Detoxification and oxidative stress responses along with microcystins accumulation in Japanese quail exposed to cyanobacterial biomass

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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / s c i t o t e n v

Detoxification and oxidative stress responses along with microcystins accumulation in Japanese quail exposed to cyanobacterial biomass Veronika Paškováa,b , Ondřej Adamovskýa,b , Jiří Pikulac , Blanka Skočovskác , Hana Band'ouchovác , Jana Horákovác , Pavel Babicaa,b , Blahoslav Maršáleka,b , Klára Hilscherováa,b,⁎ a

Centre for Cyanobacteria and Their Toxins (Institute of Botany, The Academy of Sciences of the Czech Republic & RECETOX, Masaryk University), Kamenice 126/3, CZ62500, Brno, Czech Republic b RECETOX, Research Centre for Environmental Chemistry and Ecotoxicology, Masaryk University, Kamenice 3, CZ 625 00 Brno, Czech Republic c University of Veterinary and Pharmaceutical Sciences Brno, Faculty of Veterinary Hygiene and Ecology, Palackeho 1/3, 612 42 Brno, Czech Republic

AR TIC LE I N FO

ABS TR ACT

Article history:

The cyanobacterial exposure has been implicated in mass mortalities of wild birds, but

Received 19 December 2007

information on the actual effects of cyanobacteria on birds in controlled studies is missing.

Received in revised form

Effects on detoxification and antioxidant parameters as well as bioaccumulation of

4 March 2008

microcystins (MCs) were studied in birds after sub-lethal exposure to natural cyanobacterial

Accepted 4 March 2008

biomass. Four treatment groups of model species Japanese quail (Coturnix coturnix japonica) were exposed to controlled doses of cyanobacterial bloom during acute (10 days) and sub-

Keywords:

chronic (30 days) experiment. The daily doses of cyanobacterial biomass corresponded to 0.2–

Avian dietary toxicity test

224.6 ng MCs/g body weight. Significant accumulation of MCs was observed in the liver for both

Coturnix coturnix japonica

test durations and slight accumulation also in the muscles of the highest treatment group from

Cyanobacteria

acute test. The greatest accumulation was observed in the liver of the highest treatment group

Microcystin

in the acute test reaching average concentration of 43.7 ng MCs/g fresh weight. The parameters

Detoxification

of detoxification metabolism and oxidative stress were studied in the liver, heart and brain.

Oxidative stress

The cyanobacterial exposure caused an increase of activity of cytochrome P-450-dependent 7ethoxyresorufin O-deethylase representing the activation phase of detoxification metabolism. Also the conjugation phase of detoxification, namely the activity of glutathione-S-transferase, was altered. Cyanobacterial exposure also modulated oxidative stress responses including the level of glutathione and activities of glutathione-related enzymes and caused increase in lipid peroxidation. The overall pattern of detoxification parameters and oxidative stress responses clearly separated the control and the lowest exposure group from all the higher exposed groups. This is the first controlled study documenting the induction of oxidative stress along with MCs accumulation in birds exposed to natural cyanobacterial biomass. The data also suggest that increased activities of detoxification enzymes could lead to greater biotransformation and elimination of the MCs at the longer exposure time. © 2008 Elsevier B.V. All rights reserved.

⁎ Corresponding author. CCT & RECETOX, Kamenice 126/3, CZ62500, Brno, Czech Republic. Tel.: +420 54949 3256; fax: +420 54949 2840. E-mail address: [email protected] (K. Hilscherová). 0048-9697/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.scitotenv.2008.03.001

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1.

Introduction

Cyanobacteria are known to produce secondary metabolites, which have been recognized as human and animal health hazards, since they have been shown to cause adverse effects in mammals, birds, fish, invertebrates as well as plants (Codd, 1996; Figueiredo et al., 2004; Wiegand and Pflugmacher, 2005; Malbrouck and Kestemont, 2006; Babica et al., 2006; Skocovska et al., 2007). The most frequently occurring cyanobacterial toxins are monocyclic heptapeptides called microcystins (MCs) (Carmichael, 1997). MCs or MC-producing cyanobacterial strains have been associated with poisonings of wildlife and especially with the mass mortalities of wild birds over recent years. MCs and cyanobacterial hepato- and neurotoxins contributed probably to mass deaths of Lesser Flamingos in Kenya (Krienitz et al., 2003; Ballot et al., 2004, 2005; Ndetei and Muhandiki, 2005) or Tanzania (Lugomela et al., 2006). Greater Flamingo chick deaths, attributed to MCs, occurred at wetlands lagoon in Spain after the sudden development of a bloom with prevailing Microcystis aeruginosa and Anabaena floss-aquae (Alonso-Andicoberry et al., 2002). MCs have been also detected in cyanobacterial blooms in Belgian (Wirsing et al., 1998), Japanese (Matsunaga et al., 1999) or Canadian (Murphy et al., 2000, 2003; Park et al., 2001) lakes where conspicuous deaths of wild birds occurred. MCs primarily act as hepatotoxins (Wiegand and Pflugmacher, 2005), because they are predominantly absorbed via illeum and transported via iliac vein and portal vein into liver (Dahlem et al., 1989; Bury et al., 1998) and also lungs and heart (Ito et al., 2000; Liu et al., 2002). The hepatocytes highly express organic anion transport proteins, which are responsible for active cellular uptake of MCs from blood (Runnegar et al., 1995). However, various organic anion transport proteins are present also in other organs than liver, e.g., in gastrointestinal tract, kidney or brain (Hagenbuch and Meier, 2003). Correspondingly, accumulation of MCs (or structurally related nodularins) has been reported not only in the liver, but also in intestines, kidneys, brain, heart, gonads and muscles of fish (Kankaanpaa et al., 2005; Cazenave et al., 2005; Adamovsky et al., 2007; Kagalou et al., in press) or mammals, and there is an increasing evidence about neurological or renal toxicity of MCs in vertebrates (Dietrich and Hoeger, 2005). It has been suggested that organ-specific distribution and toxic effects of MCs are governed by the presence/absence, type and expression level of organic anion transport proteins (Dietrich and Hoeger, 2005). Exposure to cyanobacterial biomass and/or purified MCs has been shown to cause oxidative stress in various organisms (Ding et al., 2000; Pietsch et al., 2001; Li et al., 2003; Wiegand and Pflugmacher, 2005). Formation of reactive oxygen species (ROS) and oxidative stress is associated with the development of many pathological states. Oxidative stress may occur either due to the decrease of cellular antioxidant level or to the overproduction of ROS (Ding and Ong, 2003). Exposure to MC-LR has been linked with increase of ROS production in mammals and fish (Ding et al., 2000; Li et al., 2003). Liver as the general detoxifying organ is considered the main region of ROS generation in mammals and birds (Prieto et al., 2006). Endogenous antioxidant defenses of enzymatic and non-enzymatic nature are critical for the control of ROS-

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mediated oxidative damage of biomolecules, including proteins, RNA, DNA and membrane polyunsaturated lipids (Halliwell and Gutterdige, 1999). The main defense mechanisms against ROS and their toxic by-products include enzymes, above all glutathione-S-transferases (GST), glutathione reductase (GR), glutathione peroxidase (GPX), catalase (CAT) and superoxide dismutases (SOD), and also nonenzymatic compounds such as glutathione (GSH). Moreover, GSTs are enzymes catalyzing a conjugation of MCs with GSH and therefore responsible for detoxification of MCs (Fu and Xie, 2005). Significant modulations of the antioxidative and detoxification system (GST) or increased production of lipid peroxides upon the exposure to pure MCs, MC-containing cyanobacteria or cyanobacterial extracts have been demonstrated by numerous studies with plants (Babica et al., 2006; Pflugmacher et al., 2006), invertebrates (Pietsch et al., 2001; Chen et al., 2005; Rosa et al., 2005) or fish (Malbrouck and Kestemont, 2006; Fu and Xie, 2005). However, little data is available for adult warm-blooded vertebrates. Only few studies have been carried out with mammalian cell lines (Ding and Ong 2003; Bouaicha et al., 2004) or with mammals in vivo (Gupta et al., 2003; Gehringer et al., 2004; Moreno et al., 2005; Maidana et al., 2006), and there is no information on the potential oxidative stress or detoxification in cyanobacteriaexposed birds. Our previous report indicated histopathological hepatic changes, modification of the biochemical parameters in blood and bioaccumulation of MCs in the liver of Japanese quails (Coturnix coturnix japonica) exposed for 10 or 30 days to controlled doses of natural cyanobacterial bloom with major content of MC-LR and MC-RR (Skocovska et al., 2007). In this part of the study, we investigated the effect of cyanobacterial exposure on activation (P450-dependent 7-ethoxyresorufin-Odeethylase activity) and conjugation (GST, GSH) phase of detoxification metabolism, antioxidant activities and lipid peroxidation as a measure of oxidative damage in the exposed birds. We also studied MC levels in the liver as the primary target organ and in the muscles as the tissue that can be used for human consumption. This study brings more information about the effects of cyanobacteria on birds in connection with detoxification and oxidative stress responses.

2.

Materials and methods

2.1.

Bioassay

The sub-lethal effects of cyanobacterial biomass were studied in Japanese quails after exposure performed according to the Organization for Economic Co-operation and Development (OECD) Guideline for the testing of chemicals 205 — Avian Dietary Toxicity Test (OECD, 1984) with some minor modifications described in detail in our previous paper (Skocovska et al., 2007).

2.2.

Cyanobacterial biomass

Cyanobacterial biomass with domination of Microcystis sp. was collected with plankton net (25 μm) from Brno reservoir (Czech Republic) in autumn 2004. Biomass concentration was

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determined by cell counting under a fluorescent microscope after disintegration of colonies using an ultrasonic probe (Bandelin Sonopuls UW2070, Bandelin Electronics, Berlin, Germany) for 10 min (70% cycle, 70% power). Dry weight of the biomass was determined by drying at 50°C. MCs were extracted from 5 mL of fresh cyanobacterial biomass after addition of equivalent volume of methanol using ultrasonication (Bandelin Sonopuls UW2070, twice 30 s, 80% cycle, 100% power). The extract was centrifuged (10 min, 2800 g) and concentration of MCs in supernatant was measured by HPLC Agilent 1100 Series coupled with PDA detector (Agilent Technologies, Waldbronn, Germany) on Supelcosil ABZ+ Plus column, 150 × 4.6 mm, 5 μm (Supelco, Bellefonte, PA, USA) at 30°C. Binary gradient of mobile phase consisted of H2O + 0.1% TFA (A) and acetonitrile + 0.1% TFA (B); linear increase from 20% B at 0 min to 59% B at 30 min, flow rate was 1 mL min− 1. UV spectra were recorded from 200 to 300 nm and chromatograms were evaluated at 238 nm. MCs were identified by comparison of UV spectra and retention times with standards of MC-LR, -LF, -LW, -RR and -YR (Alexis Biochemicals, Laeufingen, Switzerland). Concentrations of MCs in natural cyanobacterial biomass were 141.8 μg/g DW of MC-RR, 141.7 μg/g DW of MC-LR, 33.7 μg/g DW of MC-YR and 56.1 μg/ g DW of unidentified compound with MC-like UV spectrum. The total concentration of MCs in studied biomass was 373.3 μg/g DW. The homogenization of biomass for dosing was performed by repeated freezing and thawing (two times) and by ultrasonication (Bandelin Sonopuls UW2070, 10 min, 70% cycle, 70% power). Four biomass concentrations were prepared by dilution of biomass with drinking water, aliquoted into plastic cups and stored frozen. Drinking water for the control group of quails was handled the same way.

2.3.

Exposure

Experiment was conducted with 4 months old individuals of Coturnix coturnix japonica (Japanese quail, gallinaceous bird species). Japanese quail belongs to the common experimental bird species. Quails were held in standard lab cages and were fed with commercial bird food and drinking water ad libitum. The exposure design has been described in detail in the previously published part of our study (Skocovska et al., 2007). Briefly, the birds (mean weight 205 g) were divided into five experimental groups (control group C, exposure groups E1–E4) fed various daily doses (Table 1) of the cyanobacterial biomass. The daily doses of 10 mL contained from 3 × 106 (group E1) to 3 × 109 (group E4) cyanobacterial cells, which is equivalent to 0.123 mg to 123 mg dry biomass, respectively. The same daily doses have been administered and the same experimental design has been carried out during the acute (10 days) and subchronic (30 days) exposure. After the experiment, the animals were sacrificed by decapitation. Selected organs (liver, brain, heart and major pectoral muscles) were dissected and stored at −80°C for analyses of MC concentration and measurement of biochemical parameters.

2.4.

Determination of microcystin concentration in tissues

MC concentrations were determined in the liver tissue and major pectoral muscles. Liver and muscles (400 mg of fresh

Table 1 – Characterization of biomass dilutions prepared for each exposure group and recalculated daily doses of cyanobacterial biomass related to the average weight of experimental birds Biomass

Daily dose/body weight

Group Amount mg ∑ μg Amount of μg ∑ ng of cells/L DW/L MCs/L cells/g DW/g MCs/g C E1 E2 E3 E4

– 3 × 108 3 × 109 3 × 1010 3 × 1011

– 12.3 123.3 1233.6 12334.8

– 4.5 46.1 460.5 4605.4

– 14.6 × 103 14.6 × 104 14.6 × 105 14.6 × 106

– 0.6 6.0 60.1 601.7

– 0.2 2.24 22.46 224.6

Experimental birds consumed 10 mL of biomass dilution per day during acute (10 days) and sub-chronic (30 days) study.

weight) were extracted three times with 3 mL methanol using ultrasonication bath (30 min) and centrifuged (10 min, 2800 g). Distilled water (2 mL) was added to combined supernatants and extracts were portioned three times with 1 mL of hexane. The hexane layers were discarded and methanolic fraction was evaporated to dryness at 50°C. The residues were redissolved in 1 mL of distilled water on ultrasonic bath (15 min) and MC concentration was analysed by direct competitive ELISA (modified from Zeck et al., 2001). High proteinbinding 96-well microplates (Nunc, Wiesbaden, Germany) were pre-incubated overnight with 2000-fold diluted antimouse anti-Fc-IgG (MP Biomedicals, Ohio, USA). Free IgG was then removed by washing with phosphate buffer saline (PBS, pH 7.3), and the plates were coated for 1 h with 5000-fold diluted monoclonal IgG (MC10E7, Alexis Biomedicals, San Diego, USA) developed against MC-LR. The plate was then washed five times with 0.05% (v/v) Tween-20 in PBS, and nonspecific interactions were blocked by adding 20 μL of the block solution to each well (1% v/v EDTA, 1% v/v bovine serum albumin in 1 M TRIS–HCl, pH 7.4). The filtered samples, standards and controls were immediately added to the wells (200 μL per well) and the plate was incubated for 40 min at room temperature. Finally, 50 μL of MC-LR conjugated with HRP prepared and purified according to Zeck et al. (2001) was added to each well. The reaction was then incubated at room temperature for another 15 min, the plates were washed five times with 0.05% (v/v) Tween-20 in PBS, and 175 μL of the HRP substrate 3,3′,5,5′-tetramethylbenzidine was added. Development of the coloured product was stopped after 10 min by adding 50 μL of 5% (v/v) sulfuric acid. The absorbance (420 nm with reference 660 nm) was determined with a microplate reader (GENios, Tecan Group, Switzerland). Each sample was analysed in three replicates and compared with 0.125–2 μg/L calibration curve of MC-LR constructed for each individual plate.

2.5.

Biochemical methods

The biochemical analysis was focused on three important organs that could be directly affected by MCs and other cyanobacterial toxins and that are known to be susceptible to oxidative stress, i.e. liver, heart and brain. The tissues were homogenized on ice in phosphate buffer saline (PBS, pH 7.2)

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using mechanical homogenizer, 100 mg of tissue in 1 mL of PBS; postmitochondrial supernatant was collected after centrifugation (15 min at 10 000 g at 4°C) and stored frozen at −80 °C until biochemical analyses. All biochemicals and enzymes were purchased from Sigma-Aldrich (Prague, CR), other chemicals used for preparation of buffers were of the highest commercial grade available. Glutathione-S-transferase (GST) activity was measured spectrophotometrically at 340 nm using 1 mM 1-chloro-2,4dinitrobenzene (CDNB) and 2 mM GSH in PBS (Habig et al., 1974). Specific activity was expressed as nmol of evolved product per minute per milligram protein. The concentration of reduced glutathione was determined by spectrophotometric method using 5,5′-dithiobis-2-nitrobenzoic acid (DTNB) as a substrate (Ellmann, 1959). Tissues were treated with trichloroacetic acid (TCA, 2.5% v/v) and centrifuged (6000 g for 10 min at 4°C). Supernatant was mixed with TRIS–HCl buffer (0.6 M TRIS, 0.015 M EDTA, pH 8.9) and 0.8 mM DTNB and incubated for 5 min at room temperature. Absorbance was measured at 420/680 nm and the concentrations (nmol GSH/mg protein) were calculated according to the standard calibration with reduced GSH. Activity of glutathione peroxidase (GPX) was determined from the rate of NADPH oxidation recorded as the decline in absorbance at 340 nm (Flohé and Gunzler, 1984). The reaction mixtures contained 3 mM GSH, 1 U glutathione reductase (GR) (1 unit [U] will reduce 1.0 μmol of oxidized glutathione per min at pH 7.6 at 25°C), 0.15 mM NADPH in 0.1 M potassium phosphate/1 mM EDTA buffer (pH 7). Substrate used for the assay was 1.2 mM butylhydroperoxide. Also the activity of GR was determined by spectrophotometric measurement of NADPH oxidation (Carlberg and Mannervik, 1975). Assays for GR activity were performed in microplates, and the reaction mixtures contained 0.05 M potassium phosphate/1 mM EDTA buffer (pH 7.0), 1 mM oxidized glutathione (GSSG), 0.1 mM NADPH and the supernatant (0.25% v/v). Specific activities of both GPX and GR were expressed as nmol NADPH oxidized per minute per milligram protein. The level of lipid peroxidation in avian tissues was assessed as total thiobarbituric acid (TBA) reactive species (TBARS) (Uchiama and Mihara, 1978; Livingstone et al., 1990). The extracts were mixed with trichloroacetic acid (TCA, 6% w/v) and butylated hydroxytoluene (0.6% w/v) and centrifuged (1500 g for 20 min). Supernatant was further mixed with 0.06 N HCl and 40 mM TBA prepared in 10 mM TRIS (pH 7.4). The mixture was boiled in water bath for 45 min and then cooled to room temperature. Absorbance of the sample was measured at 550/590 nm and the concentration of TBARS (nmol TBARS per milligram protein) was calculated according to the standard calibration curve generated with malondialdehyde prepared by acidic hydrolysis of 1,1,3,3-tetraethoxypropane. The protein concentrations were determined by the method using Folin–Ciocalteu phenol reagent that forms with proteins red-coloured complex measurable at 680 nm (Lowry et al., 1951). Bovine serum albumin was used as a standard for protein calibration. The activity of cytochrome P-450-dependent 7-ethoxyresorufin O-deethylase (EROD) was analysed fluorimetrically (Prough et al., 1978). The reaction mixtures contained Hepes buffer (25 mM, pH 7.8) with dicumarol (1 mM), supernatant and 7-ethoxyresorufin (10 μM), which was used as a substrate. The reaction was started by the addition of 0.2 mM NADPH followed by incubation at 37°C for 20 min. The

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excitation and emission wavelengths were set at 530 and 585 nm, respectively. Enzyme activity results are given as pmol resorufin per milligram protein per minute. The GENios microplate reader (Tecan Group, Switzerland) was used for measurement of absorbance in all spectrophotometric assays and the POLARstar OPTIMA (BMG LABTECH, Germany) was used for measurement of fluorescence.

2.6.

Statistical evaluation

Statistical analyses were performed with Statistica for Windows® 7.0 (StatSoft, Tulsa, OK, USA). Results from different treatment groups were compared by one-way analysis of variance (ANOVA) and post-hoc analysis of means using the LSD test. Homogeneity of variances was tested by Levene's test. Parameters that were not normally distributed as determined by Shapiro–Wilk's test and/or for which the variance was not homogeneous as determined by Levene's test were log-transformed prior to analysis. In case of nonhomogeneous variances, nonparametric Kruskall–Wallis test was used for comparison of the treatment groups. Spearman rank order correlations were used to characterize the relationships among parameters. Variation of the biochemical parameters was further summarized in the principal component analysis (PCA) as a tool for simplifying the information from intercorrelated variables through linear transformation of the original variables into a few principal components. PCA based on correlation matrix enabled to reduce the dimensions of measured variables to the representative principal components. The results are presented in the component score and component weight plots showing the relationships among the parameters and their role in the evaluation of the samples as well as the potential differences among various treatment groups. The length and direction of the lines represent the significance of the associated variables for the plotted components and for the discrimination of the samples based on component scores. All statistical tests were performed with the probability of type I error (α) set to be less than 0.05.

3.

Results

In this study, four treatment groups of quails were fed 10 mL daily of 3 × 105–3 × 108 cells/mL of a natural biomass with the majority content of M. aeruginosa for 10 and 30 days. The total MC concentration in the biomass ranged from 4.5 μg/L (E1) to 4600 μg/L (E4), consisting of about 40% each of MC-LR and MCRR, 7% of MC-YR and 13% of unidentified MC-like compound (Table 1, Skocovska et al., 2007). The average weight of experimental animals was 205 g, Table 1 shows the daily doses recalculated for the body weight. ELISA measurements of MCs concentration in the liver and muscles of experimental birds showed cyanotoxin accumulation in both acute and subchronic test (Fig. 1). The background values measured in controls are caused by the unspecific matrix influence in ELISA (Orr et al., 2003; Ernst et al., 2005). Many studies use ELISA for determination of microcystins; LC-MC method is recommended for better understanding of detoxification since it can distinguish the amount of MC-LR-GSH conjugate in

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Fig. 1 – Concentration of MCs (ng/g FW tissue) in pectoral muscles and liver from acute and sub-chronic test. The results are expressed as mean ± standard error. Asterisks indicate the statistically significant difference from the control group [LSD test; * = P b 0.05; ** = P b 0.01; *** = P b 0.001].

tissue (Dai et al., 2008). Low accumulation of MCs was observed in the muscles. There has been about 40% increase of MCs concentration in the highest treatment group relative to the background values in control in the muscles of both acute and sub-chronic test, but this difference was statistically significant only for the acute test. On the other hand, significant accumulation in dependence on exposure concentration was observed in the liver for both test durations. There was greatest accumulation in the liver from the acute test, where the average concentration reached 43.7 ng MCs/g FW in the highest treatment group, while no significant MC accumulation was found for any of the other treatments in acute test. In the sub-chronic test, there has been significant accumulation of MC in the two highest treatment groups (E3, E4) with average concentration 2.7 and 7.5 ng MCs/g FW, respectively. However, there has been relatively great variability in the concentrations among individuals within the greatest exposure groups reflecting interindividual differences. Activities of cytochrome P-450-dependent 7-ethoxyresorufin O-deethylase (EROD) in the studied tissues were increased after exposure to cyanobacterial biomass namely in the acute test (Fig. 2). There was a significant increase from 0.7 to 1.15 pmol resorufin/min/mg protein in the heart from acute test, the increase of EROD activity from 3.1 to 4 pmol resorufin/ min/mg protein in the brain from acute test (both starting at the second lowest exposure group E2); similar increase was found in the brain from sub-chronic test. The levels of GST activity in tissues from sub-chronic test showed more distinct changes in comparison with acute test (Fig. 3). There was a non-significant increase in the liver GST activities from acute test (270 nmol/min/mg protein in control to 320 nmol/min/mg protein in the highest exposed group E4).

On the other hand, the GST activities have been significantly increased in the liver of birds from all cyanobacteria-exposed groups in sub-chronic exposure compared to control. The subchronic exposure to higher cyanobacterial concentration (E3) leads to the increase of GST activity also in the heart (36 to 44 nmol/min/mg protein) and brain of the birds, while there has been no effect in these two tissues after acute exposure. The cyanobacterial exposure caused an increase in GSH level in most tissues in both acute and sub-chronic test (Fig. 4). There was a significant dose-dependent increase of GSH level in the liver and brain from sub-chronic test. A more pronounced effect was observed in the liver from acute test where the GSH level increased fivefold already in the lowest biomass concentration (E1). Significant increase of GSH level (8 to 14 nmol/mg protein) was also detected in the heart from acute test. On the other hand, there was a decrease of GSH compared to control (45 to 35 nmol/mg protein in E2) in the brain of E1 and E2 groups of the acute test. Both glutathione peroxidase and reductase activities slightly increased in the liver of the lowest exposure group in the acute test (data not shown). GR activity was elevated after acute exposure also in the heart in the highest exposure group. The sub-chronic exposure caused increase of GPX activity in the group E3 in the brain. On the other hand, significant decrease of GPX activity was measured in the liver from sub-chronic test scheme. Dose-dependent increase in lipid peroxidation was observed in the heart from acute test (from 0.55 to 1.2 nmol TBARS/mg protein) (Fig. 5). The lowest tested concentration has induced lipid peroxidation also in the liver and brain of birds from the acute exposure. In sub-chronic test, there was an increase of lipid peroxidation in the heart (E1) and

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39

Fig. 2 – Activity of 7-ethoxyresorufin-O-deethylase (EROD; pmol resorufin/min/mg protein) in tested tissues. Activity of 7-ethoxyresorufin-O-deethylase (EROD; pmol resorufin/min/mg protein) in tested tissues. Box includes 50% values, middle point is a median and whiskers show non-outlier range. Asterisks indicate the statistically significant difference from control [LSD test; * = P b 0.05; ** = P b 0.01].

brain (E3). The greatest basal values of lipid peroxides (ranging from 2.5 to 3.2 nmol TBARS/mg protein) were with respect to high content of unsaturated lipids found in the avian brain. Significant correlations of responses in biochemical parameters within all tested organs were found (pb 0.05). Correlation of GSH and GST was found in both the liver from acute and subchronic test. Also significant were the correlations of TBARS with GSH and GPx in the liver from acute test, as well as the correlation

of GPx with GSH and EROD in the liver from sub-chronic test. Similar correlations and also correlation of GR to EROD and TBARS were found in the heart tissue. With respect to the brain tissue, more significant correlations were found in sub-chronic test, showing interrelation of GSH with GST and EROD at low p-level (b0.001). Interestingly, there were some inter-tissue correlations of biochemical parameters, for example GST and also GSH in the liver and heart, EROD in the heart and brain and finally TBARS in the liver and brain.

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Fig. 3 – Activity of glutathione-S-transferase (nmol/min/mg protein) in tested tissues. Box plot parameters as in Fig. 2 [LSD test; * = P b 0.05; ** = P b 0.01; *** = P b 0.001].

The PCA (Fig. 6) clearly separated the control group from all the exposed groups. Also, the lowest exposure group could be clearly distinguished from the other treatments. On the other hand, the three greatest exposure groups (E2, E3, E4) could not be clearly separated, showing thus similar biochemical responses (Fig. 6A). Fig. 6B shows the component weights of individual biochemical parameters used for the PCA and documents that the separation was driven namely by the modification of

glutathione-related parameters in the liver and also by changes in EROD activities and lipid peroxidation (TBARS) in the heart.

4.

Discussion

Cyanobacterial metabolites are known to cause adverse effects in diverse organisms including plants, mammals, fish

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Fig. 4 – Level of glutathione (nmol/mg protein) in tested tissues. Box plot parameters as in Fig. 2 [* = P b 0.05; ** = P b 0.01; ***= P b 0.001].

and other aquatic organisms (Figueiredo et al., 2004). They have also been linked with unnatural bird deaths (AlonsoAndicoberry et al., 2002; Ballot et al., 2005; Ndetei and Muhandiki, 2005; Lugomela et al., 2006). In the previously published part of our study, we have demonstrated histopathological hepatic changes including swelling of hepatocytes, vacuolar dystrophy, steatosis, hyperplasia of lymphatic centers and shrunken nuclei of hepatocytes, cristolysis within mitochondria and vacuoles with pseudomyelin structures on sub-cellular level after exposure to Microcystis biomass (Skocovska et al., 2007). Apart from hepatic changes on both the cellular and sub-cellular level, there were increased activities

of lactate dehydrogenase and a drop in the blood glucose in the group receiving the highest dose of cyanobacteria for 10 days. Our data clearly document bioaccumulation of MCs namely in the bird liver. Most studies on bioaccumulation of MCs are concerned with fish, but there are also some data available for other animal species, including zooplankton, mollusks, snails, shrimps, livestock or mice (Nishiwaki et al., 1994; Amorim and Vasconcelos, 1999; Beattie et al., 2003; Orr et al., 2003; Adamovsky et al., 2007; Chen and Xie, 2007; Xie et al., 2007). High levels of accumulated MC were found in the liver of flamingos dissected in case of mass deaths in Spain in 2002

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Fig. 5 – Level of lipid peroxidation (nmol TBARS/mg protein) in tested tissues. Box plot parameters as in Fig. 2 [*=Pb 0.05; **=Pb 0.01].

(Alonso-Andicoberry et al., 2002). The measured concentration was three orders of magnitude higher (81 µg MC equivalent/g liver) than in our study and cyanobacteria have been suggested as an important agent in the high mortality of flamingos. No lethal effects were observed in quails exposed to cyanobacterial biomass (Skocovska et al., 2007), even though the dose administered in the highest exposure group (230 ng MCs/g/day) was close to the previously published LD50 of about 250 ng/g/day MC-RR for quail after intraperitoneal injection (Takahashi and Kaya, 1993). This difference is probably related to both the oral way of exposure and the complex biomass as exposure material. Previous studies with

mammals indicate that the damage of tissues caused by MCLR is possible and the route of exposure via oral ingestion is 30–100 times less toxic than via intraperitoneal injection (Fawell et al., 1999). Moreover, it has been suggested that the mechanisms of the incorporation of MC-LR into the liver by i.p. and p.o. administrations are greatly different (Nishiwaki et al., 1994). In our experiment, group E4 in sub-chronic exposure ingested overall 1381 μg total MCs/205 g (i.e. 6737 ng/g) in thirty days and the tissue concentration was 7.5 ng MCs/g FW liver, which represents 11‰ of total ingested MCs. This observation corresponds to study with beef cattle (Orr et al., 2003), where the MC-LR equivalents in the liver represented about 12‰ of

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Fig. 6 – Component score (A) and component weight (B) plots from principal component analysis: distribution of samples from different treatment groups (A) based on the pattern of biochemical parameters (B) in the liver, heart and brain. Abbreviations: C—control, E1 to E4 —exposure groups, L—liver, H—heart, B—brain, EROD: 7-ethoxyresorufin-O-deethylase, GST: glutathione-S-transferase, GSH: glutathione content, TBARS: lipid peroxidation measured as TBARS.

ingested cyanotoxins. On the other hand, the uptake in the acute 10 day test reached 191‰ of the total dosed MC amount (43.7 ng MCs/g FW liver from the overall dose 460.44 μg total MCs/205 g, i.e. 2246 ng/g). The concentration of MCs in the hepatopancreas of various fish species after p.o. exposure ranged from 0.22 µg/g FW to 17.8 µg/g DW (Xie et al., 2004; Li et al., 2004; Soares et al., 2004; Zhao et al., 2006; Adamovsky et al., 2007), depending on the dose and duration of exposure. In the muscle of fish, the concentration ranged 0.014 µg/g FW to 1.77 µg/g DW (Xie et al., 2004; Magalhaes et al., 2003; Soares et al., 2004; Zhao et al., 2006; Adamovsky et al., 2007). The reported MC accumulation from experimental studies with various fish species is somewhat higher than the levels detected in birds in our study. In our experiment, daily doses were within the range of 0.23–225 ng MCs/g body weight and the maximal mean concentration of MCs was 0.94 and 2.3 ng MCs/g in the muscle and 7.5 and 43.7 ng MCs/g in the liver in sub-chronic and acute study, respectively. This difference in accumulation rate could be caused by the species differences. On the other hand, the ratio (liver/muscle) of MCs concentration in group E4 ranging from 8.3 to 20.5 is close to the ratio observed in various fish species (liver/muscle ratio 10 to 20) (Williams et al., 1997; Li et al., 2004; Malbrouck and Kestemont, 2006; Adamovsky et al., 2007). Next to the bioaccumulation this paper documents significant modulations of sub-lethal parameters in the exposed individuals. To our knowledge, this is the first study focused on both detoxification and antioxidant parameters in birds (see summary Table 2) after exposure to natural cyanobacterial biomass in a controlled experiment. Most of the studied parameters have shown stronger modulation at the shorter time of exposure (acute test) than in the prolonged exposure. Also the blood hematological and biochemical parameters

have shown greater changes (stronger effects) on day 10 than on day 30 as shown in our previous report (Skocovska et al., 2007). Many enzymes are involved in the first biotransformation steps by cytochrome P450 enzyme family. P450 induction has been shown as a sensitive parameter reflecting the exposure of birds to various contaminants (Walker and Ronis, 1989; Barron et al., 1995). The EROD activity studied in this work is only one representative of this large enzyme family and does not completely reflect the detoxification capacity. A good agreement between EROD levels in our experiment and plateau assessed in study of five different bird species was found (Liukkonen-Anttila et al., 2003). Our study documents an increase in EROD activity in the heart and brain after cyanobacterial exposure. However, there was no significant increase of this enzyme activity in the liver. Correspondingly, Wang et al. (2006) did not observe significant modulations of cytochrome P450 1A mRNA levels in the liver of tilapia exposed to MC-LR, whereas gene expression of GPx and sGST was increased significantly. The elevated EROD activity in the heart and brain could be thus probably linked to other cyanobacterial components than MC. Conjugation of MC-LR with GSH catalyzed by GST is a crucial part of its detoxification pathway (Pflugmacher et al., 1998; Fu and Xie, 2005). Moreover, GSH might be responsible for the higher resistance to MCs (Qiu et al., 2007). GST activities increased in all studied organs, but namely in the liver, only in the sub-chronic exposure, while GSH in the liver was increased in both the acute and sub-chronic exposure. These results agree with another report pointing out the increase of GST activity in the early stages of zebra fish embryos after 5 days of exposure to MC-LR (Wiegand et al., 1999). Contrariwise, exposure to MCs caused no effect on GST activity in

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Table 2 – Summary of the effects of cyanobacterial biomass with majority content of MC-LR and MC-RR on bird antioxidative and detoxification system GSH GST GPX GR TBARS EROD Acute test

Liver Heart Brain Sub-chronic test Liver Heart Brain

↑ ↑ ↓ ↑ – ↑

– – – ↑ ↑ –

↑ – – ↓ – ↑

↑ ↑ – – – –

↑ ↑ ↑ – ↑ ↑

– ↑ ↑ – – ↑

Statistically significant increase ↑ and decrease ↓; P b 0.05.

experiments with rats (brain), mice (liver) and hepatocytes of common carp (Cyprinus carpio) (Li et al., 2003; Gehringer et al., 2004; Maidana et al., 2006) and other experiments with fish eggs exposed to cyanobacterial extracts and fish exposed to MC-RR and MC-LR showed inhibition of GST activity (Pietsch et al., 2001; Malbrouck et al., 2003; Cazenave et al., 2006). However, the quails have been exposed to the complex cyanobacterial biomass, which contains many other components than just MCs. Modulations of MC-effects by co-exposure to cyanobacterial lipopolysaccharides or other cyanobacterial metabolites were reported (Pietsch et al., 2001; Best et al., 2002; Dvorakova et al., 2002; Wang et al., 2006) and therefore effects observed in any study with complex cyanobacterial biomass should not be simply linked to the MCs (Falconer, 2007). In correspondence with the enhanced activity of GST, there was an increased level of GSH in the liver, heart and brain from sub-chronic test confirming the importance of these two biomolecules in protection from the harmful effects of MC. The non-enzymatic compound GSH is considered the major intercellular antioxidant, which serves also as a substrate for GPX to reduce peroxides and directly acts as a free radical scavenger. A significant rise of GSH level was identically detected after exposure of rat hepatocytes to MC-LR (Bouaicha and Maatouk, 2004) and in the hepatopancreas of the silver carp (Blaha et al., 2004; Li et al., 2007) exposed to MC-producing cyanobacterial water bloom. On the contrary, decreased level of GSH in fish hepatocytes or no modulations of GSH level in Carassius auratus p.o. exposed to MC-LR were shown (Li et al., 2003; Malbrouck et al., 2004). The response of GPX activities differed in the acute and sub-chronic exposure scheme. The increased GPX activity observed in the liver from acute test corresponds with results of some other studies, including the induction of GPX activity in tilapia (Oreochromis sp.) and loach (Misgurnus mizolepis) p.o. exposed to MCs (Jos et al., 2005; Li et al., 2005) as well as GPX induction in the hepatopancreas and intestines of Corydoras paleatus exposed to 2 μg/L MC-RR (Cazenave et al., 2006) and enhanced GPX activity in mice liver after 32-hour study with MC-LR (Gehringer et al., 2004). However, no changes or decrease in GPX activity were observed in tilapia (Oreochromis sp.) after acute exposure to MCs and cyanobacterial cells containing microcystins (Prieto et al., 2006, 2007). Furthermore, the increase in the liver and heart in acute exposure and the decrease in the liver in sub-chronic exposure was observed for GR activities. Enhanced GR activity was found also in the hepatopancreas and brain of MC-LR or MC-

RR exposed fish (Cazenave et al., 2006; Prieto et al., 2006), whereas depletion of GR activity was found in the liver and kidney from rats exposed to MC-LR and in tilapia exposed to cyanobacterial cells containing microcystins (Moreno et al., 2005; Prieto et al., 2007). GPX has been shown to play an important role in protection against lipid peroxidation via removal of lipid hydroperoxides (Wang et al., 2001). Lipid peroxidation, mostly measured as TBARS, is commonly understood as oxyradical production by peroxidation of cellular lipids and it is known to be induced by cyanobacterial toxins (Halliwell and Gutterdige, 1999). In our study cyanobacterial biomass containing predominantly MCLR and MC-RR induced significant increase of TBARS level in all studied organs, mostly at the lowest exposure concentration (4.5 μg/L MCs). TBARS levels were increased also in the hepatopancreas, kidneys and gills of tilapia exposed to MCs (Prieto et al., 2006, 2007) and to crushed cyanobacterial cells (Jos et al., 2005), or in the hepatopancreas of silver carp exposed to cyanobacterial bloom dominated by M. ichthyoblabe and M. aeruginosa (Blaha et al., 2004). Significant increase of TBARS level was detected in mice exposed to 75% LD50 dose of pure MC-LR (Gehringer et al., 2004) and also in rat hippocampus after injection of MC-LR (Maidana et al., 2006). Another study, however, reported a decrease in TBARS level in the hepatopancreas and gills of MC-RR exposed fish (Cazenave et al., 2006) or no changes of lipid peroxidation in fish fed with cyanobacterial biomass (Li et al., 2005). Correlations among the oxidative stress parameters and detoxification enzymes activities illustrate the complex character of the response and interdependence among parameters. The interrelation was demonstrated in the liver as the most important place of detoxification of xenobiotics in birds (Riviere et al., 1985), with strong potential for impact from MCs known as strong hepatotoxic agents. Moreover, significant modulations of detoxification and antioxidative compounds and their relations were also found in the heart and brain, indicating that these organs are also highly affected, since they showed increased lipid peroxidation after cyanobacterial exposure. The observed effects of MC-containing cyanobacterial biomass in the brain well correspond with recent findings that MC-LR induces oxidative stress in rat brains along with behavioral changes (Maidana et al., 2006). Moreover, organic anion transport protein OATP1A2 expressed in human liver and brain has been demonstrated to mediate intracellular uptake of MC-LR (Fischer et al., 2005), which further indicates the brain as another target of MC toxicity. Birds coming to contact with eutrophicated aquatic ecosystems seem to react to the secondary metabolites of cyanobacterial blooms as to xenobiotics. The overall pattern of detoxification and oxidative stress responses clearly separates the control and the lowest exposure group from all the higher exposed groups documenting the shift in the detoxification and antioxidative balance after cyanobacterial exposure. General activation of the antioxidant enzymatic system in quails after the exposure to natural cyanobacterial biomass documents the occurrence of oxidative stress in the studied organs and their ability to produce antioxidative molecules protecting cells against adverse oxidation processes. Interesting differences were found between acute and subchronic exposure, both in the biochemical and accumulation

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parameters, which may indicate the potential adaptation of the detoxification and antioxidative system to the exposure with cyanobacterial biomass with increasing time of exposure. They are characterized namely by dose-dependent increase of GSH content as well as significant increase of GST activity in all three organs in the sub-chronic exposure. The increase of GST activity and GSH content corresponds with their role in detoxification pathway of MCs (Pflugmacher et al., 1998; Fu and Xie, 2005), which implies that with increasing concentration of MCs there is increasing need of GST and GSH providing the conjugation of MCs to less toxic compound. On the other hand, the higher accumulation in acute test has been linked with stronger changes of other detoxification (EROD) and oxidative stress parameters (GPx, GR). Taken together, these data document that increased activities of detoxification enzymes could lead to greater biotransformation and elimination of the MCs from both liver and muscle and thus lower accumulation at the longer exposure time. This inference corresponds to the six times lower accumulation of MC in the liver of the highest exposure group of the sub-chronic test compared to the acute one. These results support the hypothesis of the potential adaptation of the avian detoxification system to the sub-chronic exposure.

5.

Conclusions

The exposure of model birds to natural cyanobacterial biomass caused significant changes in levels and activities of antioxidative and detoxification compounds and accumulation of cyanotoxins mainly in the liver and little accumulation in the muscles. Cyanobacteria are thus capable to induce oxidative stress responses in birds linked with activation or inhibition of detoxification compounds. The generation of oxidative stress combined with insufficiency of defense mechanisms could in sensitive species at prolonged exposure potentially result in effects on the health status, especially if other stressors are involved at the same time, which is often the case in the environment.

Acknowledgements Supported by project No. 1 M6798593901 of the programme “Research Centres PP2 — DP01”(1 M), project AVOZ60050516 and project MSMT No. 6215712402.

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