A method for evaluating the impact of pollutants on serum metal elements

Abstract

The present invention belongs to the field of pollutant toxicity evaluation and relates to a method for evaluating the impact of pollutants on serum metal elements. 2.5 After exposure, serum samples were obtained; the metal content in serum was analyzed by ICP-MS after vortex extraction; the PM was evaluated by calculating the metal disorder index 2.5 The overall impact of exposure on metal elements in serum. This paper uses the total change of metal elements as an evaluation index and uses the MeELI index to study PM 2.5 The disturbance of exposure to serum metal elements has the advantages of being intuitive and comprehensive; the comprehensive evaluation index is more sensitive than other toxicity evaluation indicators, and can more truly reflect the actual environmental conditions when used for exposure to small doses of pollutants or sub-chronic toxic pollutants; the required sample size is small, and the data acquisition method based on mass spectrometry has the advantages of being simple, fast, highly sensitive, specific, and repeatable.

Description

Technical Field

[0001] The invention belongs to the field of pollutant toxicity evaluation and relates to a method for evaluating the impact of pollutants on serum metal elements. Background Art

[0002] Atmospheric fine particulate matter (PM 2.5 ) refers to particulate matter with an aerodynamic particle size Dp ≤ 2.5 μm. PM 2.5 The particle size is small, the residence time in the atmosphere is long, the transportation distance is long, and it has an important impact on air quality and visibility. 2.5 It is easy to accumulate heavy metals, polycyclic aromatic hydrocarbons and other toxic and harmful substances, which can enter the body through the blood and cause damage to the respiratory system, circulatory system and immune system, bringing potential harm to human health and the atmospheric environment.

[0003] In vitro and in vivo experiments have preliminarily revealed that PM 2.5 The toxicity mode and mechanism of PM 2.5 After exposure to the lungs, toxic components can enter lung cells by dissolving and then adsorbing on the cell membrane, migrating across the membrane or by cell autophagy, and cause a series of complex biological responses through blood circulation. For example, Ni in particulate matter can activate the transcription factor NF-kB in lung epithelial cells, further inducing oxidative stress response; Cu can induce cells to secrete IL-6 and IL-8, thereby producing an inflammatory response; in addition, heavy metals will be transported along with PM 2.5 Exposure to PM2.5 is deposited in the brain of rats and regulates inflammatory factors through the Nrf2 / NLRP3 signaling pathway, leading to depression. 2.5 There are relatively few studies on the internal exposure level of metal elements after exposure. 2.5 The effect of exposure on the elemental composition of model animals is still unclear. 2.5 After exposure to combined pollution, the changing trend of each element is different. Currently, there is no comprehensive and intuitive indicator to evaluate the overall trend of element changes. The changing trend of a single metal element cannot explain the PM 2.5 The overall impact of exposure.

[0004] Based on the above analysis, the present invention intends to carry out PM 2.5 The effects of exposure on the heavy metal content in the serum of rats were studied, and a method for evaluating the changes in metal elements in model animals after external stimulation or pollution exposure was established based on the detection of serum metal elements. This method calculated the PM 2.5 The MeELI values ​​of metal elements after exposure and the comparative analysis of MeELI values ​​of different dose groups comprehensively and intuitively show the overall interference of pollutant exposure on metal elements in the body. Summary of the invention

[0005] The purpose of the present invention is to provide a method for evaluating PM 2.5 A new method for the interference of exposure with metal elements in model animals. First, the metal elements in serum were vortex-extracted using an extraction solvent containing TritonX-100 and nitric acid. Then, the metal elements in serum were rapidly analyzed based on an inductively coupled plasma mass spectrometer (ICP-MS). The metal element disorder index (MeELI) was calculated using the ratio of the concentration of the metal element in the exposed group samples to the concentration of the element in the control group, and the PM 2.5 A comprehensive evaluation index for changes in serum metal elements after exposure. This method has the advantages of simple pre-treatment, fast detection speed, good repeatability, and comprehensive information. It can comprehensively and intuitively reflect the impact of pollutant exposure on metal elements in the test animals.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] Using SPF SD rats as model organisms, PM was administered by intratracheal instillation. 2.5 After exposure, serum samples were obtained; the metal content in serum was analyzed by ICP-MS after vortex extraction; PM was evaluated by calculating the metal disorder index 2.5 Overall effects of exposure on serum metals.

[0008] The specific steps include:

[0009] (a) SPF SD rats were subjected to PM by non-exposed intratracheal instillation. 2.5 Exposure: drip once every 4 days for 4 consecutive weeks as the exposure group, and drip of sterile saline as the control group. After the end of the exposure, blood was collected from the abdominal aorta and centrifuged to obtain serum samples;

[0010] (b) Serum samples were added to the extraction solvent, vortexed, and centrifuged, and the supernatant was detected by ICP-MS;

[0011] (c) Calculate the metal disorder index for PM using the concentration data of metal elements in serum obtained in step b. 2.5 The effects of exposure to metallic elements in serum were evaluated.

[0012] In step a, the temperature of the rat breeding room is 20-25° C., the relative humidity is 40-70%, and the light and dark light cycles are 12 hours each; the animals are fed with standard pellet feed and have free access to water and food; the centrifugal speed for obtaining serum samples is 3000-3500 g / min, and the centrifugal time is 5 minutes.

[0013] In step b, the extraction solvent is an aqueous solution containing 1% HNO3 and 0.01% Tirton-100 (volume percentage); the amount of serum is 200-400 μL, the amount of extraction solvent is 3.8-7.6 mL, the vortex extraction time is 5-10 min; the centrifugal speed is 4000-5000 g / min, and the centrifugal time is 5-10 min.

[0014] In step c, the metal elements detected in the blood include V, Mn, Co, Ni, Cu, Zn, As, Se, Rb, Sr, Ag, Cs, Ba, and Hg.

[0015] The calculation of MeELI in step c is carried out using the following formula:

[0016]

[0017] In the formula: A i It is the ratio of the concentration of a single element (i) in a serum sample to the average concentration of the element in the control group; ln(1) is to eliminate the background interference brought by the control group;

[0018] The concentration changes of all elements in a single sample are summed and then divided by the total number of elements in the sample (n) to obtain the MeELI value of the sample. The process is shown in formula (1-2);

[0019]

[0020] In the formula: n refers to the total number of elements in the sample;

[0021] Elements that are not detected in 80% of the samples are not included in the calculation, and individual undetected elements in a sample are not included in the calculation of that sample;

[0022] According to formula (1-2), calculate the PM at different doses 2.5 The MeELI value of the cumulative change of metal elements in rat serum caused by exposure. This disorder calculation method can be used not only for blood samples, but also for the calculation of the disorder of metal elements in other organs of model animals after pollutant exposure, such as liver, kidney, brain, spleen, lung, etc.

[0023] The MeELI value of the overall change of metal elements in rat serum obtained in step c, the box plot of the MeELI value can show the trend of changes in metal elements in the body caused by external stimulation or pollution exposure, and the T test of the MeELI value of the exposure group and the MeELI value of the control group is used to evaluate the effects of different toxic doses on metal elements in serum.

[0024] The beneficial effects of the present invention are as follows:

[0025] (1) The present invention uses the total change of metal elements as the evaluation index and adopts the MeELI index to study PM 2.5 Exposure to disturbances of serum metal elements has the advantage of being intuitive and comprehensive;

[0026] (2) Comprehensive evaluation indicators are more sensitive than other toxicity evaluation indicators and can more truly reflect the actual environmental conditions when used for exposure to small doses of pollutants or sub-chronic toxic pollutants;

[0027] (3) The present invention requires a small amount of sample, and the data acquisition method based on mass spectrometry has the advantages of being simple, rapid, highly sensitive, specific, and reproducible. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The present invention is based on MeELI calculation to evaluate PM 2.5 Exposure to disturbances in serum metal elements. DETAILED DESCRIPTION

[0029] The following examples may enable those skilled in the art to more fully understand the present invention, but are not intended to limit the present invention in any way.

[0030] Example 1

[0031] PM 2.5 The method for evaluating the effects of exposure on metal elements in rat serum includes the following steps:

[0032] Step (a): PM 2.5 The sampling membrane was ultrasonically extracted 6 times with ultrapure water as the medium. The extract was freeze-dried and then prepared into a high-concentration stock solution with sterile saline. The poisoning method adopted the non-exposed tracheal instillation method. The rats were anesthetized with ether and the tracheal tube was inserted into the trachea under direct vision through the oral cavity. PM 2.5 The suspension was injected into the airway, and the endotracheal tube was removed. The rat was removed after the suspension state was maintained for 10 seconds and placed in a heat-insulating cage in a lateral position. The rat was instilled through the trachea once every 4 days at a drip volume of 1 mL / kg body weight for 28 consecutive days as the exposure group. The control group was instilled with sterile saline 1 mL / kg body weight through the trachea. The animals were killed 24 hours after the end of the exposure, and blood was collected through the abdominal aorta. The blood was centrifuged at a speed of 3000 g / min for 5 minutes, and the upper layer of liquid was taken to obtain serum samples.

[0033] The low, medium and high doses of the exposure group were 0.01, 0.1 and 5 mg / kg bw / d, respectively;

[0034] Step (b): 200 μL of serum was added with 3.8 mL of extraction solvent, vortexed and centrifuged. The extraction solvent was an aqueous solution of 1% HNO3 and 0.01% Tirton-100 (volume percentage). The vortex extraction time was 5 min, the centrifugal speed was 4500 g / min, and the centrifugal time was 5 min. The supernatant was taken and the serum metal elements were detected by Agilent 8900 ICP-MS / MS in helium mode. The metal elements detected in the serum included V, Mn, Co, Ni, Cu, Zn, As, Se, Rb, Sr, Ag, Cs, Ba, and Hg.

[0035] The standard curve for Hg determination is 0.1, 0.2, 0.5, 1, 2 μg / L, and the linear range of the standard curve for other elements determination is 0.1, 0.5, 1, 5, 10, 20 μg / L;

[0036] Step (c): Calculation of the overall disorder of metal elements in serum By comparing the element content of the exposure group with that of the control group, the overall change of metal elements in each serum sample was calculated and summarized into an index MeELI.

[0037] MeELI is calculated using the following formula:

[0038]

[0039] In the formula, A i It refers to the ratio of the concentration of a single element (i) in a serum sample to the average concentration of the element in the control group. ln(1) is used to eliminate the background interference caused by the control group.

[0040] Next, the concentration changes of all elements in a single sample are summed and divided by the total number of elements in the sample (n) to obtain the metal disorder index of the sample. The process is shown in the following formula:

[0041]

[0042] Figure 1 The present invention is based on MeELI calculation to evaluate PM 2.5 The disturbance of exposure to serum metal elements can be seen from the box plot of MeELI values. 2.5 The trend of changes in metal elements in the body caused by exposure can be seen through T-test of MeELI values ​​of the three dose groups and the control group, which shows the significance of changes in metal elements caused by different exposure groups.

[0043] The present invention integrates the changing trends of individual metal elements in serum after exposure to pollutants and establishes an overall evaluation index for the changing trends of serum metal elements. The index can be used for the overall evaluation of metal element disorders in organisms induced by exposure to low-dose pollutants or sub-chronic toxic pollutants. The index is more sensitive than other toxicity evaluation indicators and has the advantage of obtaining intuitive and comprehensive information.

[0044] The above embodiments are only preferred embodiments of the present invention and are not intended to limit the implementation methods. The protection scope of the present invention shall be subject to the scope defined in the claims. Other different forms of changes or modifications may be made based on the above description. Obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. A method for evaluating the impact of pollutants on metal elements in serum, characterized in that: Using SPF SD rats as model organisms, PM was administered by intratracheal instillation. 2.5 After exposure, serum samples were obtained; the metal content in serum was analyzed by ICP-MS after vortex extraction; PM was evaluated by calculating the metal disorder index 2.5 the overall effect of exposure on serum metals; The metal disorder index MeELI is calculated using the following formula: In the formula: A i It is the ratio of the concentration of a single element (i) in a serum sample to the mean concentration of that element in the control group; ln(1) is to eliminate the background interference brought by the control group; In the formula: n refers to the total number of elements in the sample; Elements that are not detected in 80% of the samples are not included in the calculation, and individual undetected elements in a sample are not included in the calculation of that sample; According to formula (1-2), calculate the PM at different doses 2.5 The MeELI value of the cumulative change of metal elements in rat serum caused by exposure. The box plot of the MeELI value shows the trend of changes in metal elements in the body caused by external stimuli or pollution exposure. The T test of the MeELI value of the exposure group and the MeELI value of the control group was used to evaluate the impact of different toxic doses on metal elements in serum.

2. The evaluation method according to claim 1, characterized in that: The method specifically comprises the following steps: (a) SPF SD rats were subjected to PM by non-exposed intratracheal instillation. 2.5 Exposure: The exposure group was instilled once every 4 days for 4 consecutive weeks, and the control group was instilled with sterile saline. After the end of the exposure, blood was collected from the abdominal aorta and centrifuged to obtain serum samples. (b) Serum samples were added to the extraction solvent and vortexed for extraction, followed by centrifugation, and the supernatant was used for ICP-MS detection; (c) Calculate the metal disorder index for PM using the metal element content data in serum obtained in step b. 2.5 The effects of exposure to metallic elements in serum were evaluated.

3. The evaluation method according to claim 1, characterized in that: In step a, the temperature of the rat breeding room is 20-25° C., the relative humidity is 40-70%, and the light and dark light cycles are 12 hours each; the animals are fed with standard pellet feed and have free access to water and food; the centrifugal speed for obtaining serum samples is 3000-3500 g / min, and the centrifugal time is 5 minutes.

4. The evaluation method according to claim 1, characterized in that: In step b, the extraction solvent is an aqueous solution containing 1 vol% HNO3 and 0.01 vol% Tirton-100; the amount of serum is 200-400 μL, the amount of extraction solvent is 3.8-7.6 mL, the vortex extraction time is 5-10 min; the centrifugal speed is 4000-5000 g / min, and the centrifugal time is 5-10 min.

5. The evaluation method according to claim 1, characterized in that: In step c, the metal elements detected in the blood include V, Mn, Co, Ni, Cu, Zn, As, Se, Rb, Sr, Ag, Cs, Ba, and Hg.

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