Interconversion between Methoxylated and Hydroxylated

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Interconversion between methoxylated and hydroxylated polychlorinated biphenyls in rice plants: an important but overlooked metabolic pathway Jianteng Sun, Lili Pan, Zhenzhu Su, Yu Zhan, and Lizhong Zhu Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b00266 • Publication Date (Web): 01 Mar 2016 Downloaded from http://pubs.acs.org on March 1, 2016

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Environmental Science & Technology

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Interconversion between methoxylated and hydroxylated

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polychlorinated biphenyls in rice plants: an important but

3

overlooked metabolic pathway

4

Jianteng Sun†, ‡, Lili Pan†, ‡, Zhenzhu Su§, Yu Zhan†, ‡, and Lizhong Zhu*, †, ‡

5 6 7

†

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310058, China

9

‡

Department of Environmental Science, Zhejiang University, Hangzhou, Zhejiang

Zhejiang Provincial Key Laboratory of Organic Pollution Process and Control,

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Hangzhou, Zhejiang 310058, China

11

§

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University, Hangzhou, China

State Key Laboratory for Rice Biology, Institute of Biotechnology, Zhejiang

13 14 15 16

*

17

Phone/Fax: +86 57188273733

18

E-mail: [email protected]

Corresponding author: Lizhong Zhu

19 20 21 22 1

ACS Paragon Plus Environment


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Abstract

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To date, there is limited knowledge of the methoxylation of polychlorinated biphenyls

25

(PCBs) and the relationship between hydroxylated PCBs (OH-PCBs) and

26

methoxylated PCBs (MeO-PCBs) in organisms. In this study, rice (Oryza sativa L.)

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was chosen as the model organism to determine the metabolism of PCBs in plants.

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Limited para-substituted 4’-OH-CB-61 (major metabolite) and 4’-MeO-CB-61

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(minor metabolite) were found after a five-day exposure to CB-61, while ortho- and

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meta-substituted products were not detected. Interconversion between OH-PCBs and

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MeO-PCBs in organisms was observed for the first time. The demethylation ratio of

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4’-MeO-CB-61 was 18 times higher than the methylation ratio of 4’-OH-CB-61,

33

indicating that the formation of OH-PCBs was easier than the formation of

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MeO-PCBs. The transformation products were generated in the roots after 24 h of

35

exposure. The results of the in vivo and in vitro exposure studies show that the rice

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itself played a key role in the whole transformation processes, while endophytes were

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jointly responsible for the hydroxylation of PCBs and demethylation of MeO-PCBs.

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The metabolic pathways of PCBs, OH-PCBs and MeO-PCBs in intact rice plants

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were proposed. The findings are important in understanding the fate of PCBs and the

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source of OH-PCBs in the environment.

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2


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1. Introduction

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Polychlorinated biphenyls (PCBs) have been used in a wide range of industrial

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applications.1 Although PCBs have been banned, they are still ubiquitous in the

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environment, in media such as water, soil, sediment and biota.2-4 The high persistence,

46

bioaccumulation potential and long-range transport ability of PCBs continue to raise

47

concerns about the chronic risk to organisms.5-6

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Although recent studies on PCBs focus mainly on the environmental

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distribution7-8 and human exposure,9-10 it is important to study the biotransformation

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of PCBs to reveal their environmental fate. The metabolic pathway of PCBs in

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organisms is generally recognized to involve enzyme-mediated oxidation via direct

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hydroxylation and formation of OH-PCBs or oxidation of the phenyl rings to form of

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arene epoxides intermediates.11-14 The arene epoxides can be hydrolyzed to OH-PCBs

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or can undergo a Phase II conjugation with glutathione to form methylsulfonyl PCBs

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(MeSO2-PCBs).15-16 OH-PCBs, considered a class of environmental contaminants, are

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prevalent in various environmental media.17-20 OH-PCBs are even more toxic than

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their parent PCBs, which interrupt reproductive processes, the endocrine system and

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the brain function.21-23 Laboratory exposure experiments showed that the

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hydroxylation ratios of PCBs were generally lower than 1%.14 Therefore, the levels of

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OH-PCBs should be much lower than the levels of PCBs. However, the concentration

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of OH-PCBs in some environmental samples was found to be close or higher than the

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concentration of PCBs.19,

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suggested that the metabolic mechanism of PCBs should be further investigated.

24

This indicated overlooked sources of OH-PCBs, and

3



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The whole biotransformation process of PCBs might be more complicated than

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previously expected. The methylation, demethylation, and methoxylation reactions of

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various

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Methoxy-metabolites were observed forming from dichlorobiphenyl in vitro by

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tobacco cells.29 However, no further study was conducted to explore the pathway and

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mechanism. There has been no report on the environmental occurrence of MeO-PCBs,

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perhaps due to lack of concern or the extremely low concentration of MeO-PCBs. In

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addition, little toxicity information on MeO-PCBs is available. On the basis of their

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chemical structure and octanol-water partition coefficient (Kow), MeO-PCBs are likely

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more lipophilic and persistent than OH-PCBs, suggesting a potential chronic health

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risk.

organic

chemicals

have

been

observed

in

living

organisms.25-28

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Unraveling the relationship between MeO-PCBs and OH-PCBs is important for

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understanding the fate of PCBs. MeO-PCBs could be either intermediates or final

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metabolites of PCBs and may play an important role in the whole metabolic pathway

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of PCBs. The generation of MeO-PCBs and their transformation relationships with

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OH-PCBs are potential metabolic pathways of PCBs that have been ignored. The

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metabolic pathway of PCBs needs to be further studied.

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Plants are the primary trophic level of the food web. According to the

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“green-liver” metabolic model, metabolism of persistent toxic substances in plants is

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similar to that in animal livers.30 The contamination by PCBs in paddy fields and rice

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plants has been reported in numerous studies.31-33 To better understand the metabolic

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pathways of PCBs in intact rice plants and relevant endophytes, both in vivo and in 4


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vitro exposure experiments were conducted. This study revealed an overlooked

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metabolic pathway of PCBs in rice plants.

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2. Environmental Section

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2.1. Chemicals.

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The commercially available pairs of homologous MeO-PCBs and OH-PCBs are

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limited. In this study, three pairs of MeO-PCBs and OH-PCBs, and their parent

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compound: 2,3,4,5-tetrachlorobiphenyl (CB-61) were selected as model compounds

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for exposure. The MeO-PCB standards were 2’-methoxy-2,3,4,5-tetrachlorobiphenyl

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(2’-MeO-CB-61),

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4’-methoxy-2,3,4,5-tetrachlorobiphenyl

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OH-PCB standards were 2’-hydroxy-2,3,4,5-tetrachlorobiphenyl (2’-OH-CB-61),

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3’-hydroxy-2,3,4,5-tetrachlorobiphenyl

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4’-hydroxy-2,3,4,5-tetrachlorobiphenyl (4’-OH-CB-61). The detailed information of

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seven exposure chemicals was shown in the Supporting Information. Working

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solutions of all exposure compounds were prepared at 100 µg/mL in acetone.

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Surrogate

102

4’-OH-CB-101 for neutral and phenolic chemicals, respectively. All the exposure

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standards and surrogate standards were purchased from AccuStandard (New Haven,

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CT, USA). Acetonitrile (HPLC grade), methyl tert-butyl ether (MTBE) (HPLC grade),

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acetone (pesticide grade), hexane (pesticide grade), and dichloromethane (DCM)

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(pesticide grade) were purchased from Fisher Scientific (Pittsburgh, PA, USA). All

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other chemicals and reagents used in the experiments were of analytical reagent grade

3’-methoxy-2,3,4,5-tetrachlorobiphenyl

standards

were

(4’-MeO-CB-61).

(3’-MeO-CB-61), and The

corresponding

(3’-OH-CB-61),

2,2’,4,5,5’-pentachlorobiphenyl

5


and

(CB-101)

and


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or higher purity. Acidified silica gel was prepared by mixing 70 g activated silica with

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30 g concentrated H2SO4. The medium and nutriment used in the tissue culture and

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endophyte culture were bought from Sigma-Aldrich (St. Louis, MO, USA). Deionized

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water (18.2 MΩ) was used in all the experiments. Standards of PCBs, OH-PCBs, and

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MeO-PCBs were all analyzed to check their purity by injecting standards at

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concentration of 10 µg/mL directly into the analytical instruments, and the amounts of

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impurities and their abundances in the standard were thereafter calculated. Only a

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trace amount of 2’-OH-CB-61 (0.004%) was found as an impurity of the

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2’-MeO-CB-61 standard which however affected neither the discussion on metabolic

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reactions nor the conclusions drawn from this research.

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2.2. In Vivo Hydroponic Exposure.

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Rice (Oryza sativa L.) was purchased from the Chinese Academy of Agricultural

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Sciences, Beijing, China. Rice seeds of similar size were selected and sterilized using

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3% (v/v) H2O2 and subsequently germinated on moist filter paper. Uniform seedlings

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(approximately 10 cm in height) were used for the exposure. Each sterile glass reactor

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was filled with 50 mL of deionized water and 1.0 µg of the individual exposure

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compound, resulting in an initial concentration of 20 ng/mL. Each reactor was planted

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with five rice seedlings, which were fully wrapped with aluminum foil and Parafilm

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to prevent photolysis and volatilization of the chemicals. Reactors were placed in a

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controlled growth chamber, where the light intensity was 250 µmol/m2/s for 16 h/day,

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the temperature was 22 ± 2 °C, and the relative humidity was 80%. The duration of

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the total exposure was five days for the seven compounds mentioned above. 6


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Meanwhile, among all the groups, rice seedlings exposed to CB-61,

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4’-OH-CB-61 and 4’-MeO-CB-61 were harvested at intervals of 24, 48, 72, 96 and

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120 h. Blank controls (seedlings in the absence of the exposure compounds) and

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unplanted controls (exposure compounds in the absence of seedlings) were set up. All

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the controls and exposure groups, including the ones for different time intervals, were

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prepared in triplicate. The roots, shoots and solutions were sampled separately after

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exposure. Plant samples were thoroughly rinsed with deionized water, dried on tissue

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paper, weighed at fresh weight, and then frozen at −50 °C for further treatment. The

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calculations of the concentrations of targeted compounds were based on fresh weight.

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2.3. Tissue Culture Exposure.

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The callus of the rice plants was obtained as follows: mature unshelled seeds

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were sterilized through immersion in 75% ethanol for 2 min, shaken with NaClO

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solution (1% active Cl) for 30 min, and rinsed five times with sterile water. The seeds

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were cultured in modified nutrient broth (NB) medium (including 4.1 g/L of NB, 30

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g/L of sucrose, 3.0 g/L of phytagel, 0.5 g/L of proline, 0.5 g/L of glutamine, 0.3 mg/L

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of casein hydrolysate, and 2.5 mg/L of 2,4-D; adjusted to pH 5.8) and incubated at 28

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ºC in the dark for seven days. Then, the scutellum-derived calluses were transferred to

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the regeneration medium. After 14 days, the calluses were collected for the exposure

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experiment. The inducing of callus and subsequent exposures were conducted at 28 ºC

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in the dark.

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Calluses (approximately 1.0 g) were transferred to 50 mL of an autoclaved

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hydroponic medium that was placed in glass flasks wrapped with aluminum foil to 7



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prevent photolysis. CB-61, 4’-MeO-CB-61 and 4’-OH-CB-61 were selected as

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exposure compounds, and the initial exposure concentration was set to 20 ng/mL.

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Blank controls (in the absence of the exposure compound) and unplanted controls (in

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the absence of callus) were set simultaneously. The exposure and control groups were

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prepared in triplicate. Calluses were sampled to determine the transformation products

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after five days.

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2.4. Endophyte Exposure.

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The procedure of isolating and culturing endophytes was based on previously

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developed methods.34-35 Briefly, the healthy rice roots were sterilized in 75% ethanol

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for 1 min and 0.5% NaClO for 10 min. The roots were then rinsed three times in

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sterile distilled water. The surface-sterilized root was cut into small pieces, placed on

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potato dextrose agar, incubated at 25 ºC and checked daily for fungal isolation. The

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tips of fungal hyphae were selected and sub-cultured on potato dextrose agar at 25ºC

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in the dark until a pure culture was obtained. The four major endophytic Fusarium

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fungi isolated were labelled as A, B, C and D.

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Four types of endophytic fungi were separately transferred to 50 mL autoclaved

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hydroponic potato dextrose medium that was placed in glass flasks for further

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cultivation. A large number of endophytic fungi were then separately exposed to

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CB-61, 4’-MeO-CB-61 and 4’-OH-CB-61 under agitation at 150 rpm. The initial

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exposure concentration was set to 20 ng/mL. Blank controls (in the absence of

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exposure compounds) and amicrobic controls (in the absence of endophytes) were set.

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Experiments were performed in triplicate. The extracts of endophyte and solution 8


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were combined to determine the metabolites after a five-day exposure.

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2.5. Sample Preparation and Analysis.

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The sample pretreatment was adapted from previous methods.25, 36 In short, solid

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samples were freeze-dried, homogenized, spiked with surrogate standards, and

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repeatedly extracted with hexane/MTBE (1:1, v/v) using a Tissuelyser (QIAGEN,

179

Hilden, Germany). The extracts were combined and evaporated to dryness and

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redissolved in 30 mL of DCM. Acidified silica gel (10 g) was added, and the mixture

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was shaken vigorously for 10 min. The acidified silica gel was then removed through

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an anhydrous Na2SO4 column (15 g). An additional 30 mL of DCM was used to

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ensure that all the compounds were eluted. The collected elution was concentrated to

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dryness and redissolved with 200 µL of hexane. Half of the extract (100 µL in hexane)

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was transferred to a new vial for the subsequent analysis of PCBs and MeO-PCBs by

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gas chromatography/mass spectrometry (GC/MS). The other half of the extract (100

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µL in hexane) was dried under a gentle flow of nitrogen gas and redissolved in 100

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µL of acetonitrile for analyzing OH-PCBs using liquid chromatography-mass

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spectrometry/mass spectrometry (LC-MS/MS). All the solution samples were spiked

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and liquid-liquid extracted with a hexane/MTBE mixture (1:1, v/v). The combined

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extracts were evaporated to dryness and solvent exchanged to 30 mL of DCM before

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purification. The subsequent process was performed according to the method used for

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solid samples.

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The analysis of PCBs and MeO-PCBs was performed on GC/MS (7890B/5977A,

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Agilent Technologies, Santa Clara, CA, USA) using an EI ion source. The compounds 9



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were separated using a DB-5 MS (J& W Scientific, Folsom, CA, USA) capillary

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column (30 m, 0.25 mm i.d., 0.25 µm film thickness) with helium as the carrier gas at

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a constant flow of 1 mL/min. The oven temperature was initially 90 ºC and then

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increased at a rate of 10 ºC/min to 210 ºC. The post run was set at 300 ºC and held for

200

3 min. The selected ion monitoring (SIM) mode was used for the quantitative

201

determination. Quantification of OH-PCBs was carried out on an LC-MS/MS

202

(Agilent1260-6460) using a C18 column (100 mm × 2.1 mm, 2.2 µm particle size,

203

Thermo Fisher Scientific, Waltham, MA, USA). The mobile phase consisting of

204

acetonitrile (A) and water (B) was used with a gradient elution of A: B from 60: 40 to

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75: 25 in 20 min at a flow rate of 0.3 mL/min. Mass spectrometric detection was

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completed using an ESI source in the negative ion multiple-reaction monitoring

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(MRM) mode. The detailed parameters for analyzing the targeted compounds using

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GC/MS and LC-MS/MS are listed in the Supporting Information.

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2.6. Quality Control and Quality Assurance.

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Quality controls were implemented to ensure the correct identification and

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accurate quantification of the targeted analytes. No mutual interference was observed

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in the instrumental analysis of OH-PCBs, MeO-PCBs and PCBs. Blank samples were

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analyzed to monitor possible contamination, showing an absence of background

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interference. The average recoveries of CB-61, MeO-PCBs and OH-PCBs in the

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spiked samples were 87.2%, 86.5−93.2% and 85.5−91.2%, respectively, where the

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relative standard deviation (RSD) for the spiked samples was lower than 15% (n = 3).

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The recoveries for the surrogate standards, including CB-101 and 4’-OH-CB-101, 10


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were 86.3−102.5% and 87.2−95.1%, respectively. The concentrations were recovery

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corrected. Five-point standard calibration curves were employed for the quantitative

220

analyses. The method limits of detection (MLODs) were estimated on the basis of a

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signal-to-noise ratio of 3. The MLODs for PCB, MeO-PCBs and OH-PCBs in solid

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samples were 90 pg/g, 100−120 pg/g and 90−110 pg/g, respectively. The MLODs for

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the three groups of compounds in solution were 110 pg/L, 120−140 pg/L and

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130−150 pg/L, respectively.

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3. Results and Discussion

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3.1. In Vivo Metabolism of PCBs by Rice Plant.

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In analyzing the hydroxylated and methoxylated metabolites, one OH-PCB:

228

4’-OH-CB-61 and one MeO-PCB: 4’-MeO-CB-61 were identified in rice plants after

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the plants were exposed to CB-61 for five days (Figure 1). To the best of the authors’

230

knowledge, MeO-PCBs have not previously been reported during in vivo metabolism

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of PCBs in intact plants. The metabolites, para-substituted by hydroxyl and methoxyl

232

groups, were predominant in this study. 2’-OH-CB-61, 2’-MeO-CB-61, 3’-OH-CB-61,

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and 3’-MeO-CB-61 were not found. None of the metabolites was found in the blank

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and unplanted controls, suggesting that rice plants transformed CB-61 to

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4’-OH-CB-61 and 4’-MeO-CB-61. Meanwhile, there was no chemical transformation

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or cross-contamination between the reactors.

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Conversion ratios between the mass of the metabolite and the initial parent

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compound (M/P) are presented in Table 1. For 4’-OH-CB-61, the M/P ratios were

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0.74% ± 0.15% (mean ± standard deviation) in the roots, 0.14% ± 0.04% in the shoots, 11



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and 0.06% ± 0.02% in the solutions. The ratios were similar to in vitro reports on

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biotransformation of PCBs to OH-PCBs in mouse liver ( 3’-MeO-CB-61 (6.46%) >

283

2’-MeO-CB-61 (2.56%) > 4’-OH-CB-61 (1.01%) > 3’-OH-CB-61 (0.70%) > 13



284

2’-OH-CB-61 (0%). Among the three pairs of compounds, only the transformation

285

from 2’-OH-CB-61 to the homologous 2’-MeO-CB-61 was not found (Table 1). The

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hydroxyl and methoxyl at para-position were easier to transform than those at meta-

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and ortho-positions. As metabolites of CB-61, the conversion ratio from

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4’-MeO-CB-61 to 4’-OH-CB-61 was higher than the conversion ratio of other

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OH-/MeO-PCB congeners and the parent CB-61. The interconversion between

290

OH-PCBs and MeO-PCBs occurred when the rice plants were exposed to PCBs.

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Moreover, these reciprocal transformation processes might also occur when the plants

292

were contaminated by OH-PCBs or MeO-PCBs in polluted environmental media such

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as polluted water. Overall, the demethylation from MeO-PCBs to OH-PCBs was

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favored over the methylation from OH-PCBs to MeO-PCBs, which might explain

295

why OH-PCBs but not MeO-PCBs were widely detected in the environment.

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The concentration distribution of exposed OH-PCBs and MeO-PCBs with their

297

metabolites in different compartments was also investigated. The results are similar to

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those exposed to CB-61. Most compounds accumulated in the roots after the exposure

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(Table 2). Translocation of parent compounds and some transformation products from

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the roots to the shoots and solutions was observed. The concentration of

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4’-OH-CB-61 produced from 4’-MeO-CB-61 was 140 ± 27 ng/g in the roots, 41 ± 5.3

302

ng/g in the shoots, and 120 ± 8.0 ng/L in the solutions after the five-day exposure.

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The concentration of 4’-MeO-CB-61 produced from 4’-OH-CB-61 was 8.5 ± 1.0 ng/g

304

in the roots, 2.8 ± 1.4 ng/g in the shoots, and 4.0 ± 2.0 ng/L in the solutions after the

305

five-day exposure. The RCFs for the six exposure chemicals were different and 14


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ranged from 562 (4’-OH-CB-61) to 858 (3’-MeO-CB-61). The mean recoveries of

307

these compounds ranged from 86% to 90% for the exposure groups, which were

308

lower than those in the unplanted controls (95%). These results imply that some other

309

metabolites such as diOH-PCBs, diMeO-PCBs or OH-MeO-PCBs might be produced.

310

However, none of them have been identified.

311

Rice seedlings exposed to 4’-OH-CB-61 and 4’-MeO-CB-61 were harvested at

312

intervals of 24, 48, 72, 96, and 120 h (Figure 2B, C). The metabolites were detected in

313

rice only after being exposed for 24 h, indicating that methylation and demethylation

314

metabolism in rice also occurred rapidly. The highest mean concentrations of products

315

were detected at the exposure time of 72 h. Together with the results of the CB-61

316

metabolism, the interconversion between 4’-OH-CB-61 and 4’-MeO-CB-61 was

317

inferred to take place simultaneously in the rice plants. Nevertheless, the

318

demethylation ratio of 4’-MeO-CB-61 was 18 times higher than the methylation ratio

319

of 4’-OH-CB-61. Consequently, 4’-OH-CB-61 was identified as the major

320

transformation product of CB-61, and 4’-MeO-CB-61 was a minor product.

321

3.3. In Vitro Metabolism of PCBs, OH-PCBs and MeO-PCBs by Rice Callus.

322

The results of in vivo exposure experiments provided information on the fate of

323

compounds in intact plants. However, the metabolism might be caused by plants

324

themselves, microorganisms or both. In vitro studies using tissue culture under axenic

325

conditions is an effective way to further clarify the mechanism.

326

After the in vitro exposure, the same hydroxylated and methoxylated metabolites

327

were found in the callus samples exposed to CB-61, whereas none of the hydroxylated 15



328

and methoxylated metabolites were found in the in vitro blank and unplanted controls.

329

These results further confirm the formation of MeO-PCBs as one of the metabolites of

330

PCBs in the tissues of rice plants. As shown in Table 3, the M/P ratios from CB-61 to

331

4’-OH-CB-61 (0.15% ± 0.03%), from CB-61 to 4’-MeO-CB-61 (0.06% ± 0.01%),

332

and the product concentrations (1.2 ± 0.2 ng/g for 4’-OH-CB-61 and 0.4 ± 0.1 ng/g

333

for 4’-MeO-CB-61) in rice callus were all lower than the product concentrations

334

obtained by the intact rice plants. The 4’-OH-CB-61 and 4’-MeO-CB-61 were used as

335

the exposure compounds to explore the transformation relationship between

336

OH-PCBs and MeO-PCBs in vitro. The methylation of 4’-OH-CB-61 and the

337

demethylation of 4’-MeO-CB-61 were both observed. The transformation process

338

from MeO-PCB to OH-PCB (M/P ratio = 2.87% ± 0.30%, product concentration = 24

339

± 2.5 ng/g) was more likely to occur than the reverse process from OH-PCB to

340

MeO-PCB (M/P ratio = 0.16% ± 0.02%, product concentration = 1.3 ± 0.2 ng/g) in

341

the rice callus. These results were consistent with the in vivo study. Therefore, rice

342

plant cells play a key role in the metabolism of PCBs, OH-PCBs and MeO-PCBs.

343

3.4. Metabolism of PCBs, OH-PCBs and MeO-PCBs by Rice Endophytes.

344

To the best of our knowledge, metabolism of PCBs, OH-PCBs and MeO-PCBs

345

by plant endophytes has not been reported. In this work we found that CB-61 was

346

transformed to 4’-OH-CB-61 after being separately exposed to three types of

347

endophytic fungi (Table 3). The conversion ratio was 0.14% ± 0.04%, 0.16% ± 0.03%,

348

and 0.25% ± 0.05% for endophytic fungi A, B and C, respectively. No MeO-PCBs

349

were identified as metabolites of CB-61 by rice endophytic fungi. The transformation 16


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from 4’-MeO-CB-61 to 4’-OH-CB-61 occurred in the exposure groups of endophytic

351

fungi A (M/P ratio of 2.30% ± 0.15%), B (M/P ratio of 2.04% ± 0.23%), and C (M/P

352

ratio of 2.18% ± 0.20%). However, methylation of 4’-OH-CB-61 by rice endophytic

353

fungi was not observed. The results indicate that these endophytic fungi were capable

354

of transforming PCBs to OH-PCBs and transforming MeO-PCBs to OH-PCBs, but

355

were not able to produce MeO-PCBs as final metabolites of PCBs or OH-PCBs. Only

356

endophytic fungi D had no function in the metabolism of PCBs, OH-PCBs and

357

MeO-PCBs, possibly due to the lack of essential enzymes. No metabolite was found

358

in the blank and amicrobic controls.

359

The amount of endophytic fungi used in the endophyte exposure reactor was

360

much greater than that of endophytic fungi inside the plant in the in vivo exposure

361

reactor. Nevertheless, the transformation ratios of the targeted compounds by the

362

isolated and cultured endophytes were lower than the transformation ratios of the

363

targeted compounds by the intact plants, therefore demonstrating that endophytes

364

played an important but not primary role throughout the metabolism pathway. Plant

365

cells and endophytes were both responsible for the metabolism of PCBs, whereas

366

plants themselves dominated the processes of methoxylation of PCBs and methylation

367

of OH-PCBs.

368

3.5. Metabolism Pathways.

369

The proposed metabolism pathways of PCBs, OH-PCBs and MeO-PCBs in rice

370

plants are shown in Figure 1. The metabolism pathways of CB-61 in our study

371

suggest that hydroxylation and methoxylation preferentially occurred at the para 17



372

position on the non-chlorinated phenyl group. The products at the ortho position and

373

meta position were not detected. A similar pattern was reported in the study of

374

2,4,4′-tribrominated diphenyl ether (BDE-28).38 The mutual transformation between

375

para-substituted MeO-PCB and OH-PCB tended to be easier. Deriving from the

376

chemical structure, the hydroxyl and methoxyl groups at the para position have the

377

longest distance from the chlorinated phenyl group.39 Thus, during interactions with

378

certain enzymes, 4’-OH-CB-61 and 4’-MeO-CB-61 have the least steric hindrance

379

and need the least energy, compared with other OH-/MeO-PCB congeners, leading to

380

their formation.

381

Three metabolism pathways were hypothesized for the methoxylation and

382

hydroxylation processes: (i) PCB was first transformed to OH-PCB, and then small

383

amounts of OH-PCB were transformed to MeO-PCB via the methylation process; (ii)

384

PCB was first transformed to MeO-PCB, and then the major portion of the MeO-PCB

385

was transformed to OH-PCB via the demethylation reaction; and (iii) methoxylation

386

and hydroxylation of PCB occurred separately and simultaneously at first, followed

387

by interconversion between OH-PCBs and MeO-PCBs. The rice plant cells and

388

endophytes collaborated in the entire transformation processes of the PCBs.

389

Hydroxylation and demethylation processes were attributed to the combined effect of

390

the plant and the endophytes, whereas the methylation and methoxylation pathways

391

are dictated by the plant itself.

392

Similar hydroxylation and methoxylation pathways of polybrominated diphenyl

393

ethers (PBDEs) in pumpkin and maize have been proposed in previous studies.25, 39-40 18


Page 18 of 34

Page 19 of 34


394

Incontrast to the mutual transformation trends of OH- PCBs and MeO-PCBs observed

395

in this study, the methylation of OH-PBDEs was found to be easier than the

396

demethylation of MeO-PBDEs.39 The comprehensive results in both laboratory

397

studies explain the ubiquitous existence of MeO-PBDEs in the environment,41-42

398

while MeO-PCBs were not detected in real environmental samples. MeO-PCBs

399

should be paid more attention in the future studies of the existence and origin of the

400

toxic OH-PCBs. An increasing number of studies have demonstrated that partial

401

MeO-PBDEs were of natural origin and produced by marine organisms.43-44

402

Demethylation from MeO-PBDEs is a more important source than the hydroxylation

403

from parent PBDEs to form OH-PBDEs.45 Therefore, the close relationship of

404

MeO-PCBs and OH-PCBs found in this study is of potential research interest to

405

explain the high OH-PCB/PCB ratio frequently detected in marine animals.19

406

Formation of OH-PCBs was previously considered to occur via oxidative

407

mechanisms from their parent PCBs, such as the epoxide intermediates and direct

408

insertion of the hydroxyl group into a biphenyl, which is mediated by cytochrome

409

P450 enzymes.46-47 However, the specific enzymes that catalyze the reciprocal

410

transformation between OH-PCBs and MeO-PCBs have not been studied. Plant

411

enzymes including O-methyltransferase, nitrate reductase, glutathione S-transferase

412

and cytochrome P450 enzymes are known to be involved in plant enzymatic

413

transformation of many xenobiotic pollutants48-49 and are enzymes potentially

414

mediating the transformation between OH-PCBs and MeO-PCBs. Our ongoing

415

studies are attempting to explore the key enzymes and the genes mediating the 19



416

proposed metabolism pathway.

417

In conclusion, after rice plants were exposed to CB-61, a major metabolite

418

(4’-OH-CB-61) and a minor metabolite (4’-MeO-CB-61) were detected, but ortho-

419

and meta-substituted products were not detected. The reciprocal transformation

420

between OH-PCBs and MeO-PCBs was observed. The demethylation ratio of

421

4’-MeO-CB-61 reached 18.4%, much higher than the methylation ratio of

422

4’-OH-CB-61. The exposure compounds and transformation products accumulated

423

mainly in the roots. Based on in vivo and in vitro exposure experiments, the

424

transformation processes of PCBs were found to occur collaboratively by the rice

425

plant and the endophytes. The proposed metabolic pathways of PCBs, OH-PCBs and

426

MeO-PCBs in intact rice plants provide important information for better

427

understanding the environmental behavior and fate of PCBs.

428

20


Page 20 of 34

Page 21 of 34


429

Supporting Information

430

The information, instrumental analysis parameters and chromatograms of targeted

431

compounds. This material is available free of charge via the Internet at

432

http://pubs.acs.org.

433 434

Acknowledgments

435

This work was jointly supported by the National Natural Science Foundation of China

436

(21520102009, 21137003, and 21507111) and the National Basic Research Program

437

of China (973 Program, 2014CB441101). The authors would like to thank Ms.

438

Xiaodan Wu and Zi Wei from the Analysis and Measurement Center of Zhejiang

439

University for assistance in sample analysis.

440 441

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Page 27 of 34


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606

Figure legends

607 608

Figure 1. Metabolic pathways of CB-61 and the corresponding OH-PCBs and

609

MeO-PCBs in intact rice plants.

610 611

Figure 2. The concentrations of exposure compounds and their metabolites in intact

612

rice plants exposed to (A) CB-61, (B) 4’-MeO-CB-61 and (C) 4’-OH-CB-61.

613

28


Page 28 of 34

Page 29 of 34


614 615 616

Figure 1

617

29



618 619

Figure 2

620

30


Page 30 of 34

Page 31 of 34


621 622

TOC art

623

31



Page 32 of 34

Table 1. Detection of metabolites after intact rice plants being exposed for five days Exposure compound

Metabolites

Conversion ratio (%) Root

Shoot

Solution

4'-OH-CB-61

0.74 ± 0.15a

0.14 ± 0.04

0.06 ± 0.02

4'-MeO-CB-61

0.08 ± 0.02

ndb

nd

4'-OH-CB-61

4'-MeO-CB-61

0.94 ± 0.11

0.04 ± 0.02

0.02 ± 0.01

4'-MeO-CB-61

4'-OH-CB-61

17.1 ± 0.22

0.70 ± 0.09

0.58 ± 0.04

3'-OH-CB-61

3'-MeO-CB-61

0.70 ± 0.18

nd

nd

3'-MeO-CB-61

3'-OH-CB-61

5.39 ± 1.08

0.32 ± 0.07

0.75 ± 0.05

2'-OH-CB-61

2'-MeO-CB-61

nd

nd

nd

2'-MeO-CB-61

2'-OH-CB-61

2.56 ± 0.57

nd

nd

CB-61

a

Mean ± standard deviation (n = 3). b Nondetectable

624

32


Page 33 of 34


Table 2. Detection of parent compounds after intact rice plants being exposed for five days Exposure compound

Accumulation ratio (%) Root

Shoot

Solution

CB-61

80.5 ± 7.2a

3.36 ± 0.52

2.10 ± 0.48

4'-OH-CB-61

73.2 ± 8.1

8.55 ± 0.66

5.92 ± 0.74

4'-MeO-CB-61

61.2 ± 6.5

5.68 ± 0.91

3.13 ± 0.55

3'-OH-CB-61

76.3 ± 5.0

7.22 ± 0.55

6.10 ± 0.79

3'-MeO-CB-61

70.8 ± 9.2

4.72 ± 0.34

3.75 ± 0.67

2'-OH-CB-61

74.0 ± 5.6

7.88 ± 0.90

5.19 ± 0.35

2'-MeO-CB-61

77.1 ± 9.5

4.54 ± 0.72

4.12 ± 0.38

a

Mean ± standard deviation (n = 3).

625

33



Page 34 of 34

Table 3. Conversion ratios between metabolite and the initial parent compound (M/P) in separate exposures of tissue culture and endophyte Endophyte exposure Exposure compound

Tissue culture exposure

Fungi A

Fungi B

Fungi C

Fungi D

M/P ratio (%)

M/P ratio (%)

M/P ratio (%)

M/P ratio (%)

M/P ratio (%)

4'-OH-CB-61

0.15 ± 0.03a

0.14 ± 0.04

0.16 ± 0.03

0.25 ± 0.05

ndb

4'-MeO-CB-61

0.06 ± 0.01

nd

nd

nd

nd

4'-OH-CB-61

4'-MeO-CB-61

0.16 ± 0.02

nd

nd

nd

nd

4'-MeO-CB-61

4'-OH-CB-61

2.87 ± 0.30

2.30 ± 0.15

2.04 ± 0.23

2.18 ± 0.20

nd

CB-61

a

Metabolite

Mean ± standard deviation (n = 3). b Nondetectable

626

34


Interconversion between Methoxylated and Hydroxylated

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