J. Vet. Med. B 39, 307-310 (1992) 8 1992 Paul Parey Scientific Publishers, Berlin and Hamburg

ISSN 0931 - 1793

Short Communication Institute of Microbiology, Federal Armed Forces, Medical Academy, Munich

Cytotoxicity of Cyanobacterium Microcystis aeruginosa K. HENNING, J. CREMER and H. MEYER'! Address of authors: Institut fur Mikrobiologie, Sanitatsakademie der Bundeswehr, NeuherbergstraBe 11, D-8000 Miinchen 45, Germany

With 2 figures (Received for publication July 2, 1991)

Summary Cytotoxic effects of crude extracts and fractions of the purification steps towards MicrocystinLR (MCYST-LR) were investigated in vitro. Cytoxicity was evaluated by measure of lactate dehydrogenase liberation of Chang liver cells and by hemolysis. Crude extracts of strain P C C 7806 damaged the cells within a few minutes. In contrast, MCYST-LR did not show any detectable cytotoxic effects. The cytotoxic activity could be related to a heat-labile substance with a molecular weight of about 30,000 Da.

Introduction Several reports have documented the risks of toxic cyanobacterial water blooms for drinking-water hygiene. The most important organism in this context is Microcystis aeruginosa (3, 10, 11). Toxins of Microcystis aeruginosa are able to kill vertebrates within 1-3 h. Acute effects in vivo are characterized by massive hemorrhages and necrosis of the liver. Chronic exposure to sublethal doses results in a progressive damage of the liver (1,7). The hepatotoxic effects are caused by closely related cyclic peptides, termed Microcystins (MCYST) (4). Toxicity of different Microcystis-strains has been determined in mice by intraperitoneal injection of crude extracts of algal dry mass. Death occurred in a dosedependent manner within a few hours (2). In vitro testing of crude extracts correlates well with the toxicity in the mouse bioassay. Exposure of primary and permanent cell lines to crude algae extracts results in disruption of cell membranes with liberation of lactate dehydrogenase (LDH) (2, 9). In contrast, research groups investigating the toxicity of MCYST in cell cultures could not observe either cell lysis or liberation of L D H (8, 10, 11). This study monitored the cytotoxic effects of fractions obtained during the purification of MCYST-LR.

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Material and Methods Sample preparation for the cytotoxicity tests Biomass was obtained from batch cultures of Microcystis aeruginosa strain PCC 7806 grown in et al. BG-11 medium according to DIERSTEIN et al. (6). MCYST-LR was isolated according to CREMER (5). Freeze dried algae (5g) were suspended in distilled water (10mg/ml) and extracted twice by sonification and centrifugation (30,000 g, 60 min). The supernatants (500 ml) - termed “crude extracts” - were applied to a column packed with l o g CISsilica gel (Bakerbond column, Baker Gross-Gerau, FRG) adsorbing the hydrophobic peptide fraction to the solid phase. The solution passed through the column - termed “aqueous Clx-eluate” - was collected (500ml). The toxic peptide fraction was eluted with methanol. Further purification of the “Clx-methanoleluate” was done by anion exchange chromatography using a column packed with 5 g quartenary methylamine resin (QMA) and equilibrated with 0.1 M N H 4 H C 0 3 .MCYST-LR was detached from the matrix by elution with 0.2 M NH4HC0,. About 100% purity of MCYST-LR was achieved by differential elution of the concentrated “QMA-eluate” on a reversed-phase H R 16/10-PepRPC-colum using an acetonitrile gradient. Presence of MCYST-LR in the Clx-methanol eluate and in the QMA-eluate was proved by reversed-phase thin-layer-chromatography (HPTLC) and recording UV-absorption spectra in comparison with a standard MCYST-LR (Medor, Herrsching, FRG). From each separation step (i. e., crude extract, aqueous CIx-eluate,Ctx-methanol eluate, QMA-eluate and purified MCYST-LR) samples were taken. Organic solvents were removed from the Clx-methanol eluate, QMA-eluate and purified MCYST-LR by evaporation. After solubilizing in NaCl 0.9 % the volume was adjusted to 500 ml. Cytotoxicity test Chang liver cells (Flow Laboratories, Meckenheim, FRG) were grown in Ham’s F 12-medium supplemented with 10 % fetal calf serum. Cells were seeded in 24 well plates (Costar, Cambridge, USA) and incubated at 3 7 T , 5 % C02. After 2 days the medium was removed and 0.5 ml samples diluted 1 : 3 in cell culture medium were added. Samples investigated were: crude extract, aqueous Clx-eluate,CIx-methanol eluate, QMA-eluate, purified MCYST-LR as well as 10 pg of the MCYSTLR-standard (Medor, Herrsching, FRG). All tests were performed in duplicate. After incubation for 2 h (37 “C) cytotoxicity was scored by measurement of LDH-liberation (LDH,,,,-Monotest. Boehringer Mannheim, FRG). Gel filtration The aqueous Clx-eluatewas fractionated by gel filtration on a Sephadex G 100-column (column XK 26, Pharmacia LKB, Freiburg, FRG) (80 cm x 2.6 cm; flowrate: 0.55 ml/min; fraction size: 5.5 ml, sample volume: 5 ml).

Results and Discussion The crude extract and the aqueous C18-eluate displayed a strong cytotoxic effect. Within a few minutes blebbing, rounding and granulation of the cells could be observed. Subsequently high quantities of L D H were liberated (Fig. 1). LDH-activity could be demonstrated in a dose-dependent manner even at a dilution of 1 : 50. In contrast, other fractions (C18-methanol-eluate and QMA-eluate) did not show any cytotoxic effect. LDH-liberation was similar to the negative control (cell culture medium) (Fig. 1). The application of as much as 10 &well of a standard toxin had no detectable effect although 2.5 pg is equal to 1 LDS0mouse (10). Results obtained on Chang liver cells were confirmed by using chick erythrocytes. Whereas purified MCYST-LR had n o effect, crude extract and the aqueous C18-eluate lead to hemolysis (data not shown). After fractionating the aqueous CI8-eluate on a Sephacryl S 200-column, the cytotoxicity as measured by LDH-liberation could be assigned to certain fractions (Fig. 2). After SDS-gel electrophoresis of toxic fractions a single band could be detected only with an apparent MW of 30 kDa. O u r results point out that the cytotoxicity of crude algae extracts on Chang liver cells is not related to MCYST-LR but to a different substance. This conclusion is supported by the fact that, in contrast to MCYST-LR, this substance is sensitive to heat: 10 min at 50 “C destroyed the cytotoxic activity completely (data not shown).

Cytotoxicity of Cyanobacterium Microcystis ueruginosu

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Fig. 1. Lactate dehydrogenase activity (LDH) was determined 2 h after incubation of Chang liver cells with different fractions obtained during the purification of MCYST

ERIKSSON et al. (8) observed no cytotoxic effects of MCYST on permanent cell lines or erythrocytes. Therefore they assumed the existence of cytotoxic substances different A l l . 214 nm

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from MCYST in crude algae extracts. However, an effect of MCYST on primary cells (hepatocytes) has been described. These effects are not associated with a loss of viability o r liberation of LDH (10). The reasons for the discrepancy of the in vitro findings to the severe lesions observed in vivo are n o t known. T h e good correlation between cell lysis and mouse toxicity (2) upon testing crude lysates indicate that cytotoxic substances are widespread in Microcyrtzs- and Oscillatoriastrains. O u r study and earlier reports indicate that mouse bioassays cannot be replaced by cell culture techniques without further characterization and definition of cytotoxic substances.

References 1. AMANN,M.J., 1984: Instability and variable Toxicity of HBP-TX, A Toxin in the Cyanobacterium Microcystis aeruginosa. Toxicon 22, 107- 114. 2. BERG,K., and T. AUNE,1987: Freshly prepared rat hepatocytes used in screening the toxicity of blue-green algal blooms. J. Toxicol. and Environ. Health 20, 187-197. 3. CARMICHAEL, W. W., 1986: Microcystin. In: GREYLER (ed.) Compendium of natural product toxins, Verlag MarceUDekkar, New York. 4. CARMICHAEL, W.W., V.BEASLEY,D.L. BUNNER,J.N. ELOFF, LFALCONER,P.GORHAM, Y. MIN-JUAN,R.E. MOORE,K. RINEHART, M.T. C. RUNNEGAR, 0.M. SKULBERG, K. HARADA, and M. WATANABE, 1988: Naming of cyclic heptapeptide toxins of Cyanobacteria (blue-green algae). Toxicon 26, 971 -973. 5. CREMER, J., and K.HENNING,1991: Application of reversed-phase FPLC to the isolation, separation, and amino acid analysis of two closely related peptide toxins of the cyanobacterium Microcystis aeruginosa strain PCC 7806. J. Chromatography 587, 71 -80. 6. DIERSTEIN, R., I. KAISER,and J. WECKESSER, 1988: Rapid determination of Microcystis sp. toxins by reversed phase liquid chromatographie. FEMS Microbiol. Let. 49, 143- 147. T. C., I. R. FALCONER, A. R. B. JACKSON, and M. RUNNEGAR, 1978: Isolation, charac7. ELLEMAN, terization and pathology of the toxin from Microcystis aeruginosa (Anacystis cyanea) bloom. Aust. J. Biol. Sci. 31, 209-218. J. E., H. HAGERSTRAND, and B. ISOMAA, 1987: Cell selective cytotoxicity of a peptide 8. ERIKSSON, toxin from the cyanobacterium Microcystis aeruginosa. Biochim. Biophys. Acta 930, 304-310. and W.E. S c o n , 1982: Minocystir 9. GRABOW, W.O. K., W.C. D u RANDT,0.W. PROZESKY, aeruginosa Toxin: Cell Culture Toxicity, Hemolysis, and Mutagenicity Assays. Appl. Environ. Microbiol. 43, 1425-1433. 1982: The in vivo and in vitro biological effects of the 10. RUNNEGAR, M.T. C., and I. R. FALCONER, peptide Hepatotoxin from the blue-green alga Microcystis aeruginosa. S. Afr. J. Sci. 78, 363-366. 11. RUNNEGAR, M.T.C., and I.R. FALCONER, 1986: Effect of toxin from the cyanobacterium Minocystis aeruginosa on ultrastructural morphology and actin polymerization in isolated hepatocytes. Toxicon 24, 109- 115.

Cytotoxicity of cyanobacterium Microcystis aeruginosa.

Cytotoxic effects of crude extracts and fractions of the purification steps towards Microcystin-LR (MCYST-LR) were investigated in vitro. Cytotoxicity...
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