Review of Cyanotoxicity Studies Based on Cell Cultures

Cyanotoxins (CTs) are a large and diverse group of toxins produced by the peculiar photosynthetic prokaryotes of the domain Cyanoprokaryota. Toxin-producing aquatic cyanoprokaryotes can develop in mass, causing “water blooms” or “cyanoblooms,” which may lead to environmental disaster—water poisoning, extinction of aquatic life, and even to human death. CT studies on single cells and cells in culture are an important stage of toxicological studies with increasing impact for their further use for scientific and clinical purposes, and for policies of environmental protection. The higher cost of animal use and continuous resistance to the use of animals for scientific and toxicological studies lead to a progressive increase of cell lines use. This review aims to present (1) the important results of the effects of CT on human and animal cell lines, (2) the methods and concentrations used to obtain these results, (3) the studied cell lines and their tissues of origin, and (4) the intracellular targets of CT. CTs reviewed are presented in alphabetical order as follows: aeruginosins, anatoxins, BMAA (β-N-methylamino-L-alanine), cylindrospermopsins, depsipeptides, lipopolysaccharides, lyngbyatoxins, microcystins, nodularins, cyanobacterial retinoids, and saxitoxins. The presence of all these data in a review allows in one look to advance the research on CT using cell cultures by facilitating the selection of the most appropriate methods, conditions, and cell lines for future toxicological, pharmacological, and physiological studies.


Introduction
Cyanotoxins (CTs) are a large and diverse group of toxins produced by the peculiar photosynthetic prokaryotes of the domain Eubacteria, commonly known as cyanobacteria or blue-green algae, and since 1999 named Cyanoprokaryota [1,2]. Some aquatic cyanoprokaryotes can develop in mass, causing so-called "water blooms" or "cyanoblooms" [3]. When such blooms are formed by toxin-producing cyanoprokaryotic algae, they are considered harmful and are usually abbreviated as Cyano-HABs. e toxic substances are transported through the food webs and may reach people and animals by drinking water, or through other exposure routes, which include recreational activities or consumption of so-called "seafood", which includes both freshwater and marine organisms [3][4][5].
e excretion of toxic compounds may lead to environmental disasters-water poisoning, extinction of aquatic life, and even to human death [3][4][5]. Current climate changes and anthropogenic press can intensify and increase the frequency of these hazardous ecological events [3,6]. Although most research addresses aquatic toxin producers, there is a growing body of evidence on such producers from aeroterrestrial and extreme habitats, and among airborne algae as well, with a considerable number of detected toxins and outlining of additional exposure route through consumption of crops, which have been irrigated by contaminated water [7,8].
Different approaches have been applied to classify CT, two of which are the most common: by the target of their action, or by chemical composition. By target, CT are classified as hepatotoxins, neurotoxins, dermatoxins, and cytotoxins, whereas chemically they are divided in peptides, alkaloids, phosphorylated cyclic N-hydroxyguanine, diaminoacids, and lipopolysaccharides, the last widely recognized as endotoxins. Prolonged use of drinking water, contaminated with low-doses CTs, may have also carcinogenic effect [6].
us, microcystin-LR (MC-LR), the most toxic MC, is considered to express tumor promoting effect mainly by violating phosphorylation-dependent regulations of cellular proteins [new 9 Brozman et al., 2020]. e pleiotropic downstream mechanisms link MC-LR-dependent inhibition of eucaryotic protein phosphatases (PPs) PP1, PP2A, phospho-PP4, and phospho-PP5 [2] to tumor promotion and neoplastic transformation by cell growth induction, reactive oxygen species (ROS) generation, oxidative stress, mitochondrial DNA impairment, and by the transformation of cell phenotype [9]. Chronic proinflammatory effect of MC-LR alone or a combination with another CT-like cylindrospermopsin (CYN) may additionally stimulate the neoplastic transformation and tumor progression [6,10].
Cell cultures are very convenient for toxicological studies. ey allow to reveal the mechanisms of cytotoxic effects, the affected tissues, intracellular targets, and ways to minimize cytotoxicity [11]. e use of human cell lines in toxicological studies is a fast and effective way to investigate the damaging effects of toxins in humans and to identify the most sensitive tissues.
Although different methods are developed for testing of toxins in cell-and animal-based studies, during the last years, the trials on the use of animals have significantly decreased. is is caused by the high cost of these types of clinical trials and increasing resistance to the use of animals for scientific studies. erefore, the significance and use of cell lines is gradually increasing.
is review aims to present (1) the important results of the effects of CT on human and animal cell lines; (2) the methods and concentrations used to obtain these results, (3) the studied cell lines, and (4) the intracellular targets of CT. e presence of all these data in a review allows in one look to advance the toxicological and pharmacological studies of CT using cell cultures by facilitating the selection of the most appropriate methods, conditions, and cell lines. (Table 1). Aeruginosin CT contains as a basic structure 2-carboxy-6-hydroxyoctahydroindol that are serine protease inhibitors [12]. ey inhibit trypsin-like serine proteases and for this activity are important in the search for new anticoagulants [13]. (Table 2). Anatoxins-a are two types of low molecular bicyclic amino alkaloids: anatoxin-a (ANTX) and homoanatoxin-a (hANTX). e best known of them is ANTX, which was the first to be identified as a low molecular alkaloid (165 Da). hANTX is a homologue of anatoxin-a with molecular weight 179 Da and has propionyl instead of an acetyl group at C-2. ANTX and anatoxin-a (S) (ANTX(S)) are neurotoxins. ANTX binds competitively to acetylcholine receptors, while anatoxin-a (S) inhibits acetylcholine esterase [2].

Cytotoxicity of Nodularins
NODs have similar effects as microcystins and weak carcinogenicity [3]. (Table 10). Retinol, a novel retinoic acid (RA) analogue 7-hydroxy RA, 4-oxo-RA, and several analogues were identified in cyanobacterial blooms [110]. ey act as RA receptors that may cause different malformations, as well as to have a teratogenic effect on aqueous animals. (Table 11). Saxitoxin (SXT) is a collective name for a group of more than 20 cyclic nonribosomal peptide molecules, formed by sulphation at different sites of two basic molecules: SXT and neo-SXT. Based on their toxicology, SXT are grouped in three classes-carbamate derivatives, gonyautoxins, N-sulfocarbomoyl derivatives, and decarbomoyl derivatives-decarbamoylsaxitoxin. ey have a neurotoxic effect by blocking voltage-gated sodium channels [3].

Limitations
Studies on cell cultures cannot reveal all possible effects of toxins on the human body.

Perspectives
e use of cell cultures in toxicological studies will remain the main approach due to its speed, relatively low cost, reproducibility, precision with respect to the studied intracellular components, and ethical acceptability. e use of cell cocultures [116][117][118] and in vitro formed organ-like structures such as artificial neuronal network [119], cardiomyocyte spheroids with contractile activity [120], and organ-on-a-chip systems [121], which are functionally closer to the human body [11], will increase in the future.

Conclusion
e presence of all these data on the cytotoxicity of aeruginosins, anatoxins, cylindrospermopsin, depsipeptides, lipopolysaccharides, lyngbyatoxins, microcystins, nodularins, cyanobacterial retinoids, and saxitoxins in a review is a great advantage. It allows the advancement of research on CT using cell cultures by facilitating the selection of the most appropriate methods, conditions, and cell lines for toxicological and pharmacological studies. In addition, it could increase the use of CT in functional studies of their intracellular targets. erefore, this review allows in one look to advance the toxicological, physiological, and pharmacological studies of CT by the knowledge of their harmful effects with a focus on human and animal health as well as on environmental protection.