Bojan Sedmak

Microcystin-LR induces oxidative DNA damage in human hepatoma cell line HepG2.

Microcystins are naturally occurring hepatotoxins produced by strains of Microcystis aeruginosa. They are involved in promoting primary liver tumours and a previous study showed that they might also be tumour initiators. In this study we demonstrate that microcystin-LR (MCLR) at doses that were not cytotoxic (0.01-1 microg/ml), induced dose and time dependent DNA strand breaks in human hepatoma cell line HepG2. These DNA strand breaks were transient, reaching a maximum level after 4h of exposure and declining with further exposure. In the presence of the DNA repair inhibitors cytosine arabinoside (AraC) and hydroxyurea (HU), together with MCLR, DNA strand breaks accumulated after prolonged exposure. These results suggest that DNA strand breaks are intermediates, produced during the cellular repair of MCLR induced DNA damage. Digestion of DNA with purified, oxidative DNA damage specific enyzmes, endonuclease III (Endo III) and formamidopyrimidine-DNA glycosylase (Fpg) markedly increased DNA strand breaks in MCLR treated cells, providing evidence that a substantial portion of the MCLR induced DNA strand breaks originate from excision of oxidative DNA adducts. A hydroxyl radical scavenger (DMSO) significantly reduced MCLR induced DNA damage. From these results we conclude that MCLR induces formation of reactive oxygen species that cause DNA damage, and that MCLR may act as an initiator of liver cancer.

Nephrotoxic effects of chronic administration of microcystins -LR and -YR

Acute intoxication with MC-LR induces cytoskeletal alterations, apoptosis and necrosis of hepatocytes resulting in intrahepatic hemorrhage. Preliminary results have shown that chronic treatment of rats with intraperitoneal injections of sublethal doses of microcystins MC-LR and MC-YR could induce not only liver, but also kidney injuries. We aimed to investigate whether the induction of the cytoskeletal changes, apoptosis and necrosis could be the mechanisms involved in the injury of kidney cells in the chronic model of microcystin intoxication. Experimental rats were receiving intraperitoneal injections of MC-LR (10 microg/kg) or MC-YR (10 microg/kg) every second day for 8 months, while control rats were receiving only the vehicle. The histopathological investigation revealed collapsed glomeruli with thickened basement membranes and dilated tubuli filled with eosinophilic casts. Rhodamine-phalloidin labeling showed cytoplasmic aggregation and accumulation of fibrilar actin filaments within the epithelial tubular cells. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) showed increased number of TUNEL-positive cells in the kidney cortex and medulla. The pathological changes induced by MC-LR appeared more severe than those induced by MC-YR. The results support the view that at the cellular level, the mechanisms that underly the chronic nephrotoxicity are similar to the mechanisms of the acute hepatotoxicity of microcystins.

The role of microcystins in heavy cyanobacterial bloom formation

The presence of high microcystin concentrations in cyanobacterial blooms additionally affects species diversity. Blooms with high toxin contents can reach higher cell densities, which is also demonstrated by microcystin cell contents. In vitro experiments show that microcystins influence phytoplankton proliferation. The action is strongly dependent on the phytoplankton species tested and light conditions. We propose that the environmental impact of different microcystins depends on their enzymatic inhibition activity and thus could not be measured merely on the basis of their toxicity to vertebrate species. Their role in heavy cyanobacterial bloom and scum formation is discussed, as well as their impact on the massive proliferation of other species following toxic cyanobacterial bloom degradation. Introduction The majority of authors point out the significance of cyanobacteria as a health hazard to humans and agricultural livestock (e.g. Codd, 1994; Falconer et ai, 1994). Nevertheless, we would like to emphasize the negative impact cyanobacterial blooms have on the aquatic environment. Besides the health hazard to higher vertebrates, they have a great influence on general environmental health and must be considered as a warning of environmental degradation. Eutrophication, together with blooms as the consequence, has a strong influence on all living organisms in water bodies (e.g. Harper, 1992). There are a great number of measurable physicochemical and environmental factors that are responsible for phytoplankton distribution and species diversity, including bloom formation. These factors can affect all the organisms in a water body or only specific, more susceptible ones, having regard to the conditions, amounts or concentrations. Variations in day length, insolation, temperature, wind mixing, rainfall, flushing and nutrient loading rates affect all kinds of water bodies, but are more pronounced in smaller ones. Physicochemical parameters vary more widely in ponds and reservoirs than in deep natural lakes, and reflect more or less only temporary conditions. As blooms are frequent in smaller water bodies in Slovenia (Sedmak et al., 1994; Sedmak and Kosi, 1997), we focused our research mainly on phytoplankton species abundance and composition as an indicator of environmental status. In our opinion, phytoplankton species are the best reflection of long-lasting physicochemical and biological factors, which are themselves functions of the climatic and hydrological regimes, since the majority of freshwater phytoplankton organisms incorporate resistant benthic resting stages into their life history strategies. In this way, planktonic algae can compensate for noxious exogenic short-term effects. Despite the fact that cyanobacteria have lower maximal growth rates than the majority of other phytoplankton species, in certain conditions they are able to outstrip all other species, resulting in cyanobacterial blooms. Their chances of

The cytotoxic and cytolytic activity of equinatoxin II from the sea anemone Actinia equina.

The cytotoxic and cytolytic effects of equinatoxin II (EqT II) from the sea anemone Actinia equina L. were studied on exponentially growing and synchronized V-79-379 A cell line in culture. The cell viability test and the determination of the cytolytic effect by cell counting confirmed both cytotoxic and cytolytic activity of EqT II. Additionally, cytocidal and cytostatic effects depending on the toxin concentration were observed. The presence of fetal calf serum in the cell culture medium reduced both cytocidal and cytostatic effects by two magnitudes and prevented cytolysis. Combining EqT II and serum resulted in an insoluble complex which was cytostatic even when isolated and resuspended in the culture medium, while the supernatant retained both cytocidal and cytostatic activity. No significant difference in sensitivity between synchronized and exponentially growing cells could be detected after EqT II treatment.

Microcystins in Slovene freshwaters (Central Europe)—first report

Cyanobacterial blooms are frequent in the North-Eastern region of Slovenia, where the agricultural activities are intensive, resulting in higher water eutrophication. In a two year monitoring program we identified eighteen blooms of cyanobacteria, fifteen being hepatotoxic by intraperitoneal mouse bioassay. The approximate LD100 varied from 50-1000 mg/kg (dry cell weight/animal weight) and gross pathological signs were characteristic of cyanobacterial hepatotoxins. Frequently the blooms were dominated by the most common and cosmopolitan species Microcystis aeruginosa. Other bloom forming species were M. wesenbergii, Oscillatoria rubescens, Anabaena flos-aquae, and Aphanizomenon flos-aquae. Using high-performance liquid chromatography (HPLC), three hepatotoxins were identified, microcystin RR being the most frequent and present in highest amounts, LR, and YR. The phytoplankton analysis revealed that several different species of cyanobacteria were present in the water bodies at the time of the blooms. Although present in various water bodies, filamentous bloom-forming cyanobacteria had no chance to proliferate in the presence of the colonial genus Microcystis. In individual cases we were faced with a bloom in the bloom, meaning that various Microcystis aeruginosa blooms were heavily contaminated with another cyanobacteria, Phormidium mucicola which infested the mucilage of the chroococcal species.

Microcystin-LR affects cytoskeleton and morphology of rabbit primary whole embryo cultured cells in vitro

Microcystin-LR is the most frequently studied cyclic heptapeptide produced by different genera of cyanobacteria and is hepatotoxic to livestock and human populations. The adverse effects of microcystin-LR on morphology and cytoskeletal elements in different stages of early embryonal development have been studied in vitro. Embryos and whole embryo cultures have been exposed to microcystin-LR (10-100 microM). Actin filaments were visualized by fluorescence staining and the microtubular network labelled by immunostaining. Growth, development and cytoskeleton organization of the embryos embedded in zona pellucida are not affected by microcystin-LR in concentrations up to 100 microM, while whole embryo cell cultures are affected by the presence of microcystin-LR in the culture medium. High microcystin-LR concentrations (100 microM) cause cells to be detached and destroyed, while lower concentrations (10-20 microM) profoundly affect actin and microtubule organization. These effects are confirmed also by the presence of transformed microcystin-LR in all the media at the lowest concentrations. It seems that the changes to the cells are far more serious than that expressed in cell morphology. From our experiments we conclude that the presence of zona pellucida is an effective way of embryo protection against xenobiotics like microcystin-LR.

Occurrence ofDinophysis spp. and toxic shellfish in the Northern Adriatic

The dynamics of the toxicity of the musselMytilus galloprovincialis was compared between two different shellfish farms, 5 km apart, but using the same cultivation technique. The main differences concerned the freshwater influx and the open aspect to the Gulf of Trieste. It is suggested that a deep closed bay and abundant fresh water inflow are the two main conditions for the low toxicity levels in mussels and for shorter periods of danger. A detailed study of the phytoplankton samples revealed the presence of eight species ofDinophysis in the area of both shellfish farms. During the period of the DSP outbreak in Slovenia (autumn and winter 1989).D. fortii andD. acuminata were the most frequentDinophysis species. There was a high positive correlation between the onset of mussel toxicity and the appearance ofDinophysis spp.

Subchronic exposure of rats to sublethal dose of microcystin-YR induces DNA damage in multiple organs

Subchronic exposure of rats to sublethal dose of microcystin-YR induces DNA damage in multiple organs Background. Microcystins (MCs) are cyclic heptapeptides that are considered to be liver specific toxins. They are potent tumour promoters and recent studies indicate that they are also genotoxic. In this study we measured DNA damage in lymphocytes, liver, kidney (cortex and medulla), lung, spleen and brain cells of male Fisher F344 rats that were exposed to sublethal dose (every second day 10 μg/kg b.w.; i.p) of micro-cystin-YR (MCYR) for one month. Methods. At the end of exposure the animals were sacrificed, the lymphocytes were isolated from blood taken from jugular vein, liver cells were obtained by perfusion with collagenase A and the cells from other organs were isolated by incubating small tissue pieces with collagenase A. The DNA damage in isolated cells was measured with the single cells gel electrophoresis (SCGE) also called the comet assay. Results. A significant increase of the % tail DNA in MCYR-exposed animals compared to the nonexposed control ones was observed in brain (2.5 fold), liver (2.1 fold), kidney medulla (1.9 fold), kidney cortex (1.8 fold) and lung (1.7 fold) cells, while the DNA from lymphocytes and spleen cells was not affected. Conclusion. This study demonstrated that subchronic exposure to sublethal doses of MCs can induce systemic genotoxicity in mammals, and it affects not only the liver but also other vital organs.

Ecotoxicologically relevant cyclic peptides from cyanobacterial bloom (Planktothrix rubescens) - a threat to human and environmental health

Ecotoxicologically relevant cyclic peptides from cyanobacterial bloom (Planktothrix rubescens) - a threat to human and environmental health Background. The information of the overall production of major cyanobacterial cyclic peptides in a water body is essential for risk assessment and for the prediction of future development of the bloom. A procedure that gives a review of both toxic and non-hepatotoxic hydrophilic cyclic peptide production is important to evaluate the ecological conditions in the water environment and to predict the release of dangerous toxic and tumour promoting substances. Methods. The cyclic peptides were identified on the basis of their retention times, characteristic spectra, molecular masses and biological activity. The non-hepatotoxic cyclic peptides were characterised by their inhibition of porcine pancreatic elastase, while cytotoxicity to mammalian cells was tested with the MTT test on B16 cell line. Conclusions. The method presented gives a rapid, simultaneous assessment, preliminary identification and estimation of bioactive cyclic peptides. The synthesis of non-hepatotoxic cyclic peptides can mediate the release various toxic and otherwise biologically active substances that induce systemic genotoxicity in mammals.

The humoral and cellular immune response to a lipid attenuated pore-forming toxin from the sea anemone Actinia equina L.

The immunogenicity of a pore-forming polypeptide, equinatoxin II, from the sea anemone Actinia equina was studied after attenuation of the toxins lethal and cytolytic activity by autologous polar lipids. In BALB/c mice, the lipid-inactivated toxin was used to raise specific antibodies and cellular immunity, resulting in in vivo protection. In vitro, haemolytic activity could be diminished by both normal and immune serum, the latter being more efficient. Purified specific IgG1 and IgG2 did not or only poorly neutralized the haemolytic activity, therefore implying the marked role of serum lipoproteins in the toxin attenuation. In response at the cellular level, equinatoxin II activated specific splenocytes. Increased concanavalin A stimulation of specific splenocytes was observed in the absence of antigen.