CN102495183B - 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:

For the full fume exposure method research of tobacco toxicological evaluation, because it is being simulated human internal environment, can realize multiple coherent detection terminals, is immediately detecting online the irreplaceable advantage that the aspects such as original full flue gas have, the extensive concern that has caused each tobacco company of the world and analyzing and testing laboratory, some tobacco companies have carried out going deep into systematic research to this process for exposing.

At present, under the relevant external exposure of the full flue gas of cigarette, the existing patent of method is open.Publication 1 (application number: 200810230659.0) be a kind of cytotoxicity method of testing of cigarette mainstream flue gas, main exposing device is Tissue Culture Flask, by carrying out the fume exposure of cell in the cigarette mainstream flue gas transfered cell culture flask of pure air dilution, in this exposure chamber, flue gas and cell, also not exclusively in gas-liquid interface place, have partial fume and stay in nutrient solution and continue 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 plug-in type Tissue Culture Dish, by the object that the solution-air interface of cultured cells in flue gas and nutrient solution exposed to reach cell and cigarette smoke.The method that these two patents relate to be all by the cell of adherent state on solution-air interface, allow flue gas directly partly or completely exposing cell to reach the object of the full fume exposure of cigarette.Granted patent (the patent No.: 201120063251.6) be a kind of device for the full fume exposure of cigarette, by the full fume exposure bottle of the cigarette which is provided with exhanst gas outlet and be placed in full fume exposure bottle flue gas import pipe close form, wherein flue gas importing pipe close is made up of smoke inlet, pure air entrance, flue gas and air hybrid chamber, the rear flue gas carrying path of dilution, reaches the control of fume exposure amount by the pure air dilution of different in flow rate.This exposing device is easily connected with smoking machine.From the above-mentioned the 1st and the 2 two patent in exposing device gas-liquid interface place expose principle different be, the exposing device of granted patent (patent No.: ZL201120063251.6) is to design based on gas-liquid contact principle, use be the cell in suspended state.

At present, have been reported about the cell toxicity test research of the full fume exposure of cigarette.But do not find and the open report of content same document of the present invention by literature search.

List of references:

[1]Aufderheide?M,Mohr?U.Amodified? system?for?the?direct?exposure?of?bacteria?to?inhalable?substances[J].Exp?Toxic?Pathol,2004,55:451-454.

[2]Aufderheide?M,Ritter?D,Knebel?JW,et?al.Amethod?for?in?vitro?analysis?of?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 comparison [J] of the different tar cigarette flue gases of .3 kind. 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 cigarette smoke safety evaluatio provides technical support.

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

Object of the present invention realizes by following technical step:

A. the cultivation Chinese hamster ovary celI that goes down to posterity according to a conventional method, and prepare Chinese hamster ovary celI suspending liquid, cell concentration is (5-10) × 10 ⁴individual/mL, and it point is filled in the full fume exposure bottle that the patent No. is ZL201120063251.6 to 10mL/ bottle; The patent No. is that the flue gas importing pipe close two large divisions that the full fume exposure culture apparatus of the cigarette of ZL201120063251.6 is placed in the full fume exposure bottle of cigarette by the full fume exposure bottle of the cigarette which is provided with exhanst gas outlet and the latter half forms; Wherein: flue gas imports pipe close and is made up of smoke inlet, mixing air entrance, flue gas and air hybrid chamber, the rear flue gas carrying path of dilution;

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

C. on smoking machine control panel, set cigarette smoking parameter according to GB GB/T16450;

D. the cigarette parameter of setting by c step is carried out the suction of cigarette, then the exposure of the full flue gas of cigarette of the pure air that is 0-1000mL/min through flow velocity dilution, and open-assembly time is all 5min; Expose completely, take off full fume exposure bottle, carry out dimethyl diaminophenazine chloride cell toxicity test or MTT cell toxicity test;

E. cytotoxicity detects:

(1) dimethyl diaminophenazine chloride cell toxicity test

Be transferred in centrifuge tube centrifugal 5-10min under 1000rpm/min rotating speed by exposing complete cell suspending liquid; Remove supernatant, add fresh conventional growth medium 10mL, make cell suspending liquid, and it is seeded in 96 porocyte culture plates, be placed in CO ₂cultivate 24h in incubator, after carry out according to a conventional method dimethyl diaminophenazine chloride cell toxicity test;

(2) MTT cell toxicity test

By the operation of step e. (1), Chinese hamster ovary celI suspending liquid is planted to 96 porocyte culture plates, be placed in CO ₂cultivate 24h in incubator, after carry out according to a conventional method MTT cell toxicity test;

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 flow velocity)/flue gas flow rate of full flue gas;

(2) on linear pattern smoking machine, analysis list props up flue gas TPM TPM amount and puff number, the puff number of the TPM amount/single cigarette of the TPM amount=single cigarette of every mouthful of tested cigarette; Puff number when open-assembly time 5min is 6 mouthfuls, and the cell suspending liquid of 10mL exposes the puff number × 6 mouthful/10mL of the TPM amount/single cigarette of fume exposure amount=single cigarette when 5min;

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

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

Note: 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, for cigarette smoke safety evaluatio provides the advantage of technical support.

Brief description of the drawings:

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

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

Embodiment:

Be below embodiments of the invention, but content of the present invention is not limited to this.

The Chinese hamster ovary cell (Chinese Hamster Ovary Cell, Chinese hamster ovary celI) that embodiment relates to is laboratory normal experiment cell, buys and obtains from cell preservation both domestic and external mechanism.The device that embodiment uses is " a kind of device ZL201120063251.6 for the full fume exposure of cigarette ", and the flue gas importing pipe close two large divisions that the full fume exposure culture apparatus of this cigarette is placed in the full fume exposure bottle of cigarette by the full fume exposure bottle of the cigarette which is provided with exhanst gas outlet and the latter half forms; Wherein: flue gas imports pipe close and is made up of smoke inlet, mixing air entrance, flue gas and air hybrid chamber, the rear flue gas carrying path of dilution.

Embodiment mono-: 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., when cigarette sample suction, produce cigarette mainstream flue gas imports in the full fume exposure bottle of ZL201120063251.6 after pure air dilution, 4 different pure air flow velocitys (0mL/min, 250mL/min, 500mL/min, 750mL/min, 1000mL/min) are set respectively and carry out the dilution of cigarette mainstream flue gas, fume exposure time 5min, cigarette smoking capacity 35mL, suction duration 2s, suction interval 58s.

Fume exposure is complete, then carries out the detection of dimethyl diaminophenazine chloride cytotoxicity according to above-mentioned steps e. (1), according to light absorption value, calculates cell inhibitory rate CI.

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

Note: 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 in table 1.

Cigarette smoking capacity 35mL, under the condition of suction duration 2s, flue gas flow rate=1080mL/min (35mL/2s), according to the extension rate of the full flue gas of formula=(flue gas flow rate+pure air flow velocity)/flue gas flow rate, obtain the extension rate of different pure air flow velocitys, in table 2.On linear pattern smoking machine, analyzing and obtaining the mean value that tested cigarette list props up puff number is (8.55 ± 0.44) mouthful, the mean value (TPM) of single flue gas TPM is (8.14 ± 0.81) mg, fume exposure amount when obtaining 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, through the fume exposure amount of the pure air dilution of different in flow rate in table 2.Analyze under full fume exposure fume exposure amount and Cytotoxic dose-response relationship in the test of neutral RBC Toxicity, see Fig. 1.

Known by table 1 and Fig. 1, along with the flow velocity of pure air increases, 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 bis-: the MTT cytotoxicity under the full fume exposure of a certain blended type cigarette detects

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

From table 3 and Fig. 2, along with the flow velocity of pure air increases, 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 the method is as follows:

A. the cultivation Chinese hamster ovary celI that goes down to posterity according to a conventional method, and prepare Chinese hamster ovary celI suspending liquid, cell concentration is (5-10) × 10 ⁴individual/mL, and it point is filled in the full fume exposure bottle that the patent No. is ZL201120063251.6 to 10mL/ bottle; The patent No. is that the flue gas importing pipe close two large divisions that the full fume exposure culture apparatus of the cigarette of ZL201120063251.6 is placed in the full fume exposure bottle of cigarette by the full fume exposure bottle of the cigarette which is provided with exhanst gas outlet and the latter half forms; Wherein: flue gas imports pipe close and is made up of smoke inlet, mixing air entrance, flue gas and air hybrid chamber, the rear flue gas carrying path of dilution;

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

C. on smoking machine control panel, set cigarette smoking parameter according to GB GB/T16450;

D. the cigarette parameter of setting by c step is carried out the suction of cigarette, then the exposure of the full flue gas of cigarette of the pure air that is 0-1000mL/min through flow velocity dilution, and open-assembly time is all 5min; Expose completely, take off full fume exposure bottle, carry out dimethyl diaminophenazine chloride cell toxicity test or MTT cell toxicity test;

E. cytotoxicity detects:

(1) dimethyl diaminophenazine chloride cell toxicity test

Be transferred in centrifuge tube centrifugal 5-10min under 1000rpm/min rotating speed by exposing complete cell suspending liquid; Remove supernatant, add fresh conventional growth medium 10mL, make cell suspending liquid, and it is seeded in 96 porocyte culture plates, be placed in CO ₂cultivate 24h in incubator, after carry out according to a conventional method dimethyl diaminophenazine chloride cell toxicity test;

(2) MTT cell toxicity test

By the operation of step e. (1), Chinese hamster ovary celI suspending liquid is planted to 96 porocyte culture plates, be placed in CO ₂cultivate 24h in incubator, after carry out according to a conventional method MTT cell toxicity test;

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 flow velocity)/flue gas flow rate of full flue gas;

(2) on linear pattern smoking machine, analysis list props up flue gas TPM TPM amount and puff number, the puff number of the TPM amount/single cigarette of the TPM amount=single cigarette of every mouthful of tested cigarette; Puff number when open-assembly time 5min is 6 mouthfuls, and the cell suspending liquid of 10mL exposes the puff number × 6 mouthful/10mL of the TPM amount/single cigarette of fume exposure amount=single cigarette when 5min;

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