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

<ol><li>Fan, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Uptake+of+halogenated+organic+compounds+%28HOCs%29+into+peanut+and+corn+during+the+whole+life+cycle+grown+in+an+agricultural+field%5BTitle%5D)+AND+%22Environ+Pollut%22%5BJournal%5D)">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>
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<li>Liu, A. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tetrabromobisphenol-A%2FS+and+nine+novel+analogs+in+biological+samples+from+the+Chinese+Bohai+Sea%3A+Implications+for+trophic+transfer%5BTitle%5D)+AND+%22Environ+Sci+Technol%22%5BJournal%5D)">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/pubmed?term=((Environmental+pollution+of+electronic+waste+recycling+in+India%3A+A+critical+review%5BTitle%5D)+AND+%22Environ+Pollut%22%5BJournal%5D)">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/pubmed?term=((Plant+accumulation+and+transformation+of+brominated+and+organophosphate+flame+retardants%3A+A+review%5BTitle%5D)+AND+%22Environ+Pollut%22%5BJournal%5D)">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/pubmed?term=((Higher+plant+metabolism+of+xenobiotics%3A+the+%27green+liver%27+concept%5BTitle%5D)+AND+%22Pharmacogenetics%22%5BJournal%5D)">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/pubmed?term=((Metabolism+and+detoxification+of+pesticides+in+plants%5BTitle%5D)+AND+%22Sci+Total+Environ%22%5BJournal%5D)">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/pubmed?term=((Ecotoxicity+of+a+brominated+flame+retardant+%28tetrabromobisphenol+A%29+and+its+derivatives+to+aquatic+organisms%5BTitle%5D)+AND+%22Comp+Biochem+Phys+C%22%5BJournal%5D)">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/pubmed?term=((Microbially+mediated+O-methylation+of+bisphenol+A+results+in+metabolites+with+increased+toxicity+to+the+developing+zebrafish+%28Danio+rerio%29+embryo%5BTitle%5D)+AND+%22Environ+Sci+Technol%22%5BJournal%5D)">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/pubmed?term=((Multiple+metabolic+pathways+of+2%2C4%2C6-tribromophenol+in+rice+plants%5BTitle%5D)+AND+%22Environ+Sci+Technol%22%5BJournal%5D)">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/pubmed?term=((Glycosylation+of+tetrabromobisphenol+A+in+pumpkin%5BTitle%5D)+AND+%22Environ+Sci+Technol%22%5BJournal%5D)">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/pubmed?term=((Elucidating+the+metabolism+of+2%2C4-Dibromophenol+in+plants%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">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/pubmed?term=((An+Inter-laboratory+study+to+evaluate+the+effects+of+medium+composition+on+the+differentiation+and+barrier+function+of+Caco-2+cell+lines%5BTitle%5D)+AND+%22Atla-Altern+Lab+Anim%22%5BJournal%5D)">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/pubmed?term=((Methods+of+in+vitro+toxicology%5BTitle%5D)+AND+%22Food+Chem+Toxicol%22%5BJournal%5D)">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/pubmed?term=((The+Caco-2+cell+line+as+a+model+of+the+intestinal+barrier%3A+influence+of+cell+and+culture-related+factors+on+Caco-2+cell+functional+characteristics%5BTitle%5D)+AND+%22Cell+Biol+Toxicol%22%5BJournal%5D)">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/pubmed?term=((Uptake%2C+translocation%2C+and+subcellular+distribution+of+three+triazole+pesticides+in+rice%5BTitle%5D)+AND+%22Environ+Sci+Pollut+Res%22%5BJournal%5D)">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/pubmed?term=((Influence+of+triazole+pesticides+on+tillage+soil+microbial+populations+and+metabolic+changes%5BTitle%5D)+AND+%22Sci+Total+Environ%22%5BJournal%5D)">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/pubmed?term=((A+possible+but+unrecognized+risk+of+acceptable+daily+intake+dose+triazole+pesticides+exposure-bile+acid+disturbance+induced+pharmacokinetic+changes+of+oral+medication%5BTitle%5D)+AND+%22Chemosphere%22%5BJournal%5D)">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/pubmed?term=((Cell+viability+analysis+using+trypan+blue%3A+manual+and+automated+methods%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Cell viability analysis using trypan blue: manual and automated methods.</a> Methods Mol Biol. 740, 7-12 (2011).</li>
</ol>

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><tbody><tr><td>2,4-dichlorophenoxyacetic acid</td><td>WAKO</td><td>1 mg/L</td><td></td></tr><tr><td>20% H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;</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>CO&lt;sub&gt;2&lt;/sub&gt; 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&amp;amp;K Scientific</td><td>83657-24-3</td><td></td></tr><tr><td>Dulbecco&#039;s modified Eagle&#039;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&amp;amp;K Scientific</td><td>85509-19-9&amp;nbsp;&amp;nbsp;</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 &amp;amp; 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&amp;amp;K Scientific</td><td>60207-90-1&amp;nbsp;</td><td></td></tr><tr><td>Research plus</td><td>Eppendorf</td><td>10-1000 &amp;mu;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&amp;amp;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.

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

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

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, ...

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494 Views.  Zhejiang University of Technology. Ons protocol beschrijft een nieuwe methode voor het beoordelen van de integrale cytotoxiciteit van metabolieten van triazolpesticiden in planten. Plantaardige eeltcultuur wordt gebruikt om de metabolieten van eiwitten in planten te genereren, gevolgd door toxicologische tests met behulp van een menselijke cellijn om de integrale cytotoxiciteit van metabolieten te beoordelen. De metabolieten van xenobiotica in planten zijn uiterst complex en er kunnen slechts ...

Video: Auteur in de schijnwerpers: een nieuwe en efficiënte methode voor uitgebreide beoordeling van de cytotoxiciteit van metabolieten van triazolpesticiden in planten

Anticancer Metal Complexes: Synthesis and Cytotoxicity Evaluation by the MTT Assay

Titanium (IV) and vanadium (V) complexes are highly potent anticancer agents. A challenge in their synthesis refers to their hydrolytic instability; therefore their preparation should be conducted under an inert atmosphere. Evaluation of the anticancer activity of these complexes can be achieved by the MTT assay.
The MTT assay is a colorimetric viability assay based on enzymatic reduction of the MTT molecule to formazan when it is exposed to viable cells. The outcome of the reduction is a color change of the MTT molecule. Absorbance measurements relative to a control determine the percentage of remaining viable cancer cells following their treatment with varying concentrations of a tested compound, which is translated to the compound anticancer activity and its IC50 values. The MTT assay is widely common in cytotoxicity studies due to its accuracy, rapidity, and relative simplicity.
Herein we present a detailed protocol for the synthesis of air sensitive metal based drugs and cell viability measurements, including preparation of the cell plates, incubation of the compounds with the cells, viability measurements using the MTT assay, and determination of IC50 values.

A method for synthesis of air-sensitive titanium and vanadium anticancer agents is described, along with the evaluation of their cytotoxic activity towards human cancer cell line by the MTT Assay.

Titanium (IV) and vanadium (V) complexes are highly potent anticancer agents. A challenge in their synthesis refers to their hydrolytic instability; ...

Chemistry

Measuring Rates of Herbicide Metabolism in Dicot Weeds with an Excised Leaf Assay

In order to isolate and accurately determine rates of herbicide metabolism in an obligate-outcrossing dicot weed, waterhemp (Amaranthus tuberculatus), we developed an excised leaf assay combined with a vegetative cloning strategy to normalize herbicide uptake and remove translocation as contributing factors in herbicide-resistant (R) and &#8211;sensitive (S) waterhemp populations. Biokinetic analyses of organic pesticides in plants typically include the determination of uptake, translocation (delivery to the target site), metabolic fate, and interactions with the target site. Herbicide metabolism is an important parameter to measure in herbicide-resistant weeds and herbicide-tolerant crops, and is typically accomplished with whole-plant tests using radiolabeled herbicides. However, one difficulty with interpreting biokinetic parameters derived from whole-plant methods is that translocation is often affected by rates of herbicide metabolism, since polar metabolites are usually not mobile within the plant following herbicide detoxification reactions. Advantages of the protocol described in this manuscript include reproducible, accurate, and rapid determination of herbicide degradation rates in R and S populations, a substantial decrease in the amount of radiolabeled herbicide consumed, a large reduction in radiolabeled plant materials requiring further handling and disposal, and the ability to perform radiolabeled herbicide experiments in the lab or growth chamber instead of a greenhouse. As herbicide resistance continues to develop and spread in dicot weed populations worldwide, the excised leaf assay method developed and described herein will provide an invaluable technique for investigating non-target site-based resistance due to enhanced rates of herbicide metabolism and detoxification.

This manuscript describes how herbicide metabolism rates can be effectively quantified with excised leaves from a dicot weed, thereby reducing variability and removing any possible confounding effects of herbicide uptake or translocation typically observed in whole-plant assays.

In order to isolate and accurately determine rates of herbicide metabolism in an obligate-outcrossing dicot weed, waterhemp (Amaranthus tuberculatus), we ...

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea (Camellia Sinensis L.) Leaves

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form loose callus on Murashige and Skoog (MS) basal media with the plant hormones 2,4-dichlorophenoxyacetic acid (2,4-D, 1.0 mg L-1) and kinetin (KT, 0.1 mg L-1). Callus formed after 3 or 4 rounds of subculturing, each lasting 28 days. Loose callus (about 3 g) was then inoculated into B5 liquid media containing the same plant hormones and was cultured in a shaking incubator (120 rpm) in the dark at 25 &#177; 1 &#176;C. After 3&#8722;4 subcultures, a cell suspension derived from tea leaf was established at a subculture ratio ranging between 1:1 and 1:2 (suspension mother liquid: fresh medium). Using this platform, six insecticides (5 &#181;g mL-1 each thiamethoxam, imidacloprid, acetamiprid, imidaclothiz, dimethoate, and omethoate) were added into the tea leaf-derived cell suspension culture. The metabolism of the insecticides was tracked using liquid chromatography and gas chromatography. To validate the usefulness of the tea cell suspension culture, the metabolites of thiamethoxan and dimethoate present in treated cell cultures and intact plants were compared using mass spectrometry. In treated tea cell cultures, seven metabolites of thiamethoxan and two metabolites of dimethoate were found, while in treated intact plants, only two metabolites of thiamethoxam and one of dimethoate were found. The use of a cell suspension simplified the metabolic analysis compared to the use of intact tea plants, especially for a difficult matrix such as tea.

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea Camellia Sinensis L. Leaves

This work presents a protocol for establishing a cell suspension culture derived from tea (Camellia sinensis L.) leaves that can be used to study the metabolism of external compounds that can be taken up by the whole plant, such as insecticides.

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form ...

Biochemistry

A Strategy to Identify Compounds that Affect Cell Growth and Survival in Cultured Mammalian Cells at Low-to-Moderate Throughput

Cytotoxicity is a critical parameter that needs to be quantified when studying drugs that may have therapeutic benefits. Because of this, many drug screening assays utilize cytotoxicity as one of the critical characteristics to be profiled for individual compounds. Cells in culture are a useful model to assess cytotoxicity before proceeding to follow up on promising lead compounds in more costly and labor-intensive animal models. We describe a strategy to identify compounds that affect cell growth in a tdTomato expressing human neural stem cells (NSC) line. The strategy uses two complementary assays to assess cell number. One assay works via the reduction of 3-(4,5-dimethylthizol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan as a proxy for cell number and the other directly counts the tdTomato expressing NSCs. The two assays can be performed simultaneously in a single experiment and are not labor intensive, rapid, and inexpensive. The strategy described in this demonstration tested 57 compounds in an exploratory primary screen for toxicity in a 96-well plate format. Three of the hits were characterized further in a six-point dose response using the same assay set-up as the primary screen. In addition to providing excellent corroboration for toxicity, comparison of results from the two assays may be effective in identifying compounds affecting other aspects of cell growth.

It is often necessary to assess the potential cytotoxicity of a set of compounds on cultured cells. Here, we describe a strategy to reliably screen for toxic compounds in a 96-well format.

Cytotoxicity is a critical parameter that needs to be quantified when studying drugs that may have therapeutic benefits. Because of this, many drug ...

An Automated Differential Nuclear Staining Assay for Accurate Determination of Mitocan Cytotoxicity

The contribution of mitochondria to oncogenic transformation is a subject of wide interest and active study. As the field of cancer metabolism becomes more complex, the goal of targeting mitochondria using various compounds that inflict mitochondrial damage (so-called mitocans) is becoming quite popular. Unfortunately, many existing cytotoxicity assays, such as those based on tetrazolium salts or resazurin require functional mitochondrial enzymes for their performance. The damage inflicted by compounds that target mitochondria often compromises the accuracy of these assays. Here, we describe a modified protocol based on differential staining with two fluorescent dyes, one of which is cell-permeant (Hoechst 33342) and the other of which is not (propidium iodide). The difference in staining allows living and dead cells to be discriminated. The assay is amenable to automated microscopy and image analysis, which increases throughput and reduces bias. This also allows the assay to be used in high-throughput fashion using 96-well plates, making it a viable option for drug discovery efforts, particularly when the drugs in question have some level of mitotoxicity. Importantly, results obtained by Hoechst/PI staining assay show increased consistency, both with trypan blue exclusion results and between biological replicates when the assay is compared to other methods.

The protocol describes a rapid, high-throughput, reliable, inexpensive, and unbiased assay for efficiently determining cellular viability. This assay is particularly useful when cells&#39; mitochondria have been damaged, which interferes with other assays. The assay uses automated counting of cells stained with two nuclear dyes &#8211; Hoechst 33342 and propidium iodide.

The contribution of mitochondria to oncogenic transformation is a subject of wide interest and active study. As the field of cancer metabolism becomes ...

Cancer Research

Cytotoxicity Assays with Zebrafish Cell Lines

Fish cell lines have become increasingly used in ecotoxicity studies, and cytotoxicity assays have been proposed as methods to predict fish acute toxicity. Thus, this protocol presents cytotoxicity assays modified to evaluate cell viability in zebrafish (Danio rerio) embryo (ZEM2S) and liver (ZFL) cell lines in 96-well plates. The cytotoxicity endpoints evaluated are mitochondrial integrity (Alamar Blue [AB] and MTT assays), membrane integrity via esterase activity (CFDA-AM assay), and lysosomal membrane integrity (Neutral Red [NR] assay). After the exposure of the test substances in a 96-well plate, the cytotoxicity assays are performed; here, AB and CFDA-AM are carried out simultaneously, followed by NR on the same plate, while the MTT assay is performed on a separate plate. The readouts for these assays are taken by fluorescence for AB and CFDA-AM, and absorbance for MTT and NR. The cytotoxicity assays performed with these fish cell lines can be used to study the acute toxicity of chemical substances on fish.

This protocol presents commonly used cytotoxicity assays (Alamar Blue [AB], CFDA-AM, Neutral Red, and MTT assays) adapted for the assessment of cytotoxicity in zebrafish embryo (ZEM2S) and liver (ZFL) cell lines in 96-well plates.

Fish cell lines have become increasingly used in ecotoxicity studies, and cytotoxicity assays have been proposed as methods to predict fish acute ...

Elucidating the Metabolism of 2,4-Dibromophenol in Plants

Crops can be extensively exposed to organic pollutants, since soil is a major sink for pollutants discarded into the environment. This creates potential human exposure through the consumption of pollutant-accumulated foods. Elucidating the uptake and metabolism of xenobiotics in crops is essential for the assessment of dietary exposure risk in humans. However, for such experiments, the use of intact plants requires long-term experiments and complex sample preparation protocols that can be affected by various factors. Plant callus cultures combined with high-resolution mass spectrometry (HRMS) may provide a solution for the accurate and time-saving identification of metabolites of xenobiotics in plants, as it can avoid interference from the microbial or fungal microenvironment, shorten the treatment duration, and simplify the matrix effect of intact plants. 2,4-dibromophenol, a typical flame retardant and endocrine disrupter, was chosen as the model substance due to its widespread occurrence in soil and its uptake potential by plants. Herein, plant callus was generated from asepsis seeds and exposed to sterile 2,4-dibromophenol-containing culture medium. The results showed that eight metabolites of 2,4-dibromophenol were identified in the plant callus tissues after 120 h of incubation. This indicates that 2,4-dibromophenol was rapidly metabolized in&#160;the plant callus tissues. Thus, the plant callus culture platform is an effective method to evaluate the uptake and metabolism of xenobiotics in plants.

The present protocol describes a simple and efficient method for the identification of 2,4-dibromophenol metabolites in plants.

Crops can be extensively exposed to organic pollutants, since soil is a major sink for pollutants discarded into the environment. This creates potential ...

Protocols for Testing the Toxicity of Novel Insecticidal Chemistries to Mosquitoes

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such as Zika, dengue and malaria. Assays for rapid, high-throughput analyses of unformulated novel chemistries against mosquito larvae and adults are presented. We describe protocols for single point-dose and dose response assays to evaluate the toxicity of small molecule chemistries to the Aedes aegypti vector of Zika, dengue and yellow fever,&#160;the malaria vector, Anopheles gambiae and the northern house mosquito, Culex quinquefasciatus, on contact and via ingestion. As an example, we evaluated the toxicity of amitriptyline, a small molecule antagonist of G protein-coupled receptors, via larval, adult topical and adult blood-feeding assay. The protocols provide a starting point to investigate insecticide potential. Results are discussed in the context of additional experiments to explore product applications and mechanisms for delivery.

Protocols are described for assessing the toxicity of chemistries to immature and adult mosquitoes for development as new classes of larvicides, adulticides and endectocides. The protocols enable high-throughput testing of multiple chemistries at single-point dose and subsequent evaluation via dose response assay to determine toxicity on contact or via ingestion.

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such ...

Biology

Functionalized Spirocyclic Heterocycle Synthesis and Cytotoxicity Assay

Spirocyclic heterocycles have recently been reported in literature to be potential drugs for cancer therapy. The synthesis of these novel orthogonal ring systems is challenging. An efficient methodology to synthesize these compounds was recently published that described the solid phase synthesis in four steps rather than the previously reported five steps. The advantage of this shorter synthesis is the elimination of the use of toxic reagents. Low-loading Regenerating Michael (REM) linker-based resin was found to be crucial in the synthesis as high-loading versions prevented the addition of reagents containing bulky phenyl and aromatic side chains. The colorimetric 3-(4&#8242;,5&#8242;-dimethylthiazol-2&#8242;-yl)-2,5- diphenyltetrazolium bromide (MTT) assay was used to examine the cytotoxicity of micromolar concentrations of these novel spirocyclic molecules in vitro. MTT is readily available commercially and produces relatively fast, reliable results, making this assay ideal for these spirocyclic heterocycles. Orthogonal ring structures as well as furfurylamine (a precursor in the synthesis method containing a similar 5-member ring motif) were tested.

Here, we describe a bioassay using 3-(4&#8242;,5&#8242;-dimethylthiazol-2&#8242;-yl)-2,5- diphenyltetrazolium bromide (MTT) to test previously synthesized spirocyclic oximes.

Spirocyclic heterocycles have recently been reported in literature to be potential drugs for cancer therapy. The synthesis of these novel orthogonal ring ...

Biological Assessment of Thiophenesulfonamides in Quorum Sensing Inhibition

Synthesis and Assay of Vibrio Quorum Sensing Inhibitors

Bacteria detect local population numbers using quorum sensing, a method of cell-cell communication broadly utilized to control bacterial behaviors. In Vibrio species, the master quorum sensing regulators LuxR/HapR control hundreds of quorum sensing genes, many of which influence virulence, metabolism, motility, and more. Thiophenesulfonamides are potent inhibitors of LuxR/HapR that bind the ligand pocket in these transcription factors and block downstream quorum sensing gene expression. This class of compounds served as the basis for the development of a set of simple, robust, and educational procedures for college students to assimilate their chemistry and biology skills using a CURE model: course-based undergraduate research experience. Optimized protocols are described that comprise three learning stages in an iterative and multi-disciplinary platform to engage students in a year-long CURE: (1) design and synthesize new small molecule inhibitors based on the thiophenesulfonamide core, (2) use structural modeling to predict binding affinity to the target, and (3) assay the compounds for efficacy in microbiological assays against specific Vibrio LuxR/HapR proteins. The described reporter assay performed in E. coli successfully predicts the efficacy of the compounds against target proteins in the native Vibrio species.

Author Spotlight: Advancing Therapeutics to Treat Vibriosis in Humans and Aquatic Organisms

Thiophenesulfonamide compounds are potent and specific inhibitors of Vibrio quorum sensing regulators LuxR/HapR that block their activity in vivo, thus preventing transcription of genes for virulence, motility, and biofilms. This protocol details how these compounds are synthesized, modeled in silico, and assayed in vivo for activity against LuxR/HapR.

Bacteria detect local population numbers using quorum sensing, a method of cell-cell communication broadly utilized to control bacterial behaviors. In ...

Assessing Cytotoxicity of Metabolites of Typical Triazole Pesticides in Plants

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