The potential contribution of marine organisms to the discovery of new bioactive molecules is increasingly challenging [1,2]. Natural products have been isolated from a wide variety of taxa and tested for various biological activities. Among these taxa, cyanobacteria are regarded as good candidates for drug discovery, with applications in agriculture [3], industry [4] and especially, in pharmaceuticals [5]. Although cyanobacteria are still primarily viewed as an environmental nuisance or a source of toxins, hazardous to man and aquatic livestock, there are many potential benefits to research on chemicals produced by these organisms. Antibacterial, antiviral, antifungal, algicide and cytotoxic activities have been reported [6-11]. The role of bioactive molecules in the producer organism itself is poorly understood but, considering the wide spectrum of biological adaptations and tolerance to environmental stress revealed by cyanobacteria, some of these compounds can be produced in an attempt to confer advantages for their survival.

Research on biological active compounds produced by cyanobacteria has been focused especially on freshwater species. Studies on marine species are more restricted and are specially related to filamentous forms from tropical regions [10], Baltic Sea [12] and Mediterranean Sea [9]. In this work we describe biological activities of marine cyanobacteria strains belonging to the genera Synechocystis and Synechococcus isolated from the temperate Atlantic coast of Portugal. We screened marine cyanobacteria aqueous extracts and extracts obtained with organic solvents for antifungal and antibacterial activity. Cytotoxic activity was also investigated using primary rat hepatocytes and human HL-60 cells.

For the cytotoxic assay using HL-60 cells, strong apoptotic effects were observed when cells were exposed to the methanol and/or dichloromethane extracts (Figure 1 and Figure 2 respectively). This was specially evident for strains LEANCYA 5, 11, 13, 19 and 20. In the aqueous extracts the most notorious effect was observed for strain LEANCYA 19 (Figure 3). The HL-60 cell death resembled apoptosis in most cases, but had clearly necrotic features with some extracts. However, this is probably secondary necrosis, since early (4 to 10 h) evaluation of the cells indicated apoptosis as seen by cell shrinkage and membrane budding (not shown).

For the cytotoxic assays using primary rat hepatocytes, only the aqueous extract of the LEANCYA 11 strain showed mild toxicity (11.4% apoptotic cells). No significant apoptotic cells were registered for the treatments with the methanol and the dichloromethane extracts in any of the cyanobacterial strains tested.

With regards to the antimicrobial assays, the antibacterial effects of extracts from nine cyanobacteria strains against two Gram-positive bacteria, Clavibacter michiganensis subsp. insidiosum and Cellulomonas uda were determined. None of the tested extracts of the different cyanobacterial strains showed inhibitory effect on Gram-negative bacteria. In addition, no antifungal activity was found against the yeast Candida albicans. Table 3 summarizes all extracts that showed activity against the two gram-positive bacteria. Although the intensity of the inhibitory effects was variable between the extracts, methanolic extracts were the least toxic and the dichloromethane extract was the one with more pronounced inhibitory effects. In plates where disks were treated only with the solvents, no inhibitory growth was registered. The antibacterial substances used as control showed growth inhibition.

Marine cyanobacteria are known for the ability to produce cytotoxic compounds [17-19]. None of the tested extracts had microcystins. One of the cytotoxic effects of cyanobacteria is manifested by the capability to induce apoptosis in several eukaryotic cell lineages. Cyanobacterial extracts or purified compounds were found to cause apoptosis in hepatocytes, lymphocytes, endothelial cells, human skin fibroblasts, human embryonic intestine cells and promyelocytes HL-60 cells [7,20-22].

HL-60 cells are known to provide a useful model system to explore the ability of various substances to cause apoptosis [23]. These cells are an established model in biochemistry, as one can observe fundamental and critical signals involved in the activation of the body's immune system because of their ability to respond to foreign organisms. The cyanobacterial extracts tested in the present work against HL-60 cells clearly undergo apoptosis, as evidenced by DNA condensation. For this cell lineage the percentage of apoptotic cells was evident especially in the extracts obtained with organic solvents and, among these extracts, effects were particularly accentuated in the dichloromethane extract.

Rat hepatocytes have been extensively used to investigate the induction of apoptosis by cyanobacterial secondary metabolites, specially due to the potential production of liver specific toxins such as microcystins and nodularins. Our results showed only slight toxic effects on primary rat hepatocytes which led us to exclude the production of these liver specific toxins or its production in small quantities. It is possible that the compounds that caused apoptosis in HL-60 cells can also cause apoptosis in hepatocytes but in a slower way and so more time is needed for observing the apoptotic phenomenon. One possibility is that the toxins act on mechanisms that are needed for cell division. HL-60 cells have a short cell cycle and compounds interfering with mitosis will lead to apoptosis within 4 to 10 hours. Such compounds have been found in cyanobacteria [24,25].

Antimicrobial activity of cyanobacteria refer almost to filamentous strains belonging to a wide range of genera [6,8-10,14,26]. Rao [6], working on the production of antibiotic compounds by filamentous forms of cyanobacteria, referred that the ability to produce antibiotic compounds might prevent bacterial contamination of the filaments and consequently pathogenic bacterial colonization. In contrast with research on antimicrobial compounds produced by filamentous cyanobacteria, relatively little has been reported on the antimicrobial activity of cyanobacteria of the Chroococcales group, namely of the genera Synechoccystis and Synechoccocus. As marine cyanobacteria tested in this research belong to Synechoccystis and Synechoccocus genera, our results emphasise the potential of these genera as a source of antibiotic compounds. In terms of antibacterial activity our results fits into the picture that antibacterial activity of cyanobacteria is primarily directed against Gram-positive bacteria [9,14]. This finding can be related to the fact that most Gram-negative bacteria are resistant to toxic agents in the environment due to the barrier of lipopolysaccharides of their outer membrane [27]. The difference in toxicity against Gram-positive and Gram-negative bacteria can also indicate that the mechanism of toxicity involves different functions in the two types of cells namely different permeability to the cyanobacteria compounds. In spite of the non toxic effect of cyanobacterial extracts to Gram-negative bacteria, we can have the possibility of the compounds to change the permeability of the cell outer membrane. This alteration could enhance the uptake of antibiotics, specially the hydrophobic ones, as it was demonstrated for the cyanobacterial toxin microcystin-RR [27].

None of the marine bacteria used as target organisms was inhibited by cyanobacteria extracts. In spite of these results we can not disregard the possibility of production of antibiotic compounds against marine bacteria since, with the exception of the specie Streptococcus parauberis isolated from marine fish (Table 2), all the marine bacteria tested were Gram-negative. Antifungal activities of cyanobacteria have been less found than antibacterial activities. Moore et al. [28] found that in more than 1000 cyanobacteria strains only 9% were able to inhibit fungal growth. Also Kreitlow et al. [14] found no activity against a fungi but inhibitory activity against Gram-positive bacteria.

It is interesting to note that the cyanobacterial strains and extracts that caused higher percentage of apoptotic HL-60 cells were the ones that exhibited antibacterial activities. This is particularly true for strains LEANCYA 5, 11, 13, 19 and 20. It is possible that the same compounds can affect both eukaryotic and prokaryotic cell lines but it is also possible that the same cyanobacteria strain produces different bioactive substances targeted against different organisms or biochemical processes.

In this work we were able to show that marine cyanobacterial strains of the genera Synechocystis and Synechococcus produce substances with inhibitory effects on prokaryotic cells and with apoptotic activity in eukaryote cell lines, which highlights the importance of these organisms as potentials pharmacological agents. Since different activities were observed in extracts obtained with organic solvents and extracts obtained with water we can suggest that compounds with different polarities are involved.