, dimethyl diaminophenazine chloride is fully dissolved, draw 0.1mL-0.3mL lysate to 96 well culture plate.With the dimethyl diaminophenazine chloride lysate in contrast, with the light absorption value of readable 96 well culture plate spectrophotometric determination lysates at the 540nm place as blank, air exposure sample.Analyze with the Calcusyn Biological Statistic Analysis Software, the results are shown in Figure 2.LC
50(LC
50Being that certain main flume makes dead half the required concentration of subject cell) result of calculation is 49.2 μ g/L.
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 20 μ g/L, 60 μ g/L, 80 μ g/L, 100 μ g/L, 120 μ g/L and 150 μ g/L.The results are shown in Figure 3.LC
50Result of calculation is 78.0 μ g/L.
Embodiment 3
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 3mg, and fume exposure concentration is 60 μ g/L, 90 μ g/L, 120 μ g/L and 160 μ g/L.The results are shown in Figure 4.LC
50Result of calculation is 95.1 μ g/L.
Claims (5)
1, a kind of cell toxicity determination method in cigarette mainstream flue gas, it is characterized in that: elder generation directly inserts cultured cell in the cigarette mainstream flue gas that dilutes through pure air and exposes, dye by dimethyl diaminophenazine chloride then, take in the situation of dimethyl diaminophenazine chloride according to cell, utilizing readable porous culture plate spectrophotometer to carry out absorbance measures, expose control sample relatively with pure air, and then judge cigarette mainstream flue gas toxicity.
2, cell toxicity determination method in cigarette mainstream flue gas according to claim 1 is characterized in that: its concrete assay method may further comprise the steps:
A. cellular incubation: 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 culture flask, 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. the cell main flume exposes: smoking cigarette under the iso standard condition, the main flume that produces dilutes with pure air, fume exposure concentration is that index is controlled with TPM (TPM) concentration in the flue gas after diluting, flue gas after the dilution directly imports to grow and carries out the main flume exposure in the culture flask that cell is arranged, and exposure duration is 60min;
C. cellular incubation after the fume exposure: expose finish after, change the nutrient solution in the culture flask into fresh nutrient solution, and place CO
2Cultivate 24h down for 37 ℃ in the incubator, contrast parallel sample into feeding pure air and cultivating through the same terms;
D. dimethyl diaminophenazine chloride dyeing: behind the cellular incubation 24h that fume exposure is crossed, the sucking-off nutrient solution is with the Ca that contains of 37 ℃ of following preheatings
2+, Mg
2+Phosphate buffer 1 0mL-30mL rinsing 2 times adds 20mL dimethyl diaminophenazine chloride nutrient solution, puts into CO
2Cultivate 3h down for 37 ℃ in the incubator, then, remove the dimethyl diaminophenazine chloride nutrient solution, add 5mL-15mL 1% formaldehyde fixed liquid, place 2min-10min under the room temperature, inhale and remove immobile liquid, add 20mL dimethyl diaminophenazine chloride lysate, place 2min-10min under the room temperature, dimethyl diaminophenazine chloride is fully dissolved, draw the 0.1mL-0.3mL lysate, place 96 well culture plates,, air exposure sample blank with dimethyl diaminophenazine chloride lysate work compares, with the light absorption value of readable 96 well culture plate spectrophotometric determination lysates at the 540nm place;
E. data processing and analysis, evaluating cigarette main flume cytotoxicity.
3, cell toxicity determination method in cigarette mainstream flue gas according to claim 2 is characterized in that: the dimethyl diaminophenazine chloride nutrient solution be by dimethyl diaminophenazine chloride dye liquor and cell culture fluid by volume 1:99 ratio mixed preparing form same day preparation and filtration sterilization.
4, cell toxicity determination method in cigarette mainstream flue gas according to claim 2 is characterized in that: formaldehyde fixed liquid be by formaldehyde and distilled water by volume 1:99 mix.
5, cell toxicity determination method in cigarette mainstream flue gas according to claim 2 is characterized in that: the dimethyl diaminophenazine chloride lysate is that 50:49:1 is formulated by volume by ethanol, water and glacial acetic acid.
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101851660A (en) * | 2010-04-23 | 2010-10-06 | 云南烟草科学研究院 | Preparation method of neutral red solution used for detecting cigarette smoke cytotoxicity |
| CN102140489A (en) * | 2011-01-24 | 2011-08-03 | 中国烟草总公司郑州烟草研究院 | Method for testing cytotoxicity in full smoke contamination of cigarette |
| CN102168012A (en) * | 2011-01-08 | 2011-08-31 | 中国烟草总公司郑州烟草研究院 | Toxicity contamination testing device suitable for adding harmful components under full smoke gas toxicity contamination way of cigarettes |
| CN102384961A (en) * | 2011-10-16 | 2012-03-21 | 云南烟草科学研究院 | Method for distinguishing influence of additive on special smoke index of cigarettes |
| CN102495183A (en) * | 2011-12-16 | 2012-06-13 | 云南烟草科学研究院 | Detection method for cytotoxicity of gas-liquid contact type cigarettes under full-smoke exposure |
| CN102590111A (en) * | 2012-01-20 | 2012-07-18 | 云南烟草科学研究院 | Method for representing gas-liquid contact type cigarette full smoke exposure by using light absorption value |
| CN103808681A (en) * | 2014-03-16 | 2014-05-21 | 国家烟草质量监督检验中心 | Neutral red absorption assaying method for evaluating cell toxicity of electronic cigarette smoke solution |
| CN103834716A (en) * | 2014-03-16 | 2014-06-04 | 国家烟草质量监督检验中心 | WST-1 method for testing in-vitro cytotoxicity of tobacco juice of electronic cigarette |
| CN110726658A (en) * | 2019-11-21 | 2020-01-24 | 上海烟草集团有限责任公司 | Method for determining cigarette smoke induced apoptosis under gas-liquid interface exposure |
| CN110736696A (en) * | 2019-11-21 | 2020-01-31 | 上海烟草集团有限责任公司 | method for determining cigarette smoke cytotoxicity under exposure of gas-liquid interface |
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Cited By (17)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101851660A (en) * | 2010-04-23 | 2010-10-06 | 云南烟草科学研究院 | Preparation method of neutral red solution used for detecting cigarette smoke cytotoxicity |
| CN102168012B (en) * | 2011-01-08 | 2013-08-14 | 中国烟草总公司郑州烟草研究院 | Toxicity contamination testing device suitable for adding harmful components under full smoke gas toxicity contamination way of cigarettes |
| CN102168012A (en) * | 2011-01-08 | 2011-08-31 | 中国烟草总公司郑州烟草研究院 | Toxicity contamination testing device suitable for adding harmful components under full smoke gas toxicity contamination way of cigarettes |
| CN102140489A (en) * | 2011-01-24 | 2011-08-03 | 中国烟草总公司郑州烟草研究院 | Method for testing cytotoxicity in full smoke contamination of cigarette |
| CN102140489B (en) * | 2011-01-24 | 2012-12-05 | 中国烟草总公司郑州烟草研究院 | Method for testing cytotoxicity in full smoke contamination of cigarette |
| CN102384961A (en) * | 2011-10-16 | 2012-03-21 | 云南烟草科学研究院 | Method for distinguishing influence of additive on special smoke index of cigarettes |
| CN102384961B (en) * | 2011-10-16 | 2014-06-11 | 云南烟草科学研究院 | Method for distinguishing influence of additive on special smoke index of cigarettes |
| CN102495183B (en) * | 2011-12-16 | 2014-08-13 | 云南烟草科学研究院 | Detection method for cytotoxicity of gas-liquid contact type cigarettes under full-smoke exposure |
| CN102495183A (en) * | 2011-12-16 | 2012-06-13 | 云南烟草科学研究院 | Detection method for cytotoxicity of gas-liquid contact type cigarettes under full-smoke exposure |
| CN102590111B (en) * | 2012-01-20 | 2014-05-14 | 云南烟草科学研究院 | Method for representing gas-liquid contact type cigarette full smoke exposure by using light absorption value |
| CN102590111A (en) * | 2012-01-20 | 2012-07-18 | 云南烟草科学研究院 | Method for representing gas-liquid contact type cigarette full smoke exposure by using light absorption value |
| CN103808681A (en) * | 2014-03-16 | 2014-05-21 | 国家烟草质量监督检验中心 | Neutral red absorption assaying method for evaluating cell toxicity of electronic cigarette smoke solution |
| CN103834716A (en) * | 2014-03-16 | 2014-06-04 | 国家烟草质量监督检验中心 | WST-1 method for testing in-vitro cytotoxicity of tobacco juice of electronic cigarette |
| CN103834716B (en) * | 2014-03-16 | 2016-06-22 | 国家烟草质量监督检验中心 | A kind of tobacco juice for electronic smoke vitro cytotoxicity WST-1 method of testing |
| CN103808681B (en) * | 2014-03-16 | 2017-01-11 | 国家烟草质量监督检验中心 | Neutral red absorption assaying method for evaluating cell toxicity of electronic cigarette smoke solution |
| CN110726658A (en) * | 2019-11-21 | 2020-01-24 | 上海烟草集团有限责任公司 | Method for determining cigarette smoke induced apoptosis under gas-liquid interface exposure |
| CN110736696A (en) * | 2019-11-21 | 2020-01-31 | 上海烟草集团有限责任公司 | method for determining cigarette smoke cytotoxicity under exposure of gas-liquid interface |
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