exposure of caco-2 cells to pesticide metabolites from pesticide-treated carrot callus for cytotoxicity study

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.

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.

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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, ...