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Detection of nodularin in European flounder (Platichthys flesus) in the west coast of Sweden: Evidence of nodularin mediated oxidative stress
Harmful Algae 8 (2009) 832–838
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Detection of nodularin in European flounder (Platichthys flesus) in the west coast of Sweden: Evidence of nodularin mediated oxidative stress Karl-Johan Persson a,*, Catherine Legrand a, Thomas Olsson b a b
School of Pure and Applied Natural Sciences, Marine Science Centre, University of Kalmar, S-39182 Kalmar, Sweden Toxicon AB, 261 92 Ha¨rslo¨v, Sweden
A R T I C L E I N F O
A B S T R A C T
Article history: Received 25 July 2008 Received in revised form 25 February 2009 Accepted 8 March 2009
The brackish, bloom-forming cyanobacterium Nodularia spumigena produces a peptide called nodularin, which may induce liver damage in fish. In the summer of 2007, nodularin was detected in liver tissue of ¨ resund, within the upper salinity limit for N. spumigena. European flounder caught in Swedish waters of O Nodularin concentrations ranging between 22 and 557 mg kg1 liver (d.w.) were detected in fish liver. Nodularin was not detected in blue mussels (Mytilus edulis). Although N. spumigena blooms can occur in the area, the cyanobacteria were only present in very small amounts in 2007. Results suggested that nodularin accumulated in flounder livers during the summer of 2006, when vast N. spumigena blooms ¨ resund, and persisted over several months. Nodularin has previously been shown to were observed in O induce oxidative stress in mice, crustaceans and mollusks but work on the potential negative effects of nodularin on fish is still scarce. To examine the dynamics of nodularin induced oxidative stress in liver tissue of flounder, the differential responses of the antioxidant enzymes glutathione-S-transferase catalase (CAT) and the formation of malondialdehyde (MDA) were monitored during 14 days in flounder exposed to an intraperitoneal injection of nodularin (0, 2, 10 and 50 mg nodularin kg1 body weight). The activities of GST and CAT in the liver decreased significantly in the 50 mg nodularin kg1 exposure after 7 days, but were restored to control levels after an additional 10 days of recovery. The results suggested that nodularin induced oxidative stress in terms of decreased GST and CAT activity, which can result in increased vulnerability of the cell to reactive oxygen species (ROS). No significant changes could be found in MDA levels between the treatments. Thus, the antioxidant defense system presumably managed to prevent oxygen mediated toxicity as seen by the unchanged levels of MDA. Alteration of the enzymatic defense system may increase energetic costs, thus reducing fish growth and survival. The present study also suggests that oxidative stress biomarkers can be used in fish to detect early responses to nodularin. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Cyanobacteria Flounder Nodularin Oxidative stress
1. Introduction In the Baltic proper and in the southern Baltic including the ¨ resund, cyanobacterial blooms are often dominated by the toxic O Nodularia spumigena during the summer (Laamanen et al., 2001). N. spumigena is filamentous and produces nodularin, a cyclic pentapeptide toxin. Most N. spumigena blooms occurring in the Baltic Sea appear to be hepatotoxic using mice bioassay (Sivonen et al., 1989). Even though N. spumigena can dominate cyanobacterial blooms in the Baltic Sea, the effects of nodularin have been less investigated than microcystins (MCs), cyclic heptapeptides produced by freshwater colonial and filamentous cyanobacteria such as Microcystis, Oscillatoria, Anabaena, and Nostoc species (Malbrouck and Kestemont, 2006). Nodularins specifically inhibit
* Corresponding author. Tel.: +46 480447343. E-mail address: [email protected] (K.-J. Persson). 1568-9883/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.hal.2009.03.003
protein phosphatases 1 and 2A resulting in hyperphosphorylation of intracellular proteins, and possible liver failure in animals and humans depending on dose (Ohta et al., 1994). In the Baltic Sea, field surveys have revealed that filter feeders such as mussels accumulate nodularin confirming the toxin transfer to higher trophic levels in the aquatic food chain (Sipia¨ et al., 2002). In addition, liver and muscle tissue of European flounder (Platichthys flesus), which feed on blue mussels (Mytilus edulis), can contain nodularin during periods of blooms (July– September) (Mazur-Marzec et al., 2007; Sipia¨ et al., 2006). Nodularin has also been found in copepods (Eurytemora affinis, Acartia spp.), blue mussels, three-spined sticklebacks (Gasterosteus aculeatus), herring (Clupea harengus), sea trout (Salmo trutta), roach (Rutilus rutilus) and common eider (Somateria mollissima) (Sipia¨ et al., 2006; Karjalainen et al., 2006; Kankaanpa¨a¨ et al., 2007). However, nodularin has not yet been detected in aquatic ¨ resund region. In the northern Baltic Sea, organisms from the O reported nodularin concentrations in the liver of flounder range
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between 20 mg kg1 dry weight (d.w.) to approximately 1100 mg kg1 (d.w.) whereas the concentrations in flounder muscle can reach 100 mg kg1(d.w.) (Kankaanpa¨a¨ et al., 2002). While, the adverse impacts of MCs on fish have been extensively investigated (Kotak et al., 1996; Carbis et al., 1996; Cazenave et al., 2005; Jos et al., 2005; Prieto et al., 2006), less is known concerning the potential effects of nodularin on fish. Oxidative stress is defined as a negative reaction due to exposure of molecules, cells, or tissues to free radical oxidants, generally called reactive oxygen species (ROS), occurring in excess levels (Li et al., 2005a). Oxidative stress is often a consequence of cyanobacterial toxicity in mammals (BouaI¨cha and Maatouk, 2004; Lankoff et al., 2002), marine plants (Pflugmacher et al., 2007) and fish (Jos et al., 2005; Prieto et al., 2006). Antioxidant enzymes prevent cellular damages caused by oxidative stress. The liver is the most important organ involved in the regulation of redox metabolism. This is due to the synthesis of key enzymes responsible for ROS clearance and the production of the key antioxidant and protectant, glutathione (GSH). GSH has multiple functions in detoxification of chemicals, and its depletion is associated with increased risks of toxicity. GSH is also a substrate in both conjugation and reduction reactions, catalyzed by glutathione-S-transferase (GST) enzymes in cytosol, microsomes, and mitochondria. GSH works synergistically with other cellular antioxidants such as catalase (CAT), superoxide-dismutase (SOD) and glutathione reductase (GR). Nodularin is actively transported into hepatocytes using membrane protein carriers, which may result in oxidative stress in the liver. CAT is a constituent of the enzymatic antioxidant defence system in vertebrates and minimizes the adverse effects of ROS by breaking down H2O2 into H2O and O2. Peroxidation of lipids is mediated through chain reactions of ROS, leading to damage of the cell. Malondialdehyde (MDA) is a well known oxidation product of polyunsaturated fatty acids (PUFAs) in lipoproteins and is often used as a biomarker of oxidative stress (Almroth et al., 2005). In order to decrease the toxicity, cells have defence systems that modify toxic substances to aid their excretion. Beattie et al. (2003) showed that the initial step in detoxification of nodularin in brine shrimp (Artemia salina) is conjugation to GSH via GST, which increases the water solubility and ultimate excretion. Several studies have also described GST detoxification of nodularin in crustaceans (Beattie et al., 2003; Pflugmacher et al., 2005) and molluscs (Davies et al., 2005; Kankaanpa¨a¨ et al., 2007) but the role of GST in the detoxification of nodularin in fish has not yet been reported. The aim of the present study was to investigate the occurrence of nodularin in flounders and blue mussels caught in Swedish ¨ resund during the summer of 2007. We also performed waters of O a laboratory experiment to examine the dynamics of nodularin mediated oxidative stress in flounder liver. The long-term (7 days) changes in GST and CAT activity and the formation of MDA in the liver of European flounder were investigated after exposure to nodularin. 2. Material and methods 2.1. Field sampling Adult European flounder (P. flesus) (weight: 297 67 g length: 309 20 mm, age: 5–7 years) were caught using gill nets (depth: 5– 7 m) and blue mussels (M. edulis) (length: 44 5 mm) were collected using a bottom scraper (depth: 5–7 m) at two locations situated in ¨ resund; Sweden Gette reef (558550 3300 N, 128470 2000 E) and Rydeba¨ck O (558580 4600 N, 128440 1900 E). Sampling took place between 30 July and 4 August 2007. Cyanobacterial blooms had occurred at both sites during 2006. Twenty live flounders were caught from each location
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and were swiftly transported in seawater tanks to the laboratory. Twenty blue mussels from each site were also collected and transported to the laboratory in a cooled water-free container. All fish and mussels were killed and dissected 1–2 h from sampling. Fish liver and muscle, and mussel soft tissues were removed and pooled into groups of 5 individuals per sample set giving 4 sample sets per location. All tissues destined for nodularin analyses were kept at 20 8C. 2.2. Experimental design to determine oxidative stress Flounders (weight: 259 41 g, length: 306 17 mm, age: 5–7 years) used in the oxidative stress experiment were caught using gill nets (depth: 5–7 m) on April 8 and April 12, 2007 at Gette reef in ¨ resund, Sweden (558550 3300 N, 128470 2000 E). The flounders were O placed in 4 aquariums (350 L, 5 or 10 individuals per aquarium) with aerated seawater. Fish were acclimatized for 8 weeks under constant conditions (t = 16.70 0.04 8C, O2 = 8.30 0.04 mg l1; pH 8.70 0.03; salinity 9.5 0.6; light:dark cycle = 16:8 h). Half the water volume in the aquaria was changed every second week. Seawater was ¨ resund (558560 2000 N, 128460 1800 E), collected during April and May in O and was prefiltered through a 200 mm nylon net to remove large plankton. Flounders were fed earthworms (Lumbricus terrestris, 2% of the flounder body weight every second day). The flounders were observed daily in terms of reaction to being touched and general physiological appearance such as body colour and swollen abdomens. 2.3. Exposure to nodularin The doses of pure nodularin (product number: N5148, Sigma– Aldrich1, Sweden) employed in the oxidative stress experiment were obtained from a preliminary dose-finding experiment in which fish that were intraperitoneally (i.p-) injected with nodularin concentrations >100 mg kg body weight1 died within 48 h (data not shown). The toxin dose was calculated based on the weight of each fish, and was injected in 0.5 ml of 0.9% (w/v) NaCl solution into the abdominal cavity. The control fish (n = 5) received only saline solution (0.5 ml of 0.9%, w/v, NaCl). The experiment was run for 4 days (4 aquaria, n = 25) followed by 10 days of recovery (1 aquarium, n = 5). The recovery group consisted of 5 fish that initially received a nodularin dose of 50 mg kg1. Flounders were i.p-exposed to two successive doses of nodularin (on day 1 and 4) corresponding to a total concentration of 2 mg kg1 (n = 5), 10 mg kg1 (n = 5), 50 mg kg1 (n = 10) and control (0 mg kg1, n = 5). Half the total dose was given on day 1 and the second half was given on day 4 of the experiment. The doses were divided into two rounds due to observations of the fish in the dose-finding experiment indicating the acute toxicity of the toxin. By dividing the toxin injection into two rounds we assumed that the fish would have an increased chance of surviving the duration of the experiment. Immediately after the fish had been sacrificed, 500 mg of liver from each fish were dissected out, weighed, rinsed with ice-cold saline (0.9%), immediately frozen to 80 8C and kept at 80 8C until analysis of GST, CAT and MDA. Liver (0.2– 5 g fish1 wet weight) and muscle (10 g fish1 wet weight) tissue were also dissected out for analysis of nodularin content and kept at 20 8C. 2.4. Preparation of cytosolic liver fractions Liver tissue was homogenised in Na/K-phosphate buffer (0.15 M, pH 7.3) containing KCl (0.1 M). Homogenates were centrifuged at 10 000 g for 20 min at 4 8C. The supernatant was transferred to a new tube and centrifuged at 105 000 g for 1 h at 4 8C. Samples of the final supernatant (cytosolic) destined for
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enzymatic and MDA analysis was kept at 80 8C, whereas samples for determination of protein content were stored at 20 8C. 2.5. Protein assay Protein content was analysed in the cytosolic liver fractions using the method described by Bradford and Williams (1976). Protein reagent was purchased from Bio-Rad Laboratories Inc. (Catalog No. 500-0006) and protein standards were generated using bovine serum albumin (BSA, CAS: 9048-46-8, Sigma–Aldrich, Sweden). The protein content was analysed spectrophotometrically at 595 nm and the results were calculated into mg protein ml1. 2.6. Glutathione-S-transferase assay (GST) GST activity was analysed in cytosolic liver fractions using the method described by Habig et al. (1974). Briefly, the conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) with reduced glutathione (GSH) was measured as an increase in absorbance at 340 nm. The rate of the increase in absorbance is directly proportional to the GST activity in the sample (Habig et al., 1974). 2.7. Catalase assay (CAT) The assay was performed according to the kit descriptions provided by Cayman chemicals1 (Catalog No. 707002). Briefly, the assay utilises the peroxidatic function of catalase in order to determine the enzyme activity. The enzyme is allowed to react with methanol in the presence of an optimal concentration of H2O2 to form formaldehyde. The formed formaldehyde is used as a measure of CAT activity and measured spectrophotometrically at 540 nm using 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole as the chromogen, which turns purple after oxidation. 2.8. Malondialdehyde assay (MDA) The concentration of MDA was used as an index of lipid peroxidation and was determined according to Ohkawa et al. (1979). The analysis was performed with an assay kit purchased from Cayman chemicals1 (Catalog No. 10009055) and used the determination of thiobarbituric acid reactive substances (TBARS). The determination of TBARS was performed according to the instructions provided with the assay kit. Briefly, the MDA-TBA adduct, which is formed by the reaction between MDA and TBA under high temperature (90–100 8C) and acidic conditions was measured colorimetrically at 530–540 nm.
water was produced by Millipore (Elga Purelab Option, Germany) Milli-Q plus equipment. Nodularin concentrations in liver tissues of flounder were initially expressed on a wet weight basis but were recalculated on a dry weight basis in order to allow for comparison with published literature. Flounder liver dry:wet weight ratios ¨ resund and derived from flounder caught in the same region of O during the same period of time were used in the calculations (Lundgren, 2007). 2.10. Statistical analysis The data were tested for statistical differences using one-way ANOVA followed by Tukey’s post hoc test for differences between the treatments in the oxidative stress experiment. The homogeneity of variances was tested with Levene’s test and test of sample normal distribution was tested with the Kolmogorov– Smirnov test. The statistics were performed using SPSS software package (version 14.0). The data showed a normal distribution. The level of significance was set at p < 0.05. 3. Results Nodularin concentrations in flounder livers (all individuals) collected in the field ranged between 22 and 557 mg kg1 liver (d.w) (Gette reef = 235 218 and Rydeba¨ck = 102 57 mg kg1 liver d.w, Fig. 1). No nodularin was detected in any of the flounder muscle samples nor in the soft tissue of blue mussels collected in the field. Nodularin was detected in the liver of 17 out of 18 fishes injected with nodularin in the oxidative stress experiment (range: 0– 780 mg kg liver d.w.1, Fig. 2). The fish in which nodularin was not detected belonged to the 2 mg kg1 treatment. Nodularin was also detected in 3 of 5 control fish (103 21 mg kg liver d.w. 1). No nodularin was detected in the muscle tissue of any of the fish used in the oxidative stress experiment. There were no significant differences in nodularin concentrations between the control and fish exposed to 2–50 mg nodularin kg1 between day 1 and 7 (p = 0.133, one-way ANOVA followed by Tukey’s post hoc test). The recovery group contained significantly higher nodularin concentrations compared to the control group (p = 0.035, one-way ANOVA followed by Tukey’s post hoc test). Two fish from the 50 mg nodularin kg1 treatment in the oxidative stress experiment died, one during the period after the first injection and another during the period after the second injection. Observed symptoms for the first mortality were swollen abdomen, immobility and loss of pigmentation. Observed symptoms for the second mortality were malnutrition (weight loss), loss of pigmentation and reduced mobility.
2.9. Nodularin analyses Samples of flounder liver (n = 20), flounder muscle (n = 20) and blue mussel soft tissues (n = 20) were collected from each sampling site in the field and analyzed for nodularin according to Kankaanpa¨a¨ et al. (2002). Each sample set (n = 20) were pooled into 4 groups (5 tissue samples per group). Individual liver (n = 23) and muscle tissues (n = 23) from the flounders in the oxidative stress experiment were also analyzed for nodularin according to Kankaanpa¨a¨ et al. (2002). Tissues (w.w.) were homogenised in water, methanol and n-butanol solution (75:20:5) (1 ml g1 w.w.) and extracted in a sonicator (Transsonic T 570/H, Elma1, Germany) (35 kHz) for 8 h at 60 8C. The HPLC system consisted of a Gynkotek P580 pump, a Gina 50 autosampler and a Waters Lambda max detector (model 481). The mobile phase consisted of 400 ml acetonitrile, 600 ml HPLC grade water and 10 ml trifluoroacetic acid with an isocratic flow rate of 1 ml min1. The column used was a Macherey-Nagel, Nucleosil 10 C-18 ET 250/8/4 column. The chromatograms were recorded at 238 nm. Ultra pure deionised
Fig. 1. Nodularin concentrations in the livers of flounders caught at two locations ¨ resund during the end of July and beginning of August (n = 20 at each location) in O 2007. The bars indicate the minimum and maximum nodularin concentrations. The line inside of the box represents the median value. The areas below and above the median are the lower and upper quartiles.
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Fig. 2. Nodularin concentrations in the livers of i.p-injected flounders 0 (n = 5), 2 (n = 5), 10 (n = 5), 50 (n = 4) and 50-Recovery (n = 4) mg nodularin kg1 b.w. The bars indicate the minimum and maximum nodularin concentrations. The line inside of the box represents the median value. The areas below and above the median are the lower and upper quartiles.
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Fig. 5. Malondialdehyde levels in the liver of flounders i.p-injected with nodularin 0 (n = 5), 2 (n = 5), 10 (n = 5), 50 (n = 4) and 50-Recovery (n = 4) mg nodularin kg1 b.w. The data are presented as average MDA concentration g1 liver SD.
50 mg nodularin kg1 were restored to control values (Fig. 2 AB, GST p = 0.996 and CAT p = 0.984, one-way ANOVA followed by Tukey’s post hoc test). As for MDA, no significant differences in MDA levels were found between the treatments (p = 0.204, oneway ANOVA, Fig. 5). 4. Discussion 4.1. Accumulation of nodularin in liver and muscle tissues
Fig. 3. The activity of GST in the cytosolic liver fractions of flounder i.p-injected with nodularin 0 (n = 5), 2 (n = 5), 10 (n = 5), 50 (n = 4) and 50-Recovery (n = 4) mg nodularin kg1 b.w. Data are shown as the average enzyme activity SD after 7 and 14 days (recovery) of toxin exposure.
The detoxification and antioxidant enzyme activities measured in liver (GST and CAT) were significantly lower in fish exposed to 50 mg nodularin kg1 than controls (Figs. 3 and 4, GST p = 0.018 and CAT p = 0.001, one-way ANOVA followed by Tukey’s post hoc test). No difference was found in GST and CAT activities between controls and fish exposed to 2 or 10 mg nodularin kg1 (Figs. 3 and 4, GST p = 0.466 and CAT p = 0.103, one-way ANOVA). After 10 days recovery, both GST and CAT activities in fish exposed to
Fig. 4. The activity of CAT in the cytosolic liver fractions of flounder i.p-injected with nodularin 0 (n = 5), 2 (n = 5), 10 (n = 5), 50 (n = 4) and 50-Recovery (n = 4) mg nodularin kg1 b.w. Data are shown as the average enzyme activity SD after 7 and 14 days (recovery) of toxin exposure.
This is the first study to report nodularin accumulation in the ¨ resund (range: 22– liver of flounder caught in Swedish waters of O 557 mg kg1 liver d.w.). Nodularin accumulates in tissues of various fish species from the Baltic Sea, including flounder (Kankaanpa¨a¨ et al., 2002; Sipia¨ et al., 2006; Mazur-Marzec et al., 2007; Sipia¨ et al., 2008). Blue mussels are the primary food source for flounders and it is assumed that the majority of nodularin found in flounders originates from mussels (Kankaanpa¨a¨ et al., 2005; Sipia¨ et al., 2008). Studies of nodularin accumulation in the Baltic Sea have shown that blue mussels and flounders are the most affected aquatic organisms containing nodularin concentrations in the range of 40– 2200 mg kg soft tissue1 (d.w) and 5–1100 mg kg liver1 (d.w), respectively (Sipia¨ et al., 2002; Kankaanpa¨a¨ et al., 2005; MazurMarzec et al., 2007). Detected nodularin concentrations in salmon (Salmo salar) and herring (C. harengus) range between 0–10 and 0– 90 mg kg1 (d.w), respectively (Sipia¨ et al., 2002, 2007). In fact, nodularin concentrations in muscle tissue of flounder have been reported to exceed the tolerable daily intake (TDI) value set for human consumption of microcystins (MC-LR), and thus by analogy nodularin (Sipia¨ et al., 2006). However, during the summer of 2007 N. spumigena occurred in very small amounts in the waters of ¨ resund (Swedish Meteorological and Hydrological Institute). In O addition, nodularin was not detected in blue mussels at the two sampling sites suggesting that residual nodularin remains in the liver of flounders during the winter months. This hypothesis is strengthened by the presence of nodularin in the control fish from the oxidative stress experiment caught in April since N. spumigena blooms during this time of the year are uncommon. It is possible that the fish accumulated nodularin during July 2006 when both blooming N. spumigena and decaying assemblages, derived from ¨ resund (Swedish Meteorological the Baltic Proper, occurred in O and Hydrological Institute). The presence of nodularin in fish liver could be due to the ingestion of contaminated mussels and/or of sedimented Nodularia mats while foraging on the sea floor. Nodularin was analysed in washed up cyanobacterial mats on
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the beach (Amager Strand, Copenhagen, Denmark) during the summer 2006. The mats consisted almost exclusively of N. spumigena and concentrations as high as 16 mg nodularin per g dry weight were measured, which are among the highest nodularin concentrations ever measured in the area (P. Henriksen, DMU, Denmark, personal communication). These high nodularin concentrations also indicate that the concentrations used in our oxidative stress experiments were comparable to environmental concentrations. In previous oxidative stress experiments, two fish (Pomphorhynchus laevis) were injected with 50 mg nodularin kg1 (Ohta et al., 1994). They showed symptoms such as swollen abdomens, loss of pigmentation, reduced mobility and died. This indicated that both fish died of liver failure and that nodularin had a negative impact on hepatocytes membrane integrity. Similarly, nodularin caused loss of liver architecture in sea trout orally exposed to a single dose of food containing N. spumigena (210–620 mg nodularin kg1 b.w.) in an 8day survey (Kankaanpa¨a¨ et al., 2002). MCs were also reported to cause liver necrosis and degeneration after intraperitoneal inoculation in carp (Carbis et al., 1996). In our study, autopsy revealed that one of the fish that died had several parasitic worms penetrating the gut wall. The occurrence of these worms is a common feature in ¨ resund area and they can cause peritonitis, flounder from the O which in turn could impact the immune system. This suggests that inter individual variation is likely to be involved in vulnerability for nodularin exposure. In addition to this, individuals exposed to nodularin annually with residual nodularin stored in the liver may have a compromised immune system. The only group that showed a statistically significant increase in nodularin concentrations compared to the control group in the oxidative stress experiment was the recovery group (50 mg nodularin kg bw1). Kankaanpa¨a¨ et al. (2002) investigated the heterogeneity of nodularin accumulation in flounders from the Baltic Sea and concluded that the individual-to-individual toxin content in liver tissue varied considerably. Possible reasons for these variations in nodularin concentrations are that the fish accumulate different amounts of the toxin during their time in the sea. Fish differ in their uptake of food and thus do not acquire toxins in a uniform manner (Sipia¨ et al., 2002). Even though the recovery group in the oxidative stress experiment showed increased concentrations of nodularin compared to the control group, the activities of GST and CAT returned to control levels, after the 10 days recovery period. This suggests that although increased amounts of nodularin were present in the livers of the recovery fish, fish managed to increase the activities of GST and CAT, presumably in an attempt to detoxify the toxin and deal with formed ROS. The flounders used in the oxidative stress experiment contained background concentrations of nodularin, which raise the question whether the fish was compromised in some way from previous exposure. We recommend that future research is conducted to investigate whether/how residual nodularin can compromised the health of individual fish. 4.2. GST activity The activity of GST in the cytosolic liver fractions of flounders, i.p-injected with 50 mg nodularin kg1, significantly decreased compared to the control group. Earlier studies have shown decreases in GST activity in green mussels (Perna viridis) exposed to N. spumigena extracts (Davies et al., 2005) and in brine shrimp (A. salina) exposed to pure nodularin through immersion (Beattie et al., 2003). Similarly, previous research on MC-LR has shown decreases in GST activity in the liver of i.p-injected goldfish (Carassius auratus) (Malbrouck et al., 2003). MC-RR has been shown to cause reductions in GST activity in the liver, gills,
intestine and brain of Peppered Corydoras (Corydoras paleatus) exposed through immersion (Cazenave et al., 2005). However, Kankaanpa¨a¨ et al. (2007) did not observe any change in GST activity in blue mussels, exposed to N. spumigena extract. It was therefore concluded that GST is not significantly involved in the detoxification process of nodularin in blue mussels. Other studies have shown that the detoxification of cyanobacterial toxins in the liver of mice occur through conjugation to the tripeptide glutathione (GSH) via GSTs (Jayaraj et al., 2006). GSTs catalyze the addition of MCs to the SH (thiol) group of GSH. This leads to neutralisation of the electrophilic sites of MC, which increases the water solubility and facilitates excretion (Wiegand et al., 1999). The liver is already defined as the target organ for nodularin and several studies have demonstrated the presence of a conjugated nodularin-glutathione formed enzymatically via soluble GST in different aquatic organisms (Sipia¨ et al., 2002; Beattie et al., 2003; Davies et al., 2005). This conjugation of nodularin with GSH, catalyzed by GST, seems to be the first step of detoxification of this toxin in various aquatic organisms. In this context, GST would be a specific biomarker for contamination by nodularin. However, Mazur-Marzec et al. (2007) did not find any nodularin conjugates with reduced GSH in flounder or blue mussels from the Gulf of Gdansk and suggested that other biotransformation products of nodularin might occur. A decrease in GST activity could be explained by reduced synthesis of GST protein at a molecular level (Gallagher et al., 2000). Down regulation of gene expression for GSTs has been shown in the liver of mice i.p-exposed to MC-LR (38.31 mg kg1, Jayaraj et al., 2006). In addition, nodularin is known to cause inhibition of protein phosphatases (Falconer et al., 1983), which could lead to phosphorylation of GST subunits and consequently changes, in GST activity (Beattie et al., 2003). Reduced capability of fish to eliminate toxins could increase the risk for bioaccumulation and consequently the risk of cellular damage, physiological alterations and behavioural changes (Cazenave et al., 2006 and references therein). Modulation of detoxification enzymes could also result in adverse effects such as increased susceptibility to ROS formation, increased energetic demand, proliferation of cells, and also susceptibility to other xenobiotics (Van der Oost et al., 2003). GST was restored to control values 10 days after the last injection of nodularin. Thus, it seems as if the GSH based detoxification process using GST was able to cope with the applied toxins. Our results agree with the findings of Malbrouck et al. (2003) who found a recovery of GST activity in goldfish i.p-injected with 125 mg MC-LR kg1 (b w). 4.3. Catalase activity The activity of CAT decreased in fish injected with 50 mg nodularin kg1. This result agrees with earlier research with mice, i.p. exposed to nodularin (Lankoff et al., 2002), with MCLR (Jayaraj et al., 2006) and tilapia fish (Oreochromis niloticus) orally exposed to MC-LR (Prieto et al., 2007). The observed decrease in CAT activity suggests nodularin-induced oxidative stress in terms of increased amounts of ROS that are scavenged by CAT. Several studies have shown that CAT is inactivated by singlet oxygen and peroxyl radicals due to a direct damaging effect of ROS on the enzyme (Kono and Fridovich, 1982; Escobar et al., 1996). Jayaraj et al. (2006) suggested that a decrease in CAT could be due to increased amounts of superoxide anions as a consequence of a reduction in the activity of SOD. In contrast to other studies that have shown increases in CAT activity in fish exposed to MCs (Li et al., 2004; Jos et al., 2005; Prieto et al., 2006), flounders in the present study exposed to an i.pinjection of nodularin showed a decrease in CAT activity. Oxidative
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stress is therefore induced by nodularin and MCs in the different types of experimental models even though the antioxidant defence system shows different responses. These discrepancies could be due to differences in toxicity profiles between the two toxins, experimental conditions, doses employed and/or interspecies differences in susceptibility to the acute effects of these toxins (Kankaanpa¨a¨ et al., 2007). Cazaneve et al. (2006) have found a dual response of CAT activity in the liver of Peppered Corydoras after exposure to dissolved MC-RR, with an increase at low concentrations and depletion at higher levels. Higher toxin doses could involve damage on the enzyme proteins while lower doses administrated during a longer period of time may induce a defensive response (Prieto et al., 2007). The present study indicates that fish in the recovery group were able to restore the CAT activity to control levels after 10 days. This finding agrees with observed recovery of CAT between 24 h and 72 h post orally exposure of an acute dose of MC-LR (2400 mg MCLR kg1) in tilapia fish (Oreochromis niloticus) (Prieto et al., 2007) and a decrease in CAT activity after an initial increase due to exposure of N. spumigena extract in blue mussels (Kankaanpa¨a¨ et al., 2007). 4.4. MDA formation Earlier studies have demonstrated oxidative stress in terms of increases in MDA levels due to lipid peroxidation after exposure to nodularin in rat hepatocytes (BouaI¨cha and Maatouk, 2004) and in green mussels (Davies et al., 2005). However, MDA levels in livers of flounder exposed to nodularin did not differ from controls in the present study. The incapacity to initiate lipid peroxidation could be due to an intracellular decrease in ROS, which are known to attack lipids and initiate their peroxidation (BouaI¨cha and Maatouk, 2004). Ding et al. (2001) observed an early increase in ROS levels in primary cultured rat hepatocytes treated with low doses of MCs. The maximum ROS levels were reached after 10 min and sustained for 20 min, after which depletion in ROS levels occurred. Thus, this suggests that the increasing number of ROS, due to toxin exposure, is efficiently deactivated by the defence system of the cells, preventing MDA formation. In the present study the restoration of GST and CAT activity after 10 days and constant levels of MDA indicate that the antioxidant defence system managed to prevent oxidative stress in terms of MDA formation in flounder livers. Similarly, Li et al. (2005b) showed no increased MDA levels in the liver of loach (Misgurnus mizolepis), orally exposed to the cyanobacterium Microcystis and consequently concluded that the antioxidant enzymes were able to eliminate oxidative stress and thereby preventing increased MDA levels. 5. Conclusions ¨ resund during the summer of Livers from flounders caught in O 2007 contained nodularin, possibly originating from the summer of 2006, when in contrast to 2007, vast N. spumigena blooms were present at the sampling sites. This indicates the persistent nature of nodularin in flounder liver tissue. i.p.-injected nodularin induced reversible alterations of the activities of GST and CAT in flounder liver tissue and therefore suggests the role of oxidative stress in nodularin mediated toxicity. The restoration of GST and CAT activity after 10 days and constant levels of MDA indicated that the antioxidant defence system managed to prevent oxidative stress in terms of MDA formation in flounder livers. However, alteration of the enzymatic defence system might lead to increased energetic costs which could reduce fish growth and survival. The present study also suggests that using oxidative stress biomarkers
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could allow the detection of an early response in fish to nodularin exposure. Future studies should include more natural routes of exposure rather than i.p-injection (e.g. direct water contact, exposure to contaminated mussels/sediment) to demonstrate whether nodularin can be taken up through vectoral transfer under ‘artificial’ bloom conditions. Acknowledgements We thank Per Carlsson, Lund University and Ingrid Trulsson, Daniel Kuster, Fredrik Lundgren and Anna Wolfhagen, Toxicon AB for expert advice and excellent technical assistance. We are grateful to P. Henriksen (Danish Environmental Protection Agency, Roskilde, Denmark) for sharing unpublished data. This study was funded by the Region Ska˚ne (Per Carlsson), Toxicon AB (Thomas Olsson) and the Swedish Research Council for environment, Agricultural Sciences and Spatial Planning (Formas, Catherine Legrand).[SS] References Almroth, C.M., Sturve, J., Berglund, A˚., Fo¨rlin, L., 2005. 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Critical role of reactive oxygen species formation in microcystin-induced cytoskeleton disruption in primary cultured hepatocytes. J. Toxicol. Environ. Health Part A 64 (6), 507–519. Escobar, J.A., Rubia, M.A., Lissi, E.A., 1996. SOD and catalase inactivation by singlet oxygen and peroxyl radicals. Free Radic. Biol. Med. 20 (3), 285–290. Falconer, I.R., Beresford, A.M., Runnegar, M.T.C., 1983. Evidence of liver damage from a bloom of the blue green algae Microcystis aeruginosa. Med. J. Aust. 1, 511– 514. Gallagher, E.P., Sheehy, K.M., Lame, M.W., Segall, H.J., 2000. In vitro kinetics of hepatic glutathione S-transferase conjugation in largemouth bass and brown bullheads Environ. Toxicol. Chem. 19 (2), 319–326. Habig, W.H., Pabst, M.J., Jakoby, W.B., 1974. Glutathione S-transferases. The first enzymatic step in mercapturic acid formation. J. Biol. Chem. 249 (22), 7130– 7140. Jayaraj, R., Anand, T., Lakshmana, P.V.R., 2006. 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Accumulation and depuration of cyanobacterial toxin nodularin and biomarker responses in the mussel Mytilus edulis. Chemosphere 68 (7), 1210–1220. ¨ st, J., KankaanKarjalainen, M., Kozlowsky-Suzuki, B., Lehtiniemi, M., Engstro¨m-O pa¨a¨, H., Viitasalo, M., 2006. Nodularin accumulation during cyanobacterial
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Pflugmacher, S., Wiegand, C., Werner, S., Schroeder, H., Kankaanpa¨a¨, H., 2005. Activity and substrate specificity of cytosolic and microsomal glutathione Stransferase in Australian black tiger prawns (Penaeus monodon) after exposure to cyanobacterial toxins. Environ. Toxicol. 20 (3), 301–307. Pflugmacher, S., Olin, M., Kankaanpa¨a¨, H., 2007. Nodularin induces oxidative stress in the Baltic Sea brown alga Fucus vesiculosus (Phaeophyceae) Mar. Environ. Res. 64 (2), 149–159. Prieto, A.I., Jos, A., Pichardo, S., Moreno, I., Camea´n, A.M., 2006. Differential oxidative stress responses to microcystins LR and RR in intraperitoneally exposed tilapia fish (Oreochromis spp.). Aquat. Toxicol. 77 (3), 314–321. Prieto, A.I., Jos, A., Pichardo, S., Moreno, I., Camea´n, A.M., 2007. Time-dependent oxidative stress responses after acute exposure to toxic cyanobacterial cells containing microcystins in tilapia fish (Oreochromis niloticus) under laboratory conditions. Aquat. Toxicol. 84 (3), 337–345. Sivonen, K., Kononen, K., Esala, A.I., 1989. Toxicity and isolation of the cyanobacterium Nodularia spumigena from the southern Baltic Sea in 1986. Hydrobiology 185 (1), 3–8. Sipia¨, V.O., Kankaanpa¨a¨, H.T., Pflugmacher, S., Flinkman, J., Furey, A., James, K.J., 2002. Bioaccumulation and detoxification of nodularin in tissues of flounder (Platichthus flesus), mussels (Mytilus edulis, Dreissena polymorpha), and clams (Macoma balthica) from the northern Baltic Sea. Ecotoxicol. Environ. Safety 53 (2), 305–311. Sipia¨, V., Sjo¨vall, O., Valtonen, T., Barnaby, D.L., Codd, G.A., Metcalf, J.S., Kilpi, M., Mustonen, O., Meriluoto, J.A.O., 2006. Analysis of nodularin-r in eider (Somateria mollissima), roach (Rutilus rutilus) and flounder (Platichthus flesus) liver and muscle samples from the Western Gulf of Finland, northern Baltic Sea. Environ. Toxicol. Chem. 25 (11), 2834–2840. Sipia¨, V., Neffling, M.R., Metcalf, J., Nybom, S., Meriluoto, Codd, G., 2008. Nodularin in feathers and liver of eiders (Somateria mollissima) caught from the western Gulf of Finland in June-September 2005. Harmful Algae 7 (1), 99–105. Sipia¨, V., Kankaanpa¨a¨, H., Peltonen, H., Vinni, M., Meriluoto, J., 2007. Transfer of nodularin to three-spined stickleback (Gasterosteus aculeatus L.), herring (Clupea harengus L.), and salmon (Salmo salar L.) in the northern Baltic Sea. Ecotoxicol. Environ. Saf. V 66 (3), 421–425. Van der Oost, R., Beyer, J., Vermulen, N.P.E., 2003. Fish bioaccumulation and biomarkers in environmental risk assessment. Environ. Toxicol. Phar. 13 (2), 57–149. Wiegand, C., Pflugmacher, S., Oberemm, A., Meems, N., Beattie, K.A., Steinberg, C.E.W., Codd, G.A., 1999. Uptake and effects of microcystin-LR on detoxification enzymes of early life stages of the Zebra fish (Danio rerio). Environ. Toxicol. 14 (1), 89–95.
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Detection of nodularin in European flounder (Platichthys flesus) in the west coast of Sweden: Evidence of nodularin mediated oxidative stress Karl-Johan Persson a,*, Catherine Legrand a, Thomas Olsson b a b
School of Pure and Applied Natural Sciences, Marine Science Centre, University of Kalmar, S-39182 Kalmar, Sweden Toxicon AB, 261 92 Ha¨rslo¨v, Sweden
A R T I C L E I N F O
A B S T R A C T
Article history: Received 25 July 2008 Received in revised form 25 February 2009 Accepted 8 March 2009
The brackish, bloom-forming cyanobacterium Nodularia spumigena produces a peptide called nodularin, which may induce liver damage in fish. In the summer of 2007, nodularin was detected in liver tissue of ¨ resund, within the upper salinity limit for N. spumigena. European flounder caught in Swedish waters of O Nodularin concentrations ranging between 22 and 557 mg kg1 liver (d.w.) were detected in fish liver. Nodularin was not detected in blue mussels (Mytilus edulis). Although N. spumigena blooms can occur in the area, the cyanobacteria were only present in very small amounts in 2007. Results suggested that nodularin accumulated in flounder livers during the summer of 2006, when vast N. spumigena blooms ¨ resund, and persisted over several months. Nodularin has previously been shown to were observed in O induce oxidative stress in mice, crustaceans and mollusks but work on the potential negative effects of nodularin on fish is still scarce. To examine the dynamics of nodularin induced oxidative stress in liver tissue of flounder, the differential responses of the antioxidant enzymes glutathione-S-transferase catalase (CAT) and the formation of malondialdehyde (MDA) were monitored during 14 days in flounder exposed to an intraperitoneal injection of nodularin (0, 2, 10 and 50 mg nodularin kg1 body weight). The activities of GST and CAT in the liver decreased significantly in the 50 mg nodularin kg1 exposure after 7 days, but were restored to control levels after an additional 10 days of recovery. The results suggested that nodularin induced oxidative stress in terms of decreased GST and CAT activity, which can result in increased vulnerability of the cell to reactive oxygen species (ROS). No significant changes could be found in MDA levels between the treatments. Thus, the antioxidant defense system presumably managed to prevent oxygen mediated toxicity as seen by the unchanged levels of MDA. Alteration of the enzymatic defense system may increase energetic costs, thus reducing fish growth and survival. The present study also suggests that oxidative stress biomarkers can be used in fish to detect early responses to nodularin. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Cyanobacteria Flounder Nodularin Oxidative stress
1. Introduction In the Baltic proper and in the southern Baltic including the ¨ resund, cyanobacterial blooms are often dominated by the toxic O Nodularia spumigena during the summer (Laamanen et al., 2001). N. spumigena is filamentous and produces nodularin, a cyclic pentapeptide toxin. Most N. spumigena blooms occurring in the Baltic Sea appear to be hepatotoxic using mice bioassay (Sivonen et al., 1989). Even though N. spumigena can dominate cyanobacterial blooms in the Baltic Sea, the effects of nodularin have been less investigated than microcystins (MCs), cyclic heptapeptides produced by freshwater colonial and filamentous cyanobacteria such as Microcystis, Oscillatoria, Anabaena, and Nostoc species (Malbrouck and Kestemont, 2006). Nodularins specifically inhibit
* Corresponding author. Tel.: +46 480447343. E-mail address: [email protected] (K.-J. Persson). 1568-9883/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.hal.2009.03.003
protein phosphatases 1 and 2A resulting in hyperphosphorylation of intracellular proteins, and possible liver failure in animals and humans depending on dose (Ohta et al., 1994). In the Baltic Sea, field surveys have revealed that filter feeders such as mussels accumulate nodularin confirming the toxin transfer to higher trophic levels in the aquatic food chain (Sipia¨ et al., 2002). In addition, liver and muscle tissue of European flounder (Platichthys flesus), which feed on blue mussels (Mytilus edulis), can contain nodularin during periods of blooms (July– September) (Mazur-Marzec et al., 2007; Sipia¨ et al., 2006). Nodularin has also been found in copepods (Eurytemora affinis, Acartia spp.), blue mussels, three-spined sticklebacks (Gasterosteus aculeatus), herring (Clupea harengus), sea trout (Salmo trutta), roach (Rutilus rutilus) and common eider (Somateria mollissima) (Sipia¨ et al., 2006; Karjalainen et al., 2006; Kankaanpa¨a¨ et al., 2007). However, nodularin has not yet been detected in aquatic ¨ resund region. In the northern Baltic Sea, organisms from the O reported nodularin concentrations in the liver of flounder range
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between 20 mg kg1 dry weight (d.w.) to approximately 1100 mg kg1 (d.w.) whereas the concentrations in flounder muscle can reach 100 mg kg1(d.w.) (Kankaanpa¨a¨ et al., 2002). While, the adverse impacts of MCs on fish have been extensively investigated (Kotak et al., 1996; Carbis et al., 1996; Cazenave et al., 2005; Jos et al., 2005; Prieto et al., 2006), less is known concerning the potential effects of nodularin on fish. Oxidative stress is defined as a negative reaction due to exposure of molecules, cells, or tissues to free radical oxidants, generally called reactive oxygen species (ROS), occurring in excess levels (Li et al., 2005a). Oxidative stress is often a consequence of cyanobacterial toxicity in mammals (BouaI¨cha and Maatouk, 2004; Lankoff et al., 2002), marine plants (Pflugmacher et al., 2007) and fish (Jos et al., 2005; Prieto et al., 2006). Antioxidant enzymes prevent cellular damages caused by oxidative stress. The liver is the most important organ involved in the regulation of redox metabolism. This is due to the synthesis of key enzymes responsible for ROS clearance and the production of the key antioxidant and protectant, glutathione (GSH). GSH has multiple functions in detoxification of chemicals, and its depletion is associated with increased risks of toxicity. GSH is also a substrate in both conjugation and reduction reactions, catalyzed by glutathione-S-transferase (GST) enzymes in cytosol, microsomes, and mitochondria. GSH works synergistically with other cellular antioxidants such as catalase (CAT), superoxide-dismutase (SOD) and glutathione reductase (GR). Nodularin is actively transported into hepatocytes using membrane protein carriers, which may result in oxidative stress in the liver. CAT is a constituent of the enzymatic antioxidant defence system in vertebrates and minimizes the adverse effects of ROS by breaking down H2O2 into H2O and O2. Peroxidation of lipids is mediated through chain reactions of ROS, leading to damage of the cell. Malondialdehyde (MDA) is a well known oxidation product of polyunsaturated fatty acids (PUFAs) in lipoproteins and is often used as a biomarker of oxidative stress (Almroth et al., 2005). In order to decrease the toxicity, cells have defence systems that modify toxic substances to aid their excretion. Beattie et al. (2003) showed that the initial step in detoxification of nodularin in brine shrimp (Artemia salina) is conjugation to GSH via GST, which increases the water solubility and ultimate excretion. Several studies have also described GST detoxification of nodularin in crustaceans (Beattie et al., 2003; Pflugmacher et al., 2005) and molluscs (Davies et al., 2005; Kankaanpa¨a¨ et al., 2007) but the role of GST in the detoxification of nodularin in fish has not yet been reported. The aim of the present study was to investigate the occurrence of nodularin in flounders and blue mussels caught in Swedish ¨ resund during the summer of 2007. We also performed waters of O a laboratory experiment to examine the dynamics of nodularin mediated oxidative stress in flounder liver. The long-term (7 days) changes in GST and CAT activity and the formation of MDA in the liver of European flounder were investigated after exposure to nodularin. 2. Material and methods 2.1. Field sampling Adult European flounder (P. flesus) (weight: 297 67 g length: 309 20 mm, age: 5–7 years) were caught using gill nets (depth: 5– 7 m) and blue mussels (M. edulis) (length: 44 5 mm) were collected using a bottom scraper (depth: 5–7 m) at two locations situated in ¨ resund; Sweden Gette reef (558550 3300 N, 128470 2000 E) and Rydeba¨ck O (558580 4600 N, 128440 1900 E). Sampling took place between 30 July and 4 August 2007. Cyanobacterial blooms had occurred at both sites during 2006. Twenty live flounders were caught from each location
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and were swiftly transported in seawater tanks to the laboratory. Twenty blue mussels from each site were also collected and transported to the laboratory in a cooled water-free container. All fish and mussels were killed and dissected 1–2 h from sampling. Fish liver and muscle, and mussel soft tissues were removed and pooled into groups of 5 individuals per sample set giving 4 sample sets per location. All tissues destined for nodularin analyses were kept at 20 8C. 2.2. Experimental design to determine oxidative stress Flounders (weight: 259 41 g, length: 306 17 mm, age: 5–7 years) used in the oxidative stress experiment were caught using gill nets (depth: 5–7 m) on April 8 and April 12, 2007 at Gette reef in ¨ resund, Sweden (558550 3300 N, 128470 2000 E). The flounders were O placed in 4 aquariums (350 L, 5 or 10 individuals per aquarium) with aerated seawater. Fish were acclimatized for 8 weeks under constant conditions (t = 16.70 0.04 8C, O2 = 8.30 0.04 mg l1; pH 8.70 0.03; salinity 9.5 0.6; light:dark cycle = 16:8 h). Half the water volume in the aquaria was changed every second week. Seawater was ¨ resund (558560 2000 N, 128460 1800 E), collected during April and May in O and was prefiltered through a 200 mm nylon net to remove large plankton. Flounders were fed earthworms (Lumbricus terrestris, 2% of the flounder body weight every second day). The flounders were observed daily in terms of reaction to being touched and general physiological appearance such as body colour and swollen abdomens. 2.3. Exposure to nodularin The doses of pure nodularin (product number: N5148, Sigma– Aldrich1, Sweden) employed in the oxidative stress experiment were obtained from a preliminary dose-finding experiment in which fish that were intraperitoneally (i.p-) injected with nodularin concentrations >100 mg kg body weight1 died within 48 h (data not shown). The toxin dose was calculated based on the weight of each fish, and was injected in 0.5 ml of 0.9% (w/v) NaCl solution into the abdominal cavity. The control fish (n = 5) received only saline solution (0.5 ml of 0.9%, w/v, NaCl). The experiment was run for 4 days (4 aquaria, n = 25) followed by 10 days of recovery (1 aquarium, n = 5). The recovery group consisted of 5 fish that initially received a nodularin dose of 50 mg kg1. Flounders were i.p-exposed to two successive doses of nodularin (on day 1 and 4) corresponding to a total concentration of 2 mg kg1 (n = 5), 10 mg kg1 (n = 5), 50 mg kg1 (n = 10) and control (0 mg kg1, n = 5). Half the total dose was given on day 1 and the second half was given on day 4 of the experiment. The doses were divided into two rounds due to observations of the fish in the dose-finding experiment indicating the acute toxicity of the toxin. By dividing the toxin injection into two rounds we assumed that the fish would have an increased chance of surviving the duration of the experiment. Immediately after the fish had been sacrificed, 500 mg of liver from each fish were dissected out, weighed, rinsed with ice-cold saline (0.9%), immediately frozen to 80 8C and kept at 80 8C until analysis of GST, CAT and MDA. Liver (0.2– 5 g fish1 wet weight) and muscle (10 g fish1 wet weight) tissue were also dissected out for analysis of nodularin content and kept at 20 8C. 2.4. Preparation of cytosolic liver fractions Liver tissue was homogenised in Na/K-phosphate buffer (0.15 M, pH 7.3) containing KCl (0.1 M). Homogenates were centrifuged at 10 000 g for 20 min at 4 8C. The supernatant was transferred to a new tube and centrifuged at 105 000 g for 1 h at 4 8C. Samples of the final supernatant (cytosolic) destined for
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enzymatic and MDA analysis was kept at 80 8C, whereas samples for determination of protein content were stored at 20 8C. 2.5. Protein assay Protein content was analysed in the cytosolic liver fractions using the method described by Bradford and Williams (1976). Protein reagent was purchased from Bio-Rad Laboratories Inc. (Catalog No. 500-0006) and protein standards were generated using bovine serum albumin (BSA, CAS: 9048-46-8, Sigma–Aldrich, Sweden). The protein content was analysed spectrophotometrically at 595 nm and the results were calculated into mg protein ml1. 2.6. Glutathione-S-transferase assay (GST) GST activity was analysed in cytosolic liver fractions using the method described by Habig et al. (1974). Briefly, the conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) with reduced glutathione (GSH) was measured as an increase in absorbance at 340 nm. The rate of the increase in absorbance is directly proportional to the GST activity in the sample (Habig et al., 1974). 2.7. Catalase assay (CAT) The assay was performed according to the kit descriptions provided by Cayman chemicals1 (Catalog No. 707002). Briefly, the assay utilises the peroxidatic function of catalase in order to determine the enzyme activity. The enzyme is allowed to react with methanol in the presence of an optimal concentration of H2O2 to form formaldehyde. The formed formaldehyde is used as a measure of CAT activity and measured spectrophotometrically at 540 nm using 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole as the chromogen, which turns purple after oxidation. 2.8. Malondialdehyde assay (MDA) The concentration of MDA was used as an index of lipid peroxidation and was determined according to Ohkawa et al. (1979). The analysis was performed with an assay kit purchased from Cayman chemicals1 (Catalog No. 10009055) and used the determination of thiobarbituric acid reactive substances (TBARS). The determination of TBARS was performed according to the instructions provided with the assay kit. Briefly, the MDA-TBA adduct, which is formed by the reaction between MDA and TBA under high temperature (90–100 8C) and acidic conditions was measured colorimetrically at 530–540 nm.
water was produced by Millipore (Elga Purelab Option, Germany) Milli-Q plus equipment. Nodularin concentrations in liver tissues of flounder were initially expressed on a wet weight basis but were recalculated on a dry weight basis in order to allow for comparison with published literature. Flounder liver dry:wet weight ratios ¨ resund and derived from flounder caught in the same region of O during the same period of time were used in the calculations (Lundgren, 2007). 2.10. Statistical analysis The data were tested for statistical differences using one-way ANOVA followed by Tukey’s post hoc test for differences between the treatments in the oxidative stress experiment. The homogeneity of variances was tested with Levene’s test and test of sample normal distribution was tested with the Kolmogorov– Smirnov test. The statistics were performed using SPSS software package (version 14.0). The data showed a normal distribution. The level of significance was set at p < 0.05. 3. Results Nodularin concentrations in flounder livers (all individuals) collected in the field ranged between 22 and 557 mg kg1 liver (d.w) (Gette reef = 235 218 and Rydeba¨ck = 102 57 mg kg1 liver d.w, Fig. 1). No nodularin was detected in any of the flounder muscle samples nor in the soft tissue of blue mussels collected in the field. Nodularin was detected in the liver of 17 out of 18 fishes injected with nodularin in the oxidative stress experiment (range: 0– 780 mg kg liver d.w.1, Fig. 2). The fish in which nodularin was not detected belonged to the 2 mg kg1 treatment. Nodularin was also detected in 3 of 5 control fish (103 21 mg kg liver d.w. 1). No nodularin was detected in the muscle tissue of any of the fish used in the oxidative stress experiment. There were no significant differences in nodularin concentrations between the control and fish exposed to 2–50 mg nodularin kg1 between day 1 and 7 (p = 0.133, one-way ANOVA followed by Tukey’s post hoc test). The recovery group contained significantly higher nodularin concentrations compared to the control group (p = 0.035, one-way ANOVA followed by Tukey’s post hoc test). Two fish from the 50 mg nodularin kg1 treatment in the oxidative stress experiment died, one during the period after the first injection and another during the period after the second injection. Observed symptoms for the first mortality were swollen abdomen, immobility and loss of pigmentation. Observed symptoms for the second mortality were malnutrition (weight loss), loss of pigmentation and reduced mobility.
2.9. Nodularin analyses Samples of flounder liver (n = 20), flounder muscle (n = 20) and blue mussel soft tissues (n = 20) were collected from each sampling site in the field and analyzed for nodularin according to Kankaanpa¨a¨ et al. (2002). Each sample set (n = 20) were pooled into 4 groups (5 tissue samples per group). Individual liver (n = 23) and muscle tissues (n = 23) from the flounders in the oxidative stress experiment were also analyzed for nodularin according to Kankaanpa¨a¨ et al. (2002). Tissues (w.w.) were homogenised in water, methanol and n-butanol solution (75:20:5) (1 ml g1 w.w.) and extracted in a sonicator (Transsonic T 570/H, Elma1, Germany) (35 kHz) for 8 h at 60 8C. The HPLC system consisted of a Gynkotek P580 pump, a Gina 50 autosampler and a Waters Lambda max detector (model 481). The mobile phase consisted of 400 ml acetonitrile, 600 ml HPLC grade water and 10 ml trifluoroacetic acid with an isocratic flow rate of 1 ml min1. The column used was a Macherey-Nagel, Nucleosil 10 C-18 ET 250/8/4 column. The chromatograms were recorded at 238 nm. Ultra pure deionised
Fig. 1. Nodularin concentrations in the livers of flounders caught at two locations ¨ resund during the end of July and beginning of August (n = 20 at each location) in O 2007. The bars indicate the minimum and maximum nodularin concentrations. The line inside of the box represents the median value. The areas below and above the median are the lower and upper quartiles.
K.-J. Persson et al. / Harmful Algae 8 (2009) 832–838
Fig. 2. Nodularin concentrations in the livers of i.p-injected flounders 0 (n = 5), 2 (n = 5), 10 (n = 5), 50 (n = 4) and 50-Recovery (n = 4) mg nodularin kg1 b.w. The bars indicate the minimum and maximum nodularin concentrations. The line inside of the box represents the median value. The areas below and above the median are the lower and upper quartiles.
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Fig. 5. Malondialdehyde levels in the liver of flounders i.p-injected with nodularin 0 (n = 5), 2 (n = 5), 10 (n = 5), 50 (n = 4) and 50-Recovery (n = 4) mg nodularin kg1 b.w. The data are presented as average MDA concentration g1 liver SD.
50 mg nodularin kg1 were restored to control values (Fig. 2 AB, GST p = 0.996 and CAT p = 0.984, one-way ANOVA followed by Tukey’s post hoc test). As for MDA, no significant differences in MDA levels were found between the treatments (p = 0.204, oneway ANOVA, Fig. 5). 4. Discussion 4.1. Accumulation of nodularin in liver and muscle tissues
Fig. 3. The activity of GST in the cytosolic liver fractions of flounder i.p-injected with nodularin 0 (n = 5), 2 (n = 5), 10 (n = 5), 50 (n = 4) and 50-Recovery (n = 4) mg nodularin kg1 b.w. Data are shown as the average enzyme activity SD after 7 and 14 days (recovery) of toxin exposure.
The detoxification and antioxidant enzyme activities measured in liver (GST and CAT) were significantly lower in fish exposed to 50 mg nodularin kg1 than controls (Figs. 3 and 4, GST p = 0.018 and CAT p = 0.001, one-way ANOVA followed by Tukey’s post hoc test). No difference was found in GST and CAT activities between controls and fish exposed to 2 or 10 mg nodularin kg1 (Figs. 3 and 4, GST p = 0.466 and CAT p = 0.103, one-way ANOVA). After 10 days recovery, both GST and CAT activities in fish exposed to
Fig. 4. The activity of CAT in the cytosolic liver fractions of flounder i.p-injected with nodularin 0 (n = 5), 2 (n = 5), 10 (n = 5), 50 (n = 4) and 50-Recovery (n = 4) mg nodularin kg1 b.w. Data are shown as the average enzyme activity SD after 7 and 14 days (recovery) of toxin exposure.
This is the first study to report nodularin accumulation in the ¨ resund (range: 22– liver of flounder caught in Swedish waters of O 557 mg kg1 liver d.w.). Nodularin accumulates in tissues of various fish species from the Baltic Sea, including flounder (Kankaanpa¨a¨ et al., 2002; Sipia¨ et al., 2006; Mazur-Marzec et al., 2007; Sipia¨ et al., 2008). Blue mussels are the primary food source for flounders and it is assumed that the majority of nodularin found in flounders originates from mussels (Kankaanpa¨a¨ et al., 2005; Sipia¨ et al., 2008). Studies of nodularin accumulation in the Baltic Sea have shown that blue mussels and flounders are the most affected aquatic organisms containing nodularin concentrations in the range of 40– 2200 mg kg soft tissue1 (d.w) and 5–1100 mg kg liver1 (d.w), respectively (Sipia¨ et al., 2002; Kankaanpa¨a¨ et al., 2005; MazurMarzec et al., 2007). Detected nodularin concentrations in salmon (Salmo salar) and herring (C. harengus) range between 0–10 and 0– 90 mg kg1 (d.w), respectively (Sipia¨ et al., 2002, 2007). In fact, nodularin concentrations in muscle tissue of flounder have been reported to exceed the tolerable daily intake (TDI) value set for human consumption of microcystins (MC-LR), and thus by analogy nodularin (Sipia¨ et al., 2006). However, during the summer of 2007 N. spumigena occurred in very small amounts in the waters of ¨ resund (Swedish Meteorological and Hydrological Institute). In O addition, nodularin was not detected in blue mussels at the two sampling sites suggesting that residual nodularin remains in the liver of flounders during the winter months. This hypothesis is strengthened by the presence of nodularin in the control fish from the oxidative stress experiment caught in April since N. spumigena blooms during this time of the year are uncommon. It is possible that the fish accumulated nodularin during July 2006 when both blooming N. spumigena and decaying assemblages, derived from ¨ resund (Swedish Meteorological the Baltic Proper, occurred in O and Hydrological Institute). The presence of nodularin in fish liver could be due to the ingestion of contaminated mussels and/or of sedimented Nodularia mats while foraging on the sea floor. Nodularin was analysed in washed up cyanobacterial mats on
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the beach (Amager Strand, Copenhagen, Denmark) during the summer 2006. The mats consisted almost exclusively of N. spumigena and concentrations as high as 16 mg nodularin per g dry weight were measured, which are among the highest nodularin concentrations ever measured in the area (P. Henriksen, DMU, Denmark, personal communication). These high nodularin concentrations also indicate that the concentrations used in our oxidative stress experiments were comparable to environmental concentrations. In previous oxidative stress experiments, two fish (Pomphorhynchus laevis) were injected with 50 mg nodularin kg1 (Ohta et al., 1994). They showed symptoms such as swollen abdomens, loss of pigmentation, reduced mobility and died. This indicated that both fish died of liver failure and that nodularin had a negative impact on hepatocytes membrane integrity. Similarly, nodularin caused loss of liver architecture in sea trout orally exposed to a single dose of food containing N. spumigena (210–620 mg nodularin kg1 b.w.) in an 8day survey (Kankaanpa¨a¨ et al., 2002). MCs were also reported to cause liver necrosis and degeneration after intraperitoneal inoculation in carp (Carbis et al., 1996). In our study, autopsy revealed that one of the fish that died had several parasitic worms penetrating the gut wall. The occurrence of these worms is a common feature in ¨ resund area and they can cause peritonitis, flounder from the O which in turn could impact the immune system. This suggests that inter individual variation is likely to be involved in vulnerability for nodularin exposure. In addition to this, individuals exposed to nodularin annually with residual nodularin stored in the liver may have a compromised immune system. The only group that showed a statistically significant increase in nodularin concentrations compared to the control group in the oxidative stress experiment was the recovery group (50 mg nodularin kg bw1). Kankaanpa¨a¨ et al. (2002) investigated the heterogeneity of nodularin accumulation in flounders from the Baltic Sea and concluded that the individual-to-individual toxin content in liver tissue varied considerably. Possible reasons for these variations in nodularin concentrations are that the fish accumulate different amounts of the toxin during their time in the sea. Fish differ in their uptake of food and thus do not acquire toxins in a uniform manner (Sipia¨ et al., 2002). Even though the recovery group in the oxidative stress experiment showed increased concentrations of nodularin compared to the control group, the activities of GST and CAT returned to control levels, after the 10 days recovery period. This suggests that although increased amounts of nodularin were present in the livers of the recovery fish, fish managed to increase the activities of GST and CAT, presumably in an attempt to detoxify the toxin and deal with formed ROS. The flounders used in the oxidative stress experiment contained background concentrations of nodularin, which raise the question whether the fish was compromised in some way from previous exposure. We recommend that future research is conducted to investigate whether/how residual nodularin can compromised the health of individual fish. 4.2. GST activity The activity of GST in the cytosolic liver fractions of flounders, i.p-injected with 50 mg nodularin kg1, significantly decreased compared to the control group. Earlier studies have shown decreases in GST activity in green mussels (Perna viridis) exposed to N. spumigena extracts (Davies et al., 2005) and in brine shrimp (A. salina) exposed to pure nodularin through immersion (Beattie et al., 2003). Similarly, previous research on MC-LR has shown decreases in GST activity in the liver of i.p-injected goldfish (Carassius auratus) (Malbrouck et al., 2003). MC-RR has been shown to cause reductions in GST activity in the liver, gills,
intestine and brain of Peppered Corydoras (Corydoras paleatus) exposed through immersion (Cazenave et al., 2005). However, Kankaanpa¨a¨ et al. (2007) did not observe any change in GST activity in blue mussels, exposed to N. spumigena extract. It was therefore concluded that GST is not significantly involved in the detoxification process of nodularin in blue mussels. Other studies have shown that the detoxification of cyanobacterial toxins in the liver of mice occur through conjugation to the tripeptide glutathione (GSH) via GSTs (Jayaraj et al., 2006). GSTs catalyze the addition of MCs to the SH (thiol) group of GSH. This leads to neutralisation of the electrophilic sites of MC, which increases the water solubility and facilitates excretion (Wiegand et al., 1999). The liver is already defined as the target organ for nodularin and several studies have demonstrated the presence of a conjugated nodularin-glutathione formed enzymatically via soluble GST in different aquatic organisms (Sipia¨ et al., 2002; Beattie et al., 2003; Davies et al., 2005). This conjugation of nodularin with GSH, catalyzed by GST, seems to be the first step of detoxification of this toxin in various aquatic organisms. In this context, GST would be a specific biomarker for contamination by nodularin. However, Mazur-Marzec et al. (2007) did not find any nodularin conjugates with reduced GSH in flounder or blue mussels from the Gulf of Gdansk and suggested that other biotransformation products of nodularin might occur. A decrease in GST activity could be explained by reduced synthesis of GST protein at a molecular level (Gallagher et al., 2000). Down regulation of gene expression for GSTs has been shown in the liver of mice i.p-exposed to MC-LR (38.31 mg kg1, Jayaraj et al., 2006). In addition, nodularin is known to cause inhibition of protein phosphatases (Falconer et al., 1983), which could lead to phosphorylation of GST subunits and consequently changes, in GST activity (Beattie et al., 2003). Reduced capability of fish to eliminate toxins could increase the risk for bioaccumulation and consequently the risk of cellular damage, physiological alterations and behavioural changes (Cazenave et al., 2006 and references therein). Modulation of detoxification enzymes could also result in adverse effects such as increased susceptibility to ROS formation, increased energetic demand, proliferation of cells, and also susceptibility to other xenobiotics (Van der Oost et al., 2003). GST was restored to control values 10 days after the last injection of nodularin. Thus, it seems as if the GSH based detoxification process using GST was able to cope with the applied toxins. Our results agree with the findings of Malbrouck et al. (2003) who found a recovery of GST activity in goldfish i.p-injected with 125 mg MC-LR kg1 (b w). 4.3. Catalase activity The activity of CAT decreased in fish injected with 50 mg nodularin kg1. This result agrees with earlier research with mice, i.p. exposed to nodularin (Lankoff et al., 2002), with MCLR (Jayaraj et al., 2006) and tilapia fish (Oreochromis niloticus) orally exposed to MC-LR (Prieto et al., 2007). The observed decrease in CAT activity suggests nodularin-induced oxidative stress in terms of increased amounts of ROS that are scavenged by CAT. Several studies have shown that CAT is inactivated by singlet oxygen and peroxyl radicals due to a direct damaging effect of ROS on the enzyme (Kono and Fridovich, 1982; Escobar et al., 1996). Jayaraj et al. (2006) suggested that a decrease in CAT could be due to increased amounts of superoxide anions as a consequence of a reduction in the activity of SOD. In contrast to other studies that have shown increases in CAT activity in fish exposed to MCs (Li et al., 2004; Jos et al., 2005; Prieto et al., 2006), flounders in the present study exposed to an i.pinjection of nodularin showed a decrease in CAT activity. Oxidative
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stress is therefore induced by nodularin and MCs in the different types of experimental models even though the antioxidant defence system shows different responses. These discrepancies could be due to differences in toxicity profiles between the two toxins, experimental conditions, doses employed and/or interspecies differences in susceptibility to the acute effects of these toxins (Kankaanpa¨a¨ et al., 2007). Cazaneve et al. (2006) have found a dual response of CAT activity in the liver of Peppered Corydoras after exposure to dissolved MC-RR, with an increase at low concentrations and depletion at higher levels. Higher toxin doses could involve damage on the enzyme proteins while lower doses administrated during a longer period of time may induce a defensive response (Prieto et al., 2007). The present study indicates that fish in the recovery group were able to restore the CAT activity to control levels after 10 days. This finding agrees with observed recovery of CAT between 24 h and 72 h post orally exposure of an acute dose of MC-LR (2400 mg MCLR kg1) in tilapia fish (Oreochromis niloticus) (Prieto et al., 2007) and a decrease in CAT activity after an initial increase due to exposure of N. spumigena extract in blue mussels (Kankaanpa¨a¨ et al., 2007). 4.4. MDA formation Earlier studies have demonstrated oxidative stress in terms of increases in MDA levels due to lipid peroxidation after exposure to nodularin in rat hepatocytes (BouaI¨cha and Maatouk, 2004) and in green mussels (Davies et al., 2005). However, MDA levels in livers of flounder exposed to nodularin did not differ from controls in the present study. The incapacity to initiate lipid peroxidation could be due to an intracellular decrease in ROS, which are known to attack lipids and initiate their peroxidation (BouaI¨cha and Maatouk, 2004). Ding et al. (2001) observed an early increase in ROS levels in primary cultured rat hepatocytes treated with low doses of MCs. The maximum ROS levels were reached after 10 min and sustained for 20 min, after which depletion in ROS levels occurred. Thus, this suggests that the increasing number of ROS, due to toxin exposure, is efficiently deactivated by the defence system of the cells, preventing MDA formation. In the present study the restoration of GST and CAT activity after 10 days and constant levels of MDA indicate that the antioxidant defence system managed to prevent oxidative stress in terms of MDA formation in flounder livers. Similarly, Li et al. (2005b) showed no increased MDA levels in the liver of loach (Misgurnus mizolepis), orally exposed to the cyanobacterium Microcystis and consequently concluded that the antioxidant enzymes were able to eliminate oxidative stress and thereby preventing increased MDA levels. 5. Conclusions ¨ resund during the summer of Livers from flounders caught in O 2007 contained nodularin, possibly originating from the summer of 2006, when in contrast to 2007, vast N. spumigena blooms were present at the sampling sites. This indicates the persistent nature of nodularin in flounder liver tissue. i.p.-injected nodularin induced reversible alterations of the activities of GST and CAT in flounder liver tissue and therefore suggests the role of oxidative stress in nodularin mediated toxicity. The restoration of GST and CAT activity after 10 days and constant levels of MDA indicated that the antioxidant defence system managed to prevent oxidative stress in terms of MDA formation in flounder livers. However, alteration of the enzymatic defence system might lead to increased energetic costs which could reduce fish growth and survival. The present study also suggests that using oxidative stress biomarkers
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could allow the detection of an early response in fish to nodularin exposure. Future studies should include more natural routes of exposure rather than i.p-injection (e.g. direct water contact, exposure to contaminated mussels/sediment) to demonstrate whether nodularin can be taken up through vectoral transfer under ‘artificial’ bloom conditions. Acknowledgements We thank Per Carlsson, Lund University and Ingrid Trulsson, Daniel Kuster, Fredrik Lundgren and Anna Wolfhagen, Toxicon AB for expert advice and excellent technical assistance. We are grateful to P. Henriksen (Danish Environmental Protection Agency, Roskilde, Denmark) for sharing unpublished data. This study was funded by the Region Ska˚ne (Per Carlsson), Toxicon AB (Thomas Olsson) and the Swedish Research Council for environment, Agricultural Sciences and Spatial Planning (Formas, Catherine Legrand).[SS] References Almroth, C.M., Sturve, J., Berglund, A˚., Fo¨rlin, L., 2005. 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