Our protocol describes a new method for assessing the integral cytotoxicity of triazole pesticide metabolites in plants. Plant callus culture is used to generate the metabolites of proteins in plants, followed by toxicological tests using a human cell line to assess the integral cytotoxicity of metabolites. The metabolites of xenobiotics in plants are extremely complex and only a few stands of metabolites can be obtained.
Hence, toxicological debt of metabolites are limited, which hazards the comprehensive toxicity assessment. This protocol proposed a strategy to assess the integral cytotoxicity of metabolites of triazole pesticides in plants. In this protocol, plant carriers and the human cell models are combined to achieve the integral cytotoxicity assessment of metabolites of triazole pesticides in plants.
This avoids the complex steps of and separation of metabolites, thereby providing new debt for precise, trusted assessment. Begin by treating the collected carrot callus with the different triazole pesticides under sterile conditions. To do so, in separate glass flasks, makes three grams of carrot callus with 10 milliliters of each pesticide solution prepared in MS medium.
Also mix three grams of carrot callus with 10 milliliters of aseptic MS medium in another glass flask. Incubate the prepared carrot callus samples at 130 RPM and 26 degrees Celsius in the dark for 72 hours. Set all treatments in triplicate.
After the incubation, using 0.5 micron glass fiber filters, collect the carrot callus from the medium by filtration. Wash the callus with ultrapure water three times. Finally, freeze dry the callus using a freeze dryer at minus 55 degrees Celsius and then homogenize them using a high throughput tissue grinder at 70 hertz for three minutes.
The concentrations of all pesticides exponentially decreased in culture media. While those in carrot calluses began to increase, peaking at four or eight hours, followed by a gradual decrease. These results suggested that pesticides were quickly taken up and transformed by carrot callus.
The temporal recoveries of pesticides in the callus culture system showed that only 8.3 to 11%of pesticides remained after a 72 hour incubation, indicating that most of the pesticides had been transformed into metabolites. To begin, mix 0.2 grams of ground powder of each freeze dried, pesticide treated carrot callus sample with three milliliters of acetonitrile in a 10 milliliter centrifuge tube. Vortex the centrifuge tube for eight minutes and then sonicate it for five minutes at 150 watts and 40 kilohertz.
Next, centrifuge the tube at 8, 000 G and four degrees Celsius for 10 minutes before collecting the supernatant by pipetting. Using a nitrogen blowing concentrator at 40 degrees Celsius, concentrate the pooled extracts to dryness. Redissolve the residues of the extract in one milliliter of DMEM containing 0.3%DMSO to yield the pesticide metabolite exposure solution.
In a similar manner, redissolve the residues of the extract obtained from the blank callus samples in one milliliter of DMEM containing 0.3%DMSO. Then dissolve one milligram of each pesticide used in different tubes containing the blank sample residue solution to yield the parent pesticide exposure solutions. Proceed to passage the cells after culturing them until they reach a cell density of above 80%After discarding the culture medium, wash the cells three times with PBS.
Then add one milliliter of trypsin-EDTA solution to the flask and spread it uniformly for full cell contact. Once the cells change from an adherent shape to small round points as observed under the microscope at 100x magnification, remove the digestive solution. Gently tap the flask wall to detach the cells.
Add two milliliters of DMEM and repeat rinsing the flask wall 10 times. Gently tap the flask wall and transfer one milliliter of the cell suspension to another glass flask. Add five milliliters of DMEM to each glass flask.
Tap the flask wall gently to distribute the cells evenly. Culture the cells at 37 degrees Celsius with 5%carbon dioxide until the cell density is over 80%Proceed to perform the exposure test once enough cells are harvested for the same. Gently tap the flask wall containing the cells to distribute them uniformly and transfer the cell suspensions to a 15 milliliter centrifuge tube.
Dilute the cell suspension with DMEM in combination with cytometry to achieve the desired cell density. Gently tap the flask wall to ensure uniform distribution of cells. Next, add 100 microliters of PBS to the outer wells of the 96 well plate to prevent the culture medium from evaporating due to the edge effect.
Add 100 microliters of the cell suspension to the left wells of the plate and allow it to rest for 10 minutes. Incubate the cells at 37 degrees Celsius with 5%carbon dioxide for 48 hours. After removing the 96 well plate from the incubator, discard the culture medium.
To set up a pesticide metabolite group, add 100 microliters of the different prepared pesticide residue exposure solutions to each designated well, then add 100 microliters of the different prepared parent pesticide exposure solutions to each designated well for comparison, to establish a pesticide parent group. For setting up a blank control, add 100 microliters of DMEM containing 0.3%DMSO to each designated well. Incubate the cells at 37 degrees Celsius with 5%carbon dioxide for 24 hours.
To begin, retrieve the 96 well plate containing the cells from the incubator after allowing a 24 hour exposure to the metabolite extract solutions. Dispose of the culture medium and perform a double wash of the cells using PBS. Introduce 100 microliters of DMEM into each well, followed by the addition of 10 microliters of CCK8 reagents.
Ensure even distribution of the cells by gently shaking the plate. Incubate the cells at 37 degrees Celsius in 5%carbon dioxide for four hours. Finally, conduct an optical density measurement at 450 nanometers using a fluorescence spectrophotometer.
No significant difference was found between the viability of the cells in the pesticide metabolite and control group, indicating no cytotoxicity of pesticide metabolites. The cell viability in the pesticide parent group was significantly lower as compared to the control, indicating obvious cytotoxicity.
