Damage of cell membrane and antioxidative system in human erythrocytes incubated with microcystin-LR in vitro

Toxicon 47 (2006) 387–397 www.elsevier.com/locate/toxicon

Damage of cell membrane and antioxidative system in human erythrocytes incubated with microcystin-LR in vitro Paulina Sicin´ska a,b, Boz˙ena Bukowska a,*, Jaromir Michałowicz a, Wirgiliusz Duda a a

Department of Biophysics Environmental Pollution, University of Ło´dz´, Banacha 12/16, 90-237 Ło´dz´, Poland b Department of Molecular Biophysics, University of Łodz´, Banacha 12/16, 90–237 Łodz´, Poland Received 7 March 2005; revised 28 November 2005; accepted 6 December 2005 Available online 2 February 2006

Abstract The effect of the exposure of human erythrocytes to different concentrations of microcystin-LR were studied. Lipid peroxidation, membrane fluidity, cell morphology, haemoglobin oxidation and changes in the activity of antioxidant enzymes were investigated. Human erythrocytes were incubated with microcystin-LR at concentrations of 1–1000 nM for 1, 6, 12 and 24 h. We observed that microcystin-LR induces a significant increase of the level of thiobarbituric acid reactive substances (TBARS), formation of echinocytes, haemolysis, conversion of oxyhaemoglobin to methaemoglobin, decrease of membrane fluidity on the level of 16 carbon atom fat acids. The compound also changed antioxidative enzymes activities: catalase, superoxide dismutase and glutathione reductase and formation of reactive oxygen species (ROS). All of the observed changes point out that 100 nM of Microcistin LR is the liminal (threshold) toxic dose for human erythrocytes. This dose caused most of the described changes. Observed damages of erythrocytes membrane and antioxidative enzymes may be the result of direct covalent binding of microcystin-LR with -SH residues of proteins and indirectly be related with reactive oxygen species formation. q 2005 Elsevier Ltd. All rights reserved. Keywords: Microcystin-LR; Erythrocyte; Haemoglobin; Free radical; Antioxidative enzymes

1. Introduction Contamination of water by toxic blooms of cyanobacteria (blue–green algae) has occurred in many regions of the world and represents a serious public health problem (Carmichael, 1994; Dawson, 1998; Vieira et al., 2005). Cyanobacteria are planktonic algae and some of them produce microcystins, a group of hepatotoxins with strong cytotoxic activity. Microcystins are produced by cyanobacteria that belong to the genera Microcystis, Anabaena, Nodularia, Oscillatoria and Nostoc (Ding et al., 2000; Carmichael, 1992). Consumption of water contaminated * Corresponding author. Tel.: C48 426354475; fax: C48 426354473. E-mail address: [email protected] (B. Bukowska).

0041-0101/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2005.12.006

with cyanobacterial toxins caused death of domestic and wild animals (Carmichael, 1994). In Brazil, 52 dialysis patients died of acute failure due to microcystin contamination of the water used in the treatment (Azevedo et al., 2002). This event implicated decision undertook by World Health Organization which confessed epidemiological character of cyanobacterial toxins and established international quota of admissible concentration of microcystin in drinking water at 1 nM. Bioaccumulation of microcystins by aquatic organism including fish, shellfish and zooplankton determines the risk of health problems among animal and people that eat food contaminated with cyanotoxins (Pinho et al., 2003). The toxins have general structure cyclo-(D-Ala-L-Xerythro-b-methyl D-isoAsp-L-Y-Adda-D-isoGlu-N-methyldehydroAla), where Adda refers to the b-amino acid residue

388

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of 3-amino-9-metoxy-2,6,8-trimethyl-10-phenyl-deca-4,6dienoic acid, X and Y represents variable L-amino acid (Carmichael, 1994; Dawson, 1998). The unusual amino acid Adda is essential for expression of biological activity (Trogen et al., 1996). Up to date, more then 60 structurally different microcystins have been identified (Yongding et al., 2002). The most widely studied microcystin is microcystinLR (MC-LR) in which the variable L-amino acid are leucine and arginine. MC-LR has an LD50 in mice and rats of 36– 122 mg/kg by various routes, including aerosol inhalation (Dawson, 1998). Up to now, numerous investigations concerning the effect on microcystin-LR on hepatocytes have been performed. It is known that in hepatocytes microcystin is taken by multi-specific bile acid transporters (Runnegar et al., 1995) where it induces the production of reactive oxygen species (Ding et al., 2001), and lead to an increase of the formation of lipid peroxides (Hermansky et al., 1990; Bouaı¨cha and Maatouk, 2004). Lipid peroxides are known to be reduced by the action of glutathione peroxidase to alcohols using glutathione (Nordberg and Arner, 2001). The detoxification of microcystin-LR in the liver is known to occur via its conjugation to glutathione by glutathione S-transferase activity (Takenaka, 2001) and is transported to the kidneys and intestine for excretion (Ito et al., 2002). Increase in GST activity is critical for the detoxification of microcystin-LR that is regulated at the transcriptional level, and induces de novo synthesis of GSH (Gehringer et al., 2004). GST activity is increased in aquatic organisms and mice (Gehringer et al., 2003) exposed to MC-LR (Pietsch et al., 2001). In work the effect on microcystin-LR (the most toxic among cyanobacterial toxins) on human erythrocytes was investigated. Unfortunately, little is known about the effect of this toxin on erythrocytes. Microcystin-LR may penetrate the blood whiting ileum and then is administrated into another tissues (Dahlem et al., 1988). In work the effect of microcystin-LR on erythrocytes was examined in regard to possibility of contact of these cells with toxin and very important function of erythrocytes in organism. The main function of erythrocytes (RBC) is to provide the tissue with oxygen and remove carbon dioxide and protons produced in metabolic processes. This important function must be performed efficiently; therefore, the structure of RBC is completely subordinate to their task. Human erythrocytes are biconcave discs averaging about 7.7 mM in diameter. In mammals, the erythrocyte cytoplasm is destituted of nucleus also mitochondria, lysosomes and Golgi apparatus. Erythrocyte that has not those organelles is not a typical cell; nevertheless, its structural and functional simplicity makes it as a convenient cellular model that is suitable for studies concerning toxicity of xenobiotics. Using this simple cellular model (with effective antioxidant system) the authors tried to answer the question: does microcystin-LR cause reactive oxygen species formation and induce oxidative stress in cell.

In the present work, doses in the range of 1–1000 nM of microcystin-LR of erythrocytes were used (the concentrations comparable to that accumulated in people exposed to action of this compound) to estimate, which is the threshold toxic dose of microcystin-LR towards erythrocytes.

2. Materials and methods 2.1. Reagents 5,5 0 -Dithiobis-(2-nitrobenzoic acid), reduced and oxidized glutathione, microcystin-LR, trichloroacetic acid, NADP, NADPH, glutathione reductase were obtained from Sigma. Other chemicals were bought in POCh (Poland) and were of analytical grade. 2.2. Erythrocytes The following biological material was used: human erythrocytes were obtained from whole blood, taken from donors at the Blood Bank of Lodz. Erythrocytes were separated from blood plasma and leukocytes by centrifugation (600 g, 10 min) at 4 8C and washed three times with phosphate-buffer saline (PBS; 150 mmol/l NaCl, 1.9 mmol/l NaH2PO4, 8.1 mmolK1 Na2HPO4, pH 7.4) and incubation buffered (NaCl 8.1%, KCl 0.94%, MgCl2 0.143%, glucose 1.8%, Hepes 2.3%, Tris 1 M, gentamecin 40 mg/l) Isolated erythrocytes at a haematocrit of 5% were treated at the temperature of 37 8C for 1, 3, 12, 24 h with 1–1000 nM. After this treatment different (some) erythrocyte parameters were examined. 2.3. Changes in erythrocytes membrane 2.3.1. Lipid peroxidation Lipid peroxidation in human erythrocytes was quantified by measuring the formation of thiobarbituric acid reactive substances (TBARS) (Stock and Dormandy, 1971). Erythrocytes were mixed with 20% TCA (1:1). Samples were centrifuged (600 g!10 min). Fifteen percent TBA was added to supernatant and samples were heated at 100 8C for 15 min. The absorbance of the supernatant was measured at 532 nm. Lipid peroxidation was expressed in absorbance units. 2.3.2. Fluidity of human red blood cell membranes: a spinlabel study The membrane fluidity of red blood cells from healthy donors was measured by means of electron paramagnetic resonance (EPR) spectroscopy with the aid of two spin labels which reside at different depths within the lipid bilayer: 5-doxylstearic acid (5-DSA) and 16-doxylstearic acid (16-DSA) (Sigma, St Louis, Mo, USA). Erythrocytes in the 50%-haematocrit suspension were labeled with the spin labels 5-DSA or 16-DSA and then

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incubated for 30 min at room temperature. EPR measurements were performed at 20–21 8C in a Brucker 300 spectrometer. The order parameter S was determined using 5-DSA. The order parameter is a degree of the distribution of molecular orientations with respect to a reference axis chosen in this study to be normal to the membrane surface. An increase in order parameter reflects a decrease in the segmental flexibility of the spin sample (Schreier et al., 1978). The order parameter is given by the following Eq. (1) SZ

ðTs KTt Þa ðTZZ KTXX Þa 0

(1)

where S is the order parameter; aZ ð1=3ÞðTXX C TYY C TZZ Þ, and is the isotropic hyperfine constant for nitroxide in a crystal; a 0 Z ð1=3ÞðTs C 2Tt Þ, and is the isotropic hyperfine coupling constant for nitroxide in a membrane; TXX, TYY, TZZ are the hyperfine splitting parameters determined after the incorporation of nitroxide derivatives into a host crystal, with, TXXZ6.16; TYYZ6.1; TZZZ32.46 and Ts is the hyperfine splitting constant for the magnetic field parallel to the bilayer normal; Tt is the hyperfine splitting constant for the magnetic field perpendicular to the bilayer normal. For the 16-DSA spin-label rotational correlation times tB and tC were measured, reflecting the spin-label motion in the directions perpendicular and parallel to the long axis of a lipid molecule. The correlation time tB, which describes the motion in the direction perpendicular to the long axis, is given by Eq. (2) and tC, which describes the motion of the probe in the direction parallel to the long axis is given by (2) (Schreier et al., 1978)    1=2  h0 1=2 h tB Z kDW0 K 0 hC1 hK1  tC Z kDW0

h0 hC1

1=2

 C

h0 hK1

1=2

 K2

ð2Þ

where hC1 is the low-field line height; h0 the mid-field line height; hK1 the high-field line height; and Æ W is the midfield line width. The correlation times are inversely proportional to the flexibility of the probe. An increase in their values indicates reduced fluidity of the bilayer in the vicinity of the spin label. 2.3.3. Phase contrast microscope Erythrocytes after incubation with different concentrations of microcystin-LR-LR were fixed on the glass surface. Then samples were observed using phase contrast microscope (Olympus, Japan) at magnification of 400!. 2.3.4. Haemolysis The ratio of haemolysis was calculated from the equation Hð%Þ Z

Apb 100% Awater

389

where H%, haemolysis of erythrocytes incubated with microcystin-LR; Apb, absorbance of supernatant in all samples; Awater, absorbance after complete haemolysis. 2.4. Haemoglobin The concentration of haemoglobin was measured by the Drabkin method (1946). Absorption spectra of haemoglobin were obtained in the wavelength range of 440–700 nm using an automatic spectrophotometer connected to a computer. The percentage of met-Hb in the total Hb content was calculated from absorbance at 630 and 700 nm both for haemoglobin of control erythrocytes and haemoglobin of erythrocytes treated with microcystin-LR (pb). Haemoglobin treated with potassium ferricyanide (100% met) was used as a positive control. % met
Hb Z 100

ðApb630 KApb700 Þ ðA100%met630 KA100%met700 Þ

2.5. Enzymatic activities 2.5.1. Glutathione reductase activity Glutathione reductase (EC 1.6.4.2) recycles oxidized glutathione (GSSG) by reducing it to GSH in an NADPHdependent manner. GR-catalyzed NADPH oxidation was determined spectrophotometrically using 3Z 6.22 mMK1cmK1 in a Cary spectrophotometer. One unit of glutathione reductase activity was defined as the activity degrading 1 mmol NADPH in 1 min/g Hb. At saturating substrate concentrations (100-mM NADPH, 1 mM GSSG) conversion of NADPH was monitored continuously at 340 nm at 25 8C for 3 min (Carlberg and Mannervik, 1972). 2.5.2. Catalase activity Catalase activity (EC 1.11.1.6.) was determined by the method of Aebi (1984). To 3 ml H2O2 (54 mM H2O2 in 50 mM phosphate buffer pH 7.0) 5 ml of a catalase solution was added and the decrease in H2O2 was measured spectrophotometrically at 240 nm, at 258 for 60 s. In the erythrocyte preparations, haemolysates were centrifuged (12,000 rpm) and estimation of activity was made with 1% haemolysates. One unit of catalase activity was defined as the activity required to degrade 1 mmol of hydrogen peroxide in 60 s. 2.5.3. Superoxide dismutase (SOD) Superoxide dismutase activity was determined by the method of Misra and Fridovich (1972a,b), which is based on the ability of superoxide dismutase to inhibit the process of epinephrine self-oxidation in alkaline medium. In the reaction of coloured adrenochrome formation, the superoxide anion-radical is formed as an intermediate product. Superoxide dismutase activity was measured by monitoring the increase of absorbance at 480 nm.

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390

Control

0.12

1nM

10nM

Absorbance 532 nm

0.1

*

100nM *

*

1000nM *

*

* *

*

*

*

0.08 0.06 0.04 0.02 0 1

6

12

24

Time [h] Fig. 1. Level of lipid peroxidation (expressed in absorbance units of TBARS products) in erythrocytes incubated with microcystin-LR. MeanGSD of 11 experiments. Significantly different from control (P!0.05); paried (*) t-test.

high interindividual differences. The difference was considered to be significant for P!0.05.

2.6. Oxidation of DCFH-DA—the level of ROS The rate of 6-carboxy-2 0 ,7 0 -dichlorodihydrofluoresceine diacetate oxidation was measured by flow cytometry. 6-Carboxy-2 0 ,7 0 -dichlorodihydrofluorescein diacetate is a sensitive and widely used compound for detection of intracellular oxidants production (Grzelak, 2001; Grasso et al., 2003). Oxidation of DCFH-DA creates highly fluorescent dichlorofluorescein. The DCFH diacetate was added to erythrocytes. It diffuses across the cell membrane and is hydrolyzed by intracellular esterases to DCFH which, upon oxidation, yields highly fluorescent 6-carboxy-2 0 ,7 0 -dichlorofluorescein (DCF). The final concentration for DCFH-DA was 20 mM. The samples were incubated at 37 8C in the dark. Fluorescence was measured with Becton Dickinson, LSR II flow cytometer with exicitation of 488 nm (blue laser) and 530 emission filter. 2.7. Statistical analyses For statistical analysis Student’s paired* t-test was used which is obligatory for data presented in work because of

3. Results 3.1. Lipid peroxidation Statistically significant increase of the level of thiobarbituric acid reactive substances was noted after incubation of erythrocytes with microcystin-LR at concentrations of 1 nM for 12 and 24 h, and of 100 nM for 6 h of incubation (Fig. 1). 3.2. Fluidity of erythrocytes membrane No changes were observed in the value of the parameter S after incubation of erythrocytes with different concentrations of microcystin-LR (Table 1). Microcystin-LR caused statistically significant increase of TB and TC parameters for dose of 1000 nM in all incubation times. Moreover, the changes were noted after 6

Table 1 The effect of microcystin-LR on the order parameter S of 5-DSA in erythrocyte membranes Concentration microcystinLR (nM)

Parameter S 1h

6h

12 h

24 h

Control 1 10 100 1000

0.733G0.052 0.731G0.042 0.734G0.047 0.739G0.057 0.729G0.074

0.741G0.029 0.734G0.011 0.727G0.064 0.732G0.015 0.726G0.056

0.729G0.067 0.731G0.072 0.735G0.069 0.740G0.058 0.726G0.046

0.734G0.059 0.741G0.034 0.725G0.075 0.736G0.074 0.739G0.057

MeanGSD of eight experiments. No statistically significant effects.

P. Sicin´ska et al. / Toxicon 47 (2006) 387–397 Table 2 Effect of microcystin-LR on the rotational correlation times tC and tB for erythrocytes labelled 16-DSA

1h Control 1 10 100 1000 6h Control 1 10 100 1000 12 h Control 1 10 100 1000 24 h Control 1 10 100 1000

tC!10K10 s

tB!10K10 s

23.04G2.56 23.50G1.52 25.24G1.64 27.53G1.98 28.96G1.62*

16.12G1.02 16.21G1.12 18.07G0.99 19.55G1.15 20.41G1.62*

18.58G2.52 20.49G1.62 23.85G1.98 24.12G1.23* 25.01G1.52*

13.53G1.75 14.67G1.36 17.76G1.23 17.63G1.02* 18.13G0.98*

20.21G2.65 23.37G1.85 26.44G2.12 27.73G2.03* 27.79G1.18*

13.32G2.03 15.12G1.25 17.69G1.98 18.81G2.03* 18.52G1.18*

20.52G2.36 24.68G1.98 27.14G1.46* 28.42G1.89* 29.36G1.03*

14.52G1.89 17.29G1.32 19.21G1.46* 20.26G1.89* 20.78G1.03*

MeanGSD of eight experiments. Significantly different from control (P!0.05); paried (*) t-test.

and 12 h of incubation from a dose of 100 nM and after 24 h from a dose of 10 nM (Table 2). 3.3. Morphological transformations of erythrocytes Morphological changes in erythrocytes were observed after 6, 12, and 24 h of incubation with doses of 10 and 100 nM of microcystin-LR. Microscopic observations revealed that microcystin-LR converts the shape of blood corpuscles to echinocytes (Photo 1). 3.4. Haemolysis Microcystin-LR caused increase of haemolysis degree in erythrocytes. The highest changes were observed after 12 and 24 h of incubation doses of 1000 and 100 nM increased of haemolysis degree, respectively (Fig. 2). 3.5. Oxidation of haemoglobin The increase of methaemoglobin was observed after incubation of erythrocytes with microcystin-LR-LR-LR. Statistically significant changes for dose 1000 and 100 nM were noted after 6 h of incubation and for 10 nM the changes were observed after 12 and 24 h (Fig. 3).

391

3.6. Enzymatic activities 3.6.1. Glutathione reductase The decrease of activity of glutathione reductase was noted. Microcystin-LR caused essential changes for dose of 1000 nM for all incubation times. After 12 and 24 h of incubation the changes from dose of 10 nM were observed (Table 3). 3.6.2. Catalase Microcystin-LR increased the activity of catalase in concentration of 1 and 10 nM in all incubation times. However, doses of 100 and 1000 nM decreased catalase activity after 12 and 24 h incubation (Table 4). 3.6.3. Superoxide dismutase Microcystin-LR caused significant decrease of superoxide dismutase activity even after 1 h of incubation for dose of 1000 nM, for 6 h the changes from dose of 100 nM were noted. After 12 and 24 h of incubation essential changes were observed from dose of 10 nM (Table 5). 3.7. The level of reactive oxygen species Statistically significant increase of fluorescence after microcystin-LR influence was noted for doses of 1000 nM after 6, 12, 24 h of incubation and for dose of 100 nM after 12 and 24 h of incubation (Fig. 4).

4. Discussion In the light of the hitherto investigations the effect of cyanobacterial toxins on human erythrocytes has not been completely explained and literature data are often inconsistent and discontinuous. That is why the purpose of our work was to explain the mechanism of action of microcystin-LR in human erythrocytes and to answer the question—does microcystin-LR stimulate ROS formation and induce oxidative stress in erythrocytes. The mechanism of toxicity of many compounds concerns formation of reactive oxygen species (ROS), including superoxide anion, hydrogen peroxide, superoxide radicals and hydroxyl radical. ROS are capable to react with proteins, nucleic acids, lipids and/other molecules leading to changes in their structures and thus damage of cells. Fortunately, the cells are able to protect themselves against oxidative stress, as they developed numerous defensive mechanisms based on the antioxidative enzymes activity and action of low molecular antioxidants such as glutathione (Mates, 2000; Sweet and Blonchard, 1991). To examine if microcystin-LR changes biochemical parameters of erythrocytes and causes oxidative stress, the effect of microcystin-LR on cellular membrane (lipid peroxidation, changes in membrane fluidity, transformations of cell shape and haemolysis), oxidation of

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Photo 1. The change of shape of erythrocytes incubated with microcystin-LR (same echinocytes are marked by arrows).

haemoglobin, activity of antioxidative enzymes (SOD, CAT and GR) and the level of ROS formation in erythrocytes was evaluated. Analysis of erythrocytes concerning lipid peroxidation products formation after cells’ treatment with microcystinLR revealed a significant dose and time-dependent increase of TBARS concentrations when compared with controls (Fig. 1). It is known that lipid peroxidation products modify physiological properties of cell’s membrane. Lipid peroxidation causes membrane depolarization, disturbs asymetry of membrane’s lipids, induces inhibition of membrane enzymes, modulates transport of proteins and finally causes

lost of plasmatic membrane integrity (Bartosz, 2003). In erythrocytes (in non-nucleated cells) lipid peroxidation leads to membrane damage and thus to haemolysis and finally death of erythrocyte. The increased peroxidation of non-saturated residues of fatty acids may considerably affects on the decrease of lipids fluidity and in the consequence may stiffen the membrane. So that, membrane fluidity on the level of 5 and 16 carbon of fatty acid residue was examined. No changes in the parameter S for 5-DSA indicator was noted (Table 1) that suggests no disturbance in membrane fluidity on the level of 5 carbon of fatty acid residue. However, the increase of TB

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1.8

Control

1nM

10nM

100nM

1000nM

*

*

1.6 % of haemolysis

393

*

1.4 1.2 1 0.8 0.6 0.4 0.2 0 1

6

12

24

Time [h] Fig. 2. Percent of haemolysis in erythrocytes incubated with microcystin-LR. MeanGSD of 14 experiments. Significantly different from control (P!0.05); paried (*) t-test.

and TC parameters of 16-DSA indicator (Table 2) was observed what evidenced the decrease of membrane fluidity on the level of 16 carbon atom. Hermansky et al. (1991) administrated microcystin-LR to mice and observed time-dependent decrease of hepatic microsomal membrane fluidity. Ding et al. (2000, 2001) also related that microcystin-LR provoked the increase of membrane’s fluidity and formation of considerable amounts of ROS in rats. The changes in membrane’s fluidity lead to changes in shape of erythrocytes. We observed that microcystin-LR induced conversion of cell shape at concentrations from 10 nM (Photo 1). Changes in the shape of erythrocytes incubated with microcystin-LR may result from many reasons. Echinocytes are formed during binding of chemical Control

compounds to outer part of membrane’s monolayer (Bartosz, 2003) and may be also formed in a result of specific oxidative transformation of membrane skeleton (Bukowska and Zatorska, 2003). The investigations performed by Grabow and co-workers (1982) revealed that erythrocytes exposed to microcystin-LR isolated from Microcystis aeruginosa had significant morphological changes. The investigators observed also hemoaglutination of erythrocytes. Inconsistent results were obtained by Eriksson and co-workers (1987) which showed that microcystin-LR does not provoke morphological changes in erythrocytes at higher doses (90 mM) than admissible concentrations established for drinking water. The disturbance of bilayer lipid structure by peroxidation process and incorporation of xenobiotic into membrane

1nM

10nM

100nM

1000nM

20

*

18 16

*

met Hb [%]

14

*

12

*

10

*

*

8 6

*

4 2 0 0

6

12 Time [ h ]

18

24

Fig. 3. Percent of methaemoglobin in erythrocytes incubated with microcystin-LR. MeanGSD of 12 experiments. Significantly different from control (P!0.05); paried (*) t-test.

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394

Table 3 Activity of glutathione reductase in (mmol GSH/min/g Hb) in human erythrocytes incubated with microcystin-LR for 1, 6, 12 and 24 h Concentration microcystin-LR

1h

6h

12 h

24 h

Control 1 nM 10 nM 100 nM 1000 nM

4.64G0.27 4.60G0.25 4.34G0.22 4.32G0.26 3.86G0.28*

4.29G0.34 3.93G0.34 3.60G0.29 3.52G0.19* 3.53G0.22*

4.02G0.36 3.48G0.38 3.10G0.30* 3.08G0.23* 3.03G0.28*

4.68G0.41 3.80G0.38 3.68G0.25* 3.38G0.26* 3.34G0.30*

Table 4 Activity of erythrocyte catalase in (mmol H2O2/min/mg Hb) in human erythrocytes incubated with microcystin-LR for 1, 6, 12 and 24 h Concentration microcystin-LR

1h

6h

12 h

24 h

Control 1 nM 10 nM 100 nM 1000 nM

148G11 183G16* 169G21* 153G14 117G15*

146G12 195G24* 178G15* 165G26 121G10*

129G21 184G22* 162G31 126G21 116G22

145G18 171G24 155G12 138G16 111G12*

leads to changes in the shape of erythrocytes and finally causes lysis of cells and death of erythrocytes. That is why the level of haemolysis of erythrocytes was also evaluated. Lysis of erythrocytes—the cleavage of erythrocytes’ membrane and leakage of haemoglobin outside the cell always lead to its death. We observed that microcystin-LR induces haemolysis (Fig. 2). Essential role in erythrocytes plays their main component—haemoglobin. The physiological function of haemoglobin is to transport oxygen to tissues. This process depends on the ability of the ferrous form (Hb2C) to reversible binding of molecular oxygen. However, Table 5 Activity of superoxide dismutase in (U/g Hb) in human erythrocytes incubated with microcystin-LR for 1, 6, 12 and 24 h Concentration microcystin-LR

1h

6h

12 h

24 h

Control 1 nM 10 nM 100 nM 1000 nM

4137G608 3545G380 3200G363 2978G358 2375G265*

3866G682 3248G227 2972G570 2722G360* 2057G314*

4052G321 3551G424 2765G341* 2554G315* 2107G434*

4251G483 3314G253 2968G334* 2338G317* 2253G231*

oxyhaemoglobin may be converted to met-Hb (the Hb3C form), which is unable to transport oxygen. In work we observed the formation of methaemoglobin from the dose of 100 nM of microcystin-LR applied after 1-h incubation (Fig. 3). Haemoglobin oxidation may be provoked by the products of lipid peroxidation and be a/the result of inhibition of antioxidant enzymes. Catalase and GSH-Px are found as soluble proteins in erythrocytes, where they protect cells against oxidation of haemoglobin (Aebi, 1984; Bartosz, 2003; Bukowska, 2003). To date, there is no report of study concerning possible changes in activity of catalase, superoxide dismutase, and glutathione reductase in cells exposed to microcystinLR. Only Chen et al. (2004) reported changes in the activity of these enzymes exposed to microcystin-LR contained in plant tissue. GR appears to play a crucial role in the female reproductive system by recycling GSSG. The important function of this enzyme is to protect the living cells from accumulation of oxidized glutathione, protein-SSG and other mixed disulfide compounds (Bukowska, 2005). Incubation of human erythrocytes with MC-LR significantly decreases the GR activity (Table 3). The fall of GR activity may be induced by MC-LR action that interacts with thiols—Cys58–Cys63 of the enzyme. Takenaka (2001) showed that microcystin-LR bound covalently to –SH groups of the numerous compounds, such as L-cysteine and reduced glutathione (GSH). As we know sulphydryl residues play a key role in enzymes activity and all changes within their structure lead to protein inactivation (Bartosz, 2003). Previous studies on the inactivation of glutathione reductase have shown that Cys 58 within GR is often covalently modified (Schrimer et al., 1989) and exists as a highly reactive nucleophile. Also Vander Jagt et al. (1997) showed that GR inactivation by 4-hydroxynonenal involves modification of the redox-active cysteine residues that participate in the catalytic cycle of glutathione reductase and are sensitive to covalent modification. It seems that important role in GR inactivation caused by MC-LR plays oxidative stress provoked by ROS and lipid peroxidation products (Figs. 1 and 4). Vander Jagt suggests that oxidative damage of lipids, resulting in the formation of 4-hydroxynonenal and related a,b-unsaturated aldehydes, may amplify the oxidative stress through a positive feedback mechanism in which the key antioxidant enzyme glutathione reductase is inactivated by these aldehydes at low, physiologically relevant concentrations. Other authors also suggest that GR inactivation is provoked by oxidative damage, which may contribute to the oxidative stress (Tabatabaie and Floyd, 1994; Fujii et al., 2000). We observed also that microcystin-LR decreases the activity of catalase (in the highest doses (Table 4) and also superoxide dismutase (Table 5) two enzymes known to be able to ‘scavenge’ (dismutate) toxic reactive oxygen species

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395

Fig. 4. The histogram oxidation of 2 0 ,7 0 -dichlorodihydrofluorescein by radicals formed in control human erythrocytes and erythrocytes incubated with microcystin-LR 12 h. (*) Significantly different from control (P!0.05); Student’s t-test. MeanGSD of five individual experiments.

(ROS) such as the superoxide anion and strong oxidant— hydrogen peroxide. Mechanism of catalase and gluthatione reductase inactivation is similar and in our opinion mainly proceeds by covalent binding of MC-LR with thiol residues of these enzymes. According to Morikofer-Zwez et al. (1969) catalytic activity of catalase is associated with disulphide bridges formation at the active site of enzyme and enzymatic activity is supposedly dependent on the number of disulphide bridges present in protein part of the catalase. Superoxide dismutase is the third enzyme which activity is changed under the influence of MC-LR. It seems that observed decrease of the activity of superoxide dismutase in erythrocytes incubated with microcystin-LR has important consequences for these cells. Superoxide dismutase is thermostable and loses no activity during 1 h incubation at 70 8C or upon shaking with organic solvents. It retains its activity in 8 M urea. It is also resistant to proteolytic enzymes. Only some compounds like hydrogen peroxide, cyanides (Bartosz, 2003), chloric acid (Babu et al., 1999) or catechols (Bukowska and Kowalska, 2004) can damage catalase. SOD activity may be decreased due to direct damage of its protein structure by microcystin-LR and also be damaged by increasing amount of hydrogen peroxide. MicrocystinLR disturbs catalase and thus leads to formation of higher amounts of H2O2 (Table 4, Fig. 4). Disturbance in removal of superoxide radical (formed in high amounts during oxidation HbO2 to met-Hb) provokes further damage of the ¨ ztu¨rk enzyme and causes the increase of the level of H2O2 (O and Gu¨mu¨s¸lu¨, 2004).

Numerous investigations have revealed noxious effect of hydrogen peroxide on activity of superoxide dismutase. In high concentrations H2O2 behaves as a strong inhibitor of SOD activity (Uchida and Kawakishi, 1994; Sampson and Beckman, 2001). In an experiment the inhibitory effect of H2O2 increased with increasing dose of the compound. Strong inhibition (50%) of SOD activity occurred within 5 min with 0.76 mM of H2O2 and within 1 min with 6 mM H2O2 (20%) (Hodgson and Fridovich, 1975). It is also known that protein oxidation leads to modification of aminoacids, prosthetic residues and cleavage of polipeptyde chain that result in its fragmentation and thus degradation. The threat for cell’s existence is oxidation of sulphydryl residues of protein by reactive form of oxygen that appears in membranes and leads to hydrolysis of external fragments of surface glycoproteins. The result may be the membrane disintegration and labilization, induction of non-specific penetrability for proteins and disturbance of the balance in protease—antiprotease system (Bartosz, 2003). For example, semiquinones are able to bind to nucleophilic residues like –SH or –NH2 of proteins and nucleic acids, respectively (Emdadul Haque and Asanuma, 2003). As the result, macromolecules may undergo inactivation (Segura-Aguilar et al., 1997; Bukowska and Kowalska, 2004). The consequence of changes in key antioxidant enzymes may be the increase of ROS formation and oxidative stress induction. As the result of ROS activity several irreversible modifications of biologically fundamental macromolecules have been described, including oxidation of protein and initiation of the reactions of lipid peroxidation. That is why

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ROS concentration was determined using fluorescence indicator—dichlorodihydrofluoresceine. Performed investigations have revealed that microcystin-LR induces ROS formation (Fig. 4). To sum up, we have noted that microcystin-LR induces oxidative stress in human erythrocytes. Observed damages of erythrocytes membrane and antioxidative enzymes may induce cell death (lysis) and are the result of direct covalent binding of microcystin–LR with –SH residues of proteins and also be related with ROS formation induced by discussing toxin. The analysis of the results shows that 100 nM microcystin–LR is the threshold toxic dose for erythrocytes. This dose provoked most of the observed changes. The commonness of exposition of people towards cyanobacterial toxins reveals that the obtained data are very important and also alarming.

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