This article appeared in a journal published by Elsevier.

The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues. Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. In most cases authors are permitted to post their version of the article (e.g. in Word or Tex form) to their personal website or institutional repository. Authors requiring further information regarding Elseviers archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/copyright

Author's personal copy

Chemosphere 89 (2012) 3843

Contents lists available at SciVerse ScienceDirect

Chemosphere
journal homepage: www.elsevier.com/locate/chemosphere

Induction to oxidative stress by saxitoxin investigated through lipid peroxidation in Neuro 2A cells and Chlamydomonas reinhardtii alga
Silvia P. Melegari a, Franois Perreault b, Serge Moukha c,d, Radovan Popovic b, Edmond E. Creppy c, William G. Matias a,
a

Laboratory of Environmental Toxicology, LABTOX, Department of Sanitary and Environmental Engineering, Campus Universitrio Trindade, CEP 88040-970 Florianopolis, SC, Brazil Department of Chemistry, University of Quebec in Montreal, C.P. 8888, Succ. Centre-Ville, Montreal, Quebec, Canada H3C 3P8 Laboratory de Toxicologie et dHygine Applique, UFR des Sciences Pharmaceutiques, Universit Victor Segalen Bordeaux 2, 146 rue Lo Saignat, 33076 Bordeaux Cedex, France d INRA, Centre de Recherche de Bordeaux Aquitaine, MICA, Unit de Mycologie et de Scurit des Aliments, Domaine de la grande Ferrade, 71 avenue Edouart Bourleaux BP 81, 33883 Villenave dOrnon Cedex, France
b c

a r t i c l e

i n f o

a b s t r a c t
Saxitoxin (STX) is a cyanotoxin, which can cause neurotoxic effects and induce ecological changes in aquatic environments, a potential risk to public and environmental health. Many studies of cytotoxicity on animal cells and algae have been performed, although few compare the toxic effects between the two models. In this sense, we investigated the oxidative stress induced by STX (0.43.0 nM) in two different cellular models: Neuro-2A (N2A) cells and Chlamydomonas reinhardtii alga by quantication of malondialdehyde (MDA) levels as indicative of lipid peroxidation (LPO). Also was evaluated the antioxidant defense of these cells systems after exposure to STX by the addition of antioxidants in N2A cells culture, and by the measure of antioxidants enzymes activity in C. reinhardtii cells. The MDA levels of N2A cells increased from 15% to 113% for 0.4 and 3.0 nM of STX, respectively, as compared to control. Superoxidedismutase and catalase did not appear to protect the cell from STX effect while, in cells treated with vitamin E, the rates of MDA production decreased signicantly, except for higher concentrations of STX. No MDA productions were observed in algal cells however some effects on antioxidant enzymes activity were observed when algae were exposed to 3.0 nM STX. Our results indicate that the concentrations of STX that may induce oxidative stress through LPO are different in animal and phytoplankton communities. A combination of algal and animal bioassays should be conducted for reliable assessment of oxidative stress induced by STX. 2012 Elsevier Ltd. All rights reserved.

Article history: Received 11 January 2012 Received in revised form 22 March 2012 Accepted 3 April 2012 Available online 28 April 2012 Keywords: Saxitoxin Oxidative stress Lipid peroxidation Neuro-2A cells Chlamydomonas reinhardtii green alga

1. Introduction The frequency of toxic blooms of cyanobacteria in freshwater destined for human consumption increases evidently worldwide, probably due to the increase of environmental pollution and global warming (Lagos et al., 1999; Pereira et al., 2000; Tucci and Santanna, 2003; Hoeger et al., 2005; Molica et al., 2005; Berger et al., 2006;

Abbreviations: STX, saxitoxin; N2A, Neuro-2A; MDA, malondialdehyde; SOD, superoxide dismutase; CAT, catalase; APX, ascorbate peroxidase; GR, glutathione reductase; GST, glutathione S-transferase; GPx, glutathione peroxidase; GSH, glutathione reduced form; RS, reactive species; ROS, reactive oxygen species; LPO, lipid peroxidation; TBA, thiobarbituric acid; HPLC, high performance liquid chromatography. Corresponding author. Address: Laboratrio de Toxicologia Ambiental, LABTOX, Depto. de Engenharia Sanitria e Ambiental, Universidade Federal de Santa Catarina, Campus Universitrio Trindade, CEP 88040-970 Florianpolis, SC, Brazil. Tel.: +55 48 37217742; fax: +55 48 37219823. E-mail address: will@ens.ufsc.br (W.G. Matias). 0045-6535/$ - see front matter 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.chemosphere.2012.04.009

Costa et al., 2006). Blooms of cyanobacteria create severe practical problems to those in charge of water supply to populations because of water contamination by different toxic compounds. Cyanobacterial toxins, or cyanotoxins consist of diverse groups of chemical structures, each of them bearing specic mechanisms of cytotoxicity (Bartram et al., 1999; Sivonen and Jones, 1999; Carvalho Pinto-Silva et al., 2003). Saxitoxin (STX), which was rst identied in freshwater contaminated by Aphanizomenon os-aquae (Ikawa et al., 1982), is a neurotoxin classied among in the Paralytic Shellsh Poisoning (PSP) toxins. Apart from A. os-aquae, STX can be synthesized by other species of cyanobacteria such as Cylindrospermopsis raciborskii, Lyngbya and Anabaena (Sivonen and Jones, 1999; Bouacha, 2001). The fast action of STX, blocking sodium channels in nerve axons and causing loss of sensation and paralysis, confers to this toxin a highly neurotoxic and potentially lethal effect 225 h after ingestion. The LD50 in mice is of 810 lg kg1 i.p., 3.4 lg kg1 i.v. and 260 lg kg1 by oral route (Falconer, 2008). Data regarding the specic cytotoxic and genotoxic effects of STX are very scarce. For

Author's personal copy

S.P. Melegari et al. / Chemosphere 89 (2012) 3843

39

example, there is neither evidence of human health effects caused directly by the consumption of water containing STX-producing cyanobacteria or PSP-producing dinoagellates, and cases of human toxicity were only reported for paralytic shellsh poisoning events (Fitzgerald et al., 1999). There are no data related to the concentrations of STX-like neurotoxins in drinking water that have caused neurotoxic symptoms in populations exposed to public water supply (Falconer and Humpage, 2005). Rapala et al. (2005) reported the rst quantitication of STX in cyanobacterial blooms in Finland, with presence of STX in concentrations of 1 mg L1 in bloom samples of Finnish Lakes. Symptoms of swimmers from the sites contaminated with STX were reported as fever, eye irritation, abdominal pains, and skin rash in children aged 210 years, after exposure to the water, but these were not the adverse human symptoms typical of STX poisoning. Recent data published in the International Symposium on Cyanobacterial Harmful Algal Blooms (ISOC-HABs), organized by the Environmental Protection Agency of the United States of America (USEPA), reported that there are no data on sub chronic exposure, reproductive, teratogenic or carcinogenic effects of STX (Falconer, 2008). The European Food Safety Authority (EFSA, 2009) published the scientic opinion about STX-group in shellsh and reported that the toxicological database for STX-group toxins is limited and comprises mostly studies on their acute toxicity following intraperitoneal administration. There are many methods to investigate the oxidative stress induced by production of reactive species (RS). The in vitro test have been demonstrated to be satisfactory as an alternative to the in vivo assay, and they have the advantages of being fast and sensible, excluding the ethical questions that emerge from assays involving animal experimentation (Smart and Hodgson, 2008). Malondialdehyde (MDA) is a natural biomarker produced in the reaction of lipid peroxidation (LPO), which can be quantied and used for the evaluation of this process. It is mutagenic in bacteria and mammalian cells and carcinogenic in mice (Valko et al., 2006). It also modies DNA bases, generating bifunctional intermediates that form exocyclic DNA adducts contributing to mutagenic and carcinogenic process (Valko et al., 2006; Nair et al., 2007). Antioxidants are substances that delay and/or prevent the oxidation of cellular substratum at low concentrations. Antioxidants are widely distributed in living organisms and are extremely important as representatives in the direct removal of free radicals (Valko et al., 2006; Halliwell and Gutteridge, 2007). The main enzymatic antioxidants are superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and the enzymes involves in process of gluthatione metabolism (glutathione reductase GR, glutathione transferase GST and glutathione peroxidase GPx), which serve as primary defense in the destruction of RS (Valko et al., 2006). Non-enzymatic antioxidants, such as glutathione reduced (GSH), tocopherol (vitamin E) and ascorbic acid (vitamin C), among others, assist in the combat against reactive oxygen species (ROS) and reactive nitrogen species (Mller and Wallin, 1998; Matias et al., 1999; Valko et al., 2006; Halliwell and Gutteridge, 2007). Recently, we investigated the sensitivity of algal and animal cells towards STX effect and showed that algae are much more resistant to STX than animal cells (Perreault et al., 2011). In this study we investigated the oxidative stress induced by STX in two different cellular models to determine how the antioxidant defense system may participate in this difference of sensitivity. Here, Neuro-2A (N2A) cells and the green alga Chlamydomonas reinhardtii were exposed to STX effect and oxidative stress was investigated by quantication of MDA levels as an indicator of LPO. Also was evaluated the antioxidants defense of these cells systems after exposure to STX by addiction of some antioxidants in N2A cells culture, and by measure of antioxidants enzymes activity in C. reinhardtii cells.

2. Material and methods 2.1. Cells models and culture medium All the chemical products used for the cell culture were provided by SigmaAldrich (Saint Quentin Falavier, France). STX (NRC CRM-STX-e) was obtained from the Institute for Marine Biosciences (National Research Center, Halifax, NS Canada). N2A cells from the mouse neuroblastoma cell line were obtained from ATCC (CCL-131, not differentiated LGC Promochem, France). The cells were grown in 75 cm2 asks, with a RPMI 1640 culture medium supplemented with 10% of fetal bovine serum, 2% of L-glutamine solution (200 mM), 1% of sodium pyruvate solution (100 mM) and 1% of antibiotic solution (10 mg mL1 streptomycin and 10 000 U mL1 penicillin). The culture was maintained at 37 C in a humidied CO2 (5%) atmosphere (Adapted from Ledreux et al., 2009). C. reinhardtii wild type (CC-125) strains were obtained from the Chlamydomonas Genetic Center (Duke University, Durham, NC, USA). Cells were cultivated in batch culture of 1 L of high salt growth medium (HSM) according to Harris (1989). Cultures were exposed to continuous illumination (100 lE m2 s1) provided by white uorescent lamps (Sylvania Grolux F 36 W) at 23 C 1. The culture was permanently aerated to obtain a constant CO2 concentration in the growing medium. 2.2. Evaluation of LPO and antioxidant defense system For N2A cells, 1 105 cells mL1 were cultured in 24-well microplates for 24 h at 37 C as described above, and then cultures were separately incubated with different protective substances: vitamin C (8 and 16 lg mL1, respectively VitC8 and VitC16), vitamin E (6 lg mL1, VitE) and enzymes SOD/CAT (25 lg mL1 each). A negative control was employed using culture medium, and a positive control was employed using ochratoxin A (OTA = 10 lM). The N2A cells were intoxicated for 24 h at 37 C with STX in the concentrations of 0.4, 0.8, 1.5 and 3.0 nM. After 72 h incubation, the cells were scrapped and centrifuged at 2000g for 5 min. The cell pellets were resuspended in 250 lL SET buffer (0.1 M NaCl, 20 mM EDTA, 50 mM TrisHCl, pH 8.0). To each sample 25 lL of 7% SDS (w/v), 300 lL of 0.1 M HCl, 40 lL of 1% phosphotungstic acid (w/ v) and 300 lL of 0.67% thiobarbituric acid (TBA, w/v) were added. The tubes were shaken and incubated at 90 C for 1 h in the dark. They were further placed for 15 min in an ice bath (0 C). After this period, a volume of 300 lL of n-butanol was added and tubes were shaken vigorously. After centrifugation for 10 min at 900g at 4 C, the n-butanol phase from each sample containing the MDATBA adduct was separated and analyzed through high performance liquid chromatography (HPLC) with uorometric detection. The HPLC system consisted of an ICS (Instrumentation Consommable Service, France) chromatography pump equipped with a Shimadzu 8450 Fluorescence HPLC Monitor (Japan Spectroscopic Co.). The analysis of the MDATBA adducts was performed at room temperature on a Prontosil Eurobond C18 column (Bischoff Chromatography, Germany, 250 4.6 mm, part: 5 lm) using 40:60 methanolwater (v/v) as the mobile phase. The pH was adjusted to 8.4 through the addition of 0.5 M KOH. The ow rate was maintained at 0.5 mL min1 by an automatic injection of 90 lL. The excitation and emission wavelengths were of 515 and 553 nm, respectively (Adapted from Matias et al., 1999). The amount of MDA-TBA measured is related to the protein content of cellular homogenates, determined using Bradfords colorimetric method (Bradford, 1976). For algal culture, aliquots of 20 mL of the (2.5 105 cells mL1) in the exponential growth phase were exposed to 0.4, 0.8, 1.5 and

Author's personal copy

40

S.P. Melegari et al. / Chemosphere 89 (2012) 3843

3.0 nM of STX for 24, 48 and 72 h under the same illumination and temperature conditions used for growing stock cultures. For the control, the same growth medium and conditions were used without STX. During the exposures, changes in the ratio between cell number (biomass) and concentration of STX were negligible. All concentrations and controls were evaluated in triplicates. The LPO analysis was performed by determination of MDA level, using the uorescent dye BODIPY 581/591C11 (4,4-diuoro5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3undecanoic acid-Invitrogen Molecular Probe, USA). BODIPY is a uorescent fatty acid analog with uorescent properties in the red and green ranges of the visible spectrum (emission maximum at 595 nm), which after oxidation induced by ROS, its uorescence changes from red to green (Domnguez-Rebolledo et al., 2010). After 24, 48 and 72 h of treatment, 0.5 mL samples of algal cultures bioassays were exposed 30 min to 10 lL BODIPY (2 mM) in dark. Intracellular MDA level was determined by measuring the uorescence emission at 595 nm with a ow cytometer (FacScan; Becton Dickinson Instruments, USA). Cytometry results were analyzed using the WinMDI 2.8 software. To discriminate between algal cells and noncellular particles, all measurements were separated by using a relationship between particle size and red uorescence level, originating from chlorophyll uorescence reemission.

GST activity was evaluated by the change of absorbance at 340 nm due to conjugation of GSH to 1-chloro-2,4-dinitrobenzene (CDNB). GST activity was calculated using an extinction coefcient of 9.6 mM1 cm1 from CDNB. 1 lg protein (%50 lL of enzyme extract) was added to 25 lL GSH (40 mM), 10 lL CDNB (40 mM) and sufcient volume of sodium phosphate buffer (50 mM, pH 7.5) to a nal volume of 1 mL. The formation of GS-DNB conjugate was monitored each 30 s until 300 s. One GST unit was dened as the enzyme amount that transforms 1 mmol of CDNB per min. 2.4. Statistical analysis All the results were expressed as the mean SEM and the differences between the results of MDA levels and the control groups were compared with ANOVA one-way analysis of variance followed by Tukeys post hoc test for multiple comparisons. A significance level of p P 0.05 was accepted. 3. Results 3.1. STX effect in N2A cells After 24 h of incubation, the N2A cells in culture spontaneously produced 83.8 5.9 nM MDA mg protein1. This value was considered the control value for N2A cells without STX. After 24 h of exposure to the VitE (6 lg mL1), the N2A cells in culture spontaneously produced 59.1 1.6 nM MDA mg protein1. As for the N2A cells in culture with VitC8 (8 lg mL1) and VitC16 (16 lg mL1), they spontaneously produced 89.8 9.4 and 107.3 17.3 nM MDA mg protein1, respectively, while the N2A cells in culture with SOD/CAT spontaneously produced 102.5 10.9 nM MDA mg protein1. All these values were considered the control value for N2A cells without STX. After 24 h of incubation, with increasing STX concentrations, the MDA production in N2A cells also increased signicantly (Fig. 1). The production of MDA increased by 15113% for 0.38 and 3.0 nM of STX, respectively, when compared to controls (Table 1). When N2A cells were pretreated with VitC8, this rate increased by 66209% in presence of the same concentration of STX. When N2A cells were pretreated with VitC16, the rates of MDA production increased from 36181% for the same concentrations of STX (Table 1). The pretreatment of N2A cells with SOD/CAT showed no significant decrease of MDA production compared with other treatments with the same concentration of STX (Fig. 1), even with attenuated decline in the rate of production of MDA compared with the control group (Table 1). When the N2A cells were pretreated with VitE a signicant decrease of production rates of MDA were observed for the lowest STX concentrations, when compared to the condition without antioxidants. For VitE, the production rate of MDA was of 3%, 31%, 95% and 120% to the concentrations of STX of 0.4, 0.8, 1.5 and 3.0 nM STX (Table 1), respectively. 3.2. STX effect in C. reinhardtii cells We evaluated STX effect on ROS production in C. reinhardtii cells using BODIPY uorescent probe, which is specic to MDA production. When C. reinhardtii was exposed to STX (from 0.8 to 3.0 nM), MDA level was not signicantly altered when compared to the negative control, even after 72 h of treatment (p > 0.05, Fig. 2). Four different biomarkers were employed to verify the modication of enzymatic activity of C. reinhardtii when exposed to STX. This biomarkers act in cellular defense system under conditions of oxidative stress. Differences statistically signicant could

2.3. Enzymes activity assays in algae After 72 h of exposure to STX, the algal cultures were collected, centrifuged, then pellet was resuspended in 500 lL of 0.1 M sodium phosphate buffer (pH 7) with addition of 500 lL of glass beads (Sigma) and algal cells were mixed for 10 min at 4 C by using a vortex. Enzymes were extracted within 1 mL of sodium phosphate buffer and centrifuged at 2300g for 20 min at 4 C. For enzyme activity measurements the supernatant was separated and stored at 80 C. Protein content was determined according to Bradford (1976). CAT activity was evaluated spectrophotometrically by measuring the consumption of H2O2 at 240 nm and calculated using an extinction coefcient of 0.0436 mM1 cm1 according to Aebi (1984). 1 lg protein (%50 lL of enzyme extract) was added to 100 lL of H2O2 (200 mM) and sufcient volume of sodium phosphate buffer (50 mM, pH 7.5) to a nal volume of 1 mL. The consumption of H2O2 was monitored each 5 s until 30 s. One CAT unit was dened as the enzyme amount that transforms 1 mmol of H2O2 per min. APX activity was evaluated by the change of absorbance at 290 nm due to ascorbate oxidation and calculated using an extinction coefcient of 2.8 mM1 cm1 adapted of Nakano and Asada (1981). 1 lg protein (%50 lL of enzyme extract) was added to 50 lL Na-ascorbate (20 mM), 50 lL H2O2 (100 mM) and sufcient volume of sodium phosphate buffer (50 mM, pH 7.5) to a nal volume of 1 mL. The consumption of H2O2 was monitored each 5 s until 90 s. One APX unit was dened as the enzyme amount that transforms 1 mmol of ascorbate per min. GR activity was measured by the change of absorbance at 412 nm due to the reduction of 5, 5-dithiobis (2-nitrobenzoic acid) (DTNB) by GSH in 2-nitro-5-thiobenzoic acid (Smith et al. 1988). 1 lg protein (%50 lL of enzyme extract) was added to 100 lL NADPH (1 mM), 50 lL DNTB (15 mM), 50 lL glutathione oxidized form (GSSG) (20 mM) and sufcient volume of sodium phosphate buffer (50 mM, pH 7.5) to a nal volume of 1 mL. The consumption of DTNB was monitored each 10 s until 120 s and the GR activity was calculated using an extinction coefcient of 14.15 mM1 cm1. One GR unit was dened as the enzyme amount that transforms 1 mmol of DTNB per min.

Author's personal copy

S.P. Melegari et al. / Chemosphere 89 (2012) 3843

41

Fig. 1. LPO levels induced by increasing STX concentrations in N2A cells, and with several antioxidants, as measured by quantication of MDA levels, after 24 h of incubation (p < 0.05 in relation with the condition no antioxidants added).

Table 1 LPO measured by the increase of MDA production (%), when compared to negative control. STX (nM) Increase MDA production (%) Without antioxidants Vit. E 6 lg mL1 Vit. C 8 lg mL 0.38 0.75 1.50 3.00 Positive control 15 93 99 113 120 3 31 95 120 358 36 109 163 181 298
1

SOD + CAT 25 ug mL1 (each) 16lg mL 66 143 154 209 374

1

3 47 75 160 129

Fig. 2. Change of the intracellular MDA level in C. reinhardtii cultures exposed to STX concentrations, measured by BODIPY uorescence probe.

be observed in the activity of these antioxidants enzymes when alga was exposed to the major concentration of STX tested (p < 0.05, Fig. 3). CAT showed a tendency to decrease its activity in a dose-dependent manner upon exposure to STX, with signicant decrease in concentrations of 0.8 and 3.0 nM. The decrease of CAT activity in the STX concentration of 1.5 nM was observed visually, but it was not signicant (p = 0.087, Fig. 3). This discrepancy cannot be explained at this time, but do not affect the general understanding of defense mechanism of alga that decreases its activity in response to increasing concentrations of STX. The APX, GR and GST show signicant change in its activities only in the high STX concentration, an increase of their activities compared to control.

4. Discussion The cytotoxic and genotoxic effects of STX in N2A cells and C. reinhardtii alga have recently been shown and the data suggested that STX induces indirect genotoxic effects in N2A cells to concentrations ranging from 0.5 to 64 nM (Perreault et al., 2011). Again, the toxicological response indicates that the interaction of STX with algal cells may be very different than with animal cells (Taylor, 2009), showing that different cellular systems show signicant differences in their response towards pollutants, at similar STX concentrations. Induction of LPO by STX in neuronal and algal cells line and the correlation with antioxidant defense mechanisms have not yet

Author's personal copy

42

S.P. Melegari et al. / Chemosphere 89 (2012) 3843

Fig. 3. Effect of STX on Catalase (CAT), Ascorbate peroxidase (APX), Glutathione reductase (GR), and Glutathione S-transferase (GST) activities of C. reinhardtii at 72 h of exposure to different STX concentrations (p < 0.05).

been reported in the literature, but it is known that the nervous system are incline to oxidative stress due to the lack of an adjusted antioxidant defense system (Halliwell, 2006). LPO is characterized by the process where RS attack polyunsaturated fatty acids of the cellular membrane, inducing a chain reaction with lipid hydroperoxides as intermediate products (Halliwell, 1987). This process can also result in the destabilization and disintegration of the cellular membrane, bringing serious health problems to man, from aging to susceptibility to cancer (Valko et al., 2006). When initiated, the LPO forms peroxyl free radicals (ROO) that can be rearranged through a cyclization reaction of endoperoxides, producing, among other nal products of this process, MDA. The MDA levels, which increase as a consequence of the LPO for oxidative stress, were signicantly more elevated in N2A cells exposed to STX than in controls without toxin. All the results show that after 24 h of incubation, STX induces LPO in N2A cells. Endogenous and exogenous substances can produce ROS proceeding from LPO (Mller and Wallin, 1998). STX acts as an exogenous source of oxidative stress, yielding ROS that are probably responsible for the LPO. When produced inside of the cells, ROS are capable to virtually react with all types of biomolecules (Mller and Wallin, 1998), organelles and enzymes including mitochondria, cytochrome P450 involved in drug metabolism, peroxisomes, and inammatory cells activation systems (Inoue et al., 2003). Neurotoxins generally lead to the formation of ROS in several clinical conditions of the nervous system (Halliwell, 1987). The exposition of N2A cells to STX induced an evident ROS production and peroxidation of the lipid membranes. A MDA overproduction can be stimulated by these effects, as suggest by Halliwell (2006). The MDA can be toxic to cell, causing a inhibition of protein synthesis, some DNA adducts may be formed, which may lead to genotoxic, mutagenic and carcinogenic processes (Baudrimont et al., 1997; Matias et al., 1999; Halliwell, 2007). High levels of oxidative damage can result not just from oxidative stress, but also from the limitation of the cellular repair system, and this dysfunction may cause a deregulation of the cell defense system, leading to cell death (Halliwell, 2007). Matias et al. (1999) demonstrate the efciency of SOD/CAT combination in the protective effect of Vero cells against the LPO effects of okadaic acid, our results with SOD/CAT does not appear

to affect the defense system of N2A cell against STX, because the change in the production of MDA was not signicant compared to other treatments with the same concentration of STX (except treatment VitE). Vit E seemed efcient in antioxidant defense system to scavenge MDA. MDA levels increased just 3% in the minor concentration of STX, in relation with the negative control when VitE was present. However, Halliwell (2008) afrms that in vivo studies have no evidence of systemic pro-oxidant effects of avonoids in humans, and little or no clear evidence of antioxidant effects. The pre-treatment of N2A cells with VitE, seem efcient in the in vitro prevention against the LPO when compared to VitC. VitC was not efcient for the protection of N2A cells in our experimental conditions. This is most probably due the switch from antioxidant to pro-oxidant according to concentrations and RS. In specic conditions (in the presence of intracellular Cu, [CuZnSOD] for example in mitochondrial membrane and in cytosol) the ascorbate can be oxidized, producing cytotoxic species (Halliwell, 2006). In this case, VitC will synergize the toxic effects of STX (Kojo, 2004). VitC cooperates with VitE to regenerate a-tocopherol from the a-tocopherols radicals in the membranes and lipoproteins. A mixture of both vitamins should be tested further in the presence of STX. STX is known as a natural defense mechanism of phytoplankton to eliminate other competitors by allelopathic processes in cases of disturb of ecological balance to become the dominant organism from the environment (Pohnert, 2004). The change in enzymatic activity of algae by exposure to STX can be understood as a biological response of this microorganism to metabolize the toxin. It is speculated that the catabolism of the PSP most likely occurred via oxidation reactions catalyzed by oxidases and peroxidases into aliphatic products for subsequent use in purine and arginine metabolism (Donovan et al., 2008; Wiese et al., 2010). In this case were not observed effects of oxidative stress like LPO, but changes in enzyme activity, possibly in an attempt to neutralize free radicals formed from STX. Overall, our results suggest that high concentrations of STX (P3.0 nM) can affect the algal defense system of the species C. reinhardtii, breaking the balance of this system and causing reactions of oxidations. There are no evidences reported of this concentration

Author's personal copy

S.P. Melegari et al. / Chemosphere 89 (2012) 3843

43

on algae blooms of freshwaters on bibliography, but some authors reported in algal blooms with STX on freshwaters in several countries (Lagos et al., 1999; Kaas and Henriksen, 2000; Pereira et al., 2000). However, the concentrations reported are concern because they are available in the environment and any break on ecological balance may represent a potential risk of contamination of these reservoirs. 5. Conclusions In conclusion, this report evaluated the sensitivity of two different cell-based bioassays for reliable STX toxicity risk assessment. The present data conrmed that N2A cell line is suitable for toxicity evaluation and neurotoxin STX demonstrated that mechanisms triggering its toxic effects involve oxidative stress, leading possibly to cell death through LPO of cell membranes. When the cells were pre-treated with VitE and SOD/CAT, the production rates of MDA decreased signicantly for lower concentrations of STX. Therefore, our results conrm the initial investigation of Perreault et al. (2011) that STX may be present in aquatic ecosystems at levels that do not induce relevant toxic effects on phytoplankton, probably because he had developed mechanisms of protection/adaptation during the evolution of species, but which can still be harmful to animal and human populations. The evaluation of toxicity on algal-based bioassays does not appear reliable in cases of STX contamination and should be done in combination with animal-based bioassays. Acknowledgements The authors would like to acknowledge the Conselho Nacional de Desenvolvimento Cientco e Tecnolgico (CNPq, Brazil) for its nancial support. S.P. Melegari would also like to thank for her fellowship in the Laboratory of Toxicology, University of Bordeaux 2 (France) and Coordenao de Aperfeioamento de Pessoal de Nvel Superior (CAPES, Brazil) for your post-doctoral fellowship at UQAM (Canada). Thanks to Mr. Thophile A. Mobio to the contributions on experimental tests. References
Aebi, H., 1984. Catalase in vitro. Methods Enzymol. 105, 121126. Bartram, J., Carmichael, W.W., Chorus, I., Jones, G., Skulberg, O., 1999. Introduction. In: Bartram, J. (Ed.), Toxic Cyanobacteria in Water: A Guide to Their Public Health Consequences , Monitoring and Management. E & FN Spon, London, pp. 1224. Baudrimont, I., Ahouandjivo, R., Creppy, E.E., 1997. Prevention of lipid peroxidation induced by ochratoxin A in Vero cells in culture by several agents. Chem. Biol. Interact. 104, 2940. Berger, C., Ba, N., Gugger, M., Bouvy, M., Rusconi, F., Cout, A., Troussellier, M., Bernard, C., 2006. Seasonal dynamics and toxicity of Cylindrospermopsis raciborskii in Lake Guiers (Senegal, West Africa). FEMS Micriobiol. Ecol. 57, 355366. Bouacha, N., 2001. Impact sanitaire des toxines de cyanobacteries en milieu deau douce. Rev. Fr. Lab. 336, 3946. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248254. Carvalho Pinto-Silva, C.R., Ferreira, J.F., Costa, R.H.R., Belli Filho, P., Creppy, E.E., Matias, W.G., 2003. Micronucleus induction in mussels exposed to okadaic acid. Toxicon 41, 9397. Costa, I.A.S., Azevedo, S.M.F.O., Senna, P.A.C., Bernardo, R.R., Costa, S.M., Chellappa, N.T., 2006. Occurrence of toxin-producing cyanobacteria blooms in a Brazilian semiarid reservoir. Braz. J. Biol. 66, 211219. Domnguez-Rebolledo, .E., Martnez-Pastor, F., Fernndez-Santos, M.R., del Olmo, E., Bisbal, A., Ros-Santaella, J.L., Garde, J.J., 2010. Comparison of the TBARS assay and BODIPY C11 probes for assessing lipid peroxidation in red deer spermatozoa. Reprod. Domest. Anim. 45, e360368. Donovan, C.J., Ku, J.C., Quilliam, M.A., Gill, T.A., 2008. Bacterial degradation of paralytic shellsh toxins. Toxicon 52, 91100. EFSA, E.F.S.A., 2009. Marine biotoxins in shellsh Saxitoxin group scientic opinion of the panel on contaminants in the food chain. EFSA J. 1019, 13.

Falconer, I.R., 2008. Health effects associated with controlled exposures to cyanobacterial toxins. Adv. Exp. Med. Biol. 619, 607612. Falconer, I.R., Humpage, A.R., 2005. Health risk assessment of cyanobacterial (blue green algal) toxins in drinking water. Int. J. Environ. Res. Public Health 2, 4350. Fitzgerald, D.J., Cunliffe, D.A., Burch, M.D., 1999. Development of health alerts for cyanobacteria and related toxins in drinking water in South Australia. Environ. Toxicol. 14, 203209. Halliwell, B., 1987. Oxidants and human disease: some new concepts. FASEB J.1, pp. 358364. Halliwell, B., 2006. Oxidative stress and neurodegeneration: where are we now? J. Neurochem. 97, 16341658. Halliwell, B., 2007. Biochemistry of oxidative stress. Biochem. Soc. Trans. 35, 1147 1150. Halliwell, B., 2008. Are polyphenols antioxidants or pro-oxidants? What do we learn from cell culture and in vivo studies? Arch. Biochem. Biophys. 476, 107 112. Halliwell, B., Gutteridge, J.M.C., 2007. Free Radicals in Biology and Medicine, fourth ed. Oxford University Press, Oxford. Harris, E.H., 1989. The Chlamydomonas Sourcebook: A Comprehensive Guide to Biology and Laboratory Use. Academic Press San Diego, Academic Press. Hoeger, S.J., Hitzfeld, B.C., Dietrich, D.R., 2005. Occurrence and elimination of cyanobacterial toxins in drinking water treatment plants. Toxicon 203, 231 242. Ikawa, M., Wegener, K., Foxall, T.L., Sasner, J.J., 1982. Comparison of the toxins of the bluegreen alga Aphanizomenon os-aquae with the Gonyaulax toxins. Toxicon 20, 747752. Inoue, M., Sato, E.F., Nishikawa, M., Park, A.-M., Kira, Y., Imada, I., Utsumi, K., 2003. Mitochondrial generation of reactive oxygen species and its role in aerobic life. Curr. Med. Chem. 10, 24952505. Kaas, H., Henriksen, P., 2000. Saxitoxins (PSP toxins) in Danish lakes. Water Res. 34, 20892097. Kojo, S., 2004. Vitamin C: basic metabolism and its function as an index of oxidative stress. Curr. Med. Chem. 11, 10411064. Lagos, N., Onodera, H., Zagatto, P.A., Oshima, Y., 1999. The rst evidence of paralytic shellsh toxins in the freshwater cyanobacterium Cylindrospermopsis raciborskii, isolated from Brazil. Toxicon 37, 13591373. Ledreux, A., Krys, S., Bernard, C., 2009. Suitability of the Neuro-2a cell line for the detection of palytoxin and analogues (neurotoxic phycotoxins). Toxicon 53, 300308. Matias, W.G., Traore, A., Bonini, M., Sanni, A., Creppy, E.E., 1999. Oxygen reactive radicals production in cell culture by okadaic acid and their implication in protein synthesis inhibition. Human Exp. Toxicol. 18, 634639. Molica, R., Oliveira, E.J.A., Carvalho, P.V.V.C., Costa, A.N.S.F., Cunha, M.C.C., Melo, G.L., Azevedo, S.M.F.O., 2005. Occurrence of saxitoxins and an anatoxin-a(s)-like anticholinesterase in a Brazilian drinking water supply. Harmful Algae 4, 743 753. Mller, P., Wallin, H., 1998. Adduct formation, mutagenesis and nucleotide excision repair of DNA damage produced by reactive oxygen species and lipid peroxidation product. Mutat. Res. 410, 271290. Nair, U., Bartsch, H., Nair, J., 2007. Lipid peroxidation-induced DNA damage in cancer-prone inammatory diseases: a review of published adduct types and levels in humans. Free Radic. Biol. Med. 43, 11091120. Nakano, Y., Asada, K., 1981. Hydrogen peroxide is scavenged by ascorbate-specic peroxidase in spinach chloroplasts. Plant Cell Physiol. 22, 867880. Pereira, P., Onodera, H., Andrinolo, D., Franca, S., Arajo, F., Lagos, N., Oshima, Y., 2000. Paralytic shellsh toxins in the freshwater cyanobacterium Aphanizomenon os-aquae, isolated from Montargil reservoir, Portugal. Toxicon 33, 16891702. Perreault, F., Matias, M.S., Melegari, S.P., Creppy, E.E., Popovic, R., Matias, W.G., 2011. Investigation of animal and algal bioassays for reliable saxitoxin ecotoxicity and cytotoxicity risk evaluation. Ecotoxicol. Environ. Safety 74, 10211026. Pohnert, G., 2004. Chemical defense strategies of marine organisms. Topics Curr. Chem 239, 179219. Rapala, J., Robertson, A., Negri, A.P., Berg, K.A., Tuomi, P., Lyra, C., Erkomaa, K., Lahti, K., Hoppu, K., Lepist, L., 2005. First report of saxitoxin in Finnish lakes and possible associated effects on human health. Environ. Toxicol. 20, 331340. Sivonen, K., Jones, G., 1999. Cyanobacterial toxins. In: Chorus, I., Bartram, J. (Eds.), Toxic Cyanobacteria in Water: A Guide to Their Public Health Consequences, Monitoring and Management World Health Organization. E & FN Spon, London & New York, pp. 4191. Smart, R.C., Hodgson, E., 2008. Molecular and Biochemical Toxicology, fourth ed. Toxicology. John Wiley & Sons, New York. Smith, I.K., Vierheller, T.L., Thorne, C.A., 1988. Assay of glutathione reductase in crude tissue homogenates using 5,50 -dithiobis(2-nitrobenzoic acid). Anal. Biochem. 175, 408413. Taylor, A.R., 2009. A fast Na+/Ca2+-based action potential in a marine diatom. PloSone 4, e4966. Tucci, A., SantAnna, C.L., 2003. Cylindrospermopsis raciborskii (Woloszynska) Seenayya & Subba Raju (Cyanobacteria): variao semanal e relaes com fatores ambientais em um reservatrio eutrco, So Paulo, SP, Brasil. Rev. Bras. Bot. 2, 97112. Valko, M., Rhodes, C.J., Moncol, J., Izakovic, M., Mazur, M., 2006. Free radicals, metals and antioxidants in oxidative stress-induced cancer. Chem. Biol. Interact. 160, 140. Wiese, M., DAgostino, P.M., Mihali, T.K., Moftt, M.C., Neilan, B.A., 2010. Neurotoxic alkaloids: saxitoxin and its analogs. Mar. Drugs 8, 21852211.