Chemosphere xxx (2012) xxx–xxx

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Chemosphere

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Induction to oxidative stress by saxitoxin investigated through lipid peroxidationin Neuro 2A cells and Chlamydomonas reinhardtii alga

Silvia P. Melegari a, François 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 Universitário Trindade, CEP 88040-970 Florianopolis, SC, Brazilb Department of Chemistry, University of Quebec in Montreal, C.P. 8888, Succ. Centre-Ville, Montreal, Quebec, Canada H3C 3P8c Laboratory de Toxicologie et d’Hygiène Appliquée, UFR des Sciences Pharmaceutiques, Université Victor Segalen Bordeaux 2, 146 rue Léo Saignat, 33076 Bordeaux Cedex, Franced INRA, Centre de Recherche de Bordeaux Aquitaine, MICA, Unité de Mycologie et de Sécurité des Aliments, Domaine de la grande Ferrade, 71 avenue Edouart Bourleaux BP 81,33883 Villenave d’Ornon Cedex, France

a r t i c l e i n f o

Article history:Received 11 January 2012Received in revised form 22 March 2012Accepted 3 April 2012Available online xxxx

Keywords:SaxitoxinOxidative stressLipid peroxidationNeuro-2A cellsChlamydomonas reinhardtii green alga

0045-6535/$ - see front matter � 2012 Published byhttp://dx.doi.org/10.1016/j.chemosphere.2012.04.009

Abbreviations: STX, saxitoxin; N2A, Neuro-2A; Msuperoxide dismutase; CAT, catalase; APX, ascorbatereductase; GST, glutathione S-transferase; GPx, glglutathione reduced form; RS, reactive species; ROS,lipid peroxidation; TBA, thiobarbituric acid; HPLCchromatography.⇑ Corresponding author. Address: Laboratório de To

Depto. de Engenharia Sanitária e Ambiental, UniCatarina, Campus Universitário Trindade, CEP 88040-Tel.: +55 48 37217742; fax: +55 48 37219823.

E-mail address: will@ens.ufsc.br (W.G. Matias).

Please cite this article in press as: Melegari, S.P.and Chlamydomonas reinhardtii alga. Chemosph

a b s t r a c t

Saxitoxin (STX) is a cyanotoxin, which can cause neurotoxic effects and induce ecological changes inaquatic environments, a potential risk to public and environmental health. Many studies of cytotoxicityon animal cells and algae have been performed, although few compare the toxic effects between the twomodels. In this sense, we investigated the oxidative stress induced by STX (0.4–3.0 nM) in two differentcellular models: Neuro-2A (N2A) cells and Chlamydomonas reinhardtii alga by quantification of malondi-aldehyde (MDA) levels as indicative of lipid peroxidation (LPO). Also was evaluated the antioxidantdefense 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 cellsincreased from 15% to 113% for 0.4 and 3.0 nM of STX, respectively, as compared to control. Superoxide-dismutase and catalase did not appear to protect the cell from STX effect while, in cells treated with vita-min E, the rates of MDA production decreased significantly, except for higher concentrations of STX. NoMDA productions were observed in algal cells however some effects on antioxidant enzymes activitywere observed when algae were exposed to 3.0 nM STX. Our results indicate that the concentrations ofSTX that may induce oxidative stress through LPO are different in animal and phytoplankton communi-ties. A combination of algal and animal bioassays should be conducted for reliable assessment of oxida-tive stress induced by STX.

� 2012 Published by Elsevier Ltd.

1. Introduction

The frequency of toxic blooms of cyanobacteria in freshwaterdestined for human consumption increases evidently worldwide,probably due to the increase of environmental pollution and globalwarming (Lagos et al., 1999; Pereira et al., 2000; Tucci and Sant’anna,2003; Hoeger et al., 2005; Molica et al., 2005; Berger et al., 2006;

Elsevier Ltd.

DA, malondialdehyde; SOD,peroxidase; GR, glutathione

utathione peroxidase; GSH,reactive oxygen species; LPO,

, high performance liquid

xicologia Ambiental, LABTOX,versidade Federal de Santa970 Florianópolis, SC, Brazil.

, et al. Induction to oxidative stere (2012), http://dx.doi.org/10

Costa et al., 2006). Blooms of cyanobacteria create severe practicalproblems to those in charge of water supply to populations becauseof water contamination by different toxic compounds. Cyanobacte-rial toxins, or ‘‘cyanotoxins’’ consist of diverse groups of chemicalstructures, each of them bearing specific mechanisms of cytotoxicity(Bartram et al., 1999; Sivonen and Jones, 1999; Carvalho Pinto-Silvaet al., 2003). Saxitoxin (STX), which was first identified in freshwatercontaminated by Aphanizomenon flos-aquae (Ikawa et al., 1982), is aneurotoxin classified among in the Paralytic Shellfish Poisoning(PSP) toxins. Apart from A. flos-aquae, STX can be synthesized byother species of cyanobacteria such as Cylindrospermopsis raciborskii,Lyngbya and Anabaena (Sivonen and Jones, 1999; Bouaïcha, 2001).The fast action of STX, blocking sodium channels in nerve axonsand causing loss of sensation and paralysis, confers to this toxin ahighly neurotoxic and potentially lethal effect 2–25 h after inges-tion. The LD50 in mice is of 8–10 lg kg�1 i.p., 3.4 lg kg�1 i.v. and260 lg kg�1 by oral route (Falconer, 2008). Data regarding thespecific cytotoxic and genotoxic effects of STX are very scarce. For

ress by saxitoxin investigated through lipid peroxidation in Neuro 2A cells.1016/j.chemosphere.2012.04.009

2 S.P. Melegari et al. / Chemosphere xxx (2012) xxx–xxx

example, there is neither evidence of human health effects causeddirectly by the consumption of water containing STX-producingcyanobacteria or PSP-producing dinoflagellates, and cases of humantoxicity were only reported for paralytic shellfish poisoning events(Fitzgerald et al., 1999). There are no data related to the concentra-tions of STX-like neurotoxins in drinking water that have causedneurotoxic symptoms in populations exposed to public water sup-ply (Falconer and Humpage, 2005). Rapala et al. (2005) reportedthe first quantitication of STX in cyanobacterial blooms in Finland,with presence of STX in concentrations of 1 mg L�1 in bloom samplesof Finnish Lakes. Symptoms of swimmers from the sites contami-nated with STX were reported as fever, eye irritation, abdominalpains, and skin rash in children aged 2–10 years, after exposure tothe water, but these were not the adverse human symptoms typicalof STX poisoning. Recent data published in the ‘‘International Sym-posium on Cyanobacterial Harmful Algal Blooms’’ (ISOC-HABs), or-ganized by the Environmental Protection Agency of the UnitedStates of America (USEPA), reported that there are no data on subchronic exposure, reproductive, teratogenic or carcinogenic effectsof STX (Falconer, 2008). The European Food Safety Authority (EFSA,2009) published the scientific opinion about STX-group in shellfishand reported that the toxicological database for STX-group toxinsis limited and comprises mostly studies on their acute toxicity fol-lowing intraperitoneal administration.

There are many methods to investigate the oxidative stress in-duced by production of reactive species (RS). The in vitro test havebeen demonstrated to be satisfactory as an alternative to thein vivo assay, and they have the advantages of being fast and sen-sible, excluding the ethical questions that emerge from assaysinvolving animal experimentation (Smart and Hodgson, 2008).Malondialdehyde (MDA) is a natural biomarker produced in thereaction of lipid peroxidation (LPO), which can be quantified andused for the evaluation of this process. It is mutagenic in bacteriaand mammalian cells and carcinogenic in mice (Valko et al.,2006). It also modifies DNA bases, generating bifunctional interme-diates that form exocyclic DNA adducts contributing to mutagenicand carcinogenic process (Valko et al., 2006; Nair et al., 2007).

Antioxidants are substances that delay and/or prevent the oxi-dation of cellular substratum at low concentrations. Antioxidantsare widely distributed in living organisms and are extremelyimportant as representatives in the direct removal of free radicals(Valko et al., 2006; Halliwell and Gutteridge, 2007). The main enzy-matic antioxidants are superoxide dismutase (SOD), catalase (CAT),ascorbate peroxidase (APX) and the enzymes involves in process ofgluthatione metabolism (glutathione reductase – GR, glutathionetransferase – GST and glutathione peroxidase – GPx), which serveas 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) andreactive nitrogen species (Møller and Wallin, 1998; Matias et al.,1999; Valko et al., 2006; Halliwell and Gutteridge, 2007).

Recently, we investigated the sensitivity of algal and animalcells towards STX effect and showed that algae are much moreresistant to STX than animal cells (Perreault et al., 2011). In thisstudy we investigated the oxidative stress induced by STX in twodifferent cellular models to determine how the antioxidant defensesystem may participate in this difference of sensitivity. Here, Neu-ro-2A (N2A) cells and the green alga Chlamydomonas reinhardtiiwere exposed to STX effect and oxidative stress was investigatedby quantification of MDA levels as an indicator of LPO. Also wasevaluated the antioxidants defense of these cells systems afterexposure to STX by addiction of some antioxidants in N2A cells cul-ture, and by measure of antioxidants enzymes activity in C. rein-hardtii cells.

Please cite this article in press as: Melegari, S.P., et al. Induction to oxidative stand Chlamydomonas reinhardtii alga. Chemosphere (2012), http://dx.doi.org/10

2. Material and methods

2.1. Cells models and culture medium

All the chemical products used for the cell culture were pro-vided by Sigma–Aldrich (Saint Quentin Falavier, France). STX(NRC CRM-STX-e) was obtained from the Institute for Marine Bio-sciences (National Research Center, Halifax, NS – Canada). N2Acells from the mouse neuroblastoma cell line were obtained fromATCC (CCL-131, not differentiated – LGC Promochem, France).The cells were grown in 75 cm2 flasks, with a RPMI 1640 culturemedium supplemented with 10% of fetal bovine serum, 2% ofL-glutamine solution (200 mM), 1% of sodium pyruvate solution(100 mM) and 1% of antibiotic solution (10 mg mL�1 streptomycinand 10000 U mL�1 penicillin). The culture was maintained at 37 �Cin a humidified CO2 (5%) atmosphere (Adapted from Ledreux et al.,2009).

C. reinhardtii wild type (CC-125) strains were obtained from theChlamydomonas Genetic Center (Duke University, Durham, NC,USA). Cells were cultivated in batch culture of 1 L of high saltgrowth medium (HSM) according to Harris (1989). Cultures wereexposed to continuous illumination (100 lE m�2 s�1) provided bywhite fluorescent lamps (Sylvania Grolux F 36 W) at 23 �C ± 1.The culture was permanently aerated to obtain a constant CO2 con-centration in the growing medium.

2.2. Evaluation of LPO and antioxidant defense system

For N2A cells, 1 � 105 cells mL�1 were cultured in 24-wellmicroplates for 24 h at 37 �C as described above, and then cultureswere separately incubated with different protective substances:vitamin C (8 and 16 lg mL�1, respectively VitC8 and VitC16), vita-min E (6 lg mL�1, VitE) and enzymes SOD/CAT (25 lg mL�1 each).A negative control was employed using culture medium, and a po-sitive control was employed using ochratoxin A (OTA = 10 lM).The N2A cells were intoxicated for 24 h at 37 �C with STX in theconcentrations 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 cellpellets were resuspended in 250 lL SET buffer (0.1 M NaCl, 20 mMEDTA, 50 mM Tris–HCl, 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 thisperiod, a volume of 300 lL of n-butanol was added and tubes wereshaken vigorously. After centrifugation for 10 min at 900g at 4 �C,the n-butanol phase from each sample containing the MDA–TBAadduct was separated and analyzed through high performance li-quid chromatography (HPLC) with fluorometric detection.

The HPLC system consisted of an ICS (Instrumentation Consom-mable Service, France) chromatography pump equipped with aShimadzu 8450 Fluorescence HPLC Monitor (Japan SpectroscopicCo.). The analysis of the MDA–TBA adducts was performed at roomtemperature on a Prontosil Eurobond C18 column (Bischoff Chro-matography, Germany, 250 � 4.6 mm, £part: 5 lm) using 40:60methanol–water (v/v) as the mobile phase. The pH was adjustedto 8.4 through the addition of 0.5 M KOH. The flow rate was main-tained at 0.5 mL min�1 by an automatic injection of 90 lL. Theexcitation and emission wavelengths were of 515 and 553 nm,respectively (Adapted from Matias et al., 1999). The amount ofMDA-TBA measured is related to the protein content of cellularhomogenates, determined using Bradford’s colorimetric method(Bradford, 1976).

For algal culture, aliquots of 20 mL of the (2.5 � 105 cells mL�1)in the exponential growth phase were exposed to 0.4, 0.8, 1.5 and

ress by saxitoxin investigated through lipid peroxidation in Neuro 2A cells.1016/j.chemosphere.2012.04.009

S.P. Melegari et al. / Chemosphere xxx (2012) xxx–xxx 3

3.0 nM of STX for 24, 48 and 72 h under the same illumination andtemperature conditions used for growing stock cultures. For thecontrol, the same growth medium and conditions were used with-out STX. During the exposures, changes in the ratio between cellnumber (biomass) and concentration of STX were negligible. Allconcentrations and controls were evaluated in triplicates.

The LPO analysis was performed by determination of MDAlevel, using the fluorescent dye BODIPY 581/591C11 (4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid-Invitrogen Molecular Probe, USA). BODIPY is afluorescent fatty acid analog with fluorescent properties in thered and green ranges of the visible spectrum (emission maximumat 595 nm), which after oxidation induced by ROS, its fluorescencechanges from red to green (Domínguez-Rebolledo et al., 2010).After 24, 48 and 72 h of treatment, 0.5 mL samples of algal cul-tures bioassays were exposed 30 min to 10 lL BODIPY (2 mM)in dark. Intracellular MDA level was determined by measuringthe fluorescence emission at 595 nm with a flow cytometer (Fac-Scan; Becton Dickinson Instruments, USA). Cytometry resultswere analyzed using the WinMDI 2.8 software. To discriminatebetween algal cells and noncellular particles, all measurementswere separated by using a relationship between particle sizeand red fluorescence level, originating from chlorophyll fluores-cence reemission.

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 so-dium phosphate buffer (pH 7) with addition of 500 lL of glassbeads (Sigma) and algal cells were mixed for 10 min at 4 �C byusing a vortex. Enzymes were extracted within 1 mL of sodiumphosphate buffer and centrifuged at 2300g for 20 min at 4 �C. Forenzyme activity measurements the supernatant was separatedand stored at �80 �C. Protein content was determined accordingto Bradford (1976).

CAT activity was evaluated spectrophotometrically by measur-ing the consumption of H2O2 at 240 nm and calculated using anextinction coefficient of 0.0436 mM�1 cm�1 according to Aebi(1984). 1 lg protein (�50 lL of enzyme extract) was added to100 lL of H2O2 (200 mM) and sufficient volume of sodium phos-phate buffer (50 mM, pH 7.5) to a final volume of 1 mL. The con-sumption of H2O2 was monitored each 5 s until 30 s. One CATunit was defined as the enzyme amount that transforms 1 mmolof H2O2 per min.

APX activity was evaluated by the change of absorbance at290 nm due to ascorbate oxidation and calculated using an extinc-tion coefficient of 2.8 mM�1 cm�1 adapted of Nakano and Asada(1981). 1 lg protein (�50 lL of enzyme extract) was added to50 lL Na-ascorbate (20 mM), 50 lL H2O2 (100 mM) and sufficientvolume of sodium phosphate buffer (50 mM, pH 7.5) to a final vol-ume of 1 mL. The consumption of H2O2 was monitored each 5 s un-til 90 s. One APX unit was defined as the enzyme amount thattransforms 1 mmol of ascorbate per min.

GR activity was measured by the change of absorbance at412 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 lLNADPH (1 mM), 50 lL DNTB (15 mM), 50 lL glutathione oxidizedform (GSSG) (20 mM) and sufficient volume of sodium phosphatebuffer (50 mM, pH 7.5) to a final volume of 1 mL. The consumptionof DTNB was monitored each 10 s until 120 s and the GR activitywas calculated using an extinction coefficient of 14.15 mM�1

cm�1. One GR unit was defined as the enzyme amount that trans-forms 1 mmol of DTNB per min.

Please cite this article in press as: Melegari, S.P., et al. Induction to oxidative stand Chlamydomonas reinhardtii alga. Chemosphere (2012), http://dx.doi.org/10

GST activity was evaluated by the change of absorbance at340 nm due to conjugation of GSH to 1-chloro-2,4-dinitrobenzene(CDNB). GST activity was calculated using an extinction coefficientof 9.6 mM�1 cm�1 from CDNB. 1 lg protein (�50 lL of enzyme ex-tract) was added to 25 lL GSH (40 mM), 10 lL CDNB (40 mM) andsufficient volume of sodium phosphate buffer (50 mM, pH 7.5) to afinal volume of 1 mL. The formation of GS-DNB conjugate wasmonitored each 30 s until 300 s. One GST unit was defined as theenzyme amount that transforms 1 mmol of CDNB per min.

2.4. Statistical analysis

All the results were expressed as the mean ± SEM and the differ-ences between the results of MDA levels and the control groupswere compared with ANOVA one-way analysis of variance fol-lowed by Tukey’s post hoc test for multiple comparisons. A signif-icance 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 spontaneouslyproduced 83.8 ± 5.9 nM MDA mg protein�1. This value was consid-ered the control value for N2A cells without STX. After 24 h ofexposure to the VitE (6 lg mL�1), the N2A cells in culture sponta-neously produced 59.1 ± 1.6 nM MDA mg protein�1. As for theN2A cells in culture with VitC8 (8 lg mL�1) and VitC16 (16 lgmL�1), they spontaneously produced 89.8 ± 9.4 and 107.3 ± 17.3nM MDA mg protein�1, respectively, while the N2A cells in culturewith SOD/CAT spontaneously produced 102.5 ± 10.9 nM MDA mgprotein�1. All these values were considered the control value forN2A cells without STX.

After 24 h of incubation, with increasing STX concentrations,the MDA production in N2A cells also increased significantly(Fig. 1). The production of MDA increased by 15–113% for 0.38and 3.0 nM of STX, respectively, when compared to controls (Table1). When N2A cells were pretreated with VitC8, this rate increasedby 66–209% in presence of the same concentration of STX. WhenN2A cells were pretreated with VitC16, the rates of MDA produc-tion increased from 36–181% for the same concentrations of STX(Table 1).

The pretreatment of N2A cells with SOD/CAT showed no signif-icant decrease of MDA production compared with other treatmentswith the same concentration of STX (Fig. 1), even with attenuateddecline in the rate of production of MDA compared with the con-trol group (Table 1).

When the N2A cells were pretreated with VitE a significant de-crease of production rates of MDA were observed for the lowestSTX concentrations, when compared to the condition without anti-oxidants. 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 nMSTX (Table 1), respectively.

3.2. STX effect in C. reinhardtii cells

We evaluated STX effect on ROS production in C. reinhardtii cellsusing BODIPY fluorescent probe, which is specific to MDA produc-tion. When C. reinhardtii was exposed to STX (from 0.8 to 3.0 nM),MDA level was not significantly altered when compared to the neg-ative control, even after 72 h of treatment (p > 0.05, Fig. 2).

Four different biomarkers were employed to verify the modifi-cation of enzymatic activity of C. reinhardtii when exposed toSTX. This biomarkers act in cellular defense system under condi-tions of oxidative stress. Differences statistically significant could

ress by saxitoxin investigated through lipid peroxidation in Neuro 2A cells.1016/j.chemosphere.2012.04.009

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

Table 1LPO measured by the increase of MDA production (%), when compared to negative control.

STX (nM) Increase MDA production (%)

Without antioxidants Vit. E 6 lg mL�1 Vit. C SOD + CAT 25 ug mL�1 (each)

8 lg mL�1 16lg mL�1

0.38 15 3 36 66 30.75 93 31 109 143 471.50 99 95 163 154 753.00 113 120 181 209 160

Positive control 120 358 298 374 129

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

4 S.P. Melegari et al. / Chemosphere xxx (2012) xxx–xxx

be observed in the activity of these antioxidants enzymes whenalga 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-depen-dent manner upon exposure to STX, with significant decrease inconcentrations of 0.8 and 3.0 nM. The decrease of CAT activity inthe STX concentration of 1.5 nM was observed visually, but itwas not significant (p = 0.087, Fig. 3). This discrepancy cannot beexplained at this time, but do not affect the general understandingof defense mechanism of alga that decreases its activity in re-sponse to increasing concentrations of STX. The APX, GR and GSTshow significant change in its activities only in the high STX con-centration, an increase of their activities compared to control.

Please cite this article in press as: Melegari, S.P., et al. Induction to oxidative stand Chlamydomonas reinhardtii alga. Chemosphere (2012), http://dx.doi.org/10

4. Discussion

The cytotoxic and genotoxic effects of STX in N2A cells and C.reinhardtii alga have recently been shown and the data suggestedthat STX induces indirect genotoxic effects in N2A cells to concen-trations ranging from 0.5 to 64 nM (Perreault et al., 2011). Again,the toxicological response indicates that the interaction of STXwith algal cells may be very different than with animal cells (Tay-lor, 2009), showing that different cellular systems show significantdifferences in their response towards pollutants, at similar STXconcentrations.

Induction of LPO by STX in neuronal and algal cells line and thecorrelation with antioxidant defense mechanisms have not yet

ress by saxitoxin investigated through lipid peroxidation in Neuro 2A cells.1016/j.chemosphere.2012.04.009

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 ofexposure to different STX concentrations (�p < 0.05).

S.P. Melegari et al. / Chemosphere xxx (2012) xxx–xxx 5

been reported in the literature, but it is known that the nervoussystem are incline to oxidative stress due to the lack of an adjustedantioxidant defense system (Halliwell, 2006). LPO is characterizedby the process where RS attack polyunsaturated fatty acids of thecellular membrane, inducing a chain reaction with lipid hydroper-oxides as intermediate products (Halliwell, 1987). This process canalso result in the destabilization and disintegration of the cellularmembrane, bringing serious health problems to man, from agingto susceptibility to cancer (Valko et al., 2006). When initiated,the LPO forms peroxyl free radicals (ROO�) that can be rearrangedthrough a cyclization reaction of endoperoxides, producing, amongother final products of this process, MDA.

The MDA levels, which increase as a consequence of the LPO foroxidative stress, were significantly more elevated in N2A cells ex-posed to STX than in controls without toxin. All the results showthat after 24 h of incubation, STX induces LPO in N2A cells. Endog-enous and exogenous substances can produce ROS proceedingfrom LPO (Møller and Wallin, 1998). STX acts as an exogenoussource of oxidative stress, yielding ROS that are probably responsi-ble for the LPO. When produced inside of the cells, ROS are capableto virtually react with all types of biomolecules (Møller and Wallin,1998), organelles and enzymes including mitochondria, cyto-chrome P450 involved in drug metabolism, peroxisomes, andinflammatory cells activation systems (Inoue et al., 2003). Neuro-toxins generally lead to the formation of ROS in several clinicalconditions of the nervous system (Halliwell, 1987).

The exposition of N2A cells to STX induced an evident ROS pro-duction and peroxidation of the lipid membranes. A MDA overpro-duction can be stimulated by these effects, as suggest by Halliwell(2006). The MDA can be toxic to cell, causing a inhibition of proteinsynthesis, some DNA adducts may be formed, which may lead togenotoxic, mutagenic and carcinogenic processes (Baudrimontet al., 1997; Matias et al., 1999; Halliwell, 2007). High levels of oxi-dative damage can result not just from oxidative stress, but alsofrom the limitation of the cellular repair system, and this dysfunc-tion may cause a deregulation of the cell defense system, leading tocell death (Halliwell, 2007).

Matias et al. (1999) demonstrate the efficiency of SOD/CATcombination in the protective effect of Vero cells against the LPOeffects of okadaic acid, our results with SOD/CAT does not appear

Please cite this article in press as: Melegari, S.P., et al. Induction to oxidative stand Chlamydomonas reinhardtii alga. Chemosphere (2012), http://dx.doi.org/10

to affect the defense system of N2A cell against STX, because thechange in the production of MDA was not significant comparedto other treatments with the same concentration of STX (excepttreatment VitE).

Vit E seemed efficient in antioxidant defense system to scav-enge MDA. MDA levels increased just 3% in the minor concentra-tion of STX, in relation with the negative control when VitE waspresent. However, Halliwell (2008) affirms that in vivo studies haveno evidence of systemic pro-oxidant effects of flavonoids in hu-mans, and little or no clear evidence of antioxidant effects.

The pre-treatment of N2A cells with VitE, seem efficient in thein vitro prevention against the LPO when compared to VitC. VitCwas not efficient for the protection of N2A cells in our experimen-tal conditions. This is most probably due the switch from antioxi-dant to pro-oxidant according to concentrations and RS. In specificconditions (in the presence of intracellular Cu, [CuZnSOD] forexample in mitochondrial membrane and in cytosol) the ascorbatecan be oxidized, producing cytotoxic species (Halliwell, 2006). Inthis case, VitC will synergize the toxic effects of STX (Kojo, 2004).VitC cooperates with VitE to regenerate a-tocopherol from thea-tocopherols radicals in the membranes and lipoproteins. A mix-ture of both vitamins should be tested further in the presence ofSTX.

STX is known as a natural defense mechanism of phytoplanktonto eliminate other competitors by allelopathic processes in cases ofdisturb of ecological balance to become the dominant organismfrom the environment (Pohnert, 2004). The change in enzymaticactivity of algae by exposure to STX can be understood as a biolog-ical response of this microorganism to metabolize the toxin. It isspeculated that the catabolism of the PSP most likely occurredvia oxidation reactions catalyzed by oxidases and peroxidases intoaliphatic products for subsequent use in purine and argininemetabolism (Donovan et al., 2008; Wiese et al., 2010). In this casewere not observed effects of oxidative stress like LPO, but changesin enzyme activity, possibly in an attempt to neutralize free radi-cals 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. rein-hardtii, breaking the balance of this system and causing reactions ofoxidations. There are no evidences reported of this concentration

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on algae blooms of freshwaters on bibliography, but some authorsreported in algal blooms with STX on freshwaters in several coun-tries (Lagos et al., 1999; Kaas and Henriksen, 2000; Pereira et al.,2000). However, the concentrations reported are concern becausethey are available in the environment and any break on ecologicalbalance may represent a potential risk of contamination of thesereservoirs.

5. Conclusions

In conclusion, this report evaluated the sensitivity of two differ-ent cell-based bioassays for reliable STX toxicity risk assessment.The present data confirmed that N2A cell line is suitable for toxic-ity evaluation and neurotoxin STX demonstrated that mechanismstriggering its toxic effects involve oxidative stress, leading possiblyto cell death through LPO of cell membranes. When the cells werepre-treated with VitE and SOD/CAT, the production rates of MDAdecreased significantly for lower concentrations of STX. Therefore,our results confirm the initial investigation of Perreault et al.(2011) that STX may be present in aquatic ecosystems at levelsthat do not induce relevant toxic effects on phytoplankton, proba-bly because he had developed mechanisms of protection/adapta-tion during the evolution of species, but which can still beharmful to animal and human populations. The evaluation of tox-icity on algal-based bioassays does not appear reliable in cases ofSTX contamination and should be done in combination with ani-mal-based bioassays.

Acknowledgements

The authors would like to acknowledge the Conselho Nacional deDesenvolvimento Científico e Tecnológico (CNPq, Brazil) for its finan-cial support. S.P. Melegari would also like to thank for her fellow-ship in the Laboratory of Toxicology, University of Bordeaux 2(France) and Coordenação de Aperfeiçoamento de Pessoal de NívelSuperior (CAPES, Brazil) for your post-doctoral fellowship at UQAM(Canada). Thanks to Mr. Théophile A. Mobio to the contributions onexperimental tests.

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