Compartmentalization and Excretion of 2,4,6-Tribromophenol Sulfation and Glycosylation Conjugates in Rice Plants

Abstract

The most environmentally abundant bromophenol congener, 2,4,6-tribromophenol (2,4,6-TBP, 6.06 μmol/L), was exposed to rice for 5 d both in vivo (intact seedling) and in vitro (suspension cell) to systematically characterize the fate of its sulfation and glycosylation conjugates in rice. The 2,4,6-TBP was rapidly transformed to produce 6 [rice cells (3 h)] and 8 [rice seedlings (24 h)] sulfated and glycosylated conjugates. The predominant sulfation conjugate (TP₄₀₈, 93.0–96.7%) and glycosylation conjugate (TP₄₉₀, 77.1–90.2%) were excreted into the hydroponic solution after their formation in rice roots. However, the sulfation and glycosylation conjugates presented different translocation and compartmentalization behaviors during the subsequent Phase III metabolism. Specifically, the sulfated conjugate could be vertically transported into the leaf sheath and leaf, while the glycosylation conjugates were sequestered in cell vacuoles and walls, which resulted in exclusive compartmentalization within the rice roots. These results showed the micromechanisms of the different compartmentalization behaviors of 2,4,6-TBP conjugates in Phase III metabolism. Glycosylation and sulfation of the phenolic hydroxyl groups orchestrated by plant excretion and Phase III metabolism may reduce the accumulation of 2,4,6-TBP and its conjugates in rice plants.

Graphical Abstract

INTRODUCTION

Plant enzymes engage in the secondary metabolism of xenobiotics, in which natural polar endogenous compounds (e.g., sulfates, sugars, and amino acids) conjugate with xenobiotics or their activated Phase I metabolites (nonpolar xenobiotics in particular)^1–8 to reduce the deleterious effects of xenobiotics.³ Phase II metabolites [e.g., sulfate-, glucuronide-, and amino acid-conjugates (e.g., glutamic acid, cysteine, valine, and threonine)] of emerging organic contaminants (e.g., chlorinated paraffins, tetrabromobisphenol A (TBBPA), sulfamethoxazole, triclosan, 2,4,6-tribromophenol, mono-(2-ethylhexyl) phthalate, and nonylphenol) have been extensively identified in plants and documented in the literature.^6–12 For example, several xenobiotic conjugates for triclosan (8 conjugates),¹¹ carbamazepine (10 conjugates),¹³ naproxen (11 conjugates),¹⁴ and tetrabromobisphenol A (20 conjugates) have been reported recently in plants.⁹ The physicochemical properties of xenobiotics are dramatically altered after bioconjugation (e.g., sulfation and glycosylation processes).¹⁵ Phase II conjugates are generally more water-soluble than their parent compounds and are speculated to be easily excreted or compartmentalized.¹⁶

The translocation and compartmentalization of Phase II conjugates in the plant organelles with storage functions (e.g., vacuole) are defined as Phase III metabolism.^17,18 As one of the prominent sequestration organelles, the plant vacuole plays a key role in storing hazardous substances and wastes.^19,20 For instance, the vacuolar sequestration of excess toxic metals (e.g., Cd, Pb, Cu, As, Ni, or Fe) greatly enhances plant tolerance to harmful metals.^21–24 Phase III is also critical for the sequestration of organic contaminant conjugates in plants.²⁵ However, the mechanism of Phase III metabolism in plants is not well understood due to challenges in monitoring the exact locations of conjugates at the cellular level. In addition, the excretion of organic contaminant Phase II conjugates has not been fully characterized to date.

Emerging environmental contaminants can be introduced into crops from contaminated soil, irrigation water,^26–28 amended biosolids, and some other unintended sources (e.g., untreated wastewater overflow during heavy storms). As an important representative group of water-soluble contaminants, bromophenols have well-known anthropogenic and natural sources. Bromophenols are also transformation products of polybrominated diphenyl ethers (PBDEs), MeO-PBDEs, OH-PBDEs, TBBPA, and 1,2-bis (2,4,6-tribromophenoxy) ethane.^10,29–36 Moreover, bromophenols have the potential to bind with estrogen receptors and the thyroid hormone transport protein transthyretin, which could result in endocrine-disrupting effects.^37,38 The phenolic hydroxyls of contaminants (e.g., TBBPA, triclosan, and bromophenols) are generally bonded with sulfate and/or glucoside groups without the activation processes in Phase I metabolism (e.g., hydroxylation process), directly forming sulfation^10,11 and diverse glycosylation conjugates in pumpkins, carrots, and rice seedlings.^9–11

As a representative agricultural plant that grows in aqueous environments, rice is cultivated worldwide. Here, the most environmentally abundant congener of bromophenols, 2,4,6-tribromophenol (2,4,6-TBP), was chosen and exposed to rice seedlings (in whole plants) and suspension cells (at cellular level). The 2,4,6-TBP was rapidly sulfated and glycosylated in both incubation systems. The fates of the sulfation and glycosylation conjugates are of particular concern in rice plants. Different plant organelles were isolated from the exposed suspension rice cells to elucidate the mechanism of sequestration and plant excretion of Phase II conjugates (Phase III metabolism). Given that the available information on these synergetic effects is scant, this paper provides new insight into the fate of phenolic contaminants in plants at the cellular level.

MATERIALS AND METHODS

Chemicals.

The 2,4,6-TBP standard (purity: 98.7%) for the rice seedlings and cells exposure experiments was obtained from Tokyo Chemical Industry (Shanghai, China). The 2,4,6-TBP (99.2%) and [¹³C₆]-2,4,6-TBP (>98.0%) standards for the quantitative analysis were purchased from Wellington Laboratories (Ontario, Canada), and 2,4,6-tribromophenyl β-d-glucopyranoside (99.0%) was synthesized by Toronto Research Chemicals (Ontario, Canada). Chromatographic grade methanol and ethyl acetate were supplied by J.T. Baker (New Jersey, U.S.A.). Other reagents were purchased from Sigma-Aldrich (Missouri, U.S.A.). Ultrapure water was produced by an onsite Milli-Q system manufactured by Millipore Corporation (Massachusetts, U.S.A.).

Rice Seedling Exposure Experiment and Sample Collection.

Rice seeds were germinated at 30 °C under dark conditions. Germinated seeds were transferred into half-strength Hoagland nutrient solution and cultivated for 2 weeks under constant 70% relative humidity air and 16-h white light (28 °C) followed by an 8-h dark (25 °C) exposure cycle.¹⁰ Subsequently, 15 cm-length of rice seedlings were selected for the 2,4,6-TBP exposure experiments. A hydroponic exposure system (Figure S1 of the Supporting Information, SI) was used to ensure that the characterized plant metabolism of 2,4,6-TBP was solely from rice seedlings rather than microorganismal effects in the plant–soil systems. Although it was different from the real soil-plant systems in which the metabolic processes of 2,4,6-TBP in the plant might be affected by soil adsorption, bioavailability, and microorganism transformation, the results obtained from hydroponic exposure systems can reflect the truth to a great extent. Five rice seedlings were planted in 45 mL of 6.06 μmol/L 2,4,6-TBP aqueous solution prepared using deionized water and placed in a 55 mL brown glass flask. The flask was wrapped with aluminum foil to prevent the photodegradation of the target chemicals in the exposure systems. Concurrently, another 5 rice seedlings, which served as blank planted controls, were processed using the same procedure but without adding 2,4,6-TBP. The unplanted control contained 2,4,6-TBP but no rice plants. This control was used to evaluate the direct volatilization of 2,4,6-TBP from hydroponic solution and any possible chemical transformation during the exposure period. All the treatments and controls were placed in a growth chamber under the same conditions as the cultivation conditions. Hydroponic solution and rice tissues (roots, leaf sheaths, and leaves) were sampled separately from each glass flask at intervals of 6, 12, 24, 48, 72, and 120 h. The exposure groups were sampled in triplicate (n = 3) at each time point, while the blank and unplanted controls were only sampled at the end of the exposure time (120 h) in triplicate (n = 3).

Collection of Xylem Sap.

To determine whether the sap-carrying xylem vertically transported 2,4,6-TBP and its target conjugates into the leaves and leaf sheaths, 500 two-week-old rice seedlings in a 4.5 L volume of hydroponic growth medium (n = 3) were exposed to 2,4,6-TBP (6.06 μmol/L). Blank planted controls (n = 3, 500 rice seedlings for each) were maintained without 2,4,6-TBP under the same conditions. After 24 h-, 48 h-, 72 h-, and 120 h-exposures, the rice shoots were cut with sharp blades at 1–3 cm above the water surface,^39,40 and the liquid droplets (xylem sap) that sprang from the rice xylem were collected every half hour using a 2.5-μL pipet. Xylem sap was collected from 40 individual seedlings 3 times at half-hour intervals to ensure that the pooled sample (approximately 30 μL) was sufficient for subsequent ultra-performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS) analysis. Three samples were individually collected from both the exposure and control groups for each time point.

Exposure Experiment Using Rice Suspension Cell Culture.

To characterize Phase II metabolism of bromophenols and the excretion of those conjugates at the cellular level, rice suspension cells were selected and exposed to 2,4,6-TBP. Rice suspension cells were prepared and isolated as previously described.^41,42 Additional details were provided in Section S1.1. Briefly, a 4.0 mL-volume cell suspension (1.0 × 10⁷ particles per mL) was inoculated into a 25 mL glass conical flask with 9.0 mL of fresh liquid MS medium and subsequently treated with 2,4,6-TBP at an initial exposure concentration of 6.06 μmol/L. A series of control groups were incubated in parallel, including a blank rice cell control (without 2,4,6-TBP), a 2,4,6-TBP treated control (without suspension rice cells), and a nonviable cell control (autoclaved cells with 2,4,6-TBP). Culture medium and glass flasks were autoclaved at 121 °C for 20 min prior to use. The exposure group and three control groups were conducted in triplicate. All the groups were incubated in the dark at 28 °C under rotation at 120 rpm. To determine the temporal variations in 2,4,6-TBP and its metabolites during exposure, the rice cells and cell medium were collected by centrifugation at 5000g for 10 min at different sampling times (3, 6, 12, 24, 48, 72, and 120 h).

Extra triplicate sets of exposed cell reactors and blank medium controls (nonexposed rice cells) were also prepared, treated and sampled at 12 h, to further elucidate the distribution of intracellular and extracellular enzymes. Detailed information on the sample preparation and proteomic analysis is presented in Sections S1.2 and S1.3.

Isolation of Vacuoles, Protoplasts, and Cell Wall.

Different cell organelles were isolated and analyzed to elucidate the spatial compartmentalization of the 2,4,6-TBP conjugates in Phase III metabolism at the cellular level. Exposed suspension rice cells, which were treated with 6.06 μmol/L (same as hydroponic exposure experiments) of 2,4,6-TBP for 24 h, were enzymatically hydrolyzed according to the procedure^43,44 which is described in Section S1.4. As shown in Figure S2, vacuoles, protoplasts, and the cell wall fraction (including extractable and bound residues) were acquired for further analysis.

Exocrine Enzyme Exposure Experiment.

To assess the role of exocrine enzymes in the biological catalysis of 2,4,6-TBP, the interface between exocrine enzymes and 2,4,6-TBP was investigated. The cell medium containing the exocrine enzymes was obtained by centrifuging nonexposed suspension rice cells that were in turn exposed to [¹³C₆]-2,4,6-TBP (6.06 μmol/L) (Figure S3a). Some studies have reported that exposure to xenobiotic pollutants could activate phase II enzymes, which is probably a plant defense response.^45,46 Thus, to obtain the active phase II enzymes that were probably released from activated cells into the medium, the cell medium of rice cells, which was treated with native 2,4,6-TBP (6.06 μmol/L) for 6 h, was also collected. As shown in Figure S3b, one fraction (9.0 mL) of the cell medium containing active phase II enzymes was directly treated with ¹³C-2,4,6-TBP at a concentration of 6.06 μmol/L, while the other fraction was autoclaved in advance to perform the same exposure. All of the exposure groups were sampled in triplicate. Following incubation for 6 h with [¹³C₆]-2,4,6-TBP, 4.0 mL of aqueous media was then sampled from each treatment.

Sample Extraction of 2,4,6-TBP and Its Conjugates.

The sample preparation and storage steps are shown in Section S1.5. The freeze-dried rice tissue (approximately 0.10 g), cell and protoplast samples were spiked with 0.050 mg/mL of C₁₃-2,4,6-TBP (methanol) to a nominal concentration of 0.050 μg/g and then extracted with 5.0 mL of methanol twice. In accordance with our previous study,¹⁰ the combined extracts were dried under nitrogen, redissolved in 5.0 mL of methanol/water (2/98, v/v) and subsequently loaded onto a 6.0 mL HLB cartridge (200 mg, Massachusetts, U.S.A.) that was preconditioned with 5.0 mL of methanol and 5.0 mL of deionized water in sequence. A 10 mL volume of methanol was used to elute the analytes. The eluate was evaporated to dryness and diluted with 0.50 mL of methanol for subsequent UPLC-HRMS analysis.

A 4.0 mL volume of hydroponic solution, cell medium, enzymatic solution (which was derived from the enzymolysis of extracted cell walls and used to detect the bound residues of conjugates), and vacuoles were extracted with 5.0 mL ethyl acetate twice. The extracts were combined, dried, and diluted with 0.50 mL of methanol for UPLC-HRMS analysis. The xylem sap sample was mixed directly with an equivalent volume of methanol for UPLC-HRMS analysis.

Instrumental Analysis of 2,4,6-TBP and Its Conjugates.

A UPLC-Orbitrap Fusion MS system (Thermo Fisher Scientific Inc., Waltham, MA) with negative electrospray ionization (ESI) source was used to identify and quantify the 2,4,6-TBP and its conjugates. Analyte separation was performed on an Inertsil ODS-4 column (150 mm length × 3.0 mm i.d., 2.0 μm particle size, GL Science B.V., Shanghai, China). The instrumental conditions are described in Section S1.6 as previously reported.¹⁰

Quality Assurance and Quality Control (QA/QC).

The linearity of the calibration curves (concentration range 20–1000 ppb) for 2,4,6-TBP and 2,4,6-tribromophenyl β-d-glucopyranoside was satisfied, with correlation coefficients (R²) greater than 0.998. A pooled sample obtained by mixing 20 μL of each plant sample was measured for every batch of samples (9–12 samples). The relative standard deviations (RSDs) of 2,4,6-TBP and its conjugates were less than 5.8%. The spiking recoveries of ¹³C-2,4,6-TBP and 2,4,6-tribromo-phenyl β-d-glucopyranoside were 78.8–97.0% and 72.1–85.9%, respectively. The method detection limits (MDLs) of 2,4,6-TBP and 2,4,6-tribromophenyl β-d-glucopyranoside were 0.13–0.17 ng L⁻¹ and 1.04–1.34 ng L⁻¹ for the culture solutions (hydroponic solution, cell medium, and enzymatic solution), 5.00–6.67 ng kg⁻¹ and 41.6–53.6 ng kg⁻¹ for the rice samples, respectively.

Data Analysis.

All the data analysis was performed using Xcalibur software v.2.2 (Thermo Fisher Scientific) with a 5.0 ppm mass tolerance. The confidence levels of the discovered conjugates were evaluated based on criteria that were previously established by Schymanski et al.⁴⁷ Since a high purity reference standard is only available for one of the conjugates, 2,4,6-tribromophenyl β-d-glucopyranoside (TP₄₉₀), the other 7 conjugates (including one sulfation conjugate, TP₄₀₈, which had a lab-synthesized standard called 2,4,6-tribromophenyl sulfate and was used only for qualification but not for quantification because its purity was unknown, and the other 6 glycosylation conjugates TP₅₃₂, TP₅₇₆, TP₆₂₂, TP₆₅₂, TP₆₉₄, and TP₇₃₈) were all semiquantified using the authorized synthetic standard (TP₄₉₀). The K_ow (octanol–water partition coefficient) of those identified Phase II conjugates was evaluated using EPI Suit (KOWWIN v1.68 estimate). One-way ANOVA followed by a Tukey’s test was performed to compare the differences between sampling times. The differences were considered statistically significant when the data varied with p < 0.05.

RESULTS AND DISCUSSION

Variations in the Dissipation of 2,4,6-TBP in Hydroponic Exposure Experiments and Its Distribution in Planted Hydroponic Seedlings.

To understand the metabolism of 2,4,6-TBP in rice plants, the variations in the dissipation of 2,4,6-TBP in the entire exposure systems and its distributions in different compartments (hydroponic solution, rice roots, leaf sheaths, and leaves) were studied. As shown in Figure S4, the amounts of 2,4,6-TBP in both the hydroponic solutions and the exposure systems rapidly decreased, and only 1.93% (5.25 nmol) of the initial exposure amount (273 nmol) remained in the entire system after 120 h of exposure. In other words, 98.1% of the initial amount of 2,4,6-TBP was dissipated from the whole exposure system. The dissipation of 2,4,6-TBP from the whole exposure systems was primarily caused by volatilization and biotic transformation processes, and any possible chemical transformations, such as photolysis, were avoided by wrapping the reactors within aluminum foil. None of the metabolites were detected in the unplanted controls, confirming the nonexistence of chemical transformation. The volatilization was quantitatively evaluated using unplanted controls, in which 94.7% of the 2,4,6-TBP was recovered at the end of the exposure period. Accordingly, approximately 5.25% (14.3 nmol) of the initial amount of 2,4,6-TBP was volatilized from the hydroponic solution into the atmosphere after 120 h of incubation. These results showed that a large amount of the parent 2,4,6-TBP (92.9%) was biotransformed within the rice seedlings. None of the 2,4,6-TBP was detected in any samples of the planted blank controls, suggesting that no background or cross contamination influenced the determination of 2,4,6-TBP in the hydroponic solutions and rice tissues, further confirming the reliability of the analysis results.

In planted hydroponic seedlings, 2,4,6-TBP was rapidly adsorbed and bioaccumulated in the rice roots (1.83 nmol) after 12 h of treatment. The 2,4,6-TBP then gradually decreased to 0.148 nmol at the end of the exposure period. The amounts of 2,4,6-TBP in the leaves and leaf sheaths gradually increased during exposure, and eventually reached 0.00680 nmol and 0.0405 nmol at 120 h, respectively. A majority of the 2,4,6-TBP was distributed in the hydroponic solution for the entire exposure time, accounting for 92.1%–96.3% of the total amount in the exposure systems.

Temporal Variations in Sulfation and Glycosylation Conjugates in Planted Hydroponic Seedlings.

According to the above mass balance results, 92.9% of the initial amount of 2,4,6-TBP was transformed in the exposure systems. Our previous work demonstrated 40 Phase I and II metabolites of 2,4,6-TBP in rice seedlings through multiple metabolic pathways.¹⁰ Among those metabolites, eight conjugates formed from the phenolic hydroxyl of 2,4,6-TBP were directly conjugated with sulfates and sugar groups, had a relatively high yield and are the subject of special focus in this study. These conjugates (TP₄₀₈, TP₄₉₀, TP₅₃₂, TP₅₇₆, TP₆₂₂, TP₆₅₂, TP₆₉₄, and TP₇₃₈, as named according to their molecular ion m/z values shown in Table S1) were identified in the planted hydroponic exposure experiment. The identification information for the eight conjugates is shown in Section S2.1. None of these conjugates were detected in any blank or unplanted control sample, confirming that these conjugates were transformed from 2,4,6-TBP by rice seedlings, and the proposed sulfation and glycosylation pathways are summarized in Figure S5.

According to the quantitative and semiquantitative analysis results, 2.34–10.0% of the dissipated amount of 2,4,6-TBP was sulfated and glycosylated to form these 8 conjugates during the exposure period. The temporal variations in individual sulfation and glycosylation conjugates in the entire planted hydroponic exposure systems are summarized in Figure 1a. Sulfation conjugate (TP₄₀₈) increased continuously from 2.86 nmol to 24.3 nmol (3 vs 72 h, p < 0.05) and then remained roughly unchanged from 72 to 120 h (p > 0.05). As shown in Figure 1b–e, TP₄₀₈ was detected extensively in the hydroponic solution, rice roots, leaf sheaths, and leaves. The majority of TP₄₀₈ (Figure 1b), accounting for 93.0–96.7% of the measured amount in the exposure system, was distributed into the hydroponic solution during the exposure. The amounts of TP₄₀₈ in the rice roots were steady at the initial stage (Figure 1c), subsequently increased after an exposure time of 72 h (48 vs 72 h, p < 0.05) and then remained roughly unchanged (p > 0.05). As illustrated in Figure 1d, TP₄₀₈ was detected in rice leaves after a 24 h incubation. This compound showed same trend as that shown for the hydroponic solution. However, TP₄₀₈ was first detected in rice leaves after 72 h of incubation (Figure 1e). Ultimately, 30.0% of the TP₄₀₈ (the mass percentage of TP₄₀₈ in rice seedlings) was bioaccumulated in the aerial parts (leaf sheaths and leaves) at the end of the exposure.

Figure 1.

Amounts of sulfation conjugate (TP₄₀₈) and all glycosylation conjugates (TP₄₉₀, TP₅₃₂, TP₅₇₆, TP₆₂₂, TP₆₅₂, TP₆₉₄, and TP₇₃₈) in the entire exposure system (a). The amounts of all the conjugates in hydroponic solution (b), rice roots (c), leaf sheaths (d), and leaves (e) of the hydroponic exposure systems at different exposure times. TP₄₉₀ was detected in the hydroponic solution and rice roots, while TP₅₃₂, TP₅₇₆, TP₆₂₂, TP₆₅₂, TP₆₉₄, and TP₇₃₈ were nondetectable in the hydroponic solution, leaves and leaf sheaths. The temporal variation in the parent 2,4,6-TBP and its sulfation conjugate (TP₄₀₈) in xylem sap (f), and TP₄₉₀, TP₅₃₂, TP₅₇₆, TP₆₂₂, TP₆₅₂, TP₆₉₄, and TP₇₃₈ were nondetectable in the xylem sap.

As the most abundant glycosylation conjugate (Figure 1a), 2,4,6-tribromophenyl β-d-glucopyranoside (TP₄₉₀) reached 5.08 nmol at 24 h with a biotransformation yield of 1.86% in the whole exposure system (Figure S6). The TP₄₉₀ mass then dramatically decreased to 0.0241 nmol at 120 h (24 vs 120 h, p < 0.05), suggesting that TP₄₉₀ was sequentially converted to other conjugate forms after 24 h through further acetylation (TP₅₃₂), malonylation (TP₅₇₆) and glycosylation (TP₆₂₂ and TP₆₅₂). Therefore, the formation of TP₄₉₀ is crucial for further acetylation, malonylation and glycosylation processes within Phase II metabolism (Figure S5). TP₄₉₀ was only detected in the hydroponic solution and rice roots (Figure 1b and c). An additional 6 glycosylation conjugates (TP₅₃₂, TP₅₇₆, TP₆₂₂, TP₆₅₂, TP₆₉₄, and TP₇₃₈) were detected exclusively in the rice roots at low-levels (0.00540–0.493 nmol). Similar to the sulfation conjugate TP₄₀₈, the majority of the TP₄₉₀ (77.1–90.2%) was distributed into the hydroponic solution. However, unlike the sulfation conjugate (TP₄₀₈), none of the glycosylation conjugates (<LOD, 41.6–53.6 ng kg⁻¹) were detected in the leaf and leaf sheath, indicating that glycosylation conjugates could neither be produced in the leaf sheath and leaf nor vertically transported within the rice seedlings.

Translocation of Sulfation Conjugate in Rice Xylem.

Sulfation conjugate was detected in the leaf sheaths and leaves in sequence (Figure 1d and e). To determine whether the sap-carrying xylem vertically transported 2,4,6-TBP and its phase II conjugates, the xylem sap of rice plants in the treatment and blank planted controls were collected and analyzed. As shown in Figure 1f, only 2,4,6-TBP and the sulfation conjugate (TP₄₀₈) were detected in the xylem sap samples. The TP₄₀₈ concentration ranged from 0.778 to 1.65 nmol/mL, which is 139–268 times higher than that of 2,4,6-TBP (0.00439–0.00941 nmol/mL) in the xylem sap. If the amount of 2,4,6-TBP in the xylem sap (the translocated parts of 2,4,6-TBP) was assumed to be completely transformed to the sulfation conjugate (TP₄₀₈) in the aerial parts, then the transformed TP₄₀₈ in the leaves and leaf sheaths would only account for 0.372–0.713% of the vertically transported mass through the xylem sap, far less than the amount of sulfation conjugate found in the aerial parts. This observation suggests that a sulfation conjugate (TP₄₀₈) was rapidly formed in rice roots rather than in the aerial parts of rice tissues after 2,4,6-TBP treatment. Moreover, our findings revealed that almost all the sulfation conjugate detected in leaves and leaf sheaths was translocated from the roots. Another study also identified polychlorinated biphenyl sulfates in the woody parts of poplar,⁴⁸ while the translocation mechanism of sulfation conjugates within plants is still unclear. None of the glycosylation conjugates were detected in the xylem sap, further confirming that the glycosylation conjugates could not be translocated vertically within the rice seedlings.

Dissipation of 2,4,6-TBP in Cell Culture Exposure Experiments.

To further characterize the micromechanisms underlying the different translocation and distribution behaviors of the sulfation and glycosylation conjugates at the cellular level, Phase II metabolism of 2,4,6-TBP was investigated in rice suspension cells. The total amounts of parent 2,4,6-TBP all decreased in the two control groups (nonviable cell controls and treated medium controls) (Figure S7), suggesting that the volatilization of 2,4,6-TBP from cell medium has a significant role (p < 0.05) in its dissipation from exposure systems. The volatilization of 2,4,6-TBP accounted for 21.5% and 29.6% of the initial exposure amounts in nonviable cell controls and treated medium controls, respectively, after 120 h of incubation. The volatilization of 2,4,6-TBP in those two controls was not significantly different (p > 0.05) but was significantly different from that of the unplanted controls (5.25%, p < 0.05) in the hydroponic rice plant exposure experiment. The higher volatilization of 2,4,6-TBP in the cell culture than in the hydroponic plant solution was primarily caused by the different incubation conditions, such as a longer incubation time for rice cells at 28 °C than for rice plants (16-h at 28 °C followed by 8-h at 25 °C), and rotational shaking (120 rpm) of the cell culture but maintaining a static position for the rice plants. In the nonviable cell controls, the 2,4,6-TBP concentration in the nonviable cells was 46.5 nmol after 120 h of incubation, accounting for 43.0% of the total amount of 2,4,6-TBP in the entire systems at the corresponding sampling time. This amount was 7.97 times higher than that found in viable cells after 120 h of incubation.

In the viable rice cell treatments, the 2,4,6-TBP rapidly disappeared from the exposure system (Figure S7a), and only 1.56% (1.23 nmol) of the initial exposure amount (78.8 nmol) remained after 120 h of exposure. The distributions of 2,4,6-TBP in viable rice cells (Figure S7b) showed that the mass of the parent chemical increased during the first 3 h and dramatically decreased afterward (3 vs 6 h, p < 0.05). Concurrently, 2,4,6-TBP also rapidly disappeared in the cell medium during the exposure time (Figure S7b). These results indicated a significant role for rice cells (viable rice cell treatments) in the biotic metabolism of 2,4,6-TBP. Approximately 77.0% of the initial exposure amount of 2,4,6-TBP was biotransformed after 120 h of incubation.

Occurrence of Sulfation and Glycosylation Conjugates in Cell Culture Exposure Experiments.

Approximately 6 conjugates (TP₄₀₈, TP₄₉₀, TP₅₃₂, TP₆₅₂, TP₆₉₄, and TP₇₃₈) were found in the cell culture experiments, except for TP₅₇₆ and TP₆₂₂ (Figure S5). Rice cells and seedlings are derived from the same species and have the same gene sequences. The difference in the number and species of sulfation and glycosylation conjugates between rice cells and seedlings might be attributed to the variation in gene expression between cells and plants. Some genes might be activated, resulting in the expression of special enzymes during cell differentiation, which would help produce TP₅₇₆ and TP₆₂₂ in rice seedlings. This observation was consistent with our previous study, in which 5 methoxyl metabolites of 2,4,4′-tribrominated diphenyl ether found in intact pumpkin. In comparison, only 3 methoxyl metabolites were detected during the tissue (callus of pumpkin) culture exposure experiment.⁴⁹

Biotransformation of 2,4,6-TBP in the presence of viable rice cells occurred rapidly, and the variations in the 6 conjugation metabolites are shown in Figure 2a. Just after treating 2,4,6-TBP for 3 h, 22.6% of the dissipated 2,4,6-TBP was transformed into those 6 conjugates (mol mass). Therefore, sulfation and glycosylation are the crucial biotransformation processes contributing to the dissipation of 2,4,6-TBP in rice cells. The TP₄₀₈ level consistently increased with time during the exposure (Figure 2a), consistent with the variation trends in plant metabolism for the sulfation conjugates of other contaminants, such as diclofenac and benzotriazole, during the exposure period.^50,51 Similar to the hydroponic plant exposure experiment, TP₄₀₈ was detected in both the rice cells and cell medium (Figure 2b and c). However, the increasing TP₄₀₈ time in rice cells occurred earlier (starting from12 h) and at higher amounts (1.53 nmol at 120 h) than those measured in the cell medium (starting from 24 h, 0.69 nmol at 120 h).

Figure 2.

Temporal variations in the total amounts of conjugates in the whole viable rice suspension cell exposure system (a) and mass variations of all the conjugates in rice cells (b) and cell medium (c) of the viable cell treatments over time. TP₅₃₂, TP₆₅₂, TP₆₉₄, and TP₇₃₈ were nondetectable in the rice medium.

Two primary glycosylation conjugates (TP₄₉₀ and TP₅₃₂) were formed rapidly during the first 3 h and then dramatically decreased (3 vs 6 h, p < 0.05) (Figure 2a). Similar to the glycosylated conjugates of other contaminants (e.g., glucoside-conjugated 2-naphthol and glycosylated benzotriazoles), TP₄₉₀ was also detected in the culture and hydroponic medium.^51,52 The formation of TP₄₉₀ quickly reached the highest amounts (6.05 nmol for cells and 0.330 nmol for cell medium) at 3 h and then decreased until 120 h (Figure 2a) in the exposure system. During both in vivo and in vitro exposure events, TP₄₉₀ was the predominant glycosylation conjugate. Amounts reaching 1.86% (24 h) and 8.10% (3 h) of the 2,4,6-TBP were converted into TP₄₉₀ in the rice seedlings and cell exposure systems (Figure S6), respectively. Glycosylation was significantly greater than methylation (0.160%), a commonly concerned biotransformation procedure for phenolic contaminants in agricultural plants.^9,11,53 The other 4 glycosylation conjugates, TP₅₃₂, TP₆₅₂, TP₆₉₄, and TP₇₃₈, were only detected in the rice cells (Figure 2b and c) at 0.0452–1.572 nmol, 0.0348–0.200 nmol, 0.0293–0.0810 nmol, and 0.0305–0.105 nmol, respectively. These data demonstrate that glycosylation is a crucial biologically mediated process for the plant metabolism of phenolic chemicals.

Excretion of Sulfation and Glycosylation Conjugates from Rice Suspension Cells.

In both the rice seedling and cell exposure systems, large amounts of TP₄₀₈ and TP₄₉₀ were detected in the aqueous medium (Figure 1b and 2c). The sources of those conjugates in solution were hypothesized to be the following two important pathways: (I) the conjugates were intracellularly biosynthesized within the rice cells and then excreted from the cell or the rice roots into the aqueous medium; and (II) the conjugates were catalyzed by exocrine enzymes and directly formed in the cell medium or hydroponic solution. The increase in TP₄₀₈ occurred earlier in the rice cells than in the medium (Figure 2b and c), and the amounts of TP₄₀₈ and TP₄₉₀ were significantly higher in the rice cells than in the medium (p < 0.05), indicating that the conjugates detected in solution were more likely to be excreted from rice cells (Pathway I). An in vitro enzyme exposure experiment using stable isotope-labeled 2,4,6-TBP ([¹³C₆]-2,4,6-TBP) was conducted to identify the sources of the conjugates in the aqueous medium. The sulfation and glycosylation of 2,4,6-TBP and [¹³C₆]-2,4,6-TBP (TP₄₀₈, 13C-TP₄₀₈, TP₄₉₀, and ¹³C-TP₄₉₀) were specifically investigated in both nonautoclaved and autoclaved groups (Figure S3). Native TP₄₀₈ and TP₄₉₀ conjugates were detected in the active enzyme induction treatments (both nonautoclaved and autoclaved treatments), while none of the ¹³C-labeled TP₄₀₈ and TP₄₉₀ conjugates were found in any of the three in vitro enzyme treatments. Typical chromatograms of labeled and unlabeled conjugates detected in a nonautoclaved active enzyme induction treatment are shown in Figure S8a–d. Overall, the results indicate that extracellular enzymes have no roles in the sulfation and glycosylation of 2,4,6-TBP in the cell medium. Since TP₄₀₈ (K_ow = 1.70, Table S1) and TP₄₉₀ (K_ow = 2.17, Table S1) are more water-soluble than 2,4,6-TBP (K_ow = 4.18), the majority of the TP₄₀₈ (93.0–96.7%) and TP₄₉₀ (77.1–90.2%) are readily excreted into hydroponic solution after formation within rice roots. Thus, our findings provide profound evidence that the formation of sulfation and glycosylation conjugates significantly facilitates 2,4,6-TBP excretion.

The distributions of the sulfotransferase and glycosyltransferase, which mediate the biosynthesis of the sulfation and glycosylation conjugates, were studied in rice cells (intra-cellular enzymes) and the cell medium (extracellular enzymes) to establish the biotransformation sites in the cell exposure system. As shown in Figure S9a, 175 and 1539 different proteins were identified in the cell medium and rice cells, respectively. One sulfotransferase (P0493C06.9) and 14 glycosyltransferases were detected in the rice cells. The sequences of 14 glycosyltransferases were used to construct a phylogenetic tree with the neighbor-joining method,^54,55 and all of the relative glycosyltransferases are listed in Figure S9b. However, neither glycosyltransferase nor sulfotransferase was detected in the cell medium, suggesting that those transferases could not be excreted from rice cells into the medium. Therefore, TP₄₀₈ and TP₄₉₀ conjugates could not be biosynthesized in the cell medium without the corresponding transferases. It was further confirmed that 2 conjugates were intracellularly biosynthesized and then excreted into the solution medium.

Sequestration of Sulfation and Glycosylation Conjugates in Rice Suspension Cells.

Although sulfation and glycosylation conjugates have been widely identified in the plant metabolism of organic contaminants, no evidence maps their spatial distribution and compartmentalization at the cellular level. In rice seedlings, glycosylation conjugates bioaccumulated exclusively in the roots, while sulfation conjugate was translocated into the leaf and leaf sheath through the xylem sap. Their translocation difference might be related to their compartmentation behaviors in plant cells. To understand the compartmentation of conjugates in Phase III metabolism, exposed suspension rice cells, which were treated with 6.06 μmol/L 2,4,6-TBP for 24 h, were enzyme-hydrolyzed into several fractions (Figure S2). These identified conjugates showed markedly different spatial compartmentalization within rice cells. Figure 3a shows the 6 conjugates found in the cell exposure systems (TP_408, TP₄₉₀, TP₅₃₂, TP₆₅₂, TP₆₉₄, and TP₇₃₈) that were all detected in the protoplasts. By contrast, the sulfation (TP₄₀₈) and 3 glycosylation conjugates (TP₄₉₀, TP₅₃₂, and TP₆₅₂) were observed in the cell wall, and they consisted of extractable and bound residues (Figure 3b). Among the conjugates observed in the cell wall, only TP₄₉₀ and TP₅₃₂ were found as bound residues in the cell wall (Figure 3c). These results provided direct evidence for the speculation that glycosyl groups are inclined to bind and be sequestered in the cell wall.^6,56 Furthermore, the sequestration of TP₄₉₀ into vacuoles (Figure 3d) likely enhanced the resistance of the rice plants to 2,4,6-TBP, similar to the detoxification pathway for toxic heavy metals in higher plants.^57–59 Together, TP₄₉₀ could be exported from the cell cytosol into the vacuole and apoplast (i.e., cell medium (excretion process) and cell wall) (Figure 4). By contrast, the sulfation conjugate, TP₄₀₈, neither entered the vacuole nor acted as a bound residue in the cell wall (Figure 4). To reduce the bioaccumulation of sulfation conjugate in the rice roots, TP₄₀₈ was excreted out of the cell (of rice roots) into the cell medium (Figure 4) or into the xylem sap for vertical transport within the rice seedlings.

Figure 3.

Chromatograms of sulfation and glycosylation conjugates in the protoplasts (a), cell walls with extractable and bound residues (b), cell walls with only bound residues (c), and cell vacuoles (d).

Figure 4.

Illustration of the excretion and compartmentalization of sulfation and glycosylation conjugates at the cellular level.

Micromechanisms Underlying Differences in the Compartmentalization of 2,4,6-TBP Sulfation and Glycosylation Conjugates in Rice Plants.

Since TP₄₀₈ could not bond with the cell wall, TP₄₀₈ readily penetrated the cell wall and flowed into the xylem sap through transmembrane and symplastic pathways within the rice roots (Figure 5). Thus, the vertical transpiration of TP₄₀₈ from rice roots into the aboveground parts resulted in the ready distribution of TP₄₀₈ throughout the whole plant (i.e., rice root, leaf sheath, and leaf) (Figure 5). It was reported that sulfation conjugates formed in plants are potentially back-transformed to bioactive parent chemicals through the host microbiome in animals and humans.⁶⁰ Our results illustrate that the sulfation conjugate of 2,4,6-TBP can be vertically translocated and potentially entered the edible parts, which may adversely affect human health when those plants are used as fodder and feed. Thus, the sulfation process in the plant metabolism of bromophenols warrants great concern.

Figure 5.

Transformation of 2,4,6-TBP, and the translocation, excretion, and compartmentalization of 2,4,6-TBP conjugates in rice seedlings. 2,4,6-TBP was sulfated and glycosylated to form TP₄₀₈, TP₄₉₀, TP₅₃₂, TP₅₇₆, TP₆₂₂, TP₆₅₂, TP₆₉₄, and TP₇₃₈ conjugates in the rice roots (I), symplastic and transmembrane pathways of TP₄₀₈ in the rice roots (II), TP₄₉₀ was bonded in cell wall, and thus TP₄₉₀ could not cross the cortex into the xylem (III), the vertical transport of TP₄₀₈ through the sap-carrying xylem within the rice plants, and none of TP₄₉₀ was detected in xylem sap (IV), and plant excretion of TP₄₀₈ and TP₄₉₀ into hydroponic solution (V).

Glycosylation conjugates were sequestered in the cell walls, and they were present as extractable or bound residues (Figure 5). Although glycosylation conjugates have higher hydrophilicities (K_ow, in the range of −0.32–2.96, Table S1) than sulfation conjugate, the transportation of glycosylation conjugates in the cortex was constrained in both the symplastic and transmembrane pathways. However, glycosylation conjugates are likely hindered by the Casparian strip during apoplastic transport. These 7 glycosylation conjugates were exclusively compartmentalized in rice roots. Moreover, glycosylation conjugates transport from the cytoplasm to the vacuole (the actual occurrences of glycosylation conjugates in the cell vacuoles) was uncovered at the cellular level for the first time. This study revealed that the formation of water-soluble 2,4,6-TBP conjugates is orchestrated by plant excretion and Phase III metabolism to reduce the accumulation of 2,4,6-TBP and its conjugates in the cytoplasm and rice roots.