阐明植物中2,4-二溴苯酚的代谢
1Key Laboratory of Microbial Control Technology for Industrial Pollution in Zhejiang Province, College of Environment,Zhejiang University of Technology, 2International Joint Research Center for Persistent Toxic Substances (IJRC-PTS), College of Environment,Zhejiang University of Technology, 3Research and Teaching Center of Agriculture,Zhejiang Open University
Summary
本协议描述了一种简单有效的方法来鉴定植物中的2,4-二溴苯酚代谢物。
Abstract
作物可能广泛暴露于有机污染物，因为土壤是污染物丢弃到环境中的主要汇。这通过食用污染物积聚的食物创造了潜在的人类暴露。阐明作物中异生素的吸收和代谢对于评估人类饮食暴露风险至关重要。然而，对于此类实验，使用完整的植物需要长期实验和复杂的样品制备方案，这些方案可能受到各种因素的影响。植物愈伤组织培养结合高分辨率质谱（HRMS）可为准确、省时地鉴定植物异生素代谢物提供解决方案，避免微生物或真菌微环境的干扰，缩短处理时间，简化完整植物的基质效应。2,4-二溴苯酚是一种典型的阻燃剂和内分泌干扰剂，因其在土壤中的广泛存在及其被植物吸收的潜力而被选为模式物质。本文中，从无菌种子中生成植物愈伤组织并暴露于无菌的含2,4-二溴苯酚的培养基中。结果表明，孵育120 h后，在植物愈伤组织中鉴定出8种2,4-二溴苯酚代谢物。这表明2,4-二溴苯酚在植物愈伤组织中迅速代谢。因此，植物愈伤组织培养平台是评价植物异生素吸收代谢的有效方法。
Introduction
由于人为活动，越来越多的有机污染物被丢弃到环境中1,2，土壤被认为是这些污染物的主要汇3,4。土壤中的污染物可以被植物吸收，并可能通过作物消费直接进入人体，从而沿着食物链转移到更高的营养级生物中，从而导致意外暴露5,6。植物利用不同的途径代谢异生素进行解毒7;阐明异生素的代谢很重要，因为它控制着植物中污染物的实际命运。由于代谢物可以通过叶子（到大气）或根部排泄，因此在暴露的早期阶段确定代谢物提供了测试更多代谢物的可能性8。然而，使用完整植物的研究需要长期实验和复杂的样品制备方案，这些方案可能会受到各种因素的影响。
因此，植物愈伤组织培养是研究植物中异生素代谢的良好选择，因为它们可以大大缩短治疗时间。这些培养物排除了微生物干扰和光化学降解，简化了完整植物的基质效应，标准化了培养条件，并且需要更少的实验工作。植物愈伤组织培养已成功作为三氯生9、壬基酚10 和戊唑醇8 代谢研究的替代方法应用。这些研究表明，愈伤组织培养物中的代谢模式与完整植物中的代谢模式相似。本研究提出了一种无需复杂和耗时的方案即可有效准确地鉴定植物中异生素代谢物的方法。在这里，我们使用植物愈伤组织培养物与高分辨率质谱法相结合，以分析具有低强度信号的代谢物11,12。
为此，将胡萝卜（胡萝卜变种）愈伤组织悬浮液在130rpm和26°C的摇床中暴露于100μg/ L的2,4-二溴苯酚120小时。 选择2,4-二溴苯酚是由于其破坏性的内分泌活性13 和在土壤中广泛存在14。采用高分辨率质谱法提取并分析代谢物。这里提出的协议可以研究植物 中 可以电离的其他类型的有机化合物的代谢。
Protocol
1.胡萝卜愈伤组织的分化 注意：高压灭菌此处使用的所有设备，并在紫外线灭菌的超净工作台中执行所有操作。 通过将均匀的胡萝卜种子（胡萝卜种子）浸入4°C的去离子水中16小时来春化种子。 用75%乙醇对春化的种子进行表面消毒20分钟，然后在无菌条件下用无菌去离子水冲洗三次。 进一步用20%H2O2对种子灭菌20分钟?…
Representative Results
该协议的步骤如图 1所示。按照实验方案，我们将2,4-二溴苯酚处理的胡萝卜愈伤组织提取物的色谱图与对照组进行了比较，发现2,4-二溴苯酚处理中存在八个不同的峰，但在对照中不存在（图2）。这表明在2,4-二溴苯酚处理的胡萝卜愈伤组织中成功检测到2,4-二溴苯酚的8种代谢物（M562，M545，M661，M413，M339，M380，M424和M187）。此外，在2,4-二溴苯酚处理的?…
Discussion
该协议旨在有效识别植物中异生素的生物转化。该协议的关键步骤是植物愈伤组织的培养。最困难的部分是植物愈伤组织的分化和维持，因为植物愈伤组织容易感染并发育成植物组织。因此，重要的是要确保使用的所有设备都经过高压灭菌，并且所有操作都在无菌条件下进行。植物愈伤组织的分化和维持应在黑暗中进行，以避免自养生长和过度发育。此外，补充到MS培养基中的植物激素的剂量和?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
本研究得到了国家自然科学基金（21976160）和浙江省公益性技术应用研究项目（LGF21B070006）的支持。
Materials
| 2,4-dichlorophenoxyacetic acid | WAKO | 1 mg/L | |
| 20% H2O2 | Sinopharm Chemical Reagent Co., Ltd. | 10011218-500ML | |
| 4-n-NP, >99% | Dr. Ehrenstorfer GmbH | ||
| 4-n-NP-d4 | Pointe-Claire | ||
| 6-benzylaminopurine | WAKO | 0.5 mg/L | |
| 75% ethanol | Sinopharm Chemical Reagent Co., Ltd. | 1269101-500ML | |
| 7890A-5975 gas chromatography | Agilent | ||
| ACQULTY ultra-performance liquid chromatography | Waters | ||
| Amber glass vials | Waters | ||
| Artificial climate incubator | Ningbo DongNan Lab Equipment Co.,LTD | RDN-1000A-4 | |
| Autoclaves | STIK | MJ-Series | |
| C18 column | ACQUITY UPLC BEH | ||
| Centrifuge | Thermo Fisher | ||
| DB-5MS capillary column | Agilent | ||
| Dichloromethane | Sigma-Aldrich | 40071190-4L | |
| Freeze dryer | SCIENTZ | ||
| High-throughput tissue grinder | SCIENTZ | ||
| Methanol | Sigma-Aldrich | ||
| MicrOTOF-QII mass spectrometer | Bruker Daltonics | ||
| Milli-Q system | Millipore | MS1922801-4L | |
| Murashige & Skoog medium | HOPEBIO | HB8469-7 | |
| N-hexane | Sigma-Aldrich | H109658-4L | |
| Nitrogen blowing instrument | AOSHENG | MD200-2 | |
| NP isomers, >99% | Dr. Ehrenstorfer GmbH | ||
| Oasis HLB cartridges | Waters | 60 mg/3 mL | |
| Research plus | Eppendorf | 100-1000 µL | |
| Seeds of Little Finger carrot (Daucus carota var. sativus) | Shouguang Seed Industry Co., Ltd | ||
| Shaking Incubators | Shanghai bluepard instruments Co.,ltd. | THZ-98AB | |
| Solid phase extractor | AUTO SCIENCE | ||
| Ultrasound machine | ZKI | UC-6 | |
| UV-sterilized ultra-clean workbench | AIRTECH |
References
- Chakraborty, P., et al. 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. Environmental Pollution. 248, 1036-1045 (2019).
- Abril, C., Santos, J. L., Martin, J., Aparicio, I., Alonso, E. Occurrence, fate and environmental risk of anionic surfactants, bisphenol A, perfluorinated compounds and personal care products in sludge stabilization treatments. Science of the Total Environment. 711, 135048 (2020).
- Xu, Y. W., et al. Determination and occurrence of bisphenol A and thirteen structural analogs in soil. Chemosphere. 277, 130232 (2021).
- Cai, Q. Y., et al. Occurrence of nonylphenol and nonylphenol monoethoxylate in soil and vegetables from vegetable farms in the Pearl River Delta, South China. Archives of Environmental Contamination and Toxicology. 63 (1), 22-28 (2012).
- Wang, S. Y., et al. et al Migration and health risks of nonylphenol and bisphenol a in soil-winter wheat systems with long-term reclaimed water irrigation. Ecotoxicology and Environmental Safety. 158, 28-36 (2018).
- Gunther, K., Racker, T., Bohme, R. An isomer-specific approach to endocrine-disrupting nonylphenol in infant food. Journal of Agricultural and Food Chemistry. 65 (6), 1247-1254 (2017).
- Van Eerd, L. L., Hoagland, R. E., Zablotowicz, R. M., Hall, J. C. Pesticide metabolism in plants and microorganisms. Weed Science. 51 (4), 472-495 (2003).
- Hillebrands, L., Lamshoeft, M., Lagojda, A., Stork, A., Kayser, O. Evaluation of callus cultures to elucidate the metabolism of tebuconazole, flurtamone, fenhexamid, and metalaxyl-M in Brassica napus L., Glycine max (L.) Merr., Zea mays L., and Triticum aestivum L. Journal of Agricultural and Food Chemistry. 68 (48), 14123-14134 (2020).
- Macherius, A., et al. Metabolization of the bacteriostatic agent triclosan in edible plants and its consequences for plant uptake assessment. Environmental Science & Technology. 46 (19), 10797-10804 (2012).
- Sun, J. Q., et al. Uptake and metabolism of nonylphenol in plants: Isomer selectivity involved with direct conjugation. Environmental Pollution. 270, 116064 (2021).
- Schymanski, E. L., et al. Identifying small molecules via high resolution mass spectrometry: communicating confidence. Environmental Science & Technology. 48 (4), 2097-2098 (2014).
- Moschet, C., Anumol, T., Lew, B. M., Bennett, D. H., Young, T. M. Household dust as a repository of chemical accumulation: new insights from a comprehensive high-resolution mass spectrometric study. Environmental Science & Technology. 52 (5), 2878-2887 (2018).
- Ren, Z., et al. 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. Environmental Pollution. 177, 71-77 (2013).
- de Wit, C. A. An overview of brominated flame retardants in the environment. Chemosphere. 46 (5), 583-624 (2002).
- Sun, J. Q., Chen, Q., Qian, Z. X., Zheng, Y., Yu, S. A., Zhang, A. P. Plant Uptake and Metabolism of e,4-Dibromophenol in Carrot: In Vitro Enzymatic Direct Conjugation. Journal of Agricultural and Food Chemistry. 66 (17), 4328-4335 (2018).
- Chibwe, L., Titaley, I. A., Hoh, E., Simonich, S. L. M. Integrated framework for identifying toxic transformation products in complex environmental mixtures. Environmental Science & Technology Letters. 4 (2), 32-43 (2017).
- Hollender, J., Schymanski, E. L., Singer, H. P., Ferguson, P. L. Nontarget screening with high resolution mass spectrometry in the environment: ready to go. Environmental Science & Technology. 51 (20), 11505-11512 (2017).
- Nafisi, M., Fimognari, L., Sakuragi, Y. Interplays between the cell wall and phytohormones in interaction between plants and necrotrophic pathogens. Phytochemistry. 112, 63-71 (2015).
- Zhang, Q., et al. Multiple metabolic pathways of 2,4,6-tribromophenol in rice plants. Environmental Science & Technology. 53 (13), 7473-7482 (2019).
- Hou, X., et al. Glycosylation of tetrabromobisphenol A in pumpkin. Environmental Science & Technology. 53 (15), 8805-8812 (2019).
Cite This Article
Wu, J., Yang, X., Wang, Q., Zhou, Q., Zhang, A., Sun, J. Elucidating the Metabolism of 2,4-Dibromophenol in Plants. J. Vis. Exp. (192), e65089, doi:10.3791/65089 (2023).