Pharmacokinetics in Plants: Carbamazepine and Its Interactions with

May 22, 2018 - Carbamazepine and lamotrigine prescribed antiepileptic drugs are highly persistent in the environment and were detected in crops irriga...
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Pharmacokinetics in Plants: Carbamazepine and Its Interactions with Lamotrigine Myah Goldstein, Tomer Malchi, Moshe Shenker, and Benny Chefetz Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b01682 • Publication Date (Web): 22 May 2018 Downloaded from http://pubs.acs.org on May 23, 2018

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Pharmacokinetics in Plants:

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Carbamazepine and its Interactions with Lamotrigine

2 3

Myah Goldstein,†,‡ Tomer Malchi,†,‡ Moshe Shenker,† and Benny Chefetz†,‡,*

4 5



Department of Soil and Water Sciences, The Robert H. Smith Faculty of Agriculture,

6

Food and Environment, The Hebrew University of Jerusalem, P.O. Box 12, Rehovot

7

7610001, Israel

8



9

The Hebrew University Center of Excellence in Agriculture and Environmental

Health

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*

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Benny Chefetz

13

Tel.: 972-8-9489384; email: [email protected]

14

Corresponding Author:

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ABSTRACT

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Carbamazepine and lamotrigine prescribed antiepileptic drugs are highly

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persistent in the environment, and were detected in crops irrigated with reclaimed

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wastewater. This study reports pharmacokinetics of the two drugs and their

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metabolites in cucumber plants under hydroponic culture, testing their uptake,

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translocation and transformation over 96 h in single and bi-solute systems at varying

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pH. Ruling out root adsorption and transformations in the nutrient solution, we

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demonstrate that carbamazepine root uptake is largely affected by the concentration

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gradient across the membrane. Unlike carbamazepine, lamotrigine is adsorbed to the

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root and undergoes ion trapping in root cells thus its translocation to the shoots is

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limited. Based on that, carbamazepine uptake was not affected by the presence of

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lamotrigine, while lamotrigine uptake was enhanced in the presence of

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carbamazepine. Transformation of carbamazepine in the roots was slightly reduced in

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the presence of lamotrigine. Carbamazepine metabolism was far more pronounced in

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the shoots than in the roots, indicating that most of the metabolism occurs in the

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leaves, probably due to higher concentration and longer residence time. This study

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indicates that the uptake of small non-ionic pharmaceuticals is passive and governed

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by diffusion across the root membrane.

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INTRODUCTION

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Carbamazepine (CBZ) is the most frequently prescribed antiepileptic drug

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worldwide, used by patients of all ages.1–4 It acts as a voltage-dependent sodium-

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channel blocker.5–7 Once administered, CBZ is metabolized, mainly in the liver, into

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various different metabolites8,9 which are subsequently excreted from the body. Of the

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total administrated dose, 13.8% was reported to be excreted as the parent compound

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(mostly in the feces); 32% of the total as the metabolite 10,11-dihydro-10,11-

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dihydroxy-carbamazepine (DiOH-CBZ), 5.1% as 3-hydroxy-carbamazepine (3-OH-

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CBZ), 4.3% as 2-hydroxy-carbamazepine (2-OH-CBZ), and 1.4% as 10,11-dihydro-

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10,11-epoxy-carbamazepine (EP-CBZ), all excreted in the urine, with several other

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metabolites excreted in both feces and urine.10 Low removal efficiency and minimal

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degradability of CBZ and its metabolites have been reported in municipal wastewater-

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treatment plants.11–13 Thus CBZ and several of its metabolites are frequently detected

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in reclaimed-wastewater used for irrigation and in freshwater bodies receiving

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effluents.10,13–16 Once CBZ is introduced into the soil via irrigation with reclaimed-

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wastewater or biosolids application, it has been shown to undergo only limited

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biodegradation17, attributed to binding to soil organic matter and/or clays18, and to be

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highly resistant to microbial decomposition.17,19 The shortest half-life reported for

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CBZ in soils is approximately 42 days, whereas in most cases it is more than 200

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days.17,20 CBZ is a neutral compound within the range of environmental pH.15

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Lamotrigine (LTG), also a sodium-channel blocker,5–7 is a frequently

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prescribed antiepileptic drug which may be co-administered with other antiepileptic

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drug such as CBZ.1,3,4 LTG is excreted in urine mainly in its glucuronide form while

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only 10% is excreted as the parent compound 21,22. LTG is of low removal efficiency

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in municipal wastewater-treatment plants and is found at concentrations above 1 µg L-

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1

in water bodies and in reclaimed-wastewater.13,23 Lamotrigine-N2-glucuronide is the

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main LTG metabolite found in reclaimed-wastewater.13,23 Following reclaimed-

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wastewater irrigation LTG was found to accumulate in the top soil exhibiting little to

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no biodegradation.17,24 LTG is a weak base with a pKa of 5.7 and a Log D of 2.12 (at

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pH of 7.5).25,26

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Being a relatively restrictive barrier to organic compounds, the casparian strip

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in plant roots exhibits traits similar to those of tight junctions which make up the

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blood–brain barrier in mammals,27,28 thus implying that compounds known to cross

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the blood–brain barrier through diffusion, such as CBZ29 and LTG,22 may also be

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taken up and translocated by plant roots. We hypothesize that CBZ and LTG are taken

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up as non-ionic species by diffusion through root cell membranes and therefore are

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not expected to directly affect each other. However, based on well documented

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pharmacokinetic interactions reported in humans30, we suggest that co-introduction of

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CBZ and LTG will affect the kinetics of CBZ metabolism within the plant. The main

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objective of this study was to understand the kinetics governing CBZ and LTG

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uptake, translocation and metabolism in plants and to reveal whether these processes

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are affected by co-introduction with other drugs (i.e., the environmental scenario). We

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also aimed to reveal if the CBZ metabolites found in plant materials are a result of in-

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plant metabolism or due to direct uptake of the metabolites from the irrigation water

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and/or soil solution.

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EXPERIMENTAL SECTION

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Chemicals. CBZ (>97% purity) was purchased from Sigma-Aldrich Israel Ltd.

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(Rehovot, Israel), and LTG (>99%) from EnzoBiochem Inc. (New York, NY). The

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following CBZ metabolites were purchased from Toronto Research Chemicals Inc.

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(Toronto, Canada): EP-CBZ, DiOH-CBZ, and 2-OH-CBZ. Selected properties of the

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studied compounds are presented in Table 1. The following labeled compounds were

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purchased from Toronto Research Chemicals: CBZ-13C-D2, LTG-13C3, DiOH-D3,

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EP-CBZ-D8.

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Hydroponic Culture Setup. Cucumber seeds (Cucumis sativus, Patriot, Hazera

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Genetics Ltd., Berurim, Israel) were germinated in vermiculite moistened with

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CaSO4·H2O-saturated solution. Seven-day-old seedlings were transferred and each

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single plant was cultivated in 1-L darkened glass jars in a continuously aerated

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nutrient solution, at an initial volume of 800 ± 10 mL, with the following composition

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of macronutrients (mM): K2SO4, 0.7; KCl, 0.1; Ca(NO3)2·4H2O, 2.0; MgSO4, 0.5;

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KH2PO4, 0.1, and micronutrients (µM): Fe-EDTA, 10; MnSO4·H2O, 0.5;

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ZnSO4·7H2O, 0.5; CuSO4, 0.2; (NH4)6Mo7O24·4H2O, 0.01; H3BO3, 10. The nutrient

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solution was prepared in deionized water, with a final pH of 5.7. The plants were

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grown in a temperature-controlled chamber with a 16/8-h day/night cycle at 25 ± 1 °C

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and 20 ± 1 °C day and night, respectively31 and relative humidity of 60-70% during

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the day and 80-100% during the night. The nutrient solution was replaced every 3–4

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days. Following 29 days of cultivation in the nutrient solution, the plants were

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introduced into a fresh nutrient solution containing: (i) CBZ, 1.82 ± 0.05 µM (430 µg

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L-1); or (ii) LTG, 1.80 ± 0.03 µM (461 µg L-1); or (iii) EP-CBZ, 1.69 ± 0.00 µM (428

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µg L-1); or (iv) DiOH-CBZ, 1.78 ± 0.00 µM (480 µg L-1); or (v) a mixture of CBZ and

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LTG at concentrations of ~2 µM each.

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Experimental Design. Plants were exposed to either CBZ, LTG, EP-CBZ, or DiOH-

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CBZ (single-solute experiments), or to CBZ and LTG (bi-solute experiments) for up

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to 96 h, maintaining a pH of 6-7 in all experiments by adding 4-(2-hydroxyethyl)-1-

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piperazineethanesulfonic acid (HEPES) to the nutrient solution at a concentration of 5

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mM. During this time, the nutrient solution was not replenished and continuously

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aerated. The plants exhibited a healthy root system throughout the entire cultivation

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and experimental period. The nutrient solution was sampled at 1, 3, 6, 8, 24, 48, 72

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and 96 h for the CBZ and LTG single-solute experiments and CBZ and LTG bi-solute

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experiments, and once after 72 h for the EP-CBZ and DiOH-CBZ single-solute

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experiments. The plants exposed to CBZ in the presence or absence of LTG were

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sampled at 8, 24, 48, 72 and 96 h; the plants exposed to the metabolites were sampled

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after 72 h. The experiments were conducted in 5 replicates for each sampling time. At

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each sampling time, a whole plant was sacrificed for sap sampling and plant analyses.

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After plants were detopped, xylem sap was collected over a period of approximately

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15 min after detopping, collecting 2-3 mL of sap from each plant, while roots were

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still in the nutrient solution, first drops being discarded. Then roots and shoots were

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washed, weighed and frozen at -20 °C until extraction.

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CBZ and LTG uptake and translocation in a bi-solute experiment was also tested

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comparatively at pH of 4.5 and 7.5. In this experiment, the exposure time was 48 h.

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To maintain pH 7.5, HEPES was added to the nutrient solution at a concentration of 5

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mM. To maintain pH 4.5, 2(N-morpholino)ethane sulfonic acid (MES) was added at a

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concentration of 5 mM. Since the MES buffer alone was not capable of maintaining a

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steady, low pH, the Ca(NO3)2·4H2O (2 mM) in the nutrient solution was substituted

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with 0.8 mM NH4NO3, 1.2 mM Ca(NO3)2·4H2O and 0.8 mM CaCl2 in the low pH

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treatment, so that the N was composed of 20% NH4+ and 80% NO3-.

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Analysis. Analyte concentrations in all solutions (sap and nutrient solutions) were

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quantified using a Waters Alliance HPLC system equipped with a LiChrospher 100

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RP-18 column (25 cm × 4.6 mm, 5 µm particle size; Merck, Darmstadt, Germany)

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and photodiode array detector. The analytes were eluted from the column at a constant

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flow rate of 1 mL min-1 and constant temperature of 45 °C; sample injection volume

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was 10 µL. The initial mobile phase consisted of a mixture of methanol (14%),

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acetonitrile (20%) and water (66%) (v/v). A gradient program was applied by raising

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the level of acetonitrile from 20 to 50% over 12 min. CBZ, EP-CBZ and DiOH-CBZ

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were quantified based on absorption at 210 nm, and LTG was quantified at 307 nm.

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Limit of detection (LOD) and limit of quantification (LOQ) values, determined using

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a signal to noise ratio of 3:1 and 10:1 respectively, were 25 and 50 µg L-1,

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respectively, for CBZ and its metabolites and 50 and 100 µg L-1, respectively, for

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LTG.

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Analyte concentration in the plant material (roots and shoots) was analyzed as

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detailed in Goldstein et al.15 In brief, plant material was ground to a fine powder and

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extracted with methanol using accelerated solvent extractor (ASE 350, Dionex,

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Sunnyvale, CA) in two static 5-min cycles with 100% methanol at 80°C under a

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constant pressure of 10.34 MPa. Extracts were evaporated to dryness and

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reconstituted in 1 mL acetonitrile:water:acetic acid (20:80:0.01) spiked with 10 µL of

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a mixture of isotopically labeled internal standards, centrifuged at 13500 rpm for 20

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minutes and filtered (0.22 µm PTFE) before LC-MS analysis. Extracts were analyzed

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by Agilent 1200 Rapid Resolution LC system (Agilent Technologies Inc., Santa

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Clara, CA) equipped with a Gemini C-18 column (150 × 2 mm, 3-µm particle size;

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Phenomenex, Torrance, CA) coupled to an Agilent 6410 triple quadruple mass

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spectrometer with ESI ion source, in multiple reaction monitoring (MRM) mode, with

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positive or negative ionization. LOD and LOQ, determined using a signal to noise

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ratio of 3:1 and 10:1 respectively, were 1and 2 µg kg-1, respectively, for LTG; 0.05

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and 0.1 µg kg-1, respectively, for CBZ; 0.1 and 0.2 µg kg-1, respectively for DiOH-

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CBZ; 0.05 and 0.1 µg kg-1, respectively, for EP-CBZ; 0.2 and 0.5 µg kg-1,

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respectively for 2-OH-CBZ and 0.05 and 0.1 µg kg-1, respectively for 3-OH-CBZ.15

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Data Analysis. Statistical analysis (non-parametric Wilcoxon/Kruskal–Wallis test and

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non-parametric multiple comparisons using Dunn method for joint ranking, p < 0.05)

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was performed using JMP Pro 10 software (JMP®, Version 10. SAS Institute Inc.,

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Cary, NC, 1989-2007). Mass balance was calculated for CBZ and its metabolites as

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the ratio between the sum of CBZ and its metabolites at each sampling time (the

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amount of CBZ in the nutrient solution and CBZ and its metabolites in the plant

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material) and the initial amount of CBZ in the nutrient solution, and found to be 95.36

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± 0.76% in the single-solute system and 94.36 ± 0.77% in the bi-solute system. Mass

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balance calculation for LTG in the bi-solute system revealed a growing deficit over

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time, starting with 92.08 ± 2.18% at 8 h and declined to 57.33 ± 3.49% at 96 h.

168 169 170

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Table 1. Selected physicochemical properties of the studied compounds Compound

Structure

Carbamazepine (CBZ)

log Kow (ref)

2.77

(24)

171

pKa (ref) pKa1 = -0.5 (24)

pKa2 = 14.4 pKa1 = -0.9 (24)

10,11-dihydro-10,11-epoxy1.97

(24)

carbamazepine (EP-CBZ)

pKa2 = 14.8

pKa1 = -1.5 (24) 10,11-dihydro-10,11dihydroxy-carbamazepine

0.81

(24)

pKa2 = 11.7 pKa3 = 12.3

(DiOH-CBZ)

pKa4 = 14.0 2-hydroxy-carbamazepine 2.66 (32)

pKa = 9.30 (32)

2.66 (32)

pKa = 9.46 (32)

1.93 (24)

pKa = 5. 7 (25)

(2-OH-CBZ) 3-hydroxy-carbamazepine (3-OH-CBZ)

Lamotrigine (LTG)

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RESULTS AND DISCUSSION

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CBZ and LTG Uptake. The concentrations of CBZ in the nutrient solution,

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as well as in the sap, were similar for the single- and bi-solute systems (i.e., plants

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exposed only to CBZ, and plants exposed to both CBZ and LTG as a mixture,

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respectively) during the exposure time (Figure 1a). Therefore, the following

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discussion about CBZ uptake by the roots describes both treatments as a single

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scenario. CBZ concentration in the nutrient solution was constant (~1.76 µM) for the

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first 48 h, after which a minor, but statistically significant rise in concentration was

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observed (1.98 µM at 72 h and 2.3 µM at 96 h; Figure 1a). LTG concentration in the

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single-solute system (Figure 1b) was constant both in the nutrient solution and in the

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sap during the exposure time. However, for the bi-solute system LTG concentration in

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the nutrient solution decreased significantly with time. Unlike the steady increase in

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CBZ concentration in the nutrient solution, CBZ concentration in the plant sap

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remained constant from 24 h to the end of the experiment (Figure 1a). It is important

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to note that the CBZ concentration in the sap was always lower than in the nutrient

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solution and this difference increased with time from 24 h to the end of the

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experiment. CBZ, being neutral with an intermediate lipophilicity (Table 1), is

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relatively easily translocated from root to shoot via the sap.33,34 It is important to note

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that metabolites of CBZ were not detected in the sap. As for LTG concentration in the

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sap, it was consistently much lower than in the nutrient solutions in both the single-

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and bi-solute systems (Figure 1b). The sap LTG in the bi-solute system exhibited a

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significant decrease in concentration following 48 h of exposure.

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Figure 1. Concentrations of carbamazepine (CBZ; left) and lamotrigine (LTG; right) in the plant sap and in the nutrient solution throughout the exposure period in the absence (single-solute system) and presence (bi-solute system) of the companion compound. Averages and standard errors are shown (n = 5).

196 197 198 199 200

The kinetics analysis of CBZ and LTG influx into the cucumber roots,

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calculated from the decreased amount of each compound in the nutrient solution, and

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water uptake are presented in Figure S1. The initial CBZ uptake rate was high (11.98

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± 2.42 and 13.52 ± 1.48 µmol-1 kg root-1 h-1 in the single- and bi-solute systems

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respectively; average ± standard error) in the first 8 h, and decreased thereafter to 5.45

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± 0.33 and 6.30 ± 0.36 µmol-1 kg root-1 h-1, in the single- and bi-solute, respectively.

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The water-uptake rate largely affected the CBZ-uptake rate, but this does not mean

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that CBZ was actually taken up with the water-influx stream. Alternatively, we

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suggest different routes for these two components. Water transport across the roots

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occurs via three pathways: (i) through the cell walls (apoplastic path), (ii) from cell to

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cell through plasmodesmata (symplastic path), and (iii) across membranes

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(transcellular path). In order to pass the casparian band and reach the sap, water and

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solutes must enter the symplast and pass through root cell membranes in the

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endodermis.35,36 Water uptake is largely driven by water potential gradients, however

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studies have demonstrated the importance of water channels known as aquaporins in

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the active regulation of water uptake and for elevated water influx rates.35,37 The non-

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ionic CBZ and LTG molecules are mainly translocated by a passive diffusion

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mechanism across root-cell membranes, and is thus largely affected by the

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concentration gradient across the membrane according to Fick’s law, thus their initial

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influx rate is rapid and slows down as the concentration gradient decrease (Figure S1).

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Later on, the influx rate of CBZ depends on a steady concentration gradient that is

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maintained by the water influx. For LTG, the translocation rate in the sap is much

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slower, as is evident from its lower concentration in the sap (Figure 1b) and thus its

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influx rate diminishes with the time of exposure. It is hypothesized that CBZ will be

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transported with water in both apoplastic and symplastic pathways, and as water

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influx and transpiration increase, a larger CBZ gradient across the membrane will be

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maintained and larger rates of CBZ uptake will prevail.

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The apparent LTG uptake rate, as calculated from LTG disappearance from

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the nutrient solution, was higher than that of CBZ throughout most of the exposure

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period (Figure S1). The apparent LTG uptake rate decreased gradually over time. The

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high initial apparent uptake rate calculated for LTG may be attributed to two separate

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mechanisms, the first being the high root sorption affinity as demonstrated in the root

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adsorption experiments where LTG adsorption was shown to be substantial and rapid

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(Figure S2). The second mechanism is ion trapping within the root vacuole. The root

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vacuole has a pH of ~5.5 under which ~50% of LTG molecules are positively charged

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and thus vacuole serve as a sink for LTG,38 and the concentration gradient between

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the external concentration (nutrient solution) and the internal cytoplasm concentration

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is preserved, resulting in a greater driving force for diffusion across the root cell

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membrane.

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To demonstrate the uptake mechanism for both drugs, we calculated the

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apparent influx concentration (µM) as the ratio between CBZ or LTG uptake rate

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(µmol kg root-1 h-1) and water-uptake rate (L kg root-1 h-1). This is shown in Figure 2.

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For CBZ the initial (8 h) apparent influx concentration was about 60% higher than the

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CBZ concentration in the nutrient solution, similar to it at 24 and 48 h and diminished

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later to be lower than the nutrient solution concentration (Figure 2a). Since CBZ

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adsorption to the external root surfaces was shown to be negligible (see SI; Figure

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S2a) and CBZ transformation products were not detected in the nutrient solution, we

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conclude that the calculated apparent CBZ-influx concentration truly represents CBZ

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influx across the root membrane (i.e., uptake).

249

250

Figure 2. Concentrations of carbamazepine (CBZ; left side) and lamotrigine (LTG; right side) in the nutrient solution and the apparent CBZ and LTG influx concentrations in the absence (single-solute system) and presence (bi-solute system) of the companion compound throughout the duration of the exposure period. Average data are shown (n = 5); bars represent standard errors.

251 252 253 254 255 256

Based on the greater initial influx rate of CBZ molecules through the root cell

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membrane, followed by a slower and steady influx rate we suggest that water and

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CBZ are taken up through separate pathways, while water uptake occurs mainly

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through regulated water channels or aquaporins, and is affected by the plant's demand

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for water, the non-ionic CBZ molecule is mainly translocated by a diffusion

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mechanism across root-cell membranes.

The CBZ influx is driven by the

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concentration gradient between the outer and inner sides of the root cell membrane in

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accordance with Fick’s law: the initial gradient is steep facilitating the high apparent

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CBZ-influx concentration; this is followed by a decreasing gradient across the

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membrane and results in decreased apparent CBZ-influx concentration (Figure 2).

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This also explains the increase in CBZ concentration in the nutrient solution with time

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(Figure 1a). The apparent LTG-influx concentration was higher than the LTG

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concentration in the nutrient solution and significantly higher than the apparent CBZ-

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influx concentration throughout the entire exposure period, although declining over

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time (Figure 2b). The large initial difference between the two compounds is probably

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reflecting not only real uptake but also a rapid adsorption of LTG to the roots, as

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shown in Figure S2. After this initial step, it reflects a higher concentration gradient

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that is maintained across the root cell membranes due to the trapping mechanisms

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described above.

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The transpiration stream concentration factor (TSCF; Figure S3) is the ratio

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between the concentration in the xylem sap and the concentration in the nutrient

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solution. The TSCF indicates the efficiency of the uptake of a chemical from the

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nutrient solution and efficiency of its translocation from roots to shoot. TSCF values

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equal to 1 are usually interpreted as indicating that the compound is taken up and

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translocated with the water transportation stream, values >1 indicate active transport,

281

and values