Multibiomarker-based assessment of toxicity of central European strains of filamentous cyanobacteria Aphanizomenon gracile and Raphidiopsis raciborskii to zebrafish Danio rerio.

The global increase in cyanobacterial blooms poses environmental and health threats. Selected cyanobacterial strains reveal toxicities despite a lack of synthesis of known toxic metabolites, and the mechanisms of these toxicities are not well understood. Here we investigated the toxicity of non-cylindrospermopsin and non-microcystin producing Aphanizomenon gracile and Raphidiopsis raciborskii of Central European origin to zebrafish exposed for 14 days to their extracts. Toxicological screening revealed the presence of anabaenopeptins and a lack of anatoxin-a, ß-methylamino-L-alanine or saxitoxins in examined extracts. The responses were compared to 20 µg L-1 of common cyanobacterial toxins cylindrospermopsin (CYN) and microcystin-LR (MC-LR). The expression of the marker genes involved in apoptosis (caspase 3a and 3b, Bcl-2, BAX, p53, MAPK, Nrf2), DNA damage detection and repair (GADD45, RAD51, JUN, XPC), detoxification (CYP1A, CYP26, EPHX1), lipid metabolism (PPARa, FABP1, PLA2), phosphorylation/dephosphorylation (PPP6C, PPM1) and cytoskeleton (actin, tubulin) were examined using targeted transcriptomics. Cellular stress and toxicity biomarkers (oxidative injury, antioxidant enzymes, thiol pool status, and lactate dehydrogenase activity) were measured in the liver, and acetylcholinesterase activity was determined as an index of neurotoxicity in the brain. The extracts of three cyanobacterial strains that produce no known cyanotoxins caused marked toxicity in D. rerio, and the biomarker profiles indicate different toxic mechanisms between the bioactive compounds extracted from these strains and the purified cyanotoxins. All studied cyanobacterial extracts and purified cyanotoxins induced oxidative stress and neurotoxicity, downregulated Nrf2 and CYP26B1, disrupted phosphorylation/dephosphorylation processes and actin/tubulin cytoskeleton and upregulated apoptotic activity in the liver. The tested strains and purified toxins displayed distinctively different effects on lipid metabolism. Unlike CYN and MC-LR, the Central European strain of A. gracile and R. raciborskii did not reveal a genotoxic potential. These findings help to further understand the ecotoxicological consequences of toxic cyanobacterial blooms in freshwater ecosystems.

In Vitro Toxicological Screening of Stable and Senescing Cultures of Aphanizomenon, Planktothrix, and Raphidiopsis.

Toxicity of cyanobacteria is the subject of ongoing research, and a number of toxic metabolites have been described, their biosynthesis pathways have been elucidated, and the mechanism of their action has been established. However, several knowledge gaps still exist, e.g., some strains produce hitherto unknown toxic compounds, while the exact dynamics of exerted toxicity during cyanobacterial growth still requires further exploration. Therefore, the present study investigated the toxicity of extracts of nine freshwater strains of Aphanizomenon gracile, an Aphanizomenon sp. strain isolated from the Baltic Sea, a freshwater strain of Planktothrix agardhii, and two strains of Raphidiopsis raciborskii obtained from 25- and 70-day-old cultures. An in vitro experimental model based on Cyprinus carpio hepatocytes (oxidative stress markers, DNA fragmentation, and serine/threonine protein activity) and brain homogenate (cholinesterase activity) was employed. The studied extracts demonstrated toxicity to fish cells, and in general, all examined extracts altered at least one or more of considered parameters, indicating that they possess, to some degree, toxic potency. Although the time from which the extracts were obtained had a significant importance for the response of fish cells, we observed strong variability between the different strains and species. In some strains, extracts that originated from 25-day-old cultures triggered more harmful effects on fish cells compared to those obtained from 70-day-old cultures, whereas in other strains, we observed the opposite effect or a lack of a significant change. Our study revealed that there was no clear or common pattern regarding the degree of cyanobacterial bloom toxicity at a given stage of development. This means that young cyanobacterial blooms that are just forming can pose an equally toxic threat to aquatic vertebrates and ecosystem functioning as those that are stable or old with a tendency to collapse. This might be largely due to a high variability of strains in the bloom.