Aquatic Toxicology 68 (2004) 185–192
Anatoxin-a elicits an increase in peroxidase and glutathione S-transferase activity in aquatic plants Simon M. Mitrovic a,∗ , Stephan Pflugmacher b , Kevin J. James a , Ambrose Furey a a
b
PROTEOBIO, Department of Chemistry, Mass Spectrometry Centre for Proteomics and Biotoxin Research, Cork Institute of Technology, Bishopstown, Cork, Ireland Leibniz Institute of Freshwater Ecology and Inland Fisheries, FG Biogeochemical Regulation, Müggelseedamm 301, 12587 Berlin, Germany Received 1 February 2004; received in revised form 12 March 2004; accepted 14 March 2004
Abstract Although the toxic effects of cyanotoxins on animals have been examined extensively, little research has focused on their effects on macrophytes and macroalgae. To date only microcystins have been found to be detrimental to aquatic plants. Peroxidase activity of the free floating aquatic plant Lemna minor and the filamentous macroalga Chladophora fracta was measured after exposure to several concentrations of the cyanotoxin, anatoxin-a. Peroxidase activity (POD) was significantly (P < 0.05) increased after 4 days of exposure to an anatoxin-a concentration of 25 g mL−1 for both L. minor and C. fracta. Peroxidase activity was not significantly increased at test concentrations of 15 g mL−1 or lower. In another experiment, the effects of various concentrations of anatoxin-a on the detoxication enzyme, glutathione S-transferase (GST) in L. minor were investigated. GST activity was significantly elevated at anatoxin-a concentrations of 5 and 20 g mL−1 . Photosynthetic oxygen production by L. minor was also found to be reduced at these concentrations. This is the first report to our knowledge of the cyanotoxin anatoxin-a being harmful to aquatic plants. © 2004 Elsevier B.V. All rights reserved. Keywords: Cyanobacteria; Anatoxin-a; Toxicity; Aquatic plants; Lemna; Detoxication enzymes
1. Introduction Eutrophication of water bodies has led to increases in the number of excessive growths (blooms) ∗ Corresponding author. Present address: Water Ecosystems, Department of Infrastructure, Planning and Natural Resources, P.O. Box 39, NSW 2001, Australia. Tel.: +612-9228-6307; fax: +612-9228-6140. E-mail address:
[email protected] (S.M. Mitrovic).
of cyanobacteria (blue–green algae) and they are now common in many freshwater lakes and rivers throughout much of the world (Skulberg et al., 1984; Carmichael, 1992; Codd, 1995). Many of the bloom forming cyanobacteria are known to produce different types of toxins including neurotoxins, hepatotoxins, cytotoxins and lipopolysaccharide (LPS) endotoxins, which can cause of a variety of human and animal health, ecological and aesthetic concerns (Carmichael, 1997). Cyanotoxins can have adverse effects on humans and other mammals including sheep, cattle,
0166-445X/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aquatox.2004.03.017
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S.M. Mitrovic et al. / Aquatic Toxicology 68 (2004) 185–192
horses, birds (Carmichael, 2001, 1992; Onodera et al., 1997), fish (Liu et al., 2002), invertebrates (Delaney and Wilkins, 1995) and zooplankton (Rohrlack et al., 2001). Although the toxic effects to these organisms have been researched extensively, studies examining the potential effects of cyanotoxins on aquatic plants are scarce (Pflugmacher et al., 1999; Pflugmacher et al., 2001). Evidence exists that the abundance of submerged plants has coincidently decreased (Abe et al., 1996) and aquatic plant communities are less diverse (Casanova et al., 1999) where algal blooms occur. Recent studies have shown microcystins to induce growth inhibition in seedlings of a variety of terrestrial crop plants (Kos et al., 1995; McElhiney et al., 2001; Hamvas et al., 2002). In tests using aquatic plants, microcystins have caused increases in glutathione S-transferase activities of plants (Pflugmacher et al., 1999; Pflugmacher et al., 2001) and inhibited growth (Weiss et al., 2000; Pflugmacher, 2002; Mitrovic et al., in preparation). Another cyanotoxin, cylindrospermopsin, has also been found to inhibit seedling growth in the terrestrial plant Synapsis (Vasas et al., 2002). This suggests other cyanotoxins may have adverse effects on aquatic plants. Several freshwater bloom forming cyanobacterial genera including Anabaena, Aphanizomenon, Ocillatoria and Cylindrospermum produce the neurotoxin, anatoxin-a (Sivonen et al., 1989), an alkaloid with a high toxicity to animals (Carmichael et al., 1979). Anatoxin-a is a commonly encountered toxin that has been found throughout much of Europe, as well as Canada and Japan (Sivonen and Jones, 1999). The toxin can be found at considerably high concentrations in lakes and rivers, such as 4400 g g−1 dry weight (Sivonen et al., 1989) and 2600 g g−1 dry weight (Harada et al., 1993). The neurotoxin anatoxin-a is a potent post-synaptic depolarizing neuromuscular blocking agent that causes death within minutes to hours in animals (Carmichael and Falconer, 1993). In this series of experiments, we exposed the common aquatic plant Lemna minor and the filamentous macroalga Chladophora fracta to anatoxin-a in controlled laboratory tests to determine whether detoxication enzyme activity was altered. We also examined photosynthetic oxygen production in L. minor during exposure to anatoxin-a.
2. Methods Experiments were performed on sterile monocultures of L. minor and C. fracta that were maintained within our laboratory in SIS medium. Conditions for the experiment were temperature 21 ± 1 ◦ C and a light intensity of 50 mol m−2 s−1 with variability over the test area less than 5%. Cultures were aseptically transferred to fresh SIS medium (OECD, 2002) within a 3-mL sterile well container. A total of twelve healthy L. minor fronds or 40 mg wet weight of C. fracta were used for each test container. At test initiation either no anatoxin-a (control) or anatoxin-a concentrations 5, 15 and 25 g mL−1 for L. minor or concentrations of 10, 15 and 25 g mL−1 for C. fracta were added (treatments). Tests lasted for 4 days and treatments were performed in triplicate. Samples were rearranged daily to minimise any spatial differences in light intensity or temperature within the cabinet. At test termination, approximately 40 mg of wet weight L. minor and C. fracta from each treatment and three replicates within, was analysed for peroxidase (POD) activity. POD activity was determined according to Puetter (1975). In brief, plant material was accurately weighed and was then macerated in a cooled mortar with a cooled pestle in 2 mL cooled Sorensen phosphate buffer solution (Puetter, 1975). The mortar was rinsed with 2 mL buffer solution. The plant material was homogenised in the buffer and then passed through a porcelain frit with a paper filter. One millilitre of the extract and 1 mL of the buffer were placed in a cuvette and 0.2 mL of 2,2 -azino-bis-(3-ethylbenzothiazine-6-sulfonic acid) diammonium salt and 0.2 mL H2 O2 solution were added. The change in absorbance was measured at 405 nm for 300 s. In a second group of experiments, cultures of L. minor, normally 50 single plants, were used non-aseptically and exposed to concentrations of anatoxin-a in the range of 0.1, 1.0, 5.0 and 20 g mL−1 in glass Petri dishes with five replicates. Exposure duration was seven days, changing the exposure medium every day and conditions for the experiment were 50 mol m−2 s−1 and a temperature of 18–20 ◦ C. Enzyme extraction of L. minor plants was carried out according to Pflugmacher and Steinberg (1997). In short, after removal of cell debris, the membrane fraction was separated by centrifugation at
S.M. Mitrovic et al. / Aquatic Toxicology 68 (2004) 185–192
3. Results and discussion Exposure of L. minor to anatoxin-a resulted in significant (P < 0.05) increase in POD activity at a concentration of 25 g mL−1 (Fig. 1). Concentrations of 5 and 15 g mL−1 did not elicit an increase in activity. To confirm the increase in POD activity at 25 g mL−1 , the control and 25 g mL−1 treatments were repeated. In this confirmation experiment, POD activity was again significantly (P < 0.05) increased by exposure to 25 g mL−1 anatoxin-a (Fig. 2). Exposure of the macro alga C. fracta to anatoxin-a also
POD activity
0.15
*
0.10
0.05
0.00 Control
5
15
25
Anatoxin-a concentration Fig. 1. POD activity (U mg−1 fresh weight) for Lemna minor after 4 days of exposure to various anatoxin-a concentrations (g mL−1 ) for control and treatments. Bar indicates mean and error bars indicate standard error of three replicates. ∗ Denotes significantly different (P < 0.05) from the control treatment (ANOVA).
resulted in a significant (P < 0.05) increase in POD activity at a concentration of 25 g mL−1 (Fig. 3). Concentrations of 10 and 15 g mL−1 anatoxin-a did not elicit a significant effect although POD activity did rise relative to the control. In another experiment, exposure of L. minor to anatoxin-a resulted in a significant elevation of soluble glutathione S-transferase (GST) activity after 24 h exposure, indicating start of the biotransformation reaction in the plant (Fig. 4). As with POD activity, the catalase (CAT) activity was
300
* POD activity
105 000 × g for 60 min. The remaining supernatant, defined as the soluble (cytosolic) fraction, was precipitated between 0 and 35% (w/v) and 35 and 80% (w/v) ammonium sulfate saturation. The pellet from the 35–80% precipitation was resuspended in 20 mM sodium phosphate buffer (pH 7.0) and desalted by passage through a NAP-10 column (Pharmacia, Uppsala, Sweden). The subsequent eluate was used for enzyme measurements. From all organisms investigated the activity of soluble glutathione S-transferases (cGST, EC 2.5.1.18) was determined using the standard model substrate 1-chloro-2,4-dinitrobenzene (CDNB) (Habig et al., 1974), catalase (CAT) was measured according to Aebi (1970) at 240 nm. Protein content was determined according to Bradford (1976). The measurement of the macrophyte photosynthetic oxygen production was performed using a Phosy-Mess 4000 (INNOConcept, Berlin), a system for the determination of the vitality of plants on the basis of their oxygen release during photosynthesis. The oxygen measurement is conducted directly on the plant surface by a Clark probe WTW EO 196-1.5 oxygen electrode, measuring light from 630 to 650 nm at a light intensity of 36 E m−2 s−1 with a dark/light/dark cycle of 10/12/10 min under a constant temperature of 20 ◦ C. The rate of photosynthesis was calculated in mol O2 h−1 g fw−1 of plant material. All measurements were performed in 12 independent replicates. Enzyme activity and oxygen production of the different treatments was subjected to one-way analysis of variance (ANOVA) using Tukeys test and Newman–Keuls test to compare between individual means of treatments. Results were considered significant at a P level of
