Screening the toxic potential of Cylindrospermopsis raciborskii strains isolated from Lake Balaton, Hungary

Abstract

Cylindrospermopsis raciborskii is becoming a major concern among cyanobacteria, due to its potential ability to produce toxic metabolites. We assessed the cytotoxic potential of four C. raciborskii strains (ACT 9502, ACT 9503, ACT 9504 and ACT 9505) isolated from Lake Balaton (Hungary), by lactate dehydrogenase (LDH) leakage measurements and by detecting morphological alterations in CHO-K1 (Chinese Hamster Ovary) cells. The Australian AQS (cylindrospermopsin producer) strain of C. raciborskii and purified cylindrospermopsin (CYN) were used as positive references in both the biochemical and morphological studies. Chemical analysis for known cyanotoxins was performed on aqueous extracts of ACT and AQS strains by the HPLC-MS technique.

Comparing threshold values of LDH leakage data, different toxic potentials of cyanobacterial extracts are suggested in short term (3 h) and long (24 h) exposure regimes. In the acute (3 h) experiments the aqueous extract of the ACT 9505 strain proved to be most toxic (EC₅₀ = 7.4 mg mL⁻¹), while after 24 h the ACT 9504 extract was the most effective (EC₅₀ = 0.65 mg mL⁻¹). The extract of the AQS strain and the purified CYN exerted most of their toxic effects after 3 h exposure (EC₅₀ = 0.74 mg mL⁻¹, and 0.9 μg mL⁻¹ respectively).

The morphological changes of CHO-K1 cells induced by the crude extracts of the ACT strains included fragmentation of the actin filaments then relocation of the depolymerized actin to the perinuclear region, resulting cell rounding and loss of adhesion. Exposure of CHO-K1 cells to the crude extract of the AQS strain, moreover, resulted cell shrinking and formation of filopodia, i.e. distinctly different cytological alterations from that induced by the ACT extracts and the purified CYN.

Chemical analysis of the cyanobacterial crude extracts confirmed the presence of cylindrospermopsin in the extract of the AQS strain (8.5 mg CYN g⁻¹ dry weight), and none of the presently known cyanotoxins have been analytically confirmed in the extracts of the ACT strains isolated from the Lake Balaton.

Although a significant toxicity of all four ACT C. raciborskii strains is confirmed by both biochemical and morphological studies, our results also pointed out the necessity of further studies to identify the toxic, but still unknown metabolic components produced by these cyanobacterial members of the phytoplankton communities.

Introduction

The freshwater cyanobacterium Cylindrospermopsis raciborskii (Woloszynska) Seenayya and Subba Raju (Nostocales, Cyanopyceae) is a highly successful competitor of the phytoplankton community due to its high affinity for phosphorus and high P-storage capacity (Istvánovics et al., 2000); superior shade tolerance (Briand et al., 2002); N₂-fixation; resistance to grazing; buoyancy control; ability to form akinetes, thus allowing easy dispersal and environmental resistance (Padisák, 1997); and wide thermal tolerance (Briand et al., 2004), as demonstrated by its nowadays worldwide distribution (Bouvy et al., 2000, Chapman and Schelske, 1997, Dokulil and Mayer, 1996, Padisák, 1997, Saker et al., 2003). The increased proliferation of C. raciborskii is of particular concern due to the ability of certain strains to produce various toxins, from potent hepatotoxins (CYN: cylindrospermopsin), to neurotoxins (PSPs: paralytic shellfish poisons), (Berry et al., 2009, Saker et al., 2003, Stucken et al., 2009). Despite many strains of this cyanobacterium isolated in Europe have been found to be toxic in the mouse bioassay (Bernard et al., 2003; Fastner et al., 2003, Saker et al., 2003) the identification of the toxic metabolites of concern remained elusive.

In Hungary, the first reports of C. raciborskii were documented in the 1970s. In Lake Balaton this species appeared first in 1978 and has gradually become the dominant blue-green algae species blooming several times since 1982, with maximal biomasses between 70 and 160 μg L⁻¹ (Présing et al., 1996). Investigations to elucidate the toxic potential of C. raciborskii strains isolated from Lake Balaton resulted in various and often controversial results. The first strain studied (NIVA-CYA 225) proved to be toxic in the mouse bioassay, evolving protracted type symptoms (Underdal et al., 1999). Another isolate (ACT 9502), elicited toxicity both on invertebrate and vertebrate models (Hiripi et al., 1998), moreover electrophysiological tests suggested the production of anatoxin-a like metabolites, as revealed by the cholinergic blocking effects recorded on molluscan neurons (Kiss et al., 2002, Vehovszky et al., 2009). Other toxicological investigations carried out on the ACT 9502 strain, failed to confirm its toxicity by the mouse bioassay (Bernard et al., 2003). The continuous presence/dominance of C. raciborskii in Lake Balaton, along with the controversial reports of its toxic potential, made a comprehensive research necessary to assess the environmental risks of this cyanobacterium.

There is an increasing number of evidence, that concern on risks posed by cyanobacterial blooms are not related to the production of certain toxins only. Cyanobacteria produce compounds that, although non-toxic themselves, can modify the effects of the known toxins (Buryskova et al., 2006, Falconer, 2007, Oberemm et al., 1997, Pietsch et al., 2001). Consequently, in the health risk assessment of cyanobacterial biomasses, the expansion of assays with focus also on the mechanism of action based screening of the complex cyanobacterial material receives increasing priority (reviewed by Pegram et al., 2008).

In the present work we tested the toxic potential of four non-axenic C. raciborskii strains isolated from Lake Balaton during 1994–1995. Similar studies were carried out with the Australian cylindrospermopsin (CYN) producer AQS C. raciborskii strain (Saker et al., 2003) and purified cylindrospermopsin (CYN). The cytotoxic effects of the above substances were assessed by LDH leakage measurements and assessment of cytoskeletal alterations in Chinese Hamster Ovary (CHO-K1) cells exposed in acute (3 h) and sub-chronic (24 h) exposure regimes.