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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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.
<ol>
	<li>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.</li>
	<li>Sow the seeds on hormone-free agar-gelled (1% w/v) Murashige and Skoog (MS) medium (pH =5.8, au...

Estimating the Integral Metabolite Cytotoxicity of Triazole Pesticides in Plants

<ol>
	<li>Fan, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uptake+of+halogenated+organic+compounds+(HOCs)+into+peanut+and+corn+during+the+whole+life+cycle+grown+in+an+agricultural+field.">Uptake of halogenated organic compounds (HOCs) into peanut and corn during the whole life cycle grown in an agricultural field.</a> Environ Pollut. 263, 114400 (2020).</li><li>Wu, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trace+metals+in+e-waste+lead+to+serious+health+risk+through+consumption+of+rice+growing+near+an+abandoned+e-waste+recycling+site:+Comparisons+with+PBDEs+and+AHFRs.">Trace metals in e-waste lead to serious health risk through consumption of rice growing near an abandoned e-waste recycling site: Comparisons with PBDEs and AHFRs.</a> Environ Pollut. 247, 46-54 (2019).</li><li>Liu, A. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tetrabromobisphenol-A/S+and+nine+novel+analogs+in+biological+samples+from+the+Chinese+Bohai+Sea:+Implications+for+trophic+transfer.">Tetrabromobisphenol-A/S and nine novel analogs in biological samples from the Chinese Bohai Sea: Implications for trophic transfer.</a> Environ Sci Technol. 50 (8), 4203-4211 (2016).</li><li>Awasthi, A. K., Zeng, X., Li, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+pollution+of+electronic+waste+recycling+in+India:+A+critical+review.">Environmental pollution of electronic waste recycling in India: A critical review.</a> Environ Pollut. 211, 259-270 (2016).</li><li>Zhang, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+accumulation+and+transformation+of+brominated+and+organophosphate+flame+retardants:+A+review.">Plant accumulation and transformation of brominated and organophosphate flame retardants: A review.</a> Environ Pollut. 288, 117742 (2021).</li><li>Sandermann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Higher+plant+metabolism+of+xenobiotics:+the+'green+liver'+concept.">Higher plant metabolism of xenobiotics: the 'green liver' concept.</a> Pharmacogenetics. 4, 225-241 (1994).</li><li>Zhang, J. J., Yang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolism+and+detoxification+of+pesticides+in+plants.">Metabolism and detoxification of pesticides in plants.</a> Sci Total Environ. 790, 148034 (2021).</li><li>Debenest, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ecotoxicity+of+a+brominated+flame+retardant+(tetrabromobisphenol+A)+and+its+derivatives+to+aquatic+organisms.">Ecotoxicity of a brominated flame retardant (tetrabromobisphenol A) and its derivatives to aquatic organisms.</a> Comp Biochem Phys C. 152 (4), 407-412 (2010).</li><li>McCormick, J. M., Van Es, T., Cooper, K. R., White, L. A., Haggblom, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbially+mediated+O-methylation+of+bisphenol+A+results+in+metabolites+with+increased+toxicity+to+the+developing+zebrafish+(Danio+rerio)+embryo.">Microbially mediated O-methylation of bisphenol A results in metabolites with increased toxicity to the developing zebrafish (Danio rerio) embryo.</a> Environ Sci Technol. 45 (15), 6567-6574 (2011).</li><li>Zhang, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+metabolic+pathways+of+2,4,6-tribromophenol+in+rice+plants.">Multiple metabolic pathways of 2,4,6-tribromophenol in rice plants.</a> Environ Sci Technol. 53 (13), 7473-7482 (2019).</li><li>Hou, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycosylation+of+tetrabromobisphenol+A+in+pumpkin.">Glycosylation of tetrabromobisphenol A in pumpkin.</a> Environ Sci Technol. 53 (15), 8805-8812 (2019).</li><li>Wu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elucidating+the+metabolism+of+2,4-Dibromophenol+in+plants.">Elucidating the metabolism of 2,4-Dibromophenol in plants.</a> J Vis Exp. (192), e65089 (2023).</li><li>Zucco, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Inter-laboratory+study+to+evaluate+the+effects+of+medium+composition+on+the+differentiation+and+barrier+function+of+Caco-2+cell+lines.">An Inter-laboratory study to evaluate the effects of medium composition on the differentiation and barrier function of Caco-2 cell lines.</a> Atla-Altern Lab Anim. 33, 603-618 (2005).</li><li>Eisenbrand, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+of+in+vitro+toxicology.">Methods of in vitro toxicology.</a> Food Chem Toxicol. 40, 193-236 (2002).</li><li>Sambuy, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Caco-2+cell+line+as+a+model+of+the+intestinal+barrier:+influence+of+cell+and+culture-related+factors+on+Caco-2+cell+functional+characteristics.">The Caco-2 cell line as a model of the intestinal barrier: influence of cell and culture-related factors on Caco-2 cell functional characteristics.</a> Cell Biol Toxicol. 21, 1-26 (2005).</li><li>Li, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uptake,+translocation,+and+subcellular+distribution+of+three+triazole+pesticides+in+rice.">Uptake, translocation, and subcellular distribution of three triazole pesticides in rice.</a> Environ Sci Pollut Res. 29 (17), 25581-25590 (2022).</li><li>Satapute, P., Kamble, M. V., Adhikari, S. S., Jogaiah, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+triazole+pesticides+on+tillage+soil+microbial+populations+and+metabolic+changes.">Influence of triazole pesticides on tillage soil microbial populations and metabolic changes.</a> Sci Total Environ. 651, 2334-2344 (2019).</li><li>Xu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+possible+but+unrecognized+risk+of+acceptable+daily+intake+dose+triazole+pesticides+exposure-bile+acid+disturbance+induced+pharmacokinetic+changes+of+oral+medication.">A possible but unrecognized risk of acceptable daily intake dose triazole pesticides exposure-bile acid disturbance induced pharmacokinetic changes of oral medication.</a> Chemosphere. 322, 138209 (2023).</li><li>Louis, K. S., Siegel, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+viability+analysis+using+trypan+blue:+manual+and+automated+methods.">Cell viability analysis using trypan blue: manual and automated methods.</a> Methods Mol Biol. 740, 7-12 (2011).</li></ol>

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...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2,4-dichlorophenoxyacetic acid</td>
			<td>WAKO</td>
			<td>1 mg/L</td>
			<td></td>
		</tr>
		<tr>
			<td>20% H2O2</td>
			<td>Sinopharm Chemical Reagent Co., Ltd.</td>
			<td>10011218-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>6-benzylaminopurine</td>
			<td>WAKO</td>
			<td>0.5 mg/L</td>
			<td></td>
		</tr>
		<tr>
			<td>75% ethanol</td>
			<td>Sinopharm Chemical Reagent Co., Ltd.</td>
			<td>1269101-500 mL</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate</td>
			<td>Thermo Fisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Artificial climate incubator</td>
			<td>Ningbo DongNan Lab Equipment Co.,Ltd</td>
			<td>RDN-1000A-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Autoclaves</td>
			<td>STIK</td>
			<td>MJ-Series</td>
			<td></td>
		</tr>
		<tr>
			<td>Caco-2 cells</td>
			<td>Nuoyang Biotechnology Co.,Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CCK8 reagents</td>
			<td>Nanjing Jiancheng Bioengineering Institute, China</td>
			<td>G021-1-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Thermo Fisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td>Labtrip</td>
			<td>HWJ-3-160</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>Solarbio Life Sciences</td>
			<td>D8371</td>
			<td></td>
		</tr>
		<tr>
			<td>Diniconazole, 98.7%</td>
			<td>J&#38;K Scientific</td>
			<td>83657-24-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified Eagle&#39;s medium</td>
			<td>Solarbio Life Sciences</td>
			<td>11965-500 mL</td>
			<td></td>
		</tr>
		<tr>
			<td>electronic balance</td>
			<td>Shanghai Precision Instrument Co., Ltd</td>
			<td>FA1004B</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Cellmax</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence spectrophotometer</td>
			<td>Tecan</td>
			<td>Infinite M200</td>
			<td></td>
		</tr>
		<tr>
			<td>Flusilazole, 98.5%</td>
			<td>J&#38;K Scientific</td>
			<td>85509-19-9&#160;&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Freeze dryer</td>
			<td>SCIENTZ</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>High-throughput tissue grinder</td>
			<td>SCIENTZ</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Leica Biosystems</td>
			<td>DMi1</td>
			<td></td>
		</tr>
		<tr>
			<td>Milli-Q system</td>
			<td>Millipore</td>
			<td>MS1922801-4L</td>
			<td></td>
		</tr>
		<tr>
			<td>Murashige &#38; Skoog medium</td>
			<td>HOPEBIO</td>
			<td>HB8469-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrogen blowing concentrator</td>
			<td>AOSHENG</td>
			<td>MD200-2</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Solarbio Life Sciences</td>
			<td>P1022-500 mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin Liquid</td>
			<td>Solarbio Life Sciences</td>
			<td>P1400-100 mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Propiconazole, 100%</td>
			<td>J&#38;K Scientific</td>
			<td>60207-90-1&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Research plus</td>
			<td>Eppendorf</td>
			<td>10-1000 &#956;L</td>
			<td></td>
		</tr>
		<tr>
			<td>Seeds of Little Finger carrot (Daucus carota var. sativus)</td>
			<td>Shouguang Seed Industry Co., Ltd</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Shaking Incubators</td>
			<td>Shanghai bluepard instruments Co.,Ltd.</td>
			<td>THZ-98AB</td>
			<td></td>
		</tr>
		<tr>
			<td>Tebuconazole, 100%</td>
			<td>J&#38;K Scientific</td>
			<td>107534-96-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA solution</td>
			<td>Solarbio Life Sciences</td>
			<td>T1300-100 mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasound machine</td>
			<td>ZKI</td>
			<td>UC-6</td>
			<td></td>
		</tr>
		<tr>
			<td>UV-sterilized super clean bench</td>
			<td>AIRTECH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex instrument</td>
			<td>Wuxi Laipu Instrument Equipment Co., Ltd</td>
			<td>BV-1010</td>
			<td></td>
		</tr>
	</tbody>
</table>

assessing cytotoxicity of metabolites of typical 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.

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.

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...

Read this Scientific Article Protocol about Assessing Cytotoxicity of Metabolites of Typical Triazole Pesticides in Plants at JoVE.com

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, ...

assessing-cytotoxicity-metabolites-typical-triazole-pesticides