CBZ y Plantas

Subscriber access provided by Kaohsiung Medical University
Environmental Processes
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
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,

Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.
Page 1 of 32 Environmental Science & Technology
ACS Paragon Plus Environment

Environmental Science & Technology Page 2 of 32
Pharmacokinetics in Plants: 1
Carbamazepine and its Interactions with Lamotrigine 2
Myah Goldstein,†,‡ Tomer Malchi,†,‡ Moshe Shenker,† and Benny Chefetz†,‡,* 4
†
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
‡
The Hebrew University Center of Excellence in Agriculture and Environmental 9
Health 10
11
*
Corresponding Author: 12
Benny Chefetz 13
Tel.: 972-8-9489384; email: benny.chefetz@mail.huji.ac.il 14
1
ABSTRACT 15
Carbamazepine and lamotrigine prescribed antiepileptic drugs are highly 16
persistent in the environment, and were detected in crops irrigated with reclaimed 17
wastewater. This study reports pharmacokinetics of the two drugs and their 18
metabolites in cucumber plants under hydroponic culture, testing their uptake, 19
translocation and transformation over 96 h in single and bi-solute systems at varying 20
pH. Ruling out root adsorption and transformations in the nutrient solution, we 21
demonstrate that carbamazepine root uptake is largely affected by the concentration 22
gradient across the membrane. Unlike carbamazepine, lamotrigine is adsorbed to the 23
root and undergoes ion trapping in root cells thus its translocation to the shoots is 24
limited. Based on that, carbamazepine uptake was not affected by the presence of 25
lamotrigine, while lamotrigine uptake was enhanced in the presence of 26
carbamazepine. Transformation of carbamazepine in the roots was slightly reduced in 27
the presence of lamotrigine. Carbamazepine metabolism was far more pronounced in 28
the shoots than in the roots, indicating that most of the metabolism occurs in the 29
leaves, probably due to higher concentration and longer residence time. This study 30
indicates that the uptake of small non-ionic pharmaceuticals is passive and governed 31
by diffusion across the root membrane. 32
2
INTRODUCTION 33
Carbamazepine (CBZ) is the most frequently prescribed antiepileptic drug 34
worldwide, used by patients of all ages.1–4 It acts as a voltage-dependent sodium- 35
channel blocker.5–7 Once administered, CBZ is metabolized, mainly in the liver, into 36
various different metabolites8,9 which are subsequently excreted from the body. Of the 37
total administrated dose, 13.8% was reported to be excreted as the parent compound 38
(mostly in the feces); 32% of the total as the metabolite 10,11-dihydro-10,11- 39
dihydroxy-carbamazepine (DiOH-CBZ), 5.1% as 3-hydroxy-carbamazepine (3-OH- 40
CBZ), 4.3% as 2-hydroxy-carbamazepine (2-OH-CBZ), and 1.4% as 10,11-dihydro- 41
10,11-epoxy-carbamazepine (EP-CBZ), all excreted in the urine, with several other 42
metabolites excreted in both feces and urine.10 Low removal efficiency and minimal 43
degradability of CBZ and its metabolites have been reported in municipal wastewater- 44
treatment plants.11–13 Thus CBZ and several of its metabolites are frequently detected 45
in reclaimed-wastewater used for irrigation and in freshwater bodies receiving 46
effluents.10,13–16 Once CBZ is introduced into the soil via irrigation with reclaimed- 47
wastewater or biosolids application, it has been shown to undergo only limited 48 sufre
limitada
biodegradation17, attributed to binding to soil organic matter and/or clays18, and to be 49 degradació
n
highly resistant to microbial decomposition.17,19 The shortest half-life reported for 50
CBZ in soils is approximately 42 days, whereas in most cases it is more than 200 51
days.17,20 CBZ is a neutral compound within the range of environmental pH.15 52
Lamotrigine (LTG), also a sodium-channel blocker,5–7 is a frequently 53
prescribed antiepileptic drug which may be co-administered with other antiepileptic 54
drug such as CBZ.1,3,4 LTG is excreted in urine mainly in its glucuronide form while 55
only 10% is excreted as the parent compound 21,22. LTG is of low removal efficiency 56
in municipal wastewater-treatment plants and is found at concentrations above 1 µg L- 57
3
1
in water bodies and in reclaimed-wastewater.13,23 Lamotrigine-N2-glucuronide is the 58
main LTG metabolite found in reclaimed-wastewater.13,23 Following reclaimed- 59
wastewater irrigation LTG was found to accumulate in the top soil exhibiting little to 60
no biodegradation.17,24 LTG is a weak base with a pKa of 5.7 and a Log D of 2.12 (at 61
pH of 7.5).25,26 62
Being a relatively restrictive barrier to organic compounds, the casparian strip 63
in plant roots exhibits traits similar to those of tight junctions which make up the 64
blood–brain barrier in mammals,27,28 thus implying that compounds known to cross 65
the blood–brain barrier through diffusion, such as CBZ29 and LTG,22 may also be 66
taken up and translocated by plant roots. We hypothesize that CBZ and LTG are taken 67
up as non-ionic species by diffusion through root cell membranes and therefore are 68
not expected to directly affect each other. However, based on well documented 69
pharmacokinetic interactions reported in humans30, we suggest that co-introduction of 70
CBZ and LTG will affect the kinetics of CBZ metabolism within the plant. The main 71
objective of this study was to understand the kinetics governing CBZ and LTG 72
uptake, translocation and metabolism in plants and to reveal whether these processes 73
are affected by co-introduction with other drugs (i.e., the environmental scenario). We 74
also aimed to reveal if the CBZ metabolites found in plant materials are a result of in- 75
plant metabolism or due to direct uptake of the metabolites from the irrigation water 76
and/or soil solution. 77
78
EXPERIMENTAL SECTION 79
Chemicals. CBZ (>97% purity) was purchased from Sigma-Aldrich Israel Ltd. 80
(Rehovot, Israel), and LTG (>99%) from EnzoBiochem Inc. (New York, NY). The 81
following CBZ metabolites were purchased from Toronto Research Chemicals Inc. 82
4
(Toronto, Canada): EP-CBZ, DiOH-CBZ, and 2-OH-CBZ. Selected properties of the 83
studied compounds are presented in Table 1. The following labeled compounds were 84
purchased from Toronto Research Chemicals: CBZ-13C-D2, LTG-13C3, DiOH-D3, 85
EP-CBZ-D8. 86
Hydroponic Culture Setup. Cucumber seeds (Cucumis sativus, Patriot, Hazera 87
Genetics Ltd., Berurim, Israel) were germinated in vermiculite moistened with 88
CaSO4·H2O-saturated solution. Seven-day-old seedlings were transferred and each 89
single plant was cultivated in 1-L darkened glass jars in a continuously aerated 90
nutrient solution, at an initial volume of 800 ± 10 mL, with the following composition 91
of macronutrients (mM): K2SO4, 0.7; KCl, 0.1; Ca(NO3)2·4H2O, 2.0; MgSO4, 0.5; 92
KH2PO4, 0.1, and micronutrients (µM): Fe-EDTA, 10; MnSO4·H2O, 0.5; 93
ZnSO4·7H2O, 0.5; CuSO4, 0.2; (NH4)6Mo7O24·4H2O, 0.01; H3BO3, 10. The nutrient 94
solution was prepared in deionized water, with a final pH of 5.7. The plants were 95
grown in a temperature-controlled chamber with a 16/8-h day/night cycle at 25 ± 1 °C 96
and 20 ± 1 °C day and night, respectively31 and relative humidity of 60-70% during 97
the day and 80-100% during the night. The nutrient solution was replaced every 3–4 98
days. Following 29 days of cultivation in the nutrient solution, the plants were 99
introduced into a fresh nutrient solution containing: (i) CBZ, 1.82 ± 0.05 µM (430 µg 100
L-1); or (ii) LTG, 1.80 ± 0.03 µM (461 µg L-1); or (iii) EP-CBZ, 1.69 ± 0.00 µM (428 101
µg L-1); or (iv) DiOH-CBZ, 1.78 ± 0.00 µM (480 µg L-1); or (v) a mixture of CBZ and 102
LTG at concentrations of ~2 µM each. 103
Experimental Design. Plants were exposed to either CBZ, LTG, EP-CBZ, or DiOH- 104
CBZ (single-solute experiments), or to CBZ and LTG (bi-solute experiments) for up 105
to 96 h, maintaining a pH of 6-7 in all experiments by adding 4-(2-hydroxyethyl)-1- 106
piperazineethanesulfonic acid (HEPES) to the nutrient solution at a concentration of 5 107
5
mM. During this time, the nutrient solution was not replenished and continuously 108
aerated. The plants exhibited a healthy root system throughout the entire cultivation 109
and experimental period. The nutrient solution was sampled at 1, 3, 6, 8, 24, 48, 72 110
and 96 h for the CBZ and LTG single-solute experiments and CBZ and LTG bi-solute 111
experiments, and once after 72 h for the EP-CBZ and DiOH-CBZ single-solute 112
experiments. The plants exposed to CBZ in the presence or absence of LTG were 113
sampled at 8, 24, 48, 72 and 96 h; the plants exposed to the metabolites were sampled 114
after 72 h. The experiments were conducted in 5 replicates for each sampling time. At 115
each sampling time, a whole plant was sacrificed for sap sampling and plant analyses. 116
After plants were detopped, xylem sap was collected over a period of approximately 117
15 min after detopping, collecting 2-3 mL of sap from each plant, while roots were 118
still in the nutrient solution, first drops being discarded. Then roots and shoots were 119
washed, weighed and frozen at -20 °C until extraction. 120
CBZ and LTG uptake and translocation in a bi-solute experiment was also tested 121
comparatively at pH of 4.5 and 7.5. In this experiment, the exposure time was 48 h. 122
To maintain pH 7.5, HEPES was added to the nutrient solution at a concentration of 5 123
mM. To maintain pH 4.5, 2(N-morpholino)ethane sulfonic acid (MES) was added at a 124
concentration of 5 mM. Since the MES buffer alone was not capable of maintaining a 125
steady, low pH, the Ca(NO3)2·4H2O (2 mM) in the nutrient solution was substituted 126
with 0.8 mM NH4NO3, 1.2 mM Ca(NO3)2·4H2O and 0.8 mM CaCl2 in the low pH 127
treatment, so that the N was composed of 20% NH4+ and 80% NO3-. 128
Analysis. Analyte concentrations in all solutions (sap and nutrient solutions) were 129
quantified using a Waters Alliance HPLC system equipped with a LiChrospher 100 130
RP-18 column (25 cm × 4.6 mm, 5 µm particle size; Merck, Darmstadt, Germany) 131
and photodiode array detector. The analytes were eluted from the column at a constant 132
6
flow rate of 1 mL min-1 and constant temperature of 45 °C; sample injection volume 133
was 10 µL. The initial mobile phase consisted of a mixture of methanol (14%), 134
acetonitrile (20%) and water (66%) (v/v). A gradient program was applied by raising 135
the level of acetonitrile from 20 to 50% over 12 min. CBZ, EP-CBZ and DiOH-CBZ 136
were quantified based on absorption at 210 nm, and LTG was quantified at 307 nm. 137
Limit of detection (LOD) and limit of quantification (LOQ) values, determined using 138
a signal to noise ratio of 3:1 and 10:1 respectively, were 25 and 50 µg L-1, 139
respectively, for CBZ and its metabolites and 50 and 100 µg L-1, respectively, for 140
LTG. 141
Analyte concentration in the plant material (roots and shoots) was analyzed as 142
detailed in Goldstein et al.15 In brief, plant material was ground to a fine powder and 143
extracted with methanol using accelerated solvent extractor (ASE 350, Dionex, 144
Sunnyvale, CA) in two static 5-min cycles with 100% methanol at 80°C under a 145
constant pressure of 10.34 MPa. Extracts were evaporated to dryness and 146
reconstituted in 1 mL acetonitrile:water:acetic acid (20:80:0.01) spiked with 10 µL of 147
a mixture of isotopically labeled internal standards, centrifuged at 13500 rpm for 20 148
minutes and filtered (0.22 µm PTFE) before LC-MS analysis. Extracts were analyzed 149
by Agilent 1200 Rapid Resolution LC system (Agilent Technologies Inc., Santa 150
Clara, CA) equipped with a Gemini C-18 column (150 × 2 mm, 3-µm particle size; 151
Phenomenex, Torrance, CA) coupled to an Agilent 6410 triple quadruple mass 152
spectrometer with ESI ion source, in multiple reaction monitoring (MRM) mode, with 153
positive or negative ionization. LOD and LOQ, determined using a signal to noise 154
ratio of 3:1 and 10:1 respectively, were 1and 2 µg kg-1, respectively, for LTG; 0.05 155
and 0.1 µg kg-1, respectively, for CBZ; 0.1 and 0.2 µg kg-1, respectively for DiOH- 156
7
CBZ; 0.05 and 0.1 µg kg-1, respectively, for EP-CBZ; 0.2 and 0.5 µg kg-1, 157
respectively for 2-OH-CBZ and 0.05 and 0.1 µg kg-1, respectively for 3-OH-CBZ.15 158
Data Analysis. Statistical analysis (non-parametric Wilcoxon/Kruskal–Wallis test and 159
non-parametric multiple comparisons using Dunn method for joint ranking, p < 0.05) 160
was performed using JMP Pro 10 software (JMP®, Version 10. SAS Institute Inc., 161
Cary, NC, 1989-2007). Mass balance was calculated for CBZ and its metabolites as 162
the ratio between the sum of CBZ and its metabolites at each sampling time (the 163
amount of CBZ in the nutrient solution and CBZ and its metabolites in the plant 164
material) and the initial amount of CBZ in the nutrient solution, and found to be 95.36 165
± 0.76% in the single-solute system and 94.36 ± 0.77% in the bi-solute system. Mass 166
balance calculation for LTG in the bi-solute system revealed a growing deficit over 167
time, starting with 92.08 ± 2.18% at 8 h and declined to 57.33 ± 3.49% at 96 h. 168
169
170
8
Table 1. Selected physicochemical properties of the studied compounds 171
Compound Structure log Kow (ref) pKa (ref)
pKa1 = -0.5 (24)

(24)
Carbamazepine (CBZ) 2.77
pKa2 = 14.4
10,11-dihydro-10,11-epoxy- pKa1 = -0.9 (24)

(24)
1.97
carbamazepine (EP-CBZ)
pKa2 = 14.8
pKa1 = -1.5 (24)

10,11-dihydro-10,11-
pKa2 = 11.7
(24)
dihydroxy-carbamazepine 0.81
pKa3 = 12.3
(DiOH-CBZ)
pKa4 = 14.0
2-hydroxy-carbamazepine
2.66 (32) pKa = 9.30 (32)
(2-OH-CBZ)
3-hydroxy-carbamazepine
2.66 (32) pKa = 9.46 (32)
(3-OH-CBZ)
Lamotrigine (LTG) 1.93 (24) pKa = 5. 7 (25)
172
9
RESULTS AND DISCUSSION 173
CBZ and LTG Uptake. The concentrations of CBZ in the nutrient solution, 174
as well as in the sap, were similar for the single- and bi-solute systems (i.e., plants 175
exposed only to CBZ, and plants exposed to both CBZ and LTG as a mixture, 176
respectively) during the exposure time (Figure 1a). Therefore, the following 177
discussion about CBZ uptake by the roots describes both treatments as a single 178
scenario. CBZ concentration in the nutrient solution was constant (~1.76 µM) for the 179
first 48 h, after which a minor, but statistically significant rise in concentration was 180
observed (1.98 µM at 72 h and 2.3 µM at 96 h; Figure 1a). LTG concentration in the 181
single-solute system (Figure 1b) was constant both in the nutrient solution and in the 182
sap during the exposure time. However, for the bi-solute system LTG concentration in 183
the nutrient solution decreased significantly with time. Unlike the steady increase in 184
CBZ concentration in the nutrient solution, CBZ concentration in the plant sap 185
remained constant from 24 h to the end of the experiment (Figure 1a). It is important 186
to note that the CBZ concentration in the sap was always lower than in the nutrient 187
solution and this difference increased with time from 24 h to the end of the 188
experiment. CBZ, being neutral with an intermediate lipophilicity (Table 1), is 189
relatively easily translocated from root to shoot via the sap.33,34 It is important to note 190
that metabolites of CBZ were not detected in the sap. As for LTG concentration in the 191
sap, it was consistently much lower than in the nutrient solutions in both the single- 192
and bi-solute systems (Figure 1b). The sap LTG in the bi-solute system exhibited a 193
significant decrease in concentration following 48 h of exposure. 194
10
195
Figure 1. Concentrations of carbamazepine (CBZ; left) and lamotrigine (LTG; right) 196
in the plant sap and in the nutrient solution throughout the exposure period in the 197
absence (single-solute system) and presence (bi-solute system) of the companion 198
compound. Averages and standard errors are shown (n = 5). 199
200
The kinetics analysis of CBZ and LTG influx into the cucumber roots, 201
calculated from the decreased amount of each compound in the nutrient solution, and 202
water uptake are presented in Figure S1. The initial CBZ uptake rate was high (11.98 203
± 2.42 and 13.52 ± 1.48 µmol-1 kg root-1 h-1 in the single- and bi-solute systems 204
respectively; average ± standard error) in the first 8 h, and decreased thereafter to 5.45 205
± 0.33 and 6.30 ± 0.36 µmol-1 kg root-1 h-1, in the single- and bi-solute, respectively. 206
The water-uptake rate largely affected the CBZ-uptake rate, but this does not mean 207
that CBZ was actually taken up with the water-influx stream. Alternatively, we 208
suggest different routes for these two components. Water transport across the roots 209
occurs via three pathways: (i) through the cell walls (apoplastic path), (ii) from cell to 210
cell through plasmodesmata (symplastic path), and (iii) across membranes 211
(transcellular path). In order to pass the casparian band and reach the sap, water and 212
solutes must enter the symplast and pass through root cell membranes in the 213
endodermis.35,36 Water uptake is largely driven by water potential gradients, however 214
11
studies have demonstrated the importance of water channels known as aquaporins in 215
the active regulation of water uptake and for elevated water influx rates.35,37 The non- 216
ionic CBZ and LTG molecules are mainly translocated by a passive diffusion 217
mechanism across root-cell membranes, and is thus largely affected by the 218
concentration gradient across the membrane according to Fick’s law, thus their initial 219
influx rate is rapid and slows down as the concentration gradient decrease (Figure S1). 220
Later on, the influx rate of CBZ depends on a steady concentration gradient that is 221
maintained by the water influx. For LTG, the translocation rate in the sap is much 222
slower, as is evident from its lower concentration in the sap (Figure 1b) and thus its 223
influx rate diminishes with the time of exposure. It is hypothesized that CBZ will be 224
transported with water in both apoplastic and symplastic pathways, and as water 225
influx and transpiration increase, a larger CBZ gradient across the membrane will be 226
maintained and larger rates of CBZ uptake will prevail. 227
The apparent LTG uptake rate, as calculated from LTG disappearance from 228
the nutrient solution, was higher than that of CBZ throughout most of the exposure 229
period (Figure S1). The apparent LTG uptake rate decreased gradually over time. The 230
high initial apparent uptake rate calculated for LTG may be attributed to two separate 231
mechanisms, the first being the high root sorption affinity as demonstrated in the root 232
adsorption experiments where LTG adsorption was shown to be substantial and rapid 233
(Figure S2). The second mechanism is ion trapping within the root vacuole. The root 234
vacuole has a pH of ~5.5 under which ~50% of LTG molecules are positively charged 235
and thus vacuole serve as a sink for LTG,38 and the concentration gradient between 236
the external concentration (nutrient solution) and the internal cytoplasm concentration 237
is preserved, resulting in a greater driving force for diffusion across the root cell 238
membrane. 239
12
To demonstrate the uptake mechanism for both drugs, we calculated the 240
apparent influx concentration (µM) as the ratio between CBZ or LTG uptake rate 241
(µmol kg root-1 h-1) and water-uptake rate (L kg root-1 h-1). This is shown in Figure 2. 242
For CBZ the initial (8 h) apparent influx concentration was about 60% higher than the 243
CBZ concentration in the nutrient solution, similar to it at 24 and 48 h and diminished 244
later to be lower than the nutrient solution concentration (Figure 2a). Since CBZ 245
adsorption to the external root surfaces was shown to be negligible (see SI; Figure 246
S2a) and CBZ transformation products were not detected in the nutrient solution, we 247
conclude that the calculated apparent CBZ-influx concentration truly represents CBZ 248
influx across the root membrane (i.e., uptake). 249
250
Figure 2. Concentrations of carbamazepine (CBZ; left side) and lamotrigine (LTG; 251
right side) in the nutrient solution and the apparent CBZ and LTG influx 252
concentrations in the absence (single-solute system) and presence (bi-solute system) 253
of the companion compound throughout the duration of the exposure period. Average 254
data are shown (n = 5); bars represent standard errors. 255
256
Based on the greater initial influx rate of CBZ molecules through the root cell 257
membrane, followed by a slower and steady influx rate we suggest that water and 258
CBZ are taken up through separate pathways, while water uptake occurs mainly 259
13
through regulated water channels or aquaporins, and is affected by the plant's demand 260
for water, the non-ionic CBZ molecule is mainly translocated by a diffusion 261
mechanism across root-cell membranes. The CBZ influx is driven by the 262
concentration gradient between the outer and inner sides of the root cell membrane in 263
accordance with Fick’s law: the initial gradient is steep facilitating the high apparent 264
CBZ-influx concentration; this is followed by a decreasing gradient across the 265
membrane and results in decreased apparent CBZ-influx concentration (Figure 2). 266
This also explains the increase in CBZ concentration in the nutrient solution with time 267
(Figure 1a). The apparent LTG-influx concentration was higher than the LTG 268
concentration in the nutrient solution and significantly higher than the apparent CBZ- 269
influx concentration throughout the entire exposure period, although declining over 270
time (Figure 2b). The large initial difference between the two compounds is probably 271
reflecting not only real uptake but also a rapid adsorption of LTG to the roots, as 272
shown in Figure S2. After this initial step, it reflects a higher concentration gradient 273
that is maintained across the root cell membranes due to the trapping mechanisms 274
described above. 275
The transpiration stream concentration factor (TSCF; Figure S3) is the ratio 276
between the concentration in the xylem sap and the concentration in the nutrient 277
solution. The TSCF indicates the efficiency of the uptake of a chemical from the 278
nutrient solution and efficiency of its translocation from roots to shoot. TSCF values 279
equal to 1 are usually interpreted as indicating that the compound is taken up and 280
translocated with the water transportation stream, values >1 indicate active transport, 281
and values <1 indicate that the roots act as a barrier for uptake and/or 282
translocation.33,39 For CBZ, TSCFCBZ in both the single- and the bi-solute systems, 283
increased from ~0.7 to 0.9 at 48 h and declined back to ~0.7 at 96 h. TSCFLTG values 284
14
were significantly lower, ranging from 0.1 to 0.25. These differences may be 285
attributed to the above described mechanisms of root adsorption and ion trapping 286
within the root cells, as opposed to the CBZ that shows no root adsorption and is 287
easily transported from roots to shoots. 288
Previous studies have examined the relationship between log Kow and TSCF. 289
In barley, the TSCF was determined to be highest for compounds with a log Kow of 290
1.8. Very polar (log Kow <-0.5) and very lipophilic compounds (log Kow >4.5) were 291
found to have lower translocation rates, determining a log Kow dependent bell shaped 292
curve for translocation.33 In soybean, Hsu et al.,40 concluded a similar log Kow 293
dependent bell shaped curve however with higher Kow values (log Kow of 2.5-3.5) 294
having maximum translocation values. In a more recent study by Dettenmaier et al.,39 295
results contradicted past findings of a bell shaped curve and rather concluded a 296
sigmoid relationship between log Kow and the TSCF with compounds with low Kow 297
values having higher TSCF values approaching 1.39 According to the empirical 298
relationship suggested by the three authors, the TSCFCBZ would be 0.44, 0.52 and 299
0.68 according to Dettenmaier et al.,39 Briggs et al.33 and Hsu et al.,40 respectively. 300
The values suggested by Hsu et al.,40 provide the best fit with the measured results for 301
CBZ. The difference in empirical relationships is most likely due to the difference in 302
experimental setup. 303
LTG was non-ionic in the nutrient solution (pH 6-7), however unlike CBZ the 304
compound becomes positively charged (pKa = 5.7) in different plant compartments. 305
The uptake of LTG is thus affected by the electrochemical potential of the membrane 306
in addition to the concentration gradient, best described using the Nernst-plank 307
equation.41 If LTG was non-ionic the equations discussed above would result in a 308
TSCF of 0.64, 0.78 and 0.44 according to Dettenmaier et al.,39 Briggs et al.33 and Hsu 309
15
et al.,40 respectively. The actual values are much lower (0.1 – 0.25), supporting the 310
different behavior of ionic compounds and that LTG undergoes additional interactions 311
in the plant root system. 312
The neutral fraction of LTG is dependent on the pH. The pH of the nutrient 313
solution was 6-7. Plant root compartments vary in pH, the apoplast has a pH range of 314
~5-6.5, the cytoplasm has a pH of ~7 and cell vacuoles a pH of ~5.5.41,42 Thus, LTG 315
will be mostly neutral in the nutrient solution and cytoplasm but partially ionized in 316
the apoplast and inside cell vacuoles. The reduced concentration of the neutral 317
fraction in the vacuole and apoplast results in a concentration gradient from the 318
nutrient solution to the vacuoles, resulting in accumulation of LTG due to ion trapping 319
in the vacuole.38 In addition, the positively charged fraction of LTG may undergo 320
electrostatic interactions with the negatively charged cell membrane further increasing 321
the concentration gradient from the nutrient solution to the root cell.38 322
In order to compare the TSCF of LTG it is possible (with limitations) to use 323
the pH-dependent distribution coefficient log D. The log D parameter assumes that 324
only the non-charged fraction of the compound may partition into the lipid phase. Log 325
D is determined by the Kow and pKa using the Henderson-Hasselbalch equation to 326
determine the neutral fraction.43,44 Based on log D of 1.4 at pH of 5.5 (as assumed to 327
be in the vacuole) the TSCFLTG was recalculated using the suggested relationships, 328
providing values of 0.74, 0.74 and 0.26 according to Dettenmaier et al.,39 Briggs et 329
al.,33 and Hsu et al.,40 respectively. As found for CBZ, the relationship proposed by 330
Hsu et al.,40 provides the best fit to LTG using the log D. 331
CBZ in the Plant Biomass. Concentrations of CBZ alone and the sum of 332
CBZ and its metabolites in the root (Figure S4 and Figure 3, respectively) were 333
doubled during the experiment (from 40 to 80 nmol g-1). No differences were 334
16
observed between the single- and bi-solute systems. The increase in the sum of CBZ 335
and its metabolites in the shoots was steeper, the concentration was 17 times higher at 336
96 h as compared to 8 h of exposure (Figure 4S). This trend is supported by an 337
increase over time of the shoot-to-root CBZ concentration ratio (Figure S5). Both PCs 338
may be competing for the same CYP 450 enzymes, slightly reducing the degree of 339
CBZ metabolism in the presence of LTG and possibly vice versa, indicating 340
pharmacokinetic interactions when CBZ and LTG are co-introduced. These 341
pharmacokinetic interactions may be amplified under prolonged cultivation. 342
343
Figure 3. The sum of carbamazepine (CBZ) and its metabolites (detailed in Figure 4) 344
measured in the roots and shoots during the exposure period in the absence (single- 345
solute) and presence (bi-solute) of lamotrigine. Average data are shown (n = 3); bars 346
represent standard errors. 347
348
Among the CBZ metabolites, we identified and quantified EP-CBZ, DiOH- 349
CBZ, 2-OH-CBZ and 3-OH-CBZ in the different plant organs, roots and shoot 350
(Figure 4). EP-CBZ, 2-OH-CBZ and 3-OH-CBZ are known to be direct products of 351
CBZ metabolism via the CYP 450 family of enzymes, while DiOH-CBZ is a product 352
of EP-CBZ hydrolysis.8,9,19 EP-CBZ, a highly reactive and pharmacologically active 353
CBZ metabolite,45-47 was found in relatively large amounts, 1–2 orders of magnitude 354
17
higher than the other detected metabolites. 3-OH-CBZ concentration in the shoots and 355
roots for both treatments was 2–3 times higher than observed for 2-OH-CBZ 356
following the 48 h sampling. Riemenschneider et al.49 reported higher concentrations 357
of 2-OH-CBZ for zucchini, rucola and potato leaves compared to 3-OH-CBZ, while 358
in leaves of eggplant, parsley and carrot, all grown under field conditions, only 3-OH- 359
CBZ was quantified. Our results coincide with Pearce et al.8 who reported the 360
formation rate of 3-OH-CBZ in the human body to be at least 25 times greater than 361
that observed for 2-OH-CBZ. Both 2-OH-CBZ and 3-OH-CBZ may lead to the 362
formation of reactive metabolites which are attributed to toxicity resulting from CBZ 363
administration.8,9. DiOH-CBZ concentration was found to be significantly (p<0.05) 364
higher in the single-solute system compared to that in the bi-solute system in the 365
shoots at both 72 and 96 h and in the roots at 96 h. Accordingly, relatively high 366
concentrations of LTG were observed in these roots (71.75 ± 4.47 to 219.50 ± 14.05 367
nmol g-1 LTG between 8 and 96 h, respectively). These observations are in agreement 368
with the pharmacokinetic interactions between CBZ and LTG in the human body, 369
which may result in epoxide hydrolase inhibition, causing the lower DiOH-CBZ 370
concentrations in the bi-solute system.49 371
To confirm our hypothesis of possible interactions between LTG uptake and 372
EP-CBZ concentration in the plant material, uptake and transformation of CBZ in the 373
presence of LTG was further tested at two pH levels. It was found that at pH 4.5, 374
where 87% of the LTG is positively charged and is therefore taken up to a lesser 375
extent (Figures S6), the concentration of EP-CBZ in the shoots and in the roots were 376
significantly higher than found under pH 7.5 (Figure S7), where LTG is in its neutral 377
form and therefore taken up at a significantly higher concentration (Figure S6). Since 378
CBZ uptake was found to be unaffected by changes in nutrient solution pH (Figure 379
18
S6), these data further support the effect of LTG on CBZ metabolism. As reported by 380
Sankar and Lerner,47 although CYP 450 is not the favored pathway for LTG 381
metabolism in the human body, at high doses the drug will be metabolized by this 382
route. Since both compounds were taken up at relatively high and similar levels, it can 383
be suggested that both compounds are competing for the same CYP 450 enzymes, 384
reducing the degree of CBZ metabolism in the presence of LTG and possibly vice 385
versa. 386
387
388
389
Figure 4. Carbamazepine (CBZ) metabolites EP-CBZ, DiOH-CBZ, 2-OH-CBZ and 390
3-OH-CBZ in the roots and shoots during the exposure period in the absence (single- 391
solute) and presence (bi-solute) of lamotrigine. Average data are shown; bars 392
represent standard errors. 393
394
19
Our data on CBZ metabolism kinetics may help reveal whether CBZ 395
metabolism occurs mainly in the roots or shoots. DiOH-CBZ and 3-OH-CBZ were 396
detected and quantified in the roots starting at 8 h, while in the shoots, these 397
metabolites were not identified until the 24 h sampling, whereas EP-CBZ and 2-OH- 398
CBZ where found in both plant parts at the 8 h sampling. DiOH-CBZ is a 399
transformation product of EP-CBZ (by epoxide hydrolase) and 3-OH-CBZ is derived 400
directly from the parent compound, suggesting that the substrate for each metabolite 401
may have been below the critical enzymatic activation concentration in the shoots 402
during the first 24 h of exposure. Thereafter, shoot metabolism was far more 403
pronounced than root metabolism, indicating that most of the metabolism of CBZ by 404
CYP 450 occurs in the shoots. In support of this, the ratio between EP-CBZ 405
concentration in the shoots and CBZ concentration in the whole plant (roots + shoots) 406
was higher and constantly growing from 0.19 ± 0.02% at 8 h to 6.58 ± 0.50% at 96 h, 407
compared to this ratio in the roots, which remained relatively stable and below 1% 408
throughout the exposure period (Figure 5). Similar results were obtained for both the 409
single- and bi-solute systems with no statistical difference. 410
411
20
Figure 5. Concentration ratio between EP-CBZ and CBZ during the exposure period, 412
in the absence (single- solute) and presence (bi-solute) of lamotrigine. Average data 413
are shown; bars represent standard errors. 414
415
CBZ and its metabolites were detected in many crops,14,15,24,48,50 yet it is still 416
unclear whether the metabolites found in the plant material are a result of in-plant 417
metabolism of the parent compound or due to direct uptake of the metabolites from 418
the irrigation water and/or soil solution. Therefore the uptake of the major and most 419
stable metabolites of CBZ, EP-CBZ and DiOH-CBZ, was evaluated, each as a single 420
solute. EP-CBZ, a pharmacologically active and chemically reactive compound was 421
found to be taken up with biomass concentration of around 50 nmol g-1 and sap 422
concentration of 1.15 ± 0.05 µM. However DiOH-CBZ amount measured in the 423
nutrient solution was unchanged and its concentration almost doubled during 72 h 424
exposure time (from 1.78 to 2.99 µmol L-1). We thus conclude that while EP-CBZ can 425
be taken up by cucumber roots, DiOH-CBZ cannot. Therefore, EP-CBZ found in 426
reclaimed wastewater irrigated plants is a result of plant uptake and/or in-plant 427
metabolism, yet it is taken up to a lower extent when compared to CBZ. DiOH-CBZ 428
is unable to cross the root membranes due to its more hydrophilic nature (log Kow < 1; 429
Table 1), and therefore all DiOH-CBZ detected in plant material is a result of in-plant 430
metabolism. 431
LTG in the Plant Biomass. As opposed to the results obtained for CBZ, LTG 432
exhibited significantly higher accumulation in the cucumber roots, with a growing 433
trend throughout the experiment (data presented for the bi-solute system; Figure S8). 434
Mass balance calculated as the ratio between LTG at each sampling point (amount in 435
the nutrient solution and in the plant material) and the initial amount of LTG in the 436
nutrient solution, revealed a growing deficit over time with 92.08 ± 1.28% at 8 h, 437
declining to 57.33 ± 3.49% at 96 h. Testing the possible removal of LTG from the 438
21
nutrient solution in the absence of plants – adsorption to the glass jar or possible 439
biotic and abiotic degradation, did not result in any loss, inferring that this gap may be 440
due to in-plant metabolism. To date we are unable to quantify LTG metabolites, yet 441
are able to identify the presence of several of them and propose probable atomic 442
compositions in accordance to their measured mass (data not shown). 443
444
ENVIRONMENTAL IMPLICATIONS 445
Pharmaceutical compounds (PCs) are introduced into the agricultural 446
environment as a mixture and therefore plants irrigated with reclaimed-wastewater are 447
exposed simultaneously to several compounds. Our study shows that co-introduction 448
of PCs having similar physicochemical properties may affect their uptake rate by 449
plants, as demonstrated for the greater uptake of LTG in the presence of CBZ, under 450
pH conditions where both PCs are neutral and of similar log D, 2.23 and 2.15 for CBZ 451
and LTG, respectively. Although the concentrations applied in this study are higher 452
than concentrations found in the agro-environment, these results are relevant and 453
provide insight into the mechanisms that govern influx, translocation and metabolite 454
formation of these environmentally abundant PCs in plants. Since PCs can be 455
transformed in the plant through similar metabolic pathways and most probably by the 456
same set of enzymes, their in-plant rate of transformation is likely to be interrelated. 457
Although the metabolite DiOH-CBZ was detected at low concentration in the plant 458
biomass, we found its formation to be significantly inhibited in the presence of LTG. 459
If PCs are introduced into the plant at relatively high concentrations for long periods, 460
their transformation may be significantly reduced due to competition or enzyme 461
inhibition. However, in environmentally relevant scenarios where PCs are usually 462
found at much lower levels, enzyme bioavailability for metabolism might not be a 463
22
governing factor in transformation. Moreover, exposure to a mixture of PCs at low 464
concentrations might increase transformation due to enzymatic activation.7 465
In this study, CBZ served as a model for non-ionic PCs that can easily cross 466
the root membrane and be transported primarily in the direction of the transpiration 467
stream, and LTG as a model for PCs that may undergo ionization once taken up into 468
the roots. However, their translocation through the root might be different from the 469
water mass flow, affected by concentration gradients across the root membrane. These 470
compounds can be found in both the plant xylem and phloem and thus may 471
accumulate in all plant compartments.34 While non-ionic PCs and personal care 472
products such as CBZ, caffeine, triclosan, and dilantin, have been shown to be taken 473
up by plants and accumulate in their different compartments at relatively high 474
concentrations when compared to ionic PCs,14,15 there are only a handful of reports 475
concerning in-plant metabolism of these compounds.51,52 Although metabolism occurs 476
in both roots and leaves, our data demonstrate that it is predominant in the latter. The 477
importance of understanding and quantifying plant metabolism of PCs may be of 478
greater significance in prolonged cultivation.14,22,36 This is further emphasized by our 479
observation that more than 10% of CBZ was metabolized in only 96 h. Moreover, 480
CBZ and its metabolites were detected in urine sampled from healthy individuals who 481
consumed fresh fruits and vegetables irrigated with reclaimed wastewater.53 Also for 482
LTG this might be the case, based on the growing deficit in LTG mass balance and 483
identification of several possible LTG metabolites. Metabolites formed within the 484
plant material following uptake may be of a reactive and possibly pharmacologically 485
active nature as reported for EP-CBZ,50 thus emphasizing the importance of further 486
studying and evaluating plant metabolism of compounds with high uptake potential, 487
such as non-ionic pharmaceuticals. 488
23
489
24
ACKNOWLEDGMENTS 490
This research was partially supported by research grants from the Environment 491
and Health Fund (EHF) Jerusalem, BARD grant US-4771-14R., Deutsche 492
Forschungsgemeinschaft (DFG, Bonn, Germany) - Re1290/7-1 and by Israel Ministry 493
of Agriculture. 494
495
SUPPORTING INFORMATION 496
Supporting information includes: carbamazepine, lamotrigine and water 497
uptake rates (Figure S1); carbamazepine and lamotrigine root adsorption (Figure S2); 498
the transpiration stream concentration factor measured during the exposure period 499
(Figure S3); carbamazepine measured in the roots and shoots during the exposure 500
period (Figures S4); sum of carbamazepine and its metabolites shoot/root ratio during 501
the exposure period (Figure S5); carbamazepine, lamotrigine, and EP-CBZ 502
concentrations measured in the plant material under two external pH levels (Figures 503
S6-S7); and lamotrigine measured in the roots and shoots during the exposure period 504
(Figure S8). This information is available free of charge via the Internet at 505
http://pubs.acs.org/. 506
507
REFERENCES 508
(1) Hsia, Y.; Neubert, A.; Sturkenboom, M. C. J. M.; Murray, M. L.; Verhamme, 509
K. M. C.; Sen, F.; Giaquinto, C.; Ceci, A.; Wong, I. C. K. Comparison of 510
antiepileptic drug prescribing in children in three European countries. Epilepsia 511
2010, 51 (5), 789–796. 512
(2) Labiner, D. M.; Paradis, P. E.; Latre, D. Generic antiepileptic drugs and 513
associated medical resource utilization in the United States. Neurology 2010, 514
25
76 (6), 583–584. 515
(3) Berman, E.; Marom, E.; Ekstein, D.; Blatt, I.; Eyal, S. Utilization of 516
antiepileptic drugs in Israel. Epilepsy Behav. 2016, 61, 82–85. 517
(4) Tsiropoulos, I.; Gichangi, A.; Andersen, M.; Bjerrum, L.; Gaist, D.; Hallas, J. 518
Trends in utilization of antiepileptic drugs in Denmark. Acta Neurol Scand. 519
2006, 113, 405–411. 520
(5) Leach, M. J.; Marden, C. M.; Miller, A. A. Pharmacological studies on 521
lamotrigine, a novel potential antiepileptic drug: II. Neurochemical studies on 522
the mechanism of action. Epilepsia 1986, 27 (5), 490–497. 523
(6) Cheung, H.; Kamp, D.; Harris, E. An in vitro investigation of the action of 524
lamotrigine on neuronal voltage-activated sodium channels. Epilepsy Res. 525
1992, 13 (2), 107–112. 526
(7) Perucca, E. An introduction to antiepileptic drugs. Epilepsia 2005, 46, 31–37. 527
(8) Pearce, R. E.; Gangadhara, R. V.; Leeder, J. S. Pathways of carbamazepine 528
bioactivation in vitro I. Characterization of human cytochromes P450 for the 529
formation of 2- and 3- hydroxylated metabolites. Drug Metab. Dispos. 2002, 530
30, 1170–1179. 531
(9) Pearce, R. E.; Lu, W.; Wang, Y.; Uetrecht, J. P.; Correia, M. A.; Leeder, J. S. 532
Pathways of carbamazepine bioactivation in vitro. III. The role of human 533
cytochrome P450 enzymes in the formation of 2,3-dihydroxycarbamazepine. 534
Drug Metab Dispos. 2008, 36 (8), 1637–1649. 535
(10) Bahlmann, A.; Brack, W.; Schneider, R. J.; Krauss, M. Carbamazepine and its 536
metabolites in wastewater: Analytical pitfalls and occurrence in Germany and 537
Portugal. Water Res. 2014, 57C, 104–114. 538
(11) Ternes, T. Occurrence of drugs in German sewage treatment plants and rivers. 539
26
Water Res. 1998, 32 (11), 33245–33260. 540
(12) Miège, C.; Paoletti, E.; Choubert, J. M.; Ribeiro, L.; Eusèbe, M.; Coquery, M. 541
Fate of pharmaceuticals and personal care products in wastewater treatment 542
plants – Conception of a database and first results. Environ. Pollut. 2009, 157 543
(5), 1721–1726. 544
(13) Writer, J. H.; Ferrer, I.; Barber, L. B.; Thurman, E. M. Widespread occurrence 545
of neuro-active pharmaceuticals and metabolites in 24 Minnesota rivers and 546
wastewaters. Sci. Total Environ. 2013, 461–462, 519–527. 547
(14) Malchi, T.; Maor, Y.; Tadmor, G.; Shenker, M.; Chefetz, B. Irrigation of root 548
vegetables with treated wastewater: evaluating uptake of pharmaceuticals and 549
the associated human health risks. Environ. Sci. Technol. 2014, 48 (16), 9325– 550
9333. 551
(15) Goldstein, M.; Shenker, M.; Chefetz, B. Insights into the uptake processes of 552
wastewater-borne pharmaceuticals by vegetables. Environ. Sci. Technol. 2014, 553
48 (10), 5593–5600. 554
(16) Tixier, C.; Singer, H. P.; Oellers, S.; Müller, S. R. Occurrence and fate of 555
carbamazepine, clofibric acid, diclofenac, ibuprofen, ketoprofen, and naproxen 556
in surface waters. Environ. Sci. Technol. 2003, 37 (6), 1061–1068. 557
(17) Grossberger, A.; Hadar, Y.; Borch, T.; Chefetz, B. Biodegradability of 558
pharmaceutical compounds in agricultural soils irrigated with treated 559
wastewater. Environ. Pollut. 2014, 185C, 168–177. 560
(18) Navon, R.; Hernandez-Ruiz, S.; Chorover, J.; Chefetz, B. Interactions of 561
carbamazepine in soil: effects of dissolved organic matter. J. Environ. Qual. 562
2011, 40 (3), 942–948. 563
(19) Golan-Rozen, N.; Chefetz, B.; Hadar, Y. Transformation pathways of the 564
27
recalcitrant pharmaceutical compound carbamazepine by the white-rot fungus 565
Pleurotus Ostreatus: Effects of growth conditions. Environ. Sci. Technol. 2015, 566
49 (20), 12351–12362. 567
(20) Li, J.; Dodgen, L.; Ye, Q.; Gan, J. Degradation kinetics and metabolites of 568
carbamazepine in soil. Environ. Sci. Technol. 2013, 47 (8), 3678–3684. 569
(21) Posner, J.; Cohen, A. F.; Land, G.; Winton, C.; Peck, A. W. The 570
pharmacokinetics of lamotrigine ( BW430C ) in healthy subjects with 571
unconjugated hyperbilirubinaemia ( Gilbert's Syndrome ). Br. J. clin. Pharmac. 572
1989, 28 (1), 117–120. 573
(22) Garnett, W. R. Lamotrigine: Pharmacokinetics. J. child neuorology 1997, 12, 574
10–15. 575
(23) Zonja, B.; Pérez, S.; Barceló, D. Human metabolite lamotrigine-N2- 576
glucuronide is the principal source of lamotrigine-derived compounds in 577
wastewater treatment plants and surface water. Environ. Sci. Technol. 2016, 50 578
(1), 154–164. 579
(24) Paz, A.; Tadmor, G.; Malchi, T.; Blotevogel, J.; Borch, T.; Polubesova, T.; 580
Chefetz, B. Fate of carbamazepine, its metabolites, and lamotrigine in soils 581
irrigated with reclaimed wastewater: Sorption, leaching and plant uptake. 582
Chemosphere 2016, 160, 22–29. 583
(25) Ishihama, Y.; Nakamura, M.; Miwa, T.; Kajima, T.; Asakawa, N. A rapid 584
method for pKa determination of drugs using pressure-assisted capillary 585
electrophoresis with photodiode array detection in drug discovery. J. Pharm. 586
Sci. 2002, 91 (4), 933–942. 587
(26) ACD/I-lab. ACD/Structure Elucidator, 2013. 588
(27) Enstone, D. E.; Peterson, C. a.; Ma, F. Root endodermis and exodermis: 589
28
structure, function, and responses to the environment. J. Plant Growth Regul. 590
2003, 21 (4), 335–351. 591
(28) Chen, T.; Cai, X.; Wu, X.; Karahara, I.; Schreiber, L.; Lin, J. Casparian strip 592
development and its potential function in salt tolerance. Plant Signal. Behav. 593
2011, 6, 1499–1502. 594
(29) Sun, J.; Xie, L.; Liu, X. Transport of carbamazepine and drug interactions at 595
blood-brain. Acta Pharmacol. Sin. 2006, 27 (2), 249–253. 596
(30) Johannessen, SI.; Johannessen Landmark, C. Antiepileptic drug interactions - 597
principles and clinical implications. Curr. Neuropharmacol. 2010, 8 (3), 254– 598
267. 599
(31) Shenker, M.; Harush, D.; Ben-Ari, J.; Chefetz, B. Uptake of carbamazepine by 600
cucumber plants - a case study related to irrigation with reclaimed wastewater. 601
Chemosphere 2011, 82 (6), 905–910. 602
(32) Lee, E.; Lee, S.; Park, J.; Kim, Y.; Cho, J. Removal and transformation of 603
pharmaceuticals in wastewater treatment plants and constructed wetlands. 604
Drink. Water Eng. Sci. 2013, 6, 89–98. 605
(33) Briggs, G. G.; Bromilow, R. H.; Evans, A. A. Relationships between lipophilicity 606
and root uptake and translocation of non-ionised chemicals by barley. Pestic. 607
Sci. 1982, 13 (5), 495–504. 608
(34) Bromilow, R.; Chamberlain, K.; Evans, A. Physicochemical aspects of phloem 609
translocation of herbicides. Weed Sci. 1990, 38 (3), 305–314. 610
(35) Javot, H. The role of aquaporins in root water uptake. Ann. Bot. 2002, 90 (3), 611
301–313. 612
(36) Steudle, E. Water uptake by roots: effects of water deficit. J. Exp. Bot. 2000, 51 613
(350), 1531–1542. 614
29
(37) Maurel, C. Aquaporins and water permeability of plant membranes. Annu. Rev. 615
Plant Physiol. Plant Mol. Biol. 1997, 48, 399–429. 616
(38) Trapp, S. Plant uptake and transport models for neutral and ionic chemicals. 617
Environ. Sci. Pollut. Res. 2004, 11 (1), 33–39. 618
(39) Dettenmaier, E. M.; Doucette, W. J.; Bugbee, B. Chemical hydrophobicity and 619
uptake by plant roots. Environ. Sci. Technol. 2009, 43 (2), 324–329. 620
(40) Hsu, F. C.; Marxmiller, R. L.; Yang, A. Y. Study of root uptake and xylem 621
translocation of cinmethylin and related compounds in detopped soybean roots 622
using a pressure chamber technique. Plant Physiol. 1990, 93 (4), 1573−1578. 623
(41) Trapp, S. Modelling uptake into roots and subsequent translocation of neutral 624
and ionisable organic compounds. Pest Manag. Sci. 2000, 56 (9), 767–778. 625
(42) Grignon, C.; Sentenac, H. pH and ionic conditions in the apoplast. Annu. Rev. 626
Plant Physiol. Plant Mol. Biol. 1991, 42, 103–128. 627
(43) Trapp, S. Bioaccumulation of polar and ionizable compounds in plants. In 628
Ecotoxicology Modeling; Devillers, J., Ed.; Springer US: Boston, MA, 2009; 629
Vol. 2, pp 299−352. 630
(44) Escher, B. I.; Schwarzenbach, R. P. Partitioning of substituted phenols in 631
liposome−water, biomembrane−water, and octanol−water systems. Environ. 632
Sci. Technol. 1996, 30 (1), 260–270. 633
(45) Bernus, I.; Dickinson, R. G.; Hooper, W. D.; Eadie, M. J. Dose-dependent 634
metabolism of carbamazepine in humans. Epilepsy Res. 1996, 24 (3), 163–172. 635
(46) Bu, H.; Kang, P.; Deese, A. J.; Zhao, P.; Pool, W. F. Human in vitro 636
glutathionyl and protein adducts of carbamazepine-10,11-epoxide, a stable and 637
pharmacologically active metabolite of carbamazepine. Drug Metab Dispos. 638
2005, 33 (12), 1920–1924. 639
30
(47) Sankar, R.; Lerner, J. T. Teratogenicity of antiepileptic drugs. role of 640
pharmacogenomics. Int. Rev. Neurobiol. 2008, 83, 215–225. 641
(48) Riemenschneider, C.; Al-Raggad, M.; Moeder, M.; Seiwert, B.; Salameh, E.; 642
Reemtsma, T. Pharmaceuticals, their metabolites, and other polar pollutants in 643
field-grown vegetables irrigated with treated municipal wastewater. J. Agric. 644
Food Chem. 2016, 64 (29), 5784–5792. 645
(49) Riemenschneider, C.; Seiwert, B.; Goldstein, M.; Al-Raggad, M.; Salameh, E.; 646
Chefetz, B.; Reemtsma, T. An LC-MS/MS method for the determination of 28 647
polar environmental contaminants and metabolites in vegetables irrigated with 648
treated municipal wastewater. Anal. Methods 2017, 9 (8), 1273-1281. 649
(50) Warner, T.; Patsalos, P. N.; Prevett, M.; Elyas, A. A.; Duncan, J. S. 650
Lamotrigine-induced carbamazepine toxicity: an interaction with 651
carbamazeoine-10,11-epoxide. Epilepsy Res. 1992, 11 (2), 147–150. 652
(51) Macherius, A.; Eggen, T.; Lorenz, W.; Moeder, M.; Ondruschka, J.; 653
Reemtsma, T. Metabolization of the bacteriostatic agent triclosan in edible 654
plants and its consequences for plant uptake assessment. Environ. Sci. Technol. 655
2012, 46 (19), 10797–10804. 656
(52) Miller, E. L.; Nason, S. L.; Karthikeyan, K. G.; Pedersen, J. A. Root uptake of 657
pharmaceuticals and personal care product ingredients. Environ. Sci. Technol. 658
2015, 2 (50), 525–541. 659
(53) Paltiel, O.; Fedorova, G.; Tadmor, G.; Kleinstern, G.; Maor, Y.; Chefetz, B. 660
Human exposure to wastewater-derived pharmaceuticals in fresh produce: A 661
randomized controlled trial focusing on carbamazepine. Environ. Sci. Technol. 662
2016, 50 (8), 4476–4482. 663
664
31

CBZ y Plantas

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

CBZ y Plantas

Uploaded by

Copyright:

Available Formats

Subscriber access provided by Kaohsiung Medical University

is published by the American Chemical Society. 1155 Sixteenth Street N.W.,

ACS Paragon Plus Environment

Carbamazepine and its Interactions with Lamotrigine 2

Myah Goldstein,†,‡ Tomer Malchi,†,‡ Moshe Shenker,† and Benny Chefetz†,‡,* 4

Tel.: 972-8-9489384; email: benny.chefetz@mail.huji.ac.il 14

Carbamazepine and lamotrigine prescribed antiepileptic drugs are highly 16

metabolites in cucumber plants under hydroponic culture, testing their uptake, 19

translocation and transformation over 96 h in single and bi-solute systems at varying 20

demonstrate that carbamazepine root uptake is largely affected by the concentration 22

gradient across the membrane. Unlike carbamazepine, lamotrigine is adsorbed to the 23

lamotrigine, while lamotrigine uptake was enhanced in the presence of 26

carbamazepine. Transformation of carbamazepine in the roots was slightly reduced in 27

the presence of lamotrigine. Carbamazepine metabolism was far more pronounced in 28

by diffusion across the root membrane. 32

Carbamazepine (CBZ) is the most frequently prescribed antiepileptic drug 34

worldwide, used by patients of all ages.1–4 It acts as a voltage-dependent sodium- 35

(mostly in the feces); 32% of the total as the metabolite 10,11-dihydro-10,11- 39

dihydroxy-carbamazepine (DiOH-CBZ), 5.1% as 3-hydroxy-carbamazepine (3-OH- 40

CBZ), 4.3% as 2-hydroxy-carbamazepine (2-OH-CBZ), and 1.4% as 10,11-dihydro- 41

10,11-epoxy-carbamazepine (EP-CBZ), all excreted in the urine, with several other 42

in reclaimed-wastewater used for irrigation and in freshwater bodies receiving 46

days.17,20 CBZ is a neutral compound within the range of environmental pH.15 52

Lamotrigine (LTG), also a sodium-channel blocker,5–7 is a frequently 53

prescribed antiepileptic drug which may be co-administered with other antiepileptic 54

in municipal wastewater-treatment plants and is found at concentrations above 1 µg L- 57

main LTG metabolite found in reclaimed-wastewater.13,23 Following reclaimed- 59

Being a relatively restrictive barrier to organic compounds, the casparian strip 63

blood–brain barrier in mammals,27,28 thus implying that compounds known to cross 65

pharmacokinetic interactions reported in humans30, we suggest that co-introduction of 70

and/or soil solution. 77

(Toronto, Canada): EP-CBZ, DiOH-CBZ, and 2-OH-CBZ. Selected properties of the 83

purchased from Toronto Research Chemicals: CBZ-13C-D2, LTG-13C3, DiOH-D3, 85

Hydroponic Culture Setup. Cucumber seeds (Cucumis sativus, Patriot, Hazera 87

Genetics Ltd., Berurim, Israel) were germinated in vermiculite moistened with 88

CaSO4·H2O-saturated solution. Seven-day-old seedlings were transferred and each 89

KH2PO4, 0.1, and micronutrients (µM): Fe-EDTA, 10; MnSO4·H2O, 0.5; 93

grown in a temperature-controlled chamber with a 16/8-h day/night cycle at 25 ± 1 °C 96

LTG at concentrations of ~2 µM each. 103

to 96 h, maintaining a pH of 6-7 in all experiments by adding 4-(2-hydroxyethyl)-1- 106

piperazineethanesulfonic acid (HEPES) to the nutrient solution at a concentration of 5 107

washed, weighed and frozen at -20 °C until extraction. 120

reconstituted in 1 mL acetonitrile:water:acetic acid (20:80:0.01) spiked with 10 µL of 147

Data Analysis. Statistical analysis (non-parametric Wilcoxon/Kruskal–Wallis test and 159

Table 1. Selected physicochemical properties of the studied compounds 171

Compound Structure log Kow (ref) pKa (ref)

pKa1 = -0.5 (24)

10,11-dihydro-10,11-epoxy- pKa1 = -0.9 (24)

pKa1 = -1.5 (24)

Lamotrigine (LTG) 1.93 (24) pKa = 5. 7 (25)

RESULTS AND DISCUSSION 173

significant decrease in concentration following 48 h of exposure. 194

maintained and larger rates of CBZ uptake will prevail. 227

influx across the root membrane (i.e., uptake). 249

CBZ-influx concentration; this is followed by a decreasing gradient across the 265

described above. 275

easily transported from roots to shoots. 288

experimental setup. 303

in the plant root system. 312

96 h as compared to 8 h of exposure (Figure 4S). This trend is supported by an 337

pharmacokinetic interactions may be amplified under prolonged cultivation. 342

of EP-CBZ hydrolysis.8,9,19 EP-CBZ, a highly reactive and pharmacologically active 353

following the 48 h sampling. Riemenschneider et al.49 reported higher concentrations 357