Methods for assessing toxicity

Abstract

A method for characterizing toxicity of toxic pollutants and a method for characterizing comprehensive toxicity of water bodies. The methods include constructing a reporter gene cell line expressing CHOP gene associated with endoplasmic reticulum stress.

Claims

1. A method for constructing a reporter gene cell line and measuring sensitivity of the reporter gene cell line to a poisonous pollutant, the method comprising: ligating a CHOP promoter and a secreted embryonic alkaline phosphatase (SEAP) gene in a lentiviral plasmid to construct a lentiviral CHOP-SEAP plasmid vector; transfecting the lentiviral CHOP-SEAP plasmid vector into Hela cells to construct a reporter gene cell line comprising transfected Hela cells; using arsenic as a poisonous pollutant, diluting the arsenic to obtain arsenic dilutions having concentrations of 0, 0.05, 0.1, 0.15, 0.2, 0.5, and 1 mol/L; exposing the arsenic dilutions to the transfected Hela cells for 24 hours to obtain treated Hela cells; incubating culture supernatants of the treated Hela cells at 65 C. for 30 minutes; and detecting a SEAP activity of the culture supernatants of the treated Hela cells by a SEAP Reporter Gene Assay kit comprising a cell-based alkaline phosphatase standard and a SEAP substrate; wherein: detecting the SEAP activity comprises diluting the cell-based alkaline phosphatase standard to obtain standard dilutions; adding the standard dilutions and the culture supernatants of the treated Hela cells to a 96-well white plate and adding the SEAP substrate to each well; and incubating the 96-well white plate at room temperature for 10-30 minutes and detecting the chemiluminescence of the standard dilutions and the culture supernatants of the treated Hela cells.

2. A method for detecting water toxicity, comprising: ligating a CHOP promoter and a secreted embryonic alkaline phosphatase (SEAP) gene in a lentiviral plasmid to construct a lentiviral CHOP-SEAP plasmid vector; transfecting the lentiviral CHOP-SEAP plasmid vector into Hela cells to construct a reporter gene cell line comprising transfected Hela cells; concentrating 5 L water samples by solid phase extraction and then diluting with 2% DMSO to 1 mL to obtain concentrated water samples; diluting the concentrated water samples to four dilutions respectively comprising by volume 0, 0.02%, 1%, and 2% of the concentrated water samples; performing parallel comparison tests and exposing the transfected Hela cells to the four dilutions for 24 hours to obtain treated Hela cells; incubating culture supernatants of the treated Hela cells at 65 C. for 30 minutes; and detecting a SEAP activity of the culture supernatants of the treated Hela cells by a SEAP Reporter Gene Assay kit; wherein: detecting the SEAP activity comprises diluting the cell-based alkaline phosphatase standard to obtain standard dilutions; adding the standard dilutions and the culture supernatants of the treated Hela cells to a 96-well white plate and adding the SEAP substrate to each well; and incubating the 96-well white plate at room temperature for 10-30 minutes and detecting the chemiluminescence of the standard dilutions and the culture supernatants of the treated Hela cells.

3. A method for constructing a reporter gene cell line, the method comprising: 1) cleaving pHBLV-CMVIE-ZsGreen-Puro vector with EcoRI and XhoI enzymes, and extracting cleaved vector by gel electrophoresis; 2) amplifying a secreted embryonic alkaline phosphatase (SEAP) gene and a CHOP promoter by PCR to obtain a SEAP fragment and a CHOP promoter fragment; 3) ligating the SEAP fragment and the CHOP promoter fragment of 2) to the cleaved vector of 1) to obtain a CHOP-SEAP lentiviral plasmid vector; 4) transforming the CHOP-SEAP lentiviral plasmid vector of 3) into DH5a competent cells, cultivating the DH5a competent cells and selecting positive clones by PCR identification; 5) extracting the CHOP-SEAP lentiviral plasmid vector from the positive clones of 4); 6) transfecting the CHOP-SEAP lentiviral plasmid vector of 5) to 293T cells, cultivating the 293T cells, and collecting the CHOP-SEAP lentiviral plasmid vector from culture supernatants; 7) transfecting the CHOP-SEAP lentiviral plasmid vector of 6) to Hela cells to obtain transfected Hela cells; and 8) isolating a reporter gene cell line from the transfected Hela cells of 7) through screening for puromycin resistance.

4. A method for measuring sensitivity of the reporter gene cell line of claim 3 to a poisonous pollutant, the method comprising: using arsenic as a poisonous pollutant, diluting the arsenic to obtain arsenic dilutions having concentrations of 0, 0.05, 0.1, 0.15, 0.2, 0.5, and 1 mol/L; exposing the arsenic dilutions to Hela cells of the reporter gene cell line for 24 hours to obtain treated Hela cells; incubating culture supernatants of the treated Hela cells at 65 C. for 30 minutes; and detecting a SEAP activity of the culture supernatants of the treated Hela cells by a SEAP Reporter Gene Assay kit comprising a cell-based alkaline phosphatase standard and a SEAP substrate; wherein: detecting the SEAP activity comprises diluting the cell-based alkaline phosphatase standard to obtain standard dilutions; adding the standard dilutions and the culture supernatants of the treated Hela cells to a 96-well white plate and adding the SEAP substrate to each well; and incubating the 96-well white plate at room temperature for 10-30 minutes and detecting the chemiluminescence of the standard dilutions and the culture supernatants of the treated Hela cells.

5. A method for detecting water toxicity using the reporter gene cell line of claim 3, the method comprising: concentrating 5 L water samples by solid phase extraction and then diluting with 2% DMSO to 1 mL to obtain concentrated water samples; diluting the concentrated water samples to four dilutions respectively comprising by volume 0, 0.02%, 1%, and 2% of the concentrated water samples; performing parallel comparison tests and exposing Hela cells of the reporter gene cell line to the four dilutions for 24 hours to obtain treated Hela cells; incubating culture supernatants of the treated Hela cells at 65 C. for 30 minutes; and detecting a SEAP activity of the culture supernatants of the treated Hela cells by a SEAP Reporter Gene Assay kit, wherein: detecting the SEAP activity comprises diluting the cell-based alkaline phosphatase standard to obtain standard dilutions; adding the standard dilutions and the culture supernatants of the treated Hela cells to a 96-well white plate and adding the SEAP substrate to each well; and incubating the 96-well white plate at room temperature for 10-30 minutes and detecting the chemiluminescence of the standard dilutions and the culture supernatants of the treated Hela cells.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows the activity of SEAP after exposure to arsenic (mol/L) in Example 1, where 0 represents the SEAP activity of untransformed and untransfected Hela cells; 0+ indicates the SEAP activity of transformed and untransfected Hela cells;

(2) FIG. 2 shows the cell viability after exposure to different concentrations of arsenic (mol/L) in Comparative Example 1;

(3) FIG. 3 shows the intracellular reactive oxygen species (ROS) level after exposure to different concentrations of arsenic (mol/L) in Comparative Example 2;

(4) FIG. 4 shows the mitochondrial membrane potential changes after exposure to different concentrations of arsenic (mol/L) in Comparative Example 3;

(5) FIG. 5 shows the activity of SEAP after exposure to different concentrations of water samples. In this case, 0 and 0+ were the control group, 0 showed untransfected Hela cells that were not exposed; 0+ indicates transfected Hela cells that were not exposed; 0.02%+ represents the experimental group having the transfected Hela cells with 0.02% concentration of water samples;

(6) FIG. 6 shows the cell viability after exposure to different concentrations of water samples in Comparative Example 4;

(7) FIG. 7 shows the intracellular reactive oxygen species (ROS) level of the cell after exposure to different concentrations of water samples in Comparative Example 5; and

(8) FIG. 8 shows the change in mitochondrial membrane potential of cells after exposure to different concentrations of water samples in Comparative Example 6.

DETAILED DESCRIPTION OF THE EMBODIMENTS

(9) The technical solution of the present disclosure will be described further below with reference to the accompanying drawings.

Example 1

(10) Step 1, construct the lentivirus CHOP-SEAP plasmid vector: The pHBLV-CMVIE-ZsGreen-Puro vector was cleaved with EcoRI and XhoI enzymes, extracted by gel electrophoresis; primers were designed according to SEAP gene and CHOP gene sequences and PCR amplification was performed. The amplified fragments were ligated with the vector and then transformed into DH5a competent cells. After transformation, bacterial strains were selected from the plate and shaken at 250 rpm and 37 C. PCR identification of the bacterial strains was performed to obtain positive clonal bacterial liquid. The positive clonal bacterial liquid was sent for sequencing.

(11) Step 2, Lentivirus CHOP-SEAP Plasmid Vector Extraction: The lentiviral CHOP-SEAP plasmid vector was extracted from the positive clonal bacterial liquid obtained in step 1, and the extraction was carried out according to the instructions of the plasmid extraction and purification kit. When the lentivirus CHOP-SEAP plasmid vector concentration was more than 1 g/L, and the value of A260/280 is between 1.7 and 1.8, the virus can be packed.

(12) Step 3, lentivirus CHOP-SEAP vector packaging: 293T cells were plated for transfection. The 293T cells were incubated at 37 C. in an incubator containing 5% CO.sub.2. When the cell confluence rate reached 70-80%, the liposomes were transfected, and the transfection system of each dish 100 mm was as follows:

(13) TABLE-US-00002 pSPAX2 10 g pMD2G 5 g CHOP-SEAP plasmid 10 g LipofiterTM dosage 75 L;

(14) The medium was replaced with the fresh complete medium containing 10% fetal bovine serum after transfection. The virus supernatant was collected twice at 48 h and 72 h after transfection. The collected supernatant was centrifuged in a 50 mL centrifuge tube at 4 C. and 2000g for 10 min to remove cell debris. And then the supernatant of the collected virus solution was placed in an ultracentrifuge tube and centrifuged at 82700g for 120 min at 4 C. Finally, the lentivirus's ultra-centrifugal solution was dispensed into a sterile treated virus tube, packed and labeled well and preserved at 80 C.

(15) Step 4, Construction of stable transfected cell line: Cells were cultured in 10 cm large dish. When the bottom of the culture dish was covered with the cells, the cells were digested with trypsin and the cells density was diluted to 3.010.sup.5 cells/mL. The diluted cell fluid was seeded on a six-well plate and the inoculum volume was 2 mL so that the fusion rate of cells cultured for the next day (24 h) was about 60% which is conducive for infection. Cells were infected and the original medium was discarded. 2 mL of DMEM containing 5% FBS, 1% double antibody and an appropriate amount of virus suspension was added to each well. 24 h after infection, the virus infection solution was discarded, and the medium was replaced with fresh complete medium. After the cells were infected with the virus, the cells were transferred to 10 cm large dish when the cell aggregation rate reached 90%.

(16) Step 5, Screening of stable transfected cell line: Due to the puromycin resistance gene of virus's carrier, the cells infected with the virus will have puromycin resistance. According to this feature, repeated treatment of virus-infected cells with multiple use of puromycin can be used to screen out cells that have been successfully infected with the virus, followed by expanded culture and cryopreservation.

(17) Using arsenic as a typical toxic pollutant, dilution concentration gradient: 0, 0.05, 0.1, 0.15, 0.2, 0.5, 1 mol/L. seven groups of parallel comparison tests were carried out. The transfected Hela cells were exposed to different concentrations of arsenic for 24 h. The supernatant of Hela cells was incubated at 65 C. for 30 min, and the SEAP activity of Hela cells was detected by a SEAP Reporter Gene Assay kit comprising a cell-based alkaline phosphatase standard and a SEAP substrate (SEAP activity has the effect of indicating luminescence, easy to monitor). According to the SEAP Reporter Gene Assay kit protocol, the cell-based alkaline phosphatase standard and the sample were added to the 96-well white plate at 10 L per well. Each well was added with 50 L the SEAP substrate and the 96-well white plate was incubated for 10-30 min. The chemiluminescence was detected at room temperature. The experimental results are shown in FIG. 1.

(18) As can be seen from FIG. 1, there was a significant difference between the different exposure groups and the control group (p<0.05). The exposure group with an arsenic concentration of 0.05 mol/L demonstrates a significant difference from the control group, indicating that the cell lines constructed by the method can characterize early cell damage, and the results show a dose-effect relationship.

Example 2

(19) Using water samples as poisons, dilution concentration gradient: 0, 0.02%, 1% and 2% (The dilution concentrations are 0, 1, 50, and 100 times the original concentrations, respectively). Parallel comparison test was carried out. The transfected Hela cells were exposed to different concentrations of water samples for 24 h. The supernatant of Hela cells was incubated at 65 C. for 30 min, and the SEAP activity of Hela cells was detected by a SEAP Reporter Gene Assay kit comprising a cell-based alkaline phosphatase standard and a SEAP substrate (SEAP activity has the effect of indicating luminescence, easy to monitor). According to the SEAP Reporter Gene Assay kit protocol, the cell-based alkaline phosphatase standard and the sample were added to the 96-well white plate at 10 L per well. Each well was added with 50 L the SEAP substrate and the 96-well white plate was incubated for 10-30 min. The chemiluminescence was detected at room temperature. The experimental results are shown in FIG. 5.

(20) As can be seen from FIG. 5, there was a significant difference between the different exposure groups and the control group (p<0.05). The exposure group with a water sample concentration of 0.02% concentration demonstrates a significant difference from the control group, which indicates that the cell lines constructed by the method can characterize early cell damage, and the results show a dose-effect relationship.

Comparative Example 1

(21) Cytotoxicity TestCell Proliferation Inhibition Rate CCK-8 Test

(22) Step 1, in a 96-well plate, Hela cells were seeded at a density of 10,000 cell/well;

(23) Step 2, after the cells were cultured for 24 h, arsenic dilutions with concentrations of 0, 0.5, 1, 2, and 5 mol/L were used as toxic substrates and the Hela cells were exposed to the arsenic dilutions for 24 h;

(24) Step 3, 10 L of CCK-8 reagent was added to each well. After incubating the 96-well plate for 1-4 h, the OD value of each well at 450 nm was measured with a microplate reader.

(25) As shown in FIG. 2, the results of Comparative Example 1 show that an arsenic concentration of 0-2 mol/L do not cause significant cell proliferation inhibition, and an arsenic concentration of 5 mol/L causes significant proliferation inhibition.

Comparative Example 2

(26) Cytotoxicity TestIntracellular ROS Test

(27) Step 1, in a 96-well plate, Hela cells were seeded at a density of 10,000 cell/well;

(28) Step 2, after the cells were cultured for 24 h, arsenic dilutions with concentrations of 0, 0.5, 1, 2, and 5 mol/L were used as toxic substrates and the Hela cells were exposed to the arsenic dilutions for 24 h;

(29) Step 3, the medium was aspirated and the cells were washed with Hanks buffer. The DCF probe and Hoechst 33342 probe were diluted to 10 mol/L and 2.5 g/mL with Hanks buffer, 100 L of diluted probe was added to each well. After incubating the 96-well plate for 20 min, each well was washed with Hanks buffer and the fluorescence at 488/530 nm and 350/460 nm was measured using a microplate reader.

(30) As shown in FIG. 3, the detection result of Comparative Example 2 shows that an arsenic concentration of between 0 to 0.5 mol/L does not cause significant increase of intracellular ROS levels, and a significant increase of intracellular ROS level is shown when the arsenic concentration reaches 1 mol/L which indicates the oxidative stress.

Comparative Example 3

(31) Cytotoxicity TestMitochondrial Membrane Potential JC-1 Detection

(32) Step 1, in a 96-well plate, Hela cells were seeded at a density of 10,000 cell/well;

(33) Step 2, after the cells were cultured for 24 h, arsenic dilutions with concentrations of 0, 0.5, 1, 2, and 5 mol/L were used as toxic substrates and the Hela cells were exposed to the arsenic dilutions for 24 h;

(34) Step 3, 10 incubation fluid was diluted into 1 incubation solution with ddH.sub.2O, 1 L of JC-1 was added to each 500 L of 1 incubation solution, and the mixture was mixed with vortex to obtain JC-1 working solution, after centrifugation at 10,000 rpm for 1 min, the supernatant was taken;

(35) Step 4, the medium was aspirated and the cells were washed with Hanks buffer. 100 L of JC-1 working solution was added to each well. After incubation for 20 min, the fluorescence values at 488/530 nm and 530/590 nm were measured by a microplate reader.

(36) As shown in FIG. 4, the results of Comparative Example 3 show that arsenic at a concentration from 0 to 0.5 mol/L does not cause significant changes in mitochondrial membrane potential, and a significant mitochondrial membrane potential change occurred when the arsenic concentration is 1 mol/L which characterizes early cell apoptosis.

Comparative Example 4

(37) Cytotoxicity TestCell Proliferation Inhibition Rate CCK-8 Test

(38) Step 1, in a 96-well plate, Hela cells were seeded at a density of 10,000 cell/well;

(39) Step 2, after the cells were cultured for 24 h, water sample dilutions with concentrations of 0, 0.02%, 0.2%, 1%, and 2% were used as toxic substrates and the Hela cells were exposed to the water sample dilutions for 24 h;

(40) Step 3, 10 L of CCK-8 reagent was added to each well. After incubating the 96-well plate for 1-4 h, the OD value of each well at 450 nm was measured with a microplate reader.

(41) As shown in FIG. 6, the results of Comparative Example 4 show that the water samples do not cause significant cell proliferation inhibition at the concentration of 0 to 0.2%, and significant proliferation inhibition occurred at the water sample concentration of 1%.

Comparative Example 5

(42) Cytotoxicity TestIntracellular ROS Test

(43) Step 1, in a 96-well plate, Hela cells were seeded at a density of 10,000 cell/well;

(44) Step 2, after the cells were cultured for 24 h, water sample dilutions with concentrations of 0, 0.02%, 0.2%, 1%, and 2% were used as toxic substrates and the Hela cells were exposed to the water sample dilutions for 24 h;

(45) Step 3, the medium was aspirated and the cells were washed with Hanks buffer. The DCF probe and Hoechst 33342 probe were diluted to 10 mol/L and 2.5 g/mL with Hanks buffer, 100 L of diluted probe was added to each well. After incubating the 96-well plate for 20 min, each well plate was washed with Hanks buffer and the fluorescence at 488/530 nm and 350/460 nm were respectively measured using a microplate reader.

(46) As shown in FIG. 7, the detection result of Comparative Example 5 shows that the water samples do not cause significant intracellular ROS levels at concentrations of 0 to 0.02%, and a significant increase of intracellular ROS level occurs at the water sample concentration of 0.2% which indicates the existence of oxidative stress.

Comparative Example 6

(47) Cytotoxicity TestMitochondrial Membrane Potential JC-1 Detection

(48) Step 1, in a 96-well plate, Hela cells were seeded at a density of 10,000 cell/well;

(49) Step 2, after the cells were cultured for 24 h, water sample dilutions with concentrations of 0, 0.02%, 0.2%, 1%, and 2% were used as toxic substrates and the Hela cells were exposed to the water sample dilutions for 24 h;

(50) Step 3, 10 incubation fluid was diluted into 1 incubation solution with ddH.sub.2O, 1 L of JC-1 was added to each 500 L of 1 incubation solution, and the mixture was mixed with vortex to obtain JC-1 working solution. After centrifugation at 10,000 rpm for 1 min, the supernatant was taken;

(51) Step 4, the medium was aspirated and the cells were washed with Hanks buffer. 100 L of JC-1 working solution was added to each well. After incubation for 20 min, the fluorescence values at 488/530 nm and 530/590 nm of each well were measured by a microplate reader.

(52) As shown in FIG. 8, the results of Comparative Example 6 show that the water samples did not cause significant changes in mitochondrial membrane potential at a concentration of 0 to 0.02%, and a significant mitochondrial membrane potential change occurs at a water sample concentration of 0.2% which characterizes early cell apoptosis.

(53) Unless otherwise indicated, the numerical ranges involved in the invention include the end values. While particular embodiments of the invention have been shown and described, it will be obvious to those skilled in the art that changes and modifications may be made without departing from the invention in its broader aspects, and therefore, the aim in the appended claims is to cover all such changes and modifications as fall within the true spirit and scope of the invention.