Analysis method for detecting potential lung toxicity of chemicals based on high content screening

Abstract

The invention belongs to the technical field of environmental toxicology and ecological toxicology, and particularly relates to an analysis method for screening potential lung toxicity of chemicals based on high content screening. Comprising the following steps: (1) cell culture: culturing A549 cells in a 5% CO2 incubator at 37 DEG C; (2) exposure of chemicals with lung toxicity: exposure is carried out at the concentration of 0, 0.1, 0.3, 1, 3, 10, 30 and 100 mu M; (3) dyeing the A549 cells by using a fluorescent dye; (4) acquiring a cell image by using a high content cell analyzer; (5) constructing a cell phenotype analysis method by using Harmony5.1 software; and (6) analyzing phenotypic data to determine the strength of the pneumotoxicity of the chemicals. The method for rapidly screening and detecting the potential lung toxicity of the chemicals is established, the cost benefit is high, the sensitivity is high, the operation is simple and convenient, and a plurality of phenotypic parameters can be detected at the same time; meanwhile, no biological ethics exist, and the future toxicity test development direction is met.

Description

Technical Field

[0001] The present invention belongs to the technical field of environmental toxicology and ecotoxicology, and specifically relates to an analytical method for screening the potential lung toxicity of chemicals based on high content screening. Background Art

[0002] In addition to deaths caused by air pollution caused by chemical pollutants, many people also suffer from respiratory diseases. The organ most closely related to gas exchange function is the lung, which provides oxygen to various tissues and removes carbon dioxide. Gas exchange takes place in the alveoli of the lungs. The alveoli have a large surface area and exchange a large amount of gas, so the lungs are the main interface between environmental organisms and air toxics. In addition to direct contact with chemicals dispersed through the atmosphere, the blood of the entire body flows through the lungs one to five times per minute, which also directly exposes the lungs to chemicals in the circulatory system. Therefore, the probability of direct inhalation and chemicals from the circulatory system contacting the lungs is very high. Therefore, it is necessary to conduct screening studies on the potential pulmonary toxicity of chemicals.

[0003] Traditional chemical toxicity evaluation is generally carried out through live animal experiments. However, it cannot provide accurate and relevant information for hazard identification and dose-response evaluation; it cannot provide data on a wide range of chemicals, effect endpoints and life stages; it cannot minimize the cost and time of testing and regulatory review. In addition, live animal experiments do not comply with the "3R" principle of "reduction, substitution, optimization". With the increasing number of chemicals worldwide, traditional in vivo testing can no longer meet the needs of a large number of chemical toxicity evaluations.

[0004] Compared with in vivo experiments, cell-based in vitro tests have the advantages of high cost-effectiveness and simple operation. Type II alveolar epithelial cells have various functions to maintain the integrity of normal lung structure. Human non-small cell lung cancer cells (A549 cells) retain many characteristics of normal type II alveolar epithelial cells and are widely used in in vitro models of toxicology, pharmacology, injury and metabolic processes of type II alveolar epithelial cells in lung tissue. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide a method for screening chemicals for potential lung toxicity based on high content screening. The present invention constructs an A549 cell screening model, provides a method for screening chemicals for potential lung toxicity based on high content screening, and uses dyes to indicate the phenotypic changes of cells to determine the potential lung toxicity of chemicals. The present invention is based on the A549 cell model, and in vitro experiments using high content screening technology can simultaneously detect multiple parameters of a single cell, which has higher throughput, sensitivity and specificity than existing methods such as cell-based substrate conversion and luciferase, and can increase the efficiency and accuracy of the experiment.

[0006] The present invention adopts the following technical solutions:

[0007] The present invention provides a method for detecting the potential lung toxicity of chemicals based on high content technology. The method detects the number of live cells, mitochondrial membrane potential, reactive oxygen species (ROS), nuclear fluorescence intensity, nuclear area, mitochondrial area and cell area to which the tested chemical has been added based on a high content screening system; the number of live cells, mitochondrial membrane potential, reactive oxygen species (ROS), nuclear fluorescence intensity, nuclear area, mitochondrial area and cell area of ​​the tested chemical experimental group are significantly different from those of the blank control group (P<0.05), and a lung toxicity comprehensive data calculation formula is established at a concentration of 100 μM, and chemicals with a lung toxicity comprehensive data value of ≥0.5 are identified as having lung toxicity;

[0008] The calculation formula for the comprehensive data of pulmonary toxicity is:

[0009]

[0010] Among them, 1 is the theoretical value after standardization of the control group.

[0011] In the above technical solution, further, the comprehensive data value of lung toxicity is ≥0.5, and the larger the value is, the stronger the toxicity is.

[0012] In the above technical solution, further, the living cells are human non-small cell lung cancer cells A549; the chemicals are bleomycin sulfate, amiodarone, menadione, hexadecylpyridinium chloride, 2-tert-butyl 5-methylphenol, triisobutyl phosphate, and dibutyl phthalate.

[0013] In the above technical solution, further, the method adopts Opera Phenix Plus, PerkinElmer platform for imaging.

[0014] In the above technical solution, further, the method comprises the following steps:

[0015] (1) Inoculate cells onto well plates and incubate;

[0016] (2) Add the test chemical solution prepared in culture medium to the well plate and incubate for 24 hours;

[0017] (3) Remove the culture medium, wash with PBS, add Hoechst 33342, Calcein AM, Mito-TrackerRed CMXRos, and H2DCFDA dyes to the wells and incubate; remove the dyes in the well plate, wash with PBS, and then add HBSS to each well; HBSS is added to keep the cells in a normal state during imaging;

[0018] (4) Use a high-content cell analyzer to obtain cell images in different channels;

[0019] (5) A cell phenotype analysis method was constructed using Harmony5.1 software. The detected indicators included the number of viable cells, mitochondrial membrane potential, reactive oxygen species (ROS), nuclear fluorescence intensity, nuclear area, mitochondrial area, and cell area.

[0020] In the above technical solution, further, in step (1), the cells are A549 cells, the cell inoculation density is 7000 cells / well, and the incubation time is 12 hours; in step (2), the final concentration of the chemical to be tested is 0, 0.1, 0.3, 1, 3, 10, 30, 100 μM.

[0021] In the above technical scheme, further, the specific method of adding dye in step (3) is: 10μg / mL Hoechst 33342, 0.8μM Calcein AM, 60nM Mito-Tracker Red CMXRos combination is used for staining experiment 1, and the condition is incubation in a 37°C, 5% CO2 incubator for 30 minutes; 10μg / mL Hoechst 33342 and 20μM H2DCFDA combination is used for staining experiment 2, and the condition is incubation in a 37°C, 5% CO2 incubator for 30 minutes.

[0022] In the above technical solution, further, the specific process of washing in step (3) is: adding 100 μL PBS to each well, repeatedly sucking and spitting PBS with a pipette for washing, and then removing PBS.

[0023] In the above technical solution, further, the specific conditions of different channels in step (4) are set as follows: ①Hoechst 33342 (channel: 346 / 460nm); ②Calcein AM (channel: 494 / 514nm); ③Mito-Tracker Red CMXRos (channel: 579 / 599nm); ④H2DCFDA (channel: 495 / 517nm); the channels corresponding to Hoechst 33342, Calcein AM, and Mito-Tracker Red CMXRos are all imaged under a 40x water microscope.

[0024] In the above technical solution, further, the specific steps of the phenotypic analysis method constructed using Harmony5.1 in step (5) are: ① input image (flat field correction: none; overlay processing: single plane); ② find nuclei (channel: Hoechst33342; method: B (general threshold: 0.4; area: >25μM); 2; Splitting coefficient: 8.0; Individual threshold: 0.35; Contrast: >0.2); Output population: nucleus); ③ Calculate intensity properties (Channel: Hoechst 33342; Population: nucleus; Region: nucleus; Method: Standard; Property prefix: Intensity Nucleus Hoechst 33343); ④ Calculate morphological properties (Population: nucleus; Region: nucleus; Method: Standard (area; circularity; perimeter; width; length); Property prefix: Nucleus); ⑤ Find cytoplasm (Channel: Alexa 488; nucleus: nucleus; Method: D (individual threshold: 0.1)); ⑥ Calculate morphological properties (Population: nucleus; Region: cytosol; Method: Standard (area; circularity; perimeter; width; length); ⑦ Find mitochondria (Channel: Alexa 568; nucleus: nucleus; Method: Method: B (general threshold: 0.4; individual threshold: 0.45)); ⑧ Calculate intensity properties (Channel: Alexa 568; Population: Nuclei; Region: Cytoplasm (2); Method: Standard; Property Prefix: Intensity Mitochondria Alexa 568); ⑨ Calculate morphological properties (Population: Nuclei; Region: Cells; Method: Standard (Area; Circularity; Perimeter; Width; Length)); ⑩ Define results (Method: Output list).

[0025] The present invention also provides application of the above-mentioned method for detecting potential lung toxicity of chemicals in toxicity screening of chemicals.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The method for screening chemicals for potential lung toxicity based on high-content screening provided by the present invention is a new in vitro detection method for screening chemicals for potential lung toxicity. It is simpler and has higher throughput than existing methods such as CCK-8 and luciferase, and the experimental process is simpler, providing more options for screening chemicals for potential lung toxicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention, the drawings related to the embodiments are briefly introduced below.

[0029] Figure 1 This is the flowchart of cell morphology and fluorescence intensity analysis using Harmony5.1 software.

[0030] Figure 2 The figure shows the effects of seven chemicals on cell viability detected by the method of the present invention.

[0031] Figure 3 This is a graph showing the effects of seven chemicals on mitochondrial membrane potential detected by the method of the present invention.

[0032] Figure 4This is a graph showing the effects of seven chemicals on intracellular ROS detected by the method of the present invention.

[0033] Figure 5 The method of the present invention detects the effects of seven chemicals on cell area, cell nucleus area, cell nucleus fluorescence intensity, and mitochondrial area. DETAILED DESCRIPTION

[0034] The present invention is further described below in conjunction with specific embodiments, but the present invention is not limited in any way.

[0035] Example 1

[0036] Main equipment, reagents and consumables:

[0037] High-content cell analyzer (Opera Phenix Plus, PerkinElmer), electric pipette (E12-300XLS+, Rainin), PBS (Cat. No. 14190144, Gibco), HBSS (Cat. No. 14175095, Gibco), complete culture medium (F-12K culture medium + 10% FBS), F-12K culture medium (Cat. No. 30-2004, ATCC), FBS (Cat. No. 10099141C, made in Australia, Gibco), Trypsin-EDTA (Cat. No. 15400054, Gibco), black transparent bottom 96-well plate (Cat. No. 165305, ThermoFisher). The chemicals selected in this embodiment are bleomycin sulfate, amiodarone, menadione, cetylpyridinium chloride, 2-tert-butyl 5-methylphenol, which are all pulmonary toxic, and triisobutyl phosphate and dibutyl phthalate, which are not pulmonary toxic.

[0038] The structural formula of bleomycin sulfate (CAS: 9041-93-4) is:

[0039]

[0040] The structural formula of amiodarone (CAS: 1951-25-3) is:

[0041]

[0042] The structural formula of menadione (CAS: 58-27-5) is:

[0043]

[0044] The structural formula of hexadecylpyridinium chloride (CAS: 6004-24-6) is:

[0045]

[0046] The structural formula of 2-tert-butyl-5-methylphenol (CAS: 88-60-8) is:

[0047]

[0048] Triisobutyl phosphate (CAS: 126-71-6):

[0049]

[0050] Dibutyl phthalate (CAS: 84-74-2)

[0051]

[0052] This embodiment provides a method for high-throughput, multi-parameter screening of potential pulmonary toxicity of chemicals. The principle of this method is to use specific cell dyes to stain specific parts of A549 cells, and observe changes in the number of A549 cells, mitochondrial membrane potential, reactive oxygen species (ROS), nuclear fluorescence intensity, nuclear area, and mitochondrial area to illustrate the potential pulmonary toxicity of chemicals.

[0053] The specific experimental process is as follows:

[0054] 1.96-well plate seeding

[0055] Aspirate the culture medium in the culture dish, add 1 mL of 0.05% Trypsin-EDTA along the wall of the culture dish, shake the culture dish briefly to allow 0.05% Trypsin-EDTA to fully rinse the cell layer, and aspirate 0.05% Trypsin-EDTA. Then add 3 mL of 0.05% Trypsin-EDTA vertically above the culture dish, put the culture dish in the incubator and let it stand, take it out after 10 minutes, and add 7 mL of complete culture medium to terminate digestion. Use a pipette to transfer 10 mL of cell suspension to a centrifuge tube, centrifuge at 1000 rpm for 2 minutes, discard the supernatant, add complete culture medium to resuspend the cells, count them, dilute the cells with complete culture medium, inoculate them into a 96-well plate at a density of 7000 cells / well, and culture overnight at 37°C and 5% CO2.

[0056] 2. Cells were exposed to solutions of lung-toxic chemicals

[0057] Prepare chemical solutions (the solvent is complete culture medium) with concentrations of 0, 0.1, 0.3, 1, 3, 10, 30, 100 μM. The chemicals used in the experiment are bleomycin sulfate, amiodarone, menadione, hexadecylpyridinium chloride, 2-tert-butyl 5-methylphenol, triisobutyl phosphate, and dibutyl phthalate. After the cells are incubated overnight for 12 hours, remove the complete culture medium in the well plate and add the prepared chemical solution. Move to the incubator and culture at 37°C and 5% CO2 for 24 hours.

[0058] 3. Cell-specific staining

[0059] (a) Preparation and storage of four specific dye stock solutions

[0060] Dissolve 25 mg of Hoechst 33342 in 2.5 mL of deionized water to prepare a 10 mg / mL Hoechst 33342 stock solution, and dispense it into 5 tubes.

[0061] Dispense 0.5 mL of Calcein AM (2 mM) into 5 tubes.

[0062] Dissolve 50 μg of Mito-Tracker Red CMXRos in 470 μL of DMSO to prepare a 200 μM Mito-Tracker Red CMXRos stock solution and dispense into 5 tubes.

[0063] Dissolve 50 mg of H2DCFDA in 10.3 mL of DMSO to prepare a 10 mM H2DCFDA stock solution, and dispense it into 20 tubes. The dispensed stock solution should be stored in a -20°C refrigerator away from light.

[0064] (b) Staining experiment 1: Preparation of three specific dye mixtures

[0065] Before staining, the three dye stock solutions of Hoechst 33342, Calcein AM, and Mito-Tracker Red CMXRos were taken out of the refrigerator and thawed at room temperature in the dark. Then, 10 mg / mL Hoechst 33342 was diluted to 40 μg / mL, 2 mM Calcein AM was diluted to 2 μM, and 200 μM Mito-Tracker Red CMXRos was diluted to 200 nM with complete culture medium. Then, the diluted dyes were mixed according to a specific volume ratio (Hoechst 33342: Calcein AM: Mito-Tracker Red CMXRos: complete culture medium = 5:8:6:1) to obtain a mixed staining solution of 10 μg / mL Hoechst 33342, 0.8 μM Calcein AM, and 60 nM Mito-Tracker Red CMXRos.

[0066] (c) Staining experiment 2: Preparation of a mixture of two specific dyes

[0067] Before staining, take out the two dye stock solutions of Hoechst 33342 and H2DCFDA from the refrigerator and thaw them at room temperature in the dark. Then dilute 10 mg / mL Hoechst 33342 to 20 μg / mL and 10 mM H2DCFDA to 40 μM with complete culture medium. Then, mix the diluted dyes according to a specific volume ratio (Hoechst33342: H2DCFDA = 1:1) to obtain a mixed staining solution of 10 μg / mL Hoechst 33342 and 20 μM H2DCFDA.

[0068] (d) Dyeing

[0069] Use an electric pipette to suck out the chemical solution in each well of the 96-well plate, then slowly add 100μL of PBS with an electric pipette, slowly aspirate and spit 2-3 times with an electric pipette to wash, and then remove the PBS. Use an electric pipette to add 100μL of 10μg / mL Hoechst 33342, 0.8μM Calcein AM, 60nM Mito-Tracker RedCMXRos or 10μg / mL Hoechst 33342, 20μM H2DCFDA mixed staining solution to each well, move the well plate into a 37°C, 5% CO2 incubator and incubate for 30 minutes; remove the dye working solution in the well plate, wash it with PBS 2-3 times, and then add HBSS to each well.

[0070] 4. Obtain cell images using a high-content cell analyzer

[0071] After the cells were stained with the three specific dyes Hoechst 33342, Mito-Tracker Red CMXRos, and Calcein AM, the morphology and fluorescence changes were observed and photographed under a 40x water microscope, with 27 fields of view taken per well; Hoechst33342 and Calcein AM needed to be photographed under a 10x air microscope, with 6 fields of view taken per well; H2DCFDA dye and Hoechst 33342 were photographed under a 10x air microscope, with 6 fields of view taken per well after the cells were stained. Specific shooting parameters are shown in Table 1.

[0072] Table 1: Specific parameters of four specific dye imaging channels

[0073]

[0074] 5. Construction of phenotypic analysis method

[0075] The acquired cell images were analyzed using the Harmony 5.1 analysis system.

[0076] (1) Detect Hoechst 33342 staining at channel 346 / 460 nm to obtain the parameters of cell nucleus number, cell nucleus area, and cell nucleus fluorescence intensity;

[0077] (2) Detect Calcein AM staining at channel 494 / 514 nm to obtain cell number and cell area parameters;

[0078] (3) Detect Mito-Tracker Red CMXRos labeling staining at channel 579 / 599 nm to obtain mitochondrial membrane potential and mitochondrial area parameters;

[0079] (4) Detect H2DCFDA labeling staining at channel 346 / 460 nm to obtain reactive oxygen species parameters.

[0080] 6. Statistical analysis

[0081] The measured values ​​of each cell were standardized to achieve comparison between different targets. The main content was to divide the values ​​measured in the drug-added group and the control group in the 96-well plate by the values ​​obtained in the control group to obtain the standardized data of each indicator of each cell. All experimental results were derived from at least 3 independent repeated experiments. The experimental results were analyzed by one-way ANOVA using GraphPad Prism software, and P<0.05 indicated that the difference was statistically significant.

[0082] The experimental results are as follows:

[0083] Figure 2Amiodarone showed a significant decrease in cell survival rate at 10 μM, bleomycin sulfate showed a significant decrease in cell survival rate at 100 μM, hexadecylpyridinium chloride had dropped to 0 at 10 μM, menadione showed a significant decrease in cell survival rate at 30 μM, 2-tert-butyl-5-methylphenol showed a decrease in cell survival rate at 100 μM, although triisobutyl phosphate showed a significant difference, the cell survival rate decreased by less than 10% (it can be considered that there is no cytotoxicity), and dibutyl phthalate did not show a decrease in cell survival rate. Cell survival rate = (number of cells in the drug-added group ÷ number of cells in the control group) × 100%, this indicator is the most important indicator in toxicity detection. Because chemicals containing lung toxicity all showed a significant decrease in cell survival rate at 100 μM, 100 μM was selected as a standard for evaluating whether chemicals have lung toxicity.

[0084] Figure 3 Bleomycin sulfate showed a significant increase in mitochondrial membrane potential at 10, 30, and 100 μM, cetylpyridinium chloride showed a significant decrease in mitochondrial membrane potential at 3 μM, triisobutyl phosphate showed a significant increase in mitochondrial membrane potential at 10, 30, and 100 μM, and dibutyl phthalate showed a significant increase in mitochondrial membrane potential at 10, 30, and 100 μM.

[0085] Figure 4 Amiodarone showed a significant increase in reactive oxygen species at 3 and 10 μM, bleomycin sulfate showed a significant increase in reactive oxygen species at 10, 30, and 100 μM, cetylpyridinium chloride showed a significant increase in reactive oxygen species at 0.3, 1, and 3 μM, menadione showed a significant increase in reactive oxygen species at 30 μM, and 2-tert-butyl-5-methylphenol showed a significant increase in reactive oxygen species at 10, 30, and 100 μM.

[0086] Figure 5 Bleomycin sulfate showed the most obvious changes in the four indicators, and the other six substances also showed different changes.

[0087] Since the five chemicals with lung toxicity showed a significant decrease in cell survival rate only at 100 μM, 100 μM was used as the standard for screening lung toxicity. Here, the present invention proposes a formula for judging comprehensive lung toxicity, and stipulates that the comprehensive lung toxicity data ≥ 0.5 is lung toxic. The formula is as follows:

[0088] Comprehensive data on lung toxicity at 100 μM concentration

[0089]

[0090] Among them, 1 is the theoretical value after standardization of the control group.

[0091] The cell survival rate of amiodarone is close to 0 at 100 μM, the cell survival rate of cetylpyridinium chloride is close to 0 at 100 μM, and the cell survival rate of menadione is close to 0 at 100 μM. Therefore, the mitochondrial membrane potential, reactive oxygen species, cell area, nuclear area, mitochondrial area, and nuclear fluorescence intensity of these three substances at these high concentrations are not measurable. Here, the square of the difference of these terms in the formula is defined as 1; when the reactive oxygen species decreases, the square of the difference is defined as 0.

[0092] Calculated combined lung toxicity data values:

[0093] Amiodarone 2.64, bleomycin sulfate 1.76, cetylpyridinium chloride 2.65, menadione 2.64, 2-tert-butyl-5-methylphenol 0.5, triisobutyl phosphate 0.26, and dibutyl phthalate 0.32.

[0094] According to the measured values, these chemicals all meet the above distinction of pulmonary toxicity / non-pulmonary toxicity. The above results show that this method can predict the pulmonary toxicity of chemicals with high accuracy and high throughput, and can be used as an in vitro research method for detecting pulmonary toxicity.

Claims

1. An analytical method for detecting the potential lung toxicity of chemicals based on high content technology, characterized in that: The method is based on a high content screening system to detect the number of live cells, mitochondrial membrane potential, reactive oxygen species (ROS), nuclear fluorescence intensity, nuclear area, mitochondrial area and cell area to which the test chemical has been added; the number of live cells, mitochondrial membrane potential, reactive oxygen species (ROS), nuclear fluorescence intensity, nuclear area, mitochondrial area and cell area of ​​the test chemical experimental group are significantly different from those of the blank control group (P<0.05), and a lung toxicity comprehensive data calculation formula is established at a concentration of 100 μM, and chemicals with a lung toxicity comprehensive data value ≥0.5 are identified as having lung toxicity; The calculation formula for the comprehensive data of pulmonary toxicity is: Among them, 1 is the theoretical value after standardization of the control group.

2. The method according to claim 1, characterized in that The comprehensive data value of pulmonary toxicity is ≥0.5, and the larger the value, the stronger the toxicity.

3. The method according to claim 1, characterized in that The living cells are human non-small cell lung cancer cells A549; the chemicals are bleomycin sulfate, amiodarone, menadione, hexadecylpyridinium chloride, 2-tert-butyl 5-methylphenol, triisobutyl phosphate, and dibutyl phthalate.

4. The method according to claim 1, characterized in that The method uses Opera Phenix Plus, a PerkinElmer platform for imaging.

5. The method according to claim 1, characterized in that The method comprises the following steps: (1) Inoculate cells onto well plates and incubate; (2) Add the test chemical solution prepared in culture medium to the well plate and incubate for 24 hours; (3) Remove the culture medium, wash with PBS, add Hoechst 33342, Calcein AM, Mito-Tracker RedCMXRos, and H2DCFDA dyes to the wells and incubate; remove the dyes in the well plate, wash with PBS, and then add HBSS to each well; (4) Use a high-content cell analyzer to obtain cell images in different channels; (5) A cell phenotype analysis method was constructed using Harmony5.1 software. The detected indicators included the number of viable cells, mitochondrial membrane potential, reactive oxygen species (ROS), nuclear fluorescence intensity, nuclear area, mitochondrial area, and cell area.

6. The method according to claim 5, characterized in that In step (1), the cells are A549 cells, the cell inoculation density is 7000 cells / well, and the incubation time is 12 hours; in step (2), the final concentrations of the test chemicals are 0, 0.1, 0.3, 1, 3, 10, 30, and 100 μM.

7. The method according to claim 5, characterized in that The specific method of adding dye in step (3) is as follows: 10 μg / mL Hoechst 33342, 0.8 μM Calcein AM, and 60 nM Mito-Tracker Red CMXRos are combined for staining experiment 1, and the condition is incubation in a 37°C, 5% CO2 incubator for 30 minutes; 10 μg / mL Hoechst33342 and 20 μM H2DCFDA are combined for staining experiment 2, and the condition is incubation in a 37°C, 5% CO2 incubator for 30 minutes.

8. The method according to claim 5, characterized in that The specific conditions of different channels in step (4) are set as follows: ①Hoechst 33342 (channel: 346 / 460nm); ②Calcein AM (channel: 494 / 514nm); ③Mito-TrackerRed CMXRos (channel: 579 / 599nm); ④H2DCFDA (channel: 495 / 517nm).

9. The method according to claim 5, characterized in that The specific steps of the phenotypic analysis method constructed using Harmony5.1 in step (5) are: ① input image; ② find nuclei; ③ calculate intensity characteristics; ④ calculate morphological characteristics; ⑤ find cytoplasm; ⑥ calculate morphological characteristics; ⑦ find mitochondria; ⑧ Calculate intensity properties; ⑨ Calculate morphological properties; ⑩ Define results (method: output list).

10. Use of the method for detecting potential lung toxicity of chemicals according to any one of claims 1 to 9 in toxicity screening of chemicals.

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