Elucidating the Metabolism of 2,4-Dibromophenol in Plants

Summary

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

Abstract

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 the plant callus tissues. Thus, the plant callus culture platform is an effective method to evaluate the uptake and metabolism of xenobiotics in plants.

Introduction

An increasing number of organic pollutants have been discarded into the environment due to anthropogenic activities^1,2, and soil is considered a major sink for these contaminants^3,4. 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 exposure^5,6. Plants utilize different pathways to metabolize xenobiotics for detoxi....

Protocol

1. Differentiation of carrot callus

NOTE: Autoclave all equipment used here and perform all operations in a UV-sterilized ultra-clean workbench.

1. Vernalize the seeds by immersing the uniform carrot seeds (Daucus carota var. sativus) into deionized water at 4 °C for 16 h.
2. Surface-sterilize the vernalized seeds with 75% ethanol for 20 min, and then rinse three times with sterile deionized water under aseptic conditions.
3. Further steril.......

Discussion

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.......

Materials

Name	Company	Catalog Number	Comments
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