Biotransformation of flubendazole and fenbendazole and their effects in the ribwort plantain (Plantago lanceolata)

Ecotoxicology and Environmental Safety 147 (2018) 681–687

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Biotransformation of flubendazole and fenbendazole and their effects in the ribwort plantain (Plantago lanceolata)

MARK

Lucie Raisová Stuchlíkováa, Lenka Skálováa, Barbora Szotákováa, Eliška Syslováa,b, Ivan Vokřálc, ⁎ Tomáš Vaněkb, Radka Podlipnáb, a b c

Department of Biochemical Sciences, Faculty of Pharmacy in Hradec Králové, Charles University, Heyrovského 1203, 500 05 Hradec Králové, Czech Republic Laboratory of Plant Biotechnology, Institute of Experimental Botany, Czech Academy of Science, Rozvojová 313, 165 02 Praha 6 - Lysolaje, Czech Republic Department of Pharmacology and Toxicology, Faculty of Pharmacy in Hradec Králové, Charles University, Hradec Králové, Czech Republic

A R T I C L E I N F O

A B S T R A C T

Keywords: Flubendazole Fenbendazole Drug uptake Drug metabolism UHPLC-MS/MS Phytotoxicity

Although veterinary anthelmintics represent an important source of environmental pollution, the fate of anthelmintics and their effects in plants has not yet been studied sufficiently. The aim of our work was to identify metabolic pathways of the two benzimidazole anthelmintics fenbendazole (FBZ) and flubendazole (FLU) in the ribwort plantain (Plantago lanceolata L.). Plants cultivated as in vitro regenerants were used for this purpose. The effects of anthelmintics and their biotransformation products on plant oxidative stress parameters were also studied. The obtained results showed that the enzymatic system of the ribwort plantain was able to uptake FLU and FBZ, translocate them in leaves and transform them into several metabolites, particularly glycosides. Overall, 12 FLU and 22 FBZ metabolites were identified in the root, leaf base and leaf top of the plant. Concerning the effects of FLU and FBZ, both anthelmintics in the ribwort plantain cells caused significant increase of proline concentration (up to twice), a well-known stress marker, and significant decrease of superoxide dismutase activity (by 50%). In addition, the activities of four other antioxidant enzymes were significantly changed after either FLU or FBZ exposition. This could indicate a certain risk of oxidative damage in plants influenced by anthelmintics, particularly when they are under other stress conditions.

1. Introduction Flubendazole (FLU) and fenbendazole (FBZ) are veterinary drugs regularly used for antiparasitic control in pigs, chicken, turkeys, game birds and domestic carnivores (Bartikova et al., 2010). These drugs are not only efficient against nematodes but also against tapeworms, flukes and some protozoa. Due to the high use of anthelmintics for the treatment of livestock, concern has emerged about the potential impacts of their release into ecosystems. Anthelmintics can reach the environment via different routes, i.e. in manure dispersed in fields as crop fertilizer, following direct application in aquaculture, or through local water treatment plants in fish farms (Bartikova et al., 2016). There is a significant risk that anti-parasitic agents in the environment may influence aquatic organisms, invertebrates as well as plants. The presence of anthelmintics in several environmental compartments has already been reported. Residues of FLU were found in the leachate from agricultural manure into drainage waters and in wastewater from the pharmaceutical industry up to 671 ng/l (Wagil et al.,

2015a), as well as in seepage water after sprinkler irrigation of a manured area (Weiss et al., 2008). FLU has also been found in river water (39.2 ng/l), in fish tissue (38.5 ng/g) and in sediment samples (4.4 ng/g) (Wagil et al., 2015b). FBZ has been also found in river water (63 µg/l) (Zrncic et al., 2014). Sim et al. (2013)) detected fenbendazole in livestock wastewater treatment plants in maximal concentration 241 µg/l and simultaneously detected its metabolites (fenbendazole sulfone, p-hydroxyfenbendazole, amino fenbendazole and oxfendazole). The mobility of both benzimidazoles in soils was assessed by the determination of soil/water distribution coefficients (Kd values). After standard application, Kd values of 14C-flubendazole were 141 ± 30 l/kg for the clay soil and 92 ± 30 l/kg for the sand soil, resulting in the KOC > 8800 l/kg. Corresponding Kd values of 14C-fenbendazole were 63 ± 7 l/kg and 58 ± 3 l/kg resulting in KOC > 1500 l/kg. Special attention should be paid to the high values of octanol–water partition coefficients of FLU (2.91) and FBZ (3.93) which can influence their bioavailability and bioaccumulation (Kreuzig et al., 2007). Both FLU and FBZ have been shown to cause substantial acute toxicity in most

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Corresponding author. E-mail addresses: [email protected] (L.R. Stuchlíková), [email protected] (L. Skálová), [email protected] (B. Szotáková), [email protected] (E. Syslová), [email protected] (I. Vokřál), [email protected] (T. Vaněk), [email protected] (R. Podlipná). http://dx.doi.org/10.1016/j.ecoenv.2017.09.020 Received 26 June 2017; Received in revised form 4 September 2017; Accepted 9 September 2017 0147-6513/ © 2017 Elsevier Inc. All rights reserved.

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germinated on the agar MS medium (Murashige and Skoog, 1962) at 25 °C, with a 16-h photoperiod at 72 µmol of photons/m2/s. The whole plant regenerants were cultivated in Magenta boxes under the same conditions and regularly subcultivated every 4 weeks. Cell cultures were initiated from the leaves of the regenerants by setting the cuts on solid MS medium supplemented by synthetic phytohormones (0.225 mg/ml 2,4-dichlorphenoxyacetic acid, and 0.215 mg/ml kinetin). The callus cultures were cultivated in the dark at 25 °C and regularly subcultivated every 3 weeks. The cell culture was then transferred into liquid MS medium and cultivated in Erlenmeyer flasks on a horizontal shaker in the dark at 25 °C to obtain the suspension cultures.

freshwater macroinvertebrates (Wagil et al., 2015a; Bundschuh et al., 2016). Plants are also exposed to anthelmintics which can influence their development and/or physiology. The first signal of environmental stress is presented by an increase in the production of reactive oxygen species (ROS) (Zrncic, Gros et al.), which is associated with the direct damage of various biomolecules. To prevent the harmful effects of ROS, plants activate antioxidant systems, both non-enzymatic (e.g. proline) as well as enzymatic (antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), peroxidase (POX), ascorbate peroxidase (APX), and glutathione S-transferase (GST)). As a first line of defense against oxidative damage, SOD catalyzes the conversion or dismutation of toxic O2- radicals to H2O2 and molecular oxygen (O2). Plants tolerant to stress show a higher activity of SOD. The resultant hydrogen peroxide is subsequently detoxified to water by CAT or POX. Furthermore, the removal of hydrogen peroxide is the first step in the ascorbate glutathion cycle through which APX catalyzes the reduction of H2O2. Detoxification of membrane lipid hydroperoxides can be maintained by the activities of GSTs, a family of isozymes which are also responsible for detoxification of cytosolic lipid peroxidation products. The increased accumulation of proline is a common physiological response of plants exposed to various stresses, with proline playing an important role in plant stress tolerance. In effect, proline plays multifarious roles including adaptation, recovery and signaling related to combating stress in plants (Mhamdi et al. 2010; Anjum et al., 2016). Till now, no significant information has come forth about the effects of anthelmintics in plants, while limited information has been published about anthelmintic metabolism in plants (Dietz and Schnoor, 2001). The metabolism of xenobiotics occurs through three phases in plants: oxidation, reduction or hydrolysis of the xenobiotic represents phase I, conjugation reactions with endogenous compounds characterize phase II, and phase III is shown in the transport of metabolites to plant vacuoles or their binding to the cell wall and lignin. In our previous studies, the uptake and metabolism of benzimidazole anthelmintics (albendazole, FLU, FBZ) were tested in the reed (Phragmites australis) and harebell (Campanula rotundifolia) suspension cultures. Reed and harebell cells were shown as able to uptake benzimidazole anthelmintics and biotransform them into several metabolites. Nevertheless, considerable inter-species differences were observed (Podlipna et al., 2013; Stuchlikova et al., 2016). In our present study, the ribwort plantain (Plantago lanceolata), one of the most commonly occurring plants in pastures, was used. Whole plant regenerants served as model systems for this biotransformation study, as regenerants have been shown to represent an adequate model for the qualitative as well as quantitative evaluation of drug uptake and metabolism in plants (Stuchlikovan Raisova et al., 2017). Advanced UHPLC-MS/MS technology was used for anthelmintics metabolites identification and semiquantification. The proline concentrations in the leaves were monitored during a 9-day exposition of plants to FLU and FBZ. In addition, the FLU- and FBZ-mediated changes in the activities of five antioxidant enzymes were followed up in ribwort plantain cell suspensions with aim to evaluate the effect of these anthelmintics and their biotransformation products on parameters of oxidative stress in the plantain. Thus, the present study attempts to present a complex overview of the fate and effects of the anthelmintic drugs FLU and FBZ in the ribwort plantain.

2.2. Experimental design To evaluate the uptake and biotransformation of the anthelmintics, the medium of in vitro regenerants was supplemented with FLU (Janssen Pharmaceutica (New Brunswick, NJ, USA)) or FBZ (SigmaAldrich (St. Louis, MO, USA)) (final concentration 10 µM, pre-dissolved in DMSO). The control plants were treated by DMSO in corresponding concentration. The concentrations were selected based on preliminary experiments with this and other pharmaceuticals performed in our laboratory (Podlipna et al., 2013). Also Wagil et al. (2015a) used similar concentrations (1 mg/l) of FLU and FBZ in media solutions used in ecotoxicity tests for Lemna minor and Scenedesmus vacuolatus. Plant roots, tops of leaves (approx. 1 cm from the leaf top) and basal parts of leaves (approx. 1 cm from the bottom of rosette) were collected after 1, 3 and 6 weeks. Simultaneously 50 mg of leaves was harvested for proline assay. All samples were frozen (−80 °C) before analysis and prepared in triplicate (n=3). Chemical and biological blank samples were prepared for all the types of procedures.

2.3. Sample preparation All the samples were homogenized using the FastPrep-24 homogenizer (Santa Ana, CA, USA). The homogenized samples were subjected liquid-liquid extraction (LLE) according to the method described previously by Vokral et al. (2012)) and Podlipna et al. (2013))]. The obtained supernatants were evaporated to dryness using the concentrator Eppendorf (30 °C). The dry samples were quantitatively reconstituted in the mixture water/acetonitrile (70/30, v/v) by sonication and filtrated through syringe filters with PTFE membrane. One microliter of the samples was injected into a UHPLC-MS system.

2.4. Analytical conditions of UHPLC-MS/MS UHPLC (Nexera; Shimadzu, Japan) was optimized using a Zorbax RRHD Eclipse Plus 95 Å C18 column 150×2.1 mm, 1.8 µm (Agilent Technologies, Waldrbronn, Germany), temperature 40 °C, flow rate 0.4 ml/min and the injection volume 1 μl. The mobile phase consisted of water (A) and acetonitrile (B), both with addition of 0.1% formic acid (MS grade). The linear gradient was as follows: 0 min – 15% B, 8 min – 40% B, 10 min – 95% B followed by 1 min of isocratic elution. The QqQ mass spectrometer (LC-MS-8030 triple quadrupole mass analyzer; Shimadzu, Japan) was used with the following setting of tuning parameters: capillary voltage 4.5 kV, heat block temperature 400 °C, DL line temperature 250 °C, the flow rate and pressure of nitrogen were 12 l/ min, respectively. ESI mass spectra were recorded in the range of m/z 50 – 1000 in the positive-ion mode. The isolation width Δm/z 2 and the collision energy 25 eV (found as optimal energy for fragmentation of studied metabolite ions) using argon as the collision gas for MS/MS experiments. Mebendazole (MBZ) as an internal standard was used. All data are presented as arithmetic mean ± SD (n=3).

2. Materials and methods 2.1. Plant material and their cultivation Samples of the meadow plant ribwort plantain (Plantago lanceolata; seeds obtained from the Institute of Botany of the CAS, v. v. i., Průhonice, Czech Republic) were cultivated under controlled conditions. The regenerants were achieved from seeds sterilized in 70% ethanol for 1 min and next by 1% sodium hypochlorite supplemented by 0.02% detergent TWEEN 20 for a period of 10 min. The seeds were 682

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Based on these results, the schemes of metabolic pathways of anthelmintic drugs are proposed (Fig. 1 for FLU, Fig. 2 for FBZ). A comparison of the relative amounts of the main metabolites and parent drug in the roots as well as in basal parts and tops of leaves after 1, 3, and 6 weeks is also presented (Fig. 3A for FLU, Fig. 3B for FBZ).

2.5. Proline assay Proline content as a stress indicator was measured according to the modified method of Bates et al. (1973)). The leaves were homogenized in 3% (w/v) sulfosalicylic acid, centrifuged at 13,000 g for 10 min and ninhydrin dissolved in glacial acetic acid was added. The tubes were incubated in a water bath for 1 h at 100 °C, toluene was added and the absorbance of the toluene phase was measured at 520 nm using a microplate reader (Tecan Infinite 200, Tecan Group Ltd., Switzerland).

3.1.1. Flubendazole An MS/MS spectrum of the protonated molecule of the parent drug (retention time, 8.3 min) at m/z 314 resulted in the abundant product ion at m/z 282. Various drug-metabolizing enzymes catalyze the modification of the chemical structure of the parent drug. Three phase I reactions were identified in FLU biotransformation: carbonyl reduction, hydroxylation and hydrolysis. The only phase I metabolite in the ribwort and also the main metabolite of FLU in both model systems was FLU with a reduced carbonyl group (M5FLU). The parent drug and M5FLU possessed the characteristic neutral loss (NL) of methanol Δm/z 32. Concerning phase II biotransformation, several FLU metabolites were identified: methylation, glucosidation and acetylation. Conjugation with UDP-glucose predominated. Three different FLU Nglucosides (M7FLU, M10FLU and M11FLU), one hydrolysed FLU N-glucoside (M1FLU), and three carbonyl reduced FLU glucosides (M2FLU, M3FLU and M4FLU) were identified. The characteristic NL for glucose, Δm/z 162 (Holčapek et al., 2008), was observed in tandem mass spectra of all of these conjugates. Two metabolites (M2FLU and M3FLU) showed a different position of glucose conjugation (N- or O-); recognition was based on the typical NL of glucose plus Δm/z 18 of H2O. The NL Δm/z 32, characteristic for methanol, was found in all these conjugates with the exception of the hydrolysed metabolites. Hydrolysis of the peptide bond in the FLU structure led to the formation of FLU metabolites without a side chain. Metabolite M9FLU represented the methylation of M5FLU, with the typical NL of methanol. Another FLU metabolites (M6FLU and M8FLU), hydrolysed and hydroxylated FLU acetylglucosides with NL Δm/z 220 (O-acetyl-glucoside), were detected. All these metabolites of FLU were observed in the root with the exception of the metabolite M11FLU, 11 metabolites was shown in the basal part of leaves and 6 in the tops of leaves. In previous studies, some of these FLU metabolites were also detected in the reed and harebell (Podlipna et al., 2013; Stuchlikova et al., 2016).

2.6. Assays of enzyme activities Cell suspensions were cultivated with or without anthelmintics for 0.25-, 1-, 2-, and 9-days. The concentration of FLU or FBZ in medium was 10 µM (pre-dissolved in DMSO), with control cells incubated in medium with DMSO. At the end of cultivation, the plant cells were homogenized and centrifuged, with 14,000g supernatant obtained. Protein content was determined according to Bradford (1976) and the supernatant was used for the subsequent enzyme assays. Superoxide dismutase (SOD) activity was determined according to El-Shabrawi (2010). Peroxidase (POD) was assayed as described by Drotar et al. (1985)). Catalase (CAT) activity was determined spectrophotometrically by measuring the rate of H2O2 decrease at 240 nm (Verma and Dubey, 2003). Ascorbate peroxidase (APX) activity was assayed as described by Vanacker et al. (1998)). Glutathione S-transferase (GST) activity was determined using 1-chloro-2,4-dinitrobenzene as a substrate (Habig et al., 1974). The specific activity of SOD, CAT, POD, APX, and GST was expressed as units per mg of protein. 2.7. Statistical analysis Data were processed using the data analysis software STATISTICA.CZ version 12.0 (StatSoft, Prague, Czech Republic). Each treatment was represented by a minimum of three biological replicates. The normal distribution of the data was tested using the Shapiro-Wilk test. Following ANOVA, a post hoc Tukey test was performed for multiple pairwise comparisons. For situations in which the parametric conditions were not met, Kruskal-Wallis followed by 2-by-2 Wilcoxon post hoc comparison analyses were performed. Data are represented as mean values ± standard error, and significance was set at the 5% level.

3.1.2. Fenbendazole The protonated parent drug FBZ was detected at m/z 300 with retention time 9.5 min and with a product ion m/z 268 as the typical NL of methanol. Hydroxylation (M9FBZ) and two-step oxidation (M20FBZ) of FBZ represented metabolites of phase I biotransformation with the characteristic NL Δm/z 32 (methanol) and Δm/z 18 (H2O) found only for M9FBZ. Various oxidation, hydroxylation or hydrolysis reactions can be mentioned among possible phase I reactions followed by phase II reactions of FBZ. In some metabolites, recognition between S-oxidation and hydroxylation was not possible, thus these metabolites were recorded as –O. Conjugation with UDP-glucose was the initial step in the formation of all the FBZ conjugates, followed in some cases by Oacetylation. The NL of Δm/z 162 (glucose) was observed in tandem mass spectra of M1FBZ, M3-8FBZ, M10FBZ, M13FBZ, M17FBZ, M21FBZ and M22FBZ and Δm/z 220 (acetylglucose) of M2FBZ, M11FBZ, M12FBZ, M1416FBZ and M19FBZ. In contrast with FLU, one metabolite (M18FBZ) of FBZ O-glucosyl-N-glucoside was identified. In our experiments several types of positional isomers were found, with all the isomers providing identical MS/MS spectra, although the position of conjugation with glucose or acetylglucose cannot be distinguished based on mass spectra (Nobilis et al., 1996). Interestingly, three metabolites (M1FBZ, M9FBZ and M21FBZ) were detected in both model systems (in all parts of the regenerants) in the highest amount, see Fig. 3B. Moreover, with high probability the N-glucosides are instable and thus could be easily decomposed back to the parent anthelmintic. All these metabolites of FBZ were observed in the root, 13 in the basal part of leaves and 5 in the tops of leaves.

3. Results and discussion Veterinary drugs have the potential to enter the environment and cause adverse ecological and human health effects. The release of these contaminants into the environment has taken place for many decades, whereas methods for their detection at environmentally-relevant concentrations have only recently become available (Snow et al., 2014). 3.1. Metabolism of tested drugs in the ribwort plantain The biotransformation of two tested drugs (FLU and FBZ) was studied in the meadow plant ribwort plantain (Plantago lanceolata) in two model systems (cell suspensions and regenerants). Ribwort regenerants were incubated with FLU or FBZ for 1, 3, and 6 weeks. All samples were processed, extracted and analyzed using UHPLC-MS/MS. Retention times, theoretical m/z values of elemental composition and product ions of benzimidazole metabolites detected in MS/MS experiments are summarized in Table 1 for FLU and in Table 2 for FBZ. The detected metabolites were identified according to protonated molecules [M +H]+. A positive-ion mode was used for all measurements. Standards of the potential metabolites were generally unavailable and were not synthesized due to difficulties regarding their synthesis. Identification of the chemical structures of the metabolites revealed that the enzymatic systems of the ribwort plantain were able to biotransform the anthelmintics via several reactions of phase I and II drug metabolism. 683

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Table 1 Biotransformation of FLU in ribwort – main peaks detected by UHPLC-MS/MS. tR [min]

Theoretical m/z values of [M +H]+ ions

Elemental composition

3.19 3.58 4.04 4.10 4.64 5.23

418.1409 478.162 478.162 478.162 316.1092 476.1464

C20H20FN3O6 C22H24FN3O8 C22H24FN3O8 C22H24FN3O8 C16H14FN3O3 C22H22FN3O8

5.31 5.37

476.1464 476.1464

C22H22FN3O8 C22H22FN3O8

5.46 5.66 6.18 6.49 8.20

330.1248 476.1464 476.1464 492.1428 314.0935

C17H16FN3O3 C22H22FN3O8 C22H22FN3O8 C22H22FN3O9 C16H12FN3O3

Description of metabolite formation Phase I

Phase II

Hydrolysis Carbonyl reduction Carbonyl reduction Carbonyl reduction Carbonyl reduction Hydrolysis, hydroxylation – Hydrolysis, hydroxylation Carbonyl reduction – – Hydroxylation

N-glucosidation Glucosidation Glucosidation N-glucosidation – Glucosidation, Oacetylation N-glucosidation Glucosidation, Oacetylation Methylation N-glucosidation N-glucosidation N-glucosidation –

Product ions of [M +H]+, m/z

Metabolite designation

256, 298, 298, 316, 284, 256

123 266 266 284 238

M1FLU M2FLU M3FLU M4FLU M5FLU M6FLU

314, 282, 123 256

M7FLU M8FLU

298, 314, 314, 330, 282,

M9FLU M10LU M11FLU M12FLU FLU (parent drug)

174 282, 123 282 298 123

samples. FLU acts as a potent GST inhibitor, whereas FBZ increased activities of GST after an exposition of 0.25 and 1 days. All the results presented here indicate that the tested anthelmintics FLU and FBZ do have an effect on antioxidant enzymes. This could induce a higher risk of oxidative damage in plants influenced by anthelmintics, particularly when they are under other stress conditions.

3.2. The effect of FLU and FBZ on specific enzyme activities Plants contain several types of antioxidant enzymes, including all the enzymes determined in our study, which are important in the protection against oxidative damage. We measured the specific activities of CAT, APX, POX, SOD and GST in the control and FLU or FBZ influenced cells of the ribwort plantain. As a model system in these experiments, suspension cultures were used to facilitate simple and rapid cell cultivation. Enzyme activities were assayed after 0.25-, 1-, 2-, and 9-day exposure of the plant cells. All tested activities were detected in our experiments, with the obtained results summarized in Fig. 4. Both the tested anthelmintics decreased SOD activities after short exposition. CAT activities increased only after 0.25- and 1-day treatment with FLU, while with FBZ no significant effect was observed. Interestingly, POX activity increased after 0.25, 1 and 3 days of FLU treatment, and decreased by FBZ treatment after 1, 3 and 9 days. No significant differences in APX specific activities were observed between the influenced and control samples, with the exception of the 9-day FLU-treated

3.3. Phytotoxicity of FLU and FBZ We used the concentration of proline in leaves as an indicator of stress caused by the uptake of anthelmintics in the ribwort plantain. Although the plants did not show visual symptoms of stress, other stress signals were captured. While in the case of FLU treatment the concentration of proline in the first week was lower than in the control, after 6 weeks the proline concentration was more than 2-times higher than in the control plants. The plants treated by FBZ accumulated the highest amount of proline (284% of that in the control plants) after 3 weeks of treatment (Fig. 5). Increased proline concentration connected

Table 2 Biotransformation of FBZ in ribwort – main peaks detected by UHPLC-MS/MS. tR [min]

Theoretical m/z values of [M +H]+ ions

Elemental composition

Description of metabolite formation Phase I

Phase II N-glucosidation Glucosidation, O-acetylation N-glucosidation N-glucosidation N-glucosidation N-glucosidation N-glucosidation N-glucosidation – N-glucosidation N-glucosidation, Oacetylation N-glucosidation, Oacetylation N-glucosidation Glucosidation, O-acetylation Glucosidation, O-acetylation Glucosidation, O-acetylation N-glucosidation 2*glucosidation, Glucosidation, O-acetylation – N-glucosidation N-glucosidation –

3.30 3.32 3.83 4.03 4.53 4.63 4.79 4.85 5.05 5.38 5.45

478 478 478 478 404 494 478 404 316 478 520

C21H23N3O8S C21H23N3O8S C21H23N3O8S C21H23N3O8S C19H21N3O5S C21H23N3O9S C21H23N3O8S C19H21N3O5S C15H13N3O3S C21H23N3O8S C23H25N3O9S

+O 2*(+O), hydrolysis +O +O Hydrolysis 2*S-oxidation +O Hydrolysis Hydroxylation +O +O

5.53

520

C23H25N3O9S

+O

5.59 6.28 6.47 6.69 6.72 6.85 7.05 7.39 7.46 7.95 9.55

478 462 462 462 462 624 462 332 462 462 300

C21H23N3O8S C21H23N3O7S C21H23N3O7S C21H23N3O7S C21H23N3O7S C27H33N3O12S C21H23N3O7S C21H23N3O9S C21H23N3O7S C21H23N3O7S C15H13N3O2S

+O +O, hydrolysis +O, hydrolysis +O, hydrolysis – – +O, hydrolysis 2*(+O) – – –

684

Product ions of [M +H]+, m/z

Metabolite designation

316, 300, 316, 316, 242 332, 316, 242, 207, 316, 316,

300 284, 191 133 191 284, 191 299, 284

M1FBZ M2FBZ M3FBZ M4FBZ M5FBZ M6FBZ M7FBZ M8FBZ M9FBZ M10FBZ M11FBZ

316, 299, 284

M12FBZ

316, 242 242 242 300, 300, 242 300, 300, 300, 268,

M13FBZ M14FBZ M15FBZ M16FBZ M17FBZ M18FBZ M19FBZ M20FBZ M21FBZ M22FBZ FBZ

284, 191 258 284, 191 284, 191

284, 160

268, 159 268 159 268, 159 268, 159 159, 131

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Fig. 1. Scheme of metabolic pathways of FLU in the ribwort plantain.

Fig. 2. Scheme of metabolic pathways of FBZ in the ribwort plantain.

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Fig. 3. Changes in the relative amount of parent drug and its main metabolites in roots (R), basal parts of leaves (BL) and tops of leaves (TL) from plant regenerants after 1, 3, and 6 weekincubations. A: FLU, reduced FLU (M5) and FLU glucoside (M7); B: FBZ, hydroxylated FBZ (M9) and N-glucoside of FBZ (M21).

and biotransform these drugs into several metabolites, a way which could represent their remediation. However, most of FLU and FBZ metabolites formed in the ribwort can be decomposed back to the biologically active parent drug. Therefore, consumption of ribworts containing anthelmintic metabolites by infected livestock could support drug-resistance development in helminths. Consumption of these ribworts could represent a risk for free-living invertebrates. Moreover, in the plants themselves both anthelmintics could cause oxidative stress

with the presence of anthelmintics in plants indicates stress of plants. A positive correlation between proline accumulation and plant stress under different stress conditions has been reported (Lefevre et al., 2009; Kaur and Asthir, 2015; Sprenger et al., 2016; Vanek et al., 2016). 4. Conclusions The ribwort plantain (cell culture and regenerants) is able to uptake

Fig. 4. Specific activities of CAT, APX, POX, SOD and GST in plant cells after 0.25-, 1-, 2- and 9-days exposure of FLU or FBZ. Data represent the mean ± S.D. (n = 3). A significant difference was marked by triangles.

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Soil 39 (1), 205–207. Bradford, M.M., 1976. Rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle of protein-dye Binding. Anal. Biochem. 72 (1–2), 248–254. Bundschuh, M., et al., 2016. Acute toxicity and environmental risks of five veterinary pharmaceuticals for aquatic macroinvertebrates. Bull. Environ. Contam. Toxicol. 96 (2), 139–143. Dietz, A.C., Schnoor, J.L., 2001. Advances in phytoremediation. Environ. Health Perspect. 109 (Suppl 1), S163–S168. Drotar, A., et al., 1985. Evidence for glutathione-peroxidase activities in cultured plantcells. Plant Sci. 42 (1), 35–40. El-Shabrawi, H., et al., 2010. Redox homeostasis, antioxidant defense, and methylglyoxal detoxification as markers for salt tolerance in Pokkali rice. Protoplasma 245 (1–4), 85–96. Habig, W.H., et al., 1974. Glutathione S-transferases - first enzymatic step in mercapturic acid formation. J. Biol. Chem. 249 (22), 7130–7139. Holcapek, M., et al., 2008. High-performance liquid chromatography-tandem mass spectrometry in the identification and determination of phase I and phase II drug metabolites. Anal. Bioanal. Chem. 391 (1), 59–78. Kaur, G., Asthir, B., 2015. Proline: a key player in plant abiotic stress tolerance. Biol. Plant. 59 (4), 609–619. Kreuzig, R., et al., 2007. Fate of the benzimidazole antiparasitics flubendazole and fenbendazole in manure and manured solis. Clean. Soil Air Water 35 (5), 488–494. Lefevre, I., et al., 2009. Chloride salinity reduces cadmium accumulation by the Mediterranean halophyte species Atriplex halimus L. Environ. Exp. Bot. 65 (1), 142–152. Mhamdi, A., et al., 2010a. Catalase function in plants: a focus on Arabidopsis mutants as stress-mimic models. J. Exp. Bot. 61 (15), 4197–4220. Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol. Plant. 15 (3), 473–497. Nobilis, M., et al., 1996. Study of the biotransformation of a potential benzo c fluorene antineoplastic using high-performance liquid chromatography with high-speedscanning ultraviolet detection. J. Chromatogr. B-Biomed. Appl. 681 (1), 143–151. Podlipna, R., et al., 2013. Biotransformation of benzimidazole anthelmintics in reed (Phragmites australis) as a potential tool for their detoxification in environment. Bioresour. Technol. 144, 216–224. Sim, W.J., et al., 2013. Evaluation of pharmaceuticals and personal care products with emphasis on anthelmintics in human sanitary waste, sewage, hospital wastewater, livestock wastewater and receiving water. J. Hazard. Mater. 248, 219–227. Snow, D.D., et al., 2014. Detection, occurrence and fate of emerging contaminants in agricultural environments. Water Environ. Res. 86 (10), 865–881. Sprenger, H., et al., 2016. The drought response of potato reference cultivars with contrasting tolerance. Plant Cell Environ. 39 (11), 2370–2389. Stuchlikova, L., et al., 2016. Metabolic pathways of benzimidazole anthelmintics in harebell (Campanula rotundifolia). Chemosphere 157, 10–17. Stuchlikova Raisova, L., et al., 2017. Evaluation of drug uptake and deactivation in plant: fate of albendazole in ribwort plantain (Plantago laceolata) cells and regenerants. Ecotoxicol. Environ. Saf. 141, 37–42. Vanacker, H., et al., 1998. Pathogen-induced changes in the antioxidant status of the apoplast in barley leaves. Plant Physiol. 117 (3), 1103–1114. Vanek, T., et al., 2016. Accumulation of cadmium by halophytic and non-halophytic Juncus species. Theor. Exp. Plant Physiol. 28 (4), 415–423. Verma, S., Dubey, R.S., 2003. Lead toxicity induces lipid peroxidation and alters the activities of antioxidant enzymes in growing rice plants. Plant Sci. 164 (4), 645–655. Vokral, I., et al., 2012. The metabolism of flubendazole and the activities of selected biotransformation enzymes in Haemonchus contortus strains susceptible and resistant to anthelmintics. Parasitology 139 (10), 1309–1316. Wagil, M., et al., 2015a. Toxicity of anthelmintic drugs (fenbendazole and flubendazole) to aquatic organisms. Environ. Sci. Pollut. Res. 22 (4), 2566–2573. Wagil, M., et al., 2015b. A comprehensive approach to the determination of two benzimidazoles in environmental samples. Chemosphere 119, S35–S41. Weiss, K., et al., 2008. Sulfamethazine and flubendazole in seepage water after the sprinkling of manured areas. Chemosphere 72 (9), 1292–1297. Zrncic, M., et al., 2014. Analysis of anthelmintics in surface water by ultra high performance liquid chromatography coupled to quadrupole linear ion trap tandem mass spectrometry. Chemosphere 99, 224–232.

Fig. 5. Concentration of proline in ribwort plantain leaves in 1, 3, and 6 weeks of treatment by 10 µM FLU or FBZ. A significant difference was marked by triangles.

and decreased antioxidant defense. Taking into consideration all of these factors, both tested anthelmintics FLU and FBZ could have a negative environmental impact. Nevertheless, the experiments in real pastures conditions will be necessary to verify this hypothesis. Acknowledgement We would like to thank Daniel Paul Sampey, MFA, for English revision. This project was supported by the Czech Science Foundation (GA ČR, grant No. 15-05325S) and by Charles University in Prague (research project SVV 260 416). Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ecoenv.2017.09.020. References Anjum, N.A., et al., 2016. Catalase and ascorbate peroxidase-representative H2O2-detoxifying heme enzymes in plants. Environ. Sci. Pollut. Res. Int. 23 (19), 19002–19029. Bartikova, H., et al., 2010. Flubendazole metabolism and biotransformation enzymes activities in healthy sheep and sheep with haemonchosis. J. Vet. Pharmacol. Ther. 33 (1), 56–62. Bartikova, H., et al., 2016. Veterinary drugs in the environment and their toxicity to plants. Chemosphere 144, 2290–2301. Bates, L.S., et al., 1973. Rapid determination of free proline for water-stress Studies. Plant

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