Ecotoxicology and Environmental Safety 101 (2014) 205–212
Contents lists available at ScienceDirect
Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Uptake of the cyanobacterial neurotoxin, anatoxin-a, and alterations in oxidative stress in the submerged aquatic plant Ceratophyllum demersum Mi-Hee Ha, Valeska Contardo-Jara, Stephan Pflugmacher n Technische Universität Berlin, Department of Ecotoxicological Impact Research and Ecotoxicology, Ernst-Reuter-Platz 1, 10587 Berlin, Germany
art ic l e i nf o
a b s t r a c t
Article history: Received 10 July 2013 Received in revised form 22 December 2013 Accepted 24 December 2013
The prevalence of cyanobacterial blooms in fresh water bodies worldwide has become a serious environmental problem. The blooms can increase the occurrence of cyanobacterial neurotoxin, anatoxin-a, and this toxin can interact with aquatic plants and other pivotal components of aquatic ecosystems. Despite this, several questions regarding the uptake of the toxin by aquatic plants and its association with toxic effects still remain. This study investigated the uptake of anatoxin-a in relation to alterations in oxidative stress, estimated by changes in lipid peroxidation and tocopherol contents (alpha-, beta-, gamma-, and delta-tocopherol), in the submerged aquatic plant, Ceratophylum demersum, at environmentally relevant concentrations. Exposure to five different concentrations of anatoxin-a (0.005, 0.05, 0.5, 5 and 50 μg l 1) for 24 h increased concentrations in C. demersum in a dose-dependent manner. All four forms of tocopherols were elevated at low concentrations of anatoxin-a (0.005. 0.05. 0.5 and 5 μg l 1). However, a decline in the four tocopherol forms along with a high level of lipid peroxidation was observed at 50 μg l 1 exposure dose. During 336-h exposure to 15 μg l 1 anatoxin-a, rapid toxin uptake during the first 24 h and subsequent steady accumulation of the toxin were observed. The four tocopherol forms increased in response to anatoxin-a uptake, attaining their maximum levels together with a significant increase of lipid peroxidation after 12 or 24 h. After 24-h exposure, the four tocopherol forms decreased gradually without recovery. The results clearly indicate that anatoxin-a uptake can cause a disturbance of the oxidative stress in the aquatic plant, and depending on the concentration and exposure duration, oxidative damage occurs. & 2014 Elsevier Inc. All rights reserved.
Keywords: Anatoxin-a Ceratophyllum demersum Uptake Oxidative stress status Lipid peroxidation Tocopherol
1. Introduction Cyanobacteria encompassing diverse photosynthetic prokaryotic micro-organisms exist in balanced aquatic environments at the proper level at which their frequency and abundance are naturally controlled (Paerl, 2000; Stockner, 1999). However, accelerated eutrophication and climate change facilitate them to excessively proliferate to the extent that they provoke a disturbance (e.g. high algal turbidity and oxygen depletion) in aquatic ecosystems and deteriorate water quality (e.g. offensive odor and taste) related to human well-being (Hart et al., 1999; Paerl and Huisman, 2009; Pitois et al., 2000; Smith, 2003). Furthermore, many of cyanobaterial genera such as Anabaena, Aphanizomenon, Cylindrospermopsis, Microcystis and Planktothrix are known to produce a wide range of toxic bioactive secondary metabolites designated as cyanotoxins. n
Corresponding author. Fax: þ 49 30 314 29022. E-mail addresses: [email protected] (M.-H. Ha), [email protected] (V. Contardo-Jara), stephan.pfl[email protected] (S. Pflugmacher). 0147-6513/$ - see front matter & 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ecoenv.2013.12.023
These toxins are principally released into the surrounding water during their senescence and decomposition (Carmichael, 1997; Codd, 1995). Anatoxin-a is one of the most potent cyanobacterial neurotoxins commonly detected all over the world (Ballot et al., 2004; Gugger et al., 2005; Park et al., 1998; Stevens and Krieger, 1988). It is a powerful cholinergic agonist competing with acetylcholine for nicotinic acetylcholine receptor in neuronal signal transmission (Gugger et al., 2005; Krienitz et al., 2003; Park et al., 1998; Thomas et al., 1993). The toxin is chemically defined as a lowmolecular-weight semi-rigid alkaloid (MW¼ 165.26), labile under natural environmental conditions, especially sunlight and pH. Thus, anatoxin-a is reported to be easily degradable into two non-toxic compounds (i.e. dehydroanatoxin-a and epoxyanatoxin-a) and to have a half-life of several hours or days in strong sunlight and at high pH (Devlin et al., 1977; Smith and Sutton., 1993; Stevens and Krieger, 1991). Due to the distinctive chemical characteristics, low stability and persistence, as well as the notorious rapid neurotoxic mode of action, fatal to mammals, relatively little attention has been paid so far to the fate and impact of anatoxin-a in aquatic environments. However, considering the prevalence of the massive
206
M.-H. Ha et al. / Ecotoxicology and Environmental Safety 101 (2014) 205–212
growth of cyanobacteria leading to increased presence and amount of cyanotoxins, knowledge about anatoxin-a uptake, metabolism and the resulting effects in aquatic ecosystems containing various living organisms (e.g. plant, zooplankton, mollusk and fish) needs to be improved (Carmichael, 2008; Glibert et al., 2005). Very few studies investigated the bioconcentration and depuration of anatoxin-a in particular aquatic organisms, mussel and fish, associated with the risk of human intoxication from anatoxin-a-contaminated dietary source (Osswald et al., 2011, 2008, 2007). Plants are generally regarded as major players in the transport and degradation of many well-known contaminants in aquatic environments through their uptake and metabolic detoxification (Dhir et al., 2009; Takamura et al., 2003). Moreover, they are also involved in the transfer of pollutants via bioaccumulation along the aquatic food chain as primary producers offering food to other higher organisms (Gobas and Morrison, 2000). Indeed, there have been several reports presenting that the powerful cyanobacterial hepatotoxins microcystins (MCs) can be taken up, metabolized and eventually accumulated to a certain degree in aquatic plants such as C. demersum, Lemna gibba and Phragmites australia (Pflugmacher et al., 2004, 2001; Saqrane et al., 2007). The publication from Esterhuizen et al. (2011a) also showed that the cyanobacterial neurotoxin β-methylamino-l-alanine (BMAA) can be transferred from water media into C. demersum and can possibly give rise to biomagnification through protein association following accumulation of the toxin. Simultaneously, phytotoxic effects (e.g. reduced photosynthetic pigment contents and altered enzymatic systems) have been observed and linked to uptake of these cyanotoxins (Esterhuizen et al., 2011b; Pflugmacher, 2004; Saqrane et al., 2007). Currently, there is some evidence indicating that anatoxin-a can also affect multiple levels of biological metabolism (e.g. growth, photosynthesis and enzymatic systems) of aquatic macrophytes, Lemna minor (L. minor) and C. demersum by inducing oxidative stress (Ha and Pflugmacher, 2013a, 2013b; Mitrovic et al., 2004). Previous studies demonstrated that the activity of antioxidative enzymes including superoxide dismutase (SOD), peroxidase (POD), ascorbate peroxidase (APX), monodehydroascorbate reductase (MDAR) and glutathione reductase (GR) was changed in response to the elevated formation of hydrogen peroxide (H2O2) in C. demersum exposed to anatoxin-a (Ha and Pflugmacher, 2013a, 2013b). Therefore, the present study aimed to investigate the uptake of anatoxin-a in relation to alterations of the oxidative stress status in C. demersum, a world-wide distributed submerged aquatic plant. Oxidative stress refers to an imbalance between pro-oxidative reactions (e.g. generating reactive oxygen species (ROS)) and antioxidative defense (e.g. quenching ROS). Oxidative stress can lead to severe damage to all types of cellular molecules, DNA, proteins and lipids, depending on the degree of imbalance (Sies, 1997). Lipid peroxidation is described as the oxidative deterioration of polyunsaturated fatty acids (PUFAs), crucial components of cellular lipid membranes, and has been largely studied in the assessment of oxidative cell injury (Esterbauer et al., 1991; Gutteridge, 1995). It is well-established that plants possess effective antioxidative defense systems comprising various enzymes and non-enzymatic free-radical scavengers antioxidants like ascorbate (vitamin C), carotenoids, glutathione (GSH), and tocopherols (vitamin E) to mitigate various conditions of oxidative stress (e.g. controlling ROS production and relieving the resulting damage) (Gill and Tuteja, 2010; Mittler, 2002). Tocopherols, synthesized exclusively by plants and certain photosynthetic organisms, are believed to play important roles in the stabilization of lipid membranes by decomposing ROS and lipid peroxy radicals during the propagation of lipid peroxidation (Fryer, 1992; Munné-Bosch and Alegre, 2002). They exist in four different chemical forms, α-, β-, γ-, and δ-tocopherol, named according to the number and position of methyl groups on the chromanol ring. α-Tocopherol is known as the major form detected in green parts of plants.
Accordingly, in this study lipid peroxidation and the corresponding antioxidant, four tocopherols (α-, β-, γ-, and δ-tocopherol), were used as indicators of changes in oxidative stress status in response to anatoxin-a uptake in C. demersum.
2. Material and methods 2.1. Plant material C. demersum, obtained from Extraplant (Extragroup GmbH, Münster, Germany), was cultivated non-axenically in a glass tank (60 cm 60 cm 60 cm) filled with modified Provasoli0 s medium containing de-ionized water, 0.2 g l 1 CaCl2, 0.103 g l 1 NaHCO3 and 0.1 g l 1 sea-salt. The culture was conducted under cool white fluorescent light (35 μE m 2 s 1 irradiance) with a day/night cycle of 14/ 10 h at 22–24 1C.
2.2. Experimental design ( 7 ) Anatoxin-a fumarate was provided by Tocris Bioscience (Strathmore Road Natick, MA, USA). The toxin was dissolved to 1 g l 1 in 70 percent (v/v) methanol and further diluted with culture medium to the final exposure concentrations. The environmentally feasible concentrations of anatoxin-a were derived from monitoring studies conducted in German fresh water bodies (Bumke-Vogt et al., 1999; Sächsisches Staatsministerium für Soziales, 2009). The plant, comprising of shoot apical meristem, main stem and leaves, was cut one week before the experiments to avoid any physical stresses resulting from cutting. After this preculture for one week, 4.5 7 0.5 g fresh weight (FW) of plant material was exposed to anatoxin-a in a volume of 100 ml under the above mentioned light and temperature conditions for plant culture. Two different static exposure systems were set to investigate concentrationand time-dependent anatoxin-a-uptake in connection with subsequent changes in the levels of lipid peroxidation and tocopherols in C. demersum. Controls consisting of the plant and culture medium without anatoxin-a were prepared and sampled in parallel with the toxin treatment in order to consider the influence of the exposure conditions on the plant. Three independent replicate experiments were performed for each exposure system. In the first exposure system, plants were treated with 0.005, 0.05, 0.5, 5 and 50 mg l 1 anatoxin-a for 24 h. In the second experiment, plants were exposed to 15 mg l 1 anatoxin-a reflecting the highest concentration of the toxin in the environment for 4, 8, 12, 24, 48, 168 and 336 h. Additionally, the pre-cultured plants that were not introduced into the exposure systems were directly used as 0 h controls. Exposure medium was daily refilled with culture medium without anatoxin-a to the initial volume to avoid changes in anatoxin-a concentration due to evaporation. After exposure, plants were washed twice with de-ionized water to remove any surface-associated toxin and snap-frozen in liquid nitrogen. The frozen plant material was stored at 80 1C until toxin, tocopherol and LPO analysis, respectively. In addition to plant sampling, the exposure medium containing anatoxin-a in the time-dependent exposure system was sampled at each exposure time to determine the remaining toxin in the medium over the period of exposure.
2.3. Anatoxin-a analysis 2.3.1. Extraction of anatoxin-a Anatoxin-a was extracted from plant tissue according to Rellán et al. (2007) with slight modifications. The frozen plant material was ground in liquid nitrogen to a fine powder. Anatoxin-a was extracted by mixing 0.2 g of pulverized plant tissue in 1 ml of acidified 70 percent methanol (MeOH) containing 0.1 percent trifluoroacetic acid (TFA). Tissue samples were continuously shaken in the dark for 3 h at room temperature using overhead shaker, Intelli-Mixer RM-2 (Neolab, Heidelberg, Germany). Extracts were then centrifuged at 10,600g for 10 min at 4 1C and the supernatant was adjusted to pH 7.0 7 0.2 with 1 M sodium hydroxide (NaOH) before solid-phase extraction (SPE) using a weak cation-exchange material in a vacuum manifold processor (SPE-12G, JT Baker, Großgerau, Germany). SPE columns, Strata WCX, 3 ml with 200 mg silica gel-based packing (Phenomenex, CA, USA), were conditioned with equal volumes of MeOH and water. Sample loading and elution with 100 percent MeOH containing 0.2 percent TFA were carried out under gravity. Afterwards, eluates were completely dried under constant nitrogen flow. The final residue was reconstituted in 200 μl of 70 percent MeOH and anatoxin-a content was immediately determined by LC–MS/MS. Water samples were centrifuged at 5000g for 15 min at 4 1C and transparent water was settled at pH 7.0 7 0.2 with 1 M NaOH. The remaining anatoxin-a in the exposure media was extracted and concentrated with Strata WCX SPE columns using the same procedure as described above. Solid phase-extracted water samples were immediately processed for the toxin analysis via LC–MS/MS.
M.-H. Ha et al. / Ecotoxicology and Environmental Safety 101 (2014) 205–212
207
absorbance of the clear supernatant was determined at 586 nm. All measurements were made in triplicate for the three independent replicates of each exposure concentration or sampling event, respectively. A standard calibration curve of MDA was previously generated to calculate the amount of MDAþ4-HNE. 2.5. Tocopherol analysis 2.5.1. Extraction of tocopherols The extraction of tocopherols was carried out according to Stüven and Pflugmacher (2007). The frozen plant tissue was pulverized in liquid nitrogen. The tissue powder (0.2 g) was suspended in 3.2 ml of chilled 100 percent MeOH and constantly shaken in the dark for 10 min using an overhead shaker. The extract was centrifuged at 10,600g for 10 min at 4 1C to exclude plant debris. The resulting supernatant was diluted to 85 percent MeOH with de-ionized water, and then directly applied to the LC–MS/MS analysis of tocopherols. 2.5.2. Determination of tocopherols Four tocopherols (α-, β-, γ-, and δ-tocopherol) were individually quantified by LC–MS/MS. Pure α-, β-, γ-, and δ-tocopherol were purchased from Sigma-Aldrich and dissolved in 85 percent MeOH to establish reference standard curves. The four tocopherols forms in the samples were separated on the Kinetex PFP column (150 2.1 mm2, 2.6 μm pore size, Phenomenex) within 18 min using isocratic chromatographic run with a mixture of MeOH:H2O þ0.4 percent Formic acid (85:15, v/v) at a flow rate of 0.3 ml min 1. Column oven temperature was set to 20 1C and the injection volume was 20 μl. The retention time of α-, β-, γ-, and δ-tocopherol was 15.8, 12.8, 13.2 and 8.9 min, respectively. The mass spectra of the tocopherols were generated in the positive ion mode with the collision energy of 25 V using ESI. The mass-to-charge (m/z) ratio of each tocopherol was as follows: m/z 431-219/205/165 (α-tocopherol); m/z 417-205/191/151 (β- and γtocopherol); m/z 403-191/177/151/137 (δ-tocopherol). The ESI was operated under the following conditions: capillary voltage, 5.0 kV; cone voltage, 30 V; source temperature, 120 1C; desolvation temperature, 350 1C; cone gas flow rate, 50 l h 1; desolvation gas flow rate, 500 l h 1. 2.6. Statistical analysis
Fig. 1. HPLC chromatograms obtained using HILIC column coupled with ESI-MS/ MS. (A) Anatoxin-a standard (50 μg l 1) and (B) C. demersum extract exposed to 50 μg l 1 anatoxin-a for 24 h. 2.3.2. Determination of anatoxin-a Anatoxin-a analysis was carried out by LC–MS/MS (Alliance 2695 UHPLC) combined with a Quattro micro™ triple quadrupole mass spectrometer (Waters, Milford, MA, USA). Chromatographic separation was acquired under HILIC conditions employing the Kinetex HILIC column (100 2.1 mm2, 2.6 μm pore size, Phenomenex) with isocratic operation. The mobile phase consisted of 100 percent acetonitrile, water and 50 mM ammonium formate containing 0.32 percent formic acid (75: 22.5: 2.5, v/v) at a flow rate of 0.2 ml min 1. Column oven temperature was set to 30 1C and the injection volume was 2 μl. The clear and sharp elution peak of anatoxin-a was given at a retention time of 3.68 min (Fig. 1). Mass spectral data was achieved in the positive ion mode with the collision energy of 15 V using electrospray ionization (ESI). The parent compound and its fragment ions were scanned at following mass-to-charge (m/z) ratios: m/z 166-149/131/91. Operating parameters of ESI were set as follows: capillary voltage, 5.0 kV; cone voltage, 35 V; source temperature, 100 1C; desolvation temperature, 450 1C; cone gas flow rate, 35 l h 1; desolvation gas flow rate, 500 l h 1. The toxin was quantified by comparison with 6-point calibration curve with reference standard solutions using ( 7 ) anatoxin-a fumerate in 70 percent MeOH.
2.4. Lipid peroxidation Lipid peroxidation products, malondialdehyde (MDA) and 4-hydroxy-2-nonenal (4-HNE) in the plant tissue were measured according to Esterbauer and Cheeseman (1990) using colorimetric microplate assay kits (Oxford Biomedical Research, MI, USA). The assay is based on the irreversible reaction of the respective aldehydes in the two compounds with 1-methyl-2-phenylindole (MPI) in the presence of methanesulfonic acid (MSA) and ferric iron. The resulting stable chromophore yields maximal absorbance at 586 nm. Plant tissue was ground to a fine powder in liquid nitrogen using mortar and pestle. 0.1 g of plant tissue was then repeatedly homogenized in 1.2 ml of 20 mM sodium phosphate buffer (pH 7.0) with mechanical TissueLyser LT (Quiagen, Hilden, Germany) for effective cell lysis. Prior to homogenization, 12 μl of 0.5 M butylated hydroxyltoluene (BHT) was added to prevent sample oxidation. The precipitate and plant debris were removed by centrifugation at 10,600g for 10 min at 4 1C. 140 μl of the supernatant was mixed with 455 μl of MPI in acetonitrile. Then, 105 μl of MSA was added, mixed gently and incubated at 45 1C for 60 min. The reaction mixture was centrifuged at 15,000g for 10 min and the
One-way analysis of variance (ANOVA) was performed on the data obtained from the experiments on the concentration-dependent effects of anatoxin-a, followed by Turkey0 s HSD test. Two-way analysis of variance (ANOVA), with toxin treatment as one factor and exposure duration as the other factor, was carried out on data generated from experiments on the time-dependent effects of anatoxin-a, followed by Tukey0 s HSD test. Prior to ANOVA, all data were tested for normality and homogeneity of variance using Shapiro-Wilks W and Levene0 s test, respectively. All statistical analyses were performed using the commercial software SPSS 17.0 (SPSS Inc., Chicago, IL, USA) with a p-value o 0.05 considered as significant.
3. Results 3.1. Concentration-dependent responses of C. demersum to anatoxin-a After exposure for 24 h, anatoxin-a was detectable in plants treated with 0.5, 5 and 50 μg l 1 anatoxin-a (Fig. 2A). No anatoxin-a was detected in plants treated with the lower concentrations (0.005 and 0.05 μg l 1). The measured anatoxin-a in the plants at an exposure concentration of 0.5 μg l 1 amounted to 0.06570.017 ng gFW 1. The level of anatoxin-a in the plants exhibited a concentration-dependent increase up to the highest exposure concentration with average values of 15.8470.699 ng gFW 1 anatoxin-a. The detected amounts of anatoxin-a in the plants after 24-h exposure presented a linear relationship with the applied exposure concentrations (correlation coefficient R2 ¼ 0.979). Plants exposed to 50 μg l 1 anatoxin-a showed a highly significant increase in the formation of the two major metabolites of lipid peroxidation, MDA and 4-HNE, after 24 h (po0.005) (Fig. 2B). Significant enhancement of α-tocopherol content was detected in plants exposed to 0.005, 0.05, and 0.5 μg l 1 anatoxin-a after 24 h (p o0.05). The strongest increase reported in plants exposed to 0.05 μg l 1 anatoxin-a was 1.314-fold of the control (po 0.005) (Fig. 3A). β-Tocopherol content was significantly increased in plants exposed to 0.005, 0.05, 0.5 and 5 μg l 1 anatoxin-a (p o0.05) (Fig. 3B). The maximum elevation of β-tocopherol, being
208
M.-H. Ha et al. / Ecotoxicology and Environmental Safety 101 (2014) 205–212
factor (BCF), which was estimated by dividing anatoxin-a in the plant (μg μg FW 1) by anatoxin-a in the corresponding water media (μg ml 1) after 24-h exposure to 15 μg l 1, was 51.01 The plants exposed to 15 μg l 1 anatoxin-a for 336 h showed a significant production of lipid peroxidation metabolites, MDA and 4-HNE, after 12 and 24 h (po0.05) (Fig. 4B). After longer exposure, the amount of lipid peroxidation metabolites decreased to control levels. α-Tocopherol content was significantly increased in exposed plants within the initial period of exposure (4, 8 and 12 h) (po0.05), decreasing thereafter to control levels until 48 h of exposure (Fig. 5A). A significant reduction in α-tocopherol content in exposed plants compared to the control of the same sampling event was then detected after 168 and 336 h exposure. βTocopherol was immediately and significantly raised to 2.3-fold of the control at 24-h exposure (po0.05) (Fig. 5B). After 48 h, βtocopherol content was reduced and returned to control values until the end of the experiment. γ-Tocopherol content was significantly increased after 8 h (po0.05) (Fig. 5C). The maximum increase in γ-tocopherol content, which was 3.7-fold of the control, was detected after 12 h. After 24 h, the increase in γ-tocopherol content was still significant. Longer exposure led the increased γtocopherol content to a return to control levels. A significant elevation of δ-tocopherol content was observed after 4 h, reaching its maximum level after 12 h, which was 1.7-fold higher than that of the control (po0.05) (Fig. 5D). As observed for β- and γ-tocopherol, δ-tocopherol content also decreased to control levels after longer exposure.
4. Discussion 4.1. Concentration-dependent responses of C. demersum to anatoxin-a
Fig. 2. (A) Uptake of anatoxin-a and (B) levels of lipid peroxidation products (MDA þ 4-HNE) in C. demersum after 24-h exposure to different concentrations of anatoxin-a. All values are given as mean 7standard deviation (n¼ 3, nnhighly significantly different from control, p o 0.005).
0.6 times higher than that of the control, was noticed at an exposure concentration of 5 μg l 1 (po0.005). For γ-tocopherol content, no significant differences were observed between the toxin treatments and the control except for the exposure with 5 μg l 1 anatoxin-a (p o0.05) (Fig. 3C). A significant increase in δ-tocopherol was detected in plants exposed to 0.005 μg l 1 anatoxin-a (p o0.05) (Fig. 3D). The maximum level of δtocopherol amounting 1.6-fold of the control was observed at an exposure concentration of 5 μg l 1 (po 0.005). Both, γ- and δtocopherol content, displayed slightly lower values in plants exposed to 50 μg l 1 anatoxin-a compared to control plants. 3.2. Time-dependent responses of C. demersum to anatoxin-a Anatoxin-a uptake occurred in C. demersum exposed to 15 μg l 1 anatoxin-a within 4 h, increasing rapidly until 24 h (Fig. 4A). The highest level of anatoxin-a detected in exposed plants after 24 h was 7.0570.64 ng gFW 1. Within 48 h, the toxin amount slightly decreased by around 5 percent followed by a remarkable reduction of the toxin amount until 168 h. The concentration of dissolved anatoxin-a, initially set at a level of 15 μg l 1, drastically decreased to 3.96 μg l 1 after 4 h. Anatoxin-a concentration in the media was further reduced to 0.14 μg l 1 within 24 h. Consequently, almost the entire toxin (99 percent), given at the beginning of the experiment, was removed from water media within 24 h. The bioconcentration
Anatoxin-a started being detected in C. demersum exposed to 0.5 μg l 1 after a 24-h exposure period. The toxin amount in plants increased proportionally to the exposure concentrations up to 50 μg l 1 with a good linear correlation. The observation suggests that saturation of anatoxin-a uptake was not reached in the plant within the tested concentration range and that uptake could further continue at higher concentrations above 50 μg l 1. Although the amount of anatoxin-a detected in C. demersum was in ng g FW 1 range, it was about ten times higher than that of BMAA previously measured in the same plant under similar experimental conditions (Esterhuizen et al., 2011a). This finding implies that aquatic plants may display different uptake rates or metabolization efficiencies for each cyanotoxin. Anatoxin-a seems to be more easily taken up or to be more slowly metabolized by aquatic plants compared to BMAA. Tocopherols are synthesized from homogentistic acid and isopentenyl diphosphate in the envelope membrane of chloroplasts (Soll et al., 1985). They are able to scavenge not only ROS but also lipid peroxyl radical resulting from a reaction between molecular oxygen and the highly reactive carbon-centered lipid radical derived from PUFAs which lost their hydrogen atom by ROS. Different tocopherol forms, α-, β-, γ-, and δ-tocopherol, hamper thereby the propagation of lipid peroxidation ending in the production of MDA and 4-HNE in membrane and thus, contribute to maintaining membrane integrity (Munné-Bosch and Alegre, 2002). In spite of their noticeable activities in antioxidative defense systems, there are few reports investigating tocopherol contents as indicators of oxidative stress in aquatic plants. To the best of our knowledge, this study showed for the first time, changes in tocopherol profiles in connection with the induction of lipid peroxidation in an aquatic plant exposed to a cyanotoxin. The four tocopherol forms increased within the
M.-H. Ha et al. / Ecotoxicology and Environmental Safety 101 (2014) 205–212
209
Fig. 3. Changes in (A) α-, (B) β-, (C) γ-, and (D) δ-tocopherol contents of C. demersum after 24-h exposure to different concentrations of anatoxin-a. All values are given as mean 7 standard deviation (n¼ 3, nsignificantly different from control, p o 0.05; nnhighly significantly different from control, p o 0.005).
anatoxin-a concentration range of 0.005–5 μg l 1 with different activation patterns; however, plants exposed to the highest anatoxin-a concentration, 50 μg l 1, achieved similar or lower tocopherol levels than those observed in control plant. The altered tocopherol contents clearly indicated the promotion of oxidative stress in the plant due to anatoxin-a exposure. The enhancement of α-tocopherol content was mainly presented at lower concentrations, 0.005. 0.05 and 0.5 μg l 1 at which no or only low amounts of the toxin were detected in the plants. In contrast, β-, γ-, and δ-tocopherol exhibited their maximum elevation in plants exposed to 5 μg l 1 anatoxin-a. These results suggest that on one hand α-tocopherol seems to be the most sensitive and vulnerable to anatoxin-a uptake and that on the other hand the four tocopherols cooperate among one another to maintain a constant antioxidant function under different levels of anatoxin-a-induced oxidative stress. Peuthert and Pflugmacher (2010) showed that α- and β-tocopherol contents increased in the terrestrial plant Medicago savita seedling exposed to cyanobacterial crude extract containing microcystin-LR (MC-LR) for 72 h, and the strongest enhancement of each tocopherol form was observed with different concentrations of the toxin. α-Tocopherol content reached its maximum level in seedlings exposed to the lower MC-LR concentration (0.05 μg l 1), whereas β-tocopherol content displayed its strongest increase in seedlings exposed to 0.5 μg l 1. Furthermore, an increase in α- and γ-tocopherol contents was also observed in different spinach (Spinacia oleracea) variants after exposure to cyanobacterial crude extract containing 0.5 μg l 1 of MC-LR for six weeks (Pflugmacher et al., 2007). The tendency of changes in tocopherol contents of plants could provide insight into the activation of the antioxidative defense systems and its capacity limits in coping with oxidative stress caused by anatoxin-a. The
suppressed tocopherol contents at 50 mg l 1 anatoxin-a indicate that the capacity of tocopherols to act as antioxidant agents was overwhelmed and possibly furthermore, their synthesis affected. Consistent with the induction pattern of tocopherol contents, a significant amount of lipid peroxidation end products, MDA and 4-HNE, was observed not at the lower applied concentrations but in plants exposed to 50 mg l 1 anatoxin-a. These findings suggest that the elevation of tocopherols seems to be efficient enough to avoid or mitigate lipid peroxidation at low levels of oxidative stress triggered by anatoxin-a. Simultaneously, these result also provide further evidence that the plant have effective defense mechanisms allowing itself to maintain homeostasis to a certain extent under anatoxin-a-contaminated environments (Ha and Pflugmacher., 2013a, 2013b). The decreased tocopherol contents at higher toxin concentrations might be attributed to the destruction of chloroplasts due to the excessive amount of anatoxin-a transferred into the plant and the subsequently intensified oxidative stress associated with ROS overproduction. This is in accordance with the results of a previous study with C. demersum treated with the same concentration of anatoxin-a, evidencing a decrease in chlorophyll contents together with an increase in cell internal hydrogen peroxide formation (Ha and Pflugmacher, 2013a). 4.2. Time-dependent responses of C. demersum to anatoxin-a Anatoxin-a was taken up by C. demersum within the initial period of exposure (4 h) and the toxin amount steadily increased until 24 h. Meanwhile, the concentration of anatoxin-a in water media decreased sharply and almost the entire toxin was eliminated. These observations suggest that active anatoxin-a uptake by aquatic plants takes place within its short half-life. Hence, the uptake and
210
M.-H. Ha et al. / Ecotoxicology and Environmental Safety 101 (2014) 205–212
Fig. 4. (A) Uptake of anatoxin-a by C. demersum in parallel with changes in the remaining toxin in exposure media and (B) levels of lipid peroxidation products (MDA þ 4-HNE) in C. demersum during 336-h exposure to 15 μg l 1 anatoxin-a (ana-a). All values are given as mean7 standard deviation (n ¼3, different letters indicate significant differences at the level of p o0.05).
subsequent metabolization of anatoxin-a by aquatic plants may have considerable influence on the fate of the toxin in aquatic ecosystems. Other related studies demonstrating the rapid uptake of cyanotoxins by aquatic plants, e.g., Nimptsch et al. (2008) investigating the elimination of MC-LR from water media by using bioaccumulation potential of aquatic plants (i.e. L. minor, Myriophyllum elatinoides, Hydrilla verticillata and Ceratopteris thalictroides), could also verify the significant influence of aquatic plants on the fate of cyanotoxins in the ecosystem. Nimptsch et al. (2008) presented that an initial concentration of 2.5 μg l 1 MC-LR in exposure media containing aquatic plants decreased below 0.9–0.2 μg l 1 within only one d and that thereafter toxin removal continued at a lower rate until seven d. Esterhuizen et al. (2011a) also showed intense BMAA removal from exposure media of 100 μg l 1 within 24 h accompanied by a significant BMAA increase in C. demersum itself. All the mentioned results suggest that aquatic plants are able to take up cyanotoxins including anatoxin-a most effectively during the initial period of exposure. It seems that anatoxin-a uptake occurring within the first 24 h of exposure was followed by metabolization and/or elimination of the toxin without further continuous uptake due to reduction of the toxin in water media. Consequently, the maximum amount of anatoxin-a detected in exposed plants after 24 h decreased to a certain degree within the following 336 h of exposure. However, the removal rate of anatoxin-a tended to become slower after 168 h. These changes in the content of anatoxin-a suggest that metabolization and/or elimination processes of the toxin take place in the examined aquatic plant. Several other studies reporting accumulation of MC-LR in aquatic plants (i.e. C. demersum, L. minor and C. fracta) and subsequent metabolization via
GSH conjugation support the presumption that aquatic plants immediately attempt to metabolize and accordingly eliminate the respective toxin (Mitrovic et al., 2005; Pflugmacher et al., 1999, 1998). During the 336-h experiment of the present study using 15 μg l 1 anatoxin-a, the sum of the toxin in both the plant and water media, respectively, was less than the initial absolute toxin amount in water media. This result might be explained by metabolization by the plant itself as well as the natural degradation of the toxin due to its chemical instability (James et al., 1998; Smith and Sutton, 1993; Stevens and Krieger, 1991). However, the high loss in the toxin amount within the first 4 h of exposure might not be attributed to its chemical instability. Hence, further insight into the metabolization of anatoxin-a by aquatic plants is needed to give an exclusive explanation on primary fate of the toxin. All tocopherol forms were rapidly increased in accordance with the transfer of anatoxin-a into the plant within 4 h. However, the duration of enhancement and exposure time, at which the maximum induction took place, varied among the four tocopherol forms. In contrast to the steady increase in β-tocopherol content until 24 h, the strongest increase in α-, γ-, and δ-tocopherol content was reported already after 12 h. Longer exposure for 336 h led to a gradual decrease in the tocopherol contents without recovery. In the case of α-tocopherol, the content even decreased below control values in exposed plants. These results indicate a constant, but mild increase in oxidative stress in response to the continuous uptake of anatoxin-a until 12 h. It seems that after 24 h the overall amount of the toxin transferred exceeds the antioxidative capacity. Among the four tocopherols investigated in this study, β-tocopherol showed the corresponding activation to the increase in the amount of anatoxina in the plant, suggesting β-tocopherol to be more durable than the other tocopherol forms and to play the most important role in alleviating anatoxin-a-induced oxidative stress. Peuthert and Pflugmacher (2010) could demonstrate a similar pattern in M. sativa seedlings during 72-h exposure to cyanobacterial crude extract containing 0.5 μg l 1 MC-LR. β-tocopherol was increased faster than α-tocopherol and then exhibited continuous further elevation. In accordance with the marked decrease in α-, γ-, and δ-tocopherol, C. demersum showed accelerated and less-suppressed lipid peroxidation, indicated by a large amount of MDA and 4-HNE, after 24 h. Although the contents of all tocopherol forms were reduced after 48 h, the amount of MDA and 4-HNE also decreased to a certain level. This result might be explained by the allocation of other antioxidants such as glutathione and carotenoids working as membrane stabilizers just like tocopherols. MDA and 4-HNE can be detoxified via the conjugation to GSH catalyzed by GST. Carotenoids also participate in modifying the structure and property of lipid membranes against oxidative damages. Indeed, a previous study identified that GST activity and the carotenoids-to-total chlorophyll ratio increased slower than the four tocopherol forms in C. demersum, however, remaining elevated until the end of exposure (336 h) to 15 μg l 1 of anatoxin-a (Ha and Pflugmacher, 2013b). Moreover, the carotenoidsto-total chlorophyll ratio content started to be significantly raised after 12 h, and the highest value was reported after 48 h, at which the decrease in the contents of all tocopherols occurred. These findings suggest that the components of the antioxidative system, several involved enzymes and antioxidants, seem to collaborate among themselves by coordinating their complementary functions, having different immediacies of response and durability to anatoxina-induced oxidative stress.
5. Conclusion This is the first study demonstrating rapid uptake and subsequent partial elimination of the cyanobacterial neurotoxin anatoxin-a in an aquatic plant based on the static exposure of whole plants to purified
M.-H. Ha et al. / Ecotoxicology and Environmental Safety 101 (2014) 205–212
211
Fig. 5. Changes in (A) α-, (B) β-, (C) γ-, and (D) δ-tocopherol contents of C. demersum during 336-h exposure to 15 μg l 1 anatoxin-a (ana-a). All values are given as mean 7 standard deviation (n¼ 3, different letters indicate significant differences at the level of po 0.05).
toxin under controlled laboratory conditions. This study evidenced that anatoxin-a uptake and its metabolization can be linked to disturbances of oxidative stress metabolism of aquatic plants and, depending on severity and duration, oxidative damage arises as a consequence. Moreover, the possibility was shown that aquatic plants may become anatoxin-a reservoirs in highly contaminated water bodies, accumulating the toxin and thereby, despite the short halflife of the toxin, augmenting persistence in the environment. To understand the environmental fate and ecological risk of the toxin in aquatic environments, the use of aquatic plants in biomonitoring of anatoxin-a-contaminated waters should be considered. In addition, the potential of aquatic plants to be used for phytoremediation of water resources having moderate pollution of anatoxin-a can be taken into account in sustainable eco-friendly water management processes. Further studies concerning the maximum limits of anatoxin-a uptake by plants and accompanied toxin elimination in connection with plants resistance to oxidative stress imposed by the toxin are required to assess the efficiency of biomonitoring and phytoremediation as well as to suggest the most favorable aquatic plant species.
Acknowledgments This work was supported by the KIST Institutional Program (Project no. 2E24280). References Ballot, A., Krienitz, L., Kotut, K., Wiegand, C., Metcalf, J., Codd, G.A., Pflugmacher, S., 2004. Cyanobacteria and cyanobacterial toxins in three alkaline Rift Valley lakes of Kenya – Lakes Bogoria, Nakuru and Elmenteita. J. Plankton Res. 26, 925–935. Bumke-Vogt, C., Maliahn, W., Chprus, I., 1999. Anatoxin-a and neurotoxic cyanobacteria in German lakes and reservoirs. Environ. Toxicol. 14, 117–125.
Carmichael, W., 2008. A world overview – one-hundred-twenty-seven years of research on toxic cyanobacteria – where do we go from here?. In: Hudnell, H.K. (Ed.), Cyanobacterial Harmful Algal Blooms: State of the Science and Research Needs. Springer, New York, pp. 105–125. Carmichael, W.W., 1997. The cyanotoxins. In: Callow, J.A. (Ed.), Advances in Botanical Research, 27. Academic Press, San Diego, pp. 211–256. Codd, G.A., 1995. Cyanobacterial toxins: occurrence, properties and biological significance. Water Sci. Technol. 32, 149–156. Dhir, B., Sharmila, P., Daradhi, P.P., 2009. Potential of aquatic macrophytes for removing contaminants from the environment. Crit. Rev. Environ. Sci. Technol. 29, 754–781. Devlin, J.P., Edwards, O.E., Gorham, P.R., Hunter, N.R., Pike, R.K., Stavric, B., 1977. Anatoxin-a, a toxic alkaloid from Anabaena flos-aquae NRC-44h. Can. J. Chem. 55, 1367–1371. Esterhuizen, M., Pflugmacher, S., Downing, T.G., 2011a. β-N-Methylamino-l-alanine (BMAA) uptake by the aquatic macrophyte Ceratophyllum demersum. Ecotoxicol. Environ. Saf. 74, 74–77. Esterhuizen, M., Pflugmacher, S., Downing, T.G., 2011b. The effect of β-N-Methylamino-l-alanine (BMAA) on oxidative stress response enzymes of the macrophyte Ceratophyllum demersum. Toxicon 57, 803–810. Esterbauer, H., Schaur, R.J., Zollner, H., 1991. Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes. Free Radic. Biol. Med. 11, 81–128. Esterbauer, H., Cheeseman, K.H., 1990. Determination of aldehydic lipid peroxidation products: malonaldehyde and 4-hydroxynonenal. Methods Enzymol. 186, 407–421. Fryer, M.J., 1992. The antioxidant effects of thylakoid vitamin-E (alpha tocopherol). Plant Cell Environ. 15, 381–392. Gill, S.S., Tuteja, N., 2010. Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiol. Biochem. 48, 909–930. Gugger, M., Lenoir, S., Berger, C., Ledreux, A., Druart, J.C., Humbert, J.F., Guette, C., Bernard, C., 2005. First report in a river in France of the benthic cyanobacterium Phormidium favosum producing anatoxin-a associated with dog neurotoxicosis. Toxicon 45, 919–928. Glibert, P.M., Anderson, D.A., Gentien, P., Granéli, E., Sellner, K.G., 2005. The global, complex phenomena of harmful algal blooms. Oceanography 18, 136–147. Gobas, F.A.P.C., Morrison, H.A., 2000. Bioconcentration and biomagnification in the aquatic environment. In: Mackay, D., Boethling, R.S. (Eds.), Handbook of Property Estimation Methods for Chemicals. CRC Press, Florida, pp. 189–231.
212
M.-H. Ha et al. / Ecotoxicology and Environmental Safety 101 (2014) 205–212
Gutteridge, J.M., 1995. Lipid peroxidation and antioxidants as biomarkers of tissue damage. Clin. Chem. 41, 1819–1828. Ha, M.H., Pflugmacher, S., 2013a. Phytotoxic effects of the cyanobacterial neurotoxin anatoxin-a: morphological, physiological and biochemical responses in aquatic macrophyte, Ceratophyllum demersum. Toxicon 70, 1–8. Ha, M.H., Pflugmacher, S., 2013b. Time-dependent alterations in growth, photosynthetic pigments and enzymatic defense systems of submerged Ceratophyllum demersum during exposure to the cyanobacterial neurotoxin anatoxin-a. Aquat. Toxicol. 138–139, 26–34. Hart, B.T., Maher, W., Lawrence, I., 1999. New generation water quality guidelines for ecosystem protection. Freshw. Biol. 41, 347–359. James, K.J., Furey, A., Sherlock, I.R., Stack, M.A., Twohig, M., Caudwell, F.B., Skulberg, O.M., 1998. Sensitive determination of anatoxin-a, homoanatoxin-a and their degradation products by liquid chromatography with fluorimetric detection. J. Chromatogr. A 798, 147–157. Krienitz, L., Ballot, A., Kotut, K., Wiegand, C., Pütz, S., Metcalf, J.S., Codd, G.A., Pflugmacher, S., 2003. Contribution of hot spring cyanobacteria to the mysterious deaths of Lesser Flamingos at Lake Bogoria, Kenya. FEMS Microbiol. Ecol. 43, 141–148. Mitrovic, S.M., Allis, O., Furey, A., James, K.J., 2005. Bioaccumulation and harmful effects of microcystin-LR in the aquatic plants Lemna minor and Wolffia arrhiza and the filamentous alga Chladophora fracta. Ecotoxicol. Environ. Saf. 61, 345–352. Mitrovic, S.M., Pflugmacher, S., James, K.J., Furey, A., 2004. Anatoxin-a elicits an increase in peroxidase and glutathione S-transferase activity in aquatic plants. Aquat. Toxicol. 68, 185–192. Munné-Bosch, S., Alegre, L., 2002. The function of tocopherols and tocotrienols in plants. Crit. Rev. Plant Sci. 21, 31–57. Mittler, R., 2002. Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci. 7, 405–410. Nimptsch, J., Wiegand, C., Pflugmacher, S., 2008. Cyanobacterial toxin elimination via bioaccumulation of MC-LR in aquatic macrophytes: an application of the green liver concept. Environ. Sci. Technol. 42, 8552–8557. Osswald, J., Azevedo, J., Vasconcelos, V., Guilhermino, L., 2011. Experimental determination of the bioconcentration factors for anatoxin-a in juvenile rainbow trout (Oncorhynchus mykiss). Proc. Int. Acad. Ecol. Environ. Sci. 1, 77–86. Osswald, J., Réllan, S., Gago, A., Vasconcelos, V., 2008. Uptake and depuration of anatoxin-a by the mussel Mytilus galloprovincialis (Lamarck, 1819) under laboratory conditions. Chemosphere 72, 1235–1241. Osswald, J., Rellán, S., Carvalho, A.P., Gago, A., Vasconcelos, V., 2007. Acute effects of an anatoxin-a producing cyanobacterium on juvenile fish Cyprinus carpio L. Toxicon 49, 693–698. Peuthert, A., Pflugmacher, S., 2010. Influence of cyanotoxin microcystin-LR on tocopherol in Alfalfa seedlings (Medicago sativa). Toxicon 56, 411–417. Paerl, H.W., Huisman, J., 2009. Climate change: a Catalyst for global expansion of harmful cyanobacterial blooms. Environ. Microbiol. Rep. 1, 27–37. Pflugmacher, S., Aulhorn, M., Grimm, B., 2007. Influence of a cyanobacterial crude extract containing microcystin-LR on the physiology and antioxidative defence systems of different spinach variants. New Phytol. 175, 482–489. Pflugmacher, S., 2004. Promotion of oxidative stress in C. demersum due to exposure to cyanobacterial toxin. Aquat. Toxicol. 3, 169–178.
Pflugmacher, S., Wiegand, C., Beattie, K.A., Krause, E., Steinberg, C.E.W., Codd, G.A., 2001. Uptake, effects, and metabolism of cyanobacterial toxins in the emergent reed plant Phragmites australia (cav.). Environ. Toxicol. Chem. 20, 846–852. Paerl, H.W., 2000. Marine plankton. In: Whitton, B.A., Potts, M. (Eds.), The Ecology of Cyanobacteria. Kluwer Academic Pub., Dordrecht, Netherlands, pp. 121–148. Pitois, S., Jackson, M.H., Wood, B.J.B., 2000. Problems associated with the presence of cyanobacteria in recreational and drinking waters. Int. J. Environ. Health Res. 10, 203–218. Pflugmacher, S., Codd, G.A., Steinberg, C.E.W., 1999. Effects of the cyanobacterial toxin microcystin-LR on the detoxication enzymes in aquatic plants. Environ. Toxicol. 14, 62–66. Park, H.D., Kim, B., Kim, E., Okino, T., 1998. Hepatotoxins microcystins and neurotoxic anatoxin-a in cyanobacterial blooms form Korean lakes. Environ. Toxicol. Water Qual. 13, 225–234. Pflugmacher, S., Wiegand, C., Oberemm, A., Beattie, K.A., Krause, E., Codd, G.A., Steinberg, C.E.W., 1998. Identification of an enzymatically formed glutathione conjugate of the cyanobacterial hepatoxin microcystin-LR: the first step of detoxication. Biochem. Biophys. Acta (BBA): Gen. Subj. 1425, 527–533. Rellán, S., Osswald, J., Vasconcelos, V., Gago-Martinez, A., 2007. Analysis of anatoxin-a in biological samples using liquid chromatography with fluorescence detection after solid phase extraction and solid phase microextraction. J. Chromatogr. A 1156, 134–140. Sächsisches Staatsministerium für Soziales, 2009. Cyantoxine in sächsischen Badegewässern. Zentraler Broschürenversand der Sächsischen Staatsregierung, Dresden. Saqrane, S., Ghazali, I.E., Ouahid, Y., Hassni, M.E., Hadrami, I.E., Bouarab, L., del Campo, F.F., Oudra, B., Vasconcelos, V., 2007. Phytotoxic effects of cyanobacterial extract on the aquatic plant Lemna gibba: microsystin accumulation, detoxication and oxidative stress induction. Aquat. Toxicol. 83, 284–294. Stüven, J., Pflugmacher., S., 2007. Antioxidative stress response of Lepidium sativum due to exposure to cyanobacterial secondary metabolites. Toxicon 50, 85–93. Smith, V.H., 2003. Eutrophication of freshwater and coastal marine ecosystems: a global problem. Environ. Sci. Pollut. Res. 10, 126–139. Stockner, J.G., 1999. Phototrophic picoplankton: an overview from marine and freshwater ecosystems. Limnol. Oceanogr. 33, 765–775. Sies, H., 1997. Oxidative stress: oxidant and antioxidant. Exp. Physiol. 82, 291–295. Smith, C., Sutton, A., 1993. The Persistence of Anatoxin-a in Reservoir Water, Report no. FR0427. Foundation for Water Research, UK. Stevens, D.K., Krieger, R.I., 1991. Stability studies on the cyanobacterial nicotinic alkaloid anatoxin-a. Toxicon 29, 167–179. Stevens, D.K., Krieger, R.I., 1988. Analysis of anatoxin-a by GC/ECD. J. Anal. Toxicol. 12, 126–131. Soll, J., Schultz, G., Joyard, J., Douce, R., Block, M.A., 1985. Localization and synthesis of prenylquinones in isolated outer and inner envelope membranes from spinach chloroplasts. Arch. Biochem. Biophys. 238, 290–299. Takamura, N., Kadono, Y., Fukushima, M., Nakagawa, M., Kim, Baik-H.O., 2003. Effects of aquatic macrophytes on water quality and phytoplankton communities in shallow lakes. Ecol. Res. 18, 381–395. Thomas, P., Stephens, M., Wilkie, G., Amar, G., Lunt, G., Whiting, T., Gallagher, E., Pereira, E., Alkondon, M., Albuquerque, X., Wonnacott, S., 1993. ( þ)-Anatoxin-a is a potent agonist at neuronal nicotinic acetylcholine receptors. J. Neurochem. 60, 2308–2311.
Contents lists available at ScienceDirect
Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Uptake of the cyanobacterial neurotoxin, anatoxin-a, and alterations in oxidative stress in the submerged aquatic plant Ceratophyllum demersum Mi-Hee Ha, Valeska Contardo-Jara, Stephan Pflugmacher n Technische Universität Berlin, Department of Ecotoxicological Impact Research and Ecotoxicology, Ernst-Reuter-Platz 1, 10587 Berlin, Germany
art ic l e i nf o
a b s t r a c t
Article history: Received 10 July 2013 Received in revised form 22 December 2013 Accepted 24 December 2013
The prevalence of cyanobacterial blooms in fresh water bodies worldwide has become a serious environmental problem. The blooms can increase the occurrence of cyanobacterial neurotoxin, anatoxin-a, and this toxin can interact with aquatic plants and other pivotal components of aquatic ecosystems. Despite this, several questions regarding the uptake of the toxin by aquatic plants and its association with toxic effects still remain. This study investigated the uptake of anatoxin-a in relation to alterations in oxidative stress, estimated by changes in lipid peroxidation and tocopherol contents (alpha-, beta-, gamma-, and delta-tocopherol), in the submerged aquatic plant, Ceratophylum demersum, at environmentally relevant concentrations. Exposure to five different concentrations of anatoxin-a (0.005, 0.05, 0.5, 5 and 50 μg l 1) for 24 h increased concentrations in C. demersum in a dose-dependent manner. All four forms of tocopherols were elevated at low concentrations of anatoxin-a (0.005. 0.05. 0.5 and 5 μg l 1). However, a decline in the four tocopherol forms along with a high level of lipid peroxidation was observed at 50 μg l 1 exposure dose. During 336-h exposure to 15 μg l 1 anatoxin-a, rapid toxin uptake during the first 24 h and subsequent steady accumulation of the toxin were observed. The four tocopherol forms increased in response to anatoxin-a uptake, attaining their maximum levels together with a significant increase of lipid peroxidation after 12 or 24 h. After 24-h exposure, the four tocopherol forms decreased gradually without recovery. The results clearly indicate that anatoxin-a uptake can cause a disturbance of the oxidative stress in the aquatic plant, and depending on the concentration and exposure duration, oxidative damage occurs. & 2014 Elsevier Inc. All rights reserved.
Keywords: Anatoxin-a Ceratophyllum demersum Uptake Oxidative stress status Lipid peroxidation Tocopherol
1. Introduction Cyanobacteria encompassing diverse photosynthetic prokaryotic micro-organisms exist in balanced aquatic environments at the proper level at which their frequency and abundance are naturally controlled (Paerl, 2000; Stockner, 1999). However, accelerated eutrophication and climate change facilitate them to excessively proliferate to the extent that they provoke a disturbance (e.g. high algal turbidity and oxygen depletion) in aquatic ecosystems and deteriorate water quality (e.g. offensive odor and taste) related to human well-being (Hart et al., 1999; Paerl and Huisman, 2009; Pitois et al., 2000; Smith, 2003). Furthermore, many of cyanobaterial genera such as Anabaena, Aphanizomenon, Cylindrospermopsis, Microcystis and Planktothrix are known to produce a wide range of toxic bioactive secondary metabolites designated as cyanotoxins. n
Corresponding author. Fax: þ 49 30 314 29022. E-mail addresses: [email protected] (M.-H. Ha), [email protected] (V. Contardo-Jara), stephan.pfl[email protected] (S. Pflugmacher). 0147-6513/$ - see front matter & 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ecoenv.2013.12.023
These toxins are principally released into the surrounding water during their senescence and decomposition (Carmichael, 1997; Codd, 1995). Anatoxin-a is one of the most potent cyanobacterial neurotoxins commonly detected all over the world (Ballot et al., 2004; Gugger et al., 2005; Park et al., 1998; Stevens and Krieger, 1988). It is a powerful cholinergic agonist competing with acetylcholine for nicotinic acetylcholine receptor in neuronal signal transmission (Gugger et al., 2005; Krienitz et al., 2003; Park et al., 1998; Thomas et al., 1993). The toxin is chemically defined as a lowmolecular-weight semi-rigid alkaloid (MW¼ 165.26), labile under natural environmental conditions, especially sunlight and pH. Thus, anatoxin-a is reported to be easily degradable into two non-toxic compounds (i.e. dehydroanatoxin-a and epoxyanatoxin-a) and to have a half-life of several hours or days in strong sunlight and at high pH (Devlin et al., 1977; Smith and Sutton., 1993; Stevens and Krieger, 1991). Due to the distinctive chemical characteristics, low stability and persistence, as well as the notorious rapid neurotoxic mode of action, fatal to mammals, relatively little attention has been paid so far to the fate and impact of anatoxin-a in aquatic environments. However, considering the prevalence of the massive
206
M.-H. Ha et al. / Ecotoxicology and Environmental Safety 101 (2014) 205–212
growth of cyanobacteria leading to increased presence and amount of cyanotoxins, knowledge about anatoxin-a uptake, metabolism and the resulting effects in aquatic ecosystems containing various living organisms (e.g. plant, zooplankton, mollusk and fish) needs to be improved (Carmichael, 2008; Glibert et al., 2005). Very few studies investigated the bioconcentration and depuration of anatoxin-a in particular aquatic organisms, mussel and fish, associated with the risk of human intoxication from anatoxin-a-contaminated dietary source (Osswald et al., 2011, 2008, 2007). Plants are generally regarded as major players in the transport and degradation of many well-known contaminants in aquatic environments through their uptake and metabolic detoxification (Dhir et al., 2009; Takamura et al., 2003). Moreover, they are also involved in the transfer of pollutants via bioaccumulation along the aquatic food chain as primary producers offering food to other higher organisms (Gobas and Morrison, 2000). Indeed, there have been several reports presenting that the powerful cyanobacterial hepatotoxins microcystins (MCs) can be taken up, metabolized and eventually accumulated to a certain degree in aquatic plants such as C. demersum, Lemna gibba and Phragmites australia (Pflugmacher et al., 2004, 2001; Saqrane et al., 2007). The publication from Esterhuizen et al. (2011a) also showed that the cyanobacterial neurotoxin β-methylamino-l-alanine (BMAA) can be transferred from water media into C. demersum and can possibly give rise to biomagnification through protein association following accumulation of the toxin. Simultaneously, phytotoxic effects (e.g. reduced photosynthetic pigment contents and altered enzymatic systems) have been observed and linked to uptake of these cyanotoxins (Esterhuizen et al., 2011b; Pflugmacher, 2004; Saqrane et al., 2007). Currently, there is some evidence indicating that anatoxin-a can also affect multiple levels of biological metabolism (e.g. growth, photosynthesis and enzymatic systems) of aquatic macrophytes, Lemna minor (L. minor) and C. demersum by inducing oxidative stress (Ha and Pflugmacher, 2013a, 2013b; Mitrovic et al., 2004). Previous studies demonstrated that the activity of antioxidative enzymes including superoxide dismutase (SOD), peroxidase (POD), ascorbate peroxidase (APX), monodehydroascorbate reductase (MDAR) and glutathione reductase (GR) was changed in response to the elevated formation of hydrogen peroxide (H2O2) in C. demersum exposed to anatoxin-a (Ha and Pflugmacher, 2013a, 2013b). Therefore, the present study aimed to investigate the uptake of anatoxin-a in relation to alterations of the oxidative stress status in C. demersum, a world-wide distributed submerged aquatic plant. Oxidative stress refers to an imbalance between pro-oxidative reactions (e.g. generating reactive oxygen species (ROS)) and antioxidative defense (e.g. quenching ROS). Oxidative stress can lead to severe damage to all types of cellular molecules, DNA, proteins and lipids, depending on the degree of imbalance (Sies, 1997). Lipid peroxidation is described as the oxidative deterioration of polyunsaturated fatty acids (PUFAs), crucial components of cellular lipid membranes, and has been largely studied in the assessment of oxidative cell injury (Esterbauer et al., 1991; Gutteridge, 1995). It is well-established that plants possess effective antioxidative defense systems comprising various enzymes and non-enzymatic free-radical scavengers antioxidants like ascorbate (vitamin C), carotenoids, glutathione (GSH), and tocopherols (vitamin E) to mitigate various conditions of oxidative stress (e.g. controlling ROS production and relieving the resulting damage) (Gill and Tuteja, 2010; Mittler, 2002). Tocopherols, synthesized exclusively by plants and certain photosynthetic organisms, are believed to play important roles in the stabilization of lipid membranes by decomposing ROS and lipid peroxy radicals during the propagation of lipid peroxidation (Fryer, 1992; Munné-Bosch and Alegre, 2002). They exist in four different chemical forms, α-, β-, γ-, and δ-tocopherol, named according to the number and position of methyl groups on the chromanol ring. α-Tocopherol is known as the major form detected in green parts of plants.
Accordingly, in this study lipid peroxidation and the corresponding antioxidant, four tocopherols (α-, β-, γ-, and δ-tocopherol), were used as indicators of changes in oxidative stress status in response to anatoxin-a uptake in C. demersum.
2. Material and methods 2.1. Plant material C. demersum, obtained from Extraplant (Extragroup GmbH, Münster, Germany), was cultivated non-axenically in a glass tank (60 cm 60 cm 60 cm) filled with modified Provasoli0 s medium containing de-ionized water, 0.2 g l 1 CaCl2, 0.103 g l 1 NaHCO3 and 0.1 g l 1 sea-salt. The culture was conducted under cool white fluorescent light (35 μE m 2 s 1 irradiance) with a day/night cycle of 14/ 10 h at 22–24 1C.
2.2. Experimental design ( 7 ) Anatoxin-a fumarate was provided by Tocris Bioscience (Strathmore Road Natick, MA, USA). The toxin was dissolved to 1 g l 1 in 70 percent (v/v) methanol and further diluted with culture medium to the final exposure concentrations. The environmentally feasible concentrations of anatoxin-a were derived from monitoring studies conducted in German fresh water bodies (Bumke-Vogt et al., 1999; Sächsisches Staatsministerium für Soziales, 2009). The plant, comprising of shoot apical meristem, main stem and leaves, was cut one week before the experiments to avoid any physical stresses resulting from cutting. After this preculture for one week, 4.5 7 0.5 g fresh weight (FW) of plant material was exposed to anatoxin-a in a volume of 100 ml under the above mentioned light and temperature conditions for plant culture. Two different static exposure systems were set to investigate concentrationand time-dependent anatoxin-a-uptake in connection with subsequent changes in the levels of lipid peroxidation and tocopherols in C. demersum. Controls consisting of the plant and culture medium without anatoxin-a were prepared and sampled in parallel with the toxin treatment in order to consider the influence of the exposure conditions on the plant. Three independent replicate experiments were performed for each exposure system. In the first exposure system, plants were treated with 0.005, 0.05, 0.5, 5 and 50 mg l 1 anatoxin-a for 24 h. In the second experiment, plants were exposed to 15 mg l 1 anatoxin-a reflecting the highest concentration of the toxin in the environment for 4, 8, 12, 24, 48, 168 and 336 h. Additionally, the pre-cultured plants that were not introduced into the exposure systems were directly used as 0 h controls. Exposure medium was daily refilled with culture medium without anatoxin-a to the initial volume to avoid changes in anatoxin-a concentration due to evaporation. After exposure, plants were washed twice with de-ionized water to remove any surface-associated toxin and snap-frozen in liquid nitrogen. The frozen plant material was stored at 80 1C until toxin, tocopherol and LPO analysis, respectively. In addition to plant sampling, the exposure medium containing anatoxin-a in the time-dependent exposure system was sampled at each exposure time to determine the remaining toxin in the medium over the period of exposure.
2.3. Anatoxin-a analysis 2.3.1. Extraction of anatoxin-a Anatoxin-a was extracted from plant tissue according to Rellán et al. (2007) with slight modifications. The frozen plant material was ground in liquid nitrogen to a fine powder. Anatoxin-a was extracted by mixing 0.2 g of pulverized plant tissue in 1 ml of acidified 70 percent methanol (MeOH) containing 0.1 percent trifluoroacetic acid (TFA). Tissue samples were continuously shaken in the dark for 3 h at room temperature using overhead shaker, Intelli-Mixer RM-2 (Neolab, Heidelberg, Germany). Extracts were then centrifuged at 10,600g for 10 min at 4 1C and the supernatant was adjusted to pH 7.0 7 0.2 with 1 M sodium hydroxide (NaOH) before solid-phase extraction (SPE) using a weak cation-exchange material in a vacuum manifold processor (SPE-12G, JT Baker, Großgerau, Germany). SPE columns, Strata WCX, 3 ml with 200 mg silica gel-based packing (Phenomenex, CA, USA), were conditioned with equal volumes of MeOH and water. Sample loading and elution with 100 percent MeOH containing 0.2 percent TFA were carried out under gravity. Afterwards, eluates were completely dried under constant nitrogen flow. The final residue was reconstituted in 200 μl of 70 percent MeOH and anatoxin-a content was immediately determined by LC–MS/MS. Water samples were centrifuged at 5000g for 15 min at 4 1C and transparent water was settled at pH 7.0 7 0.2 with 1 M NaOH. The remaining anatoxin-a in the exposure media was extracted and concentrated with Strata WCX SPE columns using the same procedure as described above. Solid phase-extracted water samples were immediately processed for the toxin analysis via LC–MS/MS.
M.-H. Ha et al. / Ecotoxicology and Environmental Safety 101 (2014) 205–212
207
absorbance of the clear supernatant was determined at 586 nm. All measurements were made in triplicate for the three independent replicates of each exposure concentration or sampling event, respectively. A standard calibration curve of MDA was previously generated to calculate the amount of MDAþ4-HNE. 2.5. Tocopherol analysis 2.5.1. Extraction of tocopherols The extraction of tocopherols was carried out according to Stüven and Pflugmacher (2007). The frozen plant tissue was pulverized in liquid nitrogen. The tissue powder (0.2 g) was suspended in 3.2 ml of chilled 100 percent MeOH and constantly shaken in the dark for 10 min using an overhead shaker. The extract was centrifuged at 10,600g for 10 min at 4 1C to exclude plant debris. The resulting supernatant was diluted to 85 percent MeOH with de-ionized water, and then directly applied to the LC–MS/MS analysis of tocopherols. 2.5.2. Determination of tocopherols Four tocopherols (α-, β-, γ-, and δ-tocopherol) were individually quantified by LC–MS/MS. Pure α-, β-, γ-, and δ-tocopherol were purchased from Sigma-Aldrich and dissolved in 85 percent MeOH to establish reference standard curves. The four tocopherols forms in the samples were separated on the Kinetex PFP column (150 2.1 mm2, 2.6 μm pore size, Phenomenex) within 18 min using isocratic chromatographic run with a mixture of MeOH:H2O þ0.4 percent Formic acid (85:15, v/v) at a flow rate of 0.3 ml min 1. Column oven temperature was set to 20 1C and the injection volume was 20 μl. The retention time of α-, β-, γ-, and δ-tocopherol was 15.8, 12.8, 13.2 and 8.9 min, respectively. The mass spectra of the tocopherols were generated in the positive ion mode with the collision energy of 25 V using ESI. The mass-to-charge (m/z) ratio of each tocopherol was as follows: m/z 431-219/205/165 (α-tocopherol); m/z 417-205/191/151 (β- and γtocopherol); m/z 403-191/177/151/137 (δ-tocopherol). The ESI was operated under the following conditions: capillary voltage, 5.0 kV; cone voltage, 30 V; source temperature, 120 1C; desolvation temperature, 350 1C; cone gas flow rate, 50 l h 1; desolvation gas flow rate, 500 l h 1. 2.6. Statistical analysis
Fig. 1. HPLC chromatograms obtained using HILIC column coupled with ESI-MS/ MS. (A) Anatoxin-a standard (50 μg l 1) and (B) C. demersum extract exposed to 50 μg l 1 anatoxin-a for 24 h. 2.3.2. Determination of anatoxin-a Anatoxin-a analysis was carried out by LC–MS/MS (Alliance 2695 UHPLC) combined with a Quattro micro™ triple quadrupole mass spectrometer (Waters, Milford, MA, USA). Chromatographic separation was acquired under HILIC conditions employing the Kinetex HILIC column (100 2.1 mm2, 2.6 μm pore size, Phenomenex) with isocratic operation. The mobile phase consisted of 100 percent acetonitrile, water and 50 mM ammonium formate containing 0.32 percent formic acid (75: 22.5: 2.5, v/v) at a flow rate of 0.2 ml min 1. Column oven temperature was set to 30 1C and the injection volume was 2 μl. The clear and sharp elution peak of anatoxin-a was given at a retention time of 3.68 min (Fig. 1). Mass spectral data was achieved in the positive ion mode with the collision energy of 15 V using electrospray ionization (ESI). The parent compound and its fragment ions were scanned at following mass-to-charge (m/z) ratios: m/z 166-149/131/91. Operating parameters of ESI were set as follows: capillary voltage, 5.0 kV; cone voltage, 35 V; source temperature, 100 1C; desolvation temperature, 450 1C; cone gas flow rate, 35 l h 1; desolvation gas flow rate, 500 l h 1. The toxin was quantified by comparison with 6-point calibration curve with reference standard solutions using ( 7 ) anatoxin-a fumerate in 70 percent MeOH.
2.4. Lipid peroxidation Lipid peroxidation products, malondialdehyde (MDA) and 4-hydroxy-2-nonenal (4-HNE) in the plant tissue were measured according to Esterbauer and Cheeseman (1990) using colorimetric microplate assay kits (Oxford Biomedical Research, MI, USA). The assay is based on the irreversible reaction of the respective aldehydes in the two compounds with 1-methyl-2-phenylindole (MPI) in the presence of methanesulfonic acid (MSA) and ferric iron. The resulting stable chromophore yields maximal absorbance at 586 nm. Plant tissue was ground to a fine powder in liquid nitrogen using mortar and pestle. 0.1 g of plant tissue was then repeatedly homogenized in 1.2 ml of 20 mM sodium phosphate buffer (pH 7.0) with mechanical TissueLyser LT (Quiagen, Hilden, Germany) for effective cell lysis. Prior to homogenization, 12 μl of 0.5 M butylated hydroxyltoluene (BHT) was added to prevent sample oxidation. The precipitate and plant debris were removed by centrifugation at 10,600g for 10 min at 4 1C. 140 μl of the supernatant was mixed with 455 μl of MPI in acetonitrile. Then, 105 μl of MSA was added, mixed gently and incubated at 45 1C for 60 min. The reaction mixture was centrifuged at 15,000g for 10 min and the
One-way analysis of variance (ANOVA) was performed on the data obtained from the experiments on the concentration-dependent effects of anatoxin-a, followed by Turkey0 s HSD test. Two-way analysis of variance (ANOVA), with toxin treatment as one factor and exposure duration as the other factor, was carried out on data generated from experiments on the time-dependent effects of anatoxin-a, followed by Tukey0 s HSD test. Prior to ANOVA, all data were tested for normality and homogeneity of variance using Shapiro-Wilks W and Levene0 s test, respectively. All statistical analyses were performed using the commercial software SPSS 17.0 (SPSS Inc., Chicago, IL, USA) with a p-value o 0.05 considered as significant.
3. Results 3.1. Concentration-dependent responses of C. demersum to anatoxin-a After exposure for 24 h, anatoxin-a was detectable in plants treated with 0.5, 5 and 50 μg l 1 anatoxin-a (Fig. 2A). No anatoxin-a was detected in plants treated with the lower concentrations (0.005 and 0.05 μg l 1). The measured anatoxin-a in the plants at an exposure concentration of 0.5 μg l 1 amounted to 0.06570.017 ng gFW 1. The level of anatoxin-a in the plants exhibited a concentration-dependent increase up to the highest exposure concentration with average values of 15.8470.699 ng gFW 1 anatoxin-a. The detected amounts of anatoxin-a in the plants after 24-h exposure presented a linear relationship with the applied exposure concentrations (correlation coefficient R2 ¼ 0.979). Plants exposed to 50 μg l 1 anatoxin-a showed a highly significant increase in the formation of the two major metabolites of lipid peroxidation, MDA and 4-HNE, after 24 h (po0.005) (Fig. 2B). Significant enhancement of α-tocopherol content was detected in plants exposed to 0.005, 0.05, and 0.5 μg l 1 anatoxin-a after 24 h (p o0.05). The strongest increase reported in plants exposed to 0.05 μg l 1 anatoxin-a was 1.314-fold of the control (po 0.005) (Fig. 3A). β-Tocopherol content was significantly increased in plants exposed to 0.005, 0.05, 0.5 and 5 μg l 1 anatoxin-a (p o0.05) (Fig. 3B). The maximum elevation of β-tocopherol, being
208
M.-H. Ha et al. / Ecotoxicology and Environmental Safety 101 (2014) 205–212
factor (BCF), which was estimated by dividing anatoxin-a in the plant (μg μg FW 1) by anatoxin-a in the corresponding water media (μg ml 1) after 24-h exposure to 15 μg l 1, was 51.01 The plants exposed to 15 μg l 1 anatoxin-a for 336 h showed a significant production of lipid peroxidation metabolites, MDA and 4-HNE, after 12 and 24 h (po0.05) (Fig. 4B). After longer exposure, the amount of lipid peroxidation metabolites decreased to control levels. α-Tocopherol content was significantly increased in exposed plants within the initial period of exposure (4, 8 and 12 h) (po0.05), decreasing thereafter to control levels until 48 h of exposure (Fig. 5A). A significant reduction in α-tocopherol content in exposed plants compared to the control of the same sampling event was then detected after 168 and 336 h exposure. βTocopherol was immediately and significantly raised to 2.3-fold of the control at 24-h exposure (po0.05) (Fig. 5B). After 48 h, βtocopherol content was reduced and returned to control values until the end of the experiment. γ-Tocopherol content was significantly increased after 8 h (po0.05) (Fig. 5C). The maximum increase in γ-tocopherol content, which was 3.7-fold of the control, was detected after 12 h. After 24 h, the increase in γ-tocopherol content was still significant. Longer exposure led the increased γtocopherol content to a return to control levels. A significant elevation of δ-tocopherol content was observed after 4 h, reaching its maximum level after 12 h, which was 1.7-fold higher than that of the control (po0.05) (Fig. 5D). As observed for β- and γ-tocopherol, δ-tocopherol content also decreased to control levels after longer exposure.
4. Discussion 4.1. Concentration-dependent responses of C. demersum to anatoxin-a
Fig. 2. (A) Uptake of anatoxin-a and (B) levels of lipid peroxidation products (MDA þ 4-HNE) in C. demersum after 24-h exposure to different concentrations of anatoxin-a. All values are given as mean 7standard deviation (n¼ 3, nnhighly significantly different from control, p o 0.005).
0.6 times higher than that of the control, was noticed at an exposure concentration of 5 μg l 1 (po0.005). For γ-tocopherol content, no significant differences were observed between the toxin treatments and the control except for the exposure with 5 μg l 1 anatoxin-a (p o0.05) (Fig. 3C). A significant increase in δ-tocopherol was detected in plants exposed to 0.005 μg l 1 anatoxin-a (p o0.05) (Fig. 3D). The maximum level of δtocopherol amounting 1.6-fold of the control was observed at an exposure concentration of 5 μg l 1 (po 0.005). Both, γ- and δtocopherol content, displayed slightly lower values in plants exposed to 50 μg l 1 anatoxin-a compared to control plants. 3.2. Time-dependent responses of C. demersum to anatoxin-a Anatoxin-a uptake occurred in C. demersum exposed to 15 μg l 1 anatoxin-a within 4 h, increasing rapidly until 24 h (Fig. 4A). The highest level of anatoxin-a detected in exposed plants after 24 h was 7.0570.64 ng gFW 1. Within 48 h, the toxin amount slightly decreased by around 5 percent followed by a remarkable reduction of the toxin amount until 168 h. The concentration of dissolved anatoxin-a, initially set at a level of 15 μg l 1, drastically decreased to 3.96 μg l 1 after 4 h. Anatoxin-a concentration in the media was further reduced to 0.14 μg l 1 within 24 h. Consequently, almost the entire toxin (99 percent), given at the beginning of the experiment, was removed from water media within 24 h. The bioconcentration
Anatoxin-a started being detected in C. demersum exposed to 0.5 μg l 1 after a 24-h exposure period. The toxin amount in plants increased proportionally to the exposure concentrations up to 50 μg l 1 with a good linear correlation. The observation suggests that saturation of anatoxin-a uptake was not reached in the plant within the tested concentration range and that uptake could further continue at higher concentrations above 50 μg l 1. Although the amount of anatoxin-a detected in C. demersum was in ng g FW 1 range, it was about ten times higher than that of BMAA previously measured in the same plant under similar experimental conditions (Esterhuizen et al., 2011a). This finding implies that aquatic plants may display different uptake rates or metabolization efficiencies for each cyanotoxin. Anatoxin-a seems to be more easily taken up or to be more slowly metabolized by aquatic plants compared to BMAA. Tocopherols are synthesized from homogentistic acid and isopentenyl diphosphate in the envelope membrane of chloroplasts (Soll et al., 1985). They are able to scavenge not only ROS but also lipid peroxyl radical resulting from a reaction between molecular oxygen and the highly reactive carbon-centered lipid radical derived from PUFAs which lost their hydrogen atom by ROS. Different tocopherol forms, α-, β-, γ-, and δ-tocopherol, hamper thereby the propagation of lipid peroxidation ending in the production of MDA and 4-HNE in membrane and thus, contribute to maintaining membrane integrity (Munné-Bosch and Alegre, 2002). In spite of their noticeable activities in antioxidative defense systems, there are few reports investigating tocopherol contents as indicators of oxidative stress in aquatic plants. To the best of our knowledge, this study showed for the first time, changes in tocopherol profiles in connection with the induction of lipid peroxidation in an aquatic plant exposed to a cyanotoxin. The four tocopherol forms increased within the
M.-H. Ha et al. / Ecotoxicology and Environmental Safety 101 (2014) 205–212
209
Fig. 3. Changes in (A) α-, (B) β-, (C) γ-, and (D) δ-tocopherol contents of C. demersum after 24-h exposure to different concentrations of anatoxin-a. All values are given as mean 7 standard deviation (n¼ 3, nsignificantly different from control, p o 0.05; nnhighly significantly different from control, p o 0.005).
anatoxin-a concentration range of 0.005–5 μg l 1 with different activation patterns; however, plants exposed to the highest anatoxin-a concentration, 50 μg l 1, achieved similar or lower tocopherol levels than those observed in control plant. The altered tocopherol contents clearly indicated the promotion of oxidative stress in the plant due to anatoxin-a exposure. The enhancement of α-tocopherol content was mainly presented at lower concentrations, 0.005. 0.05 and 0.5 μg l 1 at which no or only low amounts of the toxin were detected in the plants. In contrast, β-, γ-, and δ-tocopherol exhibited their maximum elevation in plants exposed to 5 μg l 1 anatoxin-a. These results suggest that on one hand α-tocopherol seems to be the most sensitive and vulnerable to anatoxin-a uptake and that on the other hand the four tocopherols cooperate among one another to maintain a constant antioxidant function under different levels of anatoxin-a-induced oxidative stress. Peuthert and Pflugmacher (2010) showed that α- and β-tocopherol contents increased in the terrestrial plant Medicago savita seedling exposed to cyanobacterial crude extract containing microcystin-LR (MC-LR) for 72 h, and the strongest enhancement of each tocopherol form was observed with different concentrations of the toxin. α-Tocopherol content reached its maximum level in seedlings exposed to the lower MC-LR concentration (0.05 μg l 1), whereas β-tocopherol content displayed its strongest increase in seedlings exposed to 0.5 μg l 1. Furthermore, an increase in α- and γ-tocopherol contents was also observed in different spinach (Spinacia oleracea) variants after exposure to cyanobacterial crude extract containing 0.5 μg l 1 of MC-LR for six weeks (Pflugmacher et al., 2007). The tendency of changes in tocopherol contents of plants could provide insight into the activation of the antioxidative defense systems and its capacity limits in coping with oxidative stress caused by anatoxin-a. The
suppressed tocopherol contents at 50 mg l 1 anatoxin-a indicate that the capacity of tocopherols to act as antioxidant agents was overwhelmed and possibly furthermore, their synthesis affected. Consistent with the induction pattern of tocopherol contents, a significant amount of lipid peroxidation end products, MDA and 4-HNE, was observed not at the lower applied concentrations but in plants exposed to 50 mg l 1 anatoxin-a. These findings suggest that the elevation of tocopherols seems to be efficient enough to avoid or mitigate lipid peroxidation at low levels of oxidative stress triggered by anatoxin-a. Simultaneously, these result also provide further evidence that the plant have effective defense mechanisms allowing itself to maintain homeostasis to a certain extent under anatoxin-a-contaminated environments (Ha and Pflugmacher., 2013a, 2013b). The decreased tocopherol contents at higher toxin concentrations might be attributed to the destruction of chloroplasts due to the excessive amount of anatoxin-a transferred into the plant and the subsequently intensified oxidative stress associated with ROS overproduction. This is in accordance with the results of a previous study with C. demersum treated with the same concentration of anatoxin-a, evidencing a decrease in chlorophyll contents together with an increase in cell internal hydrogen peroxide formation (Ha and Pflugmacher, 2013a). 4.2. Time-dependent responses of C. demersum to anatoxin-a Anatoxin-a was taken up by C. demersum within the initial period of exposure (4 h) and the toxin amount steadily increased until 24 h. Meanwhile, the concentration of anatoxin-a in water media decreased sharply and almost the entire toxin was eliminated. These observations suggest that active anatoxin-a uptake by aquatic plants takes place within its short half-life. Hence, the uptake and
210
M.-H. Ha et al. / Ecotoxicology and Environmental Safety 101 (2014) 205–212
Fig. 4. (A) Uptake of anatoxin-a by C. demersum in parallel with changes in the remaining toxin in exposure media and (B) levels of lipid peroxidation products (MDA þ 4-HNE) in C. demersum during 336-h exposure to 15 μg l 1 anatoxin-a (ana-a). All values are given as mean7 standard deviation (n ¼3, different letters indicate significant differences at the level of p o0.05).
subsequent metabolization of anatoxin-a by aquatic plants may have considerable influence on the fate of the toxin in aquatic ecosystems. Other related studies demonstrating the rapid uptake of cyanotoxins by aquatic plants, e.g., Nimptsch et al. (2008) investigating the elimination of MC-LR from water media by using bioaccumulation potential of aquatic plants (i.e. L. minor, Myriophyllum elatinoides, Hydrilla verticillata and Ceratopteris thalictroides), could also verify the significant influence of aquatic plants on the fate of cyanotoxins in the ecosystem. Nimptsch et al. (2008) presented that an initial concentration of 2.5 μg l 1 MC-LR in exposure media containing aquatic plants decreased below 0.9–0.2 μg l 1 within only one d and that thereafter toxin removal continued at a lower rate until seven d. Esterhuizen et al. (2011a) also showed intense BMAA removal from exposure media of 100 μg l 1 within 24 h accompanied by a significant BMAA increase in C. demersum itself. All the mentioned results suggest that aquatic plants are able to take up cyanotoxins including anatoxin-a most effectively during the initial period of exposure. It seems that anatoxin-a uptake occurring within the first 24 h of exposure was followed by metabolization and/or elimination of the toxin without further continuous uptake due to reduction of the toxin in water media. Consequently, the maximum amount of anatoxin-a detected in exposed plants after 24 h decreased to a certain degree within the following 336 h of exposure. However, the removal rate of anatoxin-a tended to become slower after 168 h. These changes in the content of anatoxin-a suggest that metabolization and/or elimination processes of the toxin take place in the examined aquatic plant. Several other studies reporting accumulation of MC-LR in aquatic plants (i.e. C. demersum, L. minor and C. fracta) and subsequent metabolization via
GSH conjugation support the presumption that aquatic plants immediately attempt to metabolize and accordingly eliminate the respective toxin (Mitrovic et al., 2005; Pflugmacher et al., 1999, 1998). During the 336-h experiment of the present study using 15 μg l 1 anatoxin-a, the sum of the toxin in both the plant and water media, respectively, was less than the initial absolute toxin amount in water media. This result might be explained by metabolization by the plant itself as well as the natural degradation of the toxin due to its chemical instability (James et al., 1998; Smith and Sutton, 1993; Stevens and Krieger, 1991). However, the high loss in the toxin amount within the first 4 h of exposure might not be attributed to its chemical instability. Hence, further insight into the metabolization of anatoxin-a by aquatic plants is needed to give an exclusive explanation on primary fate of the toxin. All tocopherol forms were rapidly increased in accordance with the transfer of anatoxin-a into the plant within 4 h. However, the duration of enhancement and exposure time, at which the maximum induction took place, varied among the four tocopherol forms. In contrast to the steady increase in β-tocopherol content until 24 h, the strongest increase in α-, γ-, and δ-tocopherol content was reported already after 12 h. Longer exposure for 336 h led to a gradual decrease in the tocopherol contents without recovery. In the case of α-tocopherol, the content even decreased below control values in exposed plants. These results indicate a constant, but mild increase in oxidative stress in response to the continuous uptake of anatoxin-a until 12 h. It seems that after 24 h the overall amount of the toxin transferred exceeds the antioxidative capacity. Among the four tocopherols investigated in this study, β-tocopherol showed the corresponding activation to the increase in the amount of anatoxina in the plant, suggesting β-tocopherol to be more durable than the other tocopherol forms and to play the most important role in alleviating anatoxin-a-induced oxidative stress. Peuthert and Pflugmacher (2010) could demonstrate a similar pattern in M. sativa seedlings during 72-h exposure to cyanobacterial crude extract containing 0.5 μg l 1 MC-LR. β-tocopherol was increased faster than α-tocopherol and then exhibited continuous further elevation. In accordance with the marked decrease in α-, γ-, and δ-tocopherol, C. demersum showed accelerated and less-suppressed lipid peroxidation, indicated by a large amount of MDA and 4-HNE, after 24 h. Although the contents of all tocopherol forms were reduced after 48 h, the amount of MDA and 4-HNE also decreased to a certain level. This result might be explained by the allocation of other antioxidants such as glutathione and carotenoids working as membrane stabilizers just like tocopherols. MDA and 4-HNE can be detoxified via the conjugation to GSH catalyzed by GST. Carotenoids also participate in modifying the structure and property of lipid membranes against oxidative damages. Indeed, a previous study identified that GST activity and the carotenoids-to-total chlorophyll ratio increased slower than the four tocopherol forms in C. demersum, however, remaining elevated until the end of exposure (336 h) to 15 μg l 1 of anatoxin-a (Ha and Pflugmacher, 2013b). Moreover, the carotenoidsto-total chlorophyll ratio content started to be significantly raised after 12 h, and the highest value was reported after 48 h, at which the decrease in the contents of all tocopherols occurred. These findings suggest that the components of the antioxidative system, several involved enzymes and antioxidants, seem to collaborate among themselves by coordinating their complementary functions, having different immediacies of response and durability to anatoxina-induced oxidative stress.
5. Conclusion This is the first study demonstrating rapid uptake and subsequent partial elimination of the cyanobacterial neurotoxin anatoxin-a in an aquatic plant based on the static exposure of whole plants to purified
M.-H. Ha et al. / Ecotoxicology and Environmental Safety 101 (2014) 205–212
211
Fig. 5. Changes in (A) α-, (B) β-, (C) γ-, and (D) δ-tocopherol contents of C. demersum during 336-h exposure to 15 μg l 1 anatoxin-a (ana-a). All values are given as mean 7 standard deviation (n¼ 3, different letters indicate significant differences at the level of po 0.05).
toxin under controlled laboratory conditions. This study evidenced that anatoxin-a uptake and its metabolization can be linked to disturbances of oxidative stress metabolism of aquatic plants and, depending on severity and duration, oxidative damage arises as a consequence. Moreover, the possibility was shown that aquatic plants may become anatoxin-a reservoirs in highly contaminated water bodies, accumulating the toxin and thereby, despite the short halflife of the toxin, augmenting persistence in the environment. To understand the environmental fate and ecological risk of the toxin in aquatic environments, the use of aquatic plants in biomonitoring of anatoxin-a-contaminated waters should be considered. In addition, the potential of aquatic plants to be used for phytoremediation of water resources having moderate pollution of anatoxin-a can be taken into account in sustainable eco-friendly water management processes. Further studies concerning the maximum limits of anatoxin-a uptake by plants and accompanied toxin elimination in connection with plants resistance to oxidative stress imposed by the toxin are required to assess the efficiency of biomonitoring and phytoremediation as well as to suggest the most favorable aquatic plant species.
Acknowledgments This work was supported by the KIST Institutional Program (Project no. 2E24280). References Ballot, A., Krienitz, L., Kotut, K., Wiegand, C., Metcalf, J., Codd, G.A., Pflugmacher, S., 2004. Cyanobacteria and cyanobacterial toxins in three alkaline Rift Valley lakes of Kenya – Lakes Bogoria, Nakuru and Elmenteita. J. Plankton Res. 26, 925–935. Bumke-Vogt, C., Maliahn, W., Chprus, I., 1999. Anatoxin-a and neurotoxic cyanobacteria in German lakes and reservoirs. Environ. Toxicol. 14, 117–125.
Carmichael, W., 2008. A world overview – one-hundred-twenty-seven years of research on toxic cyanobacteria – where do we go from here?. In: Hudnell, H.K. (Ed.), Cyanobacterial Harmful Algal Blooms: State of the Science and Research Needs. Springer, New York, pp. 105–125. Carmichael, W.W., 1997. The cyanotoxins. In: Callow, J.A. (Ed.), Advances in Botanical Research, 27. Academic Press, San Diego, pp. 211–256. Codd, G.A., 1995. Cyanobacterial toxins: occurrence, properties and biological significance. Water Sci. Technol. 32, 149–156. Dhir, B., Sharmila, P., Daradhi, P.P., 2009. Potential of aquatic macrophytes for removing contaminants from the environment. Crit. Rev. Environ. Sci. Technol. 29, 754–781. Devlin, J.P., Edwards, O.E., Gorham, P.R., Hunter, N.R., Pike, R.K., Stavric, B., 1977. Anatoxin-a, a toxic alkaloid from Anabaena flos-aquae NRC-44h. Can. J. Chem. 55, 1367–1371. Esterhuizen, M., Pflugmacher, S., Downing, T.G., 2011a. β-N-Methylamino-l-alanine (BMAA) uptake by the aquatic macrophyte Ceratophyllum demersum. Ecotoxicol. Environ. Saf. 74, 74–77. Esterhuizen, M., Pflugmacher, S., Downing, T.G., 2011b. The effect of β-N-Methylamino-l-alanine (BMAA) on oxidative stress response enzymes of the macrophyte Ceratophyllum demersum. Toxicon 57, 803–810. Esterbauer, H., Schaur, R.J., Zollner, H., 1991. Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes. Free Radic. Biol. Med. 11, 81–128. Esterbauer, H., Cheeseman, K.H., 1990. Determination of aldehydic lipid peroxidation products: malonaldehyde and 4-hydroxynonenal. Methods Enzymol. 186, 407–421. Fryer, M.J., 1992. The antioxidant effects of thylakoid vitamin-E (alpha tocopherol). Plant Cell Environ. 15, 381–392. Gill, S.S., Tuteja, N., 2010. Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiol. Biochem. 48, 909–930. Gugger, M., Lenoir, S., Berger, C., Ledreux, A., Druart, J.C., Humbert, J.F., Guette, C., Bernard, C., 2005. First report in a river in France of the benthic cyanobacterium Phormidium favosum producing anatoxin-a associated with dog neurotoxicosis. Toxicon 45, 919–928. Glibert, P.M., Anderson, D.A., Gentien, P., Granéli, E., Sellner, K.G., 2005. The global, complex phenomena of harmful algal blooms. Oceanography 18, 136–147. Gobas, F.A.P.C., Morrison, H.A., 2000. Bioconcentration and biomagnification in the aquatic environment. In: Mackay, D., Boethling, R.S. (Eds.), Handbook of Property Estimation Methods for Chemicals. CRC Press, Florida, pp. 189–231.
212
M.-H. Ha et al. / Ecotoxicology and Environmental Safety 101 (2014) 205–212
Gutteridge, J.M., 1995. Lipid peroxidation and antioxidants as biomarkers of tissue damage. Clin. Chem. 41, 1819–1828. Ha, M.H., Pflugmacher, S., 2013a. Phytotoxic effects of the cyanobacterial neurotoxin anatoxin-a: morphological, physiological and biochemical responses in aquatic macrophyte, Ceratophyllum demersum. Toxicon 70, 1–8. Ha, M.H., Pflugmacher, S., 2013b. Time-dependent alterations in growth, photosynthetic pigments and enzymatic defense systems of submerged Ceratophyllum demersum during exposure to the cyanobacterial neurotoxin anatoxin-a. Aquat. Toxicol. 138–139, 26–34. Hart, B.T., Maher, W., Lawrence, I., 1999. New generation water quality guidelines for ecosystem protection. Freshw. Biol. 41, 347–359. James, K.J., Furey, A., Sherlock, I.R., Stack, M.A., Twohig, M., Caudwell, F.B., Skulberg, O.M., 1998. Sensitive determination of anatoxin-a, homoanatoxin-a and their degradation products by liquid chromatography with fluorimetric detection. J. Chromatogr. A 798, 147–157. Krienitz, L., Ballot, A., Kotut, K., Wiegand, C., Pütz, S., Metcalf, J.S., Codd, G.A., Pflugmacher, S., 2003. Contribution of hot spring cyanobacteria to the mysterious deaths of Lesser Flamingos at Lake Bogoria, Kenya. FEMS Microbiol. Ecol. 43, 141–148. Mitrovic, S.M., Allis, O., Furey, A., James, K.J., 2005. Bioaccumulation and harmful effects of microcystin-LR in the aquatic plants Lemna minor and Wolffia arrhiza and the filamentous alga Chladophora fracta. Ecotoxicol. Environ. Saf. 61, 345–352. Mitrovic, S.M., Pflugmacher, S., James, K.J., Furey, A., 2004. Anatoxin-a elicits an increase in peroxidase and glutathione S-transferase activity in aquatic plants. Aquat. Toxicol. 68, 185–192. Munné-Bosch, S., Alegre, L., 2002. The function of tocopherols and tocotrienols in plants. Crit. Rev. Plant Sci. 21, 31–57. Mittler, R., 2002. Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci. 7, 405–410. Nimptsch, J., Wiegand, C., Pflugmacher, S., 2008. Cyanobacterial toxin elimination via bioaccumulation of MC-LR in aquatic macrophytes: an application of the green liver concept. Environ. Sci. Technol. 42, 8552–8557. Osswald, J., Azevedo, J., Vasconcelos, V., Guilhermino, L., 2011. Experimental determination of the bioconcentration factors for anatoxin-a in juvenile rainbow trout (Oncorhynchus mykiss). Proc. Int. Acad. Ecol. Environ. Sci. 1, 77–86. Osswald, J., Réllan, S., Gago, A., Vasconcelos, V., 2008. Uptake and depuration of anatoxin-a by the mussel Mytilus galloprovincialis (Lamarck, 1819) under laboratory conditions. Chemosphere 72, 1235–1241. Osswald, J., Rellán, S., Carvalho, A.P., Gago, A., Vasconcelos, V., 2007. Acute effects of an anatoxin-a producing cyanobacterium on juvenile fish Cyprinus carpio L. Toxicon 49, 693–698. Peuthert, A., Pflugmacher, S., 2010. Influence of cyanotoxin microcystin-LR on tocopherol in Alfalfa seedlings (Medicago sativa). Toxicon 56, 411–417. Paerl, H.W., Huisman, J., 2009. Climate change: a Catalyst for global expansion of harmful cyanobacterial blooms. Environ. Microbiol. Rep. 1, 27–37. Pflugmacher, S., Aulhorn, M., Grimm, B., 2007. Influence of a cyanobacterial crude extract containing microcystin-LR on the physiology and antioxidative defence systems of different spinach variants. New Phytol. 175, 482–489. Pflugmacher, S., 2004. Promotion of oxidative stress in C. demersum due to exposure to cyanobacterial toxin. Aquat. Toxicol. 3, 169–178.
Pflugmacher, S., Wiegand, C., Beattie, K.A., Krause, E., Steinberg, C.E.W., Codd, G.A., 2001. Uptake, effects, and metabolism of cyanobacterial toxins in the emergent reed plant Phragmites australia (cav.). Environ. Toxicol. Chem. 20, 846–852. Paerl, H.W., 2000. Marine plankton. In: Whitton, B.A., Potts, M. (Eds.), The Ecology of Cyanobacteria. Kluwer Academic Pub., Dordrecht, Netherlands, pp. 121–148. Pitois, S., Jackson, M.H., Wood, B.J.B., 2000. Problems associated with the presence of cyanobacteria in recreational and drinking waters. Int. J. Environ. Health Res. 10, 203–218. Pflugmacher, S., Codd, G.A., Steinberg, C.E.W., 1999. Effects of the cyanobacterial toxin microcystin-LR on the detoxication enzymes in aquatic plants. Environ. Toxicol. 14, 62–66. Park, H.D., Kim, B., Kim, E., Okino, T., 1998. Hepatotoxins microcystins and neurotoxic anatoxin-a in cyanobacterial blooms form Korean lakes. Environ. Toxicol. Water Qual. 13, 225–234. Pflugmacher, S., Wiegand, C., Oberemm, A., Beattie, K.A., Krause, E., Codd, G.A., Steinberg, C.E.W., 1998. Identification of an enzymatically formed glutathione conjugate of the cyanobacterial hepatoxin microcystin-LR: the first step of detoxication. Biochem. Biophys. Acta (BBA): Gen. Subj. 1425, 527–533. Rellán, S., Osswald, J., Vasconcelos, V., Gago-Martinez, A., 2007. Analysis of anatoxin-a in biological samples using liquid chromatography with fluorescence detection after solid phase extraction and solid phase microextraction. J. Chromatogr. A 1156, 134–140. Sächsisches Staatsministerium für Soziales, 2009. Cyantoxine in sächsischen Badegewässern. Zentraler Broschürenversand der Sächsischen Staatsregierung, Dresden. Saqrane, S., Ghazali, I.E., Ouahid, Y., Hassni, M.E., Hadrami, I.E., Bouarab, L., del Campo, F.F., Oudra, B., Vasconcelos, V., 2007. Phytotoxic effects of cyanobacterial extract on the aquatic plant Lemna gibba: microsystin accumulation, detoxication and oxidative stress induction. Aquat. Toxicol. 83, 284–294. Stüven, J., Pflugmacher., S., 2007. Antioxidative stress response of Lepidium sativum due to exposure to cyanobacterial secondary metabolites. Toxicon 50, 85–93. Smith, V.H., 2003. Eutrophication of freshwater and coastal marine ecosystems: a global problem. Environ. Sci. Pollut. Res. 10, 126–139. Stockner, J.G., 1999. Phototrophic picoplankton: an overview from marine and freshwater ecosystems. Limnol. Oceanogr. 33, 765–775. Sies, H., 1997. Oxidative stress: oxidant and antioxidant. Exp. Physiol. 82, 291–295. Smith, C., Sutton, A., 1993. The Persistence of Anatoxin-a in Reservoir Water, Report no. FR0427. Foundation for Water Research, UK. Stevens, D.K., Krieger, R.I., 1991. Stability studies on the cyanobacterial nicotinic alkaloid anatoxin-a. Toxicon 29, 167–179. Stevens, D.K., Krieger, R.I., 1988. Analysis of anatoxin-a by GC/ECD. J. Anal. Toxicol. 12, 126–131. Soll, J., Schultz, G., Joyard, J., Douce, R., Block, M.A., 1985. Localization and synthesis of prenylquinones in isolated outer and inner envelope membranes from spinach chloroplasts. Arch. Biochem. Biophys. 238, 290–299. Takamura, N., Kadono, Y., Fukushima, M., Nakagawa, M., Kim, Baik-H.O., 2003. Effects of aquatic macrophytes on water quality and phytoplankton communities in shallow lakes. Ecol. Res. 18, 381–395. Thomas, P., Stephens, M., Wilkie, G., Amar, G., Lunt, G., Whiting, T., Gallagher, E., Pereira, E., Alkondon, M., Albuquerque, X., Wonnacott, S., 1993. ( þ)-Anatoxin-a is a potent agonist at neuronal nicotinic acetylcholine receptors. J. Neurochem. 60, 2308–2311.
