阅读说明：本技术 一种大鼠烟气吸入暴露后尿液的致突变检测方法 (Mutation-causing detection method for urine after rat smoke inhalation exposure ) 是由 蔡继宝 苏加坤 郭磊 徐达 罗娟敏 尚平平 李翔 谢复炜 于 2019-11-28 设计创作，主要内容包括：本发明公开了一种大鼠烟气吸入暴露后尿液的致突变检测方法,包括尿液酶解、过柱和萃取、洗脱、浓缩、Ames试验微量波动法和结果判定共六个步骤,其中尿液酶解采用乙酸钠-乙酸缓冲液调节控制pH环境,过柱时依次用甲醇和水预处理,并在去离子水冲洗后用甲醇洗脱,经浓缩后采用Ames试验微量波动法检测并判定结果,该方法可以显著降低传统Ames试验所需的尿液检测量,并提高检测灵敏度,尤其适用于大鼠烟气暴露模型的生物学检测。(The invention discloses a mutation-causing detection method of urine after rat smoke gas is inhaled and exposed, which comprises six steps of urine enzymolysis, column passing, extraction, elution, concentration, an Ames test micro-fluctuation method and result judgment, wherein the urine enzymolysis adopts sodium acetate-acetic acid buffer solution to adjust and control the pH environment, methanol and water are used for pretreatment in sequence when the urine passes through the column, the urine is washed by deionized water and then eluted by the methanol, and the result is detected and judged by adopting the Ames test micro-fluctuation method after the urine is concentrated.)

1. A method for detecting mutation caused by urine after rat smoke inhalation exposure is characterized by comprising the following steps:

s1, urine enzymolysis

Unfreezing a pre-collected urine sample to room temperature, putting 5mL into a glass bottle, sequentially adding 8 ~ 15mL of sodium acetate-acetic acid buffer solution and 50 muL of beta-glucuronidase, uniformly mixing, putting into a constant-temperature water bath, and performing enzymolysis for 14 ~ 20h at 37 ℃ in a dark place;

s2, column passing and extraction

Weighing 1.05 +/-0.05 g of XAD-2 adsorbent, placing the adsorbent into a chromatographic column, firstly soaking and extracting with 5 ~ 10mL of methanol, adding 5 ~ 10mL of water for pretreatment when the flow is nearly finished, and adding an enzymolysis urine sample when the flow is nearly finished;

s3, elution

Adding deionized water 5 ~ 8 mL to wash the column, washing histidine in urine, discarding the washing solution, adding 10 ~ 15mL methanol to the column, and eluting the urine on XAD-2 adsorbent;

s4, concentrating

Placing the eluent into a nitrogen blowing instrument until the eluent is dried, and adding 100 mu L of dimethyl sulfoxide (DMSO) to extract residues;

s5.Ames test micro-fluctuation method

After the salmonella typhimurium TA98 is cultured overnight, the bacterial liquid is diluted to 1 ~ 2 multiplied by 10⁸Mixing 0.1 mL of bacterial solution, 8.85 mL of selection indication growth medium, 0.1 mL of urine DMSO extract, 0.25 mL of 3.2mg% histidine, 0.25 mL of 0.8mg% biotin and 1.15mL of 10% S9 uniformly, adding a 96-well cell culture plate into each well, culturing at 37 ℃ for 48h, wherein the solvent control is DMSO, and the positive control is cyclophosphamide;

s6, judging results

After culturing for 48h, the culture solution turns to yellow holes as positive holes, blue-violet holes as negative holes, the judgment between yellow and blue-violet is negative, the number of the positive holes of each plate is counted, and the difference significance comparison is carried out on the results by chi-square test.

2. The method for detecting mutagenicity in urine after rat smoke inhalation exposure according to claim 1, wherein in said step S1, said sodium acetate-acetic acid buffer has a pH of 5 ± 0.1.

3. The method for detecting mutations in urine after rat smoke inhalation exposure according to claim 1, wherein in step S1, the enzymolysis time is 16 h.

4. The method for detecting mutation in urine after rat smoke inhalation exposure according to claim 1, wherein in step S5, the selection indicator medium is Davis-Mingoli buffer solution containing 5 μ L/mL bromocresol purple.

Technical Field

The invention relates to the technical field of biology, in particular to a mutation-causing detection method for urine after rat smoke is inhaled and exposed.

Background

Cigarette smoke is a complex aerosol containing 6000 or more compounds, of which 69 are identified by IARC as human carcinogens. Polycyclic aromatic hydrocarbons, various alkylating agents, benzene series substances, aromatic amine and other mutagenic substances in cigarette smoke are metabolized in vivo after entering a human body or an experimental animal body, and are mostly discharged from a kidney in the forms of prototypes, metabolites or conjugates and the like, so that the detection of the mutagenic effect of urine by adopting a short-term mutagenic test has important significance.

The human smoke exposure has great difference, so Sprague Dawley rats are used as test objects to detect the mutagenicity of the urine of the rats after different periods of smoke exposure and can directly reflect the mutagenicity effect of smoke inhalation exposure, but in the prior art, the urine pretreatment method of the urine mutagenicity test has the urine requirement of more than 50 ~ mL, so the prior art is only suitable for the mutagenicity detection of the urine of human bodies, and the urine quantity of the rats is very small, and the prior art cannot effectively evaluate the mutagenicity of the rats.

Ames et al used Salmonella typhimurium to detect mutagenicity of urine in the presence of microsomal enzyme system by the conventional plate incorporation method, which is limited to 0.1 mL of test substance per plate and also interferes with experimental results with histidine in urine, so that the conventional method can only detect mutagenic substances at high concentration and is not suitable for urine with low concentration of mutagenic substances.

Disclosure of Invention

The invention aims to establish a mutation-causing detection method for urine after rat smoke is inhaled and exposed, which treats the urine by adopting methods such as enzymolysis, column passing, extraction, concentration and the like, so that the urine detection amount is reduced by at least 10 times, and an Ames test micro-fluctuation method after the urine enzymolysis and concentration is researched and established, so that the detection sensitivity is improved, and the method can be used as a biological detection index for smoke exposure.

In order to achieve the above purpose, the invention provides the following technical scheme:

a method for detecting mutation caused by urine after rat smoke inhalation exposure comprises the following steps:

s1, urine enzymolysis

Unfreezing a pre-collected urine sample to room temperature, putting 5mL into a glass bottle, sequentially adding 8 ~ 15mL of sodium acetate-acetic acid buffer solution and 50 muL of beta-glucuronidase, uniformly mixing, putting into a constant-temperature water bath, and performing enzymolysis for 14 ~ 20h at 37 ℃ in a dark place;

s2, column passing and extraction

Weighing 1.05 +/-0.05 g of XAD-2 adsorbent, placing the adsorbent into a chromatographic column, firstly soaking and extracting with 5 ~ 10mL of methanol, adding 5 ~ 10mL of water for pretreatment when the flow is nearly finished, and adding an enzymolysis urine sample when the flow is nearly finished;

s3, elution

Adding deionized water 5 ~ 8 mL to wash the column, washing histidine in urine, discarding the washing solution, adding 10 ~ 15mL methanol to the column, and eluting the urine on XAD-2 adsorbent;

s4, concentrating

Placing the eluent into a nitrogen blowing instrument until the eluent is dried, and adding 100 mu L of dimethyl sulfoxide (DMSO) to extract residues;

s5.Ames test micro-fluctuation method

After the salmonella typhimurium TA98 is cultured overnight, the bacterial liquid is diluted to 1 ~ 2 multiplied by 10⁸one/mL. Uniformly mixing 0.1 mL of bacterial liquid, 8.85 mL of selection indication growth medium, 0.1 mL of urine DMSO extract, 0.25 mL of 3.2mg% histidine, 0.25 mL of 0.8mg% biotin and 1.15mL of 10% S9, adding a 96-well cell culture plate and a 48-well cell culture plate into each 0.2 mL/well, culturing at 37 ℃ for 48h, wherein the solvent control is DMSO, and the positive control is cyclophosphamide;

s6, judging results

After culturing for 48h, the culture solution turns to yellow holes as positive holes, blue-violet holes as negative holes, the judgment between yellow and blue-violet is negative, the number of the positive holes of each plate is counted, and the difference significance comparison is carried out on the results by chi-square test.

Preferably, in the S1 step, the pH of the sodium acetate-acetic acid buffer is 5 ± 0.1.

Preferably, in the step S1, the enzymolysis time is 16 h.

Preferably, in the step S5, the selection indicates that the growth medium is 5. mu.L/mL of Davis-Mingoli buffer containing bromocresol purple.

The chi-square test direction of the invention is as follows: the chi-square test has the formula

In the formula, n is the number of reaction plate holes, t is the number of test plate positive reaction holes, c is the number of negative control positive reaction holes, and if p is detected by chi-square test of a 2X 2 series table on the test plate and the control plate<0.05, it means that the test sample is positive for mutagenesis. Positive events among different exposure groups were checked by Chi-square of the RxC list, p<0.05, it indicates a significant difference between groups.

Compared with the prior art, the invention has the beneficial effects that:

the detection method comprises six steps of urine enzymolysis, column passing, extraction, elution, concentration, an Ames test micro-fluctuation method and result judgment, wherein the urine enzymolysis adopts sodium acetate-acetic acid buffer solution to adjust and control the pH environment, methanol and water are used for pretreatment in sequence when the urine passes through the column, methanol is used for elution after deionized water is used for washing, the result is detected and judged by adopting the Ames test micro-fluctuation method after concentration, the method can obviously reduce the urine detection amount required by the traditional Ames test by at least 10 times, improves the detection sensitivity, and is particularly suitable for biological detection of a rat smoke exposure model.

Detailed Description

The present invention will be described in further detail with reference to specific examples. It should be understood that the scope of the above-described subject matter is not limited to the following examples, and any techniques implemented based on the disclosure of the present invention are within the scope of the present invention.