passage of caco-2 cells to assess cytotoxicity of the pesticide metabolites from pesticide-treated carrot callus

Estimating the Integral Metabolite Cytotoxicity of Triazole Pesticides in Plants

Crop plants growing in farmland may be exposed to various organic pollutants originating from anthropogenic activities1,2. The pollutants can be taken up by plants, further causing threats to the ecosystem and human health through food chains3,4. The xenobiotics in plants probably undergo a series of biotransformation, such as Phase I and II metabolisms5, generating a number of metabolites. According to the green liver concept in plants, plant metabolism can reduce the toxicity of xenobiotics6,7. However, it has been revealed that the toxicity of some metabolites might be higher than that of their parents. For instance, the debrominated product of tetrabromobisphenol A (TBBPA) and the O-methylated product of bisphenol A (BPA) have been proven to be much more toxic than their parents8,9, and the debromination and O-methylation comprise the main Phase I metabolism pathways in plants. Thus, the toxicity assessment solely based on pollutant parents in plants is not accurate, while the corresponding metabolites should be taken into account.
The metabolites of xenobiotics in plants are extremely complex10,11, making it difficult to comprehensively identify and separate them. In addition, only a few standards of identified metabolites can be obtained. Hence, toxicological data of all metabolites are not available, which hinders a comprehensive toxicity assessment. This study proposed a strategy to assess the integral toxicity of pollutant metabolites in plants by treating them as a whole during toxicological tests, providing new data for precise toxicity assessment of pollutants in plants. Our previous study has revealed that plant callus culture opens a simple and effective avenue to obtain metabolites of xenobiotics in plants12. Accordingly, the plant callus culture was employed in this study to generate the metabolites of pollutants in plants, followed by chemical extraction and toxicological tests using a human cell line. The intestinal tract is one of the direct target organs of xenobiotics exposed to animals and humans. Caco-2 cell line has proved to be the best model for investigating the intestinal behaviors and toxicity of xenobiotics in vitro13,14,15. Thus, the Caco-2 cell model was selected in this study.
Triazole pesticides, a class of broad-spectrum fungicides, have been widely applied in agricultural production16. Their residue pollution in farmland has drawn increasing attention17,18. Here, four commonly used triazole pesticides, including flusilazole, diniconazole, tebuconazole, and propiconazole, were selected as the typical pollutants. Carrot was selected in this study as the representative plant for fresh, ready-to-eat vegetables. Carrot callus was initially exposed to the tested pesticides at a concentration of 100 mg/L. After exposure of 72 h, the metabolites were extracted to assess the cytotoxicity using Caco-2 cell line. This method can be readily extended to assess the integral cytotoxicity of metabolites of other types of pollutants in plants.

Author Spotlight: A New and Efficient Method for Comprehensive Metabolite Cytotoxicity Assessment of Triazole Pesticides in Plants

Assessing Cytotoxicity of Metabolites of Typical Triazole Pesticides in Plants

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 standards of metabolites can be obtained.Hence, toxicological data of metabolites are limited, which hinders 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 callus and human cell models are combined to achieve the integral cytotoxicity assessment of metabolites of triazole pesticides in plants.This avoids the complex steps of identification and the separation of metabolites, thereby providing new data for precise, trusted assessment.

Passage of Caco-2 Cells to Assess Cytotoxicity of the Pesticide Metabolites from Pesticide-Treated Carrot Callus

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%

passage-caco-2-cells-to-assess-cytotoxicity-pesticide-metabolites

Research

JoVE Journal

Environment

Various organic pollutants have been released into the environment because of anthropogenic activities. These pollutants can be taken up by crop plants, causing potential threats to the ecosystem and human health throughout the food chain. The biotransformation of pollutants in plants generates a number of metabolites that may be more toxic than their parent compounds, implying that the metabolites should be taken into account during the toxicity assessment. However, the metabolites of pollutants in plants are extremely complex, making it difficult to comprehensively obtain the toxicological information of all metabolites. This study proposed a strategy to assess the integral cytotoxicity of pollutant metabolites in plants by treating them as a whole during toxicological tests. Triazole pesticides, a class of broad-spectrum fungicides, have been widely applied in agricultural production. Their residue pollution in farmland has drawn increasing attention. Hence, four triazole pesticides, including flusilazole, diniconazole, tebuconazole, and propiconazole, were selected as the tested pollutants. The metabolites were generated by the treatment of carrot callus with tested triazole pesticides. After treatment of 72 h, the metabolites of pesticides in carrot callus were extracted, followed by toxicological tests using the Caco-2 cell line. The results showed that the metabolites of tested pesticides in carrot callus did not significantly inhibit the viability of Caco-2 cells (P>0.05), demonstrating no cytotoxicity of pesticide metabolites. This proposed method opens a new avenue to assess the cytotoxicity of pollutant metabolites in plants, which is expected to provide valuable data for precise toxicity assessment.

The protocol describes a new method to assess the integral cytotoxicity of metabolites of triazole pesticides in plants.

Various organic pollutants have been released into the environment because of anthropogenic activities. These pollutants can be taken up by crop plants, ...

Treatment of Carrot Callus with Triazole Pesticides to Assess Cytotoxicity of the Pesticide Metabolites

Begin by treating the collected carrot callus with the different triazole pesticides under sterile conditions. To do so, in separate glass flasks, mix 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 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 callous 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.

Chemical Extraction of Pesticide-Treated Carrot Callus to Assess Cytotoxicity of the Pesticide Metabolites

To begin, mix 0.2 grams of ground powder of each freeze dried, pesticide-treated carrot callous 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, 000G 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 callous 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 apparent pesticide exposure solutions.

Exposure of Caco-2 Cells to Pesticide Metabolites from Pesticide-Treated Carrot Callus for Cytotoxicity Study

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.

Assessing Cell Viability of Caco-2 Cells Treated with Pesticide Metabolite Extracts from Pesticide-Treated Carrot Callus

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 CCK-8 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.