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Assessing Cytotoxicity of Metabolites of Typical Triazole Pesticides in Plants

Published: December 22nd, 2023



1Key Laboratory of Microbial Control Technology for Industrial Pollution in Zhejiang Province, College of Environment, Zhejiang University of Technology, 2Research and Teaching Center of Agriculture, Zhejiang Open University, 3International Joint Research Center for Persistent Toxic Substances (IJRC-PTS), College of Environment, Zhejiang University of Technology

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, 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.

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,....

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1. Differentiation of carrot callus

NOTE: The detailed protocol for differentiation of carrot callus has been described in a previous study12. Here is a brief description.

  1. Sterilize the surface of vernalized seeds with 75% ethanol for 20 min followed by 20% H2O2 for 20 min. Wash the with distilled water at least 3x.
  2. Sow the seeds on hormone-free agar-gelled (1% w/v) Murashige and Skoog (MS) medium (pH =5.8, au.......

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Figure 1 represents the schematic of proposed method for generation, extraction, and cytotoxicity assessment of pesticide metabolites in carrot callus. In Figure 2, the uptake and metabolism kinetics curves of tested pesticides, from which we can find that the concentrations of pesticides in culture media were exponentially decreased, while those in carrot callus began to increase, peaking at 4 or 8 h, followed by a gradual decrease. These results suggested that.......

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This protocol was developed to assess the integral cytotoxicity of metabolites of triazole pesticides in plants by combining plant callus and human cell models. The critical steps for this proposed protocol are the culture of plant callus and Caco-2 cell. The most difficult part and relative advice for plant callus culture have been provided in our previous study12. Here, it should be noted that cell maintenance is the most difficult part for Caco-2 cell culture, because the cells are easily infec.......

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This study was supported by the National Natural Science Foundation of China (21976160) and Zhejiang Province Public Welfare Technology Application Research Project (LGF21B070006).



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NameCompanyCatalog NumberComments
2,4-dichlorophenoxyacetic acidWAKO1 mg/L
20% H2O2Sinopharm Chemical Reagent Co., Ltd.10011218-500ML
6-benzylaminopurineWAKO0.5 mg/L
75% ethanolSinopharm Chemical Reagent Co., Ltd.1269101-500 mL
96-well plateThermo Fisher
Artificial climate incubatorNingbo DongNan Lab Equipment Co.,LtdRDN-1000A-4
Caco-2 cellsNuoyang Biotechnology Co.,Ltd.
CCK8 reagentsNanjing Jiancheng Bioengineering Institute, ChinaG021-1-3
CentrifugeThermo Fisher
CO2 incubatorLabtripHWJ-3-160
Dimethyl sulfoxideSolarbio Life SciencesD8371
Diniconazole, 98.7%J&K Scientific83657-24-3
Dulbecco's modified Eagle's mediumSolarbio Life Sciences11965-500 mL
electronic balanceShanghai Precision Instrument Co., LtdFA1004B
Fetal bovine serumCellmax
Fluorescence spectrophotometerTecanInfinite M200
Flusilazole, 98.5%J&K Scientific85509-19-9  
Freeze dryerSCIENTZ
High-throughput tissue grinderSCIENTZ
Inverted microscopeLeica BiosystemsDMi1
Milli-Q systemMilliporeMS1922801-4L
Murashige & Skoog mediumHOPEBIOHB8469-7
Nitrogen blowing concentratorAOSHENGMD200-2
PBSSolarbio Life SciencesP1022-500 mL
Penicillin-Streptomycin LiquidSolarbio Life SciencesP1400-100 mL
Propiconazole, 100%J&K Scientific60207-90-1 
Research plusEppendorf10-1000 μL
Seeds of Little Finger carrot (Daucus carota var. sativus)Shouguang Seed Industry Co., Ltd
Shaking IncubatorsShanghai bluepard instruments Co.,Ltd.THZ-98AB
Tebuconazole, 100%J&K Scientific107534-96-3
Trypsin-EDTA solutionSolarbio Life SciencesT1300-100 mL
Ultrasound machineZKIUC-6
UV-sterilized super clean benchAIRTECH
Vortex instrumentWuxi Laipu Instrument Equipment Co., LtdBV-1010

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