This study was supported by the National Natural Science Foundation of China (21976160) and Zhejiang Province Public Welfare Technology Application Research Project (LGF21B070006).
&#160; &#160; &#160;

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, autoclaved at 121 &#176;C), and incubate them under 16 h photoperiod (350 &#181;mol/m2s) at 26 &#176;C for 15 days to form the seedlings.</li>
	<li>Harvest the explants by cutting the hypocotyl and cotyledon of seedlings into small pieces (0.5 cm) and incubate the explants in MS medium containing auximone of 1 mg/L 2,4-dichlorophenoxyacetic acid and phytokinin of 0.5 mg/L 6-benzylaminopurine at 26 &#176;C in the dark for 3-4 weeks to induce the callus.</li>
	<li>Collect the callus (around 1 cm diameter, compact) with scalpels and forceps.</li>
</ol>
2. Treatment of carrot callus with pesticides
<ol>
	<li>Dissolve 10 mg each of flusilazole, diniconazole, tebuconazole, and propiconazole in 100 mL of sterile MS medium with final concentrations of 100 mg/L (pH of 5.6-7.0). 
	NOTE: The treatment concentration of tested pesticides was chosen as the maximum of 50% cell growth inhibition concentration (IC50) to Caco-2 cell.</li>
	<li>Mix 3 g of carrot callus (from step 1.4) with 10 mL of prepared pesticide solutions (from step 2.1) in glass flasks under sterile conditions.</li>
	<li>Mix 3 g of carrot callus (from step 1.4) with 10 mL of aseptic MS medium in glass flasks under sterile conditions. 
	NOTE: All glass flasks were autoclaved.</li>
	<li>Incubate the carrot callus (from step 2.2 and 2.3) at 130 rpm and 26 &#176;C in the dark for 72 h. 
	Set all treatments in triplicate.</li>
	<li>Collect the carrot callus from the medium by filtration using glass fiber filters (0.45 &#181;m) after incubation of 72 h. Wash the callus with ultrapure water 3x.</li>
	<li>Freeze-dry the callus using a freeze dryer at -55 &#176;C, and then homogenize them using a high-throughput tissue grinder at 70 Hz for 3 min.</li>
</ol>
3. Chemical extraction of carrot callus
<ol>
	<li>Mix 0.2 g of grounded powder of the freeze-dried callus with 3 mL of acetonitrile in a centrifuge tube (10 mL).</li>
	<li>Vortex the centrifuge tube for 8 min, and then sonicate it for 5 min (150 W, 40 kHz).</li>
	<li>Collect the supernatants by pipetting after centrifugation at 8,000 x g at 4 &#176;C for 10 min.</li>
	<li>Repeat the extraction procedures 3x and pool the extracts in a clean centrifuge tube (10 mL).</li>
	<li>Concentrate the pooled extracts to dryness using a nitrogen blowing concentrator at 40 &#176;C.</li>
	<li>Redissolve the residues of extracts (from step 3.5) with 1 mL of Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) with 0.3% dimethyl sulfoxide (DMSO). 
	NOTE: The residues in step 3.6 were obtained from the extracts of pesticide-treated callus (from step 2.2).</li>
	<li>Redissolve the residues of extracts (from step 3.5) with 1 mL of DMEM with 0.3% DMSO, and dissolve 1 mg of flusilazole, diniconazole, tebuconazole, and propiconazole in 4 different tubes, respectively. 
	NOTE: The residues in step 3.7 were obtained from the extracts of blank callus (from step 2.3). 
	NOTE: DMEM was prepared with 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin and streptomycin).</li>
</ol>
4. Resuscitation of Caco-2 cell
NOTE: All of reagents and materials involved in Caco-2 cell tests were autoclaved for 20 min and sterilized under ultraviolet light for 2 h.
<ol>
	<li>Remove the cryopreservation tube (storing frozen cells) from liquid nitrogen tank and quickly transfer it to a water bath at 37 &#176;C. Continuously shake it to thaw the frozen solution. 
	NOTE: Avoid water flowing over the cover of cryopreservation tube when shaking.</li>
	<li>Disinfect the surface of cryopreservation tube with 75% alcohol after the frozen solution thaws. Quickly transfer the cell solution into a sterile centrifuge tube by pipetting.</li>
	<li>Remove the supernatants in cryopreservation tube after centrifugation at 1,000 x g at 4 &#176;C for 3 min. Add 1 mL of DMEM and suspend the cells by gently tapping and shaking.</li>
	<li>Transfer the cell suspension (from step 4.3) into a glass flask by pipetting. Add 5 mL of DMEM in the glass flask, and gently shake the glass flask to uniformly distribute the cells.</li>
	<li>Incubate the cells in an incubator at 37 &#176;C with 5% CO2. 
	NOTE: Steps 4.2-4.4 were performed in a super clean bench.</li>
</ol>
5. Passage of Caco-2 cell
<ol>
	<li>Remove the glass flask from the incubator and discard the culture medium when the cell density reaches over 80%. Wash the cells with PBS 3x.</li>
	<li>Add 1 mL of trypsin-EDTA solution and uniformly spread it to ensure full contact with cells.</li>
	<li>Remove the digestive solution when the cells shrink from adherent shape to small round point and have not suspended under the observation by microscope (100x magnification). Gently tap the flask wall to cause removal of cells.</li>
	<li>Add 2 mL of DMEM, repeat rinsing of the flask wall 10x, gently tap the flask wall, and transfer 1 mL of cell suspension to another glass flask.</li>
	<li>Add 5 mL of DMEM into glass flasks. Gently tap the flask wall to uniformly distribute the cells.</li>
	<li>Incubate the cells in the incubator at 37 &#176;C with 5% CO2. Replace the culture medium with fresh DMEM every 24 h until the cell density reaches over 80%.</li>
	<li>Repeat the cell passage procedures until there are enough cells (approximately 2 x 106) harvested for the following exposure tests. 
	NOTE: Steps 5.1-5.5 were performed in the super clean bench. The number of cells were determined by a hemocytometer19.</li>
</ol>
6. Exposure of Caco-2 cell
<ol>
	<li>Remove the glass flasks (from step 5.7) from the incubator and repeat steps 5.1-5.4 to collect the cells.</li>
	<li>Gently tap the flask wall to uniformly distribute the cells and transfer the cell suspensions to a centrifuge tube (15 mL).</li>
	<li>Dilute the cell suspension (from step 6.2) with DMEM in combination with cytometry to reach a cell density of approximately 1 x 105 cells/mL. Gently tap the flask wall to uniformly distribute the cells.</li>
	<li>Add 100 &#181;L of PBS in the outer wells of 96-well plate to prevent evaporation of culture medium due to the edge effect. 
	NOTE: Continuously tap the wall of centrifuge tube to keep the cell suspension uniform.</li>
	<li>Add 100 &#181;L of cell suspension (from step 6.3) in the left wells of 96-well plate, and allow to rest for 10 min.</li>
	<li>Incubate the cells in the incubator at 37 &#176;C with 5% CO2 for 48 h.</li>
	<li>Remove the 96-well plate from the incubator after incubation of 48 h and discard the culture medium.</li>
	<li>Set a pesticide metabolite group by adding 100 &#181;L of solutions (from step 3.6) in each well. 
	NOTE: In each well in Step 6.8, there were pesticide metabolites generated from 0.1 mg parents.</li>
	<li>Set a pesticide parent group by adding 100 &#181;L of solutions (from step 3.7) in each well for comparison. 
	NOTE: In each well in Step 6.9, there were 0.1 mg pesticide parents.</li>
	<li>Set a blank control by adding 100 &#181;L of DMEM with 0.3% DMSO in each well. Use six wells for each group (step 6.8-6.9).</li>
	<li>Incubate the cells in the incubator at 37 &#176;C with 5% CO2 for 24 h. 
	NOTE: Steps 6.1-6.5 and 6.7-6.10 were performed in the super clean bench.</li>
</ol>
7. Assessment of cell viability
<ol>
	<li>Remove the 96-well plate (from 6.11) from the incubator and discard the culture medium after exposure of 24 h. Wash the cells with PBS 2x.</li>
	<li>Add 100 &#181;L of DMEM in each well, and then add 10 &#181;L of CCK-8 reagents. Gently shake the plate to uniformly distribute the cells.</li>
	<li>Incubate the cells in the incubator at 37 &#176;C with 5% CO2 for 4 h.</li>
	<li>Remove the 96-well plate from the incubator after incubation of 4 h and measure the optical absorbance (OD) at 450 nm by a fluorescence spectrophotometer.</li>
	<li>Calculate the cell viability by the following equation: 
	cell viability (%) = (ODexperimental&#160;group&#160;- ODDMEM) / (ODcontrol - ODDMEM)&#215;100%. 
	NOTE: Steps 7.1-7.2 were performed in the super clean bench.</li>
</ol>

Estimating the Integral Metabolite Cytotoxicity of Triazole Pesticides in Plants

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	<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. 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The authors have nothing to disclose.

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, ...

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260 Views.  Zhejiang University of Technology. 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 h...

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

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%

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.