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A method for quantitative detection of cellular DNA damage induced by 1,3-butadiene using an air-liquid interface exposure system combined with high-content technology

A quantitative detection, liquid interface technology, applied in the field of in vitro genotoxicity assay, can solve the problems of limited solubility, changing the detection purpose, and it is difficult for cells to achieve sufficient contact.

Active Publication Date: 2019-08-27
CHINA NAT TOBACCO QUALITY SUPERVISION & TEST CENT +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

This is because some gases have limited solubility in liquid (usually water), which limits the upper limit of exposure concentration; second, some gaseous substances can react with water, which indirectly changes the purpose of detection; third, gas- Although liquid mixing exposure can make the gas dissolved in the liquid matrix fully contact with the cells for suspension cells, it is difficult to achieve sufficient contact for adherent cultured cells

Method used

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  • A method for quantitative detection of cellular DNA damage induced by 1,3-butadiene using an air-liquid interface exposure system combined with high-content technology
  • A method for quantitative detection of cellular DNA damage induced by 1,3-butadiene using an air-liquid interface exposure system combined with high-content technology
  • A method for quantitative detection of cellular DNA damage induced by 1,3-butadiene using an air-liquid interface exposure system combined with high-content technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] The γH2AX induced by 1,3-butadiene after 1 h exposure was measured when the metabolic activation system rat liver S9 was not added to the cell poisoning liquid.

[0079] Add 0.5 and 2 mL of PBS to the top and bottom of the Transwell insert, respectively, and equilibrate at 37°C for 1-2 hours. Aspirate and discard the PBS on the top and bottom of the Transwell insert, add 0.5mL to the top of the insert with a concentration of 4×10 5 The single-cell suspension of A549 cells in the logarithmic growth phase of each cell / mL was added to the bottom of the insert with 2 mL of RPMI-1640 culture medium containing 0.01moL / L HEPES, 2mmoL / LL-glutamine and 10% FBS, and incubated at 37 °C, 5% CO 2 Conditioned for 24h.

[0080] Remove the culture medium on the top of the Transwell insert, take out the top of the Transwell insert and put it into the exposure compartment of the cell exposure system, so that the permeable filter membrane in the Transwell insert and the cell exposure so...

Embodiment 2

[0086] The γH2AX induced by 1,3-butadiene after 1 h exposure was measured when the metabolic activation system rat liver S9 was added to the cell poisoning solution.

[0087] The experimental process was carried out as described in Example 1, the only difference being that the cell poisoning solution in the poisoning chamber was a mixture of cell culture medium and 10% S9 mixture, so that the cell poisoning solution contained 1% S9 mixture.

[0088] Figure 4 Shown is the dose-effect relationship curve of γH2AX induced by different concentrations of 1,3-butadiene in A549 cells after adding 1% S9 in the exposure medium, and image 3 The results shown are essentially the same.

[0089] The addition of S9 to the cell poisoning solution can enhance the metabolic transformation of 1,3-butadiene in vitro. Since 1,3-butadiene is a former genotoxic substance, the addition of the in vitro metabolic activation system S9 can avoid the possibility of false negative test results due to i...

Embodiment 3

[0091] When the metabolic activation system rat liver S9 was not added to the cell poisoning liquid, the γH2AX induced by 35.18mmoL / L 1,3-butadiene were measured after 15, 30, 45, 60 and 90min exposure respectively.

[0092] The experimental process was carried out as described in Example 1. The only difference was that the exposure concentration of 1,3-butadiene was fixed at a concentration of 35.18mmoL / L, and the exposure time was 0, 15, 30, 45, 60 and 90 minutes respectively. .

[0093] Figure 5 Shown is the time-effect relationship curve of γH2AX produced by A549 cells induced by 35.18mmoL / L 1,3-butadiene at 0, 15, 30, 45, 60 and 90min after exposure. It can be seen from the figure that as the exposure time increases, the γH2AX produced by A549 cells gradually increases, showing a significant time-effect relationship. When the exposure time was more than 60min, the induced γH2AX was 1.5 times higher than that of the normal group.

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Abstract

The invention discloses a method for quantitatively detecting 1,3-butadiene-induced cell DNA damage in combination with a gas-liquid interface exposure system and a high-content technology. The method comprises the following steps: 1) cell culture; 2) 1,3-butadiene exposure through a gas-liquid interface exposure mode; 3) immunofluorescent labeling of gamma H2AX; 4) high-content detection. The method disclosed by the invention has the advantages that the efficient exposure of gaseous 1,3-butadiene to wall-attached cells in vivo is realized by adopting the in-vitro gas-liquid interface exposure mode, so that the exposure efficiency of the 1,3-butadiene is improved; a high-content imaging system is utilized for automatic imaging, 1,3-butadiene-induced DNA double-chain fracture maker gamma H2AX proteins are quantitatively analyzed, and the direct and quick detection of the cells is realized, so that the sample treatment is more convenient; through high-resolution imaging, not only can the distribution of the gamma H2AX in cell nucleuses be directly observed, but also image data is convenient to store and re-analyze, and the quantitative analysis on the 1,3-butadiene-induced gamma H2AX in each of the cell nucleuses can be realized, so that detection results are more sensitive and accurate.

Description

technical field [0001] The invention belongs to the technical field of genotoxicity assay in vitro, and more specifically, the invention relates to a genotoxicity assay method of 1,3-butadiene. Background technique [0002] 1,3-Butadiene is a common atmospheric and environmental pollutant that can induce multi-organ tumors through the inhalation route. In addition, 1,3-butadiene epoxy metabolites can form DNA adducts, induce Hprt (hypoxanthine phosphoribosyltransferase) gene mutations, etc. At present, the International Organization for Research on Cancer (IARC) under the World Health Organization (WHO) has listed 1,3-butadiene as a class 1 carcinogen, and the World Health Organization Framework Convention on Tobacco Control (FCTC) has listed 1,3-butadiene Alkenes are listed as one of the priority control pollutants in flue gas. Therefore, accurate detection of 1,3-butadiene gas damage to cellular DNA is of great significance for the evaluation of the genotoxicity and haza...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/58G01N33/50
Inventor 胡清源张森侯宏卫陈欢王安刘勇
Owner CHINA NAT TOBACCO QUALITY SUPERVISION & TEST CENT
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