Test method for tk gene mutation and determination method for mutagenicity suitable for evaluating genotoxicity of cigarette smoke condensate

A technology for determining the strength of cigarette smoke, which is applied in the measurement/inspection of microorganisms, biochemical equipment and methods, etc., can solve the problems of narrow dose range, easy aging of smoke, and inability to use it directly, reaching the scope of application wide effect

Active Publication Date: 2021-10-01
ZHENGZHOU TOBACCO RES INST OF CNTC
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  • Application Information

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Problems solved by technology

[0004] Although there are domestic MLA methods related to food, food additives, and food contact materials, cigarette smoke is quite different from food, food additives, and food contact materials, and cannot be used directly. For example, smoke is easy to age, The narrow range of doses exhibiting toxicity makes it particularly urgent to establish an MLA applicable to cigarette smoke

Method used

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  • Test method for tk gene mutation and determination method for mutagenicity suitable for evaluating genotoxicity of cigarette smoke condensate
  • Test method for tk gene mutation and determination method for mutagenicity suitable for evaluating genotoxicity of cigarette smoke condensate
  • Test method for tk gene mutation and determination method for mutagenicity suitable for evaluating genotoxicity of cigarette smoke condensate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Present embodiment is to certain cigarette 1 # The TK gene mutagenesis test of L5178Y cells was carried out.

[0031]The in vitro genotoxicity test method suitable for evaluating the mutagenic effect of cigarette smoke condensate (CSC), specifically includes the following steps:

[0032] 1. Cell culture conditions

[0033] The L5178Y cells used are suspension growth cells, using RPMI1640 medium containing 10% horse serum and appropriate amount of antibiotics (the medium containing 20% ​​horse serum is used for 96-well plate culture), 5% CO 2 , 37°C, and conventional suspension culture under saturated humidity conditions.

[0034] Clear spontaneously mutated tk before formal experiments - / - Genotype cells. The method is to use THMG medium containing 3g / mL thymidine, 5g / mL hypoxanthine, 0.1g / mL methotrexate and 7.5g / mL glycine to treat the cells for 24 hours, centrifuge and wash the cells without methotrexate cultured in THG medium for 3 days.

[0035] 2. Preparatio...

Embodiment 2

[0069] The in vitro genotoxicity test method and method for determining the mutagenic strength applicable to the evaluation of the mutagenic effect of cigarette smoke condensate in this example are the same as those in Example 1. The test cigarette used is another cigarette 2 # .

[0070] The results are shown in Table 2: the TMF of 80 μg / mL in the CSC treatment group was 3 times higher than that of the solvent control group, and with the increase of the dose of CSC, the TMF showed an upward trend, showing a dose-response relationship, indicating that the cigarette smoke had a positive effect on the TK of L5158Y Genes are mutagenic.

[0071] Table 2 Cigarette 2 # The results of TK mutagenic frequency determination

[0072]

[0073] When inoculating a 96-well plate, add 0.2 mL of the test substance to each well, and conduct a quadratic analysis on the dose-response curve (the quadratic equation of one variable is ) linear fitting, quadratic coefficient a 1 Used to eval...

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Abstract

The invention relates to a TK gene mutation test method and a mutagenic strength determination method suitable for evaluating the genotoxicity of cigarette smoke condensate, and belongs to the technical field of cigarette hazard evaluation. The genotoxicity test method and the method for determining the mutagenicity of the present invention use the cigarette smoke condensate to directly treat the test cells, and can qualitatively and quantitatively judge the mutagenic effect of the cigarette smoke condensate, which can be judged simply and quickly The toxic effect of cigarettes overcomes the problems of few genotoxic endpoints, easy aging of smoke, and narrow dose range for toxicity in the genotoxicity detection process of cigarette smoke in the prior art, and can be applied to cigarettes on the market, and can The quantitative judgment of the genotoxicity of cigarette smoke can realize the horizontal comparison between the mutagenic effects of TK genes of different cigarettes, and has a wide range of applications.

Description

technical field [0001] The invention relates to a TK gene mutation test method and a mutagenic strength determination method suitable for evaluating the genotoxicity of cigarette smoke condensate, and belongs to the technical field of cigarette hazard evaluation. Background technique [0002] Cigarette smoke contains nearly 5,315 compounds, 158 of which have been identified to be toxic to humans or experimental animals. How to evaluate the hazards of cigarette smoke has always been the direction of research by relevant researchers at home and abroad. In 2004, the Tobacco Smoke In vitro Toxicity Testing Working Group of the International Tobacco Scientific Research Cooperation Center (Cooperation center for scientific research relation to tobacco, CORESTA) recommended three in vitro toxicity tests to evaluate the harm of cigarette smoke, namely cytotoxicity test, Salmonella typhimurium reverse mutation test (Ames test) and micronucleus test in vitro. [0003] With the in-dep...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6827
CPCC12Q1/6827
Inventor 尚平平华辰凤李翔乔梁峻谢复炜
Owner ZHENGZHOU TOBACCO RES INST OF CNTC
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