CN102495183A - Detection method for cytotoxicity of gas-liquid contact type cigarettes under full-smoke exposure - Google Patents

Abstract

The invention relates to a detection method for the cytotoxicity of gas-liquid contact type cigarettes under full-smoke exposure, belonging to the technical field of safety evaluation of cigarette smoke. The detection method provided by the invention comprises the following steps of: firstly, setting cigarette suction parameters to carry out suction on a cigarette; diluting the smoke by clean air with the flow speed of 0-1000 mL/min and transferring the diluted cigarette full smoke into a full-smoke exposure bottle disclosed in the patent with the patent number of ZL201120063251.6 to come into contact and exposure with suspension cells in the bottle, wherein the exposure time is 5 min; after the exposure, carrying out neutral erythrocyte toxicity test or MTT (Methyl Thiazolyl Tetrazolium) cytotoxicity test according to a regular method; analyzing a dosage reaction relation between smoke exposure amount and cytotoxicity according to the following formula: full-smoke dilution multiple=( flow speed of smoke + flow speed of clean air)/flow speed of smoke; smoke exposure amount under exposure of 10 mL of a cell suspension solution for 5 min=TPM (Total Particulate Matter) amount of single cigarette/suction times of single cigarette*6 suctions/10 mL; and representing the extent of cytotoxicity by a cell inhibition ratio CI. The detection method provided by the invention has the advantages of simplicity, feasibility, low cost and the capability of providing technical supports for the safety evaluation of cigarette smoke.

Description

Cytotoxicity detection method under the full fume exposure of a kind of gas-liquid contact type cigarette

Technical field:

The present invention relates to the cytotoxicity detection method under the full fume exposure of a kind of gas-liquid contact type cigarette, belong to cigarette smoke safety evaluatio technical field.

Background technology:

Be used for the full fume exposure method research of tobacco toxicological evaluation; Since its in anthropomorphic dummy's internal milieu, can realize the irreplaceable advantages that the aspect had such as a plurality of coherent detection terminal points, the original full flue gas of online instant detection; Caused each tobacco company of the world and the breadboard extensive concern of analyzing and testing, some tobacco companies have carried out going deep into systematic research to this process for exposing.

At present, the existing patent of method is open under the relevant external exposure of the full flue gas of cigarette.Publication 1 (application number: 200810230659.0) be a kind of cytotoxicity method of testing of cigarette mainstream flue gas; Main exposing device is a Tissue Culture Flask; Through will in the cigarette mainstream flue gas transfered cell culture flask of pure air dilution, carrying out the fume exposure of cell; Flue gas and cell also not exclusively are in the gas-liquid interface place in this exposure chamber, have partial fume and stay in the nutrient solution and continuation and cells contacting.Publication 2 (application number: 201110025480.3) be the complete flue toxicity contaminated cytotoxicity method of testing of a kind of cigarette; Main exposing device is the plug-in type Tissue Culture Dish, through making solution-air interface that cultured cells is in flue gas and nutrient solution to reach the purpose that cell and cigarette smoke expose.The method that these two patents relate to all is that the cell with adherent state is on the solution-air interface, let flue gas directly partly or completely exposing cell to reach the purpose of the full fume exposure of cigarette.Granted patent (the patent No.: 201120063251.6) be a kind of device that is used for the full fume exposure of cigarette; By the full fume exposure bottle of the cigarette that which is provided with exhanst gas outlet and place the flue gas of full fume exposure bottle to import pipe close to form; Wherein flue gas importing pipe close is made up of smoke inlet, pure air inlet, flue gas and air mixed chamber, dilution back flue gas carrying path, reaches the control of fume exposure amount through the pure air dilution of different in flow rate.This exposing device is prone to be connected with smoking machine.With the above-mentioned the 1st and the 2 two patent in the gas-liquid interface place of exposing device expose principle different be; Granted patent (patent No.: exposing device ZL201120063251.6) is based on the gas-liquid contact principle and designs, use be the cell that is in suspended state.

At present, about the existing report of the cell toxicity test research of the full fume exposure of cigarette.But, do not find public reported with content same document of the present invention through the document retrieval.

List of references:

[1]Aufderheide?M，Mohr?U.Amodified

system?for?the?direct?exposure?ofbacteria?to?inhalable?substances[J].Exp?Toxic?Pathol，2004，55：451-454.

[2]Aufderheide?M，Ritter?D，Knebel?JW，et?al.Amethod?for?in?vitro?analysisof?the?biological?activity?of?complex?mixtures?such?as?sidestream?cigarette?smoke[J].Exp?Toxic?Pathol，2001；53：141-152.

[3] Lu Binbin, RyaN M, Lynne W waits the cytotoxicity of the different tar cigarette flue gases of .3 kind to compare [J]. tobacco science and technology, 2007,12:38-41.

Summary of the invention:

The object of the present invention is to provide the cytotoxicity detection method under the full fume exposure of a kind of gas-liquid contact type cigarette, for the cigarette smoke safety evaluatio provides technical support.

The Chinese hamster ovary cell that the present invention relates to (Chinese Hamster Ovary Cell, Chinese hamster ovary celI) is used cell for the laboratory normal experiment, buys from cell preservation both domestic and external mechanism to obtain.The device that the present invention uses is " a kind of device ZL201120063251.6 that is used for the full fume exposure of cigarette ".

The object of the invention is realized through following technical step:

A. by the conventional method cultivation Chinese hamster ovary celI that goes down to posterity, and preparation Chinese hamster ovary celI suspending liquid, cell concentration is (5-10) * 10 ⁴Individual/mL, and it branch is filled in the full fume exposure bottle of ZL201120063251.6 the 10mL/ bottle;

B. full fume exposure bottle is placed in 37 ℃ of thermostat water baths, and be connected with pure air bottle and linear pattern smoking machine;

C. on the smoking machine control panel, set the cigarette smoking parameter according to GB GB/T 16450;

D. the cigarette parameter of setting by the c step is carried out the suction of cigarette, is the exposure of the full flue gas of cigarette of the pure air dilution of 0-1000mL/min through flow velocity again, and exposure duration all is 5min; Exposure finishes, and takes off full fume exposure bottle, carries out dimethyl diaminophenazine chloride cell toxicity test or MTT cell toxicity test;

E. cytotoxicity detects:

(1) dimethyl diaminophenazine chloride cell toxicity test

The cell suspending liquid that exposure is finished is transferred in the centrifuge tube, centrifugal 5-10min under the 1000rpm/min rotating speed; Remove supernatant, add fresh conventional growth medium 10mL, process cell suspending liquid, and it is seeded in the 96 porocyte culture plates, place CO ₂Cultivate 24h in the incubator, the dimethyl diaminophenazine chloride cell toxicity test is carried out by conventional method in the back;

(2) MTT cell toxicity test

The operation of e. (1) is planted 96 porocyte culture plates with Chinese hamster ovary celI suspending liquid set by step, places CO ₂Cultivate 24h in the incubator, the MTT cell toxicity test is carried out by conventional method in the back;

F. interpretation of result

Under this exposure chamber, fume exposure amount and Cytotoxic dose-response relationship are analyzed according to the following steps:

(1) extension rate=(flue gas flow rate+pure air the flow velocity)/flue gas flow rate of full flue gas;

(2) analysis list props up flue gas TPM TPM amount and puff number, the then puff number of the TPM amount/single cigarette of every mouthful TPM amount=single cigarette that receives the paper cigarette on the linear pattern smoking machine; Puff number during exposure duration 5min is 6 mouthfuls, puff number * 6 of the TPM amount/single cigarette of the fume exposure amount=single cigarette when then the cell suspending liquid of 10mL exposes 5min mouthful/10mL;

(3) according to the dose-response relationship between above-mentioned formula analysis fume exposure amount and cytotoxicity, Cytotoxic size is represented with cell inhibitory rate CI:

$\mathrm{CI}=(1-\frac{{\mathrm{OD}}_n-{\mathrm{OD}}_o}{{\mathrm{OD}}_c-{\mathrm{OD}}_o})\×100\%.$

Annotate: OD _nIt is the average light absorption value of sample porous; OD _oIt is the average light absorption value of blank porous; OD _cIt is the average light absorption value of porous of cell contrast.

The inventive method has simple, and cost is low, and the advantage of technical support is provided for the cigarette smoke safety evaluatio.

Description of drawings:

Fig. 1 is fume exposure amount and a Cytotoxic dose-response relationship in the dimethyl diaminophenazine chloride cell toxicity test.

Fig. 2 is fume exposure amount and a Cytotoxic dose-response relationship in the MTT cell toxicity test.

Embodiment:

Below be embodiments of the invention, but content of the present invention is not limited thereto.

The Chinese hamster ovary cell that embodiment relates to (Chinese Hamster Ovary Cell, Chinese hamster ovary celI) is used cell for the laboratory normal experiment, buys from cell preservation both domestic and external mechanism to obtain.The device that embodiment uses is " a kind of device ZL201120063251.6 that is used for the full fume exposure of cigarette ".

Embodiment one: the dimethyl diaminophenazine chloride cytotoxicity under the full fume exposure of a certain blended type cigarette detects

Prepare Chinese hamster ovary celI suspending liquid according to above-mentioned steps a.; Then according to above-mentioned steps b.; C. carry out the full fume exposure of cigarette of Chinese hamster ovary celI with d., produce cigarette mainstream flue gas during the cigarette sample suction and after the pure air dilution, import in the full fume exposure bottle of ZL201120063251.6,4 different pure air flow velocitys (0mL/min, 250mL/min, 500mL/min, 750mL/min, 1000mL/min) are set respectively carry out the dilution of cigarette mainstream flue gas; Fume exposure time 5min; Cigarette smoking capacity 35mL, suction duration 2s, suction is 58s at interval.

Fume exposure finishes, and carries out the dimethyl diaminophenazine chloride cytotoxicity according to above-mentioned steps e. (1) again and detects, and according to light absorption value, calculates cell inhibitory rate CI.

$\mathrm{CI}=(1-\frac{{\mathrm{OD}}_n-{\mathrm{OD}}_o}{{\mathrm{OD}}_c-{\mathrm{OD}}_o})\×100\%$

Annotate: OD _nIt is the average light absorption value of sample porous; OD _oIt is the average light absorption value of blank porous; OD _cIt is the average light absorption value of porous of cell contrast.

Dimethyl diaminophenazine chloride cytotoxicity testing result (cell inhibitory rate %) under the full fume exposure of a certain blended type cigarette is seen table 1.

Cigarette smoking capacity 35mL; Under the condition of suction duration 2s, flue gas flow rate=1080mL/min (35mL/2s) is according to extension rate=(flue gas flow rate+pure air the flow velocity)/flue gas flow rate of the full flue gas of formula; Obtain the extension rate of different pure air flow velocitys, see table 2.On the linear pattern smoking machine, analyzing the mean value that obtains receiving paper cigarette list to prop up puff number is (8.55 ± 0.44) mouth; The mean value of single flue gas TPM (TPM) is (8.14 ± 0.81) mg; Fume exposure amount when obtaining the pure air flow velocity and be 0mL/min according to puff number * 6 of the TPM amount/single cigarette of formula fume exposure amount=single cigarette mouthful/10mL is 571.23 μ g TPM/mL, sees table 2 through the fume exposure amount of the pure air dilution of different in flow rate.Analyze under the full fume exposure fume exposure amount and Cytotoxic dose-response relationship in the dimethyl diaminophenazine chloride cell toxicity test, see Fig. 1.

Know that by table 1 and Fig. 1 along with the flow velocity increase of pure air, the cell inhibitory rate that flue gas produces decreases, and is significant dose-response relationship.

Dimethyl diaminophenazine chloride cytotoxicity testing result under the full fume exposure of a certain blended type cigarette of table 1

Flue gas dilution multiple and cell inhibitory rate during table 2 dimethyl diaminophenazine chloride cytotoxicity detects

Embodiment two: the MTT cytotoxicity under the full fume exposure of a certain blended type cigarette detects

Remove according to step e. (2) and carry out the detection of MTT cytotoxicity, other experimental techniques are with embodiment one.MTT cytotoxicity testing result under the full fume exposure of a certain blended type cigarette is seen table 3 and table 4, and analyzes under the full fume exposure fume exposure amount and Cytotoxic dose-response relationship in the MTT cell toxicity test, sees Fig. 2.

Can know that by table 3 and Fig. 2 along with the flow velocity increase of pure air, the cell inhibitory rate that flue gas produces decreases, and is significant dose-response relationship.

MTT cytotoxicity testing result under the full fume exposure of a certain blended type cigarette of table 3

Flue gas dilution multiple and cell inhibitory rate during table 4MTT cytotoxicity detects

Claims (1)

1. the cytotoxicity detection method under the full fume exposure of gas-liquid contact type cigarette is characterized in that the step of this method is following:

A. by the conventional method cultivation Chinese hamster ovary celI that goes down to posterity, and preparation Chinese hamster ovary celI suspending liquid, cell concentration is (5-10) * 10 ⁴Individual/mL, and it branch is filled in the full fume exposure bottle that the patent No. is ZL201120063251.6 the 10mL/ bottle;

B. full fume exposure bottle is placed in 37 ℃ of thermostat water baths, and be connected with pure air bottle and linear pattern smoking machine;

C. on the smoking machine control panel, set the cigarette smoking parameter according to GB GB/T 16450;

D. the cigarette parameter of setting by the c step is carried out the suction of cigarette, is the exposure of the full flue gas of cigarette of the pure air dilution of 0-1000mL/min through flow velocity again, and exposure duration all is 5min; Exposure finishes, and takes off full fume exposure bottle, carries out dimethyl diaminophenazine chloride cell toxicity test or MTT cell toxicity test;

E. cytotoxicity detects:

(1) dimethyl diaminophenazine chloride cell toxicity test

The cell suspending liquid that exposure is finished is transferred in the centrifuge tube, centrifugal 5-10min under the 1000rpm/min rotating speed; Remove supernatant, add fresh conventional growth medium 10mL, process cell suspending liquid, and it is seeded in the 96 porocyte culture plates, place CO ₂Cultivate 24h in the incubator, the dimethyl diaminophenazine chloride cell toxicity test is carried out by conventional method in the back;

(2) MTT cell toxicity test

The operation of e. (1) is planted 96 porocyte culture plates with Chinese hamster ovary celI suspending liquid set by step, places CO ₂Cultivate 24h in the incubator, the MTT cell toxicity test is carried out by conventional method in the back;

F. interpretation of result

Under this exposure chamber, fume exposure amount and Cytotoxic dose-response relationship are analyzed according to the following steps:

(1) extension rate=(flue gas flow rate+pure air the flow velocity)/flue gas flow rate of full flue gas;

(2) analysis list props up flue gas TPM TPM amount and puff number, the then puff number of the TPM amount/single cigarette of every mouthful TPM amount=single cigarette that receives the paper cigarette on the linear pattern smoking machine; Puff number during exposure duration 5min is 6 mouthfuls, puff number * 6 of the TPM amount/single cigarette of the fume exposure amount=single cigarette when then the cell suspending liquid of 10mL exposes 5min mouthful/10mL;

(3) according to the dose-response relationship between above-mentioned formula analysis fume exposure amount and cytotoxicity, Cytotoxic size is represented with cell inhibitory rate CI.

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