CN101671733A - Improved cigarette smoke in-vitro contamination method - Google Patents

Abstract

The invention relates to an improved cigarette smoke in-vitro contamination method. The biological effect of the cigarette smoke is evaluated by mixing the gaseous phase part (GVP) and the particle phase part (TPM) of the cigarette smoke to carry out the in-vitro contamination method. Specifically, the improved cigarette smoke in-vitro contamination method comprises the following steps: a. collection of the particle phase part and the gaseous phase part of smoke; b. mixing of the particle phase part and the gaseous phase part; and c, in-vitro contamination: diluting TPM+GVP with a proportion of 1:1 into different concentration gradients, and carrying out a cytotoxicity test, a micronucleus test and an Ames test by in-vitro contamination method. The invention has the advantages that the gaseous phase part of cigarette smoke is collected by an absorption bottle; the in-vitro contamination way of combining GVP and TPM can comprehensively and truly reflect the biological effect of the cigarette smoke; based on the conventional TPM contamination way, the contamination of the gaseous phase part of cigarette smoke is added, so that the in-vitro contamination method is more close to the actual cigarette smoke exposure condition; besides, the contamination concentration is easy to control and the operation is simple.

Description

A kind of improved cigarette smoke in-vitro contamination method

Technical field

The present invention relates to a kind of in-vitro contamination method of cigarette smoke, promptly adopt cigarette smoke gas phase part (GVP) and grain mutually the mode that combines of part (TPM) carry out in-vitro contamination, make the toxic environment of study subject (cell or bacterium) more approach the cigarette smoke exposure situation of reality.

Background technology

Aspect the toxicological evaluation of cigaret additive, external some research institutions and company have carried out number of research projects.People such as Clements have carried out the research of the external toxicological evaluation of cigaret additive, research lays particular emphasis on the research and design of the external toxicological evaluation of cigaret additive, standard method according to the genetic toxicity test, mutant bacteria-Ames test, the human cell of analysis of cells of mamma animals sudden change-mouse lymph lymphoma and cultivation or the analysis of variance of Chinese hamster cell chromosome structure, in-vitro micronucleus analysis-utilization is duplicated index as toxicity index, cytotoxicity analysis-Neutral Red test, and at the flue gas grain mutually the characteristics of part carried out special test design.People such as the Massey of British American Tobacco have studied tobacco additive agent to smoke chemistry and toxic influence, for tobacco additive agent, adopt following 4 kinds of tests: 1) direct high temperature pyrolysis, assay products; 2) be added into cigarette, observe the objectionable constituent that influence-" Huffman list " listed smoke chemistry; 3) external biological determination of activity; 4) toxicity on inhalation is measured.PHILIP MORRIS company to the rule of cigaret additive evaluation is: about the chemical analysis of flue gas; based on two big groups is the recommendation of the U.S. consumer protection council (Huffman list) and international cancer research association (IARC), analyzes about 50 kinds of flue gas compositions.Use the test (Salmonella reversion test, mouse lymph sarcoma cell strain mutagenicity test, the test of rat micronucleus in erythrocytes) of three kinds of test smoke genetic toxicities, compared the contrast cigarette and the toxicological effect that is subjected to the paper cigarette that has added different amount compositions of no added ingredients.More domestic R﹠D institutions have carried out the animal vivo test of some smoke of tobacco in vitro toxicity Research of measuring and smoke of tobacco.

But, these researchs be based on more the cigarette smoke grain mutually partly (TPM) contaminating mode carry out, can only reflect the biological effect of TPM.And cigarette smoke not only contains cigarette smoke grain phase part, also contains a large amount of harmful gaseous components simultaneously, only adopt the TPM contaminating mode be can not be comprehensively the biological effect of evaluating cigarette flue gas truly.

The patent of invention close with the present invention is: CN101393190 " cell toxicity determination method in cigarette mainstream flue gas ", cultured cell directly inserted in the cigarette mainstream flue gas through uncontaminated air dilution expose, dye by toluylene red then, take in the situation of toluylene red according to cell, utilizing readable porous culture plate spectrophotometer to carry out absorbance measures, expose control sample relatively with uncontaminated air, and then judge cigarette mainstream flue gas toxicity.It is characterized in that cell directly is exposed to cigarette mainstream flue gas, though be that cigarette smoke directly exposes, the cigarette smoke gas phase of exposing cell partly remains water-soluble a part of gaseous substance, and is not easy control with the cigarette smoke concentration of cells contacting.

Summary of the invention

Purpose of the present invention is improved at above deficiency just.Collect cigarette smoke gas phase part (GVP) by certain approach, adopt GVP to carry out the method for in-vitro contamination, can reflect the biological effect of cigarette smoke comprehensively really in conjunction with TPM.The present invention has increased the contamination of cigarette smoke gas phase part on the basis of TPM contaminating mode in the past, make in-vitro contamination method more approach actual cigarette smoke and expose situation.

The objective of the invention is to be achieved through the following technical solutions: the improved cigarette smoke in-vitro contamination method of the present invention, adopt cigarette smoke gas phase part (GVP) to carry out the in-vitro contamination test after part (TPM) mixing mutually with the cigarette smoke grain, come the biological effect of evaluating cigarette flue gas, concrete grammar is as follows:

A, flue gas grain part mutually partly collect with gas phase: the mode of contacting mutually by smoking machine and cambridge filter and gas absorption bottle is collected the flue gas grain respectively mutually partly and flue gas gas phase part, add phosphate buffered saline buffer in the absorption bottle, and absorption bottle placed in the vacuum jacketed flask that contains ice cube, guarantee that damping fluid is lower than 4 ℃ in whole collection process;

B, grain are partly partially mixed with gas phase mutually: the TPM that Cambridge sheet is collected adopts DMSO to extract, and extraction concentration is 10mg/ml, adds in absorption bottle and the identical phosphate buffered saline buffer of the extraction used DMSO volume of TPM, then TPM and GVP is pressed 1: 1 proportioning;

C, in-vitro contamination: the TPM+GVP of 1: 1 proportioning is diluted to the different concns gradient, carries out cell toxicity test, micronucleus test and Salmonella reversion test by the method for in-vitro contamination.

Characteristics of the present invention are: collect cigarette smoke gas phase part by absorption bottle, adopt GVP to carry out the mode of in-vitro contamination in conjunction with TPM, the biological effect that can comprehensively reflect cigarette smoke really, on the basis of TPM contaminating mode in the past, increased the contamination of cigarette smoke gas phase part, made in-vitro contamination method more approach actual cigarette smoke and expose situation.Among the present invention grain and gas phase are collected respectively in addition, concentration of contamination is easily controlled, and collection method is operated easily.

Description of drawings

Fig. 1 is an idiographic flow synoptic diagram of the present invention.

Fig. 2 collects flue gas grain part and gas phase equipment and instrument connection diagram partly mutually for the present invention.

Among Fig. 2: 1 is Borgwaldt (Burger watt) rotating disk smoking machine; 2 for collecting the cambridge filter of TPM, and 3 is the GVP absorption bottle, and 4 is the vacuum jacketed flask ice bath, and 5 is pneumatic pump, and 6 is flue gas pipeline.

Embodiment

The present invention is described further below in conjunction with example:

Example 1

2R4F carries out in-vitro contamination to the reference cigarette.According to the method for the invention, connect absorption bottle and smoking machine, aspirate after 20 cigarette, with DMSO TPM is extracted into 10mg/mL, adding with the DMSO equal-volume toward absorption bottle does not then have calcium magnesium phosphate buffered saline buffer, and making concentration is the 10mg/mL of equivalent, with TPM and GVP1: 1 proportioning.Select Chinese hamster ovary cell (CHO) for use, carry out cell toxicity test with TPM, GVP, TPM+GVP respectively, after the contamination, the result is as follows:

Annotate: ^*" EC50 " is 50%effective eoncentration medium effective concentration

The result shows that GVP has biological effect really, and the biological action of TPM+GVP is better than TPM and the independent effect of GVP.

Example 2

A certain homemade fire-cured tobacco type brand cigarette is carried out in-vitro contamination.According to the method for the invention, according to described method, connect absorption bottle and smoking machine, aspirate after 20 cigarette, with DMSO TPM is extracted into 10mg/mL, adding with the DMSO equal-volume toward absorption bottle does not then have calcium magnesium phosphate buffered saline buffer, and making concentration is the 10mg/mL of equivalent, with TPM and GVP1: 1 proportioning.TPM+GVP concentration is formulated as: 25,50,100,125,250,500 μ g/mL.Positive control is during for no activation system: the TA98 positive control is 40 μ g/mL nitrofluorenes, and the TA100 positive control is 10 μ g/mL; When activation system was arranged: TA98, TA100 positive control were 20 μ g/mL anthranylamines.Negative control is a solvent control.

Then, draw the 0.1mL for preparing and tried thing and 0.1mL bacterium liquid mixing, under 37 ℃ of conditions, cultivate 30min in advance; Need activatory to add the 0.5mL5%S9 mixed solution in addition.Afterwards, get top layer substratum one pipe (2mL) that melts and in 45 ℃ of water-baths, be incubated, add 0.2mL and tried thing bacterium liquid mixed solution, activatory adds 0.7mL and is tried thing, bacterium liquid and S9 mixed solution, mixing is poured on the bottom platform substratum rapidly, and pivotal plate is evenly distributed on the bottom top layer substratum, square curing is cultivated 48h for 37 ℃; Count each dull and stereotyped sudden change colony number that returns.

The result shows that TPM+GVP mutagenesis ability is better than TPM.

Example 3

Select reference cigarette 2R4F for use, carry out in-vitro contamination.According to the method for the invention, connect absorption bottle and smoking machine, aspirate after 20 cigarette, with DMSO TPM is extracted into 10mg/mL, adding with the DMSO equal-volume toward absorption bottle does not then have calcium magnesium phosphate buffered saline buffer, and making concentration is the 10mg/mL of equivalent, with TPM and GVP1: 1 proportioning.

Carry out in-vitro micronucleus test then, cultivate CHO and reach 1 * 10 to concentration ⁵Individual cell/mL is inoculated in cell suspension the culturing bottle of 50mL by the 5mL/ bottle.Incubated overnight makes cell attachment.

With cell culture medium extraction liquid is diluted, TPM and TPM+GVP solution are formulated as various dose: 50 μ g/mL, 75 μ g/mL, 100 μ g/mL, 150 μ g/mL, 200 μ g/mL.After the cell attachment growth, discard original fluid, add and tried thing (TPM and TPM+GVP), two culturing bottles of each dose inoculation, and set up negative control and positive control.When needing activation, culturing bottle adds 4.625mL substratum, 0.275mLS9 mixed solution.Cultivate 27h.

After cultivating end,, carry out the slide smear, fix with stationary liquid (Glacial acetic acid: methyl alcohol is 1: 3) pair cell with cell dissociation.With the acridine carmine stain 5min that dyes, the flushing staining agent dries slide then.1000 cell count that contain micronucleus of each culturing bottle cell counting, the i.e. microkernel incidence of 2000 cells of each dose counting.The result is as follows:

The microkernel incidence of TPM+GVP is higher than TPM, and TPM+GVP is higher than TPM to chromosomal damage ability.

Claims (1)

1, a kind of improved cigarette smoke in-vitro contamination method, it is characterized in that: adopt cigarette smoke gas phase part (GVP) to carry out the in-vitro contamination test after part (TPM) mixing mutually with the cigarette smoke grain, come the biological effect of evaluating cigarette flue gas, concrete grammar is as follows:

A, flue gas grain part mutually partly collect with gas phase: the mode of contacting mutually by smoking machine and cambridge filter and gas absorption bottle is collected the flue gas grain respectively mutually partly and flue gas gas phase part, add phosphate buffered saline buffer in the absorption bottle, and absorption bottle placed in the vacuum jacketed flask that contains ice cube, guarantee that damping fluid is lower than 4 ℃ in whole collection process;

B, grain are partly partially mixed with gas phase mutually: the TPM that Cambridge sheet is collected adopts DMSO to extract, and extraction concentration is 10mg/ml, adds in absorption bottle and the identical phosphate buffered saline buffer of the extraction used DMSO volume of TPM, then TPM and GVP is pressed 1: 1 proportioning;

C, in-vitro contamination: the TPM+GVP of 1: 1 proportioning is diluted to the different concns gradient, carries out cell toxicity test, micronucleus test and Salmonella reversion test by the method for in-vitro contamination.

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