Direct Conjugation of Emerging Contaminants in Arabidopsis

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Direct Conjugation of Emerging Contaminants in Arabidopsis: Indication for an Overlooked Risk in Plants? Qiuguo Fu, Jianbo Zhang, Dan Borchardt, Daniel Schlenk, and Jay J. Gan Environ. Sci. Technol., Just Accepted Manuscript • Publication Date (Web): 14 May 2017 Downloaded from http://pubs.acs.org on May 14, 2017

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Environmental Science & Technology

TOC Art

ACS Paragon Plus Environment


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Direct Conjugation of Emerging Contaminants in Arabidopsis: Indication for an

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Overlooked Risk in Plants?

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Qiuguo Fu,†,‡ Jianbo Zhang,§ Dan Borchardt,¶ Daniel Schlenk,† and Jay Gan†,*

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†

Department of Environmental Sciences, University of California, Riverside, California 92521,

United States ‡

Eawag, Swiss Federal Institute of Aquatic Science and Technology Environmental Chemistry,

8600 Dübendorf, Switzerland §

Department of Health Sciences and Technology, Institute of Food, Nutrition and Health, ETH,

8092 Zürich, Switzerland ¶

Department of Chemistry, University of California, Riverside, California 92521, United States

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Word count: Text (5452) + Figure (4×300) + Table (1×300) = 6952

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ABSTRACT

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Agricultural use of treated wastewater, biosolids and animal wastes introduces a multitude of

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contaminants of emerging concerns (CECs) into the soil-plant system. The potential for food

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crops to accumulate CECs depends largely on their metabolism in plants, which at present is

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poorly understood. Here we evaluated the metabolism of naproxen and ibuprofen, two of the

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most used human drugs from the profen family, in Arabidopsis thaliana cells and Arabidopsis

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plant. The complementary use of high-resolution mass spectrometry and 14C labeling allowed

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characterization of both free and conjugated metabolites, as well as non-extractable residues.

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Naproxen and ibuprofen, in their parent form, were conjugated quickly and directly with

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glutamic acid and glutamine, and further with peptides, in A. thaliana cells. For example, after

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120 h, the metabolites of naproxen accounted for >90% of the extractable chemical mass, while

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the intact parent itself was negligible. The structures of glutamate and glutamine conjugates were

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confirmed using synthesized standards, and further verified in whole plants. Amino acid

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conjugates may easily deconjugate, releasing the parent molecule. This finding highlights the

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possibility that the bioactivity of such CECs may be effectively preserved through direct

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conjugation - a previously overlooked risk. Many other CECs are also carboxylic acids like the

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profens. Therefore, direct conjugation may be a common route for plant metabolism of these

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CECs, making it imperative to consider conjugates when assessing their risks.

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Keywords: Contaminants of emerging concern; amino acid conjugation; water reuse;

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pharmaceuticals; naproxen; ibuprofen

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INTRODUCTION

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Many countries and regions are experiencing unprecedented water scarcity due to

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population growth, urbanization, and climate change-induced disruptions in precipitation

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patterns, prompting the search for alternative water sources.1 Reuse of municipal wastewater for

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agricultural irrigation directly alleviates pressure on freshwater supplies, and indirectly

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replenishes groundwater storage via percolation.2 However, treated wastewater is known to

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contain many contaminants of emerging concern (CECs), including disinfection byproducts,

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pharmaceuticals and personal care products, and endocrine disrupting compounds. Additionally,

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large amounts of biosolids and animal wastes, which also contain numerous CECs, are

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increasingly recycled back into agricultural fields as fertilizers.3 These practices inevitably

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introduce CECs into the soil-plant system, posing the possibility for some CECs to enter the

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terrestrial food chains, including the human diet.4–6

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Studies over the last decade show that plants are capable of accumulating CECs from

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soil, but the levels in edible tissues are relatively low under realistic conditions.1,7,8 However, in

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these studies, researchers almost always targeted only the parent compound for analysis, while

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neglecting metabolites, particularly conjugates that may have biological activity.8,9 Previous

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research on xenobiotics such as pesticides shows that plants can transform man-made chemicals

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through Phase I, II and III metabolism.9–11 Phase II metabolism is characterized with conjugation

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of parent or metabolites with sugars, sulfates, and amino acids.12–14 Moreover, such conjugates

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may readily undergo cleavage through enzymatic hydrolysis (e.g., in human gut), releasing

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xenobiotics in the free form that may subsequently elicit biological responses.15 Therefore, a

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comprehensive risk assessment of CECs in the soil-plant system should consider metabolic

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transformations, especially conjugation, in plants.

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The goal of this study was to elucidate the biotransformation of CECs in plants to better

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understand their risks in the waste reuse-food safety nexus. We used naproxen and ibuprofen as

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model CECs to explore their metabolic fate in Arabidopsis. Naproxen and ibuprofen belong to

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the profen family of the non-steroidal anti-inflammatory drugs, and are among the most

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consumed human medicines.16 Like many other CECs, naproxen and ibuprofen are not

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completely removed at wastewater treatment plants (WWTPs),17 resulting in their ubiquitous

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occurrence in aquatic environments.18–20 For example, naproxen and ibuprofen were detected in

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WWTP and hospital effluents at levels up to 11 µg L-1 and 151 µg L-1, respectively.21,22

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In a recent study, low levels of naproxen and ibuprofen were detected in several

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vegetables grown with treated wastewater irrigation under field conditions.1 When cultivated

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hydroponically, naproxen and ibuprofen disappeared quickly from the nutrient media, but only

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trace amounts of the parent were found in plant tissues,23 implying that they were converted to

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other forms in the plant. Staswick et al. discovered that Arabidopsis was capable of conjugating

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endogenous carboxylic acids with a number of amino acids.24 Here we hypothesized that in

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plants, CECs with a carboxyl group, which encompass a large number of compounds, may form

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conjugates with plant biomolecules. If this pathway is prevalent, the biological activity of these

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contaminants may be effectively preserved, highlighting the importance to consider conjugates

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when predicting adverse risks of CECs.

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MATERIALS AND METHODS Chemicals. [Methoxy-14C]-Naproxen ((S)-6-methoxy-α-methyl-2-naphthaleneacetic

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acid, radiochemical and chemical purity >99%, specific activity 55 mCi/mmol) was purchased

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from American Radiolabeled Chemicals (Saint Louis, MO). Non-labeled naproxen and ibuprofen

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were obtained from Alfa Aesar (Ward Hill, MA). d3-Naproxen and d3-ibuprofen were obtained

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from C/D/N Isotopes (Pointe-Claire, Quebec, Canada). Standard of 6-O-desmethylnaproxen was

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acquired from Toronto Research Chemicals (Toronto, Ontario, Canada). Standards of glutamic

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acid and glutamine conjugates of naproxen and ibuprofen were obtained through custom

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synthesis (Biosynthesis, Lewisville, TX). All other chemicals were purchased from Fisher (Fair

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Lawn, NJ). Ultrapure water was prepared using a Barnstead E-Pure water purification system

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(Thermo Scientific, Dubuque, IA).

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Arabidopsis thaliana Cell Line. The A. thaliana cell line PSB-D was provided by the

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Arabidopsis Biological Resource Center at Ohio State University (Columbus, OH). Cells were

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cultured in suspension at 25 °C and 130 rpm in the dark based on the supplier’s instructions.

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Details on the medium composition and preparation are given in Text S1 in Supporting

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Information (SI).

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Incubation Experiments and Treatments. Active cells of A. thaliana were inoculated

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into fresh culture medium (10%, v/v), followed by incubation in the dark at 25 °C and 130 rpm

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for 4 d. An aliquot of 20 µl stock solution of 14C-naproxen (3.8 × 106 dpm) was spiked to the cell

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culture to yield an initial chemical concentration of 2.0 ± 0.05 µg mL-1. The use of such a

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relatively high concentration was to facilitate the identification of metabolites.21,22 For ibuprofen,

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only non-labeled standard was used. Several control treatments were included for quality control,

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including a blank cell culture control with A. thaliana cells only, a carrier solvent control with A.

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thaliana cells spiked with 20 µl methanol (i.e., without chemical), a medium control with

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naproxen (or ibuprofen) but without cells, and a medium control with non-viable cells

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(autoclaved at 121 °C for 45 min) similarly spiked with 14C-naproxen.

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Sample Preparation and Analysis. At 0, 6, 12, 24, 48, 96, and 120 h into the incubation,

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cells were immediately separated from the medium by centrifugation at 3000 rpm for 30 min. A

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0.5 mL aliquot of supernatant was mixed with 4 mL UltimaGold cocktail to measure 14C

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radioactivity on a PerkinElmer TriCarb liquid scintillation counter (LSC). The remaining

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supernatant was added with 50 µL of 10 mg L-1 d3-naproxen (or d3-ibuprofen) as surrogate, and

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the mixture was passed through a 150-mg HLB cartridge (Waters, Milford, MA) pre-conditioned

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with 7-mL methanol and 7-mL ultrapure water. The analytes were eluted with 20 mL methanol

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and reconstituted in 1.5 mL methanol after drying under N2.

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The cell matter after centrifugation was collected and freeze-dried, followed by

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sequential extractions and cleanup. Briefly, 0.10 g of the freeze-dried cells was spiked with 50

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µL of 10 mg L-1 d3-naproxen (or d3-ibuprofen), followed by extraction with 20 mL of methanol

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(twice) and acetonitrile (twice) in a sonication water bath. The extracts were pooled after

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centrifugation (30 min at 3000 rpm) and reconstituted in 1.0 mL methanol after drying under N2.

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A 20-µL aliquot of the final extract was measured for 14C radioactivity by LSC and defined as

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the extractable residue (ER). The remaining extract was diluted with 20 mL ultrapure water and

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passed through a 150-mg HLB cartridge (Waters, Milford, MA) for cleanup, as described above.

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The final samples were centrifuged at 12,000 rpm for 10 min (Eppendorf 5840R, Wesseling-

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Berzdorf, Germany) and filtered through a 0.22-µm polytetrafluoroethylene (PTFE) membrane

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(Millipore, Carrigtwohill, Cork, Ireland) to remove solids. The final samples were stored at -

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20 °C before analysis. The recoveries of naproxen and ibuprofen, as determined using the

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deuterated surrogate, ranged from 76% to 94%, suggesting the overall effectiveness of the above

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extraction procedure. More details on the recovery determination is provided as Text S2 in the

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Supporting Information (SI).

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To quantify the non-extractable residue, the cell matter after the sequential extractions

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was dried in the fume hood and then combusted on a Biological Oxidizer OX500 (R. J. Harvey

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Instruments, Hillsdale, NJ). The released 14CO2 was trapped in 15 mL Harvey Carbon-14

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Cocktail (R. J. Harvey) and the radioactivity was determined on LSC. The catalyst temperature

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was 680 °C, the combustion temperature 900 °C and the combustion cycle was 4 min. The

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recovery of the combustion procedure for 14C was determined to be 92.7 ± 2.6% (n= 3) using

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standards.

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UPLC-QqQ-MS/MS and LC-TOF-HRMS. The samples after SPE cleanup were

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analyzed first on LC-TOF-HRMS to obtain the overall metabolic profile and identify metabolite

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candidates after cross-comparison between the treatment and controls. The metabolite

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candidates, together with the parent compound, were further analyzed on UPLC-QqQ-MS/MS in

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different scan modes, including product ion scan, neutral loss scan, and multiple reactions

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monitoring (MRM).

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For the LC-TOF-HRMS analysis, an Agilent 1200 LC system coupled to Agilent 1200

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series UV detector and an Agilent 6210 time-of-flight (TOF) mass spectrometer was used, and

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the separation was achieved on an ACQUITY UPLC BEH C18 column (2.1 mm x 100 mm, 1.7

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µm particle size, Waters) at 40 °C. Water (containing 5% methanol and 0.001% formic acid) and

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methanol were used as the mobile phases A and B, respectively, which was programmed as

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below (with respect to e B): 0-1 min, 5% to 50%; 1-17 min, 50 to 100%; 17-27 min, 100%; 28-

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30 min, 100 to 5%; and 30-32 min, 5%. The flow rate was 0.17 mL/min and the injection volume

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was 5 µL. The UV absorbance was recorded at 232 nm. The instrument control and data

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acquisition were carried out with the MassHunter workstation software (Version B.01.03,

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Agilent). The instrumental settings for TOF-HRMS were as follow: capillary voltage 2.5

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kV, nebulizer 60 psig, drying gas 6 L/min, gas temperature 200 °C, fragmentor 115

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V, skimmer 60 V, Oct dc1 37.5 V, and Oct rf V 250 V. The mass axis was calibrated using the

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mixture provided by the manufacturer over the m/z 100 – 3200 range. Spectra were acquired in

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the electrospray ionization (ESI) negative mode over m/z 100−1700 Da, a scan rate of 1 scan/s

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and resolution of 10,000.

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For UPLC-QqQ-MS/MS analysis, samples were injected into a Waters ACQUITY ultra-

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performance liquid chromatography (UPLC) combined with a Waters Micromass Triple

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Quadrupole mass spectrometer (QqQ-MS) equipped with an electron spray ionization (ESI)

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interface (Waters, Milford, MA). Separation was achieved on the same column and with mobile

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phases as described above. The injection volume was 5 µL and flow rate was 0.2 mL min-1. The

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MRM transitions of authentic standards of naproxen, d3-naproxen, desmethyl-naproxen,

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ibuprofen and d3-ibuprofen were optimized and are summarized in Table S1. The specific

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instrument settings were: capillary voltage 3.2 kV, cone voltage 30 V, collision gas (Argon,

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99.99%) 0.2 ml/min, dwell time 0.02 s, source temperature 120 °C, desolvation temperature

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350 °C, desolvation gas 600 L h-1 and cone gas 50 L h-1. The cone voltage (V) and collision

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energy (eV) for each target analyte were optimized and are described in Supporting Information.

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Candidate Identification. Raw data files were converted to mzXML files with

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msConvert by PreoteoWizard25 and subsequently analyzed using the MZmine 2 software.26 The

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presence, peak areas, retention times, and exact m/z in the different treatments were analyzed and

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compared via MZmine according to the manual. Candidate metabolites were proposed in

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accordance with their unique presence in the treatment and absence in all controls. At identical

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retention times, characteristic natural isotopes were considered in structural elucidation. The

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possible chemical formula for metabolite candidates and the error of mass accuracy in part per

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million (ppm) were obtained using the Agilent Mass Hunter. After preliminary identification of

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candidate metabolites, the data were further confirmed by LC-QqQ-MS in the product ion scan,

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neutral loss scan, and MRM scan mode. The main fragmentation patterns were acquired to

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ascertain the structures of candidate metabolites by comparing with the database. Authentic

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standards were used to confirm the proposed structures, when available. In addition to the parent

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compound, custom synthesized standards of glutamic acid and glutamate conjugates of naproxen

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or ibuprofen were used in this study to confirm the structure of the proposed conjugates by cross

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comparison. Retention times, accurate masses, and fragmentation patterns were cross-compared

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for unambiguous identification. Validation in Whole Plants. The metabolites were further validated in the whole plants

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of two wild-type A. thaliana, i.e., Col-0 and Ler-0, grown in a hydroponic system. In brief, wild-

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type A. thaliana seedlings were exposed to naproxen at 1.0 mg L-1 in the nutrient solution. After

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4 d of exposure, the whole plants and nutrient medium were separately harvested. The

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preparation of plant and growth medium samples was the same as described above for cell

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cultures. LC-QqQ-MS/MS was used to target screen the proposed metabolites in the MRM

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mode.

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Statistical Analysis. Data are presented as mean ± standard deviation (SD). One-way

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analysis of variance (ANOVA) and Student’s t-test were carried out with GraphPad 6 to evaluate

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systematic differences among treatments and between two groups, respectively (α = 0.05).

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RESULTS Dissipation of Parent Compound in Cell Culture. Both naproxen and ibuprofen were

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considered in similar experiments and subjected to similar analyses. To streamline discussion,

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here we focus on naproxen results in the main text, and present the findings on ibuprofen in SI.

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To affirm biotransformations of naproxen in A. thaliana cells, negative and positive

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controls underwent the same handling and analytical procedures. In the control with non-viable

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cells and cell-free medium, no apparent disappearance of naproxen occurred after the 120-h

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incubation, indicating absence of abiotic transformation or adsorption onto the non-viable cells

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(Figure 1A). However, in the presence of viable A. thaliana cells, naproxen quickly dissipated in

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the medium. For example, naproxen concentration in the medium decreased from 2058 ± 290 ng

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mL-1 initially to 61.5 ± 19.2 ng mL-1 at 6 h, to 0.6 ± 0.2 ng mL-1 at 24 h, and remained at a

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negligible level until the end of 120-h incubation (Figure 1A). Fitting naproxen concentrations in

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the medium to a first-order decay model resulted in a half-life of only 1.6 h. As abiotic

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transformation or adsorption was negligible, naproxen disappearance in the medium containing

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viable cells may be attributed to uptake and subsequent biotransformation by A. thaliana cells.

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Figure 1B shows that naproxen appeared in the cells and that the level was the highest

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after 6 h of incubation (the first sampling interval), providing direct evidence for its uptake into

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A. thaliana cells. The concentration of naproxen in the cells, however, rapidly decreased as the

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incubation time further increased and became negligible after 48 h (Figure 1B). Moreover, even

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at 6 h, the amount of naproxen in the A. thaliana cells, in the parent form, accounted for only 1.0

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± 0.1% of the spiked amount. This large discrepancy in mass balance implied that naproxen was

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quickly and nearly completely converted to other forms from its parent form in the culture.

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Formation of Metabolites of Naproxen. Eleven metabolites of naproxen in A. thaliana

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cells were identified using LC-TOF-HRMS and UPLC-QqQ-MS/MS (Table 1 and Figure S1,

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Figure S2). The identification was based on the following criteria commonly used for metabolite

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identification27–31: a) error of measurement accuracy is < 5 ppm for [M-H] and [M+1-H]; b)

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difference of observed and calculated isotopic abundance is < 1.5%; c) the calculated empirical

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formula obeys the nitrogen rule;32 d) fragmentation pattern matches reported data in literature or

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database, or could be adequately explained; and e) retention time (tR) is identical to that of a

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standard, if available.

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The naproxen parent molecule was confirmed against its standard in LC-TOF-HRMS,

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giving an accurate mass to charge ratio (m/z) of 229.0837 and a predicted [M-H] formula of

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C10H9ON6. Further fragmentation gave fragments at m/z 185, 170, and 169, and the pattern was

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consistent with those in the database (MassBank record KO001524). The demethylated product,

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i.e., 6-O-desmethyl-naproxen, was observed and further confirmed with an authentic standard

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(Table S1, Figure S2). Therefore, according to the scheme proposed by Schymanski et al.,33

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naproxen and 6-O-desmethyl-naproxen achieved Level 1 unambiguous confirmation and they

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may be considered as “confirmed” structures.

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The metabolites N357 and N358 were identified via HRMS, fragmentation patterns,

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literature and database searching, and directly confirmed with custom-synthesized standards

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(Table 1, Text S2, Table S2-3, Figure S3-5). The accurate measurement of m/z of N357 (m/z

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357.1455, tR = 9.3 min) gave the predicted [M-H] formula of C19H21N2O5 with an error of 0.15

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ppm (Table 1), and the isotopic abundance difference (∆abundance) of [M+1-H] was 0.69%

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(Table S2). In addition, N357 was found to produce fragments at m/z 185, 170, 145, 128, 127,

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109, 84, and 74. The fragments of m/z 185 and 170 were the same as naproxen and suggested the

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presence of naproxen analog in the metabolite (Table 1). Meanwhile, the fragments at m/z 145,

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128, 127, 109, 84, and 74 were consistent with those of glutamine (Gln) as in the MassBank

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database (Figure S3, Table S3). Therefore, N357 was identified as (2S)-2-(6-methoxy-2-

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naphthyl) propanoyl-L-glutamine (Gln-naproxen). Similarly, the accurate m/z of N358 (m/z

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358.2609, tR = 10.0 min) allowed the prediction of a chemical formula C19H21NO6 at 4.46 ppm,

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with ∆abundance of [M+1-H] at 0.39%. The metabolite N358 produced fragments at m/z 185,

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170, 146, 128, 102, and 84, which were in agreement with the characteristic fragmentation

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pattern of naproxen and glutamic acid in the present study and in the database (Table 1, Table S3,

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and Figure S4). Consequently, N358 was identified as (2S)-2-(6-methoxy-2-naphthyl)

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propanoyl-L-glutamic acid (Glu-naproxen). The structures of Gln-naproxen and Glu-naproxen

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were further confirmed by comparing retention times and mass spectra against the authentic

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standards (Text S2 and Figure S5). Thus, Gln-naproxen and Glu-naproxen also achieved Level 1

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unambiguous confirmation as “confirmed” structures.33

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The identification of the other 8 metabolites achieved Level 2a confirmation as

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“probable” structures by applying the above criteria and analysis of fragmentation patterns.33 In

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brief, N486 was identified as (2S)-2-(6-methoxy-2-naphthyl) propanoyl-L-α-glutamyl-L-

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glutamine (GluGln-naproxen) (Table 1, Text S2), N401 as (2S)-2-(6-methoxy-2-naphthyl)

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propanoyl-L-α-anlanyl-L-threonine (ThrAla-naproxen), N429 as (2S)-2-(6-methoxy-2-naphthyl)

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propanoyl-L-α-anlanyl-L-glutamic acid (GluAla-naproxen) (Figure S4), and N457 as (2S)-2-(6-

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methoxy-2-naphthyl) propanoyl-L-α-glutamyl-L-valine (ValGlu-naproxen) (Table 1). The

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metabolites N471-a and N471-b were identified as (2S)-2-(6-methoxy-2-naphthyl) propanoyl-L-

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α-glutamyl-L-isoleucine (IleGlu-naproxen) and (2S)-2-(6-methoxy-2-naphthyl) propanoyl-L-α-

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glutamyl-L-leucine (LeuGlu-naproxen), respectively. The metabolite N505 was identified as

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(2S)-2-(6-methoxy-2-naphthyl) propanoyl-L-α-glutamyl-L-phenylalanine (PheGlu-naproxen),

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and N563 as (2S)-2-(6-hydroxy-2-naphthyl) propanoyl-L-α-glutaminylglycyl-L-tyrosine

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(TyrGlyGln-DMnaproxen) (Table 1, Text S2, Table S1, Figure S6).

272 273

Finally, N477 (m/z 477.0562, tR =7.2min) showed a predicted formula C19H17N4O7S2 with a mass error of 3.84 ppm. However, the fragments were not adequately explained and

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therefore it remained as an “unknown” structure in this study (Table 1). Phase I and II Transformations of Naproxen. Analysis of primary metabolites clearly

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showed that direct conjugation of naproxen with amino acids or peptides was a major route for

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naproxen biotransformation in A. thaliana cells, accounting for >90% of the metabolites detected

278

after 120 h of cultivation (Figures 2 and 3). In view of the confirmed and probable

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transformation intermediates, their abundance over time, and known plant auxin amino acid

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pathways,34 three tentative transformation pathways depicting Phase I and II metabolism are

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proposed (Figure 2). Briefly, naproxen parent undergoes direct conjugation with Glu to form

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Glu-naproxen (N358) and further conjugation with an additional amino acid to form dipeptide

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conjugates, i.e., ValGlu-naproxen (N457), IleGlu-naproxen (471-a), LeuGlu-naproxen (N471-b),

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GluGln-naproxen (N486), or PheGlu-naproxen (N505). In addition, naproxen may also

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conjugate directly with Gln, forming Gln-naproxen (N357) or undergo demethylation to form

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DMnaproxen. These two metabolites may then undergo multi-step reactions, e.g., demethylation

287

and amino acid conjugation, to form the tripeptide conjugates, i.e., TyrGlyGln-DMnaproxen

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(N563) (Figure 2). Interestingly, Gln-naproxen (N357) disappeared quickly from the cells, while

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Glu-naproxen (N358) increased concurrently (Figures 3 and S6), suggesting that Gln-naproxen

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may have been oxidized to Glu-naproxen. Additionally, naproxen may also conjugate with

291

peptides L-α-alanyl-L-threonine and L-α-alanyl-L-glutamic acid to form ThrAla-naproxen

292

(N401) and GluAla-naproxen (N429), respectively. The amounts of all metabolites tended to

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decrease over time, except for Glu-naproxen (N358) or ThrAla-naproxen (N401) (Figures S7 and

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S8), suggesting that some conjugates were further converted or incorporated into cell matter.

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Throughout the incubation, the relative fraction of Glu-naproxen (N358) increased, and that of Gln-naproxen (N357) decreased, while naproxen and the other metabolites remained at

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similar levels over time (Figure 3). As reference standards were not available for all metabolites,

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it was impossible to accurately evaluate the abundance of the conjugates in the cell extracts.

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However, given their generally similar core structures and the use of identical analytical

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conditions, the kinetics of formation and accumulation of these metabolites may be regarded as

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semi-quantitative.30 These metabolites were also observed in the whole plant of Arabidopsis (Col-0 and Ler-

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0). The concentrations of naproxen, Glu-naproxen and Gln-naproxen in Arabidopsis seedlings

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after 4 d of cultivation were 3.5 ± 0.9, 3.2±1.0, and 1.5 ± 0.4 mg kg-1, respectively, for

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Arabidopsis Col-0 and 1.8 ± 0.0, 4.6 ± 0.6, and 4.4 ± 1.3 mg kg-1, respectively, for Arabidopsis

306

Ler-0.

307

To the best of our knowledge, this is the first observation that conjugation of the parent

308

molecule with amino acids, instead of demethylation, glucuronidation, or sulfation, contributed

309

largely to the metabolic fate of naproxen in plants. The amino acid conjugates of naproxen and

310

its metabolite demethyl-naproxen were previously observed only in animals and microorganisms,

311

where the glycine conjugate was the most frequently observed, while the glucuronide conjugate

312

was the most dominant.35–40

313

Mass Balance and Phase III Compartmentation of Naproxen. To elucidate the full

314

metabolic fate of naproxen in plant cells, 14C labeled naproxen was used to quantitatively track

315

the incorporation of naproxen and its metabolites into cell matter. Figure 4 shows the dissipation

316

of extractable residues, formation of non-extractable residue and the overall mass balance of 14C

317

radioactivity. The overall mass balance ranged from 89±2 to 100±5% throughout the incubation,

318

suggesting good recoveries using the sample preparation protocol (Figure 4). The total

319

radioactivity in system did not change appreciably over time, implying that there was negligible

14



320

loss due to volatilization of naproxen or its metabolites (e.g., 14CO2), or adsorption to container

321

surfaces. At the end of incubation, the extractable residues were 74.2 ± 0.6% of the initial spiked

322

14

C activity. The non-extractable residue quickly increased to 11.1 ± 1.7% at 6 h and further to

323

21.7 ± 3.4% at 24 h, and then gradually decreased to 11.0 ± 0.7% at 120 h. Therefore, non-

324

extractable residue was a relatively small fraction of the total radioactivity. This finding was

325

similar to that for the herbicide 2,4-D (also a carboxylic acid as naproxen) in soybean (Glycine

326

max L.) and wheat (Triticum aestivum L.) cells.41,42

327

Direct Conjugation of Ibuprofen. To test whether other profens undergo similar

328

transformation pathways in Arabidopsis, we exposed Arabidopsis cells to ibuprofen and

329

identified metabolites in a similar manner as for naproxen. The results showed that ibuprofen

330

underwent very similar transformation reactions to naproxen. For example, ibuprofen parent

331

conjugated directly with Gln or Glu to form Gln-IBP (IBP333, confirmed structure) or Glu-IBP

332

(IBP334, confirmed structure) (Figure S9, Text S2). In addition, ibuprofen was hydroxylated to

333

OH-IBP, which subsequently conjugated with Glu, Gln, and Ser to form 2-OH-Glu-IBP

334

(IBP350a), 3-OH-Glu-IBP (IBP350b), OH-Gln-IBP (IBP349), and OH-Ser-IBP (IBP308)

335

(Figure S9, Table S4). These results, together with findings for naproxen, clearly show that

336

conjugation with amino acids, especially Glu and Gln, was a major route of biotransformation of

337

profens in Arabidopsis.

338

It must be noted that to facilitate the identification of metabolites,21,22 relatively high

339

concentrations of naproxen and ibuprofen were used in both the cell cultivation and whole plant

340

hydroponic experiments. For instance, the concentration of naproxen or ibuprofen in wastewater

341

effluents usually does not exceed the low µg/L level. However, when treated wastewater is used

342

to irrigate a field, it is likely that water evaporates or leaches below the root zone, rendering the

15


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343

actual concentration of chemicals in the soil porewater higher than that in the irrigation water.

344

The root zone enrichment may further increase with repeated water applications. On the other

345

hand, studies show that plant transformation of xenobiotics (e.g., 2-mercaptobenzothiazole in

346

Arabidopsis) followed a similar kinetics at different concentrations. The dependence of

347

naproxen or ibuprofen biotransformation kinetics on the treatment concentrations was

348

unfortunately not evaluated in this study. Future research should consider field relevant

349

concentrations to obtain realistic plant accumulation data and refine potential dietary exposure

350

assessment.

351 352 353

DISCUSSION This study represents the first attempt to explore the metabolic fate of commonly used

354

profen drugs in plants by considering especially the formation of conjugates. The results clearly

355

showed that plant metabolites of naproxen or ibuprofen differed from that in other biota such as

356

microorganisms,43 fish,39 rodents,44 and humans.35,45 Demethylation, and conjugation with

357

glucuronic acid, sulfate, or glycine accounted for the major biotransformation routes of naproxen

358

in animals and microorganisms.38,39,44,46 Surprisingly, even though they were targeted for

359

analysis, there was no glucuronide conjugate of naproxen or ibuprofen detected in this study. In

360

contrast, a series of amino acid and peptide conjugates of the parent compound were identified in

361

the present study as the primary phase II metabolites of naproxen or ibuprofen in Arabidopsis

362

(Figure 2 and 5, Table S4 and Table S5). This observation was consistent with LeFevre et al. who

363

found several amino acid conjugates of 2-mercaptobenzothiazole27 and benzotriazole47 in

364

Arabidopsis, while no glucuronide conjugate was detected. In addition, glutamine and glutamate

365

also conjugated with hydroxyl-ibuprofen. These findings together suggest that conjugation with

16



366 367

glutamic acid and glutamine is a common pathway for profens in Arabidopsis. Amino acid and dipeptide conjugates of other carboxylic acids have been previously

368

observed in insects, rodents, birds, mammals, and human, but this transformation route is

369

considered an “unusual” pathway in plants.48–50 For example, conjugates of carboxylic acids with

370

alanine, aspartic acid, arginine, serine, histidine, glutamic acid, glycine, glutamine, aspartic acid,

371

tryptophan, and taurine, as well as several dipeptides (i.e. glycyltaurin, glycylglycin,

372

aspartylserine, glycylvaline) have been reported in different animal species such as spiders,

373

houseflies, millipede, peripatus, rat, mouse, hamster, pigeon, hen, mallard, duck, bat, cow, horse,

374

cat, dog, and humans (Table S5 and Figure S10).48,51–53 In comparison, only sporadic studies

375

have shown amino acid conjugates of carboxylic acids in plants. Staswick et al.24 characterized

376

the conjugates of a plant auxin, indole-3-acetic acid (IAA), with Ala, Asp, Phe, and Trp in A.

377

thaliana seedlings. Feung et al.41 and Witham et al.42 studied the metabolism of the carboxylic

378

acid herbicide 2, 4-D in soybean callus tissue cultures. Seven amino acid conjugates were found,

379

with 2,4-D-Glu and 2,4-D-Asp as the major conjugates.41,42 In addition, the metabolic fate of 2,4-

380

D in plant cell cultures was found to be similar to that in whole plants, suggesting that

381

biotransformation in cells closely reflects the biotransformation pathways in intact plants.54

382

Findings from the previous studies and the current study together suggest that amino acid

383

conjugation is likely a common transformation pathway of carboxylic acids in plants.

384

Amino acid conjugation differs from glucuronidation or sulfation, as it involves one of

385

the various adenosine triphosphate (ATP)-dependent acid: CoA synthetases and acyl-CoA: amino

386

acid N-acyltransferases. The initial step in both xenobiotic and bile acid conjugation involves

387

activation of the carboxyl group to the reactive intermediate acyl-CoA, which is then transferred

388

to an amino acid residue.48,49 The most obvious physicochemical parameter potentially useful in

17


Page 18 of 42

Page 19 of 42


389

determining conjugation is probably the pKa value of the carboxylic acid. The data for three

390

monochlorobenzoic acid derivatives indicated that the extent of glycine conjugation increased

391

with pKa and a change of ca. 1 pKa unit resulted in a 10- to 12-fold increase in glycine

392

conjugation.48 However, for compounds with a narrower range of pKa values, the position of

393

substitution is of greater importance.48,55 In addition to the substitution position, steric hindrance

394

may also be a determining factor in amino acid conjugation. For example, examination of the

395

ortho-substituted benzoic acids showed that chlorine, nitro, and methyl groups may have

396

stronger steric hindrance than the other moieties such as hydrogen and amino group.48

397

Furthermore, the conjugation pattern differed between isomers 1- and 2-naphthylacetic acids,

398

where 1-naphthylacetic acid conjugated preferentially with glucuronic acid and glycine, whereas

399

2-naphthylacetic acid conjugated favorably with glutamine, taurine, and glycine (Table S5).56

400

Indeed, naproxen and ibuprofen, analogous to 2-naphthylacetic acid, were found to be

401

preferentially conjugated with glutamine in Arabidopsis at the beginning of incubation. The

402

decrease of glutamine conjugate of naproxen (Figure S7) after 6 h may be due to further

403

conversions, e.g., to glutamate conjugate via oxidation (Figure 2). It must be noted that naproxen

404

(pKa = 4.2) and ibuprofen (pKa = 4.6) have very similar pKa values, and differences in their

405

conjugate formation may be attributable more to differences in their substitutions.57 Such

406

structural effects should be considered in future research to establish quantitative structure-

407

activity (QSAR) relationships, such as for the profens that all contain a propionic acid group with

408

different substitutions.

409

The predominance of certain amino acid conjugates observed in this and previous studies

410

may be due to the abundance and substrate specificity of enzymes in Arabidopsis that catalyze

411

these reactions.24,58 Staswick et al.,24 Westfall et al.,59 and Peat et al.60 systematically identified

18



412

and characterized an Arabidopsis enzyme family, i.e., indole-3-acetic acid (IAA).

413

amidosynthetase (GH3), for conjugating IAA with various amino acids. Among the 19 GH3

414

proteins from Arabidopsis, GH3.5, GH3.2, GH3.12 and GH3.17 appeared to favor Glu over

415

other amino acids under in vitro reaction conditions.24,59 In addition, benzoate and 1-

416

naphthaleneacetic acid (NAA), structurally similar to naproxen, were found to have affinity for

417

the active site of GH3 proteins.24,60,61 These mechanistic studies, together with our results, show

418

that the GH3 enzyme family in plants may have a significant role in catalyzing the conjugation

419

of carboxylic acid compounds such as profens in plants. It is likely that differences in enzyme

420

types or their abundance may contribute to variations among plant species for their ability to

421

carry out these conjugation reactions.

422

Phase II reactions, i.e., glucuronidation, sulfation, and amino acid conjugation, are

423

generally considered detoxification pathways in animals, since the conjugates may be excreted

424

via urine and bile. However, conjugates in plant tissues may lead to underestimation of human

425

exposure, if such conjugates are deconjugated in plants or after human intake. Although it is

426

unclear how amino acid conjugates of naproxen or ibuprofen are regulated in plants, there is

427

evidence showing that glutamate conjugate of indole acetic acid (IAA-Glu) is not appreciably

428

hydrolyzed compared to other amino acid (e.g., leucine, alanine) conjugates of IAA in

429

Arabidopsis seedlings.34 In addition, human gut microbiota, enterocytes, and hepatocytes are

430

known to express enzymes such as aminoacylases, dipeptidases and glucuronidases that may

431

cleave conjugates.15,62–68 For instance, amino acid conjugates of salicylic acid were stable in the

432

upper gastrointestinal tract, but were hydrolyzed in the colon to release the active parent salicylic

433

acid.68 Glycine conjugate of naproxen was found to exhibit 78.5% reversion to naproxen by fecal

434

content in vitro.69 In general, the deconjugation rate of amino acid conjugates varied from 52.6%

19


Page 20 of 42

Page 21 of 42


435

to 96.5%.64,66,68–70 Indeed, the daily intake of naproxen, if estimated by considering the

436

conjugation fraction in Arabidopsis seedlings and deconjugation rates, would be 752 ng/kg body

437

weight/day, using the same input values as in Prosser and Sibley.71 The calculated adult hazard

438

quotient would become 0.11, which is 11 times larger than that derived solely from the parent

439

compound (Text S3).71

440

The metabolite-parent back transformation was also found for other biologically active

441

compounds such as steroidal growth promoters72 and anticancer drugs.63 In some cases,

442

hydrolysis of conjugates and the following release of the active compound may be even fatal.63

443

Irinotecan, a potent antineoplastic prodrug in clinical use, was metabolized to an active

444

metabolite, SN-38, which conjugates with glucuronic acid by the liver to form SN-38-

445

glucuronide and then excreted into the colon via bile. The SN-38-gluruconide then served as a

446

substrate for gut microbial glucuronidase, producing active metabolite SN-38, which caused

447

severe diarrhea.63

448

While the release of parent or metabolites in the free form may be a source of

449

exposure,64,73 the amino acid conjugates themselves may also confer toxicological effects.69,74

450

For example, a variety of amino acid conjugates of naproxen and ibuprofen were found to have

451

similar biological activity to the parent in vivo in rats.74 The glycine/glutamine conjugates of

452

several other carboxylic acids also showed acute toxicity to mice or rat that was comparable to

453

that of the parent on a molar basis.48,75 This preservation and/or enhancement of reactivity or

454

toxicity has also been observed for various conjugates arising from acetylation, glucuronidation,

455

and glutathione conjugation.48 However, little is known about the biological activity of amino

456

acid conjugates of plant origin. Given the dominance of amino acid conjugates as observed for

457

naproxen and ibuprofen, it is possible that the other profens or carboxylic acids may undergo a

20



458

similar transformation process. Thus, it is imperative to conduct further research to understand

459

the toxicological consequence of amino acid conjugates of such CECs from plant matrices.

460

Findings from this study showed that naproxen and ibuprofen were quickly metabolized

461

to produce a range of metabolites in Arabidopsis cells. The parent form of naproxen or ibuprofen

462

was only a negligible fraction of the chemical mass in Arabidopsis cells after 48 h. As many as

463

10 monoamino acid or peptide conjugates were confirmed or tentatively identified for naproxen,

464

of which the primary conjugates were Gln-naproxen (N357) and Glu-naproxen (N358). A similar

465

pattern was also observed for ibuprofen. The conjugates of amino acids and the parent compound

466

were also detected in whole plants of Arabidopsis. Given the importance of amino acid

467

conjugation in the plant metabolism of naproxen and ibuprofen, and that such conjugates are

468

potentially biologically active either before or after cleavage, risk assessment based on the

469

monitoring of the parent compound alone may lead to underestimations. Therefore, improved

470

risk assessment of human exposure to these compounds through practices such as wastewater

471

irrigation and solid waste reuse in agriculture should take plant biotransformation and conjugates

472

into consideration.

473

As many CECs similarly carry a carboxylic group, conjugation with amino acids may be

474

a common pathway for plant metabolism of a wide range of man-made chemicals. Further

475

research is needed to evaluate the biological activity of conjugates of CECs of plant origin and to

476

characterize the dependence of such conjugation on chemical structures as well as plant species.

477

Field and clinical observations targeting such conjugates in food produce may offer information,

478

enabling realistic risk predictions for practices, such as the use of treated wastewater, biosolids

479

and animal wastes in agriculture.

480

21


Page 22 of 42

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481

Supporting Information

482

Additional details on descriptions of cell culture medium, metabolite identification, human

483

health risk calculation, and mass spectral information of naproxen and ibuprofen metabolites.

484

This material is available free of charge via the Internet at http://pubs.acs.org.

485 486

Corresponding Author:

487

*Phone: 951-827-2712; fax: 951-827-3993; e-mail: [email protected].

488 489

ACKNOWLEDGMENTS. We appreciate Mr. Ron New for assistance in instrumental analysis.

490

This research was supported by the U.S. Environmental Protection Agency (Grant No.

491

83582901).

492 493

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32



716

Figure captions:

717 718

Figure 1. Levels of naproxen in the medium and Arabidopsis thaliana cells. (A) Concentrations

719

of naproxen in the medium cultured with nonviable cells (filled square, ■), no cells (filled

720

triangle, ▲), and viable cells (open circle, ○); and (B) Concentrations of naproxen in viable cells

721

(open diamond, ◊) and nonviable cells (open square, □).

722 723

Figure 2. Proposed formation pathways of naproxen metabolites observed in Arabidopsis

724

thaliana cells based on literature pathways of auxin storage and reactivation. Full chemical

725

names are given in Table 1 for naproxen and its metabolites. Solid line means that phase II

726

metabolites were formed, and dash line indicates that phase I metabolites were formed. Two

727

solid arrow suggests multi-step reactions.

728 729

Figure 3. Kinetics of realtive fractions (%, based on LC-MS/MS peak areas) of naproxen and its

730

metabolites in Arabidopsis thaliana cells during 120-h incubation. Full chemical names are

731

given in Table 1.

732 733

Figure 4. Extractable residue (filled triangle, ▼), bound residue (open triangle, △), and mass

734

balances (open square, □) of 14C-naproxen in Arabidopsis thaliana cell cultures. Error bars

735

represent standard deviation of triplicates. Invisible error bars are in the symbols.

736 737 738 739

33


Page 34 of 42

Page 35 of 42


740

Table 1. Identities of naproxen and its transformation products, and information of high-resolution mass spectra, fragments, retention

741

times, and identification confidence.

742

IDa

tR (min) TOF

m/z measured

Predicted formula [M-H]

m/z calculate d

NPX

11.5

229.0874

C14H13O3

229.0870

N357

9.3

357.1455

C19H21N2O5

357.1456

N358

10.0

358.1312

C19H20NO6

358.1296

N401

9.1

401.1721

C21H25N2O6

401.1718

Error (ppm)

Fragments (m/z)

*185 (naproxen-CO2), 1.7 170 (185-CH3), 169 185 (-Gln-CO), 170 (185-CH3), *145 (Gln), a 128 (Gln-NH2), 0.15 127 (Gln-OH), 109 (145-H2O-NH3), 84 (128-CO2), 74 185 (-Glu-CO), 170 (185-CH3), 146 (Glu), b 4.46 128 (Glu-H2O), *102 (Glu-CO2), 84 (128-CO2) 383 ( 401-H2O), 185 (-AlaThr-CO), 170 (185-CH3), 189 (AlaThr), 0.61 *171 (189-OH), 153 (171-H2O), 141 (171-CH2O), 128 (AlaThr-CO2-OH), 34


Structure proposed

Confidenceb Level 1 (Confirmed)

Level 1 (Confirmed)

Level 1 (Confirmed)

Level 2a (Probable)


N429

9.5

429.1675

C22H25N2O6

N457

10.2

457.1988

C24H29N2O7

N471a

10.8

471.2125

C25H31N2O7

D471b

11.2

471.2141

C25H31N2O7

110 (128-NH3), 86 (Thr-OH-CH3), 84 (128-C2H6N), 185 (-GluAla-CO), 170 (185-CH3), 155 (170-CH3), 429.1667 1.79 300 (+H2O-Glu) 199 (GluAla), *128 (Glu- H2O), *88 (Ala), c 185 (-GluVal-CO), 170 (185-CH3), 395 (-CO2-OH), 341 (-Val), d 297 (341-CO2), 457.1980 1.71 245 (ValGlu), e 228 (245-OH), 201 (228-OH), 184 (228-CO2), *128 (Glu- H2O), 116 (Val) 185 (-GluIle-CO), 128 (Glu-H2O), 457.2137 0.8 241 (GluIle-OH) 130 (Ile), f 185 (-GluLeu-CO), 170 (185-CH3), 128 (Glu-H2O), 457.2137 0.9 241 (GluLeu-OH) 130 (Leu), g

35


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Level 2a (Probable)

Level 2a (Probable)

Level 2a (Probable)

Level 2a (Probable)

Page 37 of 42


N486

8.9

486.1885

C24H28N3O8

N505

11.0

505.2003

C28H29N2O7

N563

8.5

563.2145

C29H31N4O8

185 (-GluGln-CO), 170 (185-CH3), *145 (Gln), 128 (Gln-OH/Glu-H2O), 127 (Gln-OH), 109 (145-H2O-NH3), 486.1882 0.63 146 (Glu), 102 (Glu-CO2), 86 (102-O), 274 (GluGln) 257 (274-OH), 239 (257-H2O), 213 (257-CO2), 185 (-PheGlu-CO), 170 (185-CH3), 325 (-Phe-O), 275 (PheGlu-OH), 252 (275-OH), 505.1980 4.5 *164 (Phe), h 147, (Phe-OH) 72 (Phe-Bn), i 128 (Glu-H2O), 109 (128-NH4), 171 (-TyrGlyGln-CO), *351 (TyrGlyGln-NH2), 333 (351-H2O), 383 (-Tyr), 162 (Tyr-OH), 563.2147 0.47 149 (Tyr-OH-NH), 119 (Tyr-CO2-OH), 128 (Gln-NH2), 101 (Gln-CO-NH2), 89, 85, 72,

36


Level 2a (Probable)

Level 2a (Probable)

Level 2a (Probable)


N477

743 744 745 746 747 748 749 750 751 752

7.2

477.0562

C19H17N4O7S2

477.0544

405, 360, 129, 195, 284, 119, 110, 101, 99, *97, 3.84 96, 85, 75,145, 139, 165, 203, 436, 74, 128, 175, 236

a

Page 38 of 42

Unknown structure

Level 3 (Unknown)

List of abbreviations: NPX (naproxen): (2S)-2-(6-methoxy-2-naphthyl)propanoic acid; N357, (2S)-2-(6-methoxy-2naphthyl)propanoyl-L-glutamine; N358, (2S)-2-(6-methoxy-2-naphthyl)propanoyl-L-glutamic acid; N401, (2S)-2-(6-methoxy-2naphthyl)propanoyl-L-α-anlanyl-L-threonine; N429, (2S)-2-(6-methoxy-2-naphthyl)propanoyl-L-α-anlanyl-L-glutamic acid; N457, (2S)-2-(6-methoxy-2-naphthyl)propanoyl-L-α-glutamyl-L-valine; N471-a, (2S)-2-(6-methoxy-2-naphthyl)propanoyl-L-α-glutamyl-Lisoleucine; D471-b, (2S)-2-(6-methoxy-2-naphthyl)propanoyl-L-α-glutamyl-L-leucine; N486, (2S)-2-(6-methoxy-2naphthyl)propanoyl-L-α-glutamyl-L-glutamine; N505, (2S)-2-(6-methoxy-2-naphthyl)propanoyl-L-α-glutamyl-L-phenylalanine; and N563, (2S)-2-(6-hydroxy-2-naphthyl)propanoyl-L-α-glutaminylglycyl-L-tyrosine. b Structure identification confidence was calculated according to Schymanski et al.33 ppm: part per million.

37


Page 39 of 42


753 754

755 756 757

Figure 1.

758 759 760 761 762 763 764

38



765 766 767

Figure 2.

768

39


Page 40 of 42

Page 41 of 42


769

770 771 772

Figure 3.

773 774

40



775

776 777 778

Figure 4.

41


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Direct Conjugation of Emerging Contaminants in Arabidopsis

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