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

1. Differentiation of carrot callus
NOTE: Autoclave all equipment used here and perform all operations in a UV-sterilized ultra-clean workbench.
<ol>
	<li>Vernalize the seeds by immersing the uniform carrot seeds (Daucus carota var. sativus) into deionized water at 4 &#176;C for 16 h.</li>
	<li>Surface-sterilize the vernalized seeds with 75% ethanol for 20 min, and then rinse three times with sterile deionized water under aseptic conditions.</li>
	<li>Further steril...

<ol>
	<li>Chakraborty, P., 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=Baseline+investigation+on+plasticizers,+bisphenol+A,+polycyclic+aromatic+hydrocarbons+and+heavy+metals+in+the+surface+soil+of+the+informal+electronic+waste+recycling+workshops+and+nearby+open+dumpsites+in+Indian+metropolitan+cities.">Baseline investigation on plasticizers, bisphenol A, polycyclic aromatic hydrocarbons and heavy metals in the surface soil of the informal electronic waste recycling workshops and nearby open dumpsites in Indian metropolitan cities.</a> Environmental Pollution. 248, 1036-1045 (2019).</li><li>Abril, C., Santos, J. 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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=Occurrence+of+nonylphenol+and+nonylphenol+monoethoxylate+in+soil+and+vegetables+from+vegetable+farms+in+the+Pearl+River+Delta,+South+China.">Occurrence of nonylphenol and nonylphenol monoethoxylate in soil and vegetables from vegetable farms in the Pearl River Delta, South China.</a> Archives of Environmental Contamination and Toxicology. 63 (1), 22-28 (2012).</li><li>Wang, S. 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=et+al+Migration+and+health+risks+of+nonylphenol+and+bisphenol+a+in+soil-winter+wheat+systems+with+long-term+reclaimed+water+irrigation.">et al Migration and health risks of nonylphenol and bisphenol a in soil-winter wheat systems with long-term reclaimed water irrigation.</a> Ecotoxicology and Environmental Safety. 158, 28-36 (2018).</li><li>Gunther, K., Racker, T., Bohme, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+isomer-specific+approach+to+endocrine-disrupting+nonylphenol+in+infant+food.">An isomer-specific approach to endocrine-disrupting nonylphenol in infant food.</a> Journal of Agricultural and Food Chemistry. 65 (6), 1247-1254 (2017).</li><li>Van Eerd, L. L., Hoagland, R. E., Zablotowicz, R. M., Hall, J. 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Merr., Zea mays L., and Triticum aestivum L.</a> Journal of Agricultural and Food Chemistry. 68 (48), 14123-14134 (2020).</li><li>Macherius, A., 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=Metabolization+of+the+bacteriostatic+agent+triclosan+in+edible+plants+and+its+consequences+for+plant+uptake+assessment.">Metabolization of the bacteriostatic agent triclosan in edible plants and its consequences for plant uptake assessment.</a> Environmental Science &amp; Technology. 46 (19), 10797-10804 (2012).</li><li>Sun, J. 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=Uptake+and+metabolism+of+nonylphenol+in+plants:+Isomer+selectivity+involved+with+direct+conjugation.">Uptake and metabolism of nonylphenol in plants: Isomer selectivity involved with direct conjugation.</a> Environmental Pollution. 270, 116064 (2021).</li><li>Schymanski, E. L., 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=Identifying+small+molecules+via+high+resolution+mass+spectrometry:+communicating+confidence.">Identifying small molecules via high resolution mass spectrometry: communicating confidence.</a> Environmental Science &amp; Technology. 48 (4), 2097-2098 (2014).</li><li>Moschet, C., Anumol, T., Lew, B. M., Bennett, D. H., Young, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Household+dust+as+a+repository+of+chemical+accumulation:+new+insights+from+a+comprehensive+high-resolution+mass+spectrometric+study.">Household dust as a repository of chemical accumulation: new insights from a comprehensive high-resolution mass spectrometric study.</a> Environmental Science &amp; Technology. 52 (5), 2878-2887 (2018).</li><li>Ren, Z., 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=Hydroxylated+PBDEs+and+brominated+phenolic+compounds+in+particulate+matters+emitted+during+recycling+of+waste+printed+circuit+boards+in+a+typical+e-waste+workshop+of+South+China.">Hydroxylated PBDEs and brominated phenolic compounds in particulate matters emitted during recycling of waste printed circuit boards in a typical e-waste workshop of South China.</a> Environmental Pollution. 177, 71-77 (2013).</li><li>de Wit, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+overview+of+brominated+flame+retardants+in+the+environment.">An overview of brominated flame retardants in the environment.</a> Chemosphere. 46 (5), 583-624 (2002).</li><li>Sun, J. Q., Chen, Q., Qian, Z. X., Zheng, Y., Yu, S. A., Zhang, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+Uptake+and+Metabolism+of+e,4-Dibromophenol+in+Carrot:+In+Vitro+Enzymatic+Direct+Conjugation.">Plant Uptake and Metabolism of e,4-Dibromophenol in Carrot: In Vitro Enzymatic Direct Conjugation.</a> Journal of Agricultural and Food Chemistry. 66 (17), 4328-4335 (2018).</li><li>Chibwe, L., Titaley, I. A., Hoh, E., Simonich, S. L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrated+framework+for+identifying+toxic+transformation+products+in+complex+environmental+mixtures.">Integrated framework for identifying toxic transformation products in complex environmental mixtures.</a> Environmental Science &amp; Technology Letters. 4 (2), 32-43 (2017).</li><li>Hollender, J., Schymanski, E. L., Singer, H. P., Ferguson, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nontarget+screening+with+high+resolution+mass+spectrometry+in+the+environment:+ready+to+go.">Nontarget screening with high resolution mass spectrometry in the environment: ready to go.</a> Environmental Science &amp; Technology. 51 (20), 11505-11512 (2017).</li><li>Nafisi, M., Fimognari, L., Sakuragi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interplays+between+the+cell+wall+and+phytohormones+in+interaction+between+plants+and+necrotrophic+pathogens.">Interplays between the cell wall and phytohormones in interaction between plants and necrotrophic pathogens.</a> Phytochemistry. 112, 63-71 (2015).</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> Environmental Science &amp; Technology. 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> Environmental Science &amp; Technology. 53 (15), 8805-8812 (2019).</li></ol>

The authors have nothing to disclose.

This protocol was developed to efficiently identify the biotransformation of xenobiotics in plants. The critical step of this protocol is the culture of the plant callus. The most difficult part is the differentiation and maintenance of the plant callus, because the plant callus is easily infected and developed to plant tissues. Therefore, it is important to make sure that all equipment used is autoclaved, and all operations are performed under aseptic conditions. The differentiation and maintenance of the plant callus s...

An increasing number of organic pollutants have been discarded into the environment due to anthropogenic activities1,2, and soil is considered a major sink for these contaminants3,4. The contaminants in the soil can be taken up by plants and potentially transferred to higher trophic-level organisms along food chains, by directly entering the human body through crop consumption, consequently leading to unintended exposure5,6. Plants utilize different pathways to metabolize xenobiotics for detoxification7; elucidating the metabolism of xenobiotics is important, as it controls the actual fate of contaminants in plants. As the metabolites can be excreted by leaves (to the atmosphere) or the roots, determining the metabolites in the very early phases of exposure hence provides the possibility to test an extended number of metabolites8. However, studies using intact plants require long-term experiments and complex sample preparation protocols that can be affected by various factors.
Plant callus cultures, therefore, are a good alternative for studying the metabolism of xenobiotics in planta, as they can greatly shorten the treatment time. These cultures exclude microbial interference and photochemical degradation, simplify the matrix effect of intact plants, standardize the cultivation conditions, and require less experimental effort. Plant callus cultures have been successfully applied as an alternative approach in metabolic studies of triclosan9, nonylphenol10, and tebuconazole8. These studies showed that the metabolic patterns in callus cultures were similar to those in intact plants. This study proposes a method for the efficient and accurate identification of metabolites of xenobiotics in plants without complex and time-consuming protocols. Here, we use plant callus cultures in combination with high-resolution mass spectrometry for the analysis of metabolites with low-intensity signals11,12.
To this end, carrot (Daucus carota var. sativus) callus suspensions were exposed to 100 &#181;g/L of 2,4-dibromophenol for 120 h in a shaker at 130 rpm and 26 &#176;C. 2,4-dibromophenol was chosen due to its disruptive endocrine activity13 and widespread occurrence in soil14. The metabolites were extracted and analyzed by high-resolution mass spectrometry. The protocol proposed here can investigate the in planta metabolism of other types of organic compounds that can be ionized.

<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>4-n-NP, &#62;99%</td>
			<td>Dr. Ehrenstorfer GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4-n-NP-d4</td>
			<td>Pointe-Claire</td>
			<td></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>1269101-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>7890A-5975&#160;gas chromatography</td>
			<td>Agilent</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ACQULTY ultra-performance&#160;liquid chromatography</td>
			<td>Waters</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Amber glass vials</td>
			<td>Waters</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>C18 column</td>
			<td>ACQUITY UPLC BEH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Thermo Fisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DB-5MS capillary column</td>
			<td>Agilent</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dichloromethane</td>
			<td>Sigma-Aldrich</td>
			<td>40071190-4L</td>
			<td></td>
		</tr>
		<tr>
			<td>Freeze dryer</td>
			<td>SCIENTZ&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>High-throughput tissue grinder</td>
			<td>SCIENTZ&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MicrOTOF-QII mass spectrometer</td>
			<td>Bruker Daltonics</td>
			<td></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>N-hexane</td>
			<td>Sigma-Aldrich</td>
			<td>H109658-4L</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrogen blowing instrument&#160;</td>
			<td>AOSHENG</td>
			<td>MD200-2</td>
			<td></td>
		</tr>
		<tr>
			<td>NP isomers, &#62;99%</td>
			<td>Dr. Ehrenstorfer GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Oasis HLB cartridges</td>
			<td>Waters</td>
			<td>60 mg/3 mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Research plus</td>
			<td>Eppendorf</td>
			<td></td>
			<td>100-1000 &#181;L</td>
		</tr>
		<tr>
			<td>Seeds of Little Finger carrot (Daucus carota var. sativus)&#160;</td>
			<td>Shouguang Seed Industry Co., Ltd</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Shaking&#160;Incubators</td>
			<td>Shanghai bluepard instruments Co.,ltd.</td>
			<td>THZ-98AB</td>
			<td></td>
		</tr>
		<tr>
			<td>Solid phase extractor</td>
			<td>AUTO SCIENCE</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasound machine</td>
			<td>ZKI</td>
			<td>UC-6</td>
			<td></td>
		</tr>
		<tr>
			<td>UV-sterilized ultra-clean workbench</td>
			<td>AIRTECH</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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 steps of the protocol are depicted in Figure 1. Following the protocol, we compared the chromatogram of the carrot callus extract from the 2,4-dibromophenol treatment to the controls, and found eight distinct peaks that are present in the 2,4-dibromophenol treatment but absent in the controls (Figure 2). This indicates that a total of eight metabolites of 2,4-dibromophenol (M562, M545, M661, M413, M339, M380, M424, and M187) were successfully detected in the...

Watch this Scientific Journal Video about Elucidating the Metabolism of 2,4-Dibromophenol in Plants at JoVE.com

Elucidating the Metabolism of 2,4-Dibromophenol 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 ...

Plant callus culture provides a solution for the accurate, efficient, and fast evaluation of metabolic degradation of xenobiotics in plants. Plant callus culture can exclude microbiome or fungal interference and photochemical degradation, simplify the matrix effect of intact plants, standardize the cultivation conditions, shorten the treatment duration, and require less experimental effort. The method proposed here could provide insight into the uptake behavior and metabolic mechanisms of different organic pollutants on crops and are useful towards efforts on environmental risk assessments.To begin, autoclave all the equipment and perform all operations in a UV sterilized ultra clean work bench. Sterilize the vernalized seed surface with 75%ethanol for 20 minutes. Rinse them with sterile deionized water three times and again sterilize them with 20%hydrogen peroxide for 20 minutes.After washing the seeds with sterilized deionized water six times, aseptically germinate them by sowing on autoclaved, hormone-free Murashige and Skoog medium of pH 5.8 containing 1%agar gel and incubate at 26 degrees Celsius with 16 hours photo period for 15 days. After 15 days of seed incubation, cut the hypocotyl and cotyledon of the seedling into small pieces of 0.5 centimeters to obtain the explants. Transform the explants in a Petri dish containing 15 to 20 milliliters of autoclaved Murashige and Skoog medium supplemented with Oxymon and Phycocyanin and incubate in the dark at 26 degrees Celsius for three to four weeks to induce the callus.Using a sterile scalpel and forceps, separate the callus of approximately one centimeter diameter formed from the initial explants. For treatment, dissolve 2, 4-dibromophenol in 10 milliliters of aseptic liquid Murashige and Skoog medium. Then add three grams of separated carrot callus to the prepared 2, 4-dibromophenol solution.After preparing the medium in blank control as described in the manuscript, incubate all the flasks in the dark. To prepare the sample, separate the callus carefully from the 2, 3-dibromophenol treatment and control flasks by filtration with 0.45 micron glass fiber filters. Wash the callus three times with ultrapure water before collecting it.Freeze dry all the collected callus and homogenize 0.2 grams of dried callus with a high throughput tissue grinder at 70 hertz for three minutes. Use a glass microsyringe to add 50 microliters of surrogate deuterated 4-n-nanophenol into the homogenized callus and vortex for one minute. Add five milliliters of the solution containing an equal ratio of methanol and water to the spiked callus and sonicate it for 30 minutes to extract the 2, 4-dibromophenol and metabolites.After extraction, centrifuge the suspension at 8, 000 G at four degree Celsius for 10 minutes and collect the supernatant by pipetting. Pass the extract through hydrophilic lipophilic balanced solid phase extraction or HLB-SPE cartridge with a flow rate of one milliliter per minute. Dilute the analytes by passing six milliliters of methanol through the HLB-SPE cartridge.Then concentrate the obtained eluent to one milliliter under a gentle stream of nitrogen gas for instrumental analysis. For analysis, open the column heater door. Then install the ultra performance liquid chromatography column by connecting the column inlet to the injection valve and the outlet to the mass spectrometer's inlet.Insert the end of the solvent tubes A and B into the corresponding solvent bottles. Place the sample vials by serial number in the corresponding locations of the sample trays and reinsert the sample trays into the sample chamber. In the software window, click on Instrument, then Inlet Method to edit the conditions for the liquid chromatogram.Select MS Method and set up the parameters of mass spectrum analysis. Click on File and then New to create and name the database. Load the sample program created above by selecting MS File, followed by Inlet File and Inject Volume.Save the database in the sample folder of the project by clicking File and Save. Next, select Run and Start in the main software window and click on Acquire Sample Data, followed by the OK button in the start sample list run to collect data. To process the data, select the target data row and click on the chromatogram window to view the MS scan chromatogram.In the chromatogram window, click on Display, followed by TIC. After clicking on the scan wave DS, add trace and OK to get the daughter mass spectra scan. The chromatogram of 2, 4-dibromophenol-treated carrot callus extract showed the presence of eight different metabolites compared to the control samples.Additionally, the absence of parent 2, 4-dibromophenol peak from the chromatogram indicates the rapid metabolism of 2, 4-dibromophenol in the carrot callus under experimental conditions. Furthermore, 2, 4-dibromophenol incubated in carrot callus led to the formation of metabolites by direct conjugation with glucose and amino acids. All equipment as well as the Murashige and Skoog medium should be autoclaved to make sure that the differentiation and maintenance of the plant callus are performed under aseptic conditions.Omics approaches following this procedure allow the creation of a clear phytotoxic mechanistic picture of environmental pollutants.

elucidating-the-metabolism-of-24-dibromophenol-in-plants

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718 Views.  Zhejiang University of Technology. Plant callus culture provides a solution for the accurate, efficient, and fast evaluation of metabolic degradation of xenobiotics in plants. Plant callus culture can exclude microbiome or fungal interference and photochemical degradation, simplify the matrix effect of intact plants, standardize the cultivation conditions, shorten the treatment duration, and require less experimental effort. The method proposed here could provide insight into the uptake behavior and metabolic mechanisms of dif...